Compositions and methods for epigenetic regulation of HBV gene expression

Abstract

This invention relates to compositions, methods, strategies, and treatment modalities related to the epigenetic modification of hepatitis B virus (HBV) genes.

Description

WSGR Ref. No. 59073-743.601 COMPOSITIONS AND METHODS FOR EPIGENETIC REGULATION OF HBV GENE EXPRESSION CROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 693,614,filed September 11, 2024, which is incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION

[0002] Despite available treatments, chronic hepatitis B (CHB) remains a high unmet medicalneed, with more than 250 million carriers of hepatitis B virus (HBV) worldwide and approximately 800,000 annual deaths due to HBV-related liver disease. Current approved CHB therapies elicit a functional cure rate (defined as durable HBsAg loss and undetectable serum HBV after completing a course of treatment) of less than 20%. Accordingly, there is a need for improved clinical modalities targeting HBV. SUMMARY OF THE INVENTION

[0003] Provided herein is a composition comprising: a. an RNA encoding an epigeneticeditor protein, wherein the RNA comprises a sequence with at least 95% sequence identity to SEQ ID NO: 1274; and b. a guide RNA comprises a spacer sequence, wherein the spacer sequence is complementary to a target region of a strand of an HBV gene or genome.

[0004] In some embodiments, the RNA and the guide RNA are encapsulated in a lipidnanoparticle.

[0005] In some embodiments, the RNA and the guide RNA are encapsulated in the lipidnanoparticle at a ratio from about 1:1 to about 1:3 by weight.

[0006] In some embodiments, the RNA and the guide RNA are encapsulated in the lipidnanoparticle at a ratio of about 1:1 by weight.

[0007] In some embodiments, the RNA and the guide RNA are encapsulated in the lipidnanoparticle at a ratio of about 1:1.5 by weight.

[0008] In some embodiments, the RNA and the guide RNA are encapsulated in the lipidnanoparticle at a ratio of about 1:3 by weight.

[0009] In some embodiments, the RNA and guide RNA are encapsulated in the lipidnanoparticle at a ratio from about 1:2 to about 1:4 by weight.

[0010] In some embodiments, the RNA and guide RNA are encapsulated in the lipidnanoparticle at a ratio from about 1:3 to about 1:5 by weight. -1-WSGR Ref. No. 59073-743.601

[0011] In some embodiments, the RNA comprises a sequence with at least 98% sequenceidentity to SEQ ID NO: 1274.

[0012] In some embodiments, the RNA comprises a sequence set forth in SEQ ID NO: 1274.

[0013] In some embodiments, the spacer sequence comprises a sequence set forth in SEQ IDNO: 391.

[0014] In some embodiments, the guide RNA comprises a nucleic acid base sequence,wherein the nucleic acid base sequence comprises a sequence with at least 80% sequence identity to AGGAGUUCCGCAGUAUGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 1260), or to AGGAGUUCCGCAGUAUGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 1280) wherein A, G, C and U represent adenine, guanine, cytosine and uracil, respectively. In some embodiments, the nucleic acid base sequence comprises a sequence set forth in SEQ ID NO: 1260.

[0015] In some embodiments, the guide RNA comprises one or more chemically modifiednucleotide(s). In some embodiments, the one or more chemically modified nucleotide(s) are at one or more nucleotide position(s) selected from 1-3, 30-40, 69-81 and 83-100 as numbered in SEQ ID NO: 1260 or corresponding position(s) thereof.

[0016] In some embodiments, the guide RNA comprises a nucleic acid sequence ofmA*mG*mG*rArGrUrUrCrCrGrCrArGrUrArUrGrGrArUrGrUrUrUrUrArGrArGmCmUmAm GmAmAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUr CrAmAmCmUmUmGmAmAmAmAmAmGmUmGrGmCmAmCmCmGmAmGmUmCmGmG mUmGmCmU*mU*mU*mU (SEQ ID NO: 1249), or mA*mG*mG*rArGrUrUrCrCrGrCrArGrUrArUrGrGrArUrGrUrUrUrUrArGrArGmCmUmAm GmAmAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUr CrAmAmCmUmUmGmAmAmAmAmAmGmUmGrGmCmAmCmCmGmAmGmUmCmGmG mUmGmC (SEQ ID NO: 1279), wherein (1) mA, mG, mC and mU represent a 2’-OMe modified adenosine, guanosine, cytidine and uridine, respectively, (2) rA, rG, rC and rU represent adenosine, guanosine, cytidine and uridine, respectively, and (3) * indicates a phosphorothioate bond. -2-WSGR Ref. No. 59073-743.601

[0017] In some embodiments, the epigenetic editor protein comprises: a) a DNA bindingdomain; b) two DNA methyltransferase (DNMT) domains; c) a repressor domain; d) a first peptide linker between the DNA binding domain and one of the DNMT domains, and a second peptide linker between the DNA binding domain and the repressor domain, wherein the first peptide linker is XTEN80, wherein the second peptide linker is XTEN16; and e) two bipartite nuclear localization sequences (NLS), and a simian virus (SV40) NLS.

[0018] In some embodiments, one of the two bipartite NLSs is positioned at an amino (N)terminus of the epigenetic editor protein and the other one of the two NLSs is positioned at a carboxy (C) terminus of the epigenetic editor protein, and wherein the SV40 NLS is positioned at the C terminus of the DNA binding domain.

[0019] In some embodiments, the DNA binding domain is a dCas9 DNA binding domain.

[0020] In some embodiments, the repressor domain is a ZIM3 repressor domain.

[0021] In some embodiments, one of the two DNMT domains is a DNMT3A domain.

[0022] In some embodiments, one of the two DNMT domains is a DNMT3L domain.

[0023] In some embodiments, the DNMT3L is derived from a species of the Equus genus.

[0024] In some embodiments, the species of the Equus genus is Equus prezewalskii.

[0025] In some embodiments, the epigenetic editor protein comprises a configurationcomprising: bipartite NLS-DNMT3A-DNMT3L-XTEN80-dCas9-SV40 NLS-XTEN16-ZIM3- bipartite NLS, from the N terminus to the C terminus.

[0026] In some embodiments, the epigenetic editor protein comprises a sequence with at least95% sequence identity to SEQ ID NO: 1252.

[0027] In some embodiments, the epigenetic editor protein comprises a sequence set forth inSEQ ID NO: 1252.

[0028] Provided herein is a method of treating an HBV infection in a subject comprisingadministering the composition of any one of the embodiments disclosed herein.

[0029] Provided herein is a method comprising administering the composition of any one ofthe embodiments disclosed herein to a subject, wherein the subject is characterized by the presence of detectable levels of HBV DNA, HBsAg, and / or HBeAg in the plasma of the subject.

[0030] In some embodiments, the composition is administered through peripheralintravenous infusion. In some embodiments, the composition is administered once. In some embodiments, the composition is administered multiple times. In some embodiments, the composition is administered once every four weeks. In some embodiments, the composition is administered once every four weeks for a total of three administrations. -3-WSGR Ref. No. 59073-743.601

[0031] In some embodiments, the composition is administered at a dosage of at least about0.2 mg / kg. In some embodiments, the composition is administered at a dosage of about 0.2 mg / kg. In some embodiments, the composition is administered at a dosage of about 0.4 mg / kg. In some embodiments, the composition is administered at a dosage of about 0.6 mg / kg. In some embodiments, the composition is administered at a dosage of about 0.8 mg / kg.

[0032] Provided herein is an RNA comprising a sequence set forth in SEQ ID NO: 1274.

[0033] Provided herein is an RNA comprising a sequence set forth in SEQ ID NO: 1257.BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is a diagram illustrating an exemplary structure of a circular HBV genome.HBV genes and CpG islands are indicated. Exemplary target sites for CRISPR-based epigenetic repressors (red arrows) as well as for zinc-finger-based epigenetic repressors (green arrows) are identified.

[0035] Figure 2 is a heat map showing conservation of guide RNA target domains acrossdifferent HBV genotypes.

[0036] Figure 3 is a bar graph illustrating the geographical distribution of different HBVgenotypes.

[0037] Figure 4A is a diagram describing the experimental timeline for testing differentCRISPR-based epigenetic repressors in HepAD38 cells, which express HPV in a doxycycline- inducible manner. Figure 4B is a diagram 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.

[0038] Figure 5A is a diagram describing the experimental timeline for testing differentCRISPR-based epigenetic repressors in a HepG2-NTCP infection model (see, e.g., Methods Mol Biol. 2017;1540:1-14). Figure 5B is a diagram showing the expression of HBe antigen (via ELISA) at different times after treatment of HBV-infected Hep2G-NTCT cells with different doses of CRISPR-based epigenetic repressors (ETRs), or with different doses of Cas9 nuclease targeting HBV (Cas9), plotted normalized to the expression value of HBe antigen measured for a negative control (empty). -4-WSGR Ref. No. 59073-743.601

[0039] Figure 6 is a diagram describing the experimental timeline for a guide RNA screentesting different CRISPR-based epigenetic repressor systems in a HepG2-NTCP infection model with ELISA readout for HBe and HBs antigens at day 6.

[0040] Figure 7 is a diagram showing QC results from different LNP batches used in theguide screen.

[0041] Figure 8 is a bar graph showing the expression of HBe and HBs for an exemplaryCRISPR-based epigenetic repressor (#3.2), calculated as the percentage of the expression of the respective antigen measured for a non-targeting control.

[0042] Figure 9 is a diagram showing HBe expression values measured in the guide RNAscreen for different guides (calculated as a percentage of the expression of HBe measured for a non-targeting control). Each guide / repressor combination is represented by a dot. A 50% repression cutoff is shown as a horizontal line. The position of the respective guide RNA within the HBV genome (shown at the bottom of the graph) is mapped on the X-axis. The position and the measured modulation of HBe expression for exemplary guide RNA #3.2 is indicated by red lines.

[0043] Figure 10 is a diagram showing HBs expression values measured in the guide RNAscreen for different guides (calculated as a percentage of the expression of HBs measured for a non-targeting control). Each guide / repressor combination is represented by a dot. A 50% repression cutoff is shown as a horizontal line. The position of the respective guide RNA within the HBV genome (shown at the bottom of the graph) is mapped on the X-axis. The position and the measured modulation of HBs expression for exemplary guide RNA #3.2 is indicated by red lines.

[0044] Figure 11 is a diagram showing a correlation between HBs and HBe expression forthe guides tested. The graph on the right shows HBe and HBs repression efficiencies for 25 exemplary guides.

[0045] Figure 12A is a diagram describing the experimental timeline for a guide RNA assaytesting CRISPR-off single construct epigenetic editor in combination with individual exemplary gRNAs in a HepG2-NTCP infection model with ELISA readout for HBe and HBs antigens at day 6; and Figure 12B is a graph summarizing the percentage reduction in HBV antigens at day 6 relative to non-targeting control.

[0046] Figure 13A is a diagram describing the experimental timeline for a guide RNA assaytesting CRISPR-off single construct epigenetic editor in combination with individual exemplary gRNAs in a PLC / PRF / 5 cell model with ELISA readout for HBs antigen at day 4; and Figure -5-WSGR Ref. No. 59073-743.601 13B is a graph summarizing the percentage reduction in HBs antigen at day 4 relative to non- targeting control.

[0047] Figure 14A is a diagram describing the experimental timeline for a guide RNA assaytesting CRISPR-off single construct epigenetic editor in combination with individual exemplary gRNAs in a PXB cell model with ELISA readout for HBe and HBs antigens at day 6; and Figure 14B is a graph summarizing the percentage reduction in HBV antigens at day 6 relative to non- targeting control. Figure 14C is a diagram describing the experimental timeline for a guide RNA assay testing CRISPR-off single construct epigenetic editor in combination with individual exemplary gRNAs in a PXB cell model with ELISA readout for HBe and HBs antigens at day12. Figure 14D is a graph summarizing the percentage reduction in HBV antigens at day 12relative to 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.

[0048] Figure 15 is a diagram describing the design for in vivo experiments testing CRISPR-off single construct epigenetic editor in combination with individual exemplary gRNAs in AAV- HBV mouse HBV genotype D persistent infection model, and transgenic HBV genotype A mouse persistent infection model, respectively.

[0049] Figure 16 shows time course graphs summarizing the level of serum HBV DNA, HBsand HBe antigens in transgenic mouse HBV model before and after single administration of an epigenetic editor (CRISPR-off with gRNA or ETR with gRNA), Cas9 with gRNA, or control vehicle at day 0.

[0050] Figure 17 shows time course graphs summarizing the level of serum HBV DNA, HBsand HBe antigens in AAV-HBV mouse model before and after single administration of an epigenetic editor (CRISPR-off with gRNA or ETR with gRNA), Cas9 with gRNA, or control vehicle at day 0.

[0051] Figure 18A shows time course graphs summarizing the level of serum HBV DNA,HBs and HBe antigens in transgenic mouse HBV model, and a schematic of the timeline for the experiment. All mice received a single administration of an epigenetic editor (CRISPR-off with gRNA or ETR with gRNA), Cas9 with gRNA, or control vehicle at day 0, and some mice received a designated redosing at day 35. Figure 18B shows results for the single-administration (no redosing) groups and controls to 168 days duration for HBV DNA and HBsAg. The lefthand panels shows the group data at each timepoint, whereas the righthand panels show the readouts for individual animals at two timepoints. EE=epigenetic editor (CRISPR-off with gRNA#011). -6-WSGR Ref. No. 59073-743.601

[0052] Figure 19 shows time course graphs summarizing the level of serum HBV DNA, HBsand HBe antigens in AAV-HBV mouse model, and a schematic of the timeline for the experiment. All mice received a single administration of an epigenetic editor (CRISPR-off with gRNA or ETR with gRNA), Cas9 with gRNA, or control vehicle at day 0, and some mice received a designated redosing at day 35.

[0053] Figure 20A is a diagram describing the experimental timeline for a zinc finger assaytesting ZF-off single construct epigenetic editor that contains individual exemplary zinc finger motif in a HepG2-NTCP infection model with ELISA readout for HBe and HBs antigens at day 6; and Figure 20B is a graph summarizing the percentage reduction in HBV antigens at day 6 relative to non-targeting control. "N" denotes non-targeting control, "P" denotes the positive control, and the individual numbers on the x-axis denote exemplary constructs tested in the experiment, for instance, "1" represents "mRNA0001" construct, and "20" represents "mRNA0020" construct.

[0054] Figure 21A is a graph summarizing the results of top ten ZF-off constructs fromFigure 20B. Figure 21B is a diagram showing HBsAg (top) and HBeAg (middle) expressionvalues measured in the ZF-off screen (calculated as a percentage of the expression of HBsAg or HBeAg--top and middle, respectively--measured for a non-targeting control). Each ZF-off construct is represented by a dot. 50% and 60% repression cutoffs are shown as horizontal lines. The position of the respective guide RNA within the HBV genome (bottom) is mapped on the X- axis.

[0055] Figure 22 is an experimental timeline for testing dose response (top) and two graphsshowing dose response of % HbsAg (bottom left) and % HbeAg (bottom right) in HepG2-NTCP cells upon administration of ZF epigenetic editor proteins. The mRNA corresponding to the ZF motif for each epigenetic editor protein is indicated.

[0056] Figures 23A-23C show an experimental timeline for testing durable silencing ofHBsAg (Figure 23A), a graph showing the durability of HBsAg silencing by ZF epigenetic editor proteins (Figure 23B), and a graph showing the durability of HBsAg silencing by CRISPR-off epigenetic editor proteins with guide RNAs (Figure 23C) in an integrated cell line. The mRNA corresponding to the ZF motif for each epigenetic editor protein is indicated. Error bars represent mean + / -SEM; in Figure 23C, N=3, 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). -7-WSGR Ref. No. 59073-743.601

[0057] Figure 24 is an experimental timeline for testing HBsAg silencing in a PLC / PRF / 5 invitro model (top) and a graph showing % HBsAg relative to control on Day 14 after administration of ZF epigenetic editor proteins. The mRNA corresponding to the ZF motif for each epigenetic editor protein is indicated. Information about the % match to target for each construct is also indicated.

[0058] Figure 25A is a volcano plot showing differentially expressed (DE) genes for anexemplary ZF specificity assay. DE genes are shown with dots. Figure 25B is a volcano plot showing DE for CRISPR-off and gRNA epigenetic editors. Points represent genes with their change in expression (x-axis) and 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. Also shown are results for low specificity and host target gene controls. Figures 25C-25D are scatter plots showing methylation levels between treatment (y-axis) and control (x-axis) for 935,000 CpG sites in the human genome. Lines represent thresholds for changes in methylation considered significant (absolute [methylation difference]>= 0.2). DMRs are noted on each figure. Results for a host target (PCSK9, next-to-final panel) as well as a low specificity control (final panel) are also shown. Figure 25C shows the results versus effector only; Figure 25D shows the results versus no treatment. EE1= PLA002 and gRNA#007, EE2=PLA002 and gRNA#008, EE3=PLA002 and gRNA#009, EE4=PLA002 and gRNA#015, EE5=PLA002 and gRNA#011, EE6=PLA002 and gRNA#003, and EE7=PLA002 and gRNA#016.

[0059] Figure 26 is a schematic of an in vivo experiment testing ZF-off constructs.

[0060] Figure 27 shows graphs showing log fold change, relative to baseline, for HBV DNA(left), HBsAg (middle), and HBeAg (right) in plasma of mice treated with the plasmids indicated in the experiment shown in Figure 26.

[0061] Figure 28 is an experimental schematic for an in vivo study of multiplexing ZFepigenetic editor protein effectors.

[0062] Figure 29 is a schematic for a dose response experiment using CRISPR-Off in anAAV-HBV in vivo model.

[0063] Figure 30 is a line graph of plasma HBsAg levels for a dose response experimentusing CRISPR-Off in an AAV-HBV in vivo model.

[0064] Figure 31 is a schematic for a dose response experiment using CRISPR-Off in a Tg-HBV in vivo model. -8-WSGR Ref. No. 59073-743.601

[0065] Figure 32 shows line graphs of plasma HBV DNA, HBsAg, and HBeAg levels for adose response experiment using CRISPR-Off in a Tg-HBV in vivo model.

[0066] Figure 33 is a dot plot of HBsAg levels of individual mice at the 207 day time pointof a dose response experiment using CRISPR-Off in a Tg-HBV in vivo model.

[0067] Figure 34 shows line graphs of HBV-DNA and HBsAg in plasma in AAV micetreated with CRISPR-Off mRNA with various single guide RNAs. n=5 for each guide RNA treatment group; n=4 for vehicle-only control.

[0068] Figure 35A shows line graphs of HBV-DNA and HBsAg in plasma in AAV micetreated with a single dose of ZF-Off mRNA.

[0069] Figure 35B shows line graphs of HBV-DNA and HBsAg in plasma in AAV micetreated with multiple doses of ZF-Off mRNA.

[0070] Figure 36 shows line graphs of HBV-DNA, HBsAg, and HBeAg in plasma in AAVmice treated with single versus multiple doses of 1 mg / kg CRISPR-Off mRNA with guide RNA.

[0071] Figure 37 shows line graphs of HBV-DNA and HBsAg in plasma in AAV micetreated with a single bolus dose of 3 mg / kg versus three doses of 1 mg / kg CRISPR-Off mRNA with guide RNA.

[0072] Figure 38 shows line graphs of HBsAg in plasma in response to treatment with twodifferent CRISPR-Off effectors (left, SEQ ID NO: 1248; right, SEQ ID NO: 1252) delivered via mRNA in combination with the same guide RNA.

[0073] Figures 39A-39G show methylation of the HBV genome upon treatment withCRISPR-Off with various single guide RNAs versus wild type Cas9, CRISPRi, and non- targeting controls. The box in Figure 39A represents the region 500 bp both upstream and downstream of the target site. The arrows indicate the position of the target sequence for the guide RNA used in the depicted experiment.

[0074] Figure 40 shows volcano plots of RNA-Seq (top) and methylation (bottom)experiments at Day 14 after treatment in HepG2.2.15 cells treated with ZF-Off (left, SEQ ID NO: 36; center, SEQ ID NO: 73) and CRISPR-Off (right, SEQ ID NO: 1248) constructs (delivered as mRNA) targeting HBV. DE, differentially expressed. DMR: differentially methylated region.

[0075] Figure 41 shows HBsAg levels over 14 days for the cells treated for the RNA-Seqand methylation plots in Figure 40. -9-WSGR Ref. No. 59073-743.601

[0076] Figure 42 shows a schematic (top) and dose curves (bottom) for CRISPR-Off dosecurve experiments in HepG2.2.15 cells using various single guide RNAs and measuring HBsAg and HBeAg.

[0077] Figure 43 shows dose curves for a CRISPR-Off variant, delivered with guide RNA,in HepG2.2.15 cells measuring HBsAg and HBeAg.

[0078] Figures 44A-44B show further in vitro study of a favorable CRISPR-Off variant witha favorable gRNA in HepG2.2.15 cells. Figure 44A shows a dose response curve with % HBsAg and HBeAg expression relative to control. Figure 44B shows a durability study of silencing of HBsAg up to Day 44 after administration relative to a non-targeting gRNA control.

[0079] Figures 45A-45B show further in vivo study of a favorable CRISPR-Off variant witha favorable gRNA in AAV-HBV mice. Figure 45A shows a mRNA / gRNA ratio study. The dotted line indicates one favorable ratio selected for further studies. Figure 45B shows a study of various dosages in milligrams per kilogram (mpk).

[0080] Figure 46 shows a scatter plot of anti-HBs levels in individual animals treated withdifferent dosages of an epigenetic editor targeting HBV or vehicle-only control. mIU / mL, milli- international unit per milliliter. mg / kg, milligrams per kilogram. LLOQ, lower limit of quantification.

[0081] Figure 47 shows a schematic of a histology experiment and histology images of liversfrom AAV-HBV mice treated with vehicle or 3 mg / kg HBV epigenetic editor (HBVEE). A scale bar is included in the lower right. The inset shows higher magnification.

[0082] Figures 48A-48F show the pharmacokinetic profile and pharmacodynamic activity ofHBV-EEP in HBV-infected PHH. Figures 48A-48B show mean ± standard deviation (SD) (n = 4 / concentration) ratio of gRNA and effector mRNA levels, respectively, to housekeeping gene POLR2J relative to mock treatment (0 μg / mL) 6 hours after treatment with HBV-EEP. The dotted line represents the lower limit of quantitation (LLoQ) and symbols represent biological replicates. Figure 48C shows mean ± SD effector protein concentration. Each symbol represents a technical replicate of a single pooled sample. Figure 48D shows mean ± SD (n = 3 / concentration) ratio of HBV viral transcripts to housekeeping gene POLR2J 14 days after treatment. Figures 48E-48F show Mean ± SD (n = 6 / concentration) percent HBsAg and HBeAg, respectively, relative to mock treatment at 14 days after treatment. A 4-parameter logistic regression was used to fit data from Figures 48D-48F.

[0083] Figures 49A-49E show the pharmacokinetic profile and pharmacodynamic activity ofHBV-EEP in PLC / PRF / 5 cells. Figures 49A-49B show mean ± SD (n = 3 / concentration) ratio of -10-WSGR Ref. No. 59073-743.601 gRNA and effector mRNA levels, respectively, to housekeeping gene POLR2J relative to mock treatment (0 μg / mL) 24 hours after treatment with HBV-EEP. The dotted line represents the LLoQ and symbols represent biological replicates. Figure 49C shows mean + SD (n = 2 technical replicates / treatment condition) effector protein concentration. Symbols represent technical replicates. Figure 49D shows mean ± SD (n = 3 / concentration) ratio of HBV viral transcripts to housekeeping gene POLR2J 14 days after treatment. Figure 49E shows mean ± SD (n = 6 / concentration) percent HBsAg relative to mock treatment at 14 days after treatment. A 4- parameter logistic regression was used to fit data from Figures 49D-49E.

[0084] Figures 50A-50G show the pharmacokinetic profile and pharmacodynamic activityof HBV-EEP in HepG2.2.15 cells. Figures 50A-50C show mean ± SD (n = 3 / concentration) ratio of gRNA and effector mRNA, respectively, to housekeeping gene POLR2J relative to mock treatment (0 μg / mL) and effector protein levels up to 14 days after treatment with HBV-EEP. The dotted line represents the LLoQ and symbols represent biological replicates. Figure 50D shows mean ± 95% confidence interval (CI) methylation change across the full HBV genome in HBV-EEP treated samples relative to mock treatment. Figures 50E-50G show mean ± SD (n = 3 / concentration) ratio of HBV viral transcripts to housekeeping ge POLR2J, percent HBsAg, and percent HBeAg, respectively, relative to mock treatment 14 days after treatment.

[0085] Figure 51 shows methylation profiles for individual samples of isolated covalentlyclosed circular DNA (cccDNA) from HBV-infected PHH cells mock treated or 1 μg / mL HBV- EEP treated with sequencing read subsampling to normalize read counts (n = 3 replicates / condition). Methylation is depicted as a bar plot and the value reported is the average percent methylation across the entire locus. The bar position along the X-axis represents the position of a CpG within the HBV genome, and the bar height is the methylation percentage of that CpG. The target site is located at 1285 – 1265 nucleotides. Gene annotations, CGI locations, and positions of individual CpGs for HBV genotype D are shown.

[0086] Figures 52A-52D show quantification of HBV antigens following HBV-EEPtreatment of pan genotype HBV infection or transfection of HepG2-hNTCP cells. Figures 52A- 52B show mean ± SD (n = 3 / treatment group) HBsAg and HBeAg levels in supernatant 7 days after treatment with 1 μg / mL HBV-EEP or mock treatment of HBV-infected cells. High and low infection refer to the multiplicity of genome equivalents (8,000 or 16,000 for high and 2,000 to 8,000 for low). Figures 52C-52D show HBV-transfected cells. The dotted line represents the LLoQ for each assay (0.8 IU / mL for HBsAg and 0.7 PEIU / mL for HBeAg). Asterisks denote -11-WSGR Ref. No. 59073-743.601 significance for two-way ANOVA test; ns (not significant); *P <0.05; **P <0.01; ***P <0.001, ****P <0.0001.

[0087] Figures 53A-53E show HBV biomarkers in plasma following a single IV dose ofHBV-EE 4.1 to Tg HBV mice. Figures 53A-53C show mean ± SD Log10 fold-change of circulating HBV biomarkers HBsAg (Figure 53A), HBV DNA (Figure 53B), and HBeAg (Figure 53C) in HBV-EE 4.1 treated Tg-HBV mice. Figure 53D shows HBsAg levels from individual animals (represented by symbols) measured 207 days post-dose. Statistical analysis was performed by one-way ANOVA; P = NS. (E) Mean ± SD anti HBs in HBV-EE 4.1-treated Tg-HBV mice.

[0088] Figures 54A-54D show pharmacologic activity of HBV-EE 4.1 in the livers of Tg-HBV mice following a single IV administration. Figure 54A shows the methylation profile of the integrated HBV genome from livers of HBV-EE 4.1-treated mice. Percentage values under each dose group label represent the mean methylation percentage calculated across all samples from each dose group. The HBV genome is annotated at the top of the figure, with annotations for CpG islands, bars with arrows representing HBV genes and vertical black lines representing positions of individual CpGs. Figure 54B shows the log10-fold reduction of HBV mRNA levels in liver at study termination 207 days post-dose. Results are presented as group mean ± SD with symbols representing individual animal values. Figure 54C shows the quantification of HBcAg- positive cells in liver sections. Results are presented as group mean ± SD with symbols representing individual animal values. Symbols pinpointed by an arrow in (Figure 54C) represent animals from which HBcAg immunohistochemistry (IHC) liver images in (Figure 54D) were derived.

[0089] Figures 55A-55L show HBV biomarkers in plasma of AAV-HBV mice administeredbiomarkers (HBsAg, HBV DNA, HBeAg) are shown relative to vehicle following a single dose of HBV-EEP (Figures 55A-55C), 3 doses of 0.5 mg / kg HBV-EEP compared to the same single dose of HBV-EEP (Figures 55D-55F), 3 doses of 1.0 mg / kg HBV-EEP compared to the same single dose of HBV-EEP (Figures 55G-55I), and a single dose of HBV-EEP, ETV, or combination (Figures 55J-55L). Data are presented as mean ± SD (n = 10 / group). Where applicable, vertical dashed lines indicate day of HBV-EEP dose and shading represents the window of daily ETV dosing.

[0090] Figures 56A-56C show pharmacologic activity of HBV-EEP in the livers of AAV-HBV mice administered HBV-EEP alone or in combination with ETV. Figure 56A shows the -12-WSGR Ref. No. 59073-743.601 numbers were first normalized to POLR2J mRNA levels (housekeeping gene) and subsequently normalized to the mean value of the vehicle group to calculate the log10 fold reduction. Statistical analysis was performed using a t-test (two groups). Figure 56B-56C show the total HBV DNA copies / diploid genome and episomal HBV DNA per diploid genome, respectively, from terminal liver samples. Statistical analysis was performed with either t-test (two groups) or one-way ANOVA (multiple groups). For all data, bars represent mean ± SD with symbols representing individual animal values. ns = not statistically significant; **** P < 0.0001.

[0091] Figure 57 shows an exemplary schematic of a workflow for verification ofunintended targets of HBV-EEP.

[0092] Figure 58 shows a mRNA encoding the epigenetic effector protein or editorcomprising a catalytically inactive CRISPR associated protein 9 (e.g., dSpCas9) with multiple nuclear localization sequences (NLS) fused to an N terminal DNA methylation effector domain (e.g., DNA methyltransferase 3 alpha - 3 like (DNMT3A-3L)) and a C-terminal transcriptional repressor domain (e.g., Krüppel associated box (KRAB) repressor domain) (SEQ ID NO: 1257).

[0093] Figure 59 shows a modified gRNA targeting HBV DNA sequence (SEQ ID NO:1249) DETAILED DESCRIPTION OF THE INVENTION

[0094] The present disclosure provides epigenetic editors, and strategies and methods ofusing such epigenetic editors, for regulating expression of HBV. By altering expression of HBV, and in particular, by repressing expression of HBV, e.g., of a gene comprised in the HBV genome or a gene product encoded by the HBV genome, the compositions and methods described herein are useful to suppress viral function in infected cells, e.g., in the context of treating an HBV infection in a human subject, or in the context of treating CHB.

[0095] The structure and biology of HBV as well as HBV-associated diseases have beenreported (see, for example, Yuen, MF., Chen, DS., Dusheiko, G. et al. Hepatitis B virus infection. Nat Rev Dis Primers 4, 18035 (2018), incorporated herein by reference in its entirety).

[0096] Exemplary HBV sequences can be found at various NCBI database entries, e.g.,representative sequences can be found under accession numbers NC_00397 and U95551, which are incorporated herein by reference in their entirety, and the sequences of which are provided elsewhere herein. -13-WSGR Ref. No. 59073-743.601

[0097] A number of treatment options for HBV has been reported, but there remains a needfor effective treatment of HBV infections. Genetic editing approaches targeting HBV genomes for cutting of genomic DNA are associated with a risk of off-target cutting and genomic translocations. The present epigenetic editors and related methods of use have several advantages compared to other genome engineering methods, including increased efficiency, decreased risk of translocation, and durable silencing of HBV.

[0098] The present disclosure also provides methods for treating Hepatitis D virus (HDV).HDV is the smallest pathogen known to infect humans. HDV infection is only found in patients infected with HBV, as HDV relies on HBV functions for most of its functions, including viral packaging, infectivity, transmission, and inhibition of host immunity. About 5% of patients with HBV infection also have an HDV infection. HDV uses HBV S-antigen (HBsAg) as a capsid protein, and HDV infection is therefore dependent on HBV S-antigen production. Decreasing HBV S-antigen expression also reduces HDV infectivity. The structure and biology of HDV has been reported (see, for example, Asselah and Rizzetto, Hepatitis D Virus Infection, The New England Journal of Medicine (389;1; July 6, 2023), incorporated herein by reference in its entirety). In some embodiments of the present disclosure, HDV infection is addressed through methods targeting an HBV gene or genome that reduce the level of HBsAg.

[0099] In some embodiments, an epigenetic editor as described herein may comprise one ormore epigenetic editor proteins, wherein each epigenetic editor protein comprises a DNA-binding domain linked to one or more effector domains for epigenetic modification. In certain embodiments, where the DNA-binding domain is a polynucleotide guided DNA-binding domain, the epigenetic editor may further comprise one or more guide polynucleotides. DNA-binding domains, effector domains, and guide polynucleotides of an epigenetic editor as described herein may be selected, e.g., from those described below, in any functional combination.

[0100] The epigenetic editors described herein may be expressed in a host cell transiently, ormay be integrated in a genome of the host cell; such cells and their progeny are also contemplated by the present disclosure. Both transiently expressed and integrated epigenetic editors or components thereof can effect stable epigenetic modifications. For example, after introducing to a host cell an epigenetic editor described herein, the target gene in the host cell may be stably or permanently repressed or silenced. For example, in some embodiments provided herein, a transiently expressed epigenetic editor comprising a DNMT3A domain, a DNMT3L domain, and a KRAB domain effects stable epigenetic modifications. For example, in some embodiments provided herein, a constitutively expressed epigenetic editor comprising -14-WSGR Ref. No. 59073-743.601 DNMT3A and a DNMT3L domain effects stable epigenetic modifications. In some embodiments, expression of the target gene is reduced or silenced for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 1 year, at least 2 years, or for the entire lifetime of the cell or the subject carrying the cell, as compared to the level of expression in the absence of the epigenetic editor. The epigenetic modification may be inherited by the progeny of the host cells into which the epigenetic editor was introduced. In some embodiments, the host cell is a liver cell characterized by the presence of an HBV genome in the cell.

[0101] The present epigenetic editors may be introduced to a patient in need thereof (e.g., ahuman patient), e.g., into the patient’s hepatocytes, biliary epithelial cells (cholangiocytes), stellate cells, Kupffer cells, and liver sinusoidal endothelial cells. I. DNA-Binding Domains

[0102] An epigenetic editor described herein may comprise one or more DNA-bindingdomains that direct the effector domain(s) of the epigenetic editor to target sequences within an HBV genome. A DNA-binding domain as described herein may be, e.g., a polynucleotide guided DNA-binding domain, a zinc finger protein (ZFP) domain, a transcription activator like effector (TALE) domain, a meganuclease DNA-binding domain, and the like. Examples of DNA-binding domains can be found in U.S. Patent 11,162,114, which is incorporated by refence herein in its entirety.

[0103] In some embodiments, a DNA-binding domain described herein is encoded by itsnative coding sequence. In other embodiments, the DNA-binding domain is encoded by a nucleotide sequence that has been codon-optimized for optimal expression in human cells. A. Polynucleotide Guided DNA-Binding Domains

[0104] In some embodiments, a DNA-binding domain herein may be a protein domaindirected by a guide nucleic acid sequence (e.g., a guide RNA sequence) to a target site in an HBV genome. In certain embodiments, the protein domain may be derived from a CRISPR- associated nuclease, such as a Class I or II CRISPR-associated nuclease. In some embodiments, the protein domain may 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 may be derived from a Class 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, -15-WSGR Ref. No. 59073-743.601 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 homologues and modified versions thereof. “Derived from” is used to mean that the protein domain comprises the full polypeptide sequence of the parent protein, or comprises a variant thereof (e.g., with amino acid residue deletions, insertions, and / or substitutions). The variant retains the desired function of the parent protein (e.g., the ability to form a complex with the guide nucleic acid sequence and the target DNA).

[0105] In some embodiments, the CRISPR-associated protein domain may be a Cas9 domaindescribed herein. Cas9 may, for example, refer to a polypeptide with at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence similarity to a wildtype Cas9 polypeptide described herein. In some embodiments, said wildtype polypeptide is Cas9 from Streptococcus pyogenes (NCBI Ref. No. NC_002737.2 (SEQ ID NO: 1)) and / or UniProt Ref. No. Q99ZW2 (SEQ ID NO: 2). In some embodiments, said wildtype 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 with at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence similarity to a wildtype Cpf1 polypeptide described herein (e.g., Cpf1 from Franscisella novicida (UniProt Ref. No. U2UMQ6 or SEQ ID NO: 4). In certain embodiments, the CRISPR-associated protein domain may be a modified form of the wildtype protein comprising one or more amino acid residue changes such as a deletion, an insertion, or a substitution; a fusion or chimera; or any combination thereof.

[0106] Cas9 sequences and structures of variant Cas9 orthologs have been described forvarious organisms. Exemplary organisms from which a Cas9 domain 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, Polar omonas naphthalenivorans, Polar omonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis -16-WSGR Ref. No. 59073-743.601 aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, 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, Oscillator ia sp., Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculumlavamentivorans, Coryne bacterium diphtheria, and Acaryochloris marina. Cas9 sequences alsoinclude those from the organisms and loci disclosed in Chylinski et al., RNA Biol. (2013) 10(5):726-37.

[0107] In some embodiments, the Cas9 domain is from Streptococcus pyogenes. In someembodiments, the Cas9 domain is from Staphylococcus aureus.

[0108] Other Cas domains are also contemplated for use in the epigenetic editors herein.These include, for example, those from CasX (Cas12E) (e.g., SEQ ID NO: 5), CasY (Cas12d) 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).

[0109] For epigenetic editing, the nuclease-derived protein domain (e.g., a Cas9 or Cpf1domain) may have reduced or no nuclease activity through mutations such that the protein domain does not cleave DNA or has reduced DNA-cleaving activity while retaining the ability to complex with the guide nucleic acid sequence (e.g., guide RNA) and the target DNA. For example, the nuclease activity may be reduced by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% compared to the wildtype domain. In some embodiments, a CRISPR-associated protein domain described herein is catalytically inactive (“dead”). Examples of such domains include, for example, dCas9 (“dead” Cas9), dCpf1, ddCpf1, dCasPhi, ddCas12a, dLbCpf1, and dFnCpf1. A dCas9 DNA binding domain, for example, may comprise one, two, or more mutations as compared to wildtype Cas9 that abrogate its nuclease activity. The DNA cleavage domain of Cas9 is known to include two subdomains: the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvC1 subdomain cleaves the non- complementary strand. Mutations within these subdomains can silence the nuclease activity of -17-WSGR Ref. No. 59073-743.601 Cas9. For example, the mutations D10A (in RuvC1) and H840A (in HNH) completely inactivate the nuclease activity of SpCas9. SaCas9, similarly, may be inactivated by the mutations D10A and N580A. In some embodiments, the dCas9 DNA binding domain comprises at least one mutation in the HNH subdomain and / or the RuvC1 subdomain that reduces or abrogates nuclease activity. In some embodiments, the dCas9 DNA binding domain only comprises a RuvC1 subdomain, or only comprises an HNH subdomain. It is to be understood that any mutation that inactivates the RuvC1 and / or the HNH domain may be included in a dCas9 herein, e.g., insertion, deletion, or single or multiple amino acid substitution in the RuvC1 domain and / or the HNH domain.

[0110] In some embodiments, a dCas9 DNA binding domain herein comprises a mutation atposition(s) corresponding to position D10 (e.g., D10A), H840 (e.g., H840A), or both, of a wildtype SpCas9 sequence as numbered in the sequence provided at UniProt Accession No. Q99ZW2 (SEQ ID NO: 2). In particular embodiments, the dCas9 DNA binding domain comprises the amino acid sequence of dSpCas9 (D10A and H840A) (SEQ ID NO: 12).

[0111] In some embodiments, a dCas9 DNA binding domain as described herein comprises amutation at position(s) corresponding to position D10 (e.g., D10A), N580 (e.g., N580A), or both, of a wildtype SaCas9 sequence (e.g., SEQ ID NO: 9). In particular embodiments, the dCas9 DNA binding domain comprises the amino acid sequence of dSaCas9 (D10A and N580A) (SEQ ID NO.: 13).

[0112] Additional suitable mutations that inactivate Cas9 will be apparent to those of skill inthe art based on this disclosure and knowledge in the field and are within the scope of this disclosure. Such mutations may include, but are not limited to, D839A, N863A, and / or K603R in SpCas9. The present disclosure contemplates any mutations that reduce or abrogate the nuclease activity of any Cas9 described herein (e.g., mutations corresponding to any of the Cas9 mutations described herein).

[0113] A dCpf1 protein domain may comprise one, two, or more mutations as compared towildtype Cpf1 that reduce or abrogate its nuclease activity. The Cpf1 protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9, but does not have an HNH endonuclease domain, and the N-terminal of Cpf1 does not have the alpha-helical recognition lobe of Cas9. In some embodiments, the dCpf1 comprises one or more mutations corresponding to position D917A, E1006A, or D1255A as numbered in the sequence of the Francisella novicida Cpf1 protein (FnCpf1; SEQ ID NO: 4). In certain embodiments, the dCpf1 protein comprises mutations corresponding to D917A, E1006A, D1255A, D917A / E1006A, -18-WSGR Ref. No. 59073-743.601 D917A / D1255A, E1006A / D1255A, or D917A / E1006A / D1255A, or corresponding mutation(s) in any of the Cpf1 amino acid sequences described herein. In some embodiments, the dCpf1 comprises a D917A mutation. In particular embodiments, the dCpf1 comprises the amino acid sequence of dFnCpf1 (SEQ ID NO: 14).

[0114] Further nuclease inactive CRISPR-associated protein domains contemplated hereininclude 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), dLbCpf1 (e.g., SEQ ID NO: 19), dAsCpf1 (e.g., SEQ ID NO: 20), denAsCpf1 (e.g., SEQ ID NO: 21), dHFAsCpf1 (e.g., SEQ ID NO: 22), dRVRAsCpf1 (e.g., SEQ ID NO: 23), dRRAsCpf1 (e.g., SEQ ID NO: 24), dCasX (e.g., SEQ ID NO: 25), and dCasPhi (e.g., SEQ ID NO: 26).

[0115] In some embodiments, a Cas9 domain described herein may be a high fidelity Cas9domain, e.g., comprising one or more mutations that decrease electrostatic interactions between the Cas9 domain and the sugar-phosphate backbone of DNA to confer increased target binding specificity. In certain embodiments, the high fidelity Cas9 domain may be nuclease inactive as described herein.

[0116] A CRISPR-associated protein domain described herein may recognize a protospaceradjacent motif (PAM) sequence in a target gene. A “PAM” sequence is typically 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 is not part of the target sequence. The CRISPR-associated protein domain may either recognize a naturally occurring or canonical PAM sequence or may have altered PAM specificity. CRISPR-associated protein domains that bind to non-canonical PAM sequences have been described in the art. For example, Cas9 domains that bind 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 may include, for example, those from “VRER” SpCas9, “EQR” SpCas9, “VQR” SpCas9, “SpG Cas9,” “SpRYCas9,” and “KKH” SaCas9. Nuclease inactive versions of these Cas9 domains are also contemplated, such as nuclease inactive VRER SpCas9 (e.g., SEQ ID NO: 27), nuclease inactive EQR SpCas9 (e.g., SEQ ID NO: 28), nuclease inactive VQR SpCas9 (e.g., SEQ ID NO: 29), nuclease inactive SpG Cas9 (e.g., SEQ ID NO: 30), nuclease inactive SpRY Cas9 (e.g., SEQ ID NO: 31), and nuclease inactive KKH SaCas9 (e.g., SEQ ID NO: 32). Another example is the Cas9 of Francisella novicida engineered to recognize 5’-YG-3’ (where “Y” is a pyrimidine). -19-WSGR Ref. No. 59073-743.601

[0117] Additional suitable CRISPR-associated proteins, orthologs, and variants, includingnuclease inactive variants and sequences, will be apparent to those of skill in the art based on this disclosure.

[0118] Guide RNAs that can be used in conjunction with the CRISPR-associated proteindomains herein are further described in Section II below. B. Zinc Finger Protein Domains

[0119] In some embodiments, the DNA-binding domain of an epigenetic editor describedherein comprises a zinc finger protein (ZFP) domain (or “ZF domain” as used herein). ZFPs are proteins having at least one zinc finger, and bind to DNA in a sequence-specific manner. A “zinc finger” (ZF) or “zinc finger motif” (ZF motif) refers to a polypeptide domain comprising a beta- nucleotides, typically three or four base pairs (contiguous or noncontiguous). Each ZF typically comprises approximately 30 amino acids. ZFP domains may contain multiple ZFs that make tandem contacts with their target nucleic acid sequence. A tandem array of ZFs may be engineered to generate artificial ZFPs that bind desired nucleic acid targets. ZFPs may be rationally designed by using databases comprising triplet (or quadruplet) nucleotide sequences and individual ZF amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of ZFs that bind the particular triplet or quadruplet sequence. See, e.g., U.S. Patents 6,453,242, 6,534,261, and 8,772,453.

[0120] ZFPs are widespread in eukaryotic cells, and may belong to, e.g., C2H2 class, CCHCclass, PHD class, or RING class. An exemplary motif characterizing one class of these proteins (C2H2 class) is -Cys-(X)2-4-Cys-(X)12-His-(X)3-5-His- (SEQ ID NO:1091), where X is any independently chosen amino acid. In some embodiments, a ZFP domain herein may comprise a ZF array comprising sequential C2H2-ZFs each contacting three or more sequential nucleotides. Additional architectures, e.g. as described in Paschon et al., Nat. Commun. 10, 1133 (2019), are also possible.

[0121] A ZFP domain of an epigenetic editor described herein may include 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, or more ZFs. The ZFP domain may include an array of two-finger or three-finger units, e.g., 3, 4, 5, 6, 7, 8, 9 or 10 or more units, wherein each unit binds 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 -20-WSGR Ref. No. 59073-743.601 ZFP domain comprising at least six ZFs recognizes a target DNA sequence of 18 to 21 nucleotides.

[0122] In some embodiments, ZFs in a ZFP domain described herein are connected viapeptide linkers. The peptide linkers may be, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids in length. In some embodiments, a linker comprises 5 or more amino acids. In some embodiments, a linker comprises 7-17 amino acids. The linker may be flexible or rigid.

[0123] In some embodiments a zinc finger array may have the sequence:SRPGERPFQCRICMRNFSXXXXXXXHXXTHTGEKPFQCRICMRNFSXXXXXXXHXXTH[l inker]FQCRICMRNFSXXXXXXXHXXTHTGEKPFQCRICMRNFSXXXXXXXHXXTH[li nker]PFQCRICMRNFSXXXXXXXHXXTHTGEKPFQCRICMRNFSXXXXXXXHXXTHLRG S (SEQ ID NO: 1084), or a sequence 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, which confers DNA-binding specificity upon the zinc finger; each X may be independently chosen. In the above sequence, “XX” in italics may be TR, LR or LK, and “[linker]” represents a linker sequence. In some embodiments, the linker sequence is TGSQKP (SEQ ID NO: 1085); this linker may be used when sub-sites targeted by the ZFs are adjacent. In some embodiments, the linker sequence is TGGGGSQKP (SEQ ID NO: 1086); this linker may be used when there is a base between the sub-sites targeted by the zinc fingers. The two indicated linkers may be the same or different.

[0124] ZFP domains herein may contain arrays of two or more adjacent ZFs that are directlyadjacent to one another (e.g., separated by a short (canonical) linker sequence), or are separated by longer, flexible or structured polypeptide sequences. 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 each other’s respective target triplets, which may help to strengthen or enhance the recognition of the target sequence, and leads to the binding of overlapping sequences. In some embodiments, distant ZFs within the ZFP domain may recognize (or bind to) non-contiguous nucleotide sequences.

[0125] The amino acid sequences of the ZF DNA-recognition helices of exemplary ZFPdomains herein, and their HBV target sequences, are shown below in Table 1.-21-WSGR Ref. No. 59073-743.601 Table 1. Zinc finger transcriptional repressors for silencing HBV. ZF sequences of exemplary ZFP domains are presented. SEQ ID Nos for target sequences and ZF can be found in Table 21 sequence listing.-22-WSGR Ref. No. 59073-743.601-23-WSGR Ref. No. 59073-743.601-24-WSGR Ref. No. 59073-743.601

[0126] In some embodiments, the ZFP domain of the present epigenetic editor binds to atarget sequence provided herein. In further embodiments, the ZFP domain comprises, in order, the F1-F6 amino acid sequences of any one of the zinc finger proteins as shown in Table 1 and Table 21. The F1-F6 amino acid sequences may be placed within the ZF framework sequence of SEQ ID NO: 1084, or within any other ZF framework known in the art. C. TALEs

[0127] In some embodiments, the DNA-binding domain of an epigenetic editor describedherein comprises a transcription activator-like effector (TALE) domain. The DNA-binding domain of a TALE comprises a highly conserved sequence of about 33-34 amino acids, with a repeat variable di-residue (RVD) at positions 12 and 13 that is central to the recognition of specific nucleotides. TALEs can be engineered to bind practically 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. D. Other DNA-Binding Domains

[0128] Other DNA-binding domains are contemplated for the epigenetic editors describedherein. In some embodiments, the DNA-binding domain comprises an argonaute protein domain, e.g., from Natronobacterium gregoryi (NgAgo). NgAgo is a ssDNA-guided endonuclease that is guided to its target site by 5' phosphorylated ssDNA (gDNA), where it -25-WSGR Ref. No. 59073-743.601 produces double-strand breaks. In contrast to Cas9, the NgAgo-gDNA system does not require a protospacer-adjacent motif (PAM). Thus, using a nuclease inactive NgAgo (dNgAgo) can greatly expand the bases that may be targeted. The characterization and use of NgAgo have been described, e.g., 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.

[0129] In some embodiments, the DNA-binding domain comprises an inactivated nuclease,for example, an inactivated meganuclease. Additional non-limiting examples of DNA-binding domains include tetracycline-controlled repressor (tetR) DNA-binding domains, leucine zippers, motifs, bZIP domains homeodomains, and AT-hooks. II. Guide Polynucleotides

[0130] Epigenetic editors described herein that comprise a polynucleotide guided DNA-binding domain may also include a guide polynucleotide that is capable of forming a complex with the DNA-binding domain. The guide polynucleotide may comprise RNA, DNA, or a mixture of both. For example, where the polynucleotide guided DNA-binding domain is a CRISPR-associated protein domain, the guide polynucleotide may be a guide RNA (gRNA). A “guide RNA” or “gRNA” refers to a nucleic acid that is able to hybridize to a target sequence and direct binding of the CRISPR-Cas complex to the target sequence. Methods of using guide polynucleotide sequences with programmable DNA-binding proteins (e.g., CRISPR-associated protein domains) for site-specific DNA targeting (e.g., to modify a genome) are known in the art.

[0131] A guide polynucleotide sequence (e.g., a gRNA sequence) may comprises two parts:1) a nucleotide sequence comprising a “targeting sequence” that is complementary to a target nucleic acid sequence (“target sequence”), e.g., to a nucleic acid sequence comprised in a genomic target site; and 2) a nucleotide sequence that binds a polynucleotide guided DNA- binding domain (e.g., a CRISPR-Cas protein domain). The nucleotide sequence in 1) may comprise a targeting sequence that is 100% complementary to a genomic nucleic acid sequence, e.g., a nucleic acid sequence comprised in a genomic target site, and thus may hybridize to the target nucleic acid sequence. The nucleotide sequence in 1) may be referred to as, e.g., a crispr RNA, or crRNA. The nucleotide sequence in 2) may be referred to as a scaffold sequence of a guide nucleic acid, e.g., a tracrRNA, or an activating region of a guide nucleic acid, and may comprise a stem-loop structure. Parts 1) and 2) as described above may be fused to form one single guide (e.g., a single guide RNA, or sgRNA), or may be on two separate nucleic acid -26-WSGR Ref. No. 59073-743.601 molecules. In some embodiments, a guide polynucleotide comprises parts 1) and 2) connected by a linker. In some embodiments, a guide polynucleotide comprises parts 1) and 2) connected by a non-nucleic acid linker, for example, a peptide linker or a chemical linker.

[0132] Part 2 (the scaffold sequence) of a guide polynucleotide as described herein may be,for example, as described in Jinek et al., Science (2012) 337:816-21; U.S. Patent Publication 2016 / 0208288; or U.S. Patent Publication 2016 / 0200779. Variants of part 2) are also contemplated by the present disclosure. For example, the tetraloop and stem loop of a gRNA scaffold (tracrRNA) sequence may be modified to include RNA aptamers, which can be bound by specific protein domains. In some embodiments, such modified gRNAs can be used to facilitate the recruitment of repressive or activating domains fused to the protein-interacting RNA aptamers.

[0133] A gRNA as provided herein typically comprises a targeting domain and a bindingdomain. The targeting domain (also termed “targeting sequence”) may comprise a nucleic acid sequence that binds to a target site, e.g., to a genomic nucleic acid molecule within a cell. The target site may be a double-stranded DNA sequence comprising a PAM sequence as well as the target sequence, which is located on the same strand as, and directly adjacent to, the PAM sequence. The targeting domain of the gRNA may comprise an RNA sequence that corresponds to the target sequence, i.e., it resembles the sequence of the target domain, sometimes with one or more mismatches, but typically comprising an RNA sequence instead of a DNA sequence. The targeting domain of the gRNA thus may base pair (in full or partial complementarity) with the sequence of the double-stranded target site that is complementary to the target sequence, and thus with the strand complementary to the strand that comprises the PAM sequence. It will be understood that the targeting domain of the gRNA typically does not include a sequence that resembles the PAM sequence. It will further be understood that the location of the PAM may be 5’ or 3’ of the target sequence, depending on the nuclease employed. For example, the PAM is typically 3’ of the target sequence for Cas9 nucleases, and 5’ of the target sequence for Casl2a nucleases. For an illustration of the location of the PAM and the mechanism of gRNA binding to a target site, see, e.g., Figure 1 of Vanegas et al., Fungal Biol Biotechnol. (2019) 6:6, which is incorporated by reference herein. For additional illustration and description of the mechanism of gRNA targeting of 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. -27-WSGR Ref. No. 59073-743.601

[0134] In some embodiments, the targeting domain sequence comprises between 17 and 30nucleotides and corresponds fully to the target sequence (i.e., without any mismatch nucleotides). In some embodiments, however, the targeting domain sequence may comprise one or more, but typically not more than 4, mismatches, e.g., 1, 2, 3, or 4 mismatches. As the targeting domain is part of gRNA, which is an RNA molecule, it will typically comprise ribonucleotides, while the DNA targeting domain will comprise deoxyribonucleotides.

[0135] An exemplary illustration of a Cas9 target site, comprising a 22 nucleotide targetdomain, and an NGG PAM sequence, as well as of a gRNA comprising a targeting domain that fully corresponds to the target sequence (and thus base pairs with full complementarity with the DNA strand complementary to the strand comprising the target sequence and PAM) is provided below: [ target domain (DNA) ][ PAM ] 5'-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-G-G-3' (DNA) 3'-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-C-C-5' (DNA) | | | | | | | | | | | | | | | | | | | | | | 5'-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-[ gRNA scaffold]-3' (RNA) [ targeting domain ( RNA) ][ binding domain ]

[0136] An exemplary illustration of a Casl2a target site, comprising a 22 nucleotide targetdomain, and a TTN PAM sequence, as well as of a gRNA comprising a targeting domain that fully corresponds to the target sequence (and thus base pairs with full complementarity with the DNA strand complementary to the strand comprising the target sequence and PAM) is provided below: [ PAM ][ target domain ( DNA) ] 5'-T-T-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-3' (DNA) 3'-A-A-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-5' (DNA) | | | | | | | | | | | | | | | | | | | | | | 5'-[gRNA scaffold]-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-3' (RNA) [ binding domain ][ targeting domain ( RNA) ]

[0137] While not wishing to be bound by theory, at least in some embodiments, it is believedthat the length and complementarity of the targeting domain with the target sequence contributes to specificity of the interaction of the gRNA / Cas9 molecule complex with a target nucleic acid. In some embodiments, the targeting domain of a gRNA provided herein is 5 to 50 nucleotides in length. In some embodiments, the targeting domain is 15 to 25 nucleotides in length. In some embodiments, the targeting domain is 18 to 22 nucleotides in length. In some embodiments, the targeting domain is 19-21 nucleotides in length. In some embodiments, the targeting domain is -28-WSGR Ref. No. 59073-743.601 15 nucleotides in length. In some embodiments, the targeting domain is 16 nucleotides in length. In some embodiments, the targeting domain is 17 nucleotides in length. In some embodiments, the targeting domain is 18 nucleotides in length. In some embodiments, the targeting domain is 19 nucleotides in length. In some embodiments, the targeting domain is 20 nucleotides in length. In some embodiments, the targeting domain is 21 nucleotides in length. In some embodiments, the targeting domain is 22 nucleotides in length. In some embodiments, the targeting domain is 23 nucleotides in length. In some embodiments, the targeting domain is 24 nucleotides in length. In some embodiments, the targeting domain is 25 nucleotides in length. In certain embodiments, the targeting domain fully corresponds, without mismatch, to a target sequence provided herein, or a part thereof. In some embodiments, the targeting domain of a gRNA provided herein comprises 1 mismatch relative to a target sequence provided herein. In some embodiments, the targetindg domain comprises 2 mismatches relative to the target sequence. In some embodiments, the target domain comprises 3 mismatches relative to the target sequence.

[0138] Methods for designing, selecting, and validating gRNAs are described herein andknown in the art. Software tools can be used to optimize the gRNAs corresponding to a target DNA sequence, e.g., to minimize total off-target activity across the genome. For example, DNA sequence searching algorithms can be used to identify a target sequence in crRNAs of a gRNA for use with Cas9. Exemplary gRNA design tools include the ones described in Bae et al., Bioinformatics (2014) 30:1473-5.

[0139] Guide polynucleotides (e.g., gRNAs) described herein may be of various lengths. Insome embodiments, the length of the spacer or targeting sequence depends on the CRISPR- associated protein component of the epigenetic editor system used. For example, Cas proteins from different bacterial species have varying optimal targeting sequence lengths. Accordingly, the spacer sequence may comprise, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more than 50 nucleotides in length. In some embodiments, the spacer comprises 10-24, 11-20, 11-16, 18-24, 19-21, or 20 nucleotides in length. In some embodiments, a guide polynucleotide (e.g., gRNA) is from 15-100 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length and 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) contiguous nucleotides complementary to the target sequence. In some -29-WSGR Ref. No. 59073-743.601 embodiments, a guide polynucleotide described herein may be truncated, e.g., by 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50 or more nucleotides.

[0140] In certain embodiments, the 3’ end of the HBV target sequence is immediatelyadjacent to a PAM sequence (e.g., a canonical PAM sequence such as NGG for SpCas9). The degree of complementarity between the targeting sequence of the guide polynucleotide (e.g., the spacer sequence of a gRNA) and the target sequence may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In particular embodiments, the targeting and the target sequence may be 100% complementary. In other embodiments, the targeting sequence and the target sequence may contain, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches.

[0141] A guide polynucleotide (e.g., gRNA, sgRNA) may be modified with, for example,chemical alterations and synthetic modifications. A modified gRNA, for instance, can include an alteration or replacement of one or both of the non-linking phosphate oxygens and / or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage, an alteration of the ribose sugar (e.g., of the 2’ hydroxyl on the ribose sugar), an alteration of the phosphate moiety, modification or replacement of a naturally occurring nucleobase, modification or replacement of the ribose-phosphate backbone, modification of the 3’ end and / or 5’ end of the oligonucleotide, replacement of a terminal phosphate group or conjugation of a moiety, cap, or linker, or any combination thereof.

[0142] In some embodiments, one or more ribose groups of the gRNA (e.g., sgRNA) may bemodified. Examples of chemical modifications to the ribose group include, but are not limited to, 2’-O-methyl (2’-OMe), 2’-fluoro (2’-F), 2’-deoxy, 2’-O-(2-methoxyethyl) (2’-MOE), 2’-NH2, 2’-O-allyl, 2’-O-ethylamine, 2’-O-cyanoethyl, 2’-O-acetalester, or a bicyclic nucleotide 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). 2’-O-methyl modification and / or 2’- fluoro modification may increase binding affinity and / or nuclease stability of the gRNA (e.g., sgRNA)oligonucleotides. The terms “mA,” “mC,” “mU,” or “mG” can be used to represent a nucleotide that has been modified with 2’O-Me.

[0143] In some embodiments, one or more phosphate groups of the gRNA may bechemically modified. Examples of chemical modifications to a phosphate group include, but are not limited to, a phosphorothioate (PS), phosphonoacetate (PACE), thiophosphonoacetate (thioPACE), amide, triazole, phosphonate, and phosphotriester modification. In some embodiments, a guide polynucleotide described herein may comprise one, two, three, or more PS -30-WSGR Ref. No. 59073-743.601 linkages at or near the 5’ end and / or the 3’ end; the PS linkages may be contiguous or noncontiguous.

[0144] Phosphorothioate (PS) linkage or bond refers to a bond where a sulfur is substitutedfor one nonbridging phosphate oxygen in a phosphodiester linkage (e.g., bond between nucleotide bases). In some embodiments, the phosphorothioate linkage is used to generate modified gRNA. In some embodiments, modified gRNA with phosphorothioate linkage is referred to as S-oligos. A “*” used herein can depict a PS modification. In some embodiments, the terms A*, C*, U*, or G* can denote a nucleotide that is linked to the next (e.g., 3') nucleotide with a PS linkage. As described herein, the terms “mA*,” “mC*,” “mU*,” or “mG*” are used to denote a nucleotide with 2’-O-Me modification and linked to the next (e.g., 3') nucleotide with a PS linkage.

[0145] In some embodiments, the gRNA (e.g., sgRNA) herein comprises a mixture ofribonucleotides and deoxyribonucleotides and / or one or more PS linkages.

[0146] In some embodiments, one or more nucleobases of the gRNA (e.g., sgRNA) may bechemically modified. Examples of chemically modified nucleobases include, but are not limited to, 2-thiouridine, 4-thiouridine, N6-methyladenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5-methylcytosine, 5-substituted pyrimidine, isoguanine, isocytosine, and nucleobases with halogenated aromatic groups. Chemical modifications can be made in the spacer region, the tracr RNA region, the stem loop, or any combination thereof.

[0147] In some embodiments, the gRNA (e.g., sgRNA) comprises at least about 75%, at leastabout 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to a sequence set forth in any one of SEQ ID NOs: 1093- 1235. In some embodiments, the gRNA (e.g., sgRNA) comprises at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to a sequence set forth in SEQ ID NO: 1249. In some embodiments, the gRNA (e.g., sgRNA) comprises at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least -31-WSGR Ref. No. 59073-743.601 about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to a sequence set forth in SEQ ID NO: 1260.

[0148] In some embodiments, the gRNA (e.g., sgRNA) comprises a nucleotide basesequence. In some embodiments, the nucleotide base sequence comprises at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to a sequence set forth in SEQ ID NO: 1260, wherein A, G, C and U represent adenine, guanine, cytosine and uracil, respectively.

[0149] In some embodiments, the guide RNA disclosed herein comprises one or morechemically modified nucleotide(s). In some embodiments, the one or more chemically modified nucleotide(s) is at any one of nucleotide position(s) 1, 2, 3, or combinations thereof as numbered in SEQ ID NO: 1260 or corresponding position(s) thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at any one of nucleotide position(s) 30-40, or combinations thereof as numbered in SEQ ID NO: 1260 or corresponding position(s) thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at any one of nucleotide position(s) 69-81, or combinations thereof as numbered in SEQ ID NO: 1260 or corresponding position(s) thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at any one of nucleotide position(s) 83-100, or combinations thereof as numbered in SEQ ID NO: 1260 or corresponding position(s) thereof. In some embodiments, the one or more chemically modified nucleotide(s) are at one or more nucleotide position(s) selected from 1-3, 30-40, 69-81 and 83-100 as numbered in SEQ ID NO: 1260 or corresponding position(s) thereof.

[0150] In some embodiments, the one or more chemically modified nucleotide(s) is atnucleotide position 1 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 2 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 3 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 30 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 31 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at -32-WSGR Ref. No. 59073-743.601 nucleotide position 32 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 33 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 34 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 35 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 36 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 37 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 38 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 39 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 40 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 69 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 70 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 71 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 72 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 73 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 74 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 75 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 76 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 77 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 78 as numbered in SEQ ID NO: 1260 -33-WSGR Ref. No. 59073-743.601 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 79 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 80 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 81 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 83 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 84 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 85 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 86 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 87 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 88 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 89 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 90 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 91 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 92 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 93 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 94 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 95 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 96 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 97 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, -34-WSGR Ref. No. 59073-743.601 the one or more chemically modified nucleotide(s) is at nucleotide position 98 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 99 as numbered in SEQ ID NO: 1260 or corresponding position thereof. In some embodiments, the one or more chemically modified nucleotide(s) is at nucleotide position 100 as numbered in SEQ ID NO: 1260 or corresponding position thereof.

[0151] Table 2 below lists exemplary target sequences for epigenetic modification of HBV,as well as the coordinates of the start and end positions of the targeted site on the HBV genome. Table 2. Targeting Domain Sequences of Exemplary gRNAs Targeting HBV. The following target sites were identified as suitable for targeting with an epigenetic repressor:-35-WSGR Ref. No. 59073-743.601-36-WSGR Ref. No. 59073-743.601-37-WSGR Ref. No. 59073-743.601-38-WSGR Ref. No. 59073-743.601

[0152] Target domains identified above that are adjacent to a PAM sequence, e.g., an S.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 sites 1-143 are suitable for dCas9-based epigenetic repressor targeting.

[0153] A suitable gRNA for targeting any of the target domain sequences would, in someembodiments, comprise a target domain sequence that is the RNA-equivalent sequence of the provided DNA sequence of the targeting domain sequence (i.e., an RNA nucleotide of that sequence instead of the provided DNA nucleotide, with uracil instead of thymine), and a suitable tracr RNA sequence.

[0154] Any tracr sequence known in the art is contemplated for a gRNA described herein.Some exemplary suitable tracr sequences are provided herein, and additional suitable tracr sequences will be apparent to the skilled artisan, including, but not limited to, tracr sequences described in DeWeirdt et al., Accounting for small variations in the tracrRNA sequence improves sgRNA activity predictions for CRISPR screening. Nat Commun 13, 5255 (2022); Scott et al., Improved Cas9 activity by specific modifications of the tracrRNA. Sci Rep 9, 16104 (2019); Riesenberg et al., Improved gRNA secondary structures allow editing of target sites resistant to CRISPR-Cas9 cleavage. Nat Commun 13, 489 (2022); and Liao C, Beisel CL. The tracrRNA in CRISPR Biology and Technologies. Annu Rev Genet. 2021 Nov 23;55:161-181, the contents of each of which are incorporated herein in their entirety. In some embodiments, a gRNA described -39-WSGR Ref. No. 59073-743.601 herein has a tracr sequence shown in Table 3 below, or a tracr sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the tracr sequence shown below (SEQ: SEQ ID NO). Table 3. Exemplary TRACR Sequences

[0155] In some embodiments, the gRNA herein is provided to the cell directly (e.g., throughan RNP complex together with the CRISPR-associated protein domain). In some embodiments, the gRNA is provided to the cell through an expression vector (e.g., a plasmid vector or a viral vector) introduced into the cell, where the cell then expresses the gRNA from the expression vector. Methods of introducing gRNAs and expression vectors into cells are well known in the art. III. Effector Domains

[0156] Epigenetic editors described herein include one or more effector protein domains(also “epigenetic effector domains,” or “effector domains,” as used herein) that effect epigenetic modification of a target gene. An epigenetic editor with one or more effector domains may modulate expression of a target gene without altering its nucleobase sequence. In some embodiments, an effector domain described herein may provide repression or silencing of expression of HBV or an HBV gene, e.g., by repressing transcription or by modifying or remodeling HBV chromatin. Such effector domains are also referred to herein as “repression domains,” “repressor domains,” “epigenetic repressor domains,” or “epigenetic repression -40-WSGR Ref. No. 59073-743.601 domains.” Non-limiting examples of chemical modifications that may be mediated by effector domains include methylation, demethylation, acetylation, deacetylation, phosphorylation, SUMOylation and / or ubiquitination of DNA or histone residues.

[0157] In some embodiments, an effector domain of an epigenetic editor described hereinmay make histone tail modifications, e.g., by adding or removing active marks on histone tails.

[0158] In some embodiments, an effector domain of an epigenetic editor described hereinmay comprise or recruit a transcription-related protein, e.g., a transcription repressor. The transcription-related protein may be endogenous or exogenous.

[0159] In some embodiments, an effector domain of an epigenetic editor described hereinmay, for example, comprise a protein that directly or indirectly blocks access of a transcription factor to the gene of interest harboring the target sequence.

[0160] An effector domain may be a full-length protein or a fragment thereof that retains theepigenetic effector function (a “functional domain”). Functional domains that are capable of modulating (e.g., repressing) gene expression can be derived from a larger protein. For example, functional domains that can reduce target gene expression may be identified based on sequences of repressor proteins. Amino acid sequences of gene expression-modulating proteins may be obtained from available genome browsers, such as the UCSD genome browser or Ensembl genome browser. Protein annotation databases such as UniProt or Pfam can be used to identify functional domains within the full protein sequence. As a starting point, the largest sequence, encompassing all regions identified by different databases, may be tested for gene expression modulation activity. Various truncations then may be tested to identify the minimal functional unit.

[0161] Variants of effector domains described herein are also contemplated by the presentdisclosure. A variant may, for example, refer to a polypeptide with at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence similarity to a wildtype effector domain described herein. In particular 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 wildtype effector domain.

[0162] In some embodiments, an epigenetic editor described herein may comprise 1 effectordomain, 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. In certain embodiments, the epigenetic editor comprises one or more epigenetic editor proteins (e.g., one, two, or three epigenetic editor proteins), each with one or -41-WSGR Ref. No. 59073-743.601 more effector domains (e.g., one, two, or three effector domains) linked to a DNA-binding domain. In some embodiments, the effector domains may induce a combination of epigenetic modifications, e.g., transcription 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 ubiquitylation, DNA methylation, and histone SUMOylation.

[0163] In certain embodiments, an effector domain described herein (e.g., DNMT3A domainand / or DNMT3L domain) is encoded by a nucleotide sequence as found in the native genome (e.g., human or murine) for that effector domain. In other embodiments, an effector domain described herein is encoded by a nucleotide sequence that has been codon-optimized for optimal expression in human cells.

[0164] Effector domains described herein may include, for example, transcriptionalrepressors, DNA methyltransferases, and / or histone modifiers, as further detailed below. A. Transcriptional Repressors

[0165] In some embodiments, an epigenetic effector domain described herein mediatesrepression of a target gene’s expression (e.g., transcription). The effector domain may comprise, e.g., 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 rhabdosarcoma 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), a WRPW motif of the hairy-related basic helix-loop-helix (bHLH) repressor proteins, an HP1 alpha chromo-shadow repression domain, an HP1 beta repression domain, or any combination thereof. The effector domain may recruit one or more protein domains that repress expression of the target gene, e.g., through a scaffold protein. In some embodiments, the effector domain may recruit or interact with a scaffold protein domain that recruits a PRMT protein, a HDAC protein, a SETDB1 protein, or a NuRD protein domain.

[0166] In some embodiments, the effector domain comprises a functional domain derivedfrom a zinc finger repressor protein, such as a KRAB domain. KRAB domains are found in approximately 400 human ZFP-based transcription factors. Descriptions of KRAB domains may be found, for example, in Ecco et al., Development (2017) 144(15):2719-29 and Lambert et al., Cell (2018) 172:650-65. -42-WSGR Ref. No. 59073-743.601

[0167] In certain embodiments, the effector domain comprises a repression domain (e.g.,KRAB) derived from KOX1 / ZNF10, KOX8 / ZNF708, ZNF43, ZNF184, ZNF91, HPF4, HTF10, or HTF34. In some embodiments, the effector domain comprises a repression 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 repression domain may be a KRAB domain derived from KOX1, ZIM3, ZFP28, or ZN627. In particular embodiments, the repression domain is a ZIM3 KRAB domain. In some embodiments, the repressor domain is a ZIM3 repressor domain. In further embodiments, the effector domain is derived from a human protein, e.g., a human ZIM3, a human KOX1, a human ZFP28, or a human ZN627.

[0168] Exemplary effector domains that may reduce or silence target gene expression areprovided in Table 4 below (SEQ: SEQ ID NO, see Table 21 for sequences of exemplary effector domains). Further examples of repressors and transcriptional repressor domains can be found, e.g., 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. Table 4. Exemplary Effector Domains Suitable for Silencing Gene Expression-43-WSGR Ref. No.59073-743.601-44-WSGR Ref. No.59073-743.601-45-WSGR Ref. No.59073-743.601-46-WSGR Ref. No.59073-743.601-47-WSGR Ref. No.59073-743.601-48-WSGR Ref. No.59073-743.601-49-WSGR Ref. No.59073-743.601-50-WSGR Ref. No. 59073-743.601

[0169] A functional analog of any one of the above-listed proteins, i.e., a molecule having thesame or substantially the same biological function (e.g., retaining 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more) of the protein’s transcription factor function) is encompassed by the present disclosure. For example, the functional analog may be an isoform or a variant of the above-listed protein, e.g., containing a portion of the above protein with or without additional amino acid residues and / or containing mutations relative to the above protein. In some embodiments, the functional analog has a sequence identity that is at least 75, 80, 85, 90, 95, 98, or 99% to one of the sequences listed in Table 4. Homologs, orthologs, and mutants of the above-listed proteins are also contemplated.

[0170] In certain embodiments, an epigenetic editor described herein comprises a KRABdomain derived from KOX1, ZIM3, ZFP28, or ZN627, and / or an effector domain derived from KAP1, MECP2, HP1a, HP1b, CBX8, CDYL2, TOX, TOX3, TOX4, EED, EZH2, RBBP4, RCOR1, or SCML2, optionally wherein the parental protein is a human protein. In particular embodiments, an epigenetic editor described herein comprises 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 may comprise a KRAB domain derived from KOX1 (ZNF10), e.g., a human KOX1. In certain embodiments, the epigenetic editor may comprise a KRAB domain derived from ZIM3 (ZNF657 or ZNF264), e.g., a human ZIM3. In certain embodiments, the epigenetic editor may comprise a KRAB domain derived from ZFP28, e.g., a human ZFP28. In certain embodiments, the epigenetic editor may comprise a KRAB domain derived from ZN627, e.g., a human ZN627. In certain embodiments, an epigenetic editor -51-WSGR Ref. No. 59073-743.601 described herein may comprise a CDYL2, e.g., a human CDYL2, and / or a TOX domain (e.g., a human TOX domain) in combination with a KOX1 KRAB domain (e.g., a human KOX1 KRAB domain).

[0171] In certain embodiments, an epigenetic effector described herein comprises arepression domain derived from ZNF10 (SEQ ID NO: 1024). For example, the repression domain may comprise the sequence of SEQ ID NO: 1024, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1024. B. DNA Methyltransferases

[0172] In some embodiments, an effector domain of an epigenetic editor described hereinalters target gene expression through DNA modification, such as methylation. Highly methylated areas of DNA tend to be less transcriptionally active than less methylated areas. DNA methylation occurs primarily at CpG sites (shorthand for “C-phosphate-G-” or “cytosine- phosphate-guanine” sites). Many mammalian genes have promoter regions near or including CpG islands (nucleic acid regions with a high frequency of CpG dinucleotides).

[0173] An effector domain described herein may be, e.g., a DNA methyltransferase (DNMT)or a catalytic domain thereof, or may be capable of recruiting a DNA methyltransferase. DNMTs encompass enzymes that catalyze the transfer of a methyl group to a DNA nucleotide, such as canonical cytosine-5 DNMTs that catalyze the addition of methyl groups to genomic DNA (e.g., DNMT1, DNMT3A, DNMT3B, and DNMT3C). This term also encompasses non-canonical family members that do not catalyze methylation themselves but that recruit (including activate) catalytically active DNMTs; a non-limiting example of such a DNMT domain is DNMT3Ldomain. See, e.g., Lyko, Nat Review (2018) 19:81-92. Unless otherwise indicated, a DNMTdomain may refer to a polypeptide domain derived from a catalytically active DNMT domain (e.g., DNMT1 domain, DNMT3A domain, and DNMT3B domain) or from a catalytically inactive DNMT domain (e.g., DNMT3L domain). A DNMT domain may repress expression of the target gene through the recruitment of repressive regulatory proteins. In some embodiments, the methylation is at a CG (or CpG) dinucleotide sequence. In some embodiments, the methylation is at a CHG or CHH sequence, where H is any one of A, T, or C.In some embodiments, DNMTs in the epigenetic editors may include, e.g., DNMT1 domain, DNMT3A domain, DNMT3B domain, and / or DNMT3C domain. In some embodiments, the DNMT domain is a mammalian (e.g., human or murine) DNMT domain. In particular embodiments, the DNMT domain is DNMT3A domain (e.g., human DNMT3A domain). In certain embodiments, an epigenetic editor described herein comprises a DNMT3A domain comprising SEQ ID NO: -52-WSGR Ref. No. 59073-743.601 1028, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1028. In certain embodiments, an epigenetic editor described herein comprises a DNMT3A domain comprising SEQ ID NO: 1029, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1029. In some embodiments, the DNMT3A domain may have, e.g., a mutation at position H739 (such as H739A or H739E), R771 (such as R771L) and / or R836 (such as R836A or R836Q), or any combination thereof (numbering according to SEQ ID NO: 1028).

[0174] In some embodiments, an effector domain described herein may be a DNMT-likedomain. As used herein a “DNMT-like domain” is a regulatory factor of DNA methyltransferase that may activate or recruit other DNMT domains, but does not itself possess 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 domain, which may be, for example, human DNMT3L domain or mouse DNMT3L domain. In certain embodiments, an epigenetic editor described herein comprises a DNMT3L domain comprising SEQ ID NO: 1032, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1032. In certain embodiments, an epigenetic editor herein comprises a DNMT3L domain comprising SEQ ID NO: 1033, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1033. In certain embodiments, an epigenetic editor described herein comprises a DNMT3L domain comprising SEQ ID NO: 1034, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1034. In certain embodiments, an epigenetic editor described herein comprises a DNMT3L domain comprising SEQ ID NO: 1035, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1035. In some embodiments, the DNMT3L domain may have, e.g., a mutation corresponding to that at position D226 (such as D226V), Q268 (such as Q268K), or both (numbering according to SEQ ID NO: 1032).

[0175] In certain embodiments, an epigenetic editor herein may comprise comprising bothDNMT and DNMT-like effector domains. For example, the epigenetic editor may comprise a DNMT3A-3L domain, wherein DNMT3A domain and DNMT3L domain may be covalently linked. In other embodiments, an epigenetic editor described herein may comprise an effector domain that comprises only a DNMT3A domain (e.g., human DNMT3A domain), or only a DNMT-like domain (e.g., DNMT3L domain, which may be human or mouse DNMT3L domain). -53-WSGR Ref. No. 59073-743.601

[0176] In some embodiments a DNMT domain can be an animal DNMT domain. In someembodiments, a DNMT domain can be a mammalian DNMT domain. In some embodiments, a DNMT domain can be a primate DNMT domain. In some embodiments, a DNMT domain can be a human DNMT domain. In some embodiments, a DNMT domain can be a human DNMT1 domain. In some embodiments, a DNMT domain can be a human DNMT3A domain. In some embodiments, a DNMT domain can be a catalytic domain of a human DNMT3A. In some embodiments, a DNMT domain can be a human DNMT3B domain. In some embodiments, a DNMT domain can be a mouse DNMT domain. In some embodiments, a DNMT domain can be a mouse DNMTC domain. In some embodiments, a DNMT domain can be a mouse DNMT3L domain. In some embodiments, a DNMT domain can be a catalytic domain of a mouse DNMT3L domain. In some embodiments, a DNMT domain can be a horse DNMT domain. In some embodiments, a DNMT domain can be a Equus przewalskii DNMT domain. In some embodiments, a DNMT domain can be derived from an Equus species. In some embodiments, a DNMT domain can be a Equus przewalskii DNMT3L domain. In some embodiments, a DNMT domain can be a catalytic domain of a Equus przewalskii DNMT3L domain.

[0177] Table 5 below provides exemplary methyltransferases from which an effector domainof an epigenetic editor protein described herein may be derived. See Table 21 for sequences of these exemplary methyltransferases. Table 5. Exemplary DNA Methyltransferase Sequences-54-WSGR Ref. No. 59073-743.601

[0178] A functional analog of any one of the above-listed proteins, i.e., a molecule having thesame or substantially the same biological function (e.g., retaining 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more) of the protein’s DNA methylation function or recruiting function) is encompassed by the present disclosure. For example, the functional analog may be an isoform or a variant of the above-listed protein, e.g., containing a portion of the above protein with or without additional amino acid residues and / or containing mutations relative to the above protein. In some embodiments, the functional analog has a sequence identity that is at least 75, 80, 85, 90, 95, 98, or 99% to one of the sequences listed in Table 5. In some -55-WSGR Ref. No. 59073-743.601 embodiments, the effector domain herein comprises only the functional domain (or functional analog thereof), e.g., the catalytical domain or recruiting domain, of the above-listed proteins.

[0179] 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., a human or murine DNMT3A domain or DNMT3L domain) or a functional analog thereof (e.g., having a functional fragment, such as a catalytic fragment or recruiting fragment, of the parental protein; and / or having mutations that improve the activity of the DNMT protein).

[0180] An epigenetic editor herein may effect methylation at, e.g., 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 the target gene or chromosome. The CpG dinucleotide sequences may be located within or near the target gene in CpG islands, or may be located in a region that is not a CpG island. A CpG island generally refers to a nucleic acid sequence or chromosome region that comprises a high frequency of CpG dinucleotides. For example, a CpG island may comprise at least 50% GC content. The CpG island may have a high observed-to-expected CpG ratio, for example, an observed-to-expected CpG ratio of at least 60%. As used herein, an observed-to-expected CpG ratio is determined by Number of CpG * (sequence length) / (Number of C * Number of G). In some embodiments, the CpG island has an observed-to-expected CpG ratio of at least 60%, 70%, 80%, 90% or more. A CpG island may be a sequence or region of, e.g., at least 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 nucleotides. In some embodiments, only 1, or less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50 CpG dinucleotides are methylated by the epigenetic editor.

[0181] In some embodiments, an epigenetic editor herein effects methylation at ahypomethylated nucleic acid sequence, i.e., a sequence that may lack methyl groups on the 5- methyl cytosine nucleotides (e.g., in CpG) as compared to a standard control. Hypomethylation may occur, for example, in aging cells or in cancer (e.g., early stages of neoplasia) relative to a younger cell or non-cancer cell, respectively.

[0182] In some embodiments, an epigenetic editor described herein induces methylation at ahypermethylated nucleic acid sequence.

[0183] In some embodiments, methylation may be introduced by the epigenetic editor at asite other than a CpG dinucleotide. For example, the target gene sequence may be methylated at the C nucleotide of CpA, CpT, or CpC sequences. In some embodiments, an epigenetic editor comprises a DNMT3A domain and effects methylation at CpG, CpA, CpT, CpC sequences, or -56-WSGR Ref. No. 59073-743.601 any combination thereof. In some embodiments, an epigenetic editor comprises a DNMT3A domain that lacks a regulatory subdomain and only maintains a catalytic domain. In some embodiments, the epigenetic editor comprising a DNMT3A catalytic domain effects methylation exclusively at CpG sequences. In some embodiments, an epigenetic editor comprising a DNMT3A domain that comprises a mutation, e.g. a R836A or R836Q mutation (numbering according to SEQ ID NO: 1028), has higher methylation activity at CpA, CpC, and / or CpT sequences as compared to an epigenetic editor comprising a wildtype DNMT3A domain. C. Histone Modifiers

[0184] In some embodiments, an effector domain of an epigenetic editor herein mediateshistone modification. Histone modifications play a structural and biochemical role in gene transcription, such as by formation or disruption of the nucleosome structure that binds to the histone and prevents gene transcription. Histone modifications may include, for example, acetylation, deacetylation, methylation, phosphorylation, ubiquitination, SUMOylation and the like, e.g., at their N-terminal ends (“histone tails”). These modifications maintain or specifically convert chromatin structure, thereby controlling responses such as gene expression, DNA replication, DNA repair, and the like, which occur on chromosomal DNA. Post-translational modification of histones is an epigenetic regulatory mechanism and is considered essential for the genetic regulation of eukaryotic cells. Recent studies have revealed that chromatin remodeling factors such as SWI / SNF, RSC, NURF, NRD, and the like, which facilitate transcription factor access to DNA by modifying the nucleosome structure; histone acetyltransferases (HATs) that regulate the acetylation state of histones; and histone deacetylases (HDACs), act as important regulators.

[0185] In particular, the unstructured N-termini of histones may be modified by acetylation,deacetylation, methylation, ubiquitylation, 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 epsilon amino group of the lysine side chains. This neutralizes the lysine’s positive charge and weakens the interactions between histones and DNA, thus opening the chromosomes for transcription factors to bind and initiate transcription. Acetylation of K14 and K9 lysines of histone H3 by histone acetyltransferase enzymes may be linked to 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 -57-WSGR Ref. No. 59073-743.601 methylation of lysine 9 of histone H3 may be associated with heterochromatin, or transcriptionally silent chromatin.

[0186] In certain embodiments, an effector domain of an epigenetic editor described hereincomprises a histone methyltransferase domain. The effector domain may comprise, for example, a DOT1L domain, a SET domain, a SUV39H1 domain, a G9a / EHMT2 protein domain, an EZH1 domain, an EZH2 domain, a SETDB1 domain, or any combination thereof. In particular embodiments, the effector domain comprises a histone-lysine-N-methyltransferase SETDB1 domain.

[0187] In some embodiments, the effector domain comprises a histone deacetylase proteindomain. In certain embodiments, the effector domain comprises a HDAC family protein domain, for example, a HDAC1, HDAC3, HDAC5, HDAC7, or HDAC9 protein domain. In particular embodiments, the effector domain comprises a nucleosome remodeling and deacetylase complex (NURD), which removes acetyl groups from histones. D. Other Effector Domains

[0188] In some embodiments, the effector domain comprises a tripartite motif containingprotein (TRIM28, TIF1-beta, or KAP1). In certain embodiments, the effector domain comprises one or more KAP1 proteins. A KAP1 protein in an epigenetic editor herein may form a complex with one or more other effector domains of the epigenetic editor or one or more proteins involved in modulation of gene expression in a cellular environment. For example, KAP1 may be recruited by a KRAB domain of a transcriptional repressor. A KAP1 protein domain may interact with or recruit one or more protein complexes that reduces or silences gene expression. In some embodiments, KAP1 interacts with or recruits a histone deacetylase protein, a histone- lysine methyltransferase protein, a chromatin remodeling protein, and / or a heterochromatin protein. For example, a KAP1 protein domain may interact with or recruit a heterochromatin protein 1 (HP1) protein, a SETDB1 protein, an HDAC protein, and / or a NuRD protein complex component. In some embodiments, a KAP1 protein domain interacts with or recruits a ZFP90 protein (e.g., isoform 2 of ZFP90), and / or a FOXP3 protein. An exemplary KAP1 amino acid sequence is shown in SEQ ID NO: 1062.

[0189] In some embodiments, the effector domain comprises a protein domain that interactswith or is recruited by one or more DNA epigenetic marks. For example, the effector domain may comprise a methyl CpG binding protein 2 (MECP2) protein that interacts with methylated DNA nucleotides in the target gene (which may or may not be at a CpG island of the target gene). An MECP2 protein domain in an epigenetic editor described herein may induce -58-WSGR Ref. No. 59073-743.601 condensed chromatin structure, thereby reducing or silencing expression of the target gene. In some embodiments, an MECP2 protein domain in an epigenetic editor described herein may interact with a histone deacetylase (e.g. HDAC), thereby repressing or silencing expression of the target gene. In some embodiments, an MECP2 protein domain in an epigenetic editor described herein may block access of a transcription factor or transcriptional activator to the target sequence, thereby repressing or silencing expression of the target gene. An exemplary MECP2 amino acid sequence is shown in SEQ ID NO: 1063.

[0190] Also contemplated as effector domains for the epigenetic editors described herein are,e.g., a chromoshadow domain, a ubiquitin-2 like Rad60 SUMO-like (Rad60-SLD / SUMO) domain, a chromatin organization modifier domain (Chromo) domain, a Yaf2 / RYBP C-terminal binding motif domain (YAF2_RYBP), a CBX family C-terminal motif domain (CBX7_C), a zinc finger C3HC4 type (RING finger) domain (ZF-C3HC4_2), a cytochrome b5 domain (Cyt-b5), a helix-loop-helix domain (HLH), a helix-hairpin-helix motif domain (e.g., HHH_3), a high mobility group box domain (HMG-box), a basic leucine zipper domain (e.g., bZIP_1 or bZIP_2), a Myb_DNA-binding domain, a homeodomain, a MYM-type Zinc finger with FCS sequence domain (ZF-FCS), an interferon regulatory factor 2-binding protein zinc finger domain (IRF- 2BP1_2), an SSX repression domain (SSXRD), a B-box-type zinc finger domain (ZF-B_box), a CXXC zinc finger domain (ZF-CXXC), a regulator of chromosome condensation 1 domain (RCC1), an SRC homology 3 domain (SH3_9), a sterile alpha motif domain (SAM_1), a sterile alpha motif domain (SAM_2), a sterile alpha motif / Pointed domain (SAM_PNT), a Vestigial / Tondu family domain (Vg_Tdu), a LIM domain, an RNA recognition motif domain (RRM_1), a paired amphipathic helix domain (PAH), a proteasomal ATPase OB C-terminal domain (Prot_ATP_ID_OB), a nervy homology 2 domain (NHR2), a hinge domain of cleavage stimulation factor subunit 2 (CSTF2_hinge), a PPAR gamma N-terminal region domain (PPARgamma_N), a CDC48 N-terminal domain (CDC48_2), a WD40 repeat domain (WD40), a Fip1 motif domain (Fip1), a PDZ domain (PDZ_6), a Von Willebrand factor type C domain (VWC), a NAB conserved region 1 domain (NCD1), an S1 RNA-binding domain (S1), an HNF3 C-terminal domain (HNF_C), a Tudor domain (Tudor_2), a histone-like transcription factor (CBF / NF-Y) and archaeal histone domain (CBFD_NFYB_HMF), a zinc finger protein domain (DUF3669), an EGF-like domain (cEGF), a GATA zinc finger domain (GATA), a TEA / ATTS domain (TEA), a phorbol esters / diacylglycerol binding domain (C1-1), polycomb-like MTF2 factor 2 domain (Mtf2_C), a transactivation domain of FOXO protein family (FOXO-TAD), a homeobox KN domain (Homeobox_KN), a BED zinc finger domain (ZF-BED), a zinc finger of -59-WSGR Ref. No. 59073-743.601 C3HC4-type RING domain (ZF-C3HC4_4), a RAD51 interacting motif domain (RAD51_interact), a p55-binding region of a nethyl-CpG-binding domain protein MBD (MBDa), a Notch domain, a Raf-like Ras-binding domain (RBD), a Spin / Ssty family domain (Spin-Ssty), a PHD finger domain (PHD_3), a Low-density lipoprotein receptor domain class A (Ldl_recept_a), a CS domain, a DM DNA-binding domain, and a QLQ domain.

[0191] In some embodiments, the effector domain is a protein domain comprising aYAF2_RYBP domain or homeodomain or any combination thereof. In certain embodiments, the homeodomain of the YAF2_RYBP domain is a PRD domain, an NKL domain, a HOXL domain, or a LIM domain. In particular embodiments, the YAF2_RYBP domain may comprise a 32 amino acid Yaf2 / RYBP C-terminal binding motif domain (32 aa RYBP).

[0192] In some embodiments, the effector domain comprises a protein domain selected froma group consisting of SUMO3 domain, Chromo domain from M phase phosphoprotein 8 (MPP8), chromoshadow domain from Chromobox 1 (CBX1), and SAM_1 / SPM domain from Scm Polycomb Group Protein Homolog 1 (SCMH1).

[0193] In some embodiments, the effector domain comprises an HNF3 C-terminal domain(HNF_C). The HNF_C domain may be from FOXA1 or FOXA2. In certain embodiments, the HNF_C domain comprises an EH1 (engrailed homology 1) motif.

[0194] In some embodiments, the effector domain may comprise an interferon regulatoryfactor 2-binding protein zinc finger domain (IRF-2BP1_2), a Cyt-b5 domain from DNA repair factor HERC2 E3 ligase, a variant SH3 domain (SH3_9) from Bridging Integrator 1 (BIN1), an HMG-box domain from transcription factor TOX or ZF-C3HC4_2 RING finger domain from the polycomb component PCGF2, a Chromodomain-helicase-DNA binding protein 3 (CHD3) domain, or a ZNF783 domain. IV. Epigenetic Editors

[0195] Provided herein are epigenetic editors, also referred to herein as epigenetic editingsystems, that direct epigenetic modification(s) to a target sequence in a gene of interest, e.g., using one or more DNA-binding domains as described herein and one or more effector domains (e.g., epigenetic repression domains) as described herein, in any combination. The DNA-binding domain (in concert with a guide polynucleotide such as one 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 that may be -60-WSGR Ref. No. 59073-743.601 durable and inheritable across cell generations. In some aspects, the epigenetic editors described herein can repress or silence genes reversibly or irreversibly in cells.

[0196] In particular embodiments, an epigenetic editor described herein comprises one ormore epigenetic editor proteins, each comprising (1) DNA-binding domain(s) and (2) effector domain(s). The effector domains may be on one or more epigenetic editor proteins comprised by the epigenetic editor. For example, a single epigenetic editor protein may comprise all of the effector domains with a DNA-binding domain. Alternatively, the effector domains or subsets thereof may be on separate epigenetic editor proteins, each with a DNA-binding domain (which may be the same or different). An epigenetic editor protein described herein may further comprise one or more linkers (e.g., peptide linkers), detectable tags, nuclear localization signals (NLSs), or any combination thereof. As used herein, a “epigenetic editor protein” refers to a chimeric protein in which two or more coding sequences (e.g., for DNA-binding domain(s) and / or effector domain(s)) are covalently or non-covalently joined, directly or indirectly.

[0197] In some embodiments, an epigenetic editor described herein comprises 2, 3, 4, 5, 6, 7,8, 9, 10, or more effector (e.g., repression) domains, which may be identical or different. In certain embodiments, two or more of said effector domains function synergistically. Combinations of effector domains may comprise DNA methylation domains, histone deacetylation domains, histone methylation domains, and / or scaffold domains that recruit any of the above. For example, an epigenetic editor described herein may comprise one or more transcriptional repressor domains (e.g., a KRAB domain such as KOX1, ZIM3, ZFP28, or ZN627 KRAB) in combination with one or more DNA methylation domains (e.g., a DNMT domain) and / or recruiter domain (e.g., a DNMT3L domain). Such an epigenetic editor may comprise, for instance, a KRAB domain, a DNMT3A domain, and a DNMT3L domain. An epigenetic editor can comprise a DNMT3A domain and a DNMT3L domain and preferably further comprise a KRAB domain. In some embodiments, the epigenetic editor further comprises an additional effector domain (e.g., a KAP1, MECP2, HP1b, CBX8, CDYL2, TOX, TOX3, TOX4, EED, RBBP4, RCOR1, or SCML2 domain). In some embodiments, the additional effector domain is a CDYL2, TOX, TOX3, TOX4, or HP1a domain. For example, an epigenetic editor described herein may comprise a CDYL2 and / or a TOX domain in combination with a KRAB domain (e.g., a KOX1 KRAB domain). -61-WSGR Ref. No. 59073-743.601 A. Linkers

[0198] An epigenetic editor protein as described herein may comprise one or more linkersthat connect components of the epigenetic editor. A linker may be a peptide or non-peptide linker.

[0199] In some embodiments, one or more linkers utilized in an epigenetic editor providedherein is a peptide linker, i.e., a linker comprising a peptide moiety. A peptide linker can be any length applicable to the epigenetic editor proteins described herein. In some embodiments, the linker can comprise a peptide between 1 and 200 (e.g., between 1 and 80) amino acids. In some embodiments, the linker comprises from 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 6050 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 in length. Longer or shorter linkers are also contemplated. In some embodiments, the peptide linker is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 25, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length. For example, the peptide linker may be 4, 5, 16, 20, 24, 27, 32, 40, 64, 92, or 104 amino acids in length. The peptide linker may be a flexible or rigid linker. In particular embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 1064-1068 or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0200] In certain embodiments, the peptide linker is an XTEN linker. Such a linker maycomprise part of the XTEN sequence (Schellenberger et al., Nat Biotechnol (2009) 27(1):1186- 90), an unstructured hydrophilic polypeptide consisting only of residues G, S, P, T, E, and A. The term “XTEN” as used herein refers to a recombinant peptide or polypeptide lacking hydrophobic amino acid residues. XTEN linkers typically are unstructured and comprise a limited set of natural amino acids. Fusion of XTEN to proteins alters its hydrodynamic properties and reduces the rate of clearance and degradation of the epigenetic editor protein. These XTEN epigenetic editor proteins are produced using recombinant technology, without the need for chemical modifications, and degraded by natural pathways. The XTEN linker may be, for example, 5, 10, 16, 20, 26, or 80 amino acids in length. In some embodiments, the XTEN linker is 16 amino acids in length. In some embodiments, the XTEN linker is 80 amino acids in -62-WSGR Ref. No. 59073-743.601 length. In certain embodiments, the XTEN linker may be XTEN10, XTEN16, XTEN20, or XTEN80. In certain embodiments, the XTEN linker may comprise the amino acid sequence of any one of SEQ ID NOs: 1069-1073 and 1092 or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the XTEN linker may be XTEN10, XTEN16, XTEN20, or XTEN80.

[0201] In some embodiments, one or more linkers utilized in an epigenetic editor providedherein is a non-peptide linker. For example, the linker may be a carbon bond, a disulfide bond, or carbon-heteroatom bond. In certain embodiments, the linker is a carbon-nitrogen bond of an amide linkage. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, or branched or unbranched aliphatic or heteroaliphatic linker.

[0202] In some embodiments, one or more linkers utilized in an epigenetic editor providedherein is polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). The linker may comprise, for example, a monomer, dimer, or polymer of aminoalkanoic acid; an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4- aminobutanoic acid, 5-pentanoic acid, etc.); a monomer, dimer, or polymer of aminohexanoic acid (Ahx); or a polyethylene glycol moiety (PEG); or an aryl or heteroaryl moiety. In certain embodiments, the linker may be based on a carbocyclic moiety (e.g., cyclopentane or cyclohexane) or a phenyl ring. The linker may include functionalized moieties to facilitate attachment of a nucleophile (e.g., thiol, amino) from the peptide to the linker. Any electrophile may be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, alkyl halides, aryl halides, acyl halides, and isothiocyanates.

[0203] Various linker lengths and flexibilities can be employed between any two componentsof an 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 linkers may range from very flexible linkers, such as glycine / serine-rich linkers, to more rigid linkers, in order to achieve the optimal length for effector domain activity for the specific application. In some embodiments, the more flexible linkers are glycine / serine- rich linkers (GS-rich linkers), 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 the GS-rich linkers are (GGGGS)n (SEQ ID NO: 485), (G)n, and W linker (SEQ ID NO: 486). In some embodiments, the more rigid linkers are in the form of the form (EAAAK)n (SEQ ID NO: 487), (SGGS)n (SEQ ID NO: 488), and (XP)n (SEQ ID NO: 489). In the aforementioned formulae of flexible and rigid linkers, n may be any integer between 1 and 30. In some embodiments, n is 1, -63-WSGR Ref. No. 59073-743.601 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, wherein n is 1, 3, or 7 (SEQ ID NO: 490). In some embodiments, the linker comprises a (GGGGS)n motif, wherein n is 4 (SEQ ID NO: 491).

[0204] In some embodiments, a linker in an epigenetic editor described herein comprises anuclear localization signal, for example, with the amino acid sequence of any one of SEQ ID NOs: 1074-1079. In some embodiments, a linker in an epigenetic editor described herein comprises an expression tag, e.g., a detectable tag such as a green fluorescence protein.

[0205] In some embodiments, the linker disclosed herein (e.g., XTEN linker) is positionedbetween two DNMT domain. In some embodiments, the linker disclosed herein (e.g., XTEN linker) is positioned between a DNA binding domain and a DNMT domain. In some embodiments, the linker disclosed herein (e.g., XTEN linker) is positioned between a DNA binding domain and a repressor domain. B. Nuclear Localization Signals

[0206] An epigenetic editor protein described herein may comprise one or more nuclearlocalization signals, and in certain embodiments, may comprise two or more nuclear localization signals. For example, the epigenetic editor 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 proteins to the nucleus. In certain embodiments, the NLS may be an SV40 NLS. In some embodiments, the NLS is a bipartite NLS. The epigenetic editor protein may comprise an NLS at its N-terminus, C-terminus, or both, and / or an NLS may be embedded in the middle of the epigenetic editor protein (e.g., at the N- or C- terminus of a DNA-binding domain or an effector domain). In certain embodiments, an NLS comprises the amino acid sequence of any one of SEQ ID NOs: 1074-1079, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the selected sequence. Additional NLSs are known in the art. C. Tags

[0207] Epigenetic editors provided herein may comprise one or more additional sequences(“tags”) for tracking, detection, and localization of the editors. In some embodiments, the epigenetic editor comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more detectable tags. Each of the detectable tags may be the same or different.

[0208] For example, an epigenetic editor protein may comprise cytoplasmic localizationsequences, export sequences, such as nuclear export sequences, or other localization sequences, as well as sequence tags that are useful for solubilization, purification, or detection of the -64-WSGR Ref. No. 59073-743.601 epigenetic editor proteins. Suitable protein tags provided herein include, but are not limited to, biotin carboxylase carrier protein (BCCP) tags, myc-tags, calmodulin-tags, FLAG-tags, hemagglutinin (HA)-tags, poly-histidine tags (also referred to 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, Softags (e.g., Softag 1 or Softag 3), strep-tags, biotin ligase tags, FlAsH tags, V5 tags, and SBP-tags. Additional suitable sequences will be apparent to those of skill in the art. Sequences disclosed herein that are presented with tag sequences included are also contemplated without the presented tag sequences; similarly, sequences disclosed herein without tag sequences are also contemplated to include the addition of suitable tag sequences apparent to those of skill in the art. D. Epigenetic Editor Protein Configurations

[0209] An epigenetic editor protein described herein may have its components structured indifferent configurations. For example, the DNA-binding domain may be at the C-terminus, the N-terminus, or in between two or more epigenetic effector domains or additional domains. In some embodiments, the DNA-binding domain is at the C-terminus of the epigenetic editor protein. In some embodiments, the DNA-binding domain is at the N-terminus of the epigenetic editor protein. 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 an epigenetic effector domain and / or an additional domain on both sides. In some embodiments, where “DBD” indicates DNA-binding domain and “ED” indicates effector domain, the epigenetic editor protein comprises the configuration of: - N’]-[ED1]-[DBD]-[ED2]-[C’ - N’]-[ED1]-[DBD]-[ED2]-[ED3]-[C’ - N’]-[ED1]-[ED2]-[DBD]-[ED3]-[C’ or - N’]-[ED1]-[ED2]-DBD]-[ED3]-[ED4]-[C’.

[0210] In some embodiments, an epigenetic editor protein comprises a DNA-binding domain(DBD), a DNA methyltransferase (DNMT) domain, and a transcriptional repressor (“repressor”) domain that represses or silences expression of a target gene. The DBD, DNMT, and transcriptional repressor domains may be any as described herein, in any combination. For example, an epigenetic editor protein can comprise a DBD, a DNMT3A domain, and a DNMT3L domain. An epigenetic editor protein can comprise a DBD, a DNMT3A domain, a DNMT3L -65-WSGR Ref. No. 59073-743.601 domain, and preferably further comprise a KRAB domain. In some embodiments, the epigenetic editor comprises an epigenetic editor protein with the configuration of: N’]-[DNA methyltransferase domain]-[DBD]-[repressor domain]-[C’ N’]-[repressor domain]-[DBD]-[DNA methyltransferase domain]-[C’ N’]-[DNA methyltransferase domain]-[repressor domain]- [DBD]-[C’ or N’]-[repressor domain]-[DNA methyltransferase domain]- [DBD]-[C’.

[0211] In some embodiments, a connecting structure “]-[“in any one of the epigenetic editorprotein structures is a linker, e.g., a peptide linker; a detectable tag; a peptide bond; a nuclear localization signal; and / or a promoter or regulatory sequence. In an epigenetic editor protein structure, the multiple connecting structures “]-[“ may be the same or may each be a different linker, tag, NLS, or peptide bond. In particular embodiments, the DNA methyltransferase domain comprises DNMT3A domain, DNMT3L domain, or both. In particular embodiments, the DBD is a catalytically inactive polynucleotide guided DNA-binding domain (e.g., a dCas9) or a ZFP domain. In particular embodiments, the repressor domain is a KRAB domain.

[0212] In some embodiments, the epigenetic editor protein comprises a configurationselected from N’]-[DNMT3A-DNMT3L]-[DBD]-[KRAB]-[C’ N’]-[KRAB]-[DBD]-[DNMT3A-DNMT3L]-[C’ N’]-[KRAB]-[DBD]-[DNMT3A]-[C’ N’]-[DNMT3A]-[DBD]-[KRAB]-[C’ N’]-[KRAB]-[DBD]-[DNMT3A]-[DNMT3L]-[C’ N’]-[DNMT3A]- [DNMT3L]- [DBD]-[KRAB]-[C’ N’]-[DNMT3A]-[DBD]-[C’ N’]-[DBD]-[DNMT3A]-[C’ N’]-[DNMT3L]-[DBD]-[C’ N’]-[DBD]-[DNMT3L]-[C’ wherein [DNMT3A-DNMT3L] indicates that the DNMT3A and DNMT3L domains are directly fused via a peptide bond, and wherein the connecting structure ]-[ is any one of the linkers as described herein, a detectable tag, an affinity domain, a peptide bond, a nuclear localization signal, a promoter, and / or a regulatory sequence. The DBD, KRAB, DNMT3A, and DNMT3L domains may be any as described herein, in any combination. In particular embodiments, the DBD is a CRISPR-associated protein domain (e.g., dCas9 DNA binding domain) or a ZFP -66-WSGR Ref. No. 59073-743.601 domain; the KRAB domain is derived from KOX1, ZIM3, ZFP28, or ZN627; the DNMT3A domain is a human DNMT3A domain; and the DNMT3L domain is a human or mouse DNMT3L domain; any combination of these components is also contemplated by the present disclosure.

[0213] In some embodiments, the epigenetic editor protein comprises a configurationselected from N’]-[DNMT3A]-[DBD]-[SETDB1]-[C’ N’]-[DNMT3A]- [DNMT3L]- [DBD]-[SETDB1]-[C’ N’]- [DNMT3A-DNMT3L]- [DBD]- [SETDB1]- [C’ N’]-[SETDB1]-[DBD]-[DNMT3A]-[DNMT3L]-[C’ N’]-[SETDB1]-[DBD]-[DNMT3A]-[C’ wherein [DNMT3A-DNMT3L] indicates that the DNMT3A and DNMT3L domains are directly fused via a peptide bond, and wherein the connecting structure ]-[ is any one of the linkers as described herein, a detectable tag, an affinity domain, a peptide bond, a nuclear localization signal, a promoter, and / or a regulatory sequence. The DBD, SETDB1, DNMT3A, and DNMT3L domains may be any as described herein, in any combination. In particular embodiments, the DBD is a CRISPR-associated protein domain (e.g., dCas9 DNA binding domain) or a ZFP domain; the SETDB1 domain is derived from human SETDB1, ZIM3, ZFP28, or ZN627; the DNMT3A domain is a human DNMT3A domain; and the DNMT3L domain is a human or mouse DNMT3L domain; any combination of these components is also contemplated by the present disclosure.

[0214] Particular constructs contemplated herein include:DNMT3A-DNMT3L-XTEN80-NLS-dCas9-NLS-XTEN16-KOX1 KRAB (Configuration 1), and DNMT3A-DNMT3L-XTEN80-NLS-ZFP domain-NLS-XTEN16-KOX1 KRAB (Configuration 2). In particular embodiments, the DNMT3L domain and DNMT3A domain are both derived from human parental proteins. In particular embodiments, the DNMT3L domain and DNMT3A domain are derived from human and mouse parental proteins, respectively. In particular embodiments, the DNMT3L domain and DNMT3A domain are derived from mouse and human parental proteins, respectively. In particular embodiments, the DNMT3L domain and DNMT3A domain are both derived from mouse parental proteins. In some embodiments, the dCas9 DNA binding domain is dSpCas9 DNA binding domain. In some embodiments, the KOX1 is human KOX1. -67-WSGR Ref. No. 59073-743.601

[0215] In particular embodiments, an epigenetic editor protein described herein may haveConfiguration 1 and comprise SEQ ID NO: 1080, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In SEQ ID NO: 1080 below, the XTEN linkers are underlined, the NLS sequences are bolded, the DNMT3A domain sequence is italicized, the DNMT3L domain sequence is underlined and italicized, the dCas9 DNA binding domain is bolded and italicized, and the KOX1 KRAB domain is underlined and bolded: MNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSI TVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRL FFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHR ARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPV FMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFA CVSSGNSNANSRGPSFSSGLVPLSLRGSHMGPMEIYKTVSAWKRQPVRVLSLFRNIDKVL KSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGWYMF QFHRILQYALPRQESQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAM RVWSNIPGLKSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQN SLPLGGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT STEEGTSTEPSEGSAPGTSTEPSEPKKKRKVYMDKKYSIGLAIGTNSVGWAVITDEYKVP SKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSN EMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDK ADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDA KAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSK DTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEH HQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEE LLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVL PKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDY FKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFED REMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGF ANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELV KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQN EKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSD NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQIT KHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAY LNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELAL PSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLD KVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI HQSITGLYETRIDLSQLGGDPKKKRKVSGSETPGTSESATPESTGRTLVTFKDVFVDFTR-68-WSGR Ref. No. 59073-743.601

[0216] In particular embodiments, an epigenetic editor protein described herein may haveConfiguration 2 and comprise SEQ ID NO: 1081, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In SEQ ID NO: 1081 below, the XTEN linkers are underlined, the NLS sequences are bolded and underlined, the DNMT3A domain sequence is italicized, the DNMT3L domain sequence is underlined and italicized, the ZFP domain is bolded, and the KOX1 KRAB domain is underlined and bolded. Variable amino acids represented by Xs are the amino acids of the DNA-recognition helix of the zinc finger and XX in italics may be either TR, LR or LK. MNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSI TVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRL FFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHR ARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPV FMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFA CVSSGNSNANSRGPSFSSGLVPLSLRGSHMGPMEIYKTVSAWKRQPVRVLSLFRNIDKVL KSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGWYMF QFHRILQYALPRQESQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAM RVWSNIPGLKSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQN SLPLGGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT STEEGTSTEPSEGSAPGTSTEPSEPKKKRKVYSRPGERPFQCRICMRNFSXXXXXXXHXX THTGEKPFQCRICMRNFSXXXXXXXHXXTH[linker]PFQCRICMRNFSXXXXXXXHXX THTGEKPFQCRICMRNFSXXXXXXXHXXTH[linker]PFQCRICMRNFSXXXXXXXHXX THTGEKPFQCRICMRNFSXXXXXXXHXXTHLRGSPKKKRKVSGSETPGTSESATPESTGR TLVTFKDVFVDFTREEWKLLDTAQQ VYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEE P (SEQ ID NO: 1081)

[0217] In certain embodiments, the six “XXXXXXX” regions in SEQ ID NO: 1081comprise, in order, the F1-F6 amino acid sequences shown in Table 1. [linker] represents a linker sequence. In some embodiments, one or both linker sequences may be TGSQKP (SEQ ID NO: 1085). In some embodiments, one or both linker sequences may be TGGGGSQKP (SEQ ID NO: 1086). In some embodiments, one linker sequence may have the amino acid sequence of SEQ ID NO: 1085 and the other linker sequence may have the amino acid sequence of SEQ ID NO: 1086.

[0218] In some embodiments, the epigenetic editor has an epigenetic editor protein with aconfiguration of the following: nucleoplasmin bipartite NLS-DNMT3A-DNMT3L (Equus przewalskii)-XTEN80-dCas9-SV40 NLS-XTEN16-ZIM3-nucleoplasmin bipartite NLS. In some embodiments, an epigenetic editor protein comprises a configuration as annotated in Table 6. In -69-WSGR Ref. No. 59073-743.601 some embodiments, a polynucleotide sequence encodes an epigenetic editor protein comprising a configuration as annotated in Table 7.

[0219] In some embodiments, the epigenetic editor protein is encoded in an mRNA. In someembodiments, an RNA (e.g., mRNA) encodes an epigenetic editor protein, wherein the RNA (e.g., mRNA) comprises a 5’ cap, a 5’ untranslated region (UTR), a coding region, a 3’ UTR, and a poly A tail. In some embodiments, the 5' cap of the RNA comprises the structure below.

[0220] In some embodiments, the RNA comprises a 5' UTR. In some embodiments, 5’ UTRdescribed herein comprises an amino acid sequence set forth in SEQ ID NO: 1261, or a sequence at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, the RNA comprises a 3' UTR. In some embodiments, the 3' UTR is derived from fibrinogen beta chain (FGB) gene. In some embodiments, 5’ UTR described herein comprises an amino acid sequence set forth in SEQ ID NO: 1262, or a sequence at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto.

[0221] In some embodiments, an RNA (e.g., mRNA) encodes an epigenetic editor proteinwith a configuration as annotated in Table 8. In some embodiments, an epigenetic editor protein described herein comprises an amino acid sequence set forth in SEQ ID NO: 1252, or a sequence at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, -70-WSGR Ref. No. 59073-743.601 at least about 97%, at least about 98%, or at least about 99% identical thereto. In some embodiments, an RNA (e.g., mRNA) encodes an epigenetic editor protein, wherein the RNA (e.g., mRNA) comprises a sequence set forth in SEQ ID NO: 1257, or a sequence at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical thereto.

[0222] In some embodiments, an RNA (e.g., mRNA) disclosed herein comprises at least 1, atleast 2, at least 3, at least 5, at least 10, at least 20, at least 30, at least 40, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 500, at least 700, at least 800, at least 900, or at least 1000 uridines substituted with N1-methylpseudouridine. In some embodiments, an RNA (e.g., mRNA) disclosed herein has all uridines substituted with N1- methylpseudouridine. Table 6. Annotation of Amino Acid Sequence (SEQ ID NO: 1252)-71-WSGR Ref. No. 59073-743.601 Table 7. Annotation of Polynucleotide Sequence (SEQ ID NO: 1251)Table 8. Annotation of RNA sequence (SEQ ID NO: 1257)-72-WSGR Ref. No. 59073-743.601

[0223] Multiple epigenetic editors may be used to effect activation or repression of a targetgene or multiple target genes. For example, an epigenetic editor protein comprising a DNA- binding domain (e.g., a dCas9 DNA binding domain) and an effector domain may be co- delivered with two or more guide polynucleotides (e.g., gRNAs), each targeting a different target DNA sequence. The target sites for two of the DNA-binding domains may be the same or in the vicinity of 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. In addition, when targeting double-strand DNA, such as an endogenous gene locus, the guide polynucleotides may target the same or different strands (one or more to the positive strand and / or one or more to the negative strand). V. Target Sequences

[0224] An epigenetic editor herein may be directed to an HBV target sequence to effectepigenetic modification of HBV or an HBV gene. As used herein, a “target sequence,” a “target site,” or a “target region” is a nucleic acid sequence present in a genome or gene of interest, e.g., in an HBV genome or an HBV gene; in some instances, the target sequence may be outside but in the vicinity of the gene of interest wherein methylation or binding by a repressor of the target sequence represses expression of the gene. In some embodiments, the target sequence may be a hypomethylated or hypermethylated nucleic acid sequence. -73-WSGR Ref. No. 59073-743.601

[0225] The structure and biology of HBV as well as HBV-associated diseases have beenreported (see, for example, Yuen, MF., Chen, DS., Dusheiko, G. et al. Hepatitis B virus infection. Nat Rev Dis Primers 4, 18035 (2018); R. Koshy and W.H. Caselman (Eds.), Hepatitis B Virus: Molecular Mechanism in Disease and Novel Strategies for Antiviral Therapy, Imperial College Press, London (1998), ISBN 1783262737; the entire contents of each of which are incorporated herein by reference). HBV genotypes and sub-types, as well as their genomic, transcript, and protein sequences have been described and are known to the skilled artisan. Some exemplary HBV sequences, e.g., those under accession numbers NC_00397 and U95551 are provided elsewhere herein, and the entire content of each such database entry is incorporated herein by reference.

[0226] Without wishing to be bound by any particular theory, it has been reported that HBVpersists as a covalently closed circular DNA (cccDNA) of approximately 3.2 kb, as well as in an integrated form. The HBV genome has been extensively characterized. The HBV genome has been shown to comprise four genes (the S gene, the P gene, the C gene, and the X gene), regulated by four promoter elements (sp1, sp2, cp and xp) and two enhancer elements (Enh I and Enh II) that control the expression of four defined (and overlapping) protein-encoding open reading frames (S, C, X, and P). See Figure 1. The HBV genome has been described to express six major viral RNA transcripts encoding the viral proteins: (1) the preCore (preC) RNA, which encodes the C protein (also referred to as Core protein, HBe Antigen, or HBeAg); (2), the pre- genomic (pg)RNA, which encodes the two viral proteins C (core) and P (polymerase), and also serves as the template for the synthesis of viral DNA, which is mediated by the reverse transcriptase activity of the viral P protein once pg RNA and the P protein are encapsidated into the nucleocapsids formed by the C protein; (3) the large surface protein (preS1) RNA, which encodes the Large S Antigen (also referred to as L-HBsAg); (4) the middle surface protein (preS2) RNA, which encodes the Middle S Antigen (also referred to as M-HBsAg); (5) the small surface protein (S) RNA, which encodes the Small S Antigen (also referred to as S-HBsAg); and (6) the X protein (HBx) RNA, which encodes the X protein. Transcription start sites (TSSs) as well as the termination site of the HBV transcripts have been mapped in various HBV genotypes and sub-types. Notably, HBV transcripts have been described to terminate at a single termination / polyadenylation signal located downstream of the Hbx CDS and comprising a canonical ATAAA motif. It has further been reported that HBV DNA may be methylated by infected cells and such methylation has been postulated to correlate with inhibition of viral gene expression. However, naturally occurring cell-mediated methylation of viral DNA is typically -74-WSGR Ref. No. 59073-743.601 insufficient to silence viral expression to a level that would result in control of HBV infection. DNA methylation typically occurs at CpG dinucleotides. Several CpG-rich genomic regions, also referred to as CpG islands or CGIs, have been identified in the HBV genome. CGIs are typically identified in HBV genomic sequences as sequences of a specific minimal length (e.g., at least 100bp) that comprise a minimum percentage of G and C nucleotides (e.g., at least 50% or at least 60% GC content) and a ratio of observed vs. expected CpG dinucleotides of at least 0.6. CGIs satisfying these criteria have been identified in all HBV genotypes, and it has been demonstrated that HBV genomes typically contain three CpG islands (CGI-I, CHI-II, and CGI- III, respectively), which are also sometimes referred to as ‘conventional’ HBV CpG islands. Some HBV genotypes or sub-types have been reported to comprise additional, ‘non- conventional’ CGIs. Figure 1 is a diagram illustrating an exemplary structure of a circular HBV genome (the underlying sequence of which is provided herein as SEQ ID NO: 1082), identifying the coding regions of HBV genes and CpG islands CGI-I-III. See, for example, M.J. Kosovsky, et al., The regulation of hepatitis B virus gene expression: an overview of the cis- and trans- acting components in R. Koshy and W.H. Caselman (Eds.), Hepatitis B Virus: Molecular Mechanism in Disease and Novel Strategies for Antiviral Therapy, Imperial College Press, London (1998), ISBN 1783262737; Miller et al Compact organization of the hepatitis B virus genome. Hepatology. 1989 Feb;9(2):322-7; Stadelmayer et al., Full-length 5'RACE identifies all major HBV transcripts in HBV-infected hepatocytes and patient serum. J Hepatol. 2020 Jul;73(1):40-51; Meier-Stephenson et al., Comprehensive Analysis of Hepatitis B Virus Promoter Region Mutations. Viruses. 2018 Nov 1;10(11):603; Vivekanandan et al., Hepatitis B viral DNA is methylated in liver tissues. J Viral Hepat. 2008, 15(2):103–7; Chen et al., Detection of hepatitis B virus DNA in hepatocellular carcinoma: methylation of integrated viral DNA. J Virol Methods. 1988, 19(3–4):257–63; Zhang et al., Comparative Analysis of CpG Islands among HBV Genotypes. PLOS ONE 2013, 8(2):e56711; Jain et al., Comprehensive DNA methylation analysis of hepatitis B virus genome in infected liver tissues. Sci Rep 5, 10478 (2015); Low et al., Hepatitis B virus DNA methylation and its potential role in chronic hepatitis B. Expert Reviews in Molecular Medicine. 2023;25:e11; Hou et al., CpG islands of hepatitis B virus genome isolated from Chinese patients. Gene (2015) 561:261-267; Mouzannar et al., The Post- Transcriptional Regulatory Element of Hepatitis B Virus: From Discovery to Therapy. Viruses. 2024 Mar 29;16(4):528; Peng et al., Nonproductive Hepatitis B Virus Covalently Closed Circular DNA Generates HBx-Related Transcripts from the HBx / Enhancer I Region and Acquires Reactivation by Superinfection in Single Cells. J Virol. 2023 Jan 31;97(1):e0171722; Altinel et -75-WSGR Ref. No. 59073-743.601 al., Single-Nucleotide Resolution Mapping of Hepatitis B Virus Promoters in Infected Human Livers and Hepatocellular Carcinoma. J Virol. 2016 Nov 14;90(23):10811-10822; the entire contents of each of which, and, where applicable, including any supplemental information, are incorporated herein by reference.

[0227] The target sequence (also referred to herein as target site or target region) of anepigenetic editor provided herein may be any suitable HBV sequence.

[0228] The target sequence may be in any part of a target gene. In some embodiments, thetarget sequence is part of or near a noncoding 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 an enhancer. In some embodiments, the target sequence is adjacent to, overlaps with, or encompasses a CpG island, e.g., 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 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 an HBV TSS. In certain embodiments, the target sequence is within 500 bp flanking the HBV TSS. In certain embodiments, the target sequence is within 1000 bp flanking the HBV TSS.

[0229] Some exemplary embodiments in which the target sequence is part of a target geneare provided herein and additional embodiments will be apparent to the skilled artisan based on the present disclosure and the knowledge of the genomic structure of HBV in the art. For example, in some embodiments, the target sequence is part of the HBV S gene, the HBV P gene, the HBV C gene, or the HBV X gene. In some embodiments, the target sequence is part of the HBV S gene. In some embodiments, the target sequence is part of the HBV P gene. In some embodiments, the target sequence is part of the HBV C gene. In some embodiments, the target sequence is part of the HBV X gene.Some exemplary embodiments in which the target sequence is part of a noncoding sequence of a target gene are provided herein and additional embodiments will be apparent to the skilled artisan based on the present disclosure and the knowledge of the genomic structure of HBV in the art. For example, in some embodiments the target sequence is part of a noncoding sequence of the HBV S gene, of the HBV P gene, of the HBV C gene, or of the HBV X gene. For example, in some embodiments, the target sequence is part of a noncoding sequence of the HBV S gene. In some embodiments, the target sequence is part of a noncoding sequence of the HBV P gene. In some embodiments, the target sequence is part of a noncoding -76-WSGR Ref. No. 59073-743.601 sequence of the HBV C gene. In some embodiments, the target sequence is part of a noncoding sequence of the HBV X gene. Noncoding sequences of the various HBV genes are known in the art and include, for example, the promoter and enhancer sequences of the HBV genome. Accordingly, in some embodiments, the target sequence is part of an HBV promoter sequence (e.g., of a promoter sequence within the HBV genome driving the transcription of one of the HBV transcripts described elsewhere herein, including, for example, of a sequence of the sp1, the sp2, the cp, and the xp promoter elements). In some embodiments, the target sequences is part of an HBV enhancer sequence (e.g., of the Enh I or of the Enh II sequence).

[0230] Some exemplary embodiments, in which the target sequence is adjacent to, overlapswith, or encompasses a CpG island, e.g., a CpG island identified within the HBV genome include embodiments in which the target sequence is adjacent to, overlaps with, or encompasses a conventional CGI of HBV, e.g., CGI I, CGI II, or CGI III. CGIs of HBV have been identified and described in numerous publications and are thus known to the skilled artisan. Bioinformatics tools for the identification of CGIs in any specific HBV sequence, e.g., in a sequence of a specific HBV genotype or sub-type, or in an HBV sequence isolated from a patient, are known in the art, including, for example, EMBOSS CpG plot (EMBL-EBI) and Methprimer (Li LC and Dahiya R. MethPrimer: designing primers for methylation PCRs. Bioinformatics. 2002 Nov;18(11):1427-31). Conventional CGIs of HBV include CGI I, which overlaps the S and the P gene ORFs; CGI-II, which overlaps the P gene and X gene ORFs; and CGI III, which overlaps the C gene and P gene ORFs (see Figure 1). In some embodiments, an HBV CGI is identified as a sequence within the HBV genome that is (1) at least 100 nucleotides long; (2) is characterized by a GC content of at least 50%; and (3) is characterized by an observed-to-expected CpG dinucleotide ratio of at least 0.6. According to these criteria, in the exemplary HBV genome referenced in Figure 1, i.e., NC_003977 (provided herein as SEQ ID NO: 1082), CGI I spans nucleotides 186-288, CGI II spans nucleotides 1,217-1,670, and CGI III spans nucleotides 2,282- 2,448 (see Figure 1). CGIs of HBV fulfilling these criteria, including conventional HBV CGIs I- III, of other HBV sequences, including other genotypes, sub-types, or specific HBV sequences, will be apparent to the skilled artisan. In some embodiments, the target sequence overlaps with HBV CGI I. In some embodiments, the target sequence overlaps with HBV CGI II. In some embodiments, the target sequence overlaps with CGI III.

[0231] Exemplary embodiments in which the target sequence is within about 3000, 2900,2800, 2700, 2600, 2500, 2400, 2300, 2200, 2100, 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 base pairs (bp) flanking -77-WSGR Ref. No. 59073-743.601 an HBV TSS (transcription start site) include embodiments, in which the target sequence is within the respective number of base pairs of the TSS of any of the six major viral RNA transcripts, i.e., the TSS of the preCore (pre-C) RNA, the TSS of the pre-genomic (pg)RNA, the TSS of the large surface protein (preS1) RNA, the TSS of the middle surface protein (preS2) RNA, the TSS of the the small surface protein (S) RNA, and the TSS of the X protein (HBx) RNA. The positions of the transcription start sites of the various HBV transcripts have been identified in various HBV genotypes and sub-types and are thus known to the skilled artisan. For example, for HBV of genotype D, as exemplified by NCBI database entries NC_003977 and U95551.1 (provided as SEQ ID NOs 1082 and 1083 herein), the TSS of the pg RNA transcript has been identified as nucleotide 1820, the TSS of the pre-C RNA as nucleotide 1791, and the TSS of the pre-S2 RNA as nucleotide 3159. The initiation of HBx RNA transcripts encoded by HBV genomes has been reported to not be limited to a single nucleotide, but to be spread over a short sequence. For example, TSSs for canonical HBx transcripts have been reported to initiate closely upstream of the first ATG in the sequence encoding the X protein, with HBx transcript TSS positions having been mapped to nucleotides 1243-1338 of HBV of genotype D, as exemplified by NCBI database entries NC_003977 and U95551.1 (provided as SEQ ID NOs 1082 and 1083 herein). TSSs for additional transcripts have also been identified and TSSs have been mapped to various HBV genotypes and sub-types.

[0232] In some embodiments in which the target sequence is within about 3000, 2900, 2800,2700, 2600, 2500, 2400, 2300, 2200, 2100, 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 base pairs (bp) flanking an HBV TSS, the HBV TSS is an HBV pg RNA TSS. For example, in some embodiments provided herein, the target sequence of an epigenetic editor is within 100 bp flanking an HBV pg RNA TSS, e.g., within 100 bp of nucleotide 1820 of SEQ ID NO: 1082 or 1083. In some embodiments provided herein, the target sequence of an epigenetic editor is within 200 bp flanking an HBV pg RNA TSS, e.g., within 200 bp of nucleotide 1820 of SEQ ID NO: 1082 or 1083. In some embodiments provided herein, the target sequence of an epigenetic editor is within 300 bp flanking an HBV pg RNA TSS, e.g., within 300 bp of nucleotide 1820 of SEQ ID NO: 1082 or 1083. In some embodiments provided herein, the target sequence of an epigenetic editor is within 400 bp flanking an HBV pg RNA TSS, e.g., within 400 bp of nucleotide 1820 of SEQ ID NO: 1082 or 1083. In some embodiments provided herein, the target sequence of an epigenetic editor is within 500 bp flanking an HBV pg RNA TSS, e.g., within 500 bp of nucleotide 1820 of SEQ ID NO: 1082 or 1083. In some embodiments provided herein, the target sequence of an -78-WSGR Ref. No. 59073-743.601 epigenetic editor is within 600 bp flanking an HBV pg RNA TSS, e.g., within 600 bp of nucleotide 1820 of SEQ ID NO: 1082 or 1083.

[0233] In some embodiments in which the target sequence is within about 3000, 2900, 2800,2700, 2600, 2500, 2400, 2300, 2200, 2100, 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 base pairs (bp) flanking an HBV TSS, the HBV TSS is an HBV preCore (preC) RNA TSS. For example, in some embodiments provided herein, the target sequence of an epigenetic editor is within 100 bp flanking an HBV preC RNA TSS, e.g., within 100 bp of nucleotide 1791 of SEQ ID NO: 1082 or 1083. In some embodiments provided herein, the target sequence of an epigenetic editor is within 200 bp flanking an HBV preC RNA TSS, e.g., within 200 bp of nucleotide 1791 of SEQ ID NO: 1082 or 1083. In some embodiments provided herein, the target sequence of an epigenetic editor is within 300 bp flanking an HBV preC RNA TSS, e.g., within 300 bp of nucleotide 1791of SEQ ID NO: 1082 or 1083. In some embodiments provided herein, the target sequence of an epigenetic editor is within 400 bp flanking an HBV preC RNA TSS, e.g., within 400 bp of nucleotide 1791 of SEQ ID NO: 1082 or 1083. In some embodiments provided herein, the target sequence of an epigenetic editor is within 500 bp flanking an HBV preC RNA TSS, e.g., within 500 bp of nucleotide 1791 of SEQ ID NO: 1082 or 1083.

[0234] In some embodiments in which the target sequence is within about 3000, 2900, 2800,2700, 2600, 2500, 2400, 2300, 2200, 2100, 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 base pairs (bp) flanking an HBV TSS, the HBV TSS is an HBV preS2 RNA TSS. For example, in some embodiments provided herein, the target sequence of an epigenetic editor is within 100 bp flanking an HBV preS2 RNA TSS, e.g., within 100 bp of nucleotide 3159 of SEQ ID NO: 1082 or 1083. In some embodiments provided herein, the target sequence of an epigenetic editor is within 200 bp flanking an HBV preS2 RNA TSS, e.g., within 200 bp of nucleotide 3159 of SEQ ID NO: 1082 or 1083. In some embodiments provided herein, the target sequence of an epigenetic editor is within 300 bp flanking an HBV preS2 RNA TSS, e.g., within 300 bp of nucleotide 3159 of SEQ ID NO: 1082 or 1083. In some embodiments provided herein, the target sequence of an epigenetic editor is within 400 bp flanking an HBV preS2 RNA TSS, e.g., within 400 bp of nucleotide 3159 of SEQ ID NO: 1082 or 1083. In some embodiments provided herein, the target sequence of an epigenetic editor is within 500 bp flanking an HBV preS2 RNA TSS, e.g., within 500 bp of nucleotide 3159 of SEQ ID NO: 1082 or 1083. -79-WSGR Ref. No. 59073-743.601

[0235] In some embodiments in which the target sequence is within about 3000, 2900, 2800,2700, 2600, 2500, 2400, 2300, 2200, 2100, 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 base pairs (bp) flanking an HBV TSS, the HBV TSS is an HBV HBx RNA TSS. For example, in some embodiments provided herein, the target sequence of an epigenetic editor is within 100 bp flanking an HBV HBx RNA TSS, e.g., within 100 bp of nucleotide 1243 of SEQ ID NO: 1082 or 1083. In some embodiments provided herein, the target sequence of an epigenetic editor is within 200 bp flanking an HBV HBx RNA TSS, e.g., within 200 bp of nucleotide 1243 of SEQ ID NO: 1082 or 1083. In some embodiments provided herein, the target sequence of an epigenetic editor is within 300 bp flanking an HBV HBx RNA TSS, e.g., within 300 bp of nucleotide 1243 of SEQ ID NO: 1082 or 1083. In some embodiments provided herein, the target sequence of an epigenetic editor is within 400 bp flanking an HBV HBx RNA TSS, e.g., within 400 bp of nucleotide 1243 of SEQ ID NO: 1082 or 1083. In some embodiments provided herein, the target sequence of an epigenetic editor is within 500 bp flanking an HBV HBx RNA TSS, e.g., within 500 bp of nucleotide 1243 of SEQ ID NO: 1082 or 1083. In some embodiments provided herein, the target sequence of an epigenetic editor is within 600 bp flanking an HBV HBx RNA TSS, e.g., within 600 bp of nucleotide 1243 of SEQ ID NO: 1082 or 1083.

[0236] In some embodiments in which the target sequence is within about 3000, 2900, 2800,2700, 2600, 2500, 2400, 2300, 2200, 2100, 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 base pairs (bp) flanking an HBV TSS, the HBV TSS is an HBV HBx RNA TSS. For example, in some embodiments provided herein, the target sequence of an epigenetic editor is within 100 bp flanking an HBV HBx RNA TSS, e.g., within 100 bp of nucleotide 1338 of SEQ ID NO: 1082 or 1083. In some embodiments provided herein, the target sequence of an epigenetic editor is within 200 bp flanking an HBV HBx RNA TSS, e.g., within 200 bp of nucleotide 1338 of SEQ ID NO: 1082 or 1083. In some embodiments provided herein, the target sequence of an epigenetic editor is within 300 bp flanking an HBV HBx RNA TSS, e.g., within 300 bp of nucleotide 1338 of SEQ ID NO: 1082 or 1083. In some embodiments provided herein, the target sequence of an epigenetic editor is within 400 bp flanking an HBV HBx RNA TSS, e.g., within 400 bp of nucleotide 1338 of SEQ ID NO: 1082 or 1083. In some embodiments provided herein, the target sequence of an epigenetic editor is within 500 bp flanking an HBV HBx RNA TSS, e.g., within 500 bp of nucleotide 1338 of SEQ ID NO: 1082 or 1083. In some embodiments provided herein, -80-WSGR Ref. No. 59073-743.601 the target sequence of an epigenetic editor is within 600 bp flanking an HBV HBx RNA TSS, e.g., within 600 bp of nucleotide 1338 of SEQ ID NO: 1082 or 1083.

[0237] In some embodiments in which the target sequence is within about 3000, 2900, 2800,2700, 2600, 2500, 2400, 2300, 2200, 2100, 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 base pairs (bp) flanking an HBV TSS, the HBV TSS is an HBV HBx RNA TSS. For example, in some embodiments provided herein, the target sequence of an epigenetic editor is within 100 bp flanking an HBV HBx RNA TSS, e.g., within 100 bp of nucleotide 1243 and within 100 bp of nucleotide 1338 of SEQ ID NO: 1082 or 1083. In some embodiments provided herein, the target sequence of an epigenetic editor is within 200 bp flanking an HBV HBx RNA TSS, e.g., within 200 bp of nucleotide 1243 and within 200 bp of nucleotide 1338 of SEQ ID NO: 1082 or 1083. In some embodiments provided herein, the target sequence of an epigenetic editor is within 300 bp flanking an HBV HBx RNA TSS, e.g., within 300 bp of nucleotide 1243 and within 300 bp of nucleotide 1338 of SEQ ID NO: 1082 or 1083. In some embodiments provided herein, the target sequence of an epigenetic editor is within 400 bp flanking an HBV HBx RNA TSS, e.g., within 400 bp of nucleotide 1243 and within 400 bp of nucleotide 1338 of SEQ ID NO: 1082 or 1083. In some embodiments provided herein, the target sequence of an epigenetic editor is within 500 bp flanking an HBV HBx RNA TSS, e.g., within 500 bp of nucleotide 1243 and within 500 bp of nucleotide 1338 of SEQ ID NO: 1082 or 1083. In some embodiments provided herein, the target sequence of an epigenetic editor is within 600 bp flanking an HBV HBx RNA TSS, e.g., within 600 bp of nucleotide 1243 and within 600 bp of nucleotide 1338 of SEQ ID NO: 1082 or 1083.

[0238] In some embodiments, the target sequence may hybridize to a guide polynucleotidesequence (e.g., gRNA) complexed with an epigenetic editor protein comprising a polynucleotide guided DNA-binding domain (e.g., a CRISPR protein such as dCas9 DNA binding domain) and effector domain(s). The guide polynucleotide sequence may be designed to have complementarity to the target sequence, or identity to the opposing strand of the target sequence. In some embodiments, the guide polynucleotide comprises a spacer sequence that is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a protospacer sequence in the target sequence. In particular embodiments, the guide polynucleotide comprises a spacer sequence that is 100% identical to a protospacer sequence in the target sequence.

[0239] In some embodiments, where the DNA-binding domain of an epigenetic editordescribed herein is a zinc finger array, the target sequence may be recognized by said zinc finger array. -81-WSGR Ref. No. 59073-743.601

[0240] In some embodiments, where the DNA-binding domain of an epigenetic editordescribed herein is a TALE, the target sequence may be recognized by said TALE.

[0241] A target sequence described herein may be specific to one genotype of HBV, to onecopy of am HBV target gene, or may be specific to one allele of an HBV target gene. In some embodiments, however, the target sequence may be conserved across two or more HBV genotypes, across two or more copies of an HBV gene, and across alleles of an HBV gene. Accordingly, the epigenetic modification and modulation of expression thereof may be specific to one copy or one allele of the target gene, or, in other embodiments, may be universal to different HBV genotypes, or HBV gene copies or alleles.

[0242] In some embodiments, the target sequence is comprised in the following sequence:>NC_003977.2 Hepatitis B virus (strain ayw) genome AATTCCACAACCTTCCACCAAACTCTGCAAGATCCCAGAGTGAGAGGCCTGTATTTCCCTGCTGGTGGCTCCAGTTCA GGAACAGTAAACCCTGTTCTGACTACTGCCTCTCCCTTATCGTCAATCTTCTCGAGGATTGGGGACCCTGCGCTGAAC ATGGAGAACATCACATCAGGATTCCTAGGACCCCTTCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTC ACAATACCGCAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAACTACCGTGTGTCTTGGCCAAAAT TCGCAGTCCCCAACCTCCAATCACTCACCAACCTCTTGTCCTCCAACTTGTCCTGGTTATCGCTGGATGTGTCTGCGG CGTTTTATCATCTTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTG CCCGTTTGTCCTCTAATTCCAGGATCCTCAACAACCAGCACGGGACCATGCCGGACCTGCATGACTACTGCTCAAGGA ACCTCTATGTATCCCTCCTGTTGCTGTACCAAACCTTCGGACGGAAATTGCACCTGTATTCCCATCCCATCATCCTGG GCTTTCGGAAAATTCCTATGGGAGTGGGCCTCAGCCCGTTTCTCCTGGCTCAGTTTACTAGTGCCATTTGTTCAGTGG TTCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGTTATATGGATGATGTGGTATTGGGGGCCAAGTCTGTACAGCATC TTGAGTCCCTTTTTACCGCTGTTACCAATTTTCTTTTGTCTTTGGGTATACATTTAAACCCTAACAAAACAAAGAGAT GGGGTTACTCTCTAAATTTTATGGGTTATGTCATTGGATGTTATGGGTCCTTGCCACAAGAACACATCATACAAAAAA TCAAAGAATGTTTTAGAAAACTTCCTATTAACAGGCCTATTGATTGGAAAGTATGTCAACGAATTGTGGGTCTTTTGG GTTTTGCTGCCCCTTTTACACAATGTGGTTATCCTGCGTTGATGCCTTTGTATGCATGTATTCAATCTAAGCAGGCTT TCACTTTCTCGCCAACTTACAAGGCCTTTCTGTGTAAACAATACCTGAACCTTTACCCCGTTGCCCGGCAACGGCCAG GTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCTTGGTCATGGGCCATCAGCGCATGCGTGGAACCT TTTCGGCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGCTCGCAGCAGGTCTGGAGCAAACATTA TCGGGACTGATAACTCTGTTGTCCTATCCCGCAAATATACATCGTTTCCATGGCTGCTAGGCTGTGCTGCCAACTGGA TCCTGCGCGGGACGTCCTTTGTTTACGTCCCGTCGGCGCTGAATCCTGCGGACGACCCTTCTCGGGGTCGCTTGGGAC TCTCTCGTCCCCTTCTCCGTCTGCCGTTCCGACCGACCACGGGGCGCACCTCTCTTTACGCGGACTCCCCGTCTGTGC CTTCTCATCTGCCGGACCGTGTGCACTTCGCTTCACCTCTGCACGTCGCATGGAGACCACCGTGAACGCCCACCAAAT ATTGCCCAAGGTCTTACATAAGAGGACTCTTGGACTCTCAGCAATGTCAACGACCGACCTTGAGGCATACTTCAAAGA CTGTTTGTTTAAAGACTGGGAGGAGTTGGGGGAGGAGATTAGGTTAAAGGTCTTTGTACTAGGAGGCTGTAGGCATAA ATTGGTCTGCGCACCAGCACCATGCAACTTTTTCACCTCTGCCTAATCATCTCTTGTTCATGTCCTACTGTTCAAGCC TCCAAGCTGTGCCTTGGGTGGCTTTGGGGCATGGACATCGACCCTTATAAAGAATTTGGAGCTACTGTGGAGTTACTC TCGTTTTTGCCTTCTGACTTCTTTCCTTCAGTACGAGATCTTCTAGATACCGCCTCAGCTCTGTATCGGGAAGCCTTA GAGTCTCCTGAGCATTGTTCACCTCACCATACTGCACTCAGGCAAGCAATTCTTTGCTGGGGGGAACTAATGACTCTA -82-WSGR Ref. No. 59073-743.601 GCTACCTGGGTGGGTGTTAATTTGGAAGATCCAGCGTCTAGAGACCTAGTAGTCAGTTATGTCAACACTAATATGGGC CTAAAGTTCAGGCAACTCTTGTGGTTTCACATTTCTTGTCTCACTTTTGGAAGAGAAACAGTTATAGAGTATTTGGTG TCTTTCGGAGTGTGGATTCGCACTCCTCCAGCTTATAGACCACCAAATGCCCCTATCCTATCAACACTTCCGGAGACT ACTGTTGTTAGACGACGAGGCAGGTCCCCTAGAAGAAGAACTCCCTCGCCTCGCAGACGAAGGTCTCAATCGCCGCGT CGCAGAAGATCTCAATCTCGGGAATCTCAATGTTAGTATTCCTTGGACTCATAAGGTGGGGAACTTTACTGGGCTTTA TTCTTCTACTGTACCTGTCTTTAATCCTCATTGGAAAACACCATCTTTTCCTAATATACATTTACACCAAGACATTAT CAAAAAATGTGAACAGTTTGTAGGCCCACTCACAGTTAATGAGAAAAGAAGATTGCAATTGATTATGCCTGCCAGGTT TTATCCAAAGGTTACCAAATATTTACCATTGGATAAGGGTATTAAACCTTATTATCCAGAACATCTAGTTAATCATTA CTTCCAAACTAGACACTATTTACACACTCTATGGAAGGCGGGTATATTATATAAGAGAGAAACAACACATAGCGCCTC ATTTTGTGGGTCACCATATTCTTGGGAACAAGATCTACAGCATGGGGCAGAATCTTTCCACCAGCAATCCTCTGGGAT TCTTTCCCGACCACCAGTTGGATCCAGCCTTCAGAGCAAACACCGCAAATCCAGATTGGGACTTCAATCCCAACAAGG ACACCTGGCCAGACGCCAACAAGGTAGGAGCTGGAGCATTCGGGCTGGGTTTCACCCCACCGCACGGAGGCCTTTTGG GGTGGAGCCCTCAGGCTCAGGGCATACTACAAACTTTGCCAGCAAATCCGCCTCCTGCCTCCACCAATCGCCAGTCAG GAAGGCAGCCTACCCCGCTGTCTCCACCTTTGAGAAACACTCATCCTCAGGCCATGCAGTGG (SEQ ID No. 1082). Figure 1 provides a diagram illustrating the structure of a circular HBV genome comprising SEQ ID NO: 1082. The coding regions of the HBV genes and CpG islands CGI-I-III are identified. Nucleotides 2309-1625 of SEQ ID NO: 1082 encode the P protein (NCBI reference number YP_009173866.1). Nucleotides 2850-837 of SEQ ID NO: 1082 encode the long surface protein (L-HBsAG or LHBS; NCBI reference number YP_009173869.1). Nucleotides 3174-837 of SEQ ID NO: 1082 encode the middle surface protein (M-HBsAg or MHBS; NCBI reference number YP_009173870.1). Nucleotides 157-837 of SEQ ID NO: 1082 encode the small surface protein (S-HBsAg or SHBs; NCBI reference number YP_009173871.1). Nucleotides 1816-2454 of SEQ ID NO: 1082 encode the C Protein (core protein, NCBI reference number AAB59971.1). Nucleotides 1376-1840 of SEQ ID NO: 1082 encode the X protein (HBx, NCBI reference number YP_009173867.1). CGI I spans nucleotides 186-288, CGI II spans nucleotides 1,217-1,670, and CGI III spans nucleotides 2,282-2,448. See, NCBI database entry NC_003977.2. TSSs of various transcripts have been mapped: pg RNA TSS: 1820; pre-C RNA TSS: 1791; pre-S2 RNA TSS: 3159; HBx RNA TSSs: 1243-1338. The ATAAA motif of the transcription termination / polyadenylation site is located at nucleotide 1919. See references cited elsewhere herein. See also, e.g., Abraham,T.M. and Loeb,D.D., The topology of hepatitis B virus pregenomic RNA promotes its replication, J. Virol. 81 (21), 11577- 11584 (2007); Chen,A., Kao,Y.F. and Brown,C.M., Translation of the first upstream ORF in the hepatitis B virus pregenomic RNA modulates translation at the core and polymerase initiation codons, Nucleic Acids Res. 33 (4), 1169-1181 (2005); Borisova,G.P., Pumpen,P.P., Bychko,V.V., Pushko,P.M., Kalis,Y.V., Dishler,A.V., Gren,E.Y., Tsibinogin,V.V. and -83-WSGR Ref. No. 59073-743.601 Kukain,R.A., Structure and expression of the gene of the core antigen of human hepatitis B virus (HBV) in Escherichia coli cells, Dokl. Biochem. 279, 386-390 (1985); Galibert,F., Mandart,E., Fitoussi,F., Tiollais,P. and Charnay,P., Nucleotide sequence of the hepatitis B virus genome (subtype ayw) cloned in E. coli, Nature 281 (5733), 646-650 (1979), the entire contents of each of which are incorporated herein by reference.

[0243] In some embodiments, the target sequence is comprised in the following sequence:>U95551.1 Hepatitis B virus subtype ayw, complete genome AATTCCACAACCTTTCACCAAACTCTGCAAGATCCCAGAGTGAGAGGCCTGTATTTCCCTGCTGGTGGCTCCAGTTCA GGAGCAGTAAACCCTGTTCCGACTACTGCCTCTCCCTTATCGTCAATCTTCTCGAGGATTGGGGACCCTGCGCTGAAC ATGGAGAACATCACATCAGGATTCCTAGGACCCCTTCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTC ACAATACCGCAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGAACTACCGTGTGTCTTGGCCAAAAT TCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTCCTCCAACTTGTCCTGGTTATCGCTGGATGTGTCTGCGG CGTTTTATCATCTTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTG CCCGTTTGTCCTCTAATTCCAGGATCCTCAACCACCAGCACGGGACCATGCCGAACCTGCATGACTACTGCTCAAGGA ACCTCTATGTATCCCTCCTGTTGCTGTACCAAACCTTCGGACGGAAATTGCACCTGTATTCCCATCCCATCATCCTGG GCTTTCGGAAAATTCCTATGGGAGTGGGCCTCAGCCCGTTTCTCCTGGCTCAGTTTACTAGTGCCATTTGTTCAGTGG TTCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGTTATATGGATGATGTGGTATTGGGGGCCAAGTCTGTACAGCATC TTGAGTCCCTTTTTACCGCTGTTACCAATTTTCTTTTGTCTTTGGGTATACATTTAAACCCTAACAAAACAAAGAGAT GGGGTTACTCTCTGAATTTTATGGGTTATGTCATTGGAAGTTATGGGTCCTTGCCACAAGAACACATCATACAAAAAA TCAAAGAATGTTTTAGAAAACTTCCTATTAACAGGCCTATTGATTGGAAAGTATGTCAACGAATTGTGGGTCTTTTGG GTTTTGCTGCCCCATTTACACAATGTGGTTATCCTGCGTTAATGCCCTTGTATGCATGTATTCAATCTAAGCAGGCTT TCACTTTCTCGCCAACTTACAAGGCCTTTCTGTGTAAACAATACCTGAACCTTTACCCCGTTGCCCGGCAACGGCCAG GTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCTTGGTCATGGGCCATCAGCGCGTGCGTGGAACCT TTTCGGCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGCTCGCAGCAGGTCTGGAGCAAACATTA TCGGGACTGATAACTCTGTTGTCCTCTCCCGCAAATATACATCGTATCCATGGCTGCTAGGCTGTGCTGCCAACTGGA TCCTGCGCGGGACGTCCTTTGTTTACGTCCCGTCGGCGCTGAATCCTGCGGACGACCCTTCTCGGGGTCGCTTGGGAC TCTCTCGTCCCCTTCTCCGTCTGCCGTTCCGACCGACCACGGGGCGCACCTCTCTTTACGCGGACTCCCCGTCTGTGC CTTCTCATCTGCCGGACCGTGTGCACTTCGCTTCACCTCTGCACGTCGCATGGAGACCACCGTGAACGCCCACCGAAT GTTGCCCAAGGTCTTACATAAGAGGACTCTTGGACTCTCTGCAATGTCAACGACCGACCTTGAGGCATACTTCAAAGA CTGTTTGTTTAAAGACTGGGAGGAGTTGGGGGAGGAGATTAGATTAAAGGTCTTTGTACTAGGAGGCTGTAGGCATAA ATTGGTCTGCGCACCAGCACCATGCAACTTTTTCACCTCTGCCTAATCATCTCTTGTTCATGTCCTACTGTTCAAGCC TCCAAGCTGTGCCTTGGGTGGCTTTGGGGCATGGACATCGACCCTTATAAAGAATTTGGAGCTACTGTGGAGTTACTC TCGTTTTTGCCTTCTGACTTCTTTCCTTCAGTACGAGATCTTCTAGATACCGCCTCAGCTCTGTATCGGGAAGCCTTA GAGTCTCCTGAGCATTGTTCACCTCACCATACTGCACTCAGGCAAGCAATTCTTTGCTGGGGGGAACTAATGACTCTA GCTACCTGGGTGGGTGTTAATTTGGAAGATCCAGCATCTAGAGACCTAGTAGTCAGTTATGTCAACACTAATATGGGC CTAAAGTTCAGGCAACTCTTGTGGTTTCACATTTCTTGTCTCACTTTTGGAAGAGAAACCGTTATAGAGTATTTGGTG TCTTTCGGAGTGTGGATTCGCACTCCTCCAGCTTATAGACCACCAAATGCCCCTATCCTATCAACACTTCCGGAAACT ACTGTTGTTAGACGACGAGGCAGGTCCCCTAGAAGAAGAACTCCCTCGCCTCGCAGACGAAGGTCTCAATCGCCGCGT CGCAGAAGATCTCAATCTCGGGAACCTCAATGTTAGTATTCCTTGGACTCATAAGGTGGGGAACTTTACTGGTCTTTA TTCTTCTACTGTACCTGTCTTTAATCCTCATTGGAAAACACCATCTTTTCCTAATATACATTTACACCAAGACATTAT CAAAAAATGTGAACAGTTTGTAGGCCCACTTACAGTTAATGAGAAAAGAAGATTGCAATTGATTATGCCTGCTAGGTT TTATCCAAAGGTTACCAAATATTTACCATTGGATAAGGGTATTAAACCTTATTATCCAGAACATCTAGTTAATCATTA CTTCCAAACTAGACACTATTTACACACTCTATGGAAGGCGGGTATATTATATAAGAGAGAAACAACACATAGCGCCTC ATTTTGTGGGTCACCATATTCTTGGGAACAAGATCTACAGCATGGGGCAGAATCTTTCCACCAGCAATCCTCTGGGAT TCTTTCCCGACCACCAGTTGGATCCAGCCTTCAGAGCAAACACAGCAAATCCAGATTGGGACTTCAATCCCAACAAGG ACACCTGGCCAGACGCCAACAAGGTAGGAGCTGGAGCATTCGGGCTGGGTTTCACCCCACCGCACGGAGGCCTTTTGG GGTGGAGCCCTCAGGCTCAGGGCATACTACAAACTTTGCCAGCAAATCCGCCTCCTGCCTCCACCAATCGCCAGACAG GAAGGCAGCCTACCCCGCTGTCTCCACCTTTGAGAAACACTCATCCTCAGGCCATGCAGTGG (SEQ ID No. 1083).

[0244] Annotation of SEQ ID NO: 1083: P protein CDS: 2309-1625; L-HBsAG CDS: 2850-837; M-HBsAg CDS: 3174-837; S-HBsAg CDS: 157-837; C Protein CDS: 1816-2454; X protein CDS: 1376-1840; CGI I: 186-288; CGI II: 1,217-1,670; CGI III: 2,282-2,448; pg RNA TSS: -84-WSGR Ref. No. 59073-743.601 1820; pre-C RNA TSS: 1791; pre-S2 RNA TSS: 3159; HBx RNA TSSs: 1243-1338; termination / polyA site: 1919. See references cited elsewhere herein. VI. Epigenetic Modifications

[0245] An epigenetic editor described herein may perform sequence-specific epigeneticmodification(s) (e.g., alteration of chemical modification(s)) of a target gene that harbors the target sequence. Such epigenetic modulation may be safer and more easily reversible than modulation due to gene editing, e.g., with generation of DNA double-strand breaks. In some embodiments, the epigenetic modulation may reduce or silence the target gene. In some embodiments, the modification is at a specific site of the target sequence. In some embodiments, the modification is at a specific allele of the target gene. Accordingly, the epigenetic modification may result in modulated (e.g., reduced) expression of one copy of a target gene harboring a specific allele, and not the other copy of the target gene. In some embodiments, the specific allele is associated with a disease, condition, or disorder.

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

[0247] The effector domain of an epigenetic editor described herein may alter (e.g., depositor remove) a chemical modification at a nucleotide of the target gene or at a histone associated with the target gene. The chemical modification may be altered at a single nucleotide or a single histone, or may 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, an effector domain of an epigenetic editor described hereinmay alter a CpG dinucleotide within the target gene. In some embodiments, all CpG dinucleotides within 2000, 1500, 1000, 500, or 200 bps flanking a target sequence (e.g., in an alteration site as described herein) are altered according to a modification type described herein, as compared to the original state of the gene or the gene in a comparable cell not contacted with -85-WSGR Ref. No. 59073-743.601 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 of the CpG dinucleotides are altered as 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 as compared to the original state of the gene or the gene in a comparable cell not contacted with the epigenetic editor. In some embodiments, one single CpG dinucleotide is altered, as compared to the original state of the gene or the gene in a comparable cell not contacted with the epigenetic editor.

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

[0250] In some embodiments, all histone tails of histones bound to DNA nucleotides within2000, 1500, 1000, 500, or 200 bps flanking the target sequence are altered according to a modification type as described herein, as compared to the original state of the chromosome or the chromosome 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, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 or more histone tails of the bound histones are altered as compared to the original state of the chromosome or the chromosome 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 histone tails of the bound histones are altered as compared to the original state of the chromosome or the chromosome in a comparable cell not contacted with the epigenetic editor. For example, one single histone tail of the bound histones may be altered as compared to the original state of the -86-WSGR Ref. No. 59073-743.601 chromosome or the chromosome in a comparable cell not contacted with the epigenetic editor. As another example, one single bound histone octamer may be altered as compared to the original state of the chromosome or the chromosome in a comparable cell not contacted with the epigenetic editor.

[0251] The chemical modification deposited at target gene DNA nucleotides or histoneresidues may be at or in close proximity to a target sequence in the target gene. In some embodiments, an effector domain of an epigenetic editor described herein alters a chemical modification state of a nucleotide or histone tail bound to a nucleotide 100-200, 200-300, 300- 400, 400-55, 500-600, 600-700, or 700-800 nucleotides 5’ or 3’ to the target sequence in the target gene. In some embodiments, an effector domain alters a chemical modification state of a nucleotide or histone tail bound to a nucleotide 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 nucleotide positions 5’ to the 5’ end of and 3’ to the 3’ end of a particular sequence, e.g. a target sequence.

[0252] In some embodiments, an effector domain mediates or induces a chemicalmodification change of a nucleotide or a histone tail bound to a nucleotide distant from a target sequence. Such modification may be initiated near the target sequence, and may subsequently spread to one or more nucleotides in the target gene distant from the target sequence. For example, an effector domain may initiate alteration of a chemical modification state of one or more nucleotides or one or more histone residues bound 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 the chemical modification state alteration may spread to one or more nucleotides 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 from the target sequence in the target gene, either upstream or downstream of the target sequence. In certain embodiments, the chemical modification may be initiated 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 nucleotides in the entire target gene. Additional proteins or transcription factors, for example, transcription repressors, methyltransferases, or transcription regulation scaffold proteins, may be involved in the spreading of the chemical modification. Alternatively, the epigenetic editor alone may be involved. -87-WSGR Ref. No. 59073-743.601

[0253] In some embodiments, an epigenetic editor described herein reduces expression of atarget 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, a tissue, or a subject as compared to a control cell, control tissue, or a control subject (e.g., in the absence of the epigenetic editor). In some embodiments, the epigenetic editors described herein reduces expression of a copy of 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%, at least about 99.9%, or more, as measured by transcription of the copy of the target gene in a cell, a tissue, or a subject as compared to a control cell, control tissue, or a control subject. For example, in some embodiments, an epigenetic editor described herein reduces expression of an HBV target gene in vitro or in vivo (e.g., as measured as the level of an HBV biomarker in a subject), by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, at least 99.9%, or more, as measured for example, by transcription of the target gene, or by assessing an HBV biomarker (e.g., plasma HBV DNA, plasma HBVsAg, or plasma HBVeAg) in a cell, a tissue, or a subject contacted or administered with the epigenetic editor as compared to a control cell, control tissue, or a control subject (e.g., in the absence of the epigenetic editor). In certain embodiments, the copy of the target gene harbors a specific sequence or allele recognized by the epigenetic editor. In particular embodiments, the epigenetically modified copy encodes a functional protein, and accordingly an epigenetic editor disclosed herein may reduce or abolish expression and / or function of the protein. For example, an epigenetic editor described herein may reduce expression and / or function of a protein encoded by the target gene by at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100 fold in a cell, a tissue, or a subject as compared to a control cell, control tissue, or a control subject.

[0254] Modulation of target gene expression can be assayed by determining any parameterthat is indirectly or directly affected by the expression of the target gene. Such parameters include, e.g., changes in RNA or protein levels; changes in protein activity; changes in product levels; changes in downstream gene expression; changes in transcription or activity of reporter genes such as, for example, luciferase, CAT, beta-galactosidase, or GFP; changes in signal -88-WSGR Ref. No. 59073-743.601 transduction; changes in phosphorylation and dephosphorylation; changes in receptor-ligand interactions; changes in concentrations of second messengers such as, for example, cGMP, cAMP, IP3, and Ca2+; changes in cell growth; changes in neovascularization; and / or changes in any functional effect of gene expression. Measurements can be made in vitro, in vivo, and / or ex vivo, and can be made by conventional methods, e.g., measurement of RNA or protein levels, measurement of RNA stability, and / or identification of downstream or reporter gene expression. Readout can be by way of, for example, chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, ligand binding assays, changes in intracellular second messengers such as cGMP and inositol triphosphate (IP3), changes in intracellular calcium levels; cytokine release, and the like.

[0255] Methods for determining the expression level of a gene, for example the target of anepigenetic editor, may include, e.g., determining the transcript level of a gene by reverse transcription PCR, quantitative RT-PCR, droplet digital PCR (ddPCR), Northern blot, RNA sequencing, DNA sequencing (e.g., sequencing of complementary deoxyribonucleic acid (cDNA) obtained from RNA); next generation (Next-Gen) sequencing, nanopore sequencing, pyrosequencing, or Nanostring sequencing. Levels of protein expressed from a gene may be determined, e.g., by Western blotting, enzyme linked immuno-absorbance assays, mass- spectrometry, immunohistochemistry, or flow cytometry analysis. Gene expression product levels may be normalized to an internal standard such as total messenger ribonucleic acid (mRNA) or the expression level of a particular gene, e.g., a housekeeping gene.

[0256] In some embodiments, the effect of an epigenetic editor in modulating target geneexpression may be examined using a reporter system. For example, an epigenetic editor may be designed to target a reporter gene encoding a reporter protein, such as a fluorescent protein. Expression of the reporter gene in such a model system may be monitored by, e.g., flow cytometry, fluorescence-activated cell sorting (FACS), or fluorescence microscopy. In some embodiments, a population of cells may be transfected with a vector that harbors a reporter gene. The vector may be constructed such that the reporter gene is expressed when the vector transfects a cell. Suitable reporter genes include genes encoding fluorescent proteins, for example green, yellow, cherry, cyan or orange fluorescent proteins. The population of cells carrying the reporter system may be transfected with DNA, mRNA, or vectors encoding the epigenetic editor targeting the reporter gene. VII. Pharmaceutical Compositions -89-WSGR Ref. No. 59073-743.601

[0257] Another aspect of the present disclosure is a pharmaceutical composition comprisingas an active ingredient (or as the sole active ingredient) one or more epigenetic editors described herein or component(s) (e.g., epigenetic editor proteins and / or guide polynucleotides) thereof, or nucleic acid molecule(s) encoding said epigenetic editors or component(s) thereof. For example, a pharmaceutical composition may comprise nucleic acid molecule(s) encoding the epigenetic editor protein(s) (and guide polynucleotides, where applicable) of an epigenetic editor described herein. In some embodiments, separate pharmaceutical compositions comprise the epigenetic editor protein(s) and the guide polynucleotide(s). In some embodiments, multiple pharmaceutical compositions, each comprising one epigenetic editor, are administered simultaneously. A pharmaceutical composition may also comprise cells that have undergone epigenetic modification(s) mediated or induced by an epigenetic editor provided herein.

[0258] Generally, the epigenetic editors described herein or component(s) thereof, or nucleicacid molecule(s) encoding said epigenetic editors or component(s) thereof, of the present disclosure are suitable to be administered as a formulation in association with one or more pharmaceutically acceptable excipient(s), e.g., as described below.

[0259] The term “excipient” is used herein to describe any ingredient other than thecompound(s) of the present disclosure. The choice of excipient(s) will to a large extent depend on 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 solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Additional examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives, or buffers, which enhance the shelf life or effectiveness of the antibody.

[0260] Formulations of a pharmaceutical composition suitable for parenteral administrationtypically comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. In some embodiments, the epigenetic editor or its component(s) are introduced to target cells in the form -90-WSGR Ref. No. 59073-743.601 of nucleic acid molecule(s) encoding the epigenetic editor or its component(s); accordingly, the pharmaceutical compositions herein comprise the nucleic acid molecule(s). Such nucleic acid molecule(s) may be, for example, DNA, RNA or mRNA, and / or modified nucleic acid sequence(s) (e.g., with chemical modifications, a 5’ cap, or one or more 3’ modifications). In some embodiments, the nucleic acid molecule(s) may be delivered as naked DNA or RNA, for instance by means of transfection or electroporation, or can be conjugated to molecules (e.g., N- acetylgalactosamine) promoting uptake by target cells. In some embodiments, the nucleic acid molecule(s) may be in nucleic acid expression vector(s), which may include expression control sequences such as promoters, enhancers, transcription signal sequences, transcription termination sequences, introns, polyadenylation signals, Kozak consensus sequences, internal ribosome entry sites (IRES), etc. Such expression control sequences are well known in the art. A vector may also comprise a sequence encoding a signal peptide (e.g., for nuclear localization, nucleolar localization, or mitochondrial localization), associated with (e.g., inserted into or fused to) a sequence coding for a protein.

[0261] Examples of vectors include, but are not limited to, plasmid vectors; viral vectorsbased on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, retrovirus (e.g., Murine Leukemia Virus, or spleen necrosis virus, vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and other recombinant vectors. In certain embodiments, the vector is a plasmid or a viral vector. Viral particles may also be used to deliver nucleic acid molecule(s) encoding epigenetic editors or component(s) thereof as described herein. For example, “empty” viral particles can be assembled to contain any suitable cargo. Viral vectors and viral particles may also be engineered to incorporate targeting ligands to alter target tissue specificity.

[0262] In certain embodiments, an epigenetic editor as described herein or component(s)thereof are encoded by nucleic acid sequence(s) 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 herpesvirus vectors (e.g., HSV-2). -91-WSGR Ref. No. 59073-743.601

[0263] In some embodiments, delivery involves an adeno-associated virus (AAV) vector.AAV vector delivery may be particularly useful where the DNA-binding domain of an epigenetic editor protein is a zinc finger array. Without wishing to be bound by any theory, the smaller size of zinc finger arrays compared to larger DNA-binding domains such as Cas protein domains may allow such an epigenetic editor protein to be conveniently packed in viral vectors such as an AAV vector.

[0264] Any AAV serotype, e.g., human AAV serotype, can be used for an AAV vector asdescribed 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), as well as variants thereof. In some embodiments, an AAV variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to a wildtype AAV. In certain embodiments, the AAV variant may be engineered such that its capsid proteins have reduced immunogenicity or enhanced transduction ability in humans. In some instances, one or more regions of at least two different AAV serotype viruses are shuffled and reassembled to generate a chimeric variant. For example, a chimeric AAV may comprise inverted terminal repeats (ITRs) that are of a heterologous serotype compared to the serotype of the capsid. The resulting chimeric AAV can have a different antigenic reactivity or recognition compared to its parental serotypes. In some embodiments, a chimeric variant of an AAV includes amino acid sequences from 2, 3, 4, 5, or more different AAV serotypes.

[0265] Non-viral systems are also contemplated for delivery as described herein. Non-viralsystems include, but are not limited to, nucleic acid transfection methods including electroporation, sonoporation, calcium phosphate transfection, microinjection, DNA biolistics, lipid-mediated transfection, transfection through heat shock, compacted DNA-mediated transfection, lipofection, cationic agent-mediated transfection, and transfection with liposomes, immunoliposomes, or cationic facial amphiphiles (CFAs). In certain embodiments, one or more mRNAs encoding epigenetic editor proteins as described herein may be co-electroporated with one or more guide polynucleotides (e.g., gRNAs) as described herein. One important category of non-viral nucleic acid vectors is nanoparticles, which can be organic (e.g., lipid) or inorganic (e.g., gold). For instance, organic (e.g. lipid and / or polymer) nanoparticles can be suitable for use as delivery vehicles in certain embodiments of this disclosure. -92-WSGR Ref. No. 59073-743.601

[0266] In some embodiments, delivery is accomplished using a lipid nanoparticle (LNP). Insome embodiments, an RNA encoding an epigenetic editor protein disclosed herein and a guide RNA disclosed herein are encapsulated in an LNP for delivery. In some embodiments, the ratio of the RNA encoding an epigenetic editor protein disclosed herein and a guide RNA disclosed herein encapsulated in an LNP is from about 1:1 to about 1:3 by weight. In some embodiments, the ratio of the RNA encoding an epigenetic editor protein disclosed herein and a guide RNA disclosed herein encapsulated in an LNP is from about 1:2 to about 1:4 by weight. In some embodiments, the ratio of the RNA encoding an epigenetic editor protein disclosed herein and a guide RNA disclosed herein encapsulated in an LNP is from about 1:3 to about 1:5 by weight. In some embodiments, the ratio of the RNA encoding an epigenetic editor protein disclosed herein and a guide RNA disclosed herein encapsulated in an LNP is from about 1:1 to about 3:1 by weight. In some embodiments, the ratio of the RNA encoding an epigenetic editor protein disclosed herein and a guide RNA disclosed herein encapsulated in an LNP is from about 2:1 to about 4:1 by weight. In some embodiments, the ratio of the RNA encoding an epigenetic editor protein disclosed herein and a guide RNA disclosed herein encapsulated in an LNP is from about 3:1 to about 5:1 by weight. In some embodiments, the ratio of the RNA encoding an epigenetic editor protein disclosed herein and a guide RNA disclosed herein encapsulated in an LNP is from about 1:1.5 by weight. In some embodiments, the RNA and the guide RNA are encapsulated in the lipid nanoparticle at a ratio of about 1:1 by weight. In some embodiments, the RNA and the guide RNA are encapsulated in the lipid nanoparticle at a ratio of about 1:3 by weight. LNP compositions are typically sized on the order of micrometers or smaller and may include a lipid bilayer. In some embodiments, a LNP refers to any particle that has a diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm. Nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes.

[0267] An LNP as described herein may be made from cationic, anionic, or neutral lipids. Insome embodiments, an LNP may comprise neutral lipids, such as the fusogenic phospholipid 1,2- Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or the membrane component cholesterol, as helper lipids to enhance transfection activity and nanoparticle stability. In some embodiments, an LNP may comprise hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids. Any lipid or combination of lipids that are known in the art can be used to produce an LNP. The lipids may be combined in any molar ratios to produce the LNP. In some -93-WSGR Ref. No. 59073-743.601 embodiments, the LNP is a liver-targeting (e.g., preferentially or specifically targeting the liver) LNP.

[0268] LNP formulations and methods of LNP delivery that can be used will be apparent tothose skilled in the art based on the present disclosure and the state of the art. 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-13978-3319107103; Ziegler, S., Lipid Nanoparticles: Advances in Research and Applications, Nova Science Pub., Inc, ISBN-13978-1536186536; 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 202219 (6), 1669-1686; Cullis PR, Hope MJ. Lipid Nanoparticle Systems for Enabling Gene Therapies, Mol Ther. 2017 Jul 5;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 primates, 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 2021121 (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 -94-WSGR Ref. No. 59073-743.601 (2021); US Patent No. 9,364,435; US Patent No. 8,058,069; US Patent No. 8,822,668; US Patent No. 8,492,359; US Patent No. 11,141,378; US Patent No. 9,518,272; US Patent No. 9,404,127; US Patent No. 9,006,417; US Patent No. 7,901,708; US Patent No. 9,005,654; US Patent No. 9,878,042; US Patent No. 9,682,139; US Patent No. 8,642,076; US Patent No. 9,593,077; US Patent No. 9,415,109; US Patent No. 9,701,623; US Patent No. 10,369,226; US Patent No. 9,999,673; US Patent No. 9,301,923; US Patent No. 10,342,761; US Patent No. 10,137,201; International Patent Application PCT / US2014 / 070882; International Publication No. WO2015199952A1; International Publication No. WO2017075531A1; International Publication No. WO2018081480A1; International Publication No. WO2016081029A1; European Application No. EP3852911A2; each of which are incorporated herein by reference in their entirety. The ordinarily skilled artisan will be able to identify an appropriate LNP and method of delivery based on the present disclosure and the state of the art. The present disclosure is not limited in this respect.

[0269] Other methods of delivery to target cells will be known to those skilled in the art andcan be used with the compositions of the present disclosure.

[0270] Any type of cell may be targeted for delivery of an epigenetic editor or component(s)thereof as described herein. For example, the cells may be eukaryotic or prokaryotic. In some embodiments, the cells are mammalian (e.g., human) cells. Human cells may include, for example, hepatocytes, biliary epithelial cells (cholangiocytes), stellate cells, Kupffer cells, and liver sinusoidal endothelial cells.

[0271] In some embodiments, an epigenetic editor described herein, or component(s) thereof,are delivered to a host cell for transient expression, e.g., via a transient expression vector. Transient expression of the epigenetic editor or its component(s) may result in prolonged or permanent epigenetic modification of the target gene. For example, the epigenetic modification may be stable for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. 11, or 12 weeks or more; or 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more, after introduction of the epigenetic editor into the host cell. The epigenetic modification may be maintained after one or more mitotic and / or meiotic events of the host cell. In particular embodiments, the epigenetic modification is maintained across generations in offspring generated or derived from the host cell. VIII. Therapeutic Uses of Epigenetic Editors

[0272] The present disclosure also provides methods for treating or preventing a condition ina subject, comprising administering to the subject an epigenetic editor or pharmaceutical -95-WSGR Ref. No. 59073-743.601 composition as described herein. The epigenetic editor may effectuate an epigenetic modification of a target polynucleotide sequence in a target gene associated with a disease, condition, or disorder in the subject, thereby modulating expression of the target gene to treat or prevent the disease, condition, or disorder. In some embodiments, the epigenetic editor reduces the expression of the target gene to an extent sufficient to achieve a desired effect, e.g., a therapeutically relevant effect such as the prevention or treatment of the disease, condition, or disorder.

[0273] In some embodiments, a subject is administered a system for modulating (e.g.,repressing) expression of HBV or of an HBV gene, wherein the system comprises (1) the epigenetic editor protein(s) and, where relevant, guide polynucleotide(s) of an epigenetic editor as described herein, or (2) nucleic acid molecules encoding said epigenetic editor protein(s) and, where relevant, guide polynucleotide(s).

[0274] “Treat,” “treating” and “treatment” refer to a method of alleviating or abrogating abiological disorder and / or at least one of its attendant symptoms. As used herein, to “alleviate” a disease, disorder or condition means reducing the severity and / or occurrence frequency of the symptoms of the disease, disorder, or condition. Further, references herein to “treatment” include references to curative, palliative and prophylactic treatment. In some embodiments, as compared with an equivalent untreated control, alleviating a symptom may involve reduction of the symptom by at least 3%, 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.9%, or 100% as measured by any standard technique.

[0275] In some embodiments, the subject may be a mammal, e.g., a human. In someembodiments, the subject is selected from a non-human primate such as chimpanzee, cynomolgus monkey, or macaque, and other apes and monkey species.

[0276] Some aspects of this disclosure provide methods comprising administering anepigenetic editing system to a subject characterized by the presence of detectable levels of HBV DNA, HBsAg, and / or HBeAg in the plasma of the subject, for example, a subject having a chronic HBV infection. In some such embodiments, 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 same, wherein the first DNA binding domain binds a first target region of an HBV gene or genome, and the administering results in a reduction of the level of HBV DNA, the level of HBsAg, and / or the level of HBsAg in the plasma of the subject, and the reduction of the level of HBV DNA, of the level of HBsAg, and / or of the level of HBsAg in the plasma of the subject, is at least 90% (a 1-log reduction) compared to the -96-WSGR Ref. No. 59073-743.601 respective level observed or observable in the plasma of the subject prior to the administering, and the 1-log reduction is maintained for at least 14 days after the administering. In some embodiments, the reduction of the level of HBV DNA in the plasma of the subject is at least 90% (a 1-log reduction). In some embodiments, the reduction of the level of HBV DNA in the plasma of the subject is at least 99% (a 2-log reduction). In some embodiments, the reduction of the level of HBsAg in the plasma of the subject is at least 90% (a 1-log reduction). In some embodiments, the reduction of the level of HBsAg in the plasma of the subject is at least 99% (a 2-log reduction). In some embodiments, the reduction of the level of HBeAg in the plasma of the subject is at least 90% (a 1-log reduction). In some embodiments, the reduction of the level of HBeAg in the plasma of the subject is at least 99% (a 2-log reduction). In some embodiments, the reduction is maintained for at least 21 days. In some embodiments, the reduction is maintained for at least 28 days. In some embodiments, the reduction is maintained for at least 35 days. In some embodiments, the reduction is maintained for at least 42 days. In some embodiments, the reduction is maintained for at least 56 days. In some embodiments, the reduction is maintained for at least 70 days. In some embodiments, the reduction is maintained for at least 84 days. In some embodiments, the reduction is maintained for at least 112 days. In some embodiments, the reduction is maintained for at least 140 days. In some embodiments, the reduction is maintained for at least 168 days. In some embodiments, the reduction is maintained for at least 6 months. In some embodiments, the reduction is maintained for at least 9 months. In some embodiments, the reduction is maintained for at least 12 months. In some embodiments, the reduction is maintained for at least 24 months. In some embodiments, the HBV genome comprises HBV genotype A. In some embodiments, the HBV genome comprises HBV genotype B. In some embodiments, the HBV genome comprises HBV genotype C. In some embodiments, the HBV genome comprises, HBV genotype D. In some embodiments, the HBV genome comprises HBV genotype E. In some embodiments, the HBV genome comprises HBV genotype F. In some embodiments, the HBV genome comprises HBV genotype G. In some embodiments, the HBV genome comprises HBV genotype H. In some embodiments, the HBV genome comprises a sequence with at least 80%, at least 90%, at least 95%, at least 99%, or greater than 99% sequence identity to an HBV genome sequence provided herein. In some embodiments, the first target region is located in a region of the HBV genome within nucleotides 0-303 of an HBV genome provided herein. In some embodiments, the first target region is located within nucleotides 0-303 of SEQ ID NO: 1082. In some embodiments, the first target region is located within nucleotides 0-303 of SEQ ID NO: 1083. In some embodiments, the first target region is -97-WSGR Ref. No. 59073-743.601 located in a region of the HBV genome within nucleotides 1000-2448 of an HBV genome provided herein. In some embodiments, the first target region is located within nucleotides 1000- 2448 of SEQ ID NO: 1082. In some embodiments, the first target region is located within nucleotides 1000-2448 of SEQ ID NO: 1083. In some embodiments, the first target region is located in a region of the HBV genome within nucleotides 2802-3182 of an HBV genome provided herein. In some embodiments, the first target region is located within nucleotides 2802- 3182 of SEQ ID NO: 1082. In some embodiments, the first target region is located within nucleotides 2802-3182 of SEQ ID NO: 1083. In some embodiments, the first target region of the HBV genome is located in an HBV CpG island (CGI). In some embodiments, the CGI is an HBV canonical CGI. In some embodiments, the CGI is canonical CGI-I. In some embodiments, CGI is canonical CGI-I of HBV genotype D. In some embodiments, CGI-I spans nucleotides 186-288 of SEQ ID NO: 1082In some embodiments, CGI-I spans nucleotides 186-288 of SEQ ID NO: 1083In some embodiments, the CGI is canonical CGI-II. In some embodiments, the CGI is canonical CGI-II HBV genotype D. In some embodiments, the CGI is CGI II spans nucleotides 1,217-1,670 of SEQ ID NO: 1082. In some embodiments, the CGI is CGI II spans nucleotides 1,217-1,670 of SEQ ID NO: 1083. In some embodiments, the CGI is canonical CGI-III. In some embodiments, the CGI is canonical CGI-III HBV genotype D. In some embodiments, the CGI is CGI-III spans nucleotides 2,282-2,448 of SEQ ID NO: 1082. In some embodiments, the CGI is CGI-III spans nucleotides 2,282-2,448 of SEQ ID NO: 1083. In some embodiments, the first target region of the HBV genome is located in a promotor. In some embodiments, the first target region of the HBV genome is located in the sp1 promoter. In some embodiments, the first target region of the HBV genome is located in sp2 promoter. In some embodiments, the first target region of the HBV genome is located in cp promoter. In some embodiments, the first target region of the HBV genome is located in xp promoter. In some embodiments, the first target region of the HBV genome is located in an enhancer region. In some embodiments, the first target region of the HBV genome is located in Enh I. In some embodiments, the first target region of the HBV genome is located in Enh II. In some embodiments, the first target region of the HBV genome is located in a section of the HBV genome that encodes a transcript. In some embodiments, the first target region of the HBV genome is located in a section of the HBV genome that encodes a pgRNA transcript. In some embodiments, the first target region of the HBV genome is located in a section of the HBV genome that encodes a preCore RNA transcript. In some embodiments, the first target region of the HBV genome is located in a section of the HBV genome that encodes a preS RNA transcript. In some embodiments, the first target region -98-WSGR Ref. No. 59073-743.601 of the HBV genome is located in a section of the HBV genome that encodes an S RNA transcript. In some embodiments, the first target region of the HBV genome is located in a section of the HBV genome that encodes an HBx RNA transcript. In some embodiments, the first target region of the HBV genome is within 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 base pairs (bp) of an HBV transcription start site (TSS). In some embodiments, the TSS is a pg RNA TSS. In some embodiments, the first target region is within 600, within 500, within 400, within 300, within 200, or within 100 base pairs of the pg RNA TSS. In some embodiments, the pg RNA TSS is located at nucleotide 1820 of SEQ ID NO: 1082 or at nucleotide 1820 of SEQ ID NO: 1083. In some embodiments, the first target region is within 600 base pairs of nucleotide 1820 in SEQ ID NO: 1082. In some embodiments, the first target region is within 600 base pairs of nucleotide 1820 in SEQ ID NO: 1083. In some embodiments, the first target region is within 500 base pairs of nucleotide 1820 in SEQ ID NO: 1082. In some embodiments, the first target region is within 500 base pairs of nucleotide 1820 in SEQ ID NO: 1083. In some embodiments, the first target region is within 400 base pairs of nucleotide 1820 in SEQ ID NO: 1082. In some embodiments, the first target region is within 400 base pairs of nucleotide 1820 in SEQ ID NO: 1083. In some embodiments, the first target region is within 300 base pairs of nucleotide 1820 in SEQ ID NO: 1082. In some embodiments, the first target region is within 300 base pairs of nucleotide 1820 in SEQ ID NO: 1083. In some embodiments, the first target region is within 200 base pairs of nucleotide 1820 in SEQ ID NO: 1082. In some embodiments, the first target region is within 200 base pairs of nucleotide 1820 in SEQ ID NO: 1083. In some embodiments, the first target region is within 100 base pairs of nucleotide 1820 in SEQ ID NO: 1082. In some embodiments, the first target region is within 100 base pairs of nucleotide 1820 in SEQ ID NO: 1083. In some embodiments, the TSS is a preC RNA TSS. In some embodiments, the first target region is within 600, within 500, within 400, within 300, within 200, or within 100 base pairs of the preC RNA TSS. In some embodiments, the preC RNA TSS is located at nucleotide 1791 of SEQ ID NO: 1082 or at nucleotide 1791 of SEQ ID NO: 1083. In some embodiments, the first target region is within 600 base pairs of nucleotide 1791 in SEQ ID NO: 1082. In some embodiments, the first target region is within 600 base pairs of nucleotide 1791 in SEQ ID NO: 1083. In some embodiments, the first target region is within 500 base pairs of nucleotide 1791 in SEQ ID NO: 1082. In some embodiments, the first target region is within 500 base pairs of nucleotide 1791 in SEQ ID NO: 1083. In some embodiments, the first target region is within 400 base pairs of nucleotide 1791 in SEQ ID NO: 1082. In some embodiments, the first target region is within 400 base pairs of nucleotide 1791 in SEQ ID NO: 1083. In some embodiments, the first -99-WSGR Ref. No. 59073-743.601 target region is within 300 base pairs of nucleotide 1791 in SEQ ID NO: 1082. In some embodiments, the first target region is within 300 base pairs of nucleotide 1791 in SEQ ID NO: 1083. In some embodiments, the first target region is within 200 base pairs of nucleotide 1791 in SEQ ID NO: 1082. In some embodiments, the first target region is within 200 base pairs of nucleotide 1791 in SEQ ID NO: 1083. In some embodiments, the first target region is within 100 base pairs of nucleotide 1791 in SEQ ID NO: 1082. In some embodiments, the first target region is within 100 base pairs of nucleotide 1791 in SEQ ID NO: 1083. In some embodiments, the TSS is a preS2 RNA TSS. In some embodiments, the first target region is within 600, within 500, within 400, within 300, within 200, or within 100 base pairs of the preS2 RNA TSS. In some embodiments, the preS2 RNA TSS is located at nucleotide 3159 of SEQ ID NO: 1082 or at nucleotide 3159 of SEQ ID NO: 1083. In some embodiments, the first target region is within 600 base pairs of nucleotide 3159 in SEQ ID NO: 1082. In some embodiments, the first target region is within 600 base pairs of nucleotide 3159 in SEQ ID NO: 1083. In some embodiments, the first target region is within 500 base pairs of nucleotide 3159 in SEQ ID NO: 1082. In some embodiments, the first target region is within 500 base pairs of nucleotide 3159 in SEQ ID NO: 1083. In some embodiments, the first target region is within 400 base pairs of nucleotide 3159 in SEQ ID NO: 1082. In some embodiments, the first target region is within 400 base pairs of nucleotide 3159 in SEQ ID NO: 1083. In some embodiments, the first target region is within 300 base pairs of nucleotide 3159 in SEQ ID NO: 1082. In some embodiments, the first target region is within 300 base pairs of nucleotide 3159 in SEQ ID NO: 1083. In some embodiments, the first target region is within 200 base pairs of nucleotide 3159 in SEQ ID NO: 1082. In some embodiments, the first target region is within 200 base pairs of nucleotide 3159 in SEQ ID NO: 1083. In some embodiments, the first target region is within 100 base pairs of nucleotide 3159 in SEQ ID NO: 1082. In some embodiments, the first target region is within 100 base pairs of nucleotide 3159 in SEQ ID NO: 1083. In some embodiments, the TSS is an HBx RNA TSSs. In some embodiments, the first target region is within 600, within 500, within 400, within 300, within 200, or within 100 base pairs of the HBx RNA TSS. In some embodiments, the HBx RNA TSS is located at a nucleotide within the sequence of nucleotides 1243-1338 of SEQ ID NO: 1082 or nucleotides 1243-1338 of SEQ ID NO: 1083. In some embodiments, the first target region is within 600 base pairs of nucleotide 1243 in SEQ ID NO: 1082. In some embodiments, the first target region is within 600 base pairs of nucleotide 1243 in SEQ ID NO: 1083. In some embodiments, the first target region is within 500 base pairs of nucleotide 1243 in SEQ ID NO: 1082. In some embodiments, the first target region is within 500 base pairs of nucleotide 1243 in -100-WSGR Ref. No. 59073-743.601 SEQ ID NO: 1083. In some embodiments, the first target region is within 400 base pairs of nucleotide 1243 in SEQ ID NO: 1082. In some embodiments, the first target region is within 400 base pairs of nucleotide 1243 in SEQ ID NO: 1083. In some embodiments, the first target region is within 300 base pairs of nucleotide 1243 in SEQ ID NO: 1082. In some embodiments, the first target region is within 300 base pairs of nucleotide 1243 in SEQ ID NO: 1083. In some embodiments, the first target region is within 200 base pairs of nucleotide 1243 in SEQ ID NO: 1082. In some embodiments, the first target region is within 200 base pairs of nucleotide 1243 in SEQ ID NO: 1083. In some embodiments, the first target region is within 100 base pairs of nucleotide 1243 in SEQ ID NO: 1082. In some embodiments, the first target region is within 100 base pairs of nucleotide 1243 in SEQ ID NO: 1083. In some embodiments, the first target region is within 600 base pairs of nucleotide 1338 in SEQ ID NO: 1083. In some embodiments, the first target region is within 500 base pairs of nucleotide 1338 in SEQ ID NO: 1082. In some embodiments, the first target region is within 500 base pairs of nucleotide 1338 in SEQ ID NO: 1083. In some embodiments, first target region is within 400 base pairs of nucleotide 1338 in SEQ ID NO: 1082. In some embodiments, the first target region is within 400 base pairs of nucleotide 1338 in SEQ ID NO: 1083. In some embodiments, the first target region is within 300 base pairs of nucleotide 1338 in SEQ ID NO: 1082. In some embodiments, the first target region is within 300 base pairs of nucleotide 1338 in SEQ ID NO: 1083. In some embodiments, the first target region is within 200 base pairs of nucleotide 1338 in SEQ ID NO: 1082. In some embodiments, the first target region is within 200 base pairs of nucleotide 1338 in SEQ ID NO: 1083. In some embodiments, the first target region is within 100 base pairs of nucleotide 1338 in SEQ ID NO: 1082. In some embodiments, the first target region is within 100 base pairs of nucleotide 1338 in SEQ ID NO: 1083. In some embodiments, the reduction is a reduction in the number of HBV viral episomes. In some embodiments, the reduction is a reduction in the number of cccDNA genomes. In some embodiments, the reduction is a reduction in total HBV DNA. In some embodiments, the reduction is a reduction in the replication of the HBV genome. In some embodiments, the reduction is a reduction in a level of expression of a protein product encoded by the HBV genome. In some embodiments, the reduction is a reduction in a level of HBsAg. In some embodiments, the reduction is a reduction in a level of HBeAg. In some embodiments, the reduction is a reduction of total HBV DNA of at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or at least 99.9%, and the reduction is maintained for at least 14 days after the contacting or the administering. In some embodiments, the reduction is a reduction of HBeAg of at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or at least 99.9%, and the -101-WSGR Ref. No. 59073-743.601 reduction is maintained for at least 14 days after the contacting or the administering. In some embodiments, the reduction is a reduction of HBsAg of at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or at least 99.9%, and the reduction is maintained at or below that level for at least 14 days after the contacting or the administering. In some embodiments, the reduction is a reduction of at least 90%. In some embodiments, the reduction is a reduction of at least 95%. In some embodiments, the reduction is a reduction of at least 99%. In some embodiments, the reduction is a reduction of at least 99.9%. In some embodiments, the reduction is maintained for at least 14 days after the contacting or the administering. In some embodiments, the reduction is maintained for at least 21 days. In some embodiments, the reduction is maintained for at least 28 days. In some embodiments, the reduction is maintained for at least 35 days. In some embodiments, the reduction is maintained for at least 42 days. In some embodiments, the reduction is maintained for at least 56 days. In some embodiments, the reduction is maintained for at least 70 days. In some embodiments, the reduction is maintained for at least 84 days. In some embodiments, the reduction is maintained for at least 112 days. In some embodiments, the reduction is maintained for at least 140 days. In some embodiments, the reduction is maintained for at least 168 days. In some embodiments, the reduction is maintained for at least 6 months. In some embodiments, the reduction is maintained for at least 7 months. In some embodiments, the reduction is maintained for at least 8 months. In some embodiments, the reduction is maintained for at least 9 months. In some embodiments, the reduction is maintained for at least 12 months. In some embodiments, the reduction is maintained for at least 18 months. In some embodiments, the reduction is maintained for at least 24 months. In some embodiments, the epigenetic editing system is administered as a monotherapy. Accordingly, in some embodiments, the method does not comprise administering a nucleoside or nucleotide analog (NUC) to the subject. In some embodiments, the method further comprises administering a NUC to the subject. 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 a gRNA provided herein, and preferably the gRNA comprises a sequence provided in Table 15 or 16. 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 with a sequence selected from any zinc finger or zinc finger motif provided herein, e.g., in Table 1 or Table 21. In some embodiments, the zinc-finger protein comprises a sequence of any of the zinc finger epigenetic repressors provided herein. In -102-WSGR Ref. No. 59073-743.601 some embodiments, the transcriptional repressor domain comprises a ZIM3 repressor domain. 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 a DNMT domain provided herein. In some embodiments, the epigenetic editing system comprises the epigenetic editor protein provided in SEQ ID NO: 1248 or the epigenetic editor protein provided in SEQ ID NO: 1252 and at least one guide RNA provided as gRNA #003, gRNA #007, gRNA #008, gRNA #009, gRNA #011, or gRNA #015 herein. Some aspects of this disclosure provide epigenetic editing systems for use in the methods described herein. In some embodiments, the epigenetic editing system comprises an epigenetic editor protein or a nucleic acid encoding the epigenetic editor protein, and the epigenetic editor protein comprises: (a) a DNA-binding domain that binds a target region of a HBV gene or genome, (b) a first DNA methyltransferase (DNMT) domain, and (c) a transcriptional repressor domain. In some embodiments, the epigenetic editor protein comprises a sequence of an epigenetic editor protein provided herein. In some embodiments, the DNA-binding domain is a CRISPR-Cas DNA binding domain, and the epigenetic editing system comprises at least gRNA provided herein. In some embodiments, the epigenetic editing system comprises the epigenetic editor protein provided in SEQ ID NO: 1248 or the epigenetic editor protein provided in SEQ ID NO: 1252 and at least one guide RNA provided as gRNA #003, gRNA #007, gRNA #008, gRNA #009, gRNA #011, or gRNA #015 herein.

[0277] In some embodiments, the subject is a mammalian subject having, or having beendiagnosed with, a Hepatitis B virus (HBV) infection. In some embodiments, the subject is a mammalian subject having, or having been diagnosed with, a Hepatitis D virus infection.

[0278] In some embodiments, the subject is a mammalian subject, for example, a humansubject, having, or having been diagnosed with, a Hepatitis B virus (HBV) infection. In some embodiments, the subject is a mammalian subject, for example, a human subject, having, or having been diagnosed with Hepatitis B In some embodiments, the subject is a mammalian subject, for example, a human subject, having, or having been diagnosed with, a Hepatitis D virus infection. In some embodiments, a patient to be treated with an epigenetic editor of the present disclosure has received prior treatment for the condition to be treated (e.g., an HBV and / or HDV infection, or Hepatitis B). In other embodiments, the patient has not received such prior treatment. In some embodiments, the patient has failed on (or is refractory to) a prior treatment for the condition (e.g., a prior HBV treatment).

[0279] In some embodiments, contacting the HBV gene or genome or a cell with anepigenetic editor as described herein results in a reduction of: number of HBV viral episomes, -103-WSGR Ref. No. 59073-743.601 replication of the HBV gene or genome, or expression of a protein product encoded by the HBV gene or genome. In some embodiments, the reduction is 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%, or at least about 90% compared to contacting the HBV gene or genome or the cell with a suitable control or without contacting the HBV gene or genome or the cell with the epigenetic editor described herein. In some embodiments, the reduction is maintained for at least 6 days, 19 days, 27 days, 42 days, or 168 days. In some embodiments, the protein product comprises a HBe antigen or a HBs antigen.

[0280] In some embodiments, administering to the subject an epigenetic editor orpharmaceutical composition as described herein results in a reduction of: number of HBV viral episomes, replication of the HBV gene or genome, or expression of a protein product encoded by the HBV gene or genome. In some embodiments, the reduction is 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%, or at least about 90% compared to administering a suitable control or without administering the epigenetic editor or pharmaceutical composition described herein. In some embodiments, the reduction is maintained for at least 6 days, 19 days, 27 days, 42 days, or 168 days. In some embodiments, the protein product comprises a HBe antigen or a HBs antigen.

[0281] An epigenetic editor of the present disclosure may be administered in atherapeutically effective amount to a patient with a condition described herein. “Therapeutically effective amount,” as used herein, refers to an amount of the therapeutic agent being administered that will relieve to some extent one or more of the symptoms of the disorder being treated, and / or result in clinical endpoint(s) desired by healthcare professionals. An effective amount for therapy may be measured by its ability to stabilize disease progression and / or ameliorate symptoms in a patient, and preferably to reverse disease progression. The ability of an epigenetic editor of the present disclosure to reduce or silence HBV expression may be evaluated by in vitro assays, e.g., as described herein, as well as in suitable animal models that are predictive of the efficacy in humans. Suitable dosage regimens will be selected in order to provide an optimum therapeutic response in each particular situation, for example, administered as a single bolus or as a continuous infusion, and with possible adjustment of the dosage as indicated by the exigencies of each case.

[0282] An epigenetic editor of the present disclosure may be administered without additionaltherapeutic treatments, i.e., as a stand-alone therapy (monotherapy). Alternatively, treatment with an epigenetic editor of the present disclosure may include at least one additional therapeutic -104-WSGR Ref. No. 59073-743.601 treatment (combination therapy). In some embodiments, the additional therapeutic agent is any known in the art to treat an HBV infection. The current standard therapy for HBV employs nucleoside / nucleotide analogs (NUCs) and interferon (IFN). NUCs are viral polymerase and reverse transcriptase inhibitors that can efficiently suppress HBV viral replication, resulting in rapid HBV DNA reduction. NUCs do not directly target HBV cccDNA transcription, but NUC treatment of human HBV patients has been reported to reduce plasma HBV biomarkers such as HBeAg and HBsAg tp some extent. Prolonged therapy with NUCs is frequently associated with the pathogen developing a resistance to the treatment, but some NUCs have been reported to be able to achieve long-term viral suppression and halt disease progression. IFN-based therapy has both direct antiviral and immunomodulatory effects, and has been reported to prevent the formation of replication-competent pregenomic RNA-containing HBV capsids, or otherwise accelerates their degradation, thereby inhibiting HBV replication. See, e.g., Su et al., Improving clinical outcomes of chronic hepatitis B virus infection. Expert Rev Gastroenterol Hepatol. 2015;9:141–154; European Association for the Study of the Liver. EASL clinical practice guidelines: management of chronic hepatitis B virus infection. J Hepatol. 2012;57:167–185; Wieland et al., Intrahepatic induction of alpha / beta interferon eliminates viral RNA-containing capsids in hepatitis B virus transgenic mice. J Virol. 2000; and Wieland et al., Interferon prevents formation of replication-competent hepatitis B virus RNA-containing nucleocapsids. Proc Natl Acad Sci U S A. 2005;102:9913–9917, the entire contents of each of which are incorporated herein by reference.

[0283] In some embodiments, an epigenetic editor of the present disclosure is administered toa subject in need thereof, e.g., a subject having an HBV infection, without additional therapeutic treatment, e.g., without the co-administration of NUCs or IFN, or any other therapeutic treatment aimed at HBV, i.e., as a stand-alone therapy (monotherapy). In some such embodiments, a durable reduction of an HBV biomarker (e.g., as measured as the plasma level of HBV DNA, HBsAg, or HBeAG) by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, at least 99.9%, or more, is achieved over a time period of at least 14 days, at least 21 days, at least 28 days, at least 35 days, at least 42 days, at least 56 days, at least 70 days, at least 84 days, at least 112 days, at least 140 days, at least 168 days, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer, after a single-dose administration of the epigenetic editor to the subject. -105-WSGR Ref. No. 59073-743.601

[0284] In some embodiments, an epigenetic editor of the present disclosure is administered toa subject in need thereof, e.g., a subject having an HBV infection, in combination with (i.e., in temporal proximity) at least one additional HBV therapeutics, e.g., with NUCs and / or IFN therapeutics, or with any other therapeutic treatment aimed at HBV, i.e., as a combination therapy (monotherapy). In some such embodiments, a durable reduction of an HBV biomarker (e.g., as measured as the plasma level of HBV DNA, HBsAg, or HBeAG) by by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, at least 99.9%, or more, is achieved over a time period of at least 14 days, at least 21 days, at least 28 days, at least 35 days, at least 42 days, at least 56 days, at least 70 days, at least 84 days, at least 112 days, at least 140 days, at least 168 days, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer.

[0285] An epigenetic editor of the present disclosure may be administered without additionaltherapeutic treatments, i.e., as a stand-alone therapy (monotherapy). Alternatively, treatment with an epigenetic editor of the present disclosure may include at least one additional therapeutic treatment (combination therapy). In some embodiments, the additional therapeutic agent is any known in the art to HBV and / or HDV. In some embodiments, therapeutic agents include, but are not limited to, antivirals, such as entecavir, tenofovir, lamivudine, telvivudine, bictegravir, emtricitabine, or defovir, as well as immune modulators, such as pegylated interferon and interferon alpha.

[0286] The epigenetic editors or components thereof (or nucleic acid molecules encoding theepigenetic editors or components thereof) of the present disclosure may be administered by any method accepted in the art (e.g., parenterally, intravenously, intradermally, or intramuscularly). The epigenetic editors or components thereof (or nucleic acid molecules encoding the epigenetic editors or components thereof) of the present disclosure may be administered intravenously (e.g., peripheral intravenous infusion).

[0287] The epigenetic editors or components thereof (or nucleic acid molecules encoding theepigenetic editors or components thereof) of the present disclosure may be administered to a subject once, twice, three times, or 4, 5, 6, 7, 8, 9, 10, or more times. In some embodiments, the one, two, three, or 4, 5, 6, 7, 8, 9, 10, or more administrations of epigenetic editors or components thereof (or nucleic acid molecules encoding the epigenetic editors or components thereof) are in temporal proximity, e.g., within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 4 weeks, 1 month or two months of each other. In some embodiments, a subject -106-WSGR Ref. No. 59073-743.601 is re-dosed with the epigenetic editors or components thereof (or nucleic acid molecules encoding the epigenetic editors or components thereof) of the present disclosure for at least one more time after an initial dose. In some cases, a subject is administered with a subsequent dose of the epigenetic editors or components thereof (or nucleic acid molecules encoding the epigenetic editors or components thereof) of the present disclosure, which target a different DNA region of the HBV genome than the DNA region of the HBV genome that is targeted by the epigenetic editors or components thereof that the subject receives at the initial dose. In some cases, a subject is administered with multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of the same epigenetic editors or components thereof (or nucleic acid molecules encoding the epigenetic editors or components thereof) of the present disclosure. In some cases, a subject is administered with a single dose of different epigenetic editors or components thereof (or nucleic acid molecules encoding the epigenetic editors or components thereof) of the present disclosure, at least two of which target different DNA regions of the HBV genome. In some cases, a subject is administered with multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of different epigenetic editors or components thereof (or nucleic acid molecules encoding the epigenetic editors or components thereof) of the present disclosure, at least two of which target different DNA regions of the HBV genome. In some embodiments, redosing of the epigenetic editors or components thereof (or nucleic acid molecules encoding the epigenetic editors or components thereof) of the present disclosure has a better therapeutic efficacy than a single dose of the same, e.g., more potent suppression of HBV replication, or more profound reduction in HBV DNA and / or HBV antigens (e.g., HBsAg, HBeAg, and / or HBV core antigen (HBcAg)) present in the subject, e.g., in the circulation system and / or liver of the subject.

[0288] In some cases, the epigenetic editors or components thereof (or nucleic acidmolecules encoding the epigenetic editors or components thereof) of the present disclosure is administered at a dosage of at least about 0.2 mg / kg, 0.4 mg / kg, 0.6 mg / kg, 0.8 mg / kg. 1.0 mg / kg, 1.2 mg / kg. 1.4 mg / kg, 1.6 mg / kg, 1.8 mg / kg, 2.0 mg / kg, 4.0 mg / kg, 6 mg / kg, 8 mg / kg, or 10 mg / kg. In some cases, the epigenetic editors or components thereof (or nucleic acid molecules encoding the epigenetic editors or components thereof) of the present disclosure is administered once daily, twice daily, or three times daily. In some cases, the epigenetic editors or components thereof (or nucleic acid molecules encoding the epigenetic editors or components thereof) of the present disclosure is administered once daily for about 3, 4, 5, 10, 15, 20, 25, 30, or 35 administrations. In some cases, the epigenetic editors or components thereof (or nucleic acid molecules encoding the epigenetic editors or components thereof) of the present disclosure is -107-WSGR Ref. No. 59073-743.601 administered once daily for 28 administrations. In some cases, the epigenetic editors or components thereof (or nucleic acid molecules encoding the epigenetic editors or components thereof) of the present disclosure is administered once every four weeks (Q4W). In some cases, the epigenetic editors or components thereof (or nucleic acid molecules encoding the epigenetic editors or components thereof) of the present disclosure is administered once every four weeks (Q4W) for about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35 administrations.

[0289] In some cases, a subject administered with an epigenetic editor described herein isassessed for HBV antigen clearance from blood or serum and / or for the development of antibodies directed against one or more HBV antigens, e.g., anti-HBs antibodies or anti-HBe antibodies. Accordingly, in some embodiments, therapeutic methods provided herein comprise administering an epigenetic editor as described herein to a subject with detectable levels of an HBV antigen, e.g., of HBsAg or HBeAg, in their blood or serum, and assessing the subjects blood or serum for levels of HBsAg or HBeAg subsequent to such administration. In some embodiments, therapeutic methods provided herein comprise administering an epigenetic editor as described herein to a subject with detectable levels of an HBV antigen, e.g., of HBsAg or HBeAg, in their blood or serum, and assessing the subjects blood or serum for levels of anti- HBsAg (anti-HBs) or anti-HBeAg (anti-HBe) antibodies subsequent to such administration. In some such embodiments, loss of one or more HBV antigens, e.g., HBsAg and / or HBeAg is observed in the subject. In some such embodiments, detectable levels of anti-HBs and / or anti- HBe antibodies is observed in the subject. The emergence of antibodies directed against an HBV antigen in a subject is also sometimes referred to as seroconversion and is considered to indicate development of immune control of HBV in a subject. In some embodiments, the administration of an epigenetic editor provided herein results in anti-HBsAg and / or anti-HBeAg seroconversion in the recipient subject. XII. Definitions

[0290] The term “nucleic acid” as used herein refers to any oligonucleotide or polynucleotidecontaining nucleotides (e.g., deoxyribonucleotides or ribonucleotides) in either single- or double- strand form, and includes DNA and RNA. “Nucleotides” contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group, and are linked together through the phosphate groups. “Bases” include purines and pyrimidines, which include natural compounds such as adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs; as well as synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modified versions -108-WSGR Ref. No. 59073-743.601 which place new reactive groups such as amines, alcohols, thiols, carboxylates, alkylhalides, etc. Nucleic acids may contain known nucleotide analogs and / or modified backbone residues or linkages, which may be synthetic, naturally occurring, and non-naturally occurring. Such nucleotide analogs, modified residues, and modified linkages are well known in the art, and may provide a nucleic acid molecule with enhanced cellular uptake, reduced immunogenicity, and / or increased stability in the presence of nucleases.

[0291] As used herein, an “isolated” or “purified” nucleic acid molecule is a nucleic acidmolecule that exists apart from its native environment. For example, an “isolated” or “purified” nucleic acid molecule (1) has been separated away from the nucleic acids of the genomic DNA or cellular RNA of its source of origin; and / or (2) does not occur in nature. In some embodiments, an “isolated” or “purified” nucleic acid molecule is a recombinant nucleic acid molecule.

[0292] It will be understood that in addition to the specific proteins and nucleic acidmolecules mentioned herein, the present disclosure also contemplates the use of variants, derivatives, homologs, and fragments thereof. A variant of any given sequence may have the specific sequence of residues (whether amino acid or nucleic acid residues) modified in such a manner that the polypeptide or polynucleotide in question substantially retains at least one of its endogenous functions. A variant sequence can be obtained by addition, deletion, substitution, modification, replacement and / or variation of 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 specific proteins described herein (e.g., KRAB, dCas9, DNMT3A, and DNMT3L proteins described herein), the present disclosure also contemplates any of the protein’s naturally occurring forms, 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 its function as compared to the specific protein described).

[0293] As used herein, a homologue of any polypeptide or nucleic acid sequencecontemplated herein includes sequences having a certain homology with the wildtype amino acid and nucleic sequence. A homologous sequence may include a sequence, e.g. an amino acid sequence which may be at least 50%, 55%, 65%, 75%, 85%, 90%, 91%, 92%< 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the subject sequence. The term “percent identical” in the context of amino acid or nucleotide sequences refers to the percent of residues in two sequences that are the same when aligned for maximum correspondence. In some embodiments, the length of a reference sequence aligned for comparison purposes is at least 30%, (e.g., at least 40, 50, 60, 70, 80, or 90%, or 100%) of the reference sequence. Sequence identity may be measured using -109-WSGR Ref. No. 59073-743.601 sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e-3 and e- 100 indicating a closely related sequence.

[0294] The percent identity of two nucleotide or polypeptide sequences is determined by, e.g.,BLAST® using default parameters (available at the U.S. National Library of Medicine’s National Center for Biotechnology Information website). In some embodiments, the length of a reference sequence aligned for comparison purposes is at least 30%, (e.g., at least 40, 50, 60, 70, 80, or 90%) of the reference sequence.

[0295] It will be understood that the numbering of the specific positions or residues inpolypeptide sequences depends on the particular protein and numbering scheme used. Numbering might be different, e.g., in precursors of a mature protein and the mature protein itself, and differences in sequences from species to species may affect numbering. One of skill in the art will be able to identify the respective residue in any homologous protein and in the respective encoding nucleic acid by methods well known in the art, e.g., by sequence alignment and determination of homologous residues.

[0296] The term “modulate” or “alter” refers to a change in the quantity, degree, or extent ofa function. For example, an epigenetic editor as described herein may modulate the activity of a promoter sequence by binding to a motif within the promoter, thereby inducing, enhancing, or suppressing transcription of a gene operatively linked to the promoter sequence. As other examples, an epigenetic editor as described herein may block RNA polymerase from transcribing a gene, or may inhibit translation of an mRNA transcript. The terms “inhibit,” “repress,” “suppress,” “silence” and the like, when used in reference to an epigenetic editor or a component thereof as described herein, refers to decreasing or preventing the activity (e.g., transcription) of a nucleic acid sequence (e.g., a target gene) or protein relative to the activity of the nucleic acid sequence or protein in the absence of the epigenetic editor or component thereof. The term may include partially or totally blocking activity, or preventing or delaying activity. The inhibited activity may be, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% less than that of a control, or may be, e.g., at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold less than that of a control. For example, in -110-WSGR Ref. No. 59073-743.601 some embodiments, the inhibited activity (e.g., the transcription or expression of an HBV target gene, or the level of an HBV biomarker) may be at least 70% less than that of a control. In some embodiments, the inhibited activity may be at least 80% less than that of a control. In some embodiments, the inhibited activity may be at least 90% less than that of a control (1 log reduction). In some embodiments, the inhibited activity may be at least 91% less than that of a control. In some embodiments, the inhibited activity may be at least 92% less than that of a control. In some embodiments, the inhibited activity may be at least 93% less than that of a control. In some embodiments, the inhibited activity may be at least 94% less than that of a control. In some embodiments, the inhibited activity may be at least 95% less than that of a control. In some embodiments, the inhibited activity may be at least 96% less than that of a control. In some embodiments, the inhibited activity may be at least 97% less than that of a control. In some embodiments, the inhibited activity may be at least 98% less than that of a control. In some embodiments, the inhibited activity may be at least 99% less than that of a control (2 log reduction). In some embodiments, the inhibited activity may be at least 99.9% less than that of a control (3 log reduction).

[0297] The term “about” or “approximately” means within an acceptable error range for theparticular 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 than one standard deviation, per the practice in the given value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” should be assumed to mean an acceptable error range for the particular value.

[0298] Ranges provided herein are understood to be shorthand for all of the values within therange. For example, a range of 1 to 50 is 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 intervening decimal values between the aforementioned integers such as, for example, 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” that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction. -111-WSGR Ref. No. 59073-743.601

[0299] Unless otherwise defined herein, scientific and technical terms used in connectionwith the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. The recitation of a listing of elements herein includes any of the elements singly or in any combination. The recitation of an embodiment herein includes that embodiment as a single embodiment, or in combination with any other embodiment(s) herein. All publications, patents, patent applications, and other references mentioned herein, including, where applicable, any supplementary information, are incorporated by reference in their entirety. To the extent that references incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art. LISTINGS OF EXEMPLARY EMBODIMENTS

[0300] In order that the present disclosure may be better understood, the following listings ofexemplary embodiments is provided. This listing is for purposes of illustration of certain embodiments only. Additional embodiments will be apparent to the skilled artisan based on the present disclosure, and the listing below is not to be construed as limiting the scope of the present disclosure.1. A composition comprising:a. an RNA encoding an epigenetic editor protein, wherein the RNA comprises a 5’ cap, a 5’untranslated region (UTR), a coding region, a 3’ UTR, and a poly A tail, wherein the coding region encodes the epigenetic editor protein; and b. a guide RNA comprising a nucleic acid base sequence, wherein the nucleic acid basesequence comprises AGGAGUUCCGCAGUAUGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU -112-WSGR Ref. No. 59073-743.601 AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 1260), wherein A, G, C and U represent adenine, guanine, cytosine and uracil, respectively, wherein the RNA and the guide RNA are encapsulated in a lipid nanoparticle.2. The composition of embodiment 1, wherein the RNA and the guide RNA are encapsulated inthe lipid nanoparticle at a ratio from about 1:1 to about 1:3 by weight.3. The composition of embodiment 2, wherein the RNA and the guide RNA are encapsulated inthe lipid nanoparticle at a ratio of about 1:1 by weight.4. The composition of embodiment 2, wherein the RNA and the guide RNA are encapsulated inthe lipid nanoparticle at a ratio of about 1:1.5 by weight.5. The composition of embodiment 2, wherein the RNA and the guide RNA are encapsulated inthe lipid nanoparticle at a ratio of about 1:3 by weight.6. The composition of embodiment 1, wherein the RNA and guide RNA are encapsulated in thelipid nanoparticle at a ratio from about 1:2 to about 1:4 by weight.7. The composition of embodiment 1, wherein the RNA and guide RNA are encapsulated in thelipid nanoparticle at a ratio from about 1:3 to about 1:5 by weight.8. The composition of embodiment 1, wherein the 5' cap comprises the below structure:9. The composition of embodiment 1, wherein the 5' UTR comprises a sequence with at least95% sequence identity to SEQ ID NO: 1261.10. The composition of embodiment 1, wherein the 5' UTR comprises a sequence set forth inSEQ ID NO: 1261.11. The composition of embodiment 1, wherein the 3' UTR is derived from a fibrinogen betachain (FGB) gene.12. The composition of embodiment 11, wherein the 3' UTR comprises a sequence with at least95% sequence identity to SEQ ID NO: 1262. -113-WSGR Ref. No. 59073-743.60113. The composition of embodiment 11, wherein the 3' UTR comprises a sequence set forth inSEQ ID NO: 1262.14. The composition of any one of embodiments 1-13, wherein the RNA comprises a sequencewith at least 95% sequence identity to SEQ ID NO: 1257.15. The composition of any one of embodiments 1-13, wherein the RNA comprises a sequenceset forth in SEQ ID NO: 1257.16. The composition of embodiment 1, wherein the guide RNA comprises a nucleic acidsequence set forth in SEQ ID NO: 1249.17. The composition of any one of embodiments 1-16, wherein the epigenetic editor proteincomprises: a) a DNA binding domain; b) two DNA methyltransferase (DNMT) domains; c) a repressor domain; d) a first peptide linker between the DNA binding domain and one of the DNMT domains, and a second peptide linker between the DNA binding domain and the repressor domain, wherein the first peptide linker is XTEN80, wherein the second peptide linker is XTEN16; and e) two bipartite nuclear localization sequences (NLS), and a simian virus (SV40) NLS.18. The composition of embodiment 17, wherein one of the two bipartite NLSs is positioned atan amino (N) terminus of the epigenetic editor protein and the other one of the two NLSs is positioned at a carboxy (C) terminus of the epigenetic editor protein, and wherein the SV40 NLS is positioned at the C terminus of the DNA binding domain.19. The composition of embodiment 17 or 18, wherein the DNA binding domain is a dCas9 DNAbinding domain.20. The composition of any one of embodiments 17-19, wherein the repressor domain is a ZIM3repressor domain.21. The composition of any one of embodiments 17-20, wherein one of the two DNMT domainsis a DNMT3A domain.22. The composition of any one of embodiments 17-21, wherein one of the two DNMT domainsis a DNMT3L domain.23. The composition of embodiment 22, wherein the DNMT3L domain is derived from a speciesof the Equus genus. -114-WSGR Ref. No. 59073-743.60124. The composition of embodiment 23, wherein the species of the Equus genus is Equusprezewalskii.25. The composition of any one of embodiments1-24, wherein the epigenetic editor proteincomprises a configuration comprising: bipartite NLS-DNMT3A-DNMT3L-XTEN80-dCas9- SV40 NLS-XTEN16-ZIM3-bipartite NLS, from the N terminus to the C terminus.26. The composition of any one of embodiments 1-25, wherein the epigenetic editor proteincomprises a sequence with at least 95% sequence identity to SEQ ID NO: 1252.27. The composition of any one of embodiments 1-26, wherein the epigenetic editor proteincomprises a sequence set forth in SEQ ID NO: 1252.28. A method of treating an HBV infection in a subject comprising administering thecomposition of any one of embodiments 1-27. 29. A method comprising administering the composition of any one of embodiments 1-27 to a subject, wherein the subject is characterized by the presence of detectable levels of HBV DNA, HBsAg, and / or HBeAg in the plasma of the subject.

[0301] In order that the present disclosure may be better understood, the following examplesare set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the present disclosure in any manner. EXAMPLES Example 1: Selection of Target HBV Sequences for Epigenetic Silencing

[0302] Target sequences were manually and computationally designed using therepresentative HBV genome sequences (SEQ ID Nos. 1082, 1083) as a reference:

[0303] While target site design focused on CpG islands identified within the HBV genome,target sites outside of HBV CpG islands were also considered.

[0304] Table 2 presents some representative target sites that were identified as suitable fortargeting with an epigenetic repressor.

[0305] Target domains identified above that are adjacent to a PAM sequence, e.g., an S.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 sites 1-143 are suitable for dCas9-based epigenetic repressor targeting. Figure 1 provides an overview over the position of the target sites identified in the HBV genome.

[0306] Target sites were analyzed for conservation across HBV genotypes A-E (Figures 2and 3). Some target sites were identified that were well conserved across two or more, or in -115-WSGR Ref. No. 59073-743.601 some cases all, HBV genotypes. Targeting such conserved sites allows for silencing different genotypes with the same epigenetic repressor. Example 2: Guide RNA Assays in HepAD38 HBV cells

[0307] The HepAD38 cell line expresses the HBV genome under a doxycycline-induciblepromoter (see, e.g., Ladner et al., Inducible expression of human hepatitis B virus (HBV) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of HBV replication. Antimicrob. Agents Chemother. 41:1715-1720(1997), incorporated herein by reference).

[0308] Results are shown in Figure 4A and B.Example 3: Guide RNA Assays in HepG2-NTCP cells

[0309] HepG2 cells were engineered by lentiviral transduction to express the human NTCPreceptor which is used by hepatitis B virus (HBV) to infect the cells.

[0310] HBV viral particles were produced using the HepAD38 cell line. HepAD38 is asubclone, derived from HepG2 cell line, that expresses HBV genome (genotype D subtype ayw) under the transcriptional control of a tetracycline-responsive promoter in a TET-OFF system.

[0311] A triple combination of Engineered Transcriptional Repressors (ETRs) consisting ofthree plasmids expressing dCas9-KRAB, dCas9-DNMT3A and dCas9-DNMT3L was used in combination with one or more of the designed sgRNAs.

[0312] LNPs were formulated using GENVOY ILM Lipid Mix (Precision Nanosystem) andthe formulator Nanoassemblr Spark (Precision Nanosystem). LNPs were formulated according to the manufacturer’s recommendations with Nitrogen:Phosphate (NP) ratio equal to 6 and flow rate ratio (FRR) 2:1. The RNA payload was diluted to a final concentration of 350 ng / uL in the PNI formulation buffer. The ETRs, dCas9-KRAB, dCas9-DNMT3A, dCas9-DNMT3L and each of the 121 sgRNA were mixed at 1:1:1:4 ratio. The RNA mix, the Genvoy lipid mix (25 mM) and PBS were loaded each in the dedicated chambers of the Spark cartridge and formulated. The quality of the formulated LNPs was evaluated quantifying the packaged mRNA using Quant-it RiboGreen RNA Assay Kit (Thermo Fisher) and sizing the LNP by Dynamic Light Scattering (Zetasizer, Malvern Panalytic).

[0313] HepG2-NTCP cells were plated at 20,000 cells / well in collagen coated 96 well plates.After 24h cells were infected with HBV at 5,000 multiplicity of genome equivalent (MGE) and 16h after viral inoculum was removed, cells were washed with PBS, and fresh media was added. -116-WSGR Ref. No. 59073-743.601 Three days post-infection, using LNPs, each sgRNA and the mRNAs encoding each of the components of the triple constructs of ETRs (dCas9-KRAB, dCas9-DNMT3A, dCas9-DNMT3L) were delivered. Three days after, LNP was removed, medium was replaced, and cells were maintained in complete medium for three days.

[0314] Viral antigens HBeAg and HBsAg were quantified 6 days after LNP removal usingELISA assays. Data were normalized to a non-targeting guide designed against the mouse PCSK9 and control 3.2 gRNA was used as positive control. Cells viability assay were performed and normalized to non-targeting control.

[0315] The Table 9 below provides amino acid sequences of exemplary epigenetic editorsused in the gRNA screen (the ETR constructs): TABLE 9: amino acid sequences of exemplary epigenetic editor proteins-117-WSGR Ref. No. 59073-743.601-118-WSGR Ref. No. 59073-743.601

[0316] The Table 10 below provides amino acid sequences and polynucleotide sequences ofexemplary epigenetic editors TABLE 10: sequences of exemplary epigenetic editor constructs-119-WSGR Ref. No. 59073-743.601-120-WSGR Ref. No. 59073-743.601-121-WSGR Ref. No. 59073-743.601-122-WSGR Ref. No. 59073-743.601-123-WSGR Ref. No. 59073-743.601-124-WSGR Ref. No. 59073-743.601-125-WSGR Ref. No. 59073-743.601-126-WSGR Ref. No. 59073-743.601-127-WSGR Ref. No. 59073-743.601-128-WSGR Ref. No. 59073-743.601-129-WSGR Ref. No. 59073-743.601-130-WSGR Ref. No. 59073-743.601-131-WSGR Ref. No. 59073-743.601

[0317] Table 11 below lists components of the epigenetic editor polypeptide PLA001 andtheir corresponding amino acid position in the epigenetic editor polypeptide sequence (SEQ ID No. 481) set forth in Table 10. TABLE 11: annotation of PLA001 amino acid sequence

[0318] Table 12 below lists components of the polynucleotide encoding the epigenetic editorpolypeptide PLA001 and their corresponding nucleotide position in the polynucleotide sequence (SEQ ID No. 482) set forth in Table 10. TABLE 12: annotation of PLA001 polynucleotide sequence-132-WSGR Ref. No. 59073-743.601

[0319] Table 13 below lists components of the epigenetic editor polypeptide PLA002 andtheir corresponding amino acid position in the epigenetic editor polypeptide sequence (SEQ ID No. 483) set forth in Table 10. TABLE 13: annotation of PLA002 amino acid sequence

[0320] Table 14 below lists components of the polynucleotide encoding the epigenetic editorpolypeptide PLA002 and their corresponding nucleotide position in the polynucleotide sequence (SEQ ID No. 484) set forth in Table 10. TABLE 14: annotation of PLA002 polynucleotide sequence -133-WSGR Ref. No. 59073-743.601

[0321] Table 15 below provides gRNA sequence tested.TABLE 15: Exemplary gRNA sequences-134-WSGR Ref. No. 59073-743.601-135-WSGR Ref. No. 59073-743.601-136-WSGR Ref. No. 59073-743.601-137-WSGR Ref. No. 59073-743.601-138-WSGR Ref. No. 59073-743.601 TABLE 16: Exemplary target domain sequences and effect on HbeAg and HbsAg expression-139-WSGR Ref. No. 59073-743.601-140-WSGR Ref. No. 59073-743.601-141-WSGR Ref. No. 59073-743.601

[0322] In vitro silencing was observed in an HepG2-NTCP infection model with gRNAstargeting CpG islands with ETRs (Figure 5A–Figure 5B). A primary screen was conducted using LNPs of quality within expected parameters and a pilot experiment with a single guide (Figure 6–Figure 8). Results demonstrated that 48 gRNAs showed less than 50% expression of HBeAg at day 6 compared to non-targeting control (Figure 9) and 28 gRNAs showed less than 50% expression of HBsAg at day 6 compared to non-targeting control (Figure 10). HBsAg and HBeAg expression was positively correlated as shown in Figure 11. Example 4: Zinc finger repressors for silencing HBV

[0323] Zinc finger repressors targeting epigenetic target sites identified in the HBV genomewere designed. Table 1 above provides amino acid sequences of zinc finger and its corresponding motif sequences and target sequences of the zinc finger.

[0324] Zinc finger repressors described in Table 1 are tested in an HBV infection model, e.g.,in HepG2 cells as described herein, and efficient repression of HBV is confirmed for the zinc finger repressors provided in Table 1. Example 5: Further In vitro Evaluation of gRNAs

[0325] A CRISPR-Off single construct encoding PLA002, consisting of KRAB, DNMT3A,DNMT3L, and dCas9, was used in combination with one or more of the designed sgRNAs for the in vitro assays described in this example.

[0326] HepG2-NTCP cells were infected with HBV for 4 days, following procedures similaras those in Example 3, and were then transfected with CRISPR-off construct and individual exemplary gRNAs (as indicated in Table 16) formulated in a research-grade LNP. At Day 6 post- transfection HBsAg and HBeAg protein expression in the supernatant was evaluated by ELISA, as depicted in Figure 12A. Results from this experiment are shown in Figure 12B. All of the tested gRNAs led to reduction of HBsAg and HBeAg levels in the supernatant. Positive control used in this experiment is a gRNA against HBV genome that was previously shown to reduce antigens ~50%.

[0327] In another experiment, the integrated HBV cell line, PLC / PRF / 5, was used to evaluateactivity of gRNAs. The PLC / PRF / 5 cells were transfected with CRISPR-off (PLA002) and -142-WSGR Ref. No. 59073-743.601 individual gRNAs using a commercial lipid-based transfection reagent. As depicted in Figure 13A, four days after transfection HBsAg protein expression in the supernatant was evaluated by ELISA. Results from this experiment are shown in Figure 13B. Target conservation was evaluated in silico and target conservation was defined as 100% gRNA-DNA match.

[0328] In a further experiment, primary human hepatocytes (PHH) derived from humanizedmice were infected with HBV for 4 days and then transfected with CRISPR-off (PLA002) and individual gRNAs formulated in a research-grade LNP, GenVoy LNPs. As depicted in Figure 14A, at Day 6 post-infection HBsAg and HBeAg protein expression in the supernatant was evaluated by ELISA. Results from this experiment are shown in Figure 14B. Positive control used in this experiment is an HBV gRNA that was previously shown to reduce antigens ~50%. The data suggested strong in vitro silencing by certain gRNAs at Day 6 after transfection. In a second PHH experiment, depicted in Figure 14C, post-infection HBsAg and HBeAg protein expression in the supernatant was evaluated by ELISA at Day 12 after delivery of 100 ng of payload (1:1 effector to guide RNA ratio) in research-grade LNPs . Epigenetic editors repress HBsAg and HBeAg secretion in HBV infected PHH cells at this time point, as well. Results are shown in Figure 14D. Sequences of the exemplary gRNAs that were tested in this example are listed in Table 16. Example 6: In vivo Silencing of HBV in HBV Rodent Models

[0329] Two different HBV rodent models were tested in this study. As shown in Figure 15,in one set of experiments, a non-transgenic model of persistent HBV infection in immunocompetent mice was used, which was established by administering an adeno-associated viral vector (AAV) that contains HBV Genotype D DNA into the mice. The administration of the AAV-HBV vector resulted in expression of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and high levels of serum HBV DNA in the mice. In another set of experiments, a transgenic mouse model of persistent HBV infection was used, whose genome was engineered to integrate HBV Genotype A DNA, resulting in expression of HBsAg and HBeAg, and circulating viral DNA in the mice.

[0330] Both mouse models were used to test 6 different treatment groups as shown in Figure15. At certain times (such as 7, 14, 28, and 35 days) after single administration of 3 mg / kg of the LNPs that were loaded with the CRISPR-off construct and respective gRNAs, WT-Cas9 construct and gRNA, or control vehicle, mouse serum was extracted for analysis of HBsAg, -143-WSGR Ref. No. 59073-743.601 HBeAg, and HBV DNA. Later the mice were sacrificed, and their livers were collected for further analysis.

[0331] As shown in Figure 16, in transgenic mouse model, durable (~1 month) andefficacious (~2 Log) DNA and HBsAg reduction was observed with CRIPSR-Off / gRNA#011 treatment. And compared to Cas9 cutter, CRISPR-Off, when administered in combination with gRNA#011, showed similar circulating viral DNA reduction, but superior HBsAg and HBeAg reduction.

[0332] Reduction of HBV markers in AAV-HBV model was also observed withadministration of certain exemplary constructs. As shown in Figure 17, overall results in AAV8- HBV model are similar to the Tg-HBV mouse model. About 1 log DNA and HBsAg antigen reduction was observed with administration of CRISPR-Off and gRNA#011.

[0333] Effects of redosing of certain exemplary constructs were also tested. In the sameexperiments as above, among the six transgenic mice receiving administration of "CRISPR-off + gRNA#016" (CRISPR-off construct and gRNA gRNA#016), three were administered with a dose of "CRISPR-off + gRNA#016" on Day 35, and the other three were administered with "CRISPR-off + gRNA#011" on Day 35. As shown in Figure 18A, redosing either with a less effective gRNA (gRNA#016 in this case) or with a more effective gRNA (gRNA#011 in this case) enhanced the silencing of all HBV marker, as shown by reduction of circulating HBV DNA, HBsAg, and HBeAg on Day 42. Redosing the gRNA#016-treated group with gRNA#011 (more effective gRNA) resulted in a more substantial reduction than redosing with gRNA#016 (less effective gRNA).

[0334] Single-dose experiments were continued to 168 days, as shown in Figure 18B. Resultsshow durable and progressive reduction of viral antigens achieving -2.7 log DNA and -2.8 log HBsAg more than five months after single administration of an epigenetic editor (CRISPR-off with gRNA #011). Five out of six animals tested had undetectable HBV DNA and HBsAg 168 days after a single dose of an epigenetic editor.

[0335] Redosing experiments were also conducted in AAV-HBV mouse model, as shown inFigure 19. Dosing with two different gRNAs (gRNA#016 and gRNA#011) further decreased all HBV markers. These data suggest of a potential enhanced activity when two HBV regions are targeted.

[0336] Sequences of the exemplary gRNAs that were tested in this example are listed inTable 16. -144-WSGR Ref. No. 59073-743.601 Example 7: Evaluation of ZFP in HepG2-NTCP Cells

[0337] In this example, ZF-off single constructs encoding an epigenetic editor proteinconsisting of KRAB, DNMT3A, DNMT3L, and an exemplary zinc finger motif of choice, were tested. Sequences of the exemplary zinc fingers that were tested in this example are listed in Table 21, as are sequences for plasmids yielding a subset of the ZF-off single construct epigenetic editor proteins.

[0338] Certain exemplary ZF-off constructs were formulated in a research-grade LNP.HepG2-NTCP cells were infected with HBV for 4 days and then transfected with the ZF-off loaded LNPs. As depicted in Figure 20A, at Day 6 post-infection HBsAg and HBeAg protein expression in the supernatant was evaluated by ELISA. Figure 20B shows the results as measured by percentage reduction in HBV antigens as compared to non-targeting control. Positive control used in this experiment is a HBV gRNA previously shown to reduce antigens ~50%. Figure 21A shows the results of the top ten ZF-off constructs that lead to the most reduction in HBV antigens. Figure 21B shows the results for all constructs in the screen.

[0339] Table 17 and 18 below show the raw data from these experiments, listed with themRNA number yielding the zinc finger motif. Table 17. % HBsAg expression relative to non-targeting control-145-WSGR Ref. No. 59073-743.601-146-WSGR Ref. No. 59073-743.601Table 18. % HBeAg expression relative to non-targeting control-147-WSGR Ref. No. 59073-743.601Example 8. Dose Response Testing of Viral Antigens in HepG2-NTCP cells

[0340] In this example, top ZF epigenetic editor proteins were tested in 5-point dose responseassay for HBsAg and HBeAg. The 5 dosage points were 200ng, 150ng, 100ng, 50ng, and 25ng. Experimental schematic and results are shown in Figure 22. Example 9. Testing for Durable Repression of HBsAg in HepG2.2.15 cells

[0341] In this example, top ZF and CRISPR-off epigenetic editor proteins with guide RNAswere tested for durable repression of HBsAg. Active ZFPs and CRISPR-off editors showed -148-WSGR Ref. No. 59073-743.601durable silencing through Day 27 with 50ng treatment. Experimental schematic and results areshown in Figures 23A-23C. Example 10. Testing of Silencing of HBsAg in a Second Model for int-HBV

[0342] In this example, top ZF epigenetic editor proteins were tested for repression ofHBsAg in PLC / PRF / 5 cells. A subset of the ZFPs silenced HBsAg in this second model. Experimental schematic and results are shown in Figure 24. 1. Testing ZF Epigenetic Editor Proteins and CRISPR-off with guide RNAs for Specificity

[0343] In this example, ZF epigenetic editor proteins targeting HBV exhibiting significantsilencing were profiled for specificity in HepG2-NTCP at day 19. All comparisons were performed against a non-targeting ZFP control. An exemplary result for the ZF epigenetic editor protein with mRNA0001 zinc finger motif is shown in Figure 25A. CRISPR-off with guide RNAs were similarly profiled. HepG2-NTCP cells were transfected with 100ng of total payload using GenVoyTM LNP at a 1:1 gRNA:effector ratio. Cells were split every 3-4 days and collected at day 15 post-treatment for specificity assessments, including RNA-seq and methylation array. DESeq2 was used to identify differential gene expression. As shown in Figure 25B, little to no changes were observed above chosen thresholds (absolute[ log2[fold change]]>1 and -log10[adjusted p-value]>5) as expected for effectors targeting HBV DNA. For methylation array, the Infinium MethylationEPIC v2.0 array was used, and DMRs were identified using Bumphunter. EE3, EE4, and EE5 had a result of DMR=0. Results are shown in Figures 25C- 25D. Example 11. In vivo analysis of ZF-Off constructs

[0344] LNPs loaded with ten ZF-Off constructs as well as vehicle-only and CRISPR-Offcontrols were administered to AAV-HBV mice at 1 mg / kg as shown in the schematic in Figure 26. Table 19 shows the zinc finger motifs for each experimental group; the corresponding plasmid from Table 21, comprising the nucleic acid encoding the ZF-Off construct, was administered. Plasma from the mice was tested at Days 7, 14, 21, and 28 post dose for HBV DNA, HBsAg, and HBeAg. The livers were collected for further analysis. Results are shown in Figure 27. The ZF-Off construct with the ZF motif from mRNA0004 showed more than a 1.5 log -149-WSGR Ref. No. 59073-743.601 reduction in HBV DNA, a >2 log reduction in HSbsAg, and a >2 log reduction of HBeAg, all sustained up to 28 days from the dose. Table 19. Experimental groups for in vivo testing of ZF-Off constructs.Example 12. Zinc Finger Protein Multiplexing Study in an AAV-HBV and Tg-HBV Mouse Model

[0345] AAV-HBV mice are injected with a single administration at 0.5 mg / kg of one, two, orthree ZF epigenetic editor proteins, delivered as mRNA, in LNPs (schematic, Figure 28) in accordance with Table 20. HBV DNA, HBsAg, and HBeAg are assayed in plasma at one or more time points, and the mouse liver is collected for further analysis. Table 20. Multiplexing sample groups.-150-WSGR Ref. No. 59073-743.601Example 13. Dose response for CRISPR-Off constructs in an AAV in vivo model A single dose of CRISPR-Off (SEQ ID NO: 1248) mRNA with guide RNA #008 as well as vehicle-only control was tested via 1:1 mRNA:guide RNA administration to AAV-HBV mice at 0.5 mg / kg, 1 mg / kg, or 3 mg / kg in LNPs as shown in the schematic in Figure 29. Plasma from the mice was tested for HBsAg at thirteen time points through 186 days after injection. Results are shown in Figure 30. The highest dose administered showed an approximately 3.3 log reduction in HBsAg, sustained through 186 days after the dose. Example 14. Dose response for CRISPR Off constructs in Tg in vivo model

[0346] A single dose of CRISPR-Off (SEQ ID NO: 1248) mRNA with guide RNA #008 aswell as vehicle-only control was tested via 1:1 mRNA:guide RNA administration to Tg-HBV mice at 0.5 mg / kg, 1 mg / kg, or 3 mg / kg in LNPs as shown in the schematic in Figure 31. Plasma from the mice was tested for HBsAg at thirteen time points through 186 days after injection. Results are shown in Figure 32. The highest dose administered showed an approximately 2.6 log reduction in HBsAg, sustained through 196 days after the dose.

[0347] A second dose response experiment in Tg-HBV model using CRISPR-Off (SEQ IDNO: 1248) mRNA with guide RNA #008 formulated in LNPs was conducted, with administrations at 0.2 mg / kg, 0.5 mg / kg, 1 mg / kg, or 3 mg / kg of 1:1 mRNA:guide RNA. A -151-WSGR Ref. No. 59073-743.601 vehicle-only control was also used. In this experiment, plasma was tested for HBV DNA, HBsAg, and HBeAg at 13 time points through 207 days after injection. Results are shown in Figure 32. The HBsAg results for individual mice at the final time point of 207 days after injection are plotted in Figure 33. All of the mice in the 0.5 mg / kg, 1 mg / kg, and 3 mg / kg group had reduced HBsAg at Day 207 as compared to vehicle only control. Alanine transaminase (ALT) level in the mice was also tested at 207 days and found to be comparable to that of healthy untreated mice for all treatment groups. Example 15. Guide RNA testing in AAV-HBV mice

[0348] Six guide RNAs were tested for relative efficacy using CRISPR-Off (SEQ ID NO:1248) in a 28-day, single-dose study. CRISPR-Off construct mRNA and one of gRNA #003, gRNA #007, gRNA #008, gRNA #009, gRNA #011, and gRNA #015 was delivered at 1:1 mRNA:guide RNA at 1 mg / kg. Controls included vehicle only, CRISPRi with gRNA #008 (not shown), and wild type Cas9 with gRNA #011 (not shown). HBV DNA and HBsAg was measured over 28 days. Results are shown in Figure 34. Most of the single guide treatments tested in this experiment resulted in decreased HBV DNA and HBsAg versus vehicle only control. Example 16. Durability study for ZF-Off in AAV-HBV in vivo model: single and re-dose

[0349] Mice were injected with a single dose ZF-Off construct (SEQ ID NO: 36) mRNA at 1mg / kg in LNPs. HBV DNA and HBsAg were measured from plasma over a period of 168 days. Results are shown in Figure 35A. The treatment resulted in a sustained reduction of greater than 2 log in HBV DNA and similar sustained reduction in HBsAg.

[0350] In another study, mice were injected with the ZF-Off construct (SEQ ID NO: 36)mRNA at 1 mg / kg for three doses: Day 0, Day 21, and Day 42. HBV DNA and HBsAg were measured from plasma over a period of 225 days. Results are shown in Figure 35B. Results were similar to those of the previous single-dose experiment and in this experiment sustained over 225 days. Example 17. Re-dosing studies for CRISPR-Off in AAV-HBV in vivo model

[0351] AAV-HBV mice were dosed with either a single dose or three doses, all at 1 mg / kg inLNPs, of CRISPR-Off (SEQ ID NO: 1248) mRNA with gRNA #008 at a 1:1 ratio of mRNA: guide RNA. For the single dose condition, the dose was administered at Day 0. For the three- -152-WSGR Ref. No. 59073-743.601 dose condition, the doses were administered at Day 36, Day 57, and Day 78. A vehicle-only control was also administered. Plasma measurements of HBV DNA, HBsAg, and HBeAg were taken through Day 168 for the single-dose condition, and through Day 261 for both the three- dose condition and the vehicle control. Results are shown in Figure 36. Re-dosing with CRISPR- Off further improved and sustained the durability of the modulation of these HBV biomarkers.

[0352] In another study, AAV-HBV mice were dosed with either a single dose of CRISPR-Off (SEQ ID NO: 1248) mRNA with gRNA #008 with an updated modification pattern (SEQ ID NO: 1249) (1:1 ratio mRNA: guide RNA) in LNPs at 3 mg / kg, or three doses of the same epigenetic editor, each at 1 mg / kg. Both groups received a dose at Day 0, and the three-dose group also received a dose at Day 14 and at Day 28. A vehicle-only control was also administered. HBsAg and HBeAg were measured from plasma through 126 days. Results are shown in Figure 37. Near-additive pharmacology was demonstrated with the repeat dosing. Example 18. Testing CRISPR-Off and guide RNA modifications in an AAV-HBV in vivo model

[0353] AAV-HBV mice were dosed with a single dose of either CRISPR-Off (SEQ ID NO:1248) mRNA with gRNA #008 or an updated CRISPR-Off variant (SEQ ID NO: 1252) mRNA with gRNA #008 with an updated modification pattern (SEQ ID NO: 1249), with a 1:1 ratio of mRNA to guide RNA at either 0.5 mg / kg or 1 mg / kg, delivered in LNPs. A vehicle only control was also administered. HBsAg was measured in plasma over 28 days. Results are shown in Figure 38. The updated CRISPR-Off variant with guide RNA modifications demonstrated 1.5x potency over the previous lead epigenetic editor. Example 19. Methylation studies for CRISPR-Off with various guide RNAs

[0354] HepG2.2.15 cells were dosed at 1 nanogram (ng) / microliter (100 ng) of 1:1 CRISPR-Off (SEQ ID NO: 1248) mRNA with various single guide RNAs in LNPs with commercial apolipoprotein E (to aid LNP entry). Methylation profiles were performed on the HBV genome samples as well as controls: for gRNA #008, untreated samples and treated with CRISPRi and wild type Cas9. For other gRNAs tested, an untreated sample (APOE only) was used as a control. Results for gRNA #008, gRNA #003, gRNA #007, gRNA #009, gRNA #011, and gRNA #015 are shown in Figures 39A, 39B, 39C, 39D, 39E, and 39F, respectively. A control for the application of an off-target PCSK9 guide RNA is shown in Figure 39G. -153-WSGR Ref. No. 59073-743.601 Example 20. Specificity studies for CRISPR-Off and ZF Off

[0355] HepG2.2.15 cells were transfected with either ZF-Off (SEQ ID NOs: 36 and 73)mRNA or CRISPR-Off (SEQ ID NO: 1248) mRNA with gRNA #008 in research-grade LNPs. RNA-Seq was conducted to determine differentially expressed genes (DEGs), and the Twist panel was used to determine differentially methylated regions (DMRs) at CpG-enriched sites. Differentially expressed genes (DEG) and differentially methylated regions (DMR) are defined based on literature reviews, software recommendations, sequencing depth and controls DEGs are genes that have >= 2-fold change and with adjusted p-value <=1e-05. DMRs are defined as regions with a minimum of 10 CpGs, with 5x coverage, p-value of <=1e-10 and min average change in methylation (beta) >=20%. Results are shown in Figure 40. Silencing data for same samples was also obtained. Results are shown in Figure 41. Example 21. Dose response of guide RNAs in vitro

[0356] An 8-point dose-response (two-fold dilution with from 4 ng / μL (400ng) to 0.031ng / μL (3.1 ng)) was generated using HepG2.2.15 cells treated with LNPs with CRISPR-Off effector (SEQ ID NO: 1248), delivered as mRNA, and each of four gRNAs co-formulated in a 1:1 ratio. HBsAg and HBeAg were measured over six days. Results are shown in Figure 42. Example 22. Dose response of CRISPR-Off variant in vitro

[0357] HepG2.2.15 cells transfected via Messenger Max with CRISPR-Off effector (SEQ IDNO: 1252), delivered as mRNA, and gRNA #008 with updated modification pattern (SEQ ID NO: 1249) was used to generate a 9-point dose-response (200-0.8 ng) curve. HBsAg and HBeAg were measured over 6 days. Results are shown in Figure 43. Example 23. Multiplexing Study in AAV-HBV and Tg-HBV Mouse Models

[0358] AAV-HBV and Tg-HBV mice are injected with a single administration at 0.5 mg / kgof one, two, three, or four guide RNAs targeting regions listed in Table 15 and Table 16 with CRISPR-Off (SEQ ID NO: 1248 or 1252) mRNA formulated in LNPs.

[0359] Amongst others, the following gRNAs are combined: (1) gRNA#008 andgRNA#011;(2) gRNA#008 and gRNA#003; (3) gRNA#008 and gRNA#015; (4) gRNA#008, gRNA#011, and gRNA#015; (6) gRNA#008, gRNA#011, and gRNA#003. Treatment with a single guide -154-WSGR Ref. No. 59073-743.601 RNA, e.g., gRNA#008 or gRNA#011 serves as a positive control, and treatment with vehicle or with a non-targeting guide as a negative control.

[0360] One or more of HBV DNA, HBsAg, and HBeAg are assayed in plasma of the mice atone or more time points after administration, and the mouse liver is collected for further analysis. Combinations of multiple guides yield silencing at least as robust as treatment with single guides. In some cases, more robust silencing with multiple guides as compared to treatment with a single guide is observed. Example 24. Testing mRNA: guide RNA ratios in vivo

[0361] AAV-HBV mice are treated with CRISPR-Off effector (SEQ ID NO: 1252) mRNAwith guide RNA (SEQ ID NO: 1249) in ratios including 1:1, 1:1.5, 2:1, 1:2, and 1:3 mRNA:guide RNA formulated into LNPs and administered at 0.5 mg / kg. 5 or 6 mice per study group are used. An optimized ratio of effector and guide RNA is identified that results in durable reduction of one or more HBV biomarkers, e.g., plasma level measurements of HBV DNA, HBsAg, and HBeAg of greater than 2 log below the observed control plasma level. Example 25. Combination Treatment with Epigenetic Editor in vivo

[0362] Tg-HBV mice are dosed with Entecavir (ETV) at 0.1 mg / kg for 14 days followed byCRISPR-Off with guide RNA at 1 mg / kg in a single intravenous dose. HBV DNA and HBsAg are measured in plasma for 112 days. HBV DNA levels drop after ETV treatment and there is slight synergism in the CRISPR-Off with guide with ETV group. After ETV withdrawal, the CRISPR-Off with guide maintains sustained reduction of DNA comparable to a group treated with CRISPR-Off and guide RNA alone. The addition of ETV does not affect HBsAg.Example 26. Stable HBV Silencing via Epigenetic Editing in Non-Transgenic Mouse Modelof Persistent HBV infection

[0363] A non-transgenic model of persistent HBV infection (AAV-HBV) inimmunocompetent mice was used, which was established by administering an adeno-associated viral vector (AAV) that contains HBV Genotype D DNA into the mice. The administration of the AAV-HBV vector resulted in expression of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and high levels of serum HBV DNA in the mice. -155-WSGR Ref. No. 59073-743.601

[0364] The CRISPR-off and ZF-off constructs are tested. Constructs are delivered via IVadministration of mRNA / gRNA (CRISPR-Off) or mRNA (ZF-Off) formulated into a lipid nanoparticle (LNP) at 2.5 mg / kg and 0.5 mg / kg for CRISPR-Off and ZF-Off, respectively. Some constructs are formulated in LNP compositions as described in PCT / US2014 / 070882, US20220402862A1, and / or US20230203480A1. A subset of the mice are re-dosed at two weeks after the first dose; a second subset are re-dosed at one month after the first dose. The readouts are circulating viral DNA, HBsAg, and HBeAg, tested using mouse plasma at one or more time points (such as 7, 14, 28, and 35 days). A durable and significant reduction in the levels of one or more of HBV DNA, HBsAg, and HBeAg is observed for some constructs.

[0365] Longer-term durability is tested over three to six months using the HBV DNA,HBsAg, and HBeAg markers. Progressive and durable reduction in one or more of these markers is seen with delivery of some constructs. The mice are sacrificed and livers are collected for further analysis, and durable silencing is confirmed by at least 2 log reduction of HBsAg and HBV DNA. Example 27: Stable HBV Silencing via Epigenetic Editing in Transgenic Mice Expressing viral HBV DNA

[0366] A transgenic mouse model of persistent HBV infection (Tg-HBV) was used, whosegenome was engineered to integrate HBV Genotype A DNA, resulting in expression of HBsAg and HBeAg, and circulating viral DNA in the mice.

[0367] The CRISPR-off and ZF-off constructs are tested. Constructs are delivered via IVadministration of mRNA / gRNA (CRISPR-Off) or mRNA (ZF-Off) formulated into LNP at 2.5 mg / kg and 0.5 mg / kg for CRISPR-Off and ZF-Off, respectively. Some constructs are formulated in LNP compositions as described in US20220402862A1, and / or US20230203480A1. A subset of the mice are re-dosed at two weeks after the first dose; a second subset are re-dosed at one month after the first dose. The readouts are circulating viral DNA, HBsAg, and HBeAg, tested using mouse plasma at one or more time points (such as 7, 14, 28, and 35 days). A durable and significant reduction in the levels of one or more of HBV DNA, HBsAg, and HBeAg is observed for some constructs.

[0368] Longer-term durability is tested over three to six months using the HBV DNA,HBsAg, and HBeAg markers. Progressive and durable reduction in one or more of these markers is seen with delivery of some constructs. The mice are sacrificed and livers are collected for -156-WSGR Ref. No. 59073-743.601 further analysis, and durable silencing is confirmed by at least 2 log reduction of HBsAg and HBV DNA.Example 28: Further testing of CRISPR-Off variant and gRNA #008 with updatedmodification pattern in vitro

[0369] HepG2.2.15 cells transfected via Messenger Max with CRISPR-Off effector (SEQ IDNO: 1252), delivered as mRNA (SEQ ID NO: 1257), and gRNA #008 with updated modification pattern (SEQ ID NO: 1249) was used to generate an 8-point dose-response curve. HBsAg and HBeAg were measured over 6 days. Results are shown in Figure 44A. The same effector and gRNA were used to test % HBsAg expression in HepG2.2.15 cells relative to a non-HBV silencing control. Results are shown in Figure 44B. This combination of effector and guide RNA strongly reduces viral markers in and HepG2.2.15 cells containing full-length integrated HBV DNA. Silencing is durable up to day 44 in dividing cells. Example 29: Testing of CRISPR-Off variant: gRNA ratio and dosage in vivo

[0370] AAV-HBV mice were used to test various ratios of mRNA / gRNA and dosages of theepigenetic editor protein with gRNA in vivo using CRISPR-Off effector (SEQ ID NO: 1252), delivered as mRNA (SEQ ID NO:1257), and gRNA #008 with updated modification pattern (SEQ ID NO: 1249). The mRNA and gRNA were encapsulated in LNP. Log (HBsAg) reduction was used to determine favorable ratios and dosages. Ratio results were obtained at Day 14 after administration and are shown in Figure 45A. Dosage results were tested at Day 14, Day 21, and Day 28 and results are shown in Figure 45B. Study is ongoing. Example 30: Testing anti-HBs in Tg-HBV

[0371] A second dose response experiment in Tg-HBV model using CRISPR-Off (SEQ IDNO: 1248) mRNA with guide RNA #008 (HBV-EE) formulated in LNPs was conducted, with administrations at 0.5 mg / kg, 1 mg / kg, or 3 mg / kg of 1:1 mRNA:guide RNA. A vehicle-only control was also used. In this experiment, plasma was tested for anti-HBs through 207 days after injection. Results for individual mice at the final time point of 207 days after injection are plotted in Figure 46. HBsAg reduction correlates with the presence of anti-HBs antibodies (as shown in Figure 33) in Tg-HBV mice. All animals treated with HBV-EE with undetectable levels of HBsAg at day 207 had measur...

Claims

WSGR Ref. No. 59073-743.601 CLAIMS What is claimed is:

1. A composition comprising:a. an RNA encoding an epigenetic editor protein, wherein the RNA comprises asequence with at least 95% sequence identity to SEQ ID NO: 1274; and b. a guide RNA comprises a spacer sequence, wherein the spacer sequence iscomplementary to a target region of a strand of an HBV gene or genome.

2. The composition of claim 1, wherein the RNA and the guide RNA are encapsulated in alipid nanoparticle.

3. The composition of claim 1, wherein the RNA and the guide RNA are encapsulated in thelipid nanoparticle at a ratio from about 1:1 to about 1:3 by weight.

4. The composition of claim 3, wherein the RNA and the guide RNA are encapsulated in thelipid nanoparticle at a ratio of about 1:1 by weight.

5. The composition of claim 3, wherein the RNA and the guide RNA are encapsulated in thelipid nanoparticle at a ratio of about 1:1.5 by weight.

6. The composition of claim 3, wherein the RNA and the guide RNA are encapsulated in thelipid nanoparticle at a ratio of about 1:3 by weight.

7. The composition of claim 1, wherein the RNA and guide RNA are encapsulated in thelipid nanoparticle at a ratio from about 1:2 to about 1:4 by weight.

8. The composition of claim 1, wherein the RNA and guide RNA are encapsulated in thelipid nanoparticle at a ratio from about 1:3 to about 1:5 by weight.

9. The composition of any one of claims 1-8, wherein the RNA comprises a sequence withat least 98% sequence identity to SEQ ID NO: 1274.

10. The composition of any one of claims 1-9, wherein the RNA comprises a sequence setforth in SEQ ID NO: 1274.

11. The composition of any one of claims 1-10, wherein the spacer sequence comprises asequence set forth in SEQ ID NO: 391.

12. The composition of any one of claims 1-11, wherein the guide RNA comprises a nucleicacid base sequence, wherein the nucleic acid base sequence comprises a sequence with at least 80% sequence identity to AGGAGUUCCGCAGUAUGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 1260), or to -282-WSGR Ref. No. 59073-743.601 AGGAGUUCCGCAGUAUGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAA GGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 1280), wherein A, G, C and U represent adenine, guanine, cytosine and uracil, respectively.

13. The composition of claim 12, wherein the nucleic acid base sequence comprises asequence set forth in SEQ ID NO: 1260.

14. The composition of any one of claims 1-13, wherein the guide RNA comprises one ormore chemically modified nucleotide(s).

15. The composition of claim 14, wherein the one or more chemically modified nucleotide(s)are at one or more nucleotide position(s) selected from 1-3, 30-40, 69-81 and 83-100 as numbered in SEQ ID NO: 1260 or corresponding position(s) thereof.

16. The composition of any one of claims 1-15, wherein the guide RNA comprises a nucleicacid sequence of mA*mG*mG*rArGrUrUrCrCrGrCrArGrUrArUrGrGrArUrGrUrUrUrUrArGrArGmCm UmAmGmAmAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCr CrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAmGmUmGrGmCmAmCmCmG mAmGmUmCmGmGmUmGmCmU*mU*mU*mU (SEQ ID NO: 1249), or of mA*mG*mG*rArGrUrUrCrCrGrCrArGrUrArUrGrGrArUrGrUrUrUrUrArGrArGmCm UmAmGmAmAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCr CrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAmGmUmGrGmCmAmCmCmG mAmGmUmCmGmGmUmGmC (SEQ ID NO: 1279), wherein (1) mA, mG, mC and mU represent a 2’-OMe modified adenosine, guanosine, cytidine and uridine, respectively, (2) rA, rG, rC and rU represent adenosine, guanosine, cytidine and uridine, respectively, and (3) * indicates a phosphorothioate bond.

17. The composition of any one of claims 1-16, wherein the epigenetic editor proteincomprises: a) a DNA binding domain; b) two DNA methyltransferase (DNMT) domains; c) a repressor domain; d) a first peptide linker between the DNA binding domain and one of the DNMT domains, and a second peptide linker between the DNA binding domain and the repressor domain, wherein the first peptide linker is XTEN80, wherein the second peptide linker is XTEN16; and -283-WSGR Ref. No. 59073-743.601 e) two bipartite nuclear localization sequences (NLS), and a simian virus (SV40) NLS.

18. The composition of claim 17, wherein one of the two bipartite NLSs is positioned at anamino (N) terminus of the epigenetic editor protein and the other one of the two NLSs is positioned at a carboxy (C) terminus of the epigenetic editor protein, and wherein the SV40 NLS is positioned at the C terminus of the DNA binding domain.

19. The composition of claim 17 or 18, wherein the DNA binding domain is a dCas9 DNAbinding domain.

20. The composition of any one of claims 17-19, wherein the repressor domain is a ZIM3repressor domain.

21. The composition of any one of claims 17-20, wherein one of the two DNMT domains is aDNMT3A domain22. The composition of any one of claims 17-21, wherein one of the two DNMT domains is aDNMT3L domain.

23. The composition of claim 22, wherein the DNMT3L is derived from a species of theEquus genus.

24. The composition of claim 23, wherein the species of the Equus genus is Equusprezewalskii.

25. The composition of any one of claims 1-24, wherein the epigenetic editor proteincomprises a configuration comprising: bipartite NLS-DNMT3A-DNMT3L-XTEN80- dCas9-SV40 NLS-XTEN16-ZIM3-bipartite NLS, from the N terminus to the C terminus.

26. The composition of any one of claims 1-25, wherein the epigenetic editor proteincomprises a sequence with at least 95% sequence identity to SEQ ID NO: 1252.

27. The composition of any one of claims 1-26, wherein the epigenetic editor proteincomprises a sequence set forth in SEQ ID NO: 1252.

28. A method of treating an HBV infection in a subject comprising administering thecomposition of any one of claims 1-27.

29. A method comprising administering the composition of any one of claims 1-27 to asubject, wherein the subject is characterized by the presence of detectable levels of HBV DNA, HBsAg, and / or HBeAg in the plasma of the subject.

30. The method of claim 28 or 29, wherein the composition is administered throughperipheral intravenous infusion.

31. The method of any one of claims 28-30, wherein the composition is administered once.-284-WSGR Ref. No. 59073-743.60132. The method of any one of claims 28-30, wherein the composition is administered multipletimes.

33. The method of claim 32, wherein the composition is administered once every four weeks.

34. The method of claim 33, wherein the composition is administered once every four weeksfor a total of three administrations.

35. The method of any one of claims 30-34, wherein the composition is administered at adosage of at least about 0.2 mg / kg.

36. The method of claim 35, wherein the composition is administered at a dosage of about 0.2mg / kg.

37. The method of claim 35, wherein the composition is administered at a dosage of about 0.4mg / kg.

38. The method of claim 35, wherein the composition is administered at a dosage of about 0.6mg / kg.

39. The method of claim 35, wherein the composition is administered at a dosage of about 0.8mg / kg.

40. An RNA comprising a sequence set forth in SEQ ID NO: 1274.

41. An RNA comprising a sequence set forth in SEQ ID NO: 1257.-285-

Images