Compositions and methods for epigenetic regulation of HBV gene expression

Abstract

The present invention relates to compositions, methods, strategies and therapeutic regimens related to epigenetic modification of the hepatitis B virus (HBV) gene.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 409,607, filed on September 23, 2022, U.S. Provisional Application No. 63 / 502,328, filed on May 15, 2023, U.S. Provisional Application No. 63 / 516,063, filed on July 27, 2023, and U.S. Provisional Application No. 63 / 581,229, filed on September 7, 2023, each of which is incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION

[0003] Despite the availability of treatment options, chronic hepatitis B (CHB) remains a highly unmet medical need, with over 250 million hepatitis B virus (HBV) carriers globally and approximately 800,000 deaths per year due to HBV-related liver diseases. The functional cure rate (defined as the persistent disappearance of HBsAg and undetectable serum HBV after completion of a treatment course) of currently approved CHB therapies is less than 20%. Therefore, there is a need for improved clinical treatment modalities targeting HBV. SUMMARY OF THE INVENTION

[0004] Some aspects of the present disclosure provide systems, compositions, strategies, and methods for epigenetic modification of HBV, including HBV in host cells and organisms.

[0005] Some aspects of the present disclosure provide methods for modifying the epigenetic state of a hepatitis B virus (HBV) gene or genome, including 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 a first DNA-binding domain, a first DNMT domain, and a transcriptional repressor domain, 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: the number of HBV viral episomes, the replication of the HBV gene or genome, and / or the expression of the 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 wherein the reduction in the number of HBV viral episomes, the replication of the HBV gene or genome, or the expression of the 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 aspects of the present disclosure provide methods for treating HBV infection in a subject, including 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 a first DNA-binding domain, a first DNMT domain, and a transcriptional repressor domain, 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: the number of HBV viral episomes, the replication of the HBV gene or genome, and / or the expression of the 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 wherein the reduction in the number of HBV viral episomes, the replication of the HBV gene or genome, or the expression of the 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 aspects of the present disclosure provide methods for modulating the expression of HBV genes or genomes, including contacting the HBV genes or genomes 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 a first DNA-binding domain, a first DNMT domain, and a transcriptional repressor domain, 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 decrease in the expression of the gene product encoded by the HBV gene or genome, optionally, wherein the gene product is a nucleic acid or a protein, wherein the decrease is at least about 20% compared to contacting the HBV genome with a suitable control, and / or wherein the decrease in the gene product encoded by the HBV gene or genome is at least about 20% compared to the expression in the subject prior to administration. Some aspects of the present disclosure provide methods for inhibiting viral replication in cells infected with HBV, including 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 a first DNA-binding domain, a first DNMT domain, and a transcriptional repressor domain, wherein the first DNA-binding domain binds to a first target region of the HBV gene or genome, and wherein the epigenetic editing system targets a target region of the HBV gene or genome, and wherein the contacting results in a decrease in the number of HBV viral episomes or a decrease in the replication of the HBV gene or genome, wherein the decrease is at least about 20% compared to administering a suitable control, and / or wherein the decrease in the number of HBV viral episomes or the replication of the HBV gene or genome is at least about 20% compared to the number and / or replication in the subject prior to administration. Some aspects of the present disclosure provide methods, including 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 a first DNA-binding domain, a first DNMT domain, and a transcriptional repressor domain, 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 decrease in: the number of HBV viral episomes, the replication of the HBV gene or genome, or the expression of the protein product encoded by the HBV gene or genome, wherein the decrease is at least about 20% compared to administering a suitable control, and / or wherein the decrease in the number of HBV viral episomes, the replication of the HBV gene or genome, or the expression of the 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. In some embodiments, the HBV genome is 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 sequences 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 a portion of the HBV genome that encodes a transcript selected from 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) that comprises 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, such as the sequences in Table 14 and / or Table 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, such as the zinc finger motifs in Table 1. In some embodiments, the zinc finger protein comprises a 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 a sequence of the DNMT domain provided herein. In some embodiments, the epigenetic editing system further comprises a second DNMT domain or a nucleic acid encoding a second DNMT domain. 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 the DNMT domain provided herein. In some embodiments, the epigenetic editing system comprises a fusion protein or a nucleic acid encoding a fusion protein, and 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 the fusion protein provided herein.In some embodiments, the epigenetic editing system further comprises a second DNA binding domain or a nucleic acid encoding a second DNA binding domain, wherein the second DNA binding domain binds to a second target region of the HBV genome. In some embodiments, the second target region is 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 in a CpG island. In some embodiments, the second target region of the HBV genome is in a promoter. In some embodiments, the second target region of the HBV genome is in a portion of the HBV genome encoding transcripts selected from pgRNA, precure 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, which comprises a region complementary to the strand of the second target region. In some embodiments, the gRNA comprises a sequence selected from the gRNA sequences provided herein, such as the sequences provided and / or disclosed in Table 14 and / or Table 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 sequence selected from the zinc finger motif sequences provided herein, such as the zinc finger motifs provided in Table 1. In some embodiments, the zinc finger protein comprises the sequence of the zinc finger motif provided in Table 1. In some embodiments, the epigenetic editing system comprises a first fusion protein or a first nucleic acid encoding a first fusion protein and a second fusion protein or a second nucleic acid encoding a second fusion protein, wherein the first fusion protein comprises a first DNA binding domain and a first DNMT domain, and wherein the second fusion protein comprises a second DNA binding domain and a transcriptional repressor domain. In some embodiments, the first fusion protein comprises the sequence of the fusion protein provided herein. In some embodiments, the second fusion protein comprises the sequence of the fusion protein provided herein. In some embodiments, the epigenetic editing system further comprises a third DNA binding domain or a nucleic acid encoding a third DNA binding domain, wherein the third DNA binding domain binds to a third target region of the HBV genome. In some embodiments, the third target region is 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 in a CpG island. In some embodiments, the third target region of the HBV genome is in a promoter. In some embodiments, the third target region of the HBV genome is in a portion of the HBV genome encoding transcripts selected from pgRNA, precure 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, which comprises 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, such as the gRNA sequences provided and / or disclosed in Table 14 and / or Table 15. In some embodiments, 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 the 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 the second DNMT domain. 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 the third fusion protein, wherein the third fusion protein comprises the third DNA binding domain and the second DNMT domain. In some embodiments, the third fusion protein comprises the sequence of the fusion protein provided herein. In some embodiments, the epigenetic editing system comprises the nucleic acid sequence provided in Table 20. In some embodiments, the reduction in the number of HBV viral episomes, the replication of the HBV gene or genome, or the expression of the protein product encoded by the HBV gene or genome is at least about 20% as compared to the number of HBV viral episomes, the replication of the HBV gene or genome, or the expression of the 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, the replication of the HBV gene or genome, or the expression of the protein product encoded by the HBV gene or genome 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 more than 99.99% as compared to the number of HBV viral episomes, the replication of the HBV gene or genome, or the expression of the 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.

[0006] Some aspects of the present disclosure provide epigenetic editing systems comprising: a fusion protein or a nucleic acid encoding a fusion protein, wherein the fusion protein comprises: (a) a DNA binding domain that binds to a target region of the HBV gene or genome, (b) a first DNA methyltransferase (DNMT) domain, and (c) a transcriptional repressor domain. In some embodiments, the epigenetic editing system is capable of reducing the number of HBV viral episomes, HBV replication, or the expression of gene products encoded by the HBV gene or genome, wherein the reduction is at least about 20% as compared to contacting the HBV gene or genome with a suitable control. In some embodiments, the HBV genome is 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 sequences provided herein. In some embodiments, the target region is in a region of the HBV genome within nucleotides 0-303, 1000-2448, or 2802-3182 of the HBV genome sequences provided herein. In some embodiments, the target region of the HBV genome is in a CpG island. In some embodiments, the target region of the HBV genome is in a promoter. In some embodiments, the target region of the HBV genome is in a portion of the HBV genome encoding transcripts selected from 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 that comprises a region complementary to the strand of the target region. In some embodiments, the gRNA comprises a sequence selected from the gRNA sequences provided herein, such as the sequences in Table 14 and / or Table 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 the zinc finger motif provided in Table 1. In some embodiments, the transcriptional repressor domain comprises the sequence of the 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 the 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 the 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 wherein 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 wherein the second DNA binding domain binds to a second target region of the HBV genome. In some embodiments, the epigenetic editing system is capable of reducing the number of HBV viral episomes, HBV replication, or the 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. In some embodiments, the HBV genome is 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 wherein 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 portion of the HBV genome encoding transcripts selected from pgRNA, precuremRNA, 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 that comprises a region complementary to a strand of a first target region; a second gRNA that comprises a region complementary to a strand of a second target region; or a third RNA that comprises a region complementary to a strand of a third target region. In some embodiments, the first gRNA comprises a sequence selected from the gRNA sequences provided herein, such as the sequences provided and / or disclosed in Table 14 and / or Table 15, the second gRNA comprises a sequence selected from the gRNA sequences provided herein, such as the sequences provided and / or disclosed in Table 14 and / or Table 15, and / or the third gRNA comprises a sequence selected from the gRNA sequences provided and / or disclosed herein, such as the sequences provided and / or disclosed in Table 14 and / or Table 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 the 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 foregoing methods, the epigenetic editing system comprises the nucleic acid sequence provided in Table 20. In some embodiments, the reduction in the number of HBV viral episomes, the replication of the HBV gene or genome, or the expression of the protein product encoded by the HBV gene or genome is at least about 20% compared to that 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, the replication of the HBV gene or genome, or the expression of the protein product encoded by the HBV gene or genome 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 more than 99.99% compared to the number of HBV viral episomes, the replication of the HBV gene or genome, or the expression of the 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.

[0007] Some aspects of the present disclosure provide a method of treating HDV infection in a subject, 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 a first DNA-binding domain, a first DNMT domain, and a transcriptional repressor domain, wherein the first DNA-binding domain binds to a first target region of the HBV gene or genome, and wherein the contact results in a reduction in: the number of HDV viral episomes, the replication of the HDV gene or genome, or the expression of the protein product encoded by the HDV gene or genome, wherein the reduction is at least about 20% compared to administration of a suitable control. Some aspects of the present disclosure provide a method of inhibiting viral replication in a cell 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 a first DNA-binding domain, a first DNMT domain, and a transcriptional repressor domain, wherein the first DNA-binding domain binds to a first target region of the HBV gene or genome, and wherein the epigenetic editing system targets a target region of the HBV gene or genome, and wherein the contact results in a reduction in the number of HDV viral episomes or a reduction in the replication of the HDV gene or genome, wherein the reduction is at least about 20% compared to administration of 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) that comprises 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, such as the sequences in Table 14 and / or Table 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, such as the sequences in Table 1 or Table 20. In some embodiments, the zinc finger protein comprises the sequence of any zinc finger epigenetic repressor 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 a second DNMT domain. 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 a fusion protein, 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 the fusion protein provided herein. In some embodiments, the first DNA binding domain binds to a target region of the HBV gene or genome encoding or controlling the expression of the S antigen. In some embodiments, the epigenetic editing system comprises the nucleic acid sequence provided in Table 20. In some embodiments, the reduction in the number of HBV viral episomes, the replication of the HBV gene or genome, or the expression of the protein product encoded by the HBV gene or genome is at least about 20% compared to the number of HBV viral episomes, the replication of the HBV gene or genome, or the expression of the 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, the replication of the HBV gene or genome, or the expression of the protein product encoded by the HBV gene or genome 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 more than 99.99% compared to the number of HBV viral episomes, the replication of the HBV gene or genome, or the expression of the 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.

[0008] Other features, objects, and advantages of the invention will be apparent from the following detailed description. However, it should be understood that although the detailed description shows embodiments of the invention, it is given by way of illustration only and not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Schematic diagram showing an exemplary structure of the circular HBV genome. The HBV genes and CpG islands are indicated. Exemplary target sites for a CRISPR-based epigenetic repressor (red arrow) and a zinc finger-based epigenetic repressor (green arrow) are also indicated.

[0010] Figure 2 is a heat map showing the conservation of the guide RNA target domains among different HBV genotypes.

[0011] Figure 3 It is a bar graph showing the geographical distribution of different HBV genotypes.

[0012] Figure 4A It is a schematic diagram depicting the experimental timeline for testing different CRISPR-based epigenetic repressors in HepAD38 cells, which express HPV in a doxycycline-induced manner. Figure 4B It is a graph showing the repression of HBV by various CRISPR-based epigenetic repressors (#1.1 - 3.2). Controls: UT: untransfected control; GFP: transfection control without repressor; HBV-KO: CRISPR nuclease-mediated knockout; sgRNA scramble: CRISPR-based repressor with sgRNA not targeting HBV; B2M: CRISPR-based repressor with sgRNA targeting B2M.

[0013] Figure 5A It is a schematic diagram depicting the experimental timeline for testing different CRISPR-based epigenetic repressors in the HepG2-NTCP infection model (see, for example, Methods Mol Biol. 2017;1540:1 - 14). Figure 5B It is a graph showing the expression of HBe antigen (detected by ELISA) at different time points after treating HBV-infected Hep2G-NTCT cells with different doses of CRISPR-based epigenetic repressor (ETR) or different doses of Cas9 nuclease targeting HBV (Cas9), and the graph is plotted with the HBe antigen expression values measured in the negative control (blank) for normalization.

[0014] Figure 6 It is a schematic diagram depicting the experimental timeline for the guide RNA screening of different CRISPR-based epigenetic repressor systems in the HepG2-NTCP infection model, where ELISA readings for HBe and HBs antigens are taken on day 6.

[0015] Figure 7 It is a graph showing the QC results of different LNP batches used in the guide screening.

[0016] Figure 8 It is a bar graph showing the HBe and HBs expression of an exemplary CRISPR-based epigenetic repressor (#3.2), calculated as a percentage of the corresponding antigen expression measured against a non-targeting control.

[0017] Figure 9A graph showing the HBe expression values measured for different guides in a guide RNA screen (calculated as a percentage of HBe expression measured against a non-targeting control). Each guide / repressor combination is represented by a dot. The 50% repression cut-off is shown as a horizontal line. The position of the corresponding guide RNA in the HBV genome (shown at the bottom of the figure) is plotted on the x-axis. The red line indicates the position of exemplary guide RNA #3.2 and the modulation of the measured HBe expression.

[0018] Figure 10 A graph showing the HBs expression values measured for different guides in a guide RNA screen (calculated as a percentage of HBs expression measured against a non-targeting control). Each guide / repressor combination is represented by a dot. The 50% repression cut-off is shown as a horizontal line. The position of the corresponding guide RNA in the HBV genome (shown at the bottom of the figure) is plotted on the x-axis. The red line indicates the position of exemplary guide RNA #3.2 and the modulation of the measured HBs expression.

[0019] Figure 11 A graph showing the correlation between HBs and HBe expression among the tested guides. The right graph shows the HBe and HBs repression efficiencies for 25 exemplary guides.

[0020] Figure 12A A schematic diagram depicting the experimental timeline of a guide RNA assay testing the CRISPR-off single construct epigenetic editor in combination with each exemplary gRNA in a HepG2-NTCP infection model, where ELISA readings for HBe and HBs antigens were taken on day 6; Figure 12B A graph summarizing the percentage reduction of HBV antigens on day 6 relative to a non-targeting control.

[0021] Figure 13A A schematic diagram depicting the experimental timeline of a guide RNA assay testing the CRISPR-off single construct epigenetic editor in combination with each exemplary gRNA in a PLC / PRF / 5 cell model, where ELISA readings for HBs antigen were taken on day 4; Figure 13B A graph summarizing the percentage reduction of HBs antigen on day 4 relative to a non-targeting control.

[0022] Figure 14A A schematic diagram depicting the experimental timeline of a guide RNA assay testing the CRISPR-off single construct epigenetic editor in combination with each exemplary gRNA in a PXB cell model, where ELISA readings for HBe and HBs antigens were taken on day 6; Figure 14B A graph summarizing the percentage reduction of HBV antigens on day 6 relative to a non-targeting control. Figure 14CSchematic of the experimental timeline for the guide RNA assay that describes testing the CRISPR-off single construct epigenetic editor in combination with each exemplary gRNA in the PXB cell model, where ELISA readings for HBe and HBs antigens were taken on day 12. Figure 14D Chart summarizing the percentage reduction of HBV antigens on day 12 relative to the non-targeting control. 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.

[0023] Figure 15A Schematic of the experimental timeline for the zinc finger assay that describes testing the ZF-off single construct epigenetic editor containing each exemplary zinc finger motif in the HepG2-NTCP infection model, where ELISA readings for HBe and HBs antigens were taken on day 6; Figure 15B Chart summarizing the percentage reduction of HBV antigens on day 6 relative to the non-targeting control. "N" represents the non-targeting control, "P" represents the positive control, and the individual numbers on the x-axis represent the exemplary constructs tested in the experiment. For example, "1" represents the "mRNA0001" construct, and "20" represents the "mRNA0020" construct.

[0024] Figure 16A Summarizes Figure 15B The results of the top ten ZF-off constructs in Figure 16B Chart showing the HBsAg (top panel) and HBeAg (middle) expression values measured in the ZF-off screen (calculated as a percentage of the HBsAg or HBeAg expression measured against the non-targeting control (top and middle, respectively)). Each ZF-off construct is represented by a dot. Repression cut-off values of 50% and 60% are shown as horizontal lines. The position of the corresponding guide RNA in the HBV genome (bottom panel) is plotted on the x-axis.

[0025] Figure 17 Experimental timeline for testing the dose response (top panel), and two charts showing the dose response of the percentage of HBsAg (left bottom panel) and HBeAg (right bottom panel) in HepG2-NTCP cells after administration of the ZF fusion protein. The mRNA corresponding to the ZF motif of each fusion protein is indicated.

[0026] Figure 18Is the experimental timeline (top panel) for testing the persistent silencing of HBsAg, and a graph (bottom panel) showing the persistent silencing of HBsAg by the ZF fusion proteins. The mRNAs corresponding to the ZF motifs of each fusion protein are indicated.

[0027] Figure 19 Is the experimental timeline (top panel) for testing HBsAg silencing in the PLC / PRF / 5 in vitro model, and a graph showing the percentage of HBsAg relative to control on day 14 after administration of the ZF fusion proteins. The mRNAs corresponding to the ZF motifs of each fusion protein are indicated. Information on the target match percentage for each construct is also indicated.

[0028] Figure 20A Is a volcano plot showing differentially expressed (DE) genes for an exemplary ZF specificity assay. The DE genes are represented as points. Figure 20B Is a volcano plot showing the DE of CRISPR-off and gRNA epigenetic editors. The points represent genes and their expression changes (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 Is a scatter plot showing the methylation levels between treated (y-axis) and control (x-axis) for 935,000 CpG sites in the human genome. The line represents the threshold for methylation changes considered significant (absolute value [methylation difference] >= 0.2). The DMRs are labeled on each plot. Results for host target (PCSK9, second-to-last group) and low specificity control (last group) are also shown. Figure 20C Shows the results compared to effector only. Figure 20D Shows the results compared to untreated. 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.

[0029] Figure 21 Is a diagrammatic illustration of the experimental schematic for in vivo studies of multiplexing ZF fusion protein effectors. Detailed Description

[0030] The present disclosure provides epigenetic editors and strategies and methods for using such epigenetic editors for regulating the expression of HBV. By altering the expression of HBV, particularly by repressing the expression of HBV (e.g., the expression of genes contained within the HBV genome or gene products encoded by the HBV genome), the compositions and methods described herein can be used to suppress viral function in infected cells, such as in the case of treating HBV infection in a human subject or in the case of treating CHB.

[0031] The structure and biology of HBV and HBV-related 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), which is incorporated herein by reference in its entirety).

[0032] 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 herein by reference in their entirety, and the sequences are provided elsewhere in this document.

[0033] Multiple treatment options for HBV have been reported, but there remains a need for effective treatment of HBV infection. Gene editing methods that target the HBV genome to cleave genomic DNA are associated with risks of off-target cleavage and genomic translocation. Compared to other genome engineering methods, the epigenetic editors and related methods of use herein have several advantages, including increased efficiency, reduced translocation risk, and durable silencing of HBV.

[0034] Hepatitis D virus (HDV) is the smallest pathogen known to infect humans. HDV infection is only seen in patients infected with HBV because most functions of HDV rely on functions of HBV, including viral packaging, infectivity, transmission, and suppression of host immunity. Approximately 5% of patients with HBV infection also have HDV infection. HDV uses the HBV S-antigen (HBsAg) as a capsid protein, and thus HDV infection depends on the production of HBV S-antigen. Reducing the expression of HBV S-antigen also reduces the infectivity of HDV. The structure and biology 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 targeting HBV genes or the HBV genome.

[0035] In some embodiments, the epigenetic editors described herein can comprise one or more fusion proteins, wherein each fusion protein comprises a DNA-binding domain linked to one or more effector domains for epigenetic modification. In certain embodiments, when the DNA-binding domain is a polynucleotide-guided DNA-binding domain, the epigenetic editor can further comprise one or more guide polynucleotides. The DNA-binding domains, effector domains, and guide polynucleotides of the epigenetic editors described herein can be selected in any functional combination from those described, for example, below.

[0036] The epigenetic editors described herein can be transiently expressed in a host cell or integrated into the genome of the host cell; the present disclosure also encompasses such cells and their progeny. Both transiently expressed and integrated epigenetic editors or their components can effect stable epigenetic modifications. For example, after introducing the epigenetic editors described herein into a host cell, a target gene in the host cell can be stably or permanently repressed or silenced. For example, in some embodiments provided herein, a transiently expressed epigenetic editor comprising DNMT3A domain, DNMT3L domain, and KRAB domain effects stable epigenetic modification. For example, in some embodiments provided herein, a constitutively expressed epigenetic editor comprising DNMT3A and DNMT3L domains effects stable epigenetic modification. In some embodiments, the 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 throughout the life cycle of the cell or the subject carrying the cell, compared to the expression level in the absence of the epigenetic editor. The epigenetic modification may be inherited by progeny of the host cell into which the epigenetic editor is introduced.

[0037] The epigenetic editors of the present invention can be introduced into a patient in need (e.g., a human patient), for example, into hepatocytes, cholangiocytes (bile duct cells), stellate cells, Kupffer cells, and hepatic sinusoidal endothelial cells of the patient.

[0038] I. DNA - binding domain

[0039] The epigenetic editors described herein may comprise 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 as described herein can 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 11,162,114, which is incorporated herein by reference in its entirety.

[0040] In some embodiments, the DNA-binding domains described herein are encoded by their native coding sequences. In other embodiments, the DNA-binding domains are encoded by nucleotide sequences that have been codon-optimized for optimal expression in human cells.

[0041] A. Polynucleotide-guided DNA-binding domains

[0042] In some embodiments, the DNA-binding domain herein can be a protein domain that is guided by a guide nucleic acid sequence (e.g., a guide RNA sequence) to a target site in the HBV genome. In certain embodiments, the protein domain can be derived from a CRISPR-associated nuclease, such as a type I or type II CRISPR-associated nuclease. In some embodiments, the protein domain can be derived from a Cas nuclease, such as a type II, type IIA, type IIB, type IIC, type V, or type VI Cas nuclease. In certain embodiments, the protein domain can be derived from a type II Cas nuclease selected from 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, 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 contains the complete polypeptide sequence of the parental protein, or contains a variant thereof (e.g., having amino acid residue deletions, insertions, and / or substitutions). The variant retains the expected function of the parental protein (e.g., the ability to form a complex with the guide nucleic acid sequence and target DNA).

[0043] In some embodiments, the CRISPR - associated protein domain can be the Cas9 domain described herein. Cas9 can refer, for example, to 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 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 the wild - type Cpf1 polypeptide described herein (e.g., Cpf1 from Francisella novicida (UniProt reference number U2UMQ6 or SEQ ID NO:4)). In certain embodiments, the CRISPR - associated protein domain can be a modified form of the wild - type protein, including one or more amino acid residue changes such as deletions, insertions or substitutions; fusions or chimeras; or any combination thereof.

[0044] The structures of Cas9 sequences and variant Cas9 orthologs have been described for various organisms. Exemplary organisms from which the Cas9 domains herein can be derived include, but are not limited to, 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, Fibrobacter succinogene, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus buchneri, Treponema denticola, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp.) Acetohalobium arabaticum, Ammonifex degensii, Caldicellulosiruptor bescii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionium, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp.) Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Corynebacterium diphtheria, and Acaryochloris marina. The Cas9 sequences also include sequences from organisms and loci disclosed in Chylinski et al., RNA Biol. (2013) 10(5):726-37.

[0045] In some embodiments, the Cas9 domain is from Streptococcus pyogenes. In some embodiments, the Cas9 domain is from Staphylococcus aureus.

[0046] Other Cas domains for use in the epigenetic editors herein are also contemplated. These include, for example, those from CasX (Cas12E) (e.g., SEQ ID NO:5), CasY (Cas12d) (e.g., SEQ ID NO:6), (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).

[0047] For epigenetic editing, nuclease-derived protein domains (e.g., Cas9 or Cpf1 domains) can be mutated to have reduced or no nuclease activity, such that the protein domain does not cleave DNA or has reduced DNA cleavage activity while retaining the ability to complex with a guide nucleic acid sequence (e.g., guide RNA) and target DNA. For example, nuclease activity can 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 domains described herein are catalytically inactive ("dead"). Examples of such domains include, for example, dCas9 ("dead" Cas9), dCpf1, ddCpf1, dCasPhi, ddCas12a, dLbCpf1, and dFnCpf1. For example, the dCas9 protein domain can contain one, two, or more mutations compared to wild-type Cas9, thereby eliminating its nuclease activity. The DNA cleavage domain of Cas9 is known to contain two subdomains: the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, while 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. Similarly, SaCas9 can be inactivated by the mutations D10A and N580A. In some embodiments, dCas9 contains at least one mutation in the HNH subdomain and / or the 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, e.g., an insertion, deletion, or single or multiple amino acid substitutions in the RuvC1 domain and / or the HNH domain, can be included in the dCas9 described herein.

[0048] In some embodiments, the dCas9 protein herein contains a mutation at a position corresponding to position D10 (e.g., D10A), H840 (e.g., H840A), or both, in the wild-type SpCas9 sequence numbered in the sequence provided in UniProt accession number Q99ZW2 (SEQ ID NO:2). In certain embodiments, dCas9 contains the amino acid sequence of dSpCas9 (D10A and H840A) (SEQ ID NO:12).

[0049] In some embodiments, the dCas9 protein as described herein contains mutations at position D10 (e.g., D10A), N580 (e.g., N580A), or both, corresponding to the wild-type SaCas9 sequence (e.g., SEQ ID NO:9). In certain embodiments, the dCas9 contains the amino acid sequence of dSaCas9 (D10A and N580A) (SEQ ID NO:13).

[0050] Based on the present disclosure and knowledge in the art, those skilled in the art will appreciate other suitable mutations that inactivate Cas9, which are within the scope of the present disclosure. Such mutations can include, but are not limited to, D839A, N863A, and / or K603R in SpCas9. The present disclosure encompasses any mutation that reduces or eliminates the nuclease activity of any Cas9 described herein (e.g., a mutation corresponding to any Cas9 mutation described herein).

[0051] Compared to wild-type Cpfl, the dCpfl protein domain may contain one, two, or more mutations that reduce or eliminate its nuclease activity. The Cpfl protein has a RuvC-like endonuclease domain similar to the RuvC domain of Cas9, but does not have an HNH endonuclease domain, and the N-terminus of Cpfl does not have the α-helical recognition lobe of Cas9. In some embodiments, the dCpfl contains one or more mutations corresponding to the positions numbered D917A, E1006A, or D1255A in the Francisella novicida Cpfl protein sequence (FnCpfl; SEQ ID NO:4). In certain embodiments, the dCpf1 protein contains mutations corresponding to D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A, or D917A / E1006A / D1255A, or corresponding mutations in any of the Cpf1 amino acid sequences described herein. In some embodiments, the dCpf1 contains the D917A mutation. In certain embodiments, the dCpf1 contains the amino acid sequence of dFnCpf1 (SEQ ID NO:14).

[0052] Further nuclease-inactive CRISPR-associated protein domains covered herein include those from, for example, 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), dLbCpfl (e.g., SEQ ID NO:19), dAsCpfl (e.g., SEQ ID NO:20), denAsCpfl (e.g., SEQ ID NO:21), dHFasCpfl (e.g., SEQ ID NO:22), dRVRAsCpfl (e.g., SEQ ID NO:23), dRRAsCpfl (e.g., SEQ ID NO:24), dCasX (e.g., SEQ ID NO:25), and dCasPhi (e.g., SEQ ID NO:26).

[0053] In some embodiments, the Cas9 domains described herein can be high-fidelity Cas9 domains, e.g., comprising one or more mutations that reduce the electrostatic interaction between the Cas9 domain and the sugar-phosphate backbone of DNA, thereby conferring increased target-binding specificity. In certain embodiments, the high-fidelity Cas9 domain can be nuclease-inactive as described herein.

[0054] The CRISPR - associated protein domains described herein can recognize protospacer adjacent motif (PAM) sequences in target genes. The "PAM" sequence is generally a 2 - to - 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 it is not part of the target sequence. CRISPR - associated protein domains can recognize naturally occurring or canonical PAM sequences, or may have altered PAM specificities. 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 have been 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, domains from "VRER" SpCas9, "EQR" SpCas9, "VQR" SpCas9, "SpG Cas9", "SpRYCas9", and "KKH" SaCas9. Also encompassed are nuclease - inactive versions of these Cas9 domains, 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 Cas9 from Francisella novicida engineered to recognize 5’ - YG - 3’ (where "Y" is a pyrimidine).

[0055] Based on the present disclosure, those of ordinary skill in the art will appreciate additional suitable CRISPR - associated proteins, orthologs, and variants, including nuclease - inactive variants and sequences.

[0056] Guide RNAs that can be used in conjunction with the CRISPR - associated protein domains of the present invention are further described in Section II below.

[0057] B. Zinc - finger protein domains

[0058] 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 having at least one zinc finger and binds to DNA in a sequence-specific manner. A "zinc finger" (ZF) or "zinc finger motif" (ZF motif) refers to a polypeptide domain that comprises a β-β-α (ββα)-protein fold stabilized by a zinc ion. A ZF binds 2 to 4 nucleotide base pairs, typically 3 or 4 base pairs (consecutive or non-consecutive). Each ZF typically comprises about 30 amino acids. A ZFP domain can comprise multiple ZFs that contact its target nucleic acid sequence in tandem. The ZFs of a tandem array can be engineered to generate an artificial ZFP that binds to a desired nucleic acid target. Databases comprising triplet (or quadruplet) nucleotide sequences and the amino acid sequences of the respective ZFs can be used to rationally design ZFPs, wherein each triplet or quadruplet nucleotide sequence is associated with the amino acid sequence of one or more ZFs that can bind to that particular triplet or quadruplet sequence. See, e.g., U.S. Patents 6,453,242, 6,534,261, and 8,772,453.

[0059] ZFPs are widespread in eukaryotic cells and can belong to, for example, the C2H2 class, CCHC class, PHD class, or RING (Really Interesting New Gene) 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 an array of ZFs that comprises contiguous C2H2-ZFs, each C2H2-ZF contacting three or more contiguous nucleotides. Additional architectures, such as those described in Paschon et al., Nat. Commun. 10, 1133 (2019), are also possible.

[0060] The ZFP domain of the epigenetic editors described herein can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more ZFs. The ZFP domain can comprise an array of two-finger or three-finger units, such as 3, 4, 5, 6, 7, 8, 9, or 10 or more units, wherein 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 to 14 nucleotides. In some embodiments, a ZFP domain comprising at least six ZFs recognizes a target DNA sequence of 18 to 21 nucleotides.

[0061] In some embodiments, the ZFs in the ZFP domains described herein are linked by a peptide linker. The length of 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 some embodiments, the linker comprises 5 or more amino acids. In some embodiments, the linker comprises 7 - 17 amino acids. The linker can be flexible or rigid.

[0062] In some embodiments, the zinc finger array can have the following sequence:

[0063] SRPGERPFQCRICMRNFSXXXXXXXHXXTHTGEKPFQCRICMRNFSXXXXXXXHXXTH[linker]FQCRICMRNFSXXXXXXXHXXTHTGEKPFQCRICMRNFSXXXXXXXHXXTH[linker]PFQCRICMRNFSXXXXXXXHXXTHTGEKPFQCRICMRNFSXXXXXXXHXXTHLRGS (SEQ ID NO:1084),

[0064] or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto, where "XXXXXXX" represents the amino acids of the ZF recognition helix that confer zinc finger DNA binding specificity; each X can be independently selected. In the above sequence, the italicized "XX" can be TR, LR or LK, and "[linker]" represents the linker sequence. In some embodiments, the linker sequence is TGSQKP (SEQ ID NO:1085); this linker can be used when the sub - sites targeted by the ZFs are adjacent. In some embodiments, the linker sequence is TGGGGSQKP (SEQ ID NO:1086); this linker can be used when there are bases between the sub - sites targeted by the zinc fingers. The two indicated linkers can be the same or different.

[0065] The ZFP domains herein can comprise an array of two or more adjacent ZFs that are 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., to adjacent trinucleotides / triplets. In some embodiments, adjacent fingers cross - bind between their respective target triplets, which can help enhance or improve recognition of the target sequence and result in binding of overlapping sequences. In some embodiments, more distant ZFs within the ZFP domain can recognize (or bind) non - contiguous nucleotide sequences.

[0066] The amino acid sequences of the ZF DNA recognition helices of the exemplary ZFP domains of the present invention and their HBV target sequences are shown in Table 1 below.

[0067] Table 1. Zinc finger transcriptional repressors for silencing HBV. The ZF sequences of the exemplary ZFP domains are presented. The target sequences and the SEQ ID Nos of the ZFs can be found in the sequence listing of Table 20.

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074] In some embodiments, the ZFP domains of the epigenetic editors of the present invention bind to the target sequences provided herein. In further embodiments, the ZFP domain sequentially comprises the F1-F6 amino acid sequences of any of the zinc finger proteins shown 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 within any other ZF framework known in the art.

[0075] C. TALE

[0076] In some embodiments, the DNA binding domain of the epigenetic editors described herein comprises a transcription activator-like effector (TALE) domain. The DNA binding domain of TALE contains a highly conserved sequence of about 33-34 amino acids, with repeat variable diresidues (RVDs) at positions 12 and 13, which are crucial for the recognition of specific nucleotides. TALEs can be engineered to bind to almost 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.

[0077] D. Other DNA Binding Domains

[0078] Also covered are other DNA binding domains for the epigenetic editors described herein. In some embodiments, the DNA binding domain comprises an argonaute protein domain, such as from Natronobacterium gregoryi (NgAgo). NgAgo is a ssDNA-guided endonuclease that is guided by 5'-phosphorylated ssDNA (gDNA) to its target site where it creates a double-strand break. Compared to Cas9, the NgAgo-gDNA system does not require a protospacer adjacent motif (PAM). Thus, using nuclease-inactive NgAgo (dNgAgo) can greatly expand the bases that can be targeted. The characterization and use of NgAgo have been 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.

[0079] In some embodiments, the DNA binding domain comprises an inactivated nuclease, such as an inactivated meganuclease. Additional non-limiting examples of DNA binding domains include the tetracycline-controlled repressor (tetR) DNA binding domain, leucine zipper, helix-loop-helix (HLH) domain, helix-turn-helix domain, β-sheet motif, steroid receptor motif, bZIP domain, homeodomain, and AT-hook.

[0080] II. Guide polynucleotide

[0081] The epigenetic editors described herein that comprise a polynucleotide-guided DNA binding domain can also include a guide polynucleotide capable of forming a complex with the DNA binding domain. The guide polynucleotide can comprise RNA, DNA, or a mixture of both. For example, when 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 capable of hybridizing to a target sequence and guiding a CRISPR-Cas complex to bind to the target sequence. Methods for site-specific DNA targeting (e.g., to modify the genome) using guide polynucleotide sequences with programmable DNA binding proteins (such as CRISPR-associated protein domains) are known in the art.

[0082] A guide polynucleotide sequence (e.g., a gRNA sequence) can comprise two parts: 1) a nucleotide sequence comprising a “targeting sequence” that is complementary to a target nucleic acid sequence (the “target sequence”) (e.g., complementary to a nucleic acid sequence contained in a genomic target site); and 2) a nucleotide sequence that binds to a polynucleotide-guided DNA-binding domain (e.g., a CRISPR-Cas protein domain). The nucleotide sequence in 1) can comprise 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 thus can hybridize to the target nucleic acid sequence. The nucleotide sequence in 1) can be referred to as, for example, a crispr RNA or crRNA. The nucleotide sequence in 2) can be referred to as a scaffold sequence (e.g., tracrRNA) of the guide nucleic acid, or an activation region of the guide nucleic acid, and can comprise a stem-loop structure. The parts 1) and 2) described above can be fused to form a single guide (e.g., a single guide RNA, or sgRNA), or can be located on two separate nucleic acid molecules. In some embodiments, the guide polynucleotide comprises parts 1) and 2) linked by a linker. In some embodiments, the guide polynucleotide comprises parts 1) and 2) linked by a non-nucleic acid linker (e.g., a peptide linker or a chemical linker).

[0083] The second part (scaffold sequence) of the guide polynucleotide as described herein can be described, for example, in Jinek et al., Science (2012) 337:816-21; U.S. Patent Publication 2016 / 0208288; or U.S. Patent Publication 2016 / 0200779. This disclosure also encompasses variants of part 2). For example, the tetraloop and stem-loop of the gRNA scaffold (tracrRNA) sequence can be modified to contain 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 a repressor domain or an activator domain fused to an RNA aptamer that interacts with a protein.

[0084] The gRNAs provided herein generally comprise a targeting domain and a binding domain. The targeting domain (also referred to as the "targeting sequence") can comprise a nucleic acid sequence that binds to a target site (e.g., a genomic nucleic acid molecule within a cell). The target site can be a double-stranded DNA sequence comprising a PAM sequence and a target sequence, and the targeting sequence is on the same strand as and immediately adjacent to the PAM sequence. The targeting domain of the gRNA can comprise an RNA sequence corresponding to the target sequence, i.e., it is sequence-similar to the target domain, sometimes with one or more mismatches, but generally 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 double-stranded target site sequence complementary to the target sequence, and thus with the strand complementary to the strand comprising the PAM sequence. It should be understood that the targeting domain of the gRNA generally does not comprise a sequence similar to the PAM sequence. Further, it should be understood that the position of the PAM can be at the 5' or 3' of the target sequence, depending on the nuclease used. For example, for the Cas9 nuclease, the PAM is generally located at the 3' end of the target sequence, while for the Cas12a nuclease, the PAM is generally located at the 5' end of the target sequence. For an illustration of the PAM position and the binding mechanism of the gRNA to the target site, see, e.g., Vanegas et al., Fungal Biol Biotechnol. (2019) 6:6 in Figure 1 , which is incorporated herein by reference. For additional illustration and description of the mechanism by which the gRNA targets an RNA-guided nuclease to a target site, see Fu et al., Nat Biotechnol (2014) 32(3):279-84 and Sternberg et al., Nature (2014) 507(7490):62-7, each incorporated herein by reference.

[0085] In some embodiments, the targeting domain sequence comprises 17 to 30 nucleotides and corresponds exactly to the target sequence (i.e., without any mismatched nucleotides). However, in some embodiments, the targeting domain sequence can comprise one or more mismatches, but generally no more than 4 mismatches, such as 1, 2, 3, or 4 mismatches. Since the targeting domain is part of the gRNA (which is an RNA molecule), it generally comprises ribonucleotides, while a DNA targeting domain comprises deoxyribonucleotides.

[0086] An exemplary illustration of a Cas9 target site comprising a 22-nucleotide target domain and an NGGPAM sequence is provided below, as well as an exemplary illustration of a gRNA comprising a targeting domain that corresponds exactly to the target sequence (and thus base pairs completely complementary to the DNA strand complementary to the strand comprising the target sequence and the PAM):

[0087]

[0088] Exemplary descriptions of Casl2a target sites are provided below, which contain a 22-nucleotide target domain and a TTN PAM sequence, and exemplary descriptions of gRNAs, which contain a targeting domain that corresponds exactly to the target sequence (and thus base pairs completely complementary to the DNA strand complementary to the strand containing the target sequence and PAM):

[0089]

[0090] While not wishing to be bound by theory, in at least some embodiments, it is believed that the length and complementarity of the targeting domain to the target sequence contribute to the specificity of the interaction of the gRNA / Cas9 molecular complex with the target nucleic acid. In some embodiments, the length of the targeting domain of the gRNA provided herein is 5 to 50 nucleotides. In some embodiments, the length of the targeting domain is 15 to 25 nucleotides. In some embodiments, the length of the targeting domain is 18 to 22 nucleotides. In some embodiments, the length of the targeting domain is 19 - 21 nucleotides. In some embodiments, the length of the targeting domain is 15 nucleotides. In some embodiments, the length of the targeting domain is 16 nucleotides. In some embodiments, the length of the targeting domain is 17 nucleotides. In some embodiments, the length of the targeting domain is 18 nucleotides. In some embodiments, the length of the targeting domain is 19 nucleotides. In some embodiments, the length of the targeting domain is 20 nucleotides. In some embodiments, the length of the targeting domain is 21 nucleotides. In some embodiments, the length of the targeting domain is 22 nucleotides. In some embodiments, the length of the targeting domain is 23 nucleotides. In some embodiments, the length of the targeting domain is 24 nucleotides. In some embodiments, the length of the targeting domain is 25 nucleotides. In some embodiments, the targeting domain corresponds exactly to the target sequence provided herein or a portion thereof, with no mismatches. In some embodiments, the targeting domain of the gRNA provided herein contains 1 mismatch relative to the target sequence provided herein. In some embodiments, the targeting domain contains 2 mismatches relative to the target sequence. In some embodiments, the target domain contains 3 mismatches relative to the target sequence.

[0091] This document describes methods for designing, selecting, and validating gRNAs, and such methods are known in the art. Software tools can be used to optimize gRNAs corresponding to target DNA sequences, for example, to minimize total off-target activity across the genome. For example, DNA sequence search algorithms can be used to identify target sequences in the crRNAs of gRNAs for Cas9. Exemplary gRNA design tools include those described by Bae et al., Bioinformatics (2014) 30:1473-5.

[0092] The guide polynucleotides (e.g., gRNAs) described herein can have various lengths. In some embodiments, the length of the spacer or targeting sequence depends on the CRISPR-associated protein component in the epigenetic editor system used. For example, Cas proteins from different bacterial species have different optimal targeting sequence lengths. Thus, the length of the spacer sequence can comprise, 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 some embodiments, the length of the spacer comprises 10-24, 11-20, 11-16, 18-24, 19-21 or 20 nucleotides. In some embodiments, the guide polynucleotide (e.g., gRNA) has a length of 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 and comprises 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) consecutive nucleotides complementary to the target sequence. In some embodiments, the guide polynucleotides described herein can be truncated, for example, truncated by 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50 or more nucleotides.

[0093] In certain embodiments, the 3' end of the HBV target sequence is adjacent to the PAM sequence (e.g., the canonical PAM sequence such as NGG for SpCas9). The degree of complementarity between the targeting sequence of the guiding polynucleotide (e.g., the spacer sequence of the 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.

[0094] The guiding polynucleotide (e.g., the gRNA) can be modified by, for example, chemical alteration and synthetic modification. For example, the modified gRNA can include altering or replacing one or both non-linking phosphate oxygens and / or one or more linking phosphate oxygens in the phosphodiester backbone linkage, altering the ribose (e.g., altering the 2'-hydroxyl group on the ribose), altering the phosphate moiety, modifying or replacing the naturally occurring nucleobases, modifying or replacing the ribose-phosphate backbone, modifying the 3' end and / or 5' end of the oligonucleotide, replacing the terminal phosphate group or conjugating moieties, caps or linkers, or any combination thereof.

[0095] In some embodiments, one or more ribose groups of the gRNA can be modified. Examples of chemical modifications of ribose groups 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-acetalester or bicyclic nucleotides such as locked nucleic acid (LNA), 2'-(5-constrained ethyl (S-cEt)), constrained MOE or 2'-0,4'-C-aminomethylene bridged nucleic acid (2',4'-BNANC). The 2'-O-methyl modification and / or 2'-fluoro modification can increase the binding affinity and / or nuclease stability of the gRNA oligonucleotide.

[0096] In some embodiments, one or more phosphate groups of the gRNA can be chemically modified. Examples of chemical modifications of phosphate groups include, but are not limited to, phosphorothioate (PS), phosphonoacetate (PACE), thiophosphonoacetate (thioPACE), amide, triazole, phosphonate and phosphotriester modifications. In some embodiments, the guiding polynucleotides described herein can contain one, two, three or more PS linkages at the 5' end and / or 3' end or near thereto; the PS linkages can be continuous or discontinuous.

[0097] In some embodiments, the gRNAs herein comprise a mixture of ribonucleotides and deoxyribonucleotides and / or one or more PS linkages.

[0098] In some embodiments, one or more nucleobases of the gRNA may 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 pyrimidines, isoguanine, isocytosine, and nucleobases with halogenated aromatic groups. The chemical modification may be carried out in the spacer region, the tracr RNA region, the stem-loop, or any combination thereof.

[0099] Table 2 below lists exemplary target sequences for HBV epigenetic modification, as well as the starting and ending position coordinates of the target sites on the HBV genome.

[0100] Table 2. Targeting domain sequences of exemplary gRNAs targeting HBV. The following target sites were identified as suitable for targeting with epigenetic repressors:

[0101]

[0102]

[0103]

[0104]

[0105]

[0106] The target domains identified above that are adjacent to a PAM sequence (e.g., the Streptococcus pyogenes Cas9 PAM sequence) can be targeted by a CRISPR-based epigenetic repressor (e.g., an epigenetic repressor comprising a dCas9 DNA-binding domain). For example, target site 1-143 is suitable for targeting with a dCas9-based epigenetic repressor.

[0107] In some embodiments, a suitable gRNA for targeting any target domain sequence will comprise the target domain sequence and a suitable tracr RNA sequence, where the target domain sequence is the RNA equivalent of the DNA sequence of the provided targeting domain sequence (i.e., the RNA nucleotides of the sequence replace the provided DNA nucleotides, with uracil replacing thymine).

[0108] Any tracr sequence known in the art is encompassed for the gRNAs described herein. In some embodiments, the gRNAs described herein have a tracr sequence as shown in Table 3 below, or a tracr sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the tracr sequence shown below (SEQ: SEQ ID NO).

[0109] Table 3. Exemplary TRACR Sequences

[0110]

[0111] In some embodiments, the gRNAs herein are provided directly to the cell (e.g., as an RNP complex consisting of the CRISPR-associated protein domain). In some embodiments, the gRNAs are provided to the cell by an expression vector (e.g., a plasmid vector or a viral vector) introduced into the cell, and the cell then expresses the gRNA from the expression vector. Methods for introducing the gRNA and the expression vector into the cell are well known in the art.

[0112] III. Effector domain

[0113] The epigenetic editors described herein comprise 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. An epigenetic editor having one or more effector domains can regulate the expression of a target gene without altering its nuclear base sequence. In some embodiments, the effector domains described herein can repress or silence the expression of HBV or an HBV gene, e.g., by repressing transcription or by modifying or remodeling the HBV chromatin. Such effector domains are also referred to herein as "repressor domains", "repressor domains", "epigenetic repressor domains", or "epigenetic repression domains". Non-limiting examples of chemical modifications that can be mediated by the effector domains include methylation, demethylation, acetylation, deacetylation, phosphorylation, SUMOylation, and / or ubiquitination of DNA or histone residues.

[0114] In some embodiments, the effector domains of the epigenetic editors described herein can effect histone tail modifications, e.g., by adding or removing active marks on the histone tail.

[0115] In some embodiments, the effector domains of the epigenetic editors described herein can comprise or recruit transcription-related proteins, such as transcription repressors. The transcription-related proteins can be endogenous or exogenous.

[0116] In some embodiments, the effector domain of the epigenetic editors described herein can comprise, for example, a protein that directly or indirectly blocks access of a transcription factor to a target gene containing the target sequence.

[0117] The effector domain can be a full-length protein or a fragment thereof that retains epigenetic effector function ("functional domain"). A functional domain capable of regulating (e.g., repressing) gene expression can be derived from a larger protein. For example, a functional domain that can reduce the expression of a target gene can be identified based on the sequence of a repressor protein. The amino acid sequence of a gene expression regulatory protein can be obtained from available genomic 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 a full-length protein sequence. As a starting point, the gene expression regulatory activity of the largest sequence containing all regions identified by different databases can be tested. Then various truncations can be tested to identify the minimal functional unit.

[0118] The present disclosure also encompasses variants of the effector domains described herein. Variants can refer, for example, to polypeptides 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, the 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.

[0119] In some embodiments, the epigenetic editors described herein can comprise 1 effector domain, 2 effector domains, 3 effector domains, 4 effector domains, 5 effector domains, 6 effector domains, 7 effector domains, 8 effector domains, 9 effector domains, 10 effector domains or more effector domains. In certain embodiments, the epigenetic editor comprises one or more fusion proteins (e.g., one, two or three fusion proteins), each fusion protein 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 can induce epigenetic modifications, such as combinations of 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, DNA methylation and histone SUMOylation.

[0120] In some embodiments, the effector domains described herein (e.g., DNMT3A and / or DNMT3L) are encoded by nucleotide sequences found in the native genome (e.g., human or murine) of the effector domain. In other embodiments, the effector domains described herein are encoded by nucleotide sequences that have been codon-optimized for optimal expression in human cells.

[0121] The effector domains described herein can include, for example, transcriptional repressors, DNA methyltransferases, and / or histone modifiers, as further detailed below.

[0122] A. Transcriptional repressors

[0123] In some embodiments, the epigenetic effector domains described herein mediate repression of target gene expression (e.g., transcription). The effector domain can 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, a 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 interaction domain (SID), the WRPW motif of a hairy-related basic helix-loop-helix (bHLH) repressor protein, an HP1α chromo-shadow repression domain, an HP1β repression domain, or any combination thereof. The effector domain can recruit one or more protein domains that repress target gene expression, for example, via a scaffold protein. In some embodiments, the effector domain can recruit or interact with a scaffold protein domain that recruits a PRMT protein, an HDAC protein, a SETDB1 protein, or a NuRD protein domain.

[0124] In some embodiments, the effector domain contains a functional domain derived from a zinc finger repressor protein, such as a KRAB domain. The KRAB domain is present in approximately 400 human ZFP-based transcription factors. A description of the KRAB domain can be found, for example, in Ecco et al., Development (2017) 144(15):2719-29 and Lambert et al., Cell (2018) 172:650-65.

[0125] In certain embodiments, the effector domain comprises a repressor domain (e.g., KRAB) derived from KOX1 / ZNF10, KOX8 / ZNF708, ZNF43, ZNF184, ZNF91, HPF4, HTF10, or HTF34. In some embodiments, the effector domain comprises a repressor domain (e.g., KRAB) derived from 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, ZNF250, 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 repressor domain can be a KRAB domain derived from KOX1, ZIM3, ZFP28, or ZN627. In a particular embodiment, the repressor domain is the ZIM3KRAB domain. In a further embodiment, the effector domain is derived from a human protein, such as human ZIM3, human KOX1, human ZFP28, or human ZN627.

[0126] Table 4 below provides exemplary effector domains (SEQ: ID NO, sequences of exemplary effector domains are shown in Table 20) that can reduce or silence the expression of a target gene. Further examples of repressors and transcriptional repressor domains can be found, for example, in PCT patent publication WO 2021 / 226077 and Tycko et al., Cell (2020) 183(7): 2020-35, each of which is incorporated herein by reference in its entirety.

[0127] Table 4. Exemplary Effector Domains Suitable for Silencing Gene Expression

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136] The present disclosure encompasses functional analogs of any of the proteins listed above, i.e., molecules having the same or substantially the same biological function (e.g., protein transcription factor function with 70% or higher, 80% or higher, 90% or higher, 95% or higher, or 98% or higher retention). For example, a functional analog can be an isoform or variant of the protein listed above, e.g., comprising a portion of the above protein, which portion may or may not have additional amino acid residues and / or comprises a mutation relative to the above protein. In some embodiments, the functional analog has at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to one of the sequences listed in Table 4. Also encompassed are homologs, orthologs, and mutants of the proteins listed above.

[0137] In certain embodiments, the epigenetic editors described herein comprise a KRAB domain derived from KOX1, ZIM3, ZFP28, or ZN627, and / or an effector domain derived from KAP1, MECP2, HPla, HPlb, CBX8, CDYL2, TOX, TOX3, TOX4, EED, EZH2, RBBP4, RCOR1, or SCML2, optionally wherein the parental protein is a human protein. In certain embodiments, the epigenetic editors described herein comprise a domain derived from KOX1, ZIM3, ZFP28, and / or ZN627, optionally wherein the parental protein is a human protein. In certain embodiments, the epigenetic editor can comprise a KRAB domain derived from KOX1 (ZNF10) (e.g., human KOX1). In certain embodiments, the epigenetic editor can comprise a KRAB domain derived from ZIM3 (ZNF657 or ZNF264) (e.g., human ZIM3). In certain embodiments, the epigenetic editor can comprise a KRAB domain derived from ZFP28 (e.g., human ZFP28). In certain embodiments, the epigenetic editor can comprise a KRAB domain derived from ZN627 (e.g., human ZN627). In certain embodiments, the epigenetic editors described herein can comprise a combination of a CDYL2 (e.g., human CDYL2) and / or TOX domain (e.g., human TOX domain) with a KOX1 KRAB domain (e.g., human KOX1 KRAB domain).

[0138] In certain embodiments, the epigenetic effector described herein comprises a repressor domain derived from ZNF10 (SEQ ID NO: 1024). For example, the repressor domain can comprise the sequence of SEQ ID NO: 1024, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1024.

[0139] B. DNA Methyltransferase

[0140] In some embodiments, the effector domain of the epigenetic editors described herein alters target gene expression by DNA modification such as methylation. The transcriptional activity of highly methylated regions of DNA tends to be lower than that of less methylated regions. DNA methylation mainly occurs at CpG sites (an abbreviation for "C-phosphate-G-" or "cytosine-phosphate-guanine" sites). The promoter regions of many mammalian genes are near or include CpG islands (nucleic acid regions with a high frequency of CpG dinucleotides).

[0141] The effector domain described herein can be, for example, a DNA methyltransferase (DNMT) or its catalytic domain, or can be capable of recruiting a DNA methyltransferase. DNMT encompasses enzymes that catalyze the transfer of a methyl group to a DNA nucleotide, such as the canonical cytosine-5 DNMTs that catalyze the addition of a methyl group to genomic DNA (e.g., DNMT1, DNMT3A, DNMT3B, and DNMT3C). The term also encompasses non-canonical family members that do not themselves catalyze methylation but recruit (including 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 specified, a DNMT domain can refer 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). A DNMT can repress the expression of a target gene by recruiting repressive regulatory proteins. In some embodiments, methylation occurs at CG (or CpG) dinucleotide sequences. In some embodiments, methylation occurs at CHG or CHH sequences, where H is any one of A, T, or C. In some embodiments, the DNMT in an epigenetic editor can include, for example, DNMT1, DNMT3A, DNMT3B, and / or DNMT3C. In some embodiments, the DNMT is a mammalian (e.g., human or murine) DNMT. In certain embodiments, the DNMT is DNMT3A (e.g., human DNMT3A). In certain embodiments, the epigenetic editor described herein comprises a DNMT3A domain that comprises SEQ ID NO:1028, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:1028. In certain embodiments, the epigenetic editor described herein comprises a DNMT3A domain that comprises SEQ ID NO:1029, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:1029. In some embodiments, the DNMT3A domain can have mutations, for example, at positions: H739 (such as H739A or H739E), R771 (such as R771L), and / or R836 (such as R836A or R836Q), or any combination thereof (numbered according to SEQ ID NO:1028).

[0142] In some embodiments, the effector domain described herein can be a DNMT-like domain. As used herein, a "DNMT-like domain" is 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 that comprises SEQ ID NO: 1032, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1032. In certain embodiments, the epigenetic editor herein comprises a DNMT3L domain that comprises SEQ ID NO: 1033, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1033. In certain embodiments, the epigenetic editor described herein comprises a DNMT3L domain that comprises SEQ ID NO: 1034, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1034. In certain embodiments, the epigenetic editor described herein comprises a DNMT3L domain that comprises SEQ ID NO: 1035, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1035. In some embodiments, the DNMT3L domain can have mutations corresponding to, for example, the following positions: D226 (such as D226V), Q268 (such as Q268K), or both (numbered according to SEQ ID NO: 1032).

[0143] In certain embodiments, the epigenetic editors herein can include DNMT and DNMT-like effector domains. For example, the epigenetic editor can include the DNMT3A-3L domain, where DNMT3A and DNMT3L can be covalently linked. In other embodiments, the epigenetic editors described herein can include an effector domain that includes only the DNMT3A domain (e.g., human DNMT3A) or only the DNMT-like domain (e.g., DNMT3L, which can be human or murine DNMT3L).

[0144] Table 5 below provides exemplary methyltransferases from which the effector domains of the epigenetic editors described herein can be derived. The sequences of these exemplary methyltransferases are provided in Table 20.

[0145] Table 5. Exemplary DNA methyltransferase sequences

[0146]

[0147]

[0148] The present disclosure encompasses functional analogs of any of the proteins listed above, i.e., molecules having the same or substantially the same biological function (e.g., retaining 70% or higher, 80% or higher, 90% or higher, 95% or higher, or 98% or higher of the protein DNA methylation function or recruitment function). For example, the functional analog can be an isoform or variant of the proteins listed above, e.g., comprising a portion of the above proteins that has or does not have additional amino acid residues and / or comprising mutations relative to the above proteins. In some embodiments, the 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, the effector domains herein include only the functional domains (or functional analogs thereof) of the proteins listed above, such as catalytic domains or recruitment domains.

[0149] As used herein, a DNMT domain (e.g., a DNMT3A domain or a DNMT3L domain) refers to a protein domain that is identical to the parental protein (e.g., human or murine DNMT3A or DNMT3L) or a functional analog thereof (e.g., having a functional fragment of the parental protein, such as a catalytic fragment or a recruitment fragment; and / or having mutations that enhance the activity of the DNMT protein).

[0150] The epigenetic editors of the present invention can methylate, for example, 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 a target gene or chromosome. The CpG dinucleotide sequences can be within or near the target gene in a CpG island, or can be within a region that is not a CpG island. A CpG island generally refers to a nucleic acid sequence or chromosomal region that contains a high frequency of CpG dinucleotides. For example, a CpG island can contain at least 50% GC content. A CpG island may have a high ratio of observed to expected CpG, for example, the ratio of observed to expected CpG is at least 60%. As used herein, the ratio of observed to expected CpG is determined by the number of CpGs * (sequence length) / (number of Cs * number of Gs). In some embodiments, the ratio of observed to expected CpG of a CpG island is at least 60%, 70%, 80%, 90%, or higher. A CpG island can be a sequence or region that is, for example, at least 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 nucleotides. In some embodiments, the epigenetic editor methylates 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.

[0151] In some embodiments, the epigenetic editors of the present invention perform methylation at hypomethylated nucleic acid sequences (i.e., sequences that may lack methylation on 5-methylcytosine nucleotides (e.g., in CpGs) compared to a standard control). For example, hypomethylation can occur in senescent cells or cancer cells (e.g., early stages of tumors) relative to younger cells or non-cancerous cells, respectively.

[0152] In some embodiments, the epigenetic editors described herein induce methylation at hypermethylated nucleic acid sequences.

[0153] In some embodiments, an epigenetic editor can introduce methylation at sites other than CpG dinucleotides. For example, a target gene sequence can be methylated at the C nucleotide of a CpA, CpT, or CpC sequence. In some embodiments, the epigenetic editor comprises a DNMT3A domain and methylates at CpG, CpA, CpT, CpC sequences, or any combination thereof. In some embodiments, the epigenetic editor comprises a DNMT3A domain lacking the regulatory subdomain and retaining only the catalytic domain. In some embodiments, an epigenetic editor comprising the DNMT3A catalytic domain methylates only at CpG sequences. In some embodiments, an epigenetic editor comprising a DNMT3A domain containing a mutation (e.g., an R836A or R836Q mutation (numbered according to SEQ ID NO: 1028)) has higher methylation activity at CpA, CpC, and / or CpT sequences compared to an epigenetic editor comprising a wild-type DNMT3A domain.

[0154] C. Histone Modifying Factors

[0155] In some embodiments, the effector domain of the epigenetic editors herein mediates histone modification. Histone modification plays a structural and biochemical role in gene transcription, such as by forming or disrupting nucleosome structures that bind to histones and block gene transcription. Histone modification can include, for example, acetylation, deacetylation, methylation, phosphorylation, ubiquitination, SUMOylation, etc., for example at its N-terminus (“histone tail”). These modifications maintain or specifically alter chromatin structure, thereby controlling reactions such as gene expression, DNA replication, DNA repair, etc. that occur on chromosomal DNA. Post-translational modification of histones is an epigenetic regulatory mechanism and is considered essential for genetic regulation in eukaryotic cells. Recent studies have shown that chromatin remodeling factors such as SWI / SNF, RSC, NURF, NRD, etc. promote the entry of transcription factors into DNA by modifying nucleosome structure; histone acetyltransferases (HATs) regulate the acetylation state of histones; and histone deacetylases (HDACs) serve as important regulators.

[0156] In particular, the unstructured N-terminus of histones can be modified by acetylation, deacetylation, methylation, ubiquitination, phosphorylation, SUMOylation, ribosylation, citrullination, O-GlcNAcylation, crotonylation, or any combination thereof. For example, histone acetyltransferases (HATs) utilize acetyl-CoA as a cofactor and catalyze the transfer of an acetyl group to the ε-amino group of the lysine side chain. This neutralizes the positive charge of lysine and weakens the interaction between histones and DNA, thereby opening up the chromosome and enabling transcription factors to bind and initiate transcription. Acetylation of lysines K14 and K9 of histone H3 by histone acetyltransferases 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. On the other hand, histone methylation of lysine 9 of histone H3 may be associated with heterochromatin or transcriptionally silent chromatin.

[0157] In certain embodiments, the effector domain of the epigenetic editor described herein comprises a histone methyltransferase domain. The effector domain can 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 a particular embodiment, the effector domain comprises the histone-lysine-N-methyltransferase SETDB1 domain.

[0158] 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 an HDAC1, HDAC3, HDAC5, HDAC7, or HDAC9 protein domain. In a particular embodiment, the effector domain comprises the nucleosome remodeling and deacetylase complex (NURD), which removes acetyl groups from histones.

[0159] D. Other Effector Domains

[0160] In some embodiments, the effector domain comprises a protein containing a three-part motif (TRIM28, TIF1-β, or KAP1). In certain embodiments, the effector domain comprises one or more KAP1 proteins. The KAP1 protein in the epigenetic editors herein can form a complex with one or more other effector domains of the epigenetic editor or one or more proteins involved in regulating gene expression in the cellular environment. For example, KAP1 can be recruited by the KRAB domain of a transcriptional repressor. The KAP1 protein domain can 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 components of the NuRD protein complex. In some embodiments, the KAP1 protein domain interacts with or recruits ZFP90 protein (e.g., isoform 2 of ZFP90) and / or FOXP3 protein. An exemplary KAP1 amino acid sequence is shown in SEQ ID NO:1062.

[0161] In some embodiments, the effector domain comprises a protein domain that interacts with or is recruited by one or more DNA epigenetic marks. For example, the effector domain can comprise methyl-CpG-binding protein 2 (MECP2) protein, which interacts with methylated DNA nucleotides in the target gene (which may or may not be located in the CpG island of the target gene). The MECP2 protein domain in the epigenetic editors described herein can induce chromatin structure condensation, thereby reducing or silencing the expression of the target gene. In some embodiments, the MECP2 protein domain in the epigenetic editors described herein can interact with histone deacetylases (e.g., HDAC) to repress or silence the expression of the target gene. In some embodiments, the MECP2 protein domain in the epigenetic editors described herein can block the access of transcription factors or transcriptional activators to the target sequence, thereby repressing or silencing the expression of the target gene. An exemplary MECP2 amino acid sequence is shown in SEQ ID NO:1063.

[0162] Also encompassed as effector domains of the epigenetic editors described herein are, for example, chromosomal shadow domains, ubiquitin-2-like Rad60 SUMO-like (Rad60-SLD / SUMO) domains, chromatin organization modifier domains (Chromo) domains, Yaf2 / RYBP C-terminal binding motif domains (YAF2_RYBP), CBX family C-terminal motif domains (CBX7_C), zinc finger C3HC4 type (RING finger) domains (ZF-C3HC4_2), cytochrome b5 domains (Cyt-b5), helix-loop-helix domains (HLH), helix-hairpin-helix motif domains (e.g., HHH_3), high mobility group box domains (HMG-box), basic leucine zipper domains (e.g., bZIP_1 or bZIP_2), Myb_DNA binding domains, homeodomains, MYM-type zinc finger domains containing FCS sequences (ZF-FCS), interferon regulatory factor 2 binding protein zinc finger domains (IRF-2BP1_2), SSX repression domains (SSXRD), B-box type zinc finger domains (ZF-B_box), CXXC zinc finger domains (ZF-CXXC), regulator of chromosome condensation 1 domains (RCC1), SRC homology 3 domains (SH3_9), sterile alpha motif domains (SAM_1), sterile alpha motif domains (SAM_2), sterile alpha motif / tip domains (Pointed domain) (SAM_PNT), Vestigial / Tondu family domains (Vg_Tdu), LIM domains, RNA recognition motif domains (RRM_1), paired amphipathic helix domains (PAH), proteasome ATPase OB C-terminal domains (Prot_ATP_ID_OB), nervy homology 2 domains (NHR2), hinge domain of cleavage stimulation factor subunit 2 (CSTF2_hinge), PPARγ N-terminal region domains (PPARgamma_N), CDC48 N-terminal domains (CDC48_2), WD40 repeat domains (WD40), Fip1 motif domains (Fip1), PDZ domains (PDZ_6), Von Willebrand factor type C domains (VWC), NAB conserved region 1 domains (NCD1), S1 RNA binding domains (S1), HNF3C-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 esters / diacylglycerol binding domain (C1-1), polycomb-like MTF2 factor 2 domain (Mtf2_C), transactivation domain of FOXO protein family (FOXO-TAD), homeobox KN domain (Homeobox_KN), BED zinc finger domain (ZF-BED), zinc finger of C3HC4-type ring (RING) domain (ZF-C3HC4_4), RAD51 interaction motif domain (RAD51_interact), p55 binding region of methyl-CpG binding domain protein MBD (MBDa), Notch domain, Raf-like Ras binding domain (RBD), Spin / Ssty family domain (Spin-Ssty), PHD finger domain (PHD_3), low density lipoprotein receptor domain class A (Ldl_recept_a), CS domain, DM DNA binding domain and QLQ domain.

[0163] In some embodiments, the effector domain is a protein domain comprising the YAF2_RYBP domain or a homeodomain or any combination thereof. In certain embodiments, the homeodomain of the YAF2_RYBP domain is the PRD domain, the NKL domain, the HOXL domain or the LIM domain. In specific embodiments, the YAF2_RYBP domain may comprise the 32-amino acid Yaf2 / RYBP C-terminal binding motif domain (32aa RYBP).

[0164] In some embodiments, the effector domain includes a protein domain selected from the following: SUMO3 domain, Chromo domain from M phase phosphoprotein 8 (MPP8), Chromoshadow domain from Chromobox 1 (CBX1), and SAM_1 / SPM domain from Scm PolycombGroup Protein Homolog 1 (SCMH1).

[0165] In some embodiments, the effector domain comprises the 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 homology1) motif.

[0166] In some embodiments, the effector domain can comprise the interferon regulatory factor 2-binding protein zinc finger domain (IRF-2BP1_2), the Cyt-b5 domain from the DNA repair factor HERC2 E3 ligase, the variant SH3 domain (SH3_9) from bridging integrator 1 (BIN1), the HMG-box domain from the transcription factor TOX, or the ZF-C3HC4_2 ring (RING) finger domain, chromatin domain (Chromodomain)-helicase-DNA-binding protein 3 (CHD3) domain, or ZNF783 domain from the polycomb component PCGF2.

[0167] IV. Epigenetic editor

[0168] Disclosed herein are epigenetic editors, also referred to herein as epigenetic editing systems, that direct epigenetic modification of a target sequence in a target gene, e.g., using any combination of one or more DNA-binding domains as described herein and one or more effector domains (e.g., epigenetic repressor domains) as described herein. The DNA-binding domain (acting in concert with a guide polynucleotide such as the guide polynucleotides described herein, where the DNA-binding domain is a polynucleotide-guided DNA-binding domain) directs the effector domain to epigenetically modify the target sequence, resulting in gene repression or silencing, which repression or silencing can be persistent and heritable across cell generations. In some aspects, the epigenetic editors described herein can repress or silence genes reversibly or irreversibly in a cell.

[0169] In certain embodiments, the epigenetic editors described herein comprise one or more fusion proteins, each fusion protein comprising (1) a DNA binding domain and (2) an effector domain. The effector domain(s) can be located on one or more of the fusion proteins comprised by the epigenetic editor. For example, a single fusion protein can comprise all of the effector domains and one DNA binding domain. Alternatively, the effector domain or a subset thereof can be located on separate fusion proteins, each having a DNA binding domain (which can be the same or different). The fusion proteins described herein can 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., a DNA binding domain and / or an effector domain) are joined directly or indirectly, covalently or non-covalently.

[0170] In some embodiments, the epigenetic editors described herein comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more effector (e.g., repressor) domains, which can be the same or different. In certain embodiments, two or more of the effector domains act in concert. Combinations of effector domains can include DNA methylation domains, histone deacetylation domains, histone methylation domains, and / or scaffold domains that recruit any of the foregoing. For example, the epigenetic editors described herein can comprise a combination of one or more transcriptional repressor domains (e.g., KRAB domains such as KOX1, ZIM3, ZFP28 or ZN627 KRAB) with one or more DNA methylation domains (e.g., DNMT domains) and / or recruitment domains (e.g., DNMT3L domains). Such epigenetic editors can comprise, for example, a KRAB domain, a DNMT3A domain, and a DNMT3L domain. The epigenetic editor can comprise a DNMT3A domain and a DNMT3L domain and preferably further comprises a KRAB domain. In some embodiments, the epigenetic editor further comprises additional effector domains (e.g., KAP1, MECP2, HP1b, CBX8, CDYL2, TOX, TOX3, TOX4, EED, RBBP4, RCOR1 or SCML2 domains). In some embodiments, the additional effector domain is a CDYL2, TOX, TOX3, TOX4 or HP1a domain. For example, the epigenetic editors described herein can comprise a combination of a CDYL2 and / or TOX domain with a KRAB domain (e.g., a KOX1 KRAB domain).

[0171] A. Linker

[0172] The fusion proteins as described herein can comprise one or more linkers that connect epigenetic editor components. The linker can be a peptide linker or a non-peptide linker.

[0173] In some embodiments, one or more linkers used in the epigenetic editors provided herein are peptide linkers, i.e., linkers that contain a peptide moiety. The peptide linker can be of any length suitable for the epigenetic editor fusion proteins described herein. In some embodiments, the linker can comprise a peptide between 1 and 200 (e.g., 1 to 80) amino acids. In some embodiments, the linker comprises 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, 10 to 100, 10 to 150, 10 to 200, 20 to 30, 20 to 40, 20 to 50, 20 to 60, 20 to 80, 20 to 100, 20 to 150, 20 to 200, 30 to 40, 30 to 50, 30 to 60, 30 to 80, 30 to 100, 30 to 150, 30 to 200, 40 to 50, 40 to 60, 40 to 80, 40 to 100, 40 to 150, 40 to 200, 50 to 60, 50 to 80, 50 to 100, 50 to 150, 50 to 200, 60 to 80, 60 to 100, 60 to 150, 60 to 200, 80 to 100, 80 to 150, 80 to 200, 100 to 150, 100 to 200 or 150 to 200 amino acids. Longer or shorter linkers are also contemplated. In some embodiments, the peptide linker has a length of 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. For example, the peptide linker can have a length of 4, 5, 16, 20, 24, 27, 32, 40, 64, 92 or 104 amino acids. The peptide linker can be a flexible or a rigid linker. In certain embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NO: 1064 - 1068, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0174] In certain embodiments, the peptide linker is an XTEN linker. Such linkers can comprise portions of the XTEN sequence (Schellenberger et al., Nat Biotechnol (2009) 27(1):1186-90), i.e., an unstructured hydrophilic polypeptide consisting only of the 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 generally unstructured and contain a limited set of natural amino acids. Fusion of XTEN to a protein alters its hydrodynamic properties and reduces the clearance and degradation rate of the fusion protein. These XTEN fusion proteins are produced using recombinant techniques without chemical modification and are degraded by natural pathways. The length of the XTEN linker can be, for example, 5, 10, 16, 20, 26, or 80 amino acids. In some embodiments, the length of the XTEN linker is 16 amino acids. In some embodiments, the length of the XTEN linker is 80 amino acids. 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 having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, the XTEN linker can be XTEN10, XTEN16, XTEN20, or XTEN80.

[0175] 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 an amide-linked carbon-nitrogen bond. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, or branched or unbranched aliphatic or heteroaliphatic linker.

[0176] In some embodiments, one or more linkers used in the epigenetic editors provided herein are polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). The linker can comprise, for example, monomers, dimers, or polymers of aminoalkanoic acids; aminoalkanoic acids (e.g., glycine, acetic acid, alanine, β-alanine, 3-aminopropionic acid, 4-aminobutyric acid, 5-aminovaleric acid, etc.); monomers, dimers, or polymers of aminohexanoic acid (Ahx); or polyethylene glycol moieties (PEG); or aryl or heteroaryl moieties. In certain embodiments, the linker can be based on a carbocyclic moiety (e.g., cyclopentane or cyclohexane) or a benzene ring. The linker can comprise a functionalized moiety to facilitate attachment of a nucleophile (e.g., a thiol, an amino group) on the peptide to the linker. Any electrophile can be used as part of the linker. Electrophiles include, but are not limited to: activated esters, activated amides, alkyl halides, aryl halides, acyl halides, and isothiocyanates.

[0177] Linkers of various lengths and flexibilities can be employed between any two components of the epigenetic editor (e.g., between an effector domain (e.g., a repressor domain) and a DNA-binding domain (e.g., a Cas9 domain), between a first effector domain and a second effector domain, etc.). The linker can range from a very flexible linker (such as a glycine / serine-rich linker) to a more rigid linker to achieve an optimal length for effector domain activity for a particular application. In some embodiments, the more flexible linker is a glycine / serine-rich linker (GS-rich linker), where 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, the more rigid linker is in the form of (EAAAK)n (SEQ ID NO:487), (SGGS)n (SEQ ID NO:488), and (XP)n (SEQ ID NO:489). In the formulas of the flexible and rigid linkers above, 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).

[0178] In some embodiments, the linker in the epigenetic editors described herein comprises a nuclear localization signal, e.g., having an amino acid sequence of any one of SEQ ID NOs: 1074-1079. In some embodiments, the linker in the epigenetic editors described herein comprises an expression tag, e.g., a detectable tag such as green fluorescent protein.

[0179] B. Nuclear localization signal

[0180] The fusion proteins described herein may comprise one or more nuclear localization signals and, in certain embodiments, may comprise two or more nuclear localization signals. For example, the fusion protein may comprise 1, 2, 3, 4, or 5 nuclear localization signals. As used herein, a "nuclear localization signal" (NLS) is an amino acid sequence that directs a protein into the nucleus. In certain embodiments, the NLS can be the SV40 NLS. The fusion protein may comprise the NLS at its N-terminus, C-terminus, or both, and / or the NLS may be embedded in the middle of the fusion protein (e.g., at the N-terminus or C-terminus of the DNA binding domain or effector domain). In certain embodiments, the NLS comprises an amino acid sequence of any one of SEQ ID NOs: 1074-1079, or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the selected sequence. Additional NLSs are known in the art.

[0181] C. Tags

[0182] The epigenetic editors provided herein may comprise one or more additional sequences ("tags") for tracking, detecting, and localizing the editor. In some embodiments, the epigenetic editor comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more detectable tags. Each detectable tag may be the same or different.

[0183] For example, an epigenetic editor fusion protein can include a cytoplasmic localization sequence, an export sequence (such as a nuclear export sequence) or other localization sequences, and sequence tags that can be used to solubilize, purify, or detect the fusion protein. Suitable protein tags provided herein include, but are not limited to, biotin carboxyl carrier protein (BCCP) tags, myc tags, calmodulin tags, FLAG tags, hemagglutinin (HA) tags, polyhistidine tags (also known as 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 (such as 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. The sequences disclosed herein include tag sequences, but are also applicable without the tag sequences; similarly, sequences disclosed herein without tag sequences are also covered to include the addition of suitable sequences that are apparent to those skilled in the art. Sequences disclosed herein with tag sequences are also covered without the presented tag sequences; similarly, sequences disclosed herein without tag sequences are also covered to include the addition of suitable sequences that are apparent to those skilled in the art.

[0184] D. Fusion Protein Constructs

[0185] The fusion proteins of the epigenetic editors described herein can have components structured in different constructs. For example, the DNA binding domain can be located at the C-terminus, N-terminus, or between two or more epigenetic effector domains or additional domains. In some embodiments, the DNA binding domain is located at the C-terminus of the epigenetic editor. In some embodiments, the DNA binding domain is located 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 by epigenetic effector domains and / or additional domains. In some embodiments, when "DBD" represents the DNA binding domain and "ED" represents the effector domain, the epigenetic editor comprises the following constructs:

[0186] -N’]-[ED1]-[DBD]-[ED2]-[C’

[0187] -N’]-[ED1]-[DBD]-[ED2]-[ED3]-[C’

[0188] -N’]-[ED1]-[ED2]-[DBD]-[ED3]-[C’

[0189] or

[0190] -N']-[ED1]-[ED2]-DBD]-[ED3]-[ED4]-[C'.

[0191] In some embodiments, the epigenetic editor comprises a DNA binding domain (DBD), a DNA methyltransferase (DNMT) domain, and a transcriptional repressor ("repressor") domain that represses or silences the expression of a target gene. The DBD, DNMT, and transcriptional repressor domains can be any of the domains 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 comprises a fusion protein having the following construct:

[0192] N']-[DNA methyltransferase domain]-[DBD]-[repressor domain]-[C'

[0193] N']-[repressor domain]-[DBD]-[DNA methyltransferase domain]-[C'

[0194] N']-[DNA methyltransferase domain]-[repressor domain]-[DBD]-[C'

[0195] or

[0196] N']-[repressor domain]-[DNA methyltransferase domain]-[DBD]-[C'.

[0197] In some embodiments, the linker structure "]-[ " in any of the epigenetic editor structures is a linker, such as a peptide linker; a detectable tag; a peptide bond; a nuclear localization signal; and / or a promoter or regulatory sequence. In the epigenetic editor structure, multiple linker structures "]-[ " can be the same, or can each be a different linker, tag, NLS, or peptide bond. In certain embodiments, the DNA methyltransferase domain comprises DNMT3A, DNMT3L, or both. In certain embodiments, the DBD is a catalytically inactive polynucleotide-directed DNA binding domain (such as dCas9) or a ZFP domain. In certain embodiments, the repressor domain is a KRAB domain.

[0198] In some embodiments, the epigenetic editor comprises a construct selected from the following

[0199] N']-[DNMT3A-DNMT3L]-[DBD]-[KRAB]-[C'

[0200] N’]-[KRAB]-[DBD]-[DNMT3A-DNMT3L]-[C’

[0201] N’]-[KRAB]-[DBD]-[DNMT3A]-[C’

[0202] N’]-[DNMT3A]-[DBD]-[KRAB]-[C’

[0203] N’]-[KRAB]-[DBD]-[DNMT3A]-[DNMT3L]-[C’

[0204] N’]-[DNMT3A]-[DNMT3L]-[DBD]-[KRAB]-[C’

[0205] N’]-[DNMT3A]-[DBD]-[C’

[0206] N’]-[DBD]-[DNMT3A]-[C’

[0207] N’]-[DNMT3L]-[DBD]-[C’

[0208] N’]-[DBD]-[DNMT3L]-[C’

[0209] Wherein [DNMT3A-DNMT3L] indicates that the DNMT3A and DNMT3L domains are directly fused by a peptide bond, and wherein the linking structure ]-[ is any linker, detectable tag, affinity domain, peptide bond, nuclear localization signal, promoter and / or regulatory sequence as described herein. The DBD, KRAB, DNMT3A, and DNMT3L domains can be any domain (in any combination) as described herein. 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 the human DNMT3A domain; and the DNMT3L domain is the human or mouse DNMT3L domain; the present disclosure also encompasses any combination of these components.

[0210] In some embodiments, the epigenetic editor comprises a construct selected from the following

[0211] N’]-[DNMT3A]-[DBD]-[SETDB1]-[C’

[0212] N’]-[DNMT3A]-[DNMT3L]-[DBD]-[SETDB1]-[C’

[0213] N’]-[DNMT3A-DNMT3L]-[DBD]-[SETDB1]-[C’

[0214] N’]-[SETDB1]-[DBD]-[DNMT3A]-[DNMT3L]-[C’

[0215] N’]-[SETDB1]-[DBD]-[DNMT3A]-[C’

[0216] Wherein [DNMT3A-DNMT3L] represents that the DNMT3A and DNMT3L domains are directly fused by a peptide bond, and wherein the linker structure ]-[ is any linker, detectable tag, affinity domain, peptide bond, nuclear localization signal, promoter and / or regulatory sequence as described herein. The DBD, SETDB1, DNMT3A, and DNMT3L domains can be any domains (in any combination) as described herein. 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 the human DNMT3A domain; and the DNMT3L domain is the human or mouse DNMT3L domain; the present disclosure also encompasses any combination of these components.

[0217] Specific constructs encompassed herein include:

[0218] DNMT3A-DNMT3L-XTEN80-NLS-dCas9-NLS-XTEN16-KOX1 KRAB

[0219] (Construct 1) and

[0220] DNMT3A-DNMT3L-XTEN80-NLS-ZFP domain-NLS-XTEN16-KOX1

[0221] KRAB (Construct 2).

[0222] In certain embodiments, both DNMT3L and DNMT3A are derived from human parental proteins. In certain embodiments, DNMT3L and DNMT3A are derived from human and mouse parental proteins, respectively. In certain embodiments, DNMT3L and DNMT3A are derived from mouse and human parental proteins, respectively. In certain embodiments, both DNMT3L and DNMT3A are derived from mouse parental proteins. In some embodiments, dCas9 is dSpCas9. In some embodiments, KOX1 is human KOX1.

[0223] In certain embodiments, the fusion constructs described herein can have construct 1 and comprise SEQ ID NO:1080 or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. In SEQ ID NO:1080 below, the XTEN linker is Underline , the NLS sequence is in bold, the DNMT3A sequence is in italics, the DNMT3L sequence Underlined and italic , the dCas9 domain is in bold and italics, and the KOX1 KRAB domain is Underlined and bold :

[0224]

[0225]

[0226] In certain embodiments, the fusion constructs described herein can have construct 2 and comprise SEQ ID NO:1081 or a sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. In SEQ ID NO:1081 below, the XTEN linker is Underline , the NLS sequence is in bold and underlined, the DNMT3A sequence is in italics, the DNMT3L sequence has Underlined and italic , the ZFP domain is in bold, and the KOX1 KRAB domain has Underlined and bold . The variable amino acids represented by X are the amino acids of the zinc finger DNA recognition helix, and the italicized XX can be TR, LR, or LK.

[0227]

[0228]

[0229] In certain embodiments, the six "XXXXXXX" regions in SEQ ID NO:1081 sequentially comprise the F1-F6 amino acid sequences shown in Table 1. [Linker] represents the linker sequence. In some embodiments, one or two of the linker sequences can be TGSQKP (SEQ ID NO:1085). In some embodiments, one or two of the linker sequences can be TGGGGSQKP (SEQ ID NO:1086). In some embodiments, one linker sequence can have the amino acid sequence of SEQ ID NO:1085, and the other linker sequence can have the amino acid sequence of SEQ ID NO:1086.

[0230] Multiple epigenetic editors can be used to activate or repress one or more target genes. For example, an epigenetic editor fusion protein comprising a DNA binding domain (e.g., a dCas9 domain) and an effector domain can be co-delivered with two or more guide polynucleotides (e.g., gRNAs), each guide polynucleotide targeting a different target DNA sequence. The target sites of the two DNA binding domains can be the same or adjacent to each other, or 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. Additionally, when targeting double-stranded DNA such as an endogenous gene locus, the guide polynucleotides can target the same or different strands (one or more targeting the positive strand and / or one or more targeting the negative strand).

[0231] V. Target sequence

[0232] The epigenetic editors of the present disclosure can be directed against HBV target sequences to effect epigenetic modification of HBV or HBV genes. As used herein, "target sequence", "target site", or "target region" is a nucleic acid sequence present in a target genome or gene (e.g., the HBV genome or an HBV gene); in some cases, the target sequence may be outside but near the target gene, where methylation of the target sequence or binding to a repressor represses expression of the gene. In some embodiments, the target sequence can be a hypomethylated or hypermethylated nucleic acid sequence.

[0233] The target sequence can be located in any part of the target gene. In some embodiments, the target sequence is part of or near a 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 a transcriptional regulatory sequence of the gene (such as a promoter or enhancer). In some embodiments, the target sequence is adjacent to, overlaps with, or contains a CpG island, such as a CpG island identified within the HBV genome. In some embodiments, the target sequence is outside of a CpG island. In certain embodiments, the target sequence is located 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) flanking the HBV TSS. In certain embodiments, the target sequence is located within 500 bp flanking the HBV TSS. In certain embodiments, the target sequence is located within 1000 bp flanking the HBV TSS.

[0234] In some embodiments, the target sequence can hybridize with a guide polynucleotide sequence (e.g., gRNA) that is complexed with a fusion protein comprising a polynucleotide-guided DNA-binding domain (e.g., a CRISPR protein such as dCas9) and an effector domain. The guide polynucleotide sequence can be designed to be complementary to the target sequence or identical to the reverse strand of the target sequence. In some embodiments, the guide polynucleotide comprises a spacer sequence having about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity 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.

[0235] In some embodiments, when the DNA-binding domain of the epigenetic editor described herein is a zinc finger array, the target sequence can be recognized by the zinc finger array.

[0236] In some embodiments, when the DNA-binding domain of the epigenetic editor described herein is a TALE, the target sequence can be recognized by the TALE.

[0237] The target sequences described herein can be specific for one HBV genotype, one copy of an HBV target gene, or can be specific for an allele of an HBV target gene. However, in some embodiments, the target sequences can be conserved among two or more HBV genotypes, two or more copies of an HBV gene, and alleles of an HBV gene. Thus, the epigenetic modification and its expression regulation can be specific for one copy or one allele of a target gene or, in other embodiments, can be general for different HBV genotypes, HBV gene copies, or alleles.

[0238] In some embodiments, the target sequence is contained in the following sequences:

[0239] >NC_003977.2 Hepatitis B virus (strain ayw) genome

[0240]

[0241] In some embodiments, the target sequence is contained in the following sequences:

[0242] >U95551.1 Hepatitis B virus subtype ayw, complete genome

[0243]

[0244] VI. Epigenetic modification

[0245] The epigenetic editors described herein can perform sequence-specific epigenetic modifications (e.g., altering chemical modifications) on target genes containing a target sequence. Such epigenetic regulation may be safer and more easily reversible than regulation by gene editing (e.g., generating DNA double-strand breaks). In some embodiments, the epigenetic regulation can reduce or silence the target gene. In some embodiments, the modification is at a specific locus of the target sequence. In some embodiments, the modification is at a specific allele of the target gene. Thus, the epigenetic modification may result in the regulation (e.g., reduction) of the expression of one copy of the target gene containing a specific allele, while the expression of the other copy of the target gene remains unaffected. In some embodiments, the specific allele is associated with a disease, condition, or disorder.

[0246] In some embodiments, the epigenetic modification reduces or eliminates the transcription of the target gene containing the target sequence. In some embodiments, the epigenetic modification reduces or eliminates the transcription of the copy of the target gene containing a specific allele recognized by the epigenetic editor. In some embodiments, the epigenetic editor reduces the expression level of the protein encoded by the target gene or eliminates its expression. In some embodiments, the epigenetic editor reduces the expression level of the protein encoded by the copy of the target gene containing a specific allele recognized by the epigenetic editor or eliminates its expression. The target HBV gene can be epigenetically modified in vitro, ex vivo, or in vivo.

[0247] The effector domain of the epigenetic editors described herein can alter (e.g., deposit or remove) chemical modifications at the nucleotides of the target gene or at the histones associated with the target gene. The chemical modification can be altered at a single nucleotide or a single histone, or can be altered at 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.

[0248] In some embodiments, the effector domain of the epigenetic editors described herein can alter CpG dinucleotides within a target gene. In some embodiments, all CpG dinucleotides within 2000, 1500, 1000, 500, or 200 bp flanking the target sequence (e.g., at the site of alteration as described herein) are altered according to the type of modification 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 the epigenetic editor. In 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 the epigenetic editor.

[0249] The effector domain of the epigenetic editors described herein can alter the histone modification state of histones associated with or bound to a target gene. For example, the effector domain can deposit a modification on one or more lysine residues of the histone tail of a histone associated with the target gene. In some embodiments, the effector domain can cause deacetylation of one or more histone tails of a histone associated with the target gene, thereby reducing or silencing the expression of the target gene. In some embodiments, the histone modification state is a methylation state. For example, the effector domain can cause methylation of one or more histone tails associated with the target gene at H3K9, H3K27, or H4K20 (e.g., one or more of H3K9me2, H3K9me3, H3K27me2, H3K27me3, and H4K20me3 methylation), thereby reducing or silencing the expression of the target gene.

[0250] In some embodiments, all histone tails of histones that bind to DNA nucleotides within 2000, 1500, 1000, 500, or 200 bp flanking the target sequence are altered according to the types of modifications described herein, compared to the original state of the chromosome or the chromosome in comparable cells that have not been 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, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 or more histone tails of the histone that binds are altered, compared to the original state of the chromosome or the chromosome in comparable cells that have not been 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 histone tails of the histone that binds are altered, compared to the original state of the chromosome or the chromosome in comparable cells that have not been contacted with the epigenetic editor. For example, a single histone tail of the histone that binds can be altered, compared to the original state of the chromosome or the chromosome in comparable cells that have not been contacted with the epigenetic editor. As another example, a single bound histone octamer can be altered, compared to the original state of the chromosome or the chromosome in comparable cells that have not been contacted with the epigenetic editor.

[0251] The chemical modifications deposited at the target gene DNA nucleotides or histone residues can be at or near the target sequence in the target gene. In some embodiments, the effector domain of the epigenetic editor described herein alters the chemical modification state of nucleotides or histone tails that bind to nucleotides 100 - 200, 200 - 300, 300 - 400, 400 - 55, 500 - 600, 600 - 700, or 700 - 800 nucleotides in the 5' or 3' direction of the target sequence in the target gene. In some embodiments, the effector domain alters the chemical modification state of nucleotides or histone tails that bind 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 flanking the target sequence. As used herein, "flanking" refers to the nucleotide positions at the 5' to 5' end and 3' to 3' end of a particular sequence (e.g., the target sequence).

[0252] In some embodiments, the effector domain mediates or induces a change in the chemical modification of a nucleotide or histone tail that binds to a nucleotide distal to the target sequence. Such modifications may initiate near the target sequence and may subsequently spread to one or more nucleotides in the target gene that are distal to the target sequence. For example, the effector domain can initiate a change in the chemical modification state of one or more nucleotides or one or more histone residues that bind to one or more nucleotides within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 nucleotides flanking the target sequence, and this change in chemical modification state can spread to one or more nucleotides in the target gene that are 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 away from the target sequence, whether upstream or downstream of the target sequence. In certain embodiments, the chemical modification may initiate at less than 2, 3, 5, 10, 20, 30, 40, 50 or 100 nucleotides in the target gene and spread to at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000 or more nucleotides in the target gene. In some embodiments, the chemical modification spreads to the nucleotides of the entire target gene. Other proteins or transcription factors, such as transcriptional repressors, methyltransferases or transcriptional regulatory scaffold proteins, may be involved in the spread of the chemical modification. Alternatively, the epigenetic editor may be involved alone.

[0253] In some embodiments, compared to a control cell, control tissue, or control subject (e.g., in the absence of an epigenetic editor), the epigenetic editors described herein reduce the 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, as measured by transcription of the target gene in a cell, tissue, or subject. In some embodiments, compared to a control cell, control tissue, or control subject, the epigenetic editors described herein reduce the expression of a target gene copy 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, as measured by transcription of the target gene copy in a cell, tissue, or subject. In certain embodiments, the target gene copy contains a specific sequence or allele recognized by the epigenetic editor. In specific embodiments, the epigenetically modified copy encodes a functional protein, and thus the epigenetic editors disclosed herein can reduce or eliminate the expression and / or function of that protein. For example, compared to a control cell, control tissue, or control subject, the epigenetic editors described herein can reduce the expression and / or function of the protein encoded by the target gene in a cell, tissue, or subject 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.

[0254] Regulation of target gene expression can be assayed by determining any parameter directly or indirectly affected by the expression of the target gene. These parameters include, for example, 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 a reporter gene (such as luciferase, CAT, β-galactosidase, or GFP); changes in signal transduction; changes in phosphorylation and dephosphorylation; changes in receptor-ligand interactions; second messengers (such as cGMP, cAMP, IP3, and Ca2 +) Changes in concentration; changes in cell growth; changes in neovascularization; 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 carried out by conventional methods, e.g., measuring RNA or protein levels, measuring RNA stability, and / or identifying the expression of downstream or reporter genes. Readouts can be obtained, for example, by chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, ligand-binding assays, changes in intracellular second messengers such as cGMP and inositol trisphosphate (IP3), changes in intracellular calcium levels, cytokine release, etc.

[0255] Methods for determining the expression level of a gene (e.g., a target of an epigenetic editor) can include, for example, determining the transcript level of the gene by reverse transcription PCR, quantitative RT-PCR, droplet digital PCR (ddPCR), Northern blotting, 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 protein level of gene expression can be determined, for example, by Western blotting, enzyme-linked immunosorbent assay, mass spectrometry, immunohistochemistry, or flow cytometry analysis. The level of gene expression products can be normalized to an internal standard such as the expression level of total messenger ribonucleic acid (mRNA) or a specific gene (e.g., a housekeeping gene).

[0256] In some embodiments, a reporter system can be used to examine the role of an epigenetic editor in regulating target gene expression. For example, an epigenetic editor can be designed to target a reporter gene encoding a reporter protein (such as a fluorescent protein). The expression of the reporter gene in such model systems can be monitored by, for example, flow cytometry, fluorescence-activated cell sorting (FACS), or fluorescence microscopy. In some embodiments, a cell population can be transfected with a vector containing the reporter gene. The vector can be constructed such that the reporter gene is expressed when the vector transfects the cell. Suitable reporter genes include genes encoding fluorescent proteins (e.g., green, yellow, cherry, cyan, or orange fluorescent proteins). A cell population carrying the reporter system can be transfected with DNA, mRNA, or a vector encoding the epigenetic editor targeting the reporter gene.

[0257] VII. Pharmaceutical composition

[0258] Another aspect of the present disclosure is a pharmaceutical composition comprising one or more of the epigenetic editors or components thereof (e.g., fusion proteins and / or guide polynucleotides) described herein as the active ingredient (or as the sole active ingredient), or a nucleic acid molecule encoding the epigenetic editor or components thereof. For example, the pharmaceutical composition can comprise a nucleic acid molecule encoding a fusion protein of the epigenetic editor described herein (and a guide polynucleotide, if applicable). In some embodiments, the pharmaceutical composition separately comprises a fusion protein and a guide polynucleotide. In some embodiments, multiple pharmaceutical compositions are administered simultaneously, each pharmaceutical composition comprising an epigenetic editor. The pharmaceutical composition can also comprise a cell that has undergone an epigenetic modification mediated or induced by the epigenetic editor provided herein.

[0259] Generally, the epigenetic editors or components thereof described herein of the present disclosure, or the nucleic acid molecule encoding the epigenetic editor or components thereof, are suitable for administration as a formulation associated with one or more pharmaceutically acceptable excipients, e.g., as described below.

[0260] The term "excipient" is used herein to describe any ingredient other than the compounds of the present disclosure. The choice of excipient will to a large extent depend on various factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. As used herein, "pharmaceutically acceptable excipient" includes any and all physiologically compatible solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, etc. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate buffered saline, glucose, glycerol, ethanol, etc., and combinations thereof. In many cases, it is preferred to include an isotonic agent in the composition, such as a sugar, a polyol (such as mannitol, sorbitol) or sodium chloride. Further examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances, such as wetting agents or emulsifying agents, preservatives or buffering agents, which can extend the shelf life or effectiveness of the antibody.

[0261] Pharmaceutical composition formulations suitable for parenteral administration typically comprise a combination of an active ingredient with a pharmaceutically acceptable carrier (such as sterile water or sterile isotonic saline). Such formulations can be prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. In some embodiments, the epigenetic editor or its components are introduced into the target cell in the form of a nucleic acid molecule encoding the epigenetic editor or its components; thus, the pharmaceutical compositions herein comprise such nucleic acid molecules. Such nucleic acid molecules can be, for example, DNA, RNA, or mRNA, and / or modified nucleic acid sequences (e.g., having chemical modifications, 5' caps, or one or more 3' modifications). In some embodiments, the nucleic acid molecule can be delivered in the form of naked DNA or RNA, e.g., by transfection or electroporation, or can be conjugated with a molecule that facilitates uptake by the target cell (such as N-acetylgalactosamine). In some embodiments, the nucleic acid molecule can be present in a nucleic acid expression vector, which can include expression control sequences such as promoters, enhancers, transcriptional signal sequences, transcriptional 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 can also contain a sequence encoding a signal peptide (e.g., for nuclear localization, nucleolar localization, or mitochondrial localization), which is associated with (e.g., inserted or fused to) the sequence encoding the protein.

[0262] 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, retroviruses (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 epigenetic editors or their components as described herein. For example, "blank" viral particles can be assembled to accommodate any suitable cargo. Viral vectors and viral particles can also be engineered to bind targeting ligands to alter target tissue specificity.

[0263] In some embodiments, an epigenetic editor or a component thereof as described herein is encoded by a nucleic acid sequence present in one or more viral vectors or a suitable capsid protein 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., Maloney murine leukemia virus, MML-V), adenoviral vectors (e.g., AD100), lentiviral vectors (e.g., HIV- and FIV-based vectors), and herpes viral vectors (e.g., HSV-2).

[0264] In some embodiments, delivery involves an adeno-associated virus (AAV) vector. AAV vector delivery may be particularly useful when the DNA-binding domain of the epigenetic editor fusion protein is a zinc finger array. Without wishing to be bound by any theory, compared to larger DNA-binding domains such as Cas protein domains, the smaller size of the zinc finger array may enable such fusion proteins to be conveniently packaged in viral vectors such as AAV vectors.

[0265] Any AAV serotype, such as human AAV serotypes, can be used for the AAV vectors as 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, the AAV variant can be engineered to have reduced immunogenicity or enhanced transduction ability in the human body. 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, a chimeric AAV may contain inverted terminal repeats (ITRs) of a heterologous serotype different from the capsid serotype. The resulting chimeric AAV may have different antigen reactivity or recognition ability compared to its parental serotype. In some embodiments, the chimeric variant of AAV includes amino acid sequences from 2, 3, 4, 5, or more different AAV serotypes.

[0266] Also encompasses non-viral systems 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 particle bombardment, lipid-mediated transfection, transfection by heat shock, condensed DNA-mediated transfection, lipofection, cationic agent-mediated transfection, and transfection using liposomes, immunoliposomes, or cationic amphiphiles (CFAs). In certain embodiments, one or more mRNAs encoding an epigenetic editor fusion protein as described herein can be co-electroporated with one or more guide polynucleotides (e.g., gRNAs) as described herein. An important class of non-viral nucleic acid carriers are nanoparticles, which can be organic (e.g., lipid) or inorganic (e.g., gold). For example, organic (e.g., lipid and / or polymer) nanoparticles can be suitable for use as delivery vehicles in certain embodiments of the present disclosure.

[0267] In some embodiments, delivery is accomplished using lipid nanoparticles (LNPs). The size of the LNP composition is typically on the order of microns or smaller and can comprise a lipid bilayer. In some embodiments, an LNP refers to any particle having a diameter 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 lipid complexes.

[0268] The LNPs as described herein can be made from cationic, anionic, or neutral lipids. In some embodiments, the LNP can comprise neutral lipids, such as the fusogenic phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or the membrane component cholesterol, as co-lipids to enhance transfection activity and nanoparticle stability. In some embodiments, the LNP can comprise hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids. Any lipid or combination of lipids known in the art can be used to produce LNPs. The lipids can be combined in any molar ratio to produce LNPs. In some embodiments, the LNP is liver-targeted (e.g., preferentially or specifically targeted to the liver).

[0269] Based on the present disclosure and the prior art, those skilled in the art will be aware of the LNP formulations and LNP delivery methods that can be used. Non-limiting exemplary compositions and methods can be found in 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, M.J., Billingsley, M.M., Haley, R.M. 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 J.D. Ross. Molecular Pharmaceutics 2022 19(6), 1669-1686; Cullis PR, Hope MJ. Lipid Nanoparticle Systems for Enabling Gene Therapies, Mol Ther. July 5, 2017; 25(7):1467-1475; Hatit, M.Z.C., Lokugamage, M.P., Dobrowolski, C.N. et 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 primate, bioRxiv 2021.11.08.467731, doi: https: / / doi.org / 10.1101 / 2021.11.08.467731; Tombácz, I. et al., Highly efficient CD4+ T cell targeting and genetic recombination using engineered CD4+ cell-homing mRNA-LNPs, Molecular Therapy, Volume 29, Issue 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. 8,642,076; U.S. Patent No. 9,593,077; U.S. Patent No. 9,415,109; U.S. Patent No. 9,701,623; U.S. Patent No. 10,369,226; U.S. Patent No. 9,999,673; U.S. Patent No. 9,301,923; U.S. Patent No. 10,342,761; U.S. Patent No. 10,137,201; International Publication No. WO2016081029A1; each of the foregoing is incorporated herein by reference in its entirety. Those of ordinary skill in the art can determine suitable LNPs and delivery methods based on the present disclosure and the prior art. The present disclosure is not limited in this regard.

[0270] Other methods of delivering to target cells are known to those skilled in the art and can be used in conjunction with the compositions of the present disclosure.

[0271] Any type of cell can be targeted for delivery of an epigenetic editor or its components as described herein. For example, the cell can be a eukaryotic or prokaryotic cell. In some embodiments, the cell is a mammalian (e.g., human) cell. Human cells can include, for example, hepatocytes, cholangiocytes (bile duct cells), stellate cells, Kupffer cells, and hepatic sinusoidal endothelial cells.

[0272] In some embodiments, the epigenetic editors or components thereof described herein are delivered to a host cell for transient expression, e.g., via a transient expression vector. Transient expression of the epigenetic editor or components thereof can result in extended or permanent epigenetic modification of a target gene. For example, after introduction of the epigenetic editor into a host cell, the epigenetic modification can be stable for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks or longer; or 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer. The epigenetic modification can be maintained after one or more mitotic and / or meiotic events of the host cell. In certain embodiments, the epigenetic modification is maintained across generations in progeny produced by or derived from the host cell.

[0273] VIII. Therapeutic use of epigenetic editors

[0274] The present disclosure also provides methods for treating or preventing a condition in a subject, comprising administering to the subject an epigenetic editor or a pharmaceutical composition as described herein. The epigenetic editor can epigenetically modify a target polynucleotide sequence in a target gene associated with a disease, condition, or disorder of the subject, thereby regulating the expression of the target gene to treat or prevent the disease, condition, or disorder. In some embodiments, the epigenetic editor reduces the expression of the target gene to an extent sufficient to achieve a desired effect, e.g., a treatment-related effect such as preventing or treating the disease, condition, or disorder.

[0275] In some embodiments, a system for modulating (e.g., repressing) HBV or HBV gene expression is administered to a subject, wherein the system comprises (1) a fusion protein of an epigenetic editor as described herein and, where relevant, a guide polynucleotide, or (2) a nucleic acid molecule encoding the fusion protein and, where relevant, the guide polynucleotide.

[0276] "Treat", "treating", and "treatment" refer to methods 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 refers to reducing the severity and / or frequency of occurrence of the disease, disorder, or condition. Further, "treatment" as referred to herein includes curative treatment, palliative treatment, and prophylactic treatment. In some embodiments, alleviating symptoms can involve a reduction in symptoms of at least 3%, 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.9%, or 100% compared to an untreated control of equal status, as measured by any standard technique.

[0277] In some embodiments, the subject can be a mammal, such as a human. In some embodiments, the subject is selected from non-human primates, such as chimpanzees, cynomolgus monkeys or rhesus monkeys, as well as other apes and monkeys.

[0278] In some embodiments, the human patient has a condition characterized by HBV infection. In some embodiments, the patient has hepatitis B.

[0279] In some embodiments, the patient to be treated with the 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's prior treatment for the condition (e.g., prior HBV treatment) has failed (or is refractory to it).

[0280] The epigenetic editor of the present disclosure can be administered to a patient having a condition described herein in a therapeutically effective amount. As used herein, "therapeutically effective amount" refers to the amount of a therapeutic agent administered that will, to some extent, alleviate one or more symptoms of the treated disorder, and / or result in a clinical endpoint desired by a healthcare professional. A therapeutically effective amount can be measured by its ability to stabilize the progression of the patient's disease and / or improve symptoms, and preferably reverse the progression of the disease. The ability of the epigenetic editor of the present disclosure to reduce or silence HBV expression can be evaluated by in vitro assays (e.g., as described herein) and in suitable animal models predictive of its efficacy in humans. A suitable dosage regimen will be selected so as to provide an optimal therapeutic response in each particular case, e.g., administered as a single bolus or by continuous infusion, and the dosage may be adjusted according to the exigencies of each case.

[0281] The epigenetic editor of the present disclosure can be administered without additional treatment, i.e., as a monotherapy (single therapy). Alternatively, treatment using the epigenetic editor of the present disclosure can include at least one additional treatment (combination therapy). In some embodiments, the additional therapeutic agent is any therapeutic agent known in the art for HBV and / or HDV. In some embodiments, the therapeutic agent includes, but is not limited to, antiviral drugs such as entecavir, tenofovir, lamivudine, telvivudine, bictegravir, emtricitabine or defovir, and immunomodulators such as pegylated interferon and interferon α.

[0282] The epigenetic editor of the present disclosure or its components (or a nucleic acid molecule encoding the epigenetic editor or its components) can be administered by any method acceptable in the art (e.g., parenterally, intravenously, intradermally, or intramuscularly).

[0283] The epigenetic editor of the present disclosure or its components (or a nucleic acid molecule encoding the epigenetic editor or its components) can be administered to a subject one, two, three, or 4, 5, 6, 7, 8, 9, 10 times or more. In some embodiments, one, two, three, or 4, 5, 6, 7, 8, 9, 10 or more administrations of the epigenetic editor or its components (or a nucleic acid molecule encoding the epigenetic editor or its components) are close in time (e.g., within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 4 weeks, 1 month, or 2 months of each other). In some embodiments, after an initial dose, the epigenetic editor of the present disclosure or its components (or a nucleic acid molecule encoding the epigenetic editor or its components) is re-administered to the subject at least once. In some cases, a subsequent dose of the epigenetic editor of the present disclosure or its components (or a nucleic acid molecule encoding the epigenetic editor or its components) is administered to the subject, targeting a different DNA region of the HBV genome than the DNA region of the HBV genome targeted by the epigenetic editor or its components that the subject received at the initial dose. In some cases, multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of the same epigenetic editor of the present disclosure or its components (or a nucleic acid molecule encoding the epigenetic editor or its components) are administered to the subject. In some cases, a single dose of different epigenetic editors of the present disclosure or its components (or a nucleic acid molecule encoding the epigenetic editor or its components) is administered to the subject, where at least two target different DNA regions of the HBV genome. In some cases, multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of different epigenetic editors of the present disclosure or its components (or a nucleic acid molecule encoding the epigenetic editor or its components) are administered to the subject, where at least two target different DNA regions of the HBV genome. In some embodiments, re-administering the epigenetic editor of the present disclosure or its components (or a nucleic acid molecule encoding the epigenetic editor or its components) has better therapeutic efficacy than a single administration of the same substance, e.g., more effectively suppressing HBV replication, or more significantly reducing the 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).

[0284] XII. Definition

[0285] As used herein, the term "nucleic acid" refers to any oligonucleotide or polynucleotide containing nucleotides (e.g., deoxyribonucleotides or ribonucleotides) in single-stranded or double-stranded form, and includes DNA and RNA. A "nucleotide" contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group, and is linked together by phosphate groups. "Bases" include purines and pyrimidines, which include natural compounds such as adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs; and synthetic derivatives of purines and pyrimidines, including but not limited to modified forms that introduce new reactive groups (such as amines, alcohols, thiols, carboxylates, alkyl halides, etc.). Nucleic acids can contain known nucleotide analogs and / or modified backbone residues or linkages, which can be synthetic, naturally occurring, and non-naturally occurring. Such nucleotide analogs, modified residues, and modified linkages are well known in the art and can confer enhanced cellular uptake, reduced immunogenicity, and / or enhanced stability in the presence of nucleases to nucleic acid molecules.

[0286] As used herein, an "isolated" or "purified" nucleic acid molecule is a nucleic acid molecule that exists separate from its natural environment. For example, an "isolated" or "purified" nucleic acid molecule (1) has been separated from the nucleic acids of genomic DNA or cellular RNA from which it is derived; and / or (2) does not exist in nature. In some embodiments, an "isolated" or "purified" nucleic acid molecule is a recombinant nucleic acid molecule.

[0287] It should be understood that, in addition to the specific protein and nucleic acid molecules mentioned herein, the present disclosure also encompasses the use of their variants, derivatives, homologs, and fragments. Variants of any given sequence can have a specific residue sequence (whether amino acid residues or nucleic acid residues) that is modified such that the polypeptide or polynucleotide substantially retains at least one of its endogenous functions. Variant sequences can be obtained by adding, deleting, substituting, modifying, permuting, and / or altering at least one residue present in the naturally occurring sequence (in some embodiments, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 residues). For the specific proteins described herein (e.g., the KRAB, dCas9, DNMT3A, and DNMT3L proteins described herein), the present disclosure also encompasses any naturally occurring form of the protein, or variants or homologs that retain at least one of its endogenous functions (e.g., at least 50%, 60%, 70%, 80%, 90%, 85%, 96%, 97%, 98%, or 99% of the function compared to the specific protein described).

[0288] As used herein, homologs of any polypeptide or nucleic acid sequence covered herein include sequences having a certain degree of homology to wild-type amino acid and nucleic acid sequences. Homologous sequences can include sequences that can have at least 50%, 55%, 65%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the subject sequence, such as an amino acid sequence. In the context of an amino acid or nucleotide sequence, "percent identity" refers to the percentage of residues that are the same when the two sequences are aligned for maximum correspondence. In some embodiments, the length of the alignment reference sequence for comparison purposes is at least 30% of the reference sequence (e.g., at least 40%, 50%, 60%, 70%, 80%, 90% or 100%). Sequence identity can be measured using sequence analysis software (e.g., the sequence analysis software package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, WI 53705), BLAST, BESTFIT, GAP or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assessing homology for various substitutions, deletions and / or other modifications. In an exemplary method for determining the degree of identity, the BLAST program can be used, and a probability score between e-3 and e-100 indicates that the sequences are closely related.

[0289] The percent identity of two nucleotide or polypeptide sequences is determined, for example, using default parameters (available on the website of the National Center for Biotechnology Information of the U.S. National Library of Medicine). In some embodiments, the length of the alignment reference sequence for comparison purposes is at least 30% of the reference sequence (e.g., at least 40%, 50%, 60%, 70%, 80% or 90%).

[0290] It should be understood that the numbering of a particular position or residue in a polypeptide sequence depends on the specific protein and the numbering scheme used. The numbering may vary. For example, the numbering of the precursor of a mature protein and the mature protein itself may be different, and sequence differences among different species may also affect the numbering. Those skilled in the art can identify the corresponding residues in any homologous protein and its corresponding coding nucleic acid by methods well known in the art, such as by sequence alignment and determination of homologous residues.

[0291] The terms "modulate" or "alter" refer to a change in the quantity, degree, or extent of a function. For example, an epigenetic editor as described herein can modulate the activity of a promoter sequence by binding to a motif within the promoter, thereby inducing, enhancing, or suppressing the transcription of a gene operably linked to the promoter sequence. As other examples, an epigenetic editor as described herein can block the transcription of a gene by RNA polymerase, or can inhibit the translation of an mRNA transcript. The terms "inhibit", "repress", "suppress", "silence", etc., when used in the context of an epigenetic editor or its components as described herein, refer to a decrease or prevention of the activity (e.g., transcription) of a nucleic acid sequence (e.g., a target gene) or a protein relative to the activity of the nucleic acid sequence or protein in the absence of the epigenetic editor or its components. The term can include partial or complete blocking of the activity, or prevention or delay of the activity. The inhibited activity can be, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% lower than the activity of a control, or can be, for example, at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold lower than the activity of a control.

[0292] The terms "about" or "approximately" refer to an acceptable error range of 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 or more standard deviations in accordance with the practice for a given value. When a particular value is described in the present application and claims, unless otherwise stated, the term "about" should be assumed to refer to the acceptable error range of the particular value.

[0293] The ranges provided herein are to be understood as shorthand for all values within the range. For example, a range of 1 to 50 should be understood to include any number, combination of numbers, or sub-range 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 intermediate decimal values between the above 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 sub-ranges, "nested sub-ranges" extending from either endpoint of the range are specifically covered. For example, the nested sub-ranges of the exemplary range of 1 to 50 can include, in one direction, 1 to 10, 1 to 20, 1 to 30, and 1 to 40, or in the other direction, 50 to 40, 50 to 30, 50 to 20, and 50 to 10.

[0294] Unless otherwise defined herein, scientific and technical terms related to the present disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Exemplary methods and materials are described below, but methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification (including definitions) shall prevail. In addition, unless the context otherwise requires, singular terms shall include the plural, and plural terms shall include the singular. Throughout the specification and the embodiments, the words "have" and "comprise", or their variants, such as "has", "having", "comprises", or "comprising" shall be understood to imply the inclusion of the 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 element singly or in any combination. The recitation of embodiments herein includes the embodiments as a single embodiment, or in combination with any other embodiment herein. All publications, patents, patent applications, and other references mentioned herein are incorporated by reference in their entirety. If the references incorporated by reference conflict with the disclosure contained in the present specification, the present specification is intended to supersede and / or take precedence over any such conflicting material. Although multiple documents are cited herein, such citation does not constitute an admission that any of these documents constitutes common general knowledge in the art.

[0295] To better understand the present disclosure, the following examples are presented. These examples are for illustrative purposes only and should not be construed as limiting the scope of the present disclosure in any way.

[0296] Example

[0297] Example 1: Selection of Target HBV Sequences for Epigenetic Silencing

[0298] Using representative HBV genomic sequences (SEQ ID Nos. 1082, 1083) as a reference, target sequences were designed manually and computationally:

[0299] Although the target site design focused on the identified CpG islands within the HBV genome, target sites outside the HBV CpG islands were also considered.

[0300] Table 2 presents some representative target sites identified as suitable for targeting with epigenetic repressors.

[0301] The target domains adjacent to the PAM sequence (e.g., Streptococcus pyogenes Cas9 PAM sequence) identified above can be targeted by CRISPR-based epigenetic repressors (e.g., epigenetic repressors containing the dCas9 DNA-binding domain). For example, target site 1-143 is suitable for targeting with dCas9-based epigenetic repressors. Figure 1 An overview of the positions of the identified target sites in the HBV genome is provided.

[0302] The conservation of the target sites in HBV genotypes A - E was analyzed ( Figure 2 and Figure 3 ). Some target sites with good conservation in two or more or in some cases in all HBV genotypes were identified. Targeting such conserved sites allows the use of the same epigenetic repressor to silence different genotypes.

[0303] Example 2: Guide RNA Assay in HepAD38 HBV Cells

[0304] The HepAD38 cell line expresses the HBV genome under a doxycycline-inducible promoter (see, for example, 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).

[0305] The results are as Figure 4A and Figure 4B shown.

[0306] Example 3: Guide RNA Assay in HepG2-NTCP Cells

[0307] HepG2 cells were engineered by lentiviral transduction to express the human NTCP receptor that hepatitis B virus (HBV) uses to infect cells.

[0308] The HepAD38 cell line was used to produce HBV viral particles. HepAD38 is a subclone derived from the HepG2 cell line that expresses the HBV genome (genotype D, ayw subtype) under the transcriptional regulation of a tetracycline-responsive promoter in the TET-OFF system.

[0309] A triple-engineered transcriptional repressor (ETR) combination consisting of three plasmids expressing dCas9-KRAB, dCas9-DNMT3A, and dCas9-DNMT3L was used in combination with one or more designed sgRNAs.

[0310] LNPs were formulated using the GENVOY ILM lipid mixture (Precision Nanosystem) and the formulator NanoassemblrSpark (Precision Nanosystem). LNPs were formulated according to the manufacturer's recommendations, with 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 / μL in PNI formulation buffer. Each of the ETR, dCas9-KRAB, dCas9-DNMT3A, dCas9-DNMT3L, and 121 sgRNAs was mixed in a ratio of 1:1:1:4. The RNA mixture, Genvoy lipid mixture (25 mM), and PBS were each loaded into dedicated chambers of the Spark cartridge and formulated. The packaged mRNA was quantified using the Quant-it TM RiboGreen RNA Assay Kit (Thermo Fisher), and the quality of the formulated LNPs was evaluated by determining the size of the LNPs by dynamic light scattering (Zetasizer, Malvern Panalytic).

[0311] HepG2-NTCP cells were seeded at 20,000 cells per well in a collagen-coated 96-well plate. After 24 hours, the cells were infected with HBV at a multiplicity of genome equivalent (MGE) of 5,000 genome equivalents. Sixteen hours after removing the virus inoculum, the cells were washed with PBS and fresh medium was added. On the third day after infection, each sgRNA and the mRNA components of the triple construct encoding ETR (dCas9-KRAB, dCas9-DNMT3A, dCas9-DNMT3L) were delivered using LNP. Three days later, the LNP was removed, the medium was changed, and the cells were cultured in complete medium for three days.

[0312] Six days after LNP removal, ELISA assays were used to quantify the viral antigens HBeAg and HBsAg. Data were normalized to a non-targeting guide designed against mouse PCSK9, and a control 3.2 gRNA was used as a positive control. A cell viability assay was performed and normalized to a non-targeting control.

[0313] The following table provides the amino acid sequences of exemplary epigenetic editors (ETR constructs) for gRNA screening:

[0314] Table 6: Amino acid sequences of exemplary epigenetic editors

[0315]

[0316]

[0317]

[0318]

[0319] The following table provides the amino acid sequences and polynucleotide sequences of exemplary epigenetic editors

[0320] Table 7: Sequences of exemplary epigenetic editors

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335] Table 8 below lists the components of the fusion polypeptide PLA001 and their corresponding amino acid positions in the fusion polypeptide sequence (SEQ ID No. 481) shown in Table 7.

[0336] Table 8: Annotation of the Amino Acid Sequence of PLA001

[0337] Type Start End Length SV40 NLS CDS 2 8 7 SV40 NLS CDS 9 15 7 DNMT3A CDS 17 317 301 Linker CDS 318 344 27 DNMT3L full - length CDS 345 730 386 XTEN80 CDS 731 810 80 dCas9 CDS 811 2180 1370 NLS CDS 2181 2187 7 XTEN16 CDS 2188 2208 21 ZN627 CDS 2211 2290 80 FLAG CDS 2293 2300 8 SV40 NLS CDS 2302 2308 7 SV40 NLS CDS 2309 2315 7

[0338] Table 9 below lists the components of the polynucleotide encoding the fusion polypeptide PLA001 and their corresponding nucleotide positions in the polynucleotide sequence (SEQ ID No. 482) shown in Table 7.

[0339] Table 9: Annotation of the Polynucleotide Sequence of PLA001

[0340]

[0341]

[0342] Table 10 below lists the components of the fusion polypeptide PLA002 and their corresponding amino acid positions in the fusion polypeptide sequence (SEQ ID No. 483) shown in Table 7.

[0343] Table 10: Annotation of the Amino Acid Sequence of PLA002

[0344] Name Type Minimum Maximum Length SV40 NLS CDS 2 8 7 SV40 NLS CDS 9 15 7 DNMT3A CDS 17 317 301 Linker CDS 318 344 27 DNMT3L full - length CDS 345 730 386 XTEN80 CDS 731 810 80 dCas9 CDS 811 2180 1370 NLS CDS 2181 2187 7 XTEN16 CDS 2188 2208 21 ZIM3 CDS 2211 2310 100 FLAG CDS 2313 2320 8 SV40 NLS CDS 2322 2328 7 SV40 NLS CDS 2329 2335 7

[0345] Table 11 below lists the components of the polynucleotide encoding the fusion polypeptide PLA002 and their corresponding nucleotide positions in the polynucleotide sequence (SEQ ID No. 484) shown in Table 7.

[0346] Table 11: Annotation of the Polynucleotide Sequence of PLA002

[0347] Name Type Minimum Maximum Length SV40 NLS CDS 4 24 21 SV40 NLS CDS 25 45 21 DNMT3A CDS 49 951 903 Linker CDS 952 1032 81 DNMT3L full - length CDS 1033 2190 1158 XTEN80 CDS 2191 2430 240 dCas9 CDS 2431 6540 4110 NLS CDS 6541 6561 21 XTEN16 CDS 6562 6624 63 ZIM3 CDS 6631 6930 300 FLAG CDS 6937 6960 24 SV40NLS CDS 6964 6984 21 SV40NLS CDS 6985 7005 21 Termination (stop) Terminator 7006 7008 3

[0348] Table 12. Annotation of the Amino Acid Sequence of PLA003

[0349] Name Type Minimum Maximum Length SV40 NLS CDS 2 8 7 SV40NLS CDS 9 15 7 DNMT3A CDS 17 317 301 Linker CDS 318 344 27 DNMT3L full - length CDS 345 730 386 XTEN80 CDS 731 810 80 dCas9 CDS 811 2180 1370 NLS CDS 2181 2187 7 XTEN16 CDS 2188 2208 21 ZIM3 CDS 2211 2310 100 SV40 NLS CDS 2313 2319 7 SV40 NLS CDS 2320 2326 7

[0350] Table 13. Annotation of the Polynucleotide Sequence of PLA003

[0351]

[0352]

[0353] Table 14 below provides the gRNA sequences tested.

[0354] Table 14: Exemplary gRNA Sequences

[0355]

[0356]

[0357]

[0358]

[0359]

[0360] Table 15: Exemplary Target Domain Sequences and Their Effects on HbeAg and HbsAg Expression

[0361]

[0362]

[0363]

[0364] In the HepG2-NTCP infection model of ETR using gRNA targeting CpG islands, in vitro silencing was observed ( Figures 5A - 5B ). Preliminary screening was performed using LNPs within the expected parameter range of quality, and pilot experiments were performed using single guides ( Figures 6 - 8 ). The results showed that compared with the non-targeting control, the HBeAg expression of 48 gRNAs was less than 50% on day 6 ( Figure 9 ), and compared with the non-targeting control, the HBsAg expression of 28 gRNAs was less than 50% on day 6 ( Figure 10 ). As Figure 11 shown, there was a positive correlation between HBsAg and HBeAg expression.

[0365] Example 4: Zinc Finger Repressor for Silencing HBV

[0366] Zinc finger repressors targeting the identified epigenetic target sites in the HBV genome were designed. Table 1 above provides the amino acid sequences of the zinc fingers, their corresponding motif sequences, and the target sequences of the zinc fingers.

[0367] The zinc finger repressors described in Table 1 were tested in an HBV infection model (e.g., HepG2 cells as described herein), and it was confirmed that the zinc finger repressors provided in Table 1 were capable of effectively inhibiting HBV.

[0368] Example 5: Further in vitro evaluation of gRNAs

[0369] The CRISPR-Off single construct encoding PLA002, consisting of KRAB, DNMT3A, DNMT3L, and dCas9, was combined with one or more designed sgRNAs for the in vitro assays described in this example.

[0370] Following a procedure similar to that of Example 3, HepG2-NTCP cells were infected with HBV for 4 days and then transfected with the CRISPR-off construct formulated in research-grade LNPs and each exemplary gRNA (as shown in Table 13). On the 6th day after transfection, the expression of HBsAg and HBeAg proteins in the supernatant was evaluated by ELISA, as Figure 12A shown. The results of this experiment are as Figure 12B shown. All tested gRNAs led to a decrease in the levels of HBsAg and HBeAg in the supernatant. The positive control used in this experiment was a gRNA targeting the HBV genome, which had previously been shown to reduce the antigen by approximately 50%.

[0371] In another experiment, an integrated HBV cell line, PLC / PRF / 5, was used to evaluate the activity of the gRNAs. PLC / PRF / 5 cells were transfected with CRISPR-off (PLA002) and each gRNA using a commercially available lipid-based transfection reagent. As Figure 13A described, on the fourth day after transfection, the expression of HBsAg protein in the supernatant was evaluated by ELISA. The results of this experiment are as Figure 13B shown. Target conservation was evaluated in silico, and target conservation was defined as 100% gRNA-DNA match.

[0372] In a further experiment, primary human hepatocytes (PHHs) derived from humanized mice were infected with HBV for 4 days and then transfected with CRISPR-off (PLA002) and each gRNA formulated in research-grade LNPs (GenVoy LNPs). As Figure 14A described, on the 6th day after infection, the expression of HBsAg and HBeAg proteins in the supernatant was evaluated by ELISA. The results of this experiment are as Figure 14BAs shown. The positive control used in this experiment was an HBV gRNA that had previously been shown to reduce the antigen by approximately 50%. The data indicated that, on day 6 post-transfection, certain gRNAs exhibited strong in vitro silencing. As Figure 14C described, in the second PHH experiment, on day 12, the expression of post-infection HBsAg and HBeAg proteins in the supernatant was evaluated by ELISA after delivering 100 ng of payload (effector to guide RNA ratio of 1:1) in research-grade LNPs. The epigenetic editor also repressed the secretion of HBsAg and HBeAg in PHH cells infected with HBV at this time point. The results are as Figure 14D shown.

[0373] The sequences of the exemplary gRNAs tested in this example are listed in Table 13.

[0374] Example 6: Evaluation of ZFPs in HepG2-NTCP cells

[0375] In this example, a ZF-off single construct encoding a fusion protein composed of KRAB, DNMT3A, DNMT3L, and a selected exemplary zinc finger motif was tested. The sequences of the exemplary zinc fingers tested in this example are listed in Table 20, and the plasmid sequences generating the partial ZF-off single construct fusion proteins are also listed in Table 20.

[0376] Certain exemplary ZF-off constructs were formulated in research-grade LNPs. HepG2-NTCP cells were infected with HBV for 4 days and then transfected with ZF-off-loaded LNPs. As Figure 15A described, on day 6 post-infection, the expression of HBsAg and HBeAg proteins in the supernatant was evaluated by ELISA. Figure 15B The results measured by the percentage reduction of HBV antigen compared to the non-targeting control are shown. The positive control used in this experiment was an HBV gRNA that had previously been shown to reduce the antigen by approximately 50%. Figure 16A The results of the top ten ZF-off constructs that caused the most reduction in HBV antigen are shown. Figure 16B The results of all constructs in the screen are shown.

[0377] The following Tables 16 and 17 show the raw data for these experiments, listing the mRNA numbers that generated the zinc finger motifs.

[0378] Table 16. Percentage of HBsAg expression relative to the non-targeting control

[0379]

[0380]

[0381]

[0382] Table 17. Percentage of HBeAg expression relative to non-targeted control

[0383]

[0384]

[0385]

[0386] Example 7. Dose-response test of viral antigens in HepG2-NTCP cells

[0387] In this example, the top-ranked ZF fusion proteins were tested for HBsAg and HBeAg in a 5-point dose-response assay. The 5 dose points were 200 ng, 150 ng, 100 ng, 50 ng, and 25 ng. The experimental schematic and results are as Figure 17 shown.

[0388] Example 8. Testing the durable repression of HBsAg in HepG2.2.15 cells

[0389] In this example, the durable repression of HBsAg by the top-ranked ZF fusion proteins was tested. The active ZFP showed durable silencing until day 27 under a total treatment of 50 ng. The experimental schematic and results are as Figure 18 shown.

[0390] Example 9. Testing the silencing of HBsAg in a second int-HBV model

[0391] In this example, the repression of HBsAg by the top-ranked ZF fusion proteins in PLC / PRF / 5 cells was tested. In this second model, some ZFPs silenced HBsAg. The experimental schematic and results are as Figure 19 shown.

[0392] Example 10. Testing the specificity of ZF fusion proteins and CRISPR-off with guide RNAs

[0393] In this example, the specificity of ZF fusion proteins targeting HBV and showing significant silencing was analyzed on day 19 in HepG2-NTCP. All comparisons were made against a non-targeted ZFP control. Figure 20AShows exemplary results of ZF fusion proteins with the mRNA0001 zinc finger motif. A similar analysis was performed on CRISPR-off and guide RNAs. HepG2-NTCP cells were transfected with 100 ng of total payload at a 1:1 gRNA:effector ratio using GenVoyTM LNP. Cells were passaged every 3 - 4 days and harvested on day 15 post-treatment for specific evaluations including RNA sequencing and methylation array analysis. Differential gene expression was identified using DESeq2. As Figure 20B shown, as expected for effectors targeting HBV DNA, very few or no changes were observed above a selected threshold (absolute value [log2[fold change]] > 1 and -log10[adjusted p-value] > 5). For the methylation array, the InfiniumMethylationEPIC v2.0 array was used and DMRs were identified in silico. The DMR results for EE3, EE4, and EE5 were 0. The results are as Figures 20C - 20D shown.

[0394] Example 11. Stable HBV silencing by epigenetic editing in a non-transgenic persistent HBV infection mouse model

[0395] A non-transgenic persistent HBV infection model (AAV-HBV) of immunocompetent mice was used, which was established by administering an adeno-associated virus vector (AAV) containing HBV genotype D DNA to the mice. Administration of the AAV-HBV vector resulted in mice expressing hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and high levels of serum HBV DNA.

[0396] CRISPR-off and ZF-off constructs were tested. The constructs were delivered by intravenous administration of mRNA / gRNA (CRISPR-Off) or mRNA (ZF-Off) formulated in lipid nanoparticles (LNP) at 2.5 mg / kg of CRISPR-Off and 0.5 mg / kg of ZF-Off, respectively. Some constructs were formulated in LNP compositions as described in US20220402862A1 and / or US20230203480A1. A subgroup of mice was re-administered two weeks after the first dose; another subgroup of mice was re-administered one month after the first dose. The readouts were circulating viral DNA, HBsAg, and HBeAg, which were tested using mouse plasma at one or more time points such as 7, 14, 28, and 35 days. For some constructs, a persistent and significant decrease in one or more of the levels of HBV DNA, HBsAg, and HBeAg was observed.

[0397] Long-term persistence was tested over 3 to 6 months using HBV DNA, HBsAg, and HBeAg markers. With the delivery of some constructs, a gradual and persistent decrease in one or more of these markers was observed. Mice were sacrificed and livers were harvested for further analysis, and persistent silencing was confirmed by at least a 2-log reduction in HBsAg and HBV DNA.

[0398] Example 12: Stable HBV silencing by epigenetic editing in transgenic mice expressing viral HBV DNA

[0399] A transgenic mouse model of persistent HBV infection (Tg-HBV) was used, whose genome was engineered to integrate HBV genotype A DNA, resulting in mice expressing HBsAg and HBeAg and circulating viral DNA.

[0400] CRISPR-Off and ZF-Off constructs were tested. The constructs were delivered by intravenous administration of mRNA / gRNA (CRISPR-Off) or mRNA (ZF-Off) formulated in LNP at 2.5 mg / kg of CRISPR-Off and 0.5 mg / kg of ZF-Off, respectively. Some constructs were formulated in LNP compositions as described in US20220402862A1 and / or US20230203480A1. A subgroup of mice was redosed two weeks after the first administration; another subgroup of mice was redosed one month after the first administration. Readouts were circulating viral DNA, HBsAg, and HBeAg, which were tested using mouse plasma at one or more time points such as 7, 14, 28, and 35 days. For some constructs, a persistent and significant decrease in one or more of the levels of HBV DNA, HBsAg, and HBeAg was observed.

[0401] Long-term durability was tested over 3 to 6 months using HBV DNA, HBsAg, and HBeAg markers. With the delivery of some constructs, a gradual and persistent decrease in one or more of these markers was observed. Mice were sacrificed and livers were harvested for further analysis, and persistent silencing was confirmed by at least a 2-log reduction in HBsAg and HBV DNA.

[0402] Example 13. CRISPR-Off guide RNA multiplexing study in AAV-HBV and Tg-HBV mouse models

[0403] According to Table 18, AAV-HBV and Tg-HBV mice were injected with one, two, or three guide RNAs and CRISPR-Off fusion proteins in LNP at a single dose of 1.5 mg / kg. Samples containing CRISPR-Off from each of PLA002 and PLA003 were included. HBV DNA, HBsAg, and HBeAg in plasma were measured at one or more time points, and mouse livers were collected for further analysis. Durable silencing was confirmed by at least a 2-log reduction in HBsAg and HBV DNA.

[0404] Table 18. CRISPR-Off multiplexed sample groups

[0405] Group Guide RNA 1 Guide RNA 2 Guide RNA 3 1 gRNA#008 gRNA#011 - 2 gRNA#008 gRNA#003 - 3 gRNA#008 gRNA#015 - 4 gRNA#008 gRNA#011 gRNA#015 5 gRNA#008 gRNA#011 gRNA#003 6 gRNA#008 - - 7 Vector - -

[0406] Example 14. Zinc finger protein multiplexing study in AAV-HBV and Tg-HBV mouse models

[0407] According to Table 19, AAV-HBV and Tg-HBV mice were injected with one, two, or three ZF fusion proteins in LNP at a single dose of 0.5 mg / kg (schematic diagram, Figure 21 ). HBV DNA, HBsAg, and HBeAg in plasma were measured at one or more time points, and mouse livers were collected for further analysis. Durable silencing was confirmed by at least a 2-log reduction in HBsAg and HBV DNA.

[0408] Table 19. ZFP multiplexed sample groups.

[0409] Group ZF_Off - 1 ZF_Off - 2 ZF_Off - 3 1 mRNA0004 mRNA0021 - 2 mRNA0004 mRNA0003 - 3 mRNA0004 mRNA0038 - 4 mRNA0004 mRNA0021 mRNA0003 5 mRNA0004 mRNA0038 mRNA0003 6 mRNA0004 mRNA0021 mRNA0038 7 mRNA0004 mRNA0001 - 8 mRNA0004 mRNA0039 - 9 mRNA0004 - - 10 vector - -

[0410] Sequence

[0411] The SEQ ID NO (SEQ) of the nucleotide (nt) and amino acid (aa) sequences described in this disclosure are listed in Table 20 below.

[0412] Table 20. Sequence listing.

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425]

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

[0432]

[0433]

[0434]

[0435]

[0436]

[0437]

[0438]

[0439]

[0440]

[0441]

[0442]

[0443]

[0444]

[0445]

[0446]

[0447]

[0448]

[0449]

[0450]

[0451]

[0452]

[0453]

[0454]

[0455]

[0456]

[0457]

[0458]

[0459]

[0460]

[0461]

[0462]

[0463]

[0464]

[0465]

[0466]

[0467]

[0468]

[0469]

[0470]

[0471]

[0472]

[0473]

[0474]

Claims

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, 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 the first DNA-binding domain, the first DNMT domain, and the transcriptional repressor domain, 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 the number of HBV viral episomes, the replication of the HBV gene or genome, or the expression of the 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.

2. 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 the first DNA-binding domain, the first DNMT domain, and the transcriptional repressor domain, 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 the number of HBV viral episomes, the replication of the HBV gene or genome, or the expression of the protein product encoded by the HBV gene or genome, wherein the reduction is at least about 20% compared to administering a suitable control.

3. A method of regulating the 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 the first DNA-binding domain, the first DNMT domain, and the transcriptional repressor domain, 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 the expression of the gene product encoded by the HBV gene or genome, optionally, wherein the gene product is a nucleic acid or a protein, wherein the reduction is at least about 20% compared to contacting the HBV genome with a suitable control.

4. A method of 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 the first DNA-binding domain, the first DNMT domain, and the transcriptional repressor domain, wherein the first DNA binding domain binds to a first target region of the HBV gene or genome, and wherein the epigenetic editing system targets the target region of the HBV gene or genome, and wherein the contact results in a decrease in the number of HBV viral episomes or a decrease in the replication of the HBV gene or genome, wherein the decrease is at least about 20% compared to administration of a suitable control.

5. The method according to any one of claims 1-4, wherein the HBV genome is covalently closed circular DNA (cccDNA) or HBV integrated DNA.

6. The method according to any one of claims 1-5, 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. The method according to any one of claims 1-6, wherein the HBV genome comprises a sequence having at least 80% identity to the HBV genome sequence provided herein.

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

9. The method according to any one of claims 1-6, wherein the first target region of the HBV genome is in a CpG island.

10. The method according to any one of claims 1-6, wherein the first target region of the HBV genome is in a promoter.

11. The method according to any one of claims 1-6, wherein the first target region of the HBV genome is in a portion of the HBV genome encoding transcripts selected from pgRNA, precure mRNA, preS mRNA, S mRNA, and X mRNA.

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

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

14. The method according to claim 13, wherein the gRNA comprises a sequence selected from the gRNAs provided herein, such as the sequences in Table 14 and / or Table 15.

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

16. The method according to claim 15, wherein 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, such as the zinc finger motifs in Table 1.

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

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

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

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

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

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

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

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

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

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

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

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

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

31. The method according to claim 27, wherein the second target region of the HBV genome is located in a portion of the HBV genome encoding transcripts selected from pgRNA, precure mRNA, preS mRNA, S mRNA, and X mRNA.

32. The method according to any one of claims 27-31, wherein the second DNA binding domain comprises a CRISPR-Cas protein.

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

34. The method according to claim 33, wherein the gRNA comprises a sequence selected from the gRNA sequences provided herein, such as the sequences provided in Table 14 and / or Table 15.

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

36. The method according to claim 35, wherein the zinc finger protein comprises a zinc finger motif having a sequence selected from the zinc finger motif sequences provided herein, such as the zinc finger motifs provided in Table 1.

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

38. The method according to any one of claims 27-37, wherein 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, wherein the first fusion protein comprises the first DNA binding domain and the first DNMT domain, and wherein the second fusion protein comprises the second DNA binding domain and the transcriptional repressor domain.

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

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

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

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

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

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

45. The method according to claim 41, wherein the third target region of the HBV genome is in a portion of the HBV genome encoding transcripts selected from pgRNA, precure mRNA, preS mRNA, S mRNA, and X mRNA.

46. The method according to any one of claims 41-45, wherein the third DNA binding domain comprises a CRISPR-Cas protein.

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

48. The method according to claim 47, wherein the third gRNA comprises a sequence selected from the gRNA sequences provided herein, such as the gRNA sequences provided in Table 14 and / or Table 15.

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

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

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

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

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

54. The method according to claim 53, wherein the epigenetic editing system comprises a third fusion protein or a nucleic acid encoding the third fusion protein, wherein the third fusion protein comprises the third DNA binding domain and the second DNMT domain.

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

56. 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 the HBV gene or genome, (b) a first DNA methyltransferase (DNMT) domain, and (c) a transcriptional repressor domain.

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

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

59. The epigenetic system according to any one of claims 56-58, 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. The epigenetic system according to any one of claims 56-59, wherein the HBV genome comprises a sequence having at least 80% identity to the HBV genome sequence provided herein.

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

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

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

64. The epigenetic system according to any one of claims 56 - 60, wherein the target region of the HBV genome is in a portion of the HBV genome encoding transcripts selected from pgRNA, precure mRNA, preS mRNA, S mRNA, and X mRNA.

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

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

67. The epigenetic system according to claim 66, wherein the gRNA comprises a sequence selected from the gRNA sequences provided herein, such as the sequences in Table 14 and / or Table 15.

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

69. The epigenetic system according to claim 68, wherein the zinc - finger protein comprises zinc - finger motifs having sequences selected from the zinc - finger motifs provided herein.

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

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

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

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

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

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

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

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

78. 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 wherein 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 wherein the second DNA-binding domain binds to a second target region of the HBV genome.

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

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

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

83. The epigenetic system according to any one of claims 78-81, further comprising 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 wherein the third DNA-binding domain binds to a third target region of the HBV genome.

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

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

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

87. The epigenetic system according to claim 83, wherein the first target region, the second target region, or the third target region of the HBV genome is located in a portion of the HBV genome that encodes transcripts selected from the group consisting of pgRNA, precure mRNA, preS mRNA, S mRNA, and X mRNA.

88. The epigenetic system according to 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. The epigenetic system according to claim 88, wherein the epigenetic editing system further comprises a first gRNA that comprises a region complementary to a strand of the first target region; a second gRNA that comprises a region complementary to a strand of the second target region; or a third RNA that comprises a region complementary to a strand of the third target region.

90. The epigenetic system according to claim 89, wherein the first gRNA comprises a sequence selected from the gRNA sequences provided herein, such as the sequences provided in Table 14 and / or Table 15, the second gRNA comprises a sequence selected from the gRNA sequences provided herein, such as the sequences provided in Table 14 and / or Table 15, and / or the third gRNA comprises a sequence selected from the gRNA sequences provided herein, such as the sequences provided in Table 14 and / or Table 15.

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

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

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

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

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

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

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

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

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

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

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

103. A method of treating HDV infection in a subject, 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 the first DNA binding domain, the first DNMT domain, and the transcriptional repressor domain, wherein the first DNA binding domain binds to a first target region of the HBV gene or genome, and wherein the contact results in a reduction in the number of HDV viral episomes, the replication of the HDV gene or genome, or the expression of the protein product encoded by the HDV gene or genome, wherein the reduction is at least about 20% compared to administration of a suitable control.

104. A method of inhibiting viral replication in a cell 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 the first DNA binding domain, the first DNMT domain, and the transcriptional repressor domain, wherein the first DNA binding domain binds to a first target region of the HBV gene or genome and wherein the epigenetic editing system targets the target region of the HBV gene or genome, and wherein the contact results in a reduction in the number of HDV viral episomes or a reduction in the replication of the HDV gene or genome, wherein the reduction is at least about 20% compared to administration of a suitable control.

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

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

107. The method according to claim 106, wherein the gRNA comprises a sequence selected from the gRNAs provided herein, such as the sequences in Table 14 and / or Table 15.

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

109. The method according to claim 108, wherein 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, such as the zinc finger motif in Table 1.

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

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

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

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

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

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

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

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

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

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

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

121. A method 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 the first DNA binding domain, the first DNMT domain, and the transcriptional repressor domain, 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 the number of HBV viral episomes, the replication of the HBV gene or genome, or the expression of the protein product encoded by the HBV gene or genome, wherein the reduction is at least about 20% compared to administration of a suitable control.

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

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