Methods for treating chronic hepatitis b infection and related formulations

Abstract

Provided herein are methods for treating chronic hepatitis B (CHB) infections in human subjects. In some embodiments, the methods involve administering a lipid nanoparticle carrying an epigenetic-modifying DNA targeting system composed of polynucleotides for targeting repression of total Hepatitis B Viral (HBV) transcripts. Also provided is a lipid nanoparticle carrying the polynucleotides.

Description

22474-20039.40METHODS FOR TREATING CHRONIC HEPATITIS B INFECTION AND RELATED FORMULATIONSCross-Reference to Related Applications

[0001] This application claims priority from U.S. provisional application No. 63 / 717,275 filed November 06, 2024, entitled, “METHODS FOR TREATING CHRONIC HEPATITIS B INFECTION AND RELATED FORMULATIONS”, the contents of which are incorporated by reference in its entirety.Incorporation by Reference of Sequence Listing

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 224742003940SeqList.xml, created November 5, 2025, which is 310, 138 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.Field

[0003] The present disclosure relates in some aspects to methods for treating chronic hepatitis B (CHB) infections in human subjects. In some embodiments, the methods involve administering a lipid nanoparticle carrying an epigenetic-modifying DNA targeting system composed of polynucleotides for targeting repression of total Hepatitis B Viral (HBV) transcripts. Also provided is a lipid nanoparticle carrying the polynucleotides.Background

[0004] A large patient population, estimated at one million individuals in the US alone, and 250 million worldwide, deals with chronic Hepatitis B infection. However, current standard of care, including suppression of viral DNA transcription such as administration of nucleoside analogs, PEGylated interferon, anti-sense oligonucleotide, and siRNA approaches face challenges in efficacy and stability. Therefore, there is a need for new and improved methods to overcome these challenges. The present disclosure addresses these and other needs.Summary

[0005] Provided herein is a method of treating chronic hepatitis B (CHB) infection in a subject in need thereof, the method comprising administering to the subject a dose of lipid nanoparticles comprising an epigenetic-modifying DNA-targeting system comprising a DNA-1MF-36384587022474-20039.40 targeting module for repressing transcription of total viral Hepatitis B Viral (HBV) mRNA transcripts. In any of the embodiments herein, the DNA-targeting module comprises: (a) a gRNA for targeting to a target site in a HBV DNA sequence, and (b) an mRNA encoding a fusion protein comprising a deactivated Streptococcus pyogenes Cas9 (dSpCas9) protein and at least one transcriptional repressor effector domain comprising a KRAB domain and a DNA methyltransferase 3 (DNMT3) domain that has DNA methyltransferase activity. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 27, a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing, n any of the embodiments herein, the dose comprises (a) between at or about 0.1 mg / kg body weight of the subject and at or about 1 mg / kg body weight of the subject, or (b) between at or about 10 mg to about 100 mg.

[0006] In any of the embodiments herein, the subject has an HBV-associated viral infection. In any of the embodiments herein, the HBV-associated viral infection is hepatitis D virus infection.

[0007] Also provided herein is a method of treating hepatitis D virus (HDV) infection in a subject in need thereof, the method comprising administering to the subject a dose of lipid nanoparticles comprising an epigenetic-modifying DNA-targeting system comprising a DNA- targeting module for repressing transcription of total viral Hepatitis B Viral (HBV) mRNA transcripts, wherein the DNA-targeting module comprises: (a) a gRNA for targeting to a target site in a HBV DNA sequence, wherein the target site comprises the sequence set forth in SEQ ID NO: 27, a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing; and (b) an mRNA encoding a fusion protein comprising a deactivated Streptococcus pyogenes Cas9 (dSpCas9) protein and at least one transcriptional repressor effector domain comprising a KRAB domain and a DNA methyltransferase 3 (DNMT3) domain that has DNA methyltransferase activity, and wherein the dose comprises (a) between at or about 0.1 mg / kg body weight of the subject and at or about 1.5 mg / kg body weight of the subject, or (b) between at or about 10 mg to about 100 mg.

[0008] In any of the embodiments herein, the dose comprises (a) between at or about 0.1 mg / kg body weight of the subject and at or about 1 mg / kg body weight of the subject, or (b) between at or about 10 mg to about 100 mg.

[0009] In any of the embodiments herein, the target site comprises the sequence set forth in SEQ ID NO: 27.

[0010] In any of the embodiments herein, the fusion protein comprising the KRAB domain2MF-36384587022474-20039.40 comprises a K0X1 domain, a ZNF10 domain, or a ZIM3 domain. In any of the embodiments herein, the KRAB domain comprises a ZNF10 domain. In any of the embodiments herein, the KRAB domain comprises the sequence set forth in SEQ ID NO: 28, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:28. In any of the embodiments herein, the DNMT3 domain comprises a DNMT3A domain. In any of the embodiments herein, the DNMT3 A comprises the sequence set forth in SEQ ID NO: 22, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 22. In any of the embodiments herein, the DNMT3A comprises the sequence set forth in SEQ ID NO: 22. In any of the embodiments herein, the DNMT3 domain comprises a DNMT3L domain or a variant thereof. In any of the embodiments herein, the DNMT3 domain comprises the sequence set forth in SEQ ID NO: 23, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 23. In any of the embodiments herein, the DNMT3 domain comprises the sequence set forth in SEQ ID NO: 23.

[0011] In any of the embodiments herein, the DNMT3 domain comprises a DNMT3 A domain and a DNMT3L domain (DNMT3A / L domain). In any of the embodiments herein, the fusion protein comprises from the N-terminus to the C-terminus: a DNMT3 A domain and a DNMT3L domain (DNMT3A / L domain), a dSpCas9 domain, and a KRAB domain. In any of the embodiments herein, the DNMT3 domain is a DNMT3A / L domain and the DNMT3 A domain and the DNMT3L domain are separated by a linker. In some embodiments, the linker comprises the sequence set forth in SEQ ID NO: 34. In any of the embodiments herein, the DNMT3 domain is a DNMT3A / L domain and comprises the sequence set forth in SEQ ID NO: 42 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 42. In any of the embodiments herein, the fusion protein comprises a sequence comprising at least 90% sequence identity to SEQ ID NO: 42 and a sequence comprising at least 90% sequence identity to SEQ ID NO: 28.

[0012] In any of the embodiments herein, the fusion protein comprising the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO: 24. In any of the embodiments herein, the dSpCas9 protein comprises the sequence set forth in SEQ ID NO: 25, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 25. In any of the embodiments herein, the dSpCas9 protein comprises the sequence set forth in SEQ ID NO: 25.3MF-36384587022474-20039.40

[0013] In any of the embodiments herein, the fusion protein further comprises one or more nuclear localization signals (NLS). In some embodiments, the NLS is at the N- or C-terminus, between N- or C-terminus and the KRAB domain, between the N- or C-terminus and the DNMT3A / L domain, between the dSpCas9 and the DNMT3A / 3L domains, or between the dSpCas9 and the KRAB domain. In some embodiments, the NLS comprises the sequence set forth in any one of SEQ ID NOS: 13, 16, 18, a portion thereof, or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the foregoing. In some embodiments, the NLS comprises the sequence set forth in any one of SEQ ID NOS: 13, 16, and 18.

[0014] In any of the embodiments herein, the fusion protein further comprises one or more linkers connecting two or more of: the DNMT3 A domain, the DNMT3L domain, the dSpCas9, the KRAB domain, and the one or more nuclear localization signals. In some embodiments, the one or more linkers is independently selected from a linker comprising the sequence set forth in any one of SEQ ID NOS: 11, 12, 14, 15, or 17.

[0015] In any of the embodiments herein, the mRNA further comprises one or more untranslated regions (UTR) near the 5’ end and / or the 3’ end. In any of the embodiments, the mRNA further comprises a UTR near the 5’ end and near the 3’ end. In some embodiments, the UTR near the 5’ end comprises a sequence set forth in SEQ ID NO: 9 or a sequence that has at least 90% sequence identity to SEQ ID NO: 9 and the UTR near the 3’ end comprises a sequence set forth in SEQ ID NO: 10 or a sequence that has at least 90% sequence identity to SEQ ID NO: 10. In any of the embodiments herein, the mRNA further comprises a polyA sequence at the 3’ end. In any of the embodiments herein, the mRNA comprises from 5’ end to the 3’ end: a UTR, a nucleotide sequence encoding the fusion proteina UTR, and a polyA sequence.

[0016] In any of the embodiments herein, the mRNA encoding the fusion protein comprises from the 5’ end to the 3’ end: the UTR comprising the sequence set forth in SEQ ID NO: 9, a nucleotide sequence encoding the fusion protein, the UTR comprising the sequence set forth in SEQ ID NO: 10, and a polyA sequence comprising the sequence set forth in SEQ ID NO: 19. In some embodiments, the nucleotide sequence encoding the fusion protein comprises, in order from the 5’ end to the 3’ end, nucleotides encoding: a DNMT3A domain, a linker, the DNMT3L domain, a linker, a NLS, a linker, the dSpCas9, a linker, a NLS, a linker, the KRAB, and a NLS. In some embodiments, the nucleotide sequence encoding the fusion protein comprises, in order from the 5’ end to the 3’ end: a nucleotide sequence encoding the DNMT3A4MF-36384587022474-20039.40 domain comprising the sequence set forth in SEQ ID NO: 2, a nucleotide sequence encoding a linker comprising the sequence set forth in SEQ ID NO: 11, a nucleotide sequence encoding the DNMT3L domain comprising the sequence set forth in SEQ ID NO: 3, a nucleotide sequence encoding the linker comprising the sequence set forth in SEQ ID NO: 12, a nucleotide sequence encoding an NLS comprising the sequence set forth in SEQ ID NO: 13, a nucleotide sequence encoding a linker comprising the sequence set forth in SEQ ID NO: 14, a nucleotide sequence encoding the dSpCas9 comprising the sequence set forth in SEQ ID NO: 4, a nucleotide sequence encoding a linker comprising the sequence set forth in SEQ ID NO: 15, a nucleotide sequence encoding an NLS comprising the sequence set forth in SEQ ID NO: 16, a nucleotide sequence encoding the linker comprising the sequence set forth in SEQ ID NO: 17, a nucleotide sequence encoding the KRAB comprising the sequence set forth in SEQ ID NO: 5, and a nucleotide sequence encoding the NLS comprising the sequence set forth in SEQ ID NO: 18.

[0017] In any of the embodiments herein, the mRNA comprises the sequence set forth in SEQ ID NO: 1, or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1.

[0018] Also provided herein is a method of treating chronic hepatitis B (CHB) infection in a subject in need thereof, the method comprising administering to the subject a dose of lipid nanoparticles comprising an epigenetic-modifying DNA-targeting system comprising a DNA- targeting module for repressing total viral Hepatitis B Viral (HBV) mRNA transcripts. In any of the embodiments herein, the DNA-targeting module comprises: (a) a gRNA for targeting to a target site in a HBV DNA sequence and (b) an mRNA comprising from the 5’ end to the 3’ end a UTR comprising the sequence set forth in SEQ ID NO: 9, a nucleotide encoding a fusion protein, a UTR comprising the sequence set forth in SEQ ID NO: 10, and a polyA sequence comprising the sequence set forth in SEQ ID NO: 19. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 27. In some embodiments, the nucleotide sequence encoding the fusion protein comprises, in order from the 5’ end to the 3’ end: a nucleotide sequence encoding the DNMT3 A domain comprising the sequence set forth in SEQ ID NO: 2, a nucleotide sequence encoding a linker comprising the sequence set forth in SEQ ID NO: 11, a nucleotide sequence encoding the DNMT3L domain comprising the sequence set forth in SEQ ID NO: 3, a nucleotide sequence encoding the linker comprising the sequence set forth in SEQ ID NO: 12, a nucleotide sequence encoding an NLS comprising the sequence set forth in SEQ ID NO: 13, a nucleotide sequence encoding a linker comprising the sequence set forth in SEQ ID NO: 14, a nucleotide sequence encoding the dSpCas9 comprising the sequence5MF-36384587022474-20039.40 set forth in SEQ ID NO: 4, a nucleotide sequence encoding a linker comprising the sequence set forth in SEQ ID NO: 15, a nucleotide sequence encoding an NLS comprising the sequence set forth in SEQ ID NO: 16, a nucleotide sequence encoding the linker comprising the sequence set forth in SEQ ID NO: 17, a nucleotide sequence encoding the KRAB comprising the sequence set forth in SEQ ID NO: 5, and a nucleotide sequence encoding the NLS comprising the sequence set forth in SEQ ID NO: 18. In some embodiments, the dose comprises (a) between at or about 0.1 mg / kg and at or about 1 mg / kg, or (b) between at or about 10 mg to about 100 mg.

[0019] Also provided herein is a method of treating chronic hepatitis B (CHB) infection in a subject in need thereof, the method comprising administering to the subject a dose of lipid nanoparticles comprising (a) a gRNA for targeting to a target site in a HBV DNA sequence, and (b) a mRNA comprising the sequence set forth in SEQ ID NO: 1 or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 27. In some embodiments, the dose comprises (a) between at or about 0.1 mg / kg and at or about 1 mg / kg, or (b) between at or about 10 mg to about 100 mg.

[0020] In any of the embodiments herein, the subject has an HBV-associated viral infection. In any of the embodiments herein, the HBV-associated viral infection is hepatitis D virus infection.

[0021] Also provided herein is a method of treating a hepatitis D virus infection in a subject in need thereof, the method comprising administering to the subject a dose of lipid nanoparticles comprising an epigenetic-modifying DNA-targeting system comprising a DNA-targeting module for repressing total viral Hepatitis B Viral (HBV) mRNA transcripts, wherein the DNA- targeting module comprises: (a) a gRNA for targeting to a target site in a HBV DNA sequence, wherein the target site comprises the sequence set forth in SEQ ID NO: 27; and (b) an mRNA comprising from the 5’ end to the 3’ end a UTR comprising the sequence set forth in SEQ ID NO: 9, a nucleotide encoding a fusion protein, a UTR comprising the sequence set forth in SEQ ID NO: 10, and a polyA sequence comprising the sequence set forth in SEQ ID NO: 19, wherein the nucleotide sequence encoding the fusion protein comprises , in order from the 5’ end to the 3’ end: a nucleotide sequence encoding the DNMT3A domain comprising the sequence set forth in SEQ ID NO: 2, a nucleotide sequence encoding a linker comprising the sequence set forth in SEQ ID NO: 11, a nucleotide sequence encoding the DNMT3L domain comprising the sequence set forth in SEQ ID NO: 3, a nucleotide sequence encoding the linker comprising the sequence set forth in SEQ ID NO: 12, a nucleotide sequence encoding an NLS comprising the sequence6MF-36384587022474-20039.40 set forth in SEQ ID NO: 13, a nucleotide sequence encoding a linker comprising the sequence set forth in SEQ ID NO: 14, a nucleotide sequence encoding the dSpCas9 comprising the sequence set forth in SEQ ID NO: 4, a nucleotide sequence encoding a linker comprising the sequence set forth in SEQ ID NO: 15, a nucleotide sequence encoding an NLS comprising the sequence set forth in SEQ ID NO: 16, a nucleotide sequence encoding the linker comprising the sequence set forth in SEQ ID NO: 17, a nucleotide sequence encoding the KRAB comprising the sequence set forth in SEQ ID NO: 5, and a nucleotide sequence encoding the NLS comprising the sequence set forth in SEQ ID NO: 18, wherein the dose comprises (a) between at or about 0.1 mg / kg and at or about 1.5 mg / kg, or (b) between at or about 10 mg to about 100 mg.

[0022] Also provided herein is a method of treating a hepatitis D virus infection in a subject in need thereof, the method comprising administering to the subject a dose of lipid nanoparticles comprising (a) a gRNA for targeting to a target site in a HBV DNA sequence, wherein the target site comprises the sequence set forth in SEQ ID NO: 27; and (b) a mRNA comprising the sequence set forth in SEQ ID NO: 1 or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1, wherein the dose comprises (a) between at or about 0.1 mg / kg and at or about 1.5 mg / kg, or (b) between at or about 10 mg to about 100 mg.

[0023] In any of the embodiments herein, the dose comprises (a) between at or about 0.1 mg / kg and at or about 1.0 mg / kg, or (b) between at or about 10 mg to about 100 mg

[0024] In any of the embodiments herein, the mRNA comprises the sequence set forth in SEQ ID NO: 1.

[0025] In any of the embodiments herein, the gRNA for targeting to the target site comprises a spacer sequence comprising the sequence set forth in SEQ ID NO: 26, a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing. In some embodiments, the spacer sequence is 17 to 24 nucleotides in length. In some embodiments, the spacer sequence is at or about 20 nucleotides in length.

[0026] In any of the embodiments herein, the gRNA comprises a spacer sequence for targeting to the target site that comprises the sequence set forth in SEQ ID NO: 26. In any of the embodiments herein, the gRNA comprises a spacer sequence for targeting to the target site that is set forth in SEQ ID NO: 26.

[0027] In any of the embodiments herein, the gRNA comprises a scaffold sequence for dSpCas9. In some embodiments, the scaffold sequence comprises a sequence set forth in SEQ ID NO: 39 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,7MF-36384587022474-20039.4096%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 39. In some embodiments, the scaffold sequence is set forth in SEQ ID NO: 39.

[0028] In any of the embodiments herein, the gRNA for targeting to the target site comprises the sequence set forth in SEQ ID NO: 38 or a complementary sequence of the foregoing.

[0029] In any of the embodiments herein, the gRNA for targeting to the target site is set forth in SEQ ID NO: 38.

[0030] In any of the embodiments herein, the lipid nanoparticles comprise a cationic lipid having the following structure (I):or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. In some embodiments, R1is optionally substituted C1-C24 alkyl or optionally substituted C2-C24 alkenyl; R2and R3are each independently optionally substituted C1-C36 alkyl; R4and R5are each independently optionally substituted Ci-Ce alkyl, or R4and R5join, along with the N to which they are attached, to form a heterocyclyl or heteroaryl; L1, L2, and L3are each independently optionally substituted Ci-Cis alkylene; G1is a direct bond, -(CH2)nO(C=O)-, - (CH2)n(C=O)O-, or -(C=O)-; G2and G3are each independently -(C=O)O- or -O(C=O)-; and n is an integer greater than 0.

[0031] In some embodiments, the cationic lipid is selected from a compound in Table 1. In some embodiments herein, the cationic lipid is 1-18.

[0032] In any of the embodiments herein, the lipid nanoparticles comprise a pegylated lipid having the following structureor a pharmaceutically acceptable salt, tautomer or stereoisomer thereof. In some embodiments, R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms. In some embodiments, the8MF-36384587022474-20039.40 alkyl chain is optionally interrupted by one or more ester bonds; and z has a mean value ranging from 30 to 60.

[0033] In some embodiments, the pegylated lipid is Ila.

[0034] In any of the embodiments herein, the lipid nanoparticles comprises: an cationic lipid that is bis(2 -butyloctyl) 10-(N-decyl-4-(dimethylamino)butanamido)nonadecanedioate, l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), a PEGylated lipid that is 2-[2-(co-methoxy (polyethyleneglycol2000) ethoxy]-N,N-ditetradecylacetamide, and cholesterol. In some embodiments, the cationic lipid / DSPC / PEGylated lipid / cholesterol are formulated in a ratio of 47.5 / 10 / 2.5 / 40 mol %. In some embodiments, the ratio of cationic lipid to mRNA (N / P) is 6.0.

[0035] In any of the embodiments herein, the total RNA in the LNP is 1 mg.

[0036] In any of the embodiments herein, the ratio of the mRNA encoding the fusion protein and the gRNA is 2: 1.

[0037] In any of the embodiments herein, the lipid nanoparticle is formulated in 300 mM sucrose in phosphate buffered saline at pH about 7.0 to about 7.4.

[0038] In any of the embodiments herein, the dose is an amount between at or about 0.1 mg / kg body weight of the subject and about 1 mg / kg body weight of the subject. In any of the embodiments herein, the dose is an amount between at or about 0.15 mg / kg body weight of the subject and about 0.8 mg / kg body weight of the subject, between at or about 0.3 mg / kg body weight of the subject and at or about 0.65 mg / kg body weight of the subject, or between at or about 0.2 mg / kg body weight of the subject and at or about 0.45 mg / kg body weight of the subject.

[0039] In any of the embodiments herein, the dose is at or about 0.2 mg / kg body weight of the subject. In any of the embodiments herein, the dose is at or about 0.45 mg / kg body weight of the subject. In any of the embodiments herein, the dose is at or about 0.65 mg / kg body weight of the subject. In any of the embodiments herein, the dose is greater than 0.65 mg / kg body weight of the subject and less than 100 mg / kg body weight of the subject. In any of the embodiments herein, if the subject is 100 kg or greater then the dose is no more than 100 mg. In any of the embodiments herein, the dose is an amount between about 10 to about 100 mg. In any of the embodiments herein, the dose is an amount from at or about 12 mg to about 52 mg, from at or about 12 mg to about 39 mg, from at or about 12 mg to about 36 mg, from at or about 12 mg to about 27 mg, from at or about 12 mg to at or about 16 mg, from at or about 27 mg to at or about 100 mg, from at or about 27 mg to about 39 mg or from at or about 52 mg to at our about 100 mg.9MF-36384587022474-20039.40

[0040] In any of the embodiments herein, the dose is from about 75 mg to about 90 mg.

[0041] In any of the embodiments herein, a single dose of the lipid nanoparticles is administered to the subject.

[0042] In any of the embodiments herein, comprising administering two or more doses of the lipid nanoparticles to the subject as part of a multiple-dose regimen.

[0043] In any of the embodiments herein, two, three, four, five, or six doses of the lipid nanoparticles are administered to the subject.

[0044] In any of the embodiments herein, the multipe-dose regimen is a finite multipledosing regimen in which a finite number of discrete doses are administered over a defined treatment period.

[0045] In any of the embodiments herein, the finite multiple-dosing regimen comprises two, three, four, five, or six total doses.

[0046] In any of the embodiments herein, each dose of the multiple-dose regimen independently comprises a dosage amount or range as recited in any embodiment.

[0047] In any of the embodiments herein, each dose of the multiple-dose regimen independently is from about 0.1 to about 1 mg / kg, optionally about 0.2 mg / kg, 0.45 mg / kg, or 0.65 mg / kg.

[0048] In any of the embodiments herein, each dose of the multiple-dose regimen is a fixed amount from about 10 to 100 mg, optionally about 12 mg, 27 mg, 39 mg, 52 mg, or 100 mg, or a value between any of the foregoing.

[0049] In any of the embodiments herein, the multiple-dose regimen comprises 2 to 6 doses administered once every 14 to 60 days.

[0050] In any of the embodiments herein, each dose of the multiple-dose regimen is administered once every 21 to 42 days.

[0051] In any of the embodiments herein, the each dose of the multiple-dose regimen is administered every two weeks (Q2W), once every 28 days (Q4W), or once every six weeks (Q6W).

[0052] In any of the embodiments herein, the multiple-dose regimen is selected from three doses once every 28 days (Q4Wx3), four doses once every 28 days (Q4Wx4), three doses once every 21 days (Q3Wx3), or four doses once every six weeks (Q6Wx4).

[0053] In any of the embodiments herein, the multiple-dose regimen comprises three doses once every 28 days (Q4Wx3).

[0054] In any of the embodiments herein, the lipid nanoparticles are administered via10MF-36384587022474-20039.40 intravenous administration.

[0055] In any of the embodiments herein, the subject is a human. In any of the embodiments herein, the subject is an adult. In any of the embodiments herein, the subject is 12 years or older. In any of the embodiments herein, the subject has a weight of at least 40 kg.

[0056] In any of the embodiments herein, the subject has hepatocytes comprising cccDNA and / or integrated HBV DNA. In any of the embodiments herein, the subject has hepatocytes comprising cccDNA and integrated HBV DNA. In any of the embodiments herein, the subject has hepatocytes expressing HBV proteins. In some embodiments, the HBV proteins are HBsAg, HBeAg, or HBcrAg and combinations thereof.

[0057] In any of the embodiments herein, the subject has been diagnosed as positive for HBsAg for at least about 12 months. In any of the embodiments herein, the subject has an HBsAg titer of at least about 500 lU / mL.

[0058] In any of the embodiments herein, the subject has received treatment with a nucleoside analog for at least about 12 months. In some embodiments, the nucleoside analog comprises one or more of entecavir, tenofovir disoproxil fumarate, or tenofovir alafenamide. In some embodiments, the subject is receiving the nucleoside analog at the time of administration of the lipid nanoparticle and continues to receive the nucleoside analog. In some embodiments, the treatment with the nucleoside analog is discontinued at the time of administration of the lipid nanoparticle.

[0059] In any of the embodiments herein, the subject has or has been diagnosed as being positive for HBV DNA levels of less than about 90 U / mL for at least 6 months.

[0060] In any of the embodiments herein, the subject (a) has or has been diagnosed with chronic Hepatitis B (CHB) defined as HBsAg positive for 12 months or more than 12 months; (b) Has baseline titers of HBsAg of more than 500 lU / ml; (c) Has received treatment with a nucleoside analog. In some embodiments, the nucleoside analog is entecavir (ETV), tenofovir disoproxil fumarate (TDF) or tenofovir alafenamide (TAF) for 12 months of more than 12 months with a stable dose for 6 months or more than 6 months; and (d) Has HBV DNA of less than 90 lU / ml for 6 months or less than 6 months.

[0061] In any of the embodiments herein, the method reduces the levels of Hepatitis B virus in the subject. In any of the embodiments herein, the method reduces Hepatitis B virus RNA and / or protein levels in the subject. In any of the embodiments herein, the expression of HBV DNA and / or pre-genomic (pgRNA) is reduced. In any of the embodiments herein, the levels of HBsAg, HBeAg, HBcrAg, and / or HBeAg and / or p-HBcAg are reduced. In any of the11MF-36384587022474-20039.40 embodiments herein, the levels of stiffness and / or fat are changed. In any of the embodiments herein, the levels of anti-HBsAg, anti-HBcAg, and / or anti-HBeAg antibodies and / or HBV- specific PBMC immune markers are reduced.

[0062] Also provided herein is a lipid nanoparticle comprising an epigenetic-modifying DNA-targeting system comprising a DNA-targeting module for repressing total viral Hepatitis B Viral (HBV) mRNA transcripts. In some embodiments, the DNA-targeting module comprises: (a) a gRNA for targeting to a target site in a HBV DNA sequence, and (b) an mRNA encoding a fusion protein comprising a deactivated Streptococcus pyogenes Cas9 (dSpCas9) protein and at least one transcriptional repressor effector domain comprising a KRAB domain and a DNA methyltransferase 3 (DNMT3) domain that has DNA methyltransferase activity. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 27, a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing. In some embodiments, the lipid nanoparticles comprise a cationic lipid having the following structure (I):or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. In some embodiments, R1is optionally substituted C1-C24 alkyl or optionally substituted C2-C24 alkenyl; R2and R3are each independently optionally substituted C1-C36 alkyl; R4and R5are each independently optionally substituted Ci-Ce alkyl, or R4and R5join, along with the N to which they are attached, to form a heterocyclyl or heteroaryl; L1, L2, and L3are each independently optionally substituted Ci-Cis alkylene; G1is a direct bond, -(CH2)nO(C=O)-, - (CH2)n(C=O)O-, or -(C=O)-; G2and G3are each independently -(C=O)O- or -O(C=O)-; and n is an integer greater than 0. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 27. In some embodiments, the fusion protein comprising the KRAB domain comprises a K0X1 domain, a ZNF10 domain, or a ZIM3 domain.

[0063] In any of the embodiments herein, the KRAB domain comprises a ZNF10 domain. In any of the embodiments herein, the KRAB domain comprises the sequence set forth in SEQ ID NO: 28, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,12MF-36384587022474-20039.4097%, 98%, or 99% sequence identity to SEQ ID NO:28. In any of the embodiments herein, the DNMT3 domain comprises a DNMT3 A domain. In any of the embodiments herein, the DNMT3A comprises the sequence set forth in SEQ ID NO: 22, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 22. In some embodiments, the DNMT3A comprises the sequence set forth in SEQ ID NO: 22. In any of the embodiments herein, the DNMT3 domain comprises a DNMT3L domain or a variant thereof. In some embodiments, the DNMT3 domain comprises the sequence set forth in SEQ ID NO: 23, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 23. In some embodiments, the DNMT3 domain comprises the sequence set forth in SEQ ID NO: 23.

[0064] In any of the embodiments herein, the DNMT3 domain comprises a DNMT3 A domain and a DNMT3L domain (DNMT3A / L domain). In any of the embodiments herein, the fusion protein comprises from the N-terminus to the C-terminus: a DNMT3 A domain and a DNMT3L domain (DNMT3A / L domain), a dSpCas9 domain, and a KRAB domain. In some embodiments, the DNMT3 domain is a DNMT3A / L domain and the DNMT3 A domain and the DNMT3L domain are separated by a linker. In some embodiments, the linker comprises the sequence set forth in SEQ ID NO: 34. In any of the embodiments herein, the DNMT3 domain is a DNMT3A / L domain and comprises the sequence set forth in SEQ ID NO: 42 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 42. In any of the embodiments herein, the fusion protein comprises a sequence comprising at least 90% sequence identity to SEQ ID NO: 42 and a sequence comprising at least 90% sequence identity to SEQ ID NO: 28.

[0065] In any of the embodiments herein, the fusion protein comprising the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO: 24. In any of the embodiments herein, the dSpCas9 protein comprises the sequence set forth in SEQ ID NO: 25, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 25. In any of the embodiments herein, the dSpCas9 protein comprises the sequence set forth in SEQ ID NO: 25.

[0066] In any of the embodiments herein, the fusion protein further comprises one or more nuclear localization signals (NLS). In some embodiments, the NLS is at the N- or C-terminus, between N- or C-terminus and the KRAB domain, between the N- or C-terminus and the DNMT3A / L domain, between the dSpCas9 and the DNMT3A / 3L domains, or between the13MF-36384587022474-20039.40 dSpCas9 and the KRAB domain. In some embodiments, the NLS comprises the sequence set forth in any one of SEQ ID NOS: 13, 16, 18, a portion thereof, or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the foregoing. In some embodiments herein, the NLS comprises the sequence set forth in any one of SEQ ID NOS: 13, 16, and 18.

[0067] In any of the embodiments herein, the fusion protein further comprises one or more linkers connecting two or more of: the DNMT3 A domain, the DNMT3L domain, the dSpCas9, the KRAB domain, and the one or more nuclear localization signals. In some embodiments, the one or more linkers is independently selected from a linker comprising the sequence set forth in any one of SEQ ID NO: 11, 12, 14, 15, or 17.

[0068] In any of the embodiments herein, the mRNA further comprises one or more untranslated regions (UTR) near the 5’ end and / or the 3’ end. In some embodiments, the mRNA further comprises a UTR near the 5’ end and near the 3’ end. In some embodiments, the UTR near the 5’ end comprises a sequence set forth in SEQ ID NO: 9 or a sequence that has at least 90% sequence identity to SEQ ID NO:9 and the UTR near the 3’ end comprises a sequence set forth in SEQ ID NO: 10 or a sequence that has at least 90% sequence identity to SEQ ID NO: 10. In any of the embodiments herein, the mRNA further comprises a polyA sequence at the 3’ end. In any of the embodiments herein, the mRNA comprises from 5’ end to the 3’ end: a UTR, a nucleotide sequence encoding the fusion proteina UTR, and a polyA sequence.

[0069] In any of the embodiments herein, the mRNA encoding the fusion protein comprises from the 5’ end to the 3’ end: the UTR comprising the sequence set forth in SEQ ID NO: 9, a nucleotide sequence encoding the fusion protein, the UTR comprising the sequence set forth in SEQ ID NO: 10, and a polyA sequence comprising the sequence set forth in SEQ ID NO: 19. In some embodiments, the nucleotide sequence encoding the fusion protein comprises, in order from the 5’ end to the 3’ end, nucleotides encoding: a DNMT3A domain, a linker, the DNMT3L domain, a linker, a NLS, a linker, the dSpCas9, a linker, a NLS, a linker, the KRAB, and a NLS. In any of the embodiments herein, the nucleotide sequence encoding the fusion protein comprises, in order from the 5’ end to the 3’ end: a nucleotide sequence encoding the DNMT3A domain comprising the sequence set forth in SEQ ID NO: 2, a nucleotide sequence encoding a linker comprising the sequence set forth in SEQ ID NO: 11, a nucleotide sequence encoding the DNMT3L domain comprising the sequence set forth in SEQ ID NO: 3, a nucleotide sequence encoding the linker comprising the sequence set forth in SEQ ID NO: 12, a nucleotide sequence encoding an NLS comprising the sequence set forth in SEQ ID NO: 13, a14MF-36384587022474-20039.40 nucleotide sequence encoding a linker comprising the sequence set forth in SEQ ID NO: 14, a nucleotide sequence encoding the dSpCas9 comprising the sequence set forth in SEQ ID NO: 4, a nucleotide sequence encoding a linker comprising the sequence set forth in SEQ ID NO: 15, a nucleotide sequence encoding an NLS comprising the sequence set forth in SEQ ID NO: 16, a nucleotide sequence encoding the linker comprising the sequence set forth in SEQ ID NO: 17, a nucleotide sequence encoding the KRAB comprising the sequence set forth in SEQ ID NO: 5, and a nucleotide sequence encoding the NLS comprising the sequence set forth in SEQ ID NO: 18.

[0070] In any of the embodiments herein, the mRNA comprises the sequence set forth in SEQ ID NO: 1, or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1.

[0071] Also provided herein is a lipid nanoparticle comprising an epigenetic-modifying DNA-targeting system comprising a DNA-targeting module for repressing total viral Hepatitis B Viral (HBV) mRNA transcripts. In some embodiments, the DNA-targeting module comprises: (a) a gRNA for targeting to a target site in a HBV DNA sequence, and (b) an mRNA comprising from the 5’ end to the 3’ end: a UTR comprising the sequence set forth in SEQ ID NO: 9, a nucleotide encoding a fusion protein, a UTR comprising the sequence set forth in SEQ ID NO: 10, and a poly A sequence comprising the sequence set forth in SEQ ID NO: 19. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 27. In some embodiments, the nucleotide sequence encoding the fusion protein comprises, in order from the 5’ end to the 3’ end: a nucleotide sequence encoding the DNMT3A domain comprising the sequence set forth in SEQ ID NO: 2, a nucleotide sequence encoding a linker comprising the sequence set forth in SEQ ID NO: 11, a nucleotide sequence encoding the DNMT3L domain comprising the sequence set forth in SEQ ID NO: 3, a nucleotide sequence encoding the linker comprising the sequence set forth in SEQ ID NO: 12, a nucleotide sequence encoding an NLS comprising the sequence set forth in SEQ ID NO: 13, a nucleotide sequence encoding a linker comprising the sequence set forth in SEQ ID NO: 14, a nucleotide sequence encoding the dSpCas9 comprising the sequence set forth in SEQ ID NO: 4, a nucleotide sequence encoding a linker comprising the sequence set forth in SEQ ID NO: 15, a nucleotide sequence encoding an NLS comprising the sequence set forth in SEQ ID NO: 16, a nucleotide sequence encoding the linker comprising the sequence set forth in SEQ ID NO: 17, a nucleotide sequence encoding the KRAB comprising the sequence set forth in SEQ ID NO: 5, and a nucleotide sequence encoding the NLS comprising the sequence set forth in SEQ ID NO: 18. In some embodiments, the lipid15MF-36384587022474-20039.40 nanoparticles comprise a cationic lipid having the following structure (I):or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. In some embodiments, R1is optionally substituted C1-C24 alkyl or optionally substituted C2-C24 alkenyl; R2and R3are each independently optionally substituted C1-C36 alkyl; R4and R5are each independently optionally substituted Ci-Ce alkyl, or R4and R5join, along with the N to which they are attached, to form a heterocyclyl or heteroaryl; L1, L2, and L3are each independently optionally substituted Ci-Cis alkylene; G1is a direct bond, -(CH2)n0(C=0)-, -(CH2)n(C=0)0-, or -(C=O)-; G2and G3are each independently -(C=O)O- or -O(C=O)-; and n is an integer greater than 0.

[0072] Also provided herein is a lipid nanoparticle comprising an epigenetic-modifying DNA-targeting system comprising a DNA-targeting module for repressing total viral Hepatitis B Viral (HBV) mRNA transcripts. In some embodiments, the DNA-targeting module comprises (a) a gRNA for targeting to a target site in a HBV DNA sequence, and (b) a mRNA comprising the sequence set forth in SEQ ID NO: 1 or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 27. In some embodiments, the lipid nanoparticles comprise a cationic lipid having the following structure (I):or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. In some embodiments, R1is optionally substituted C1-C24 alkyl or optionally substituted C2-C24 alkenyl;R2and R3are each independently optionally substituted C1-C36 alkyl; R4and R5are each independently optionally substituted Ci-Ce alkyl, or R4and R5join, along with the N to which they are attached, to form a heterocyclyl or heteroaryl; L1, L2, and L3are each independently optionally16MF-36384587022474-20039.40 substituted Ci-Cis alkylene; G1is a direct bond, -(CH2)n0(C=0)-, -(CH2)n(C=0)0-, or -(C=O)-; G2and G3are each independently -(C=O)O- or -O(C=O)-; and n is an integer greater than 0.

[0073] In any of the embodiments herein, the mRNA comprises the sequence set forth in SEQ ID NO: 1.

[0074] In any of the embodiments herein, the gRNA for targeting to the target site comprises a spacer sequence comprising the sequence set forth in SEQ ID NO: 26, a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing. In some embodiments, the spacer sequence is 17 to 24 nucleotides in length. In some embodiments, the spacer sequence is at or about 20 nucleotides in length.

[0075] In any of the embodiments herein, the gRNA comprises a spacer sequence for targeting to the target site that comprises the sequence set forth in SEQ ID NO: 26. In any of the embodiments herein, the gRNA comprises a spacer sequence for targeting to the target site that is set forth in SEQ ID NO: 26.

[0076] In any of the embodiments herein, the gRNA comprises a scaffold sequence for dSpCas9. In some embodiments, the scaffold sequence comprises a sequence set forth in SEQ ID NO: 39 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 39. In some embodiments, the scaffold sequence is set forth in SEQ ID NO: 39.

[0077] In any of the embodiments herein, the gRNA for targeting to the target site comprises the sequence set forth in SEQ ID NO: 38 or a complementary sequence of the foregoing. In any of the embodiments herein, the gRNA for targeting to the target site is set forth in SEQ ID NO: 38.

[0078] In any of the embodiments herein, the cationic lipid is selected from a compound in Table 1. In any of the embodiments herein, the cationic lipid is 1-18.

[0079] In any of the embodiments herein, the lipid nanoparticles comprise a pegylated lipid having the following structureor a pharmaceutically acceptable salt, tautomer or stereoisomer thereof. In some embodiments, :R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms. In some embodiments, the17MF-36384587022474-20039.40 alkyl chain is optionally interrupted by one or more ester bonds; and z has a mean value ranging from 30 to 60. In some embodiments, the pegylated lipid is Ila.

[0080] In any of the embodiments herein, the nanoparticle comprises a cationic lipid that is bis(2 -butyloctyl) 10-(N-decyl-4-(dimethylamino)butanamido)nonadecanedioate, l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), a PEGylated lipid that is 2-[2-(co-methoxy (polyethyleneglycol2000) ethoxy]-N,N-ditetradecylacetamide, and cholesterol. In some embodiments, the cationic lipid / DSPC / PEGylated lipid / cholesterol are formulated in a ratio of 47.5 / 10 / 2.5 / 40 mol %.

[0081] In any of the embodiments herein, the ratio of cationic lipid to mRNA (N / P) is 6.0.

[0082] In any of the embodiments herein, the total RNA in the LNP is 1 mg.

[0083] In any of the embodiments herein, the ratio of the mRNA encoding the fusion protein and the sgRNA is 2: 1.

[0084] In any of the embodiments herein, the lipid nanoparticle is formulated in 300 mM sucrose in phosphate buffered saline at pH about 7.0 to about 7.4.Brief Description of the Drawings

[0085] FIG. 1 is a schematic representation of SEQ ID NO: 1 mRNA encoding the DNMT3A / 3L-dSpCas9-KRAB fusion protein.

[0086] FIGs 2A-2B show repression of integrated HB V transcription in Hep3B cells up to 50 days after exposure to 1 38 HBV LNP drug product.

[0087] FIG.s 3A-3B show the HBV DNA methylation status in Hep3B on Day 57 (FIG.3 A) and show comparisons to epi-silencer protein concentrations on Day 1.

[0088] FIGs. 4A-4B show average methylation of CpG Island 2 compared to total HBV RNA expression and extracellular HBsAg levels on Day 57.

[0089] FIGs. 5A-5B show the repression of Transcription Seven Days After Exposure of HBV-infected Primary Human Hepatocytes to 1 38 HBV LNP drug product. HBsAg protein was not evaluated for donor 3 due to insufficient sample volume.

[0090] FIGs. 6A-6B show HBV repression following 1 38 HBV LNP drug product dosing alone or co-dosing with entecavir (ETV) or tenofovir disoproxil fumarate (TDF). Relative levels of HBV DNA (left) and 2.5kb HBV RNA (right) for 1 38 HBV LNP drug product alone, ETV or TDF alone (patterned bars), or in combination. Dotted line represents the 90% repression level.18MF-36384587022474-20039.40

[0091] FIGs. 7-8 show repression of HBV RNA in hepatocytes from HBV-infected FRG mice. FIG. 7 shows a representative ISH labeling of 3.5kb HBV RNA and FIG. 8 shows quantification of ISH signal from mice (shown as a point) and human hepatocytes / mouse analyzed.

[0092] FIG. 9A-9C show durable repression of HBV following delivery of 1 38 HBV LNP as measured by reduced HBeAg protein levels (FIG. 9A), HBsAg protein levels (FIG. 9B) and serum HBV DNA (FIG. 9C) relative to predose and a Non-targeting epi-silencer-GalNAc.

[0093] FIG.10 shows PCSK9 Serum Levels After a Single Dose of the Surrogate cPCSK9- epi-silencer-1.

[0094] FIG. 11 shows liver biopsy DNA methylation at the PCSK9 CpG island after a single dose of the surrogate cPCSK9-epi-silencer-l. Animal 153 day 8 data excluded due to high fat signature measured in liver biopsy.

[0095] FIG. 12 shows PCSK9 serum levels after a single dose of the surrogate cPcsk9-epi- silencer-2.

[0096] FIG. 13A shows DNA methylation at the PCSK9 CpG island approximately 1- and 3-months after a single dose of the surrogate cPCSK9-epi-silencer-2. FIG. 13B shows the average percent methylation at Day 29 and necropsy at various doses of the LNP.

[0097] FIG. 14 shows average PCSK9 repression after a single dose of the surrogate cPCSK9-epi-silencer-2.

[0098] FIGs. 15A-15C shows potency and species factor determination for identifying a starting dose for administration to human subjects with Hepatitis B viral infection. FIG. 15A shows cccDNA measured by HBV RNA expression and human PCSK9 gene expression in PHH cells post-dosing. FIG. 15B shows repression of integrated HBV DNA and human PCSK9 in Hep3B cells post-dosing. FIG. 15C shows comparison of human PCSK9 and HNP PCSK9 repression post dosing. All gene expression was relative to TBP housekeeping gene control.

[0099] FIG. 16A and FIG. 16B depict the percent decrease in HBV pgRNA and HBsAg expression in a subject on Day 21 after administration of an HBV LNP drug product (FIG. 16A) and the percent decrease of HBsAg expression on Day 7 after administration (FIG. 16B).Detailed Description

[0100] Provided herein are methods for treatment of chronic Hepatitis B (CHB) infection that involve administering a lipid nanoparticle (LNP) carrying an epigenetic-modifying DNA targeting system (also called “epi-silencer”) composed of polynucleotides for targeting19MF-36384587022474-20039.40 repression of total Hepatitis B Viral (HBV) transcripts. In provided embodiments, the epigenetic-modifying DNA targeting system is composed of a polynucleotide encoding a fusion protein with a deactivated Cas (dCas) and a transcriptional repressor domain and a gRNA for targeting a viral HBV sequence is encapsulated in the LNP. In some embodiments, the epigenetic-modifying DNA targeting system is composed of a fusion protein of a deactivated Cas9 (dCas9) linked to a DNA methyltransferase and KRAB effector domains, and a gRNA for targeting a viral HBV sequence. In some embodiments, the polynucleotide encoding the fusion protein is an mRNA. In provided embodiments, the lipid nanoparticle carries an mRNA sequence encoding the fusion protein and the gRNA for targeting a viral HBV sequence. An exemplary LNP HBV epi-silencer therapy for use in the methods herein is an 1 38 HBV LNP drug product.

[0101] Hepatitis B is a potentially life-threatening liver infection caused by the Hepatitis B virus (HBV). HBV infection is a global public health problem causing chronic liver infection and increasing the risk for liver cirrhosis and liver cancer. The WHO estimated that 296 million people worldwide with 253 million people in the world, were living with chronic hepatitis B infections in 2022, with 1.5 million new infections each year. In 2022, hepatitis B resulted in an estimated 1.1 million deaths, mostly from cirrhosis and hepatocellular carcinoma (primary liver cancer).

[0102] In some cases, individuals with hepatitis B infections are at risk for hepatitis D virus (HDV) infections (also called Delta hepatitis), the most severe viral liver disease. HDV is a “satellite virus” meaning that only individuals infected by HBV can be infected by HDV. Individuals with hepatitis D infections can become infected with both HBV and HDV at the same time or be infected with HDV after first being infected with HBV. HDV can cause severe symptoms and serious illness that can lead to liver damage and even death.

[0103] Most of this global burden can be attributed to mother to child transmission at the time of or shortly after birth and horizontal household transmission. Considerable progress has been made towards eliminating the perinatal transmission of HBV through universal infant HBV immunization, including the timely hepatitis B birth-dose (HepBD), which has been highly effective in reducing new infections among children. However, HepBD coverage is only 45% globally, with lowest coverage (18%) in the WHO African Region (World Health Organization [WHO], 2024). For people with CHB infection, nucleoside analogue (NA) treatment with currently recommended tenofovir and entecavir is highly effective and can reduce progression of liver disease and incidence of HCC and improve long-term survival. However, a major testing20MF-36384587022474-20039.40 and treatment gap remains. In 2022, only 13% of the estimated 253 million people with CHB had been diagnosed and 3% had been treated. CHB infection can lead to severe health consequences, including liver decompensation, cirrhosis, HCC, liver transplantation, and death, resulting in both high morbidity and mortality rates. CHB is the leading risk factor for primary liver cancer globally (Russo et al., Hepatocellular Carcinoma in Chronic Viral Hepatitis: Where Do We Stand? International. Journal of Molecular Sciences, 23(1). 2022). Those living with HBV require years if not decades of regular monitoring to prevent liver complications from occurring. Generally, those who start antiviral therapy for HBV suppression can expect to be on treatment for life. Although treatment with antivirals significantly reduces the risk of liver cancer, it cannot eliminate risk or reduce it to the level of a non-infected individual. In addition, living with CHB has deleterious physical, social, and emotional impacts that often negatively affect patients’ quality of life (QOL).

[0104] HBV belongs to the Hepadnaviridae family, a family of small enveloped hepatotropic DNA viruses (Wei L. and Ploss A. Nature communications 12(1591) 1-13 (2021)). The HBV virion contains a compact, partially double-stranded, about 3.2 kb relaxed circular DNA (rcDNA) genome. The genome contains four lesions: a covalently linked HBV polymerase and a 10 nucleotide (nt) DNA flap on the 5 '-end of the minus strand; and a 5 '-capped RNA primer and single-stranded DNA (ssDNA) gap on the plus-strand. The HBV genome is an about 3.2 kilobase double-stranded DNA molecule, but can be longer or shorter depending on the particular HBV strain (e.g. up to 3300 bp or more in size). An exemplary HBV genome is the Hepatitis B Virus genome (Hepatitis B virus subtype ayw, complete genome, GenBank: U95551.1), SEQ ID NO: 21. At least 10 genotypes (A to J) have been identified with a divergence of no more than 8% between genotypes. Subgenotypes also exist including subgenotypes classified as HBV genotype A (A1-A7), genotype B (B1-B9), genotype C (Cl- 16), genotype D (D1-D8), and genotype F (F1-F4) (Zhang et al. World J Gastroenterol., 2015, 22: 126-144). Within sub-genotypes the sequence identity divergence is only about 4%. It is understood that the provided systems and methods are applicable to a plurality of HBV genomes, particularly given the high sequence similarity. For purposes herein, reference to numbering of nucleotide positions is nucleotide (base pair) numbering of HBV DNA sequence described under GenBank accession no. U95551.1, set forth in SEQ ID NO: 21. A skilled artisan understands that target site or base pair positions, such as described herein, in another HBV genome may not be at the identical position but nevertheless may be a homologous sequence or substantially homologous sequence (e.g. 1, 2 or 3 mismatches), such as determined by alignment21MF-36384587022474-20039.40 of an HBV genome sequence with the sequence set forth in SEQ ID NO: 21. A corresponding position or positions may thus be readily identified by alignment of an HBV genome sequence with the reference sequence set forth in SEQ ID NO: 21. The Hepatitis B virus contains a circular genome and therefore for the purposes herein references to numbering of nucleotide positions of a linear HBV DNA sequence may shift by a few nucleotides, such as depending on the start of a linear sequence. For instance, the sequence set forth in SEQ ID NO: 21 and SEQ ID NO: 20 are the same sequence but the start of the linear sequences shifts by two nucleotides due to differences in the start of the linerar sequence. It is well within the level of a skilled artisan to identify corresponding sequence regions between and among different sequences of an HBV genome sequence.

[0105] The HBV lifecycle includes processes, such as viral entry, cccDNA formation, transcription, replication, assembly, secretion, and integration. Following viral entry into hepatocytes via the bile acid transporter NTCP11, the viral nucleocapsid harboring the HBV rcDNA is transported to the nucleus. The rcDNA is released, and the four lesions on the rcDNA are fully repaired to form a supercoiled cccDNA molecule (also called minichromosomes). The viral repair factors are dispensable for repair and the cccDNA often relies on host DNA repair machinery, including TDP2, DNA polymerase (POL) K, POLa, DNA ligase 1 and 3, and flap endonuclease 1. The HBV hijacks host ubiquitous and liver-enriched transcription factors for cccDNA transcriptional regulation. The cccDNA is the key viral depot driving chronic HBV infection and serves as the template for all HBV viral transcripts. Another form of HBV DNA in the host is the stably integrated HBV DNA in the host genome (Zhao K., et al., Cell Press-The Innovation 1(2): 1-10 (2020). Double-stranded linear DNA (dslDNA) is the dominant substrate for integration into the host genome. As there is almost no sequence homology between the viral DNA and the cellular DNA, NHEJ DNA repair pathway is proposed as a mechanism for HBV DNA integration. HBV DNA integration occurs throughout the host genome at double stranded breaks, with terminal deletions up to 200 bp from the integrated HBV DNA being common. No specific chromosomal hot-spots or common recurring sites have been observed between patients. There is some evidence for enrichment in particular genomic sites in tumour tissues (Sung W., et al., Nature Genetics 44(7):765-9 (2012)). Although no progeny virus is produced, integrated HBV DNA can produce viral RNAs and proteins. HBV DNA integration occurs more often in hepatic cancer cells (84%) than in normal liver tissues (30%).

[0106] Current standard of care includes nucleoside analogs (e.g., lamivudine) and PEGlyated interferon therapies. Nucleoside analogs act by inhibiting HBV polymerase activity22MF-36384587022474-20039.40 resulting in a decrease in viral replication. However, prolonged treatment periods, increase in viral resistance and emergence of mutant strains, have reduced the effectiveness of nucleoside therapies (Papatheodoridis G.V. et al., Am. J. Gastroenterol 97(7): 1618-28 (2002). PEGlyated interferon therapy either alone or in combination with nucleoside analogs (e.g., lamivudine) has been tested to suppress transcription of viral DNA. PEGylated interferon therapy has been shown to mediate divergent effects on the innate and adaptive arms of the immune system, with strinkingly depleting effects on CD8 T cells, limiting the efficacy of the therapy (Micco L., et al., Journal of Hepatology 58(2): 225-233 (2013); Stelma F., et al., Journal f Infectious Disease 212(7): 1042-51 (2015) Marcellin P., et al., New England Journal of Medicine 351(12): 1206-17 (2004)). Nucleotide analogs nor PEGylated therapies are able to clear or suppress production of HBV surface antigen (HBsAg), which has been linked to poor prognosis of HBV infection. Other therapies including antisense oligonucleotide (ASO) and siRNA approaches centered at reducing HBsAg to reach functional cures (Billioud G., et al., Journal of Hepatology 64(4):781- 9 (2015); Gane E., et al., Hepatology 74(4): 1795-1808 (2021); Flisiak R., et al., Expert Opinion on Biology Therapy 18(6)609-617) have shown to be promising in inhibiting HBsAg, HBeAg, and HBV DNA synthesis. However, the functional benefit of any of these therapies on the regeneration of liver tissue is unclear.

[0107] Current antiviral therapies rarely achieve a cure as they inhibit cytoplasmic HBV genome replication and do not directly target the cccDNA form- a form that serves as an HBV replication intermediate and viral persistence reservoir (Yang G., et al., Theranostics 9(24):7345-58 (2019)). Genome engineering approaches such as nucleases or base editors target removal or mutagenesis of the cccDNA pool in order to functionally cure the infection. However, such nuclease-based therapies have a chance of generating chromosomal abnormalities and therefore are not preferred, highlighting the need for better HBV therapeutics.

[0108] The persistence of the episomal cccDNA pool in infected hepatocytes remains a critical obstacle in complete elimination by anti-HBV therapies. The cccDNA accumulates in the nucleus as a chromatin-like cccDNA minichromosome assembled by histones and nonhistones. The cccDNA shows unusual chromatin regulation due to its non-native status. For instance, changes the epigenetic states of the cccDNA have been found to dictate its transcriptional activity (Yang G., et al., Theranostics 9(24):7345-58 (2019). For example, the host nucleosome assembly machinery (HAT1 / CAF-1) acetylates histone H4 at the sites of H4K5 and H4K12 contributing to the assembly of the cccDNA. The acetylation marks on the histones of the cccDNA in turn promote HBV replication and accumulation of the cccDNA. This23MF-36384587022474-20039.40 transcriptional activity is largely driven by the presence of absence of activating epigenetic marks on the cccDNA; repressive histone marks (e.g., H3K27me3 and H3K9me3) are minute, suggesting that there is limited repression in the cccDNA (Tropberger P. et al., PNAS, 112(42):E5715-E5724 (2015), Riviere L., et al., J Hepatol 15(00450):S0168-8278 (2015)). Desirable clinical outcomes are believed to require not only targeting integrated HBV DNA but also the cccDNA minichromosome.

[0109] Results have shown potential of epigenetic regulation of cccDNA. Studies have found that cccDNA contains methylation-prone CpG islands that are connected to the behavior of HBV (Zhang Y., et al., PlosOne 9(10):el 10442 (2014), Vivekanandan P, et al., Journal of infectious diseases, 199(9): 1286-1291 (2009), Vivekanandan P, et al., Journal of Virology, 84(9):4321-4329 (2010), Vivekanandan P. et al., Journal of Viral hepatitis 15(2): 103-107 (2008), Jain S., et al., Scientific Reports 5: 10478 (2015)). Methylation of CpG islands II and III has been correlated to low levels of serum HBV DNA and HBsAg titres in patients (Zhang Y., et al., PlosOne 9(10):el 10442 (2014)). HBV genotype, HBeAg positivity, patient age, and liver fibrosis stage have been found to correlate to cccDNA CpG methylation status. In vitro methylation studies have further confirmed that CpG island II methylation can markedly reduce cccDNA transcription and subsequent viral core DNA replication (Zhang Y., et al., PlosOne 9(10):el 10442 (2014)), establishing the importance of chromatin for cccDNA regulation and as a potential target for therapy of chronic HBV infections. Anti-virals and broad epigenetic- modifying agents, such as IFNa have been attributed to reducing active histone post translational modifications thereby transcriptionally down-regulating transcription of cccDNA (Tropberger P. et al., PNAS, 112(42):E5715-E5724 (2015), Belloni L, et al., Journal of Clinical investigation 122L529-537 (2012), Allweiss L., et al., Journal of Hepatology 60:500-507 (2014), Lucifora J., et al., Science 343: 1221-1228 (2014)).

[0110] The provided embodiments are based on a recognition that epigenetically silencing HBV viral transcripts, including those from both integrated DNA and the cccDNA, is a viable therapeutic approach to curing HBV infections. Disclosed herein are approaches to achieve amelioration of infection, and in some cases potentially a functional cure, from HBV via precise epigenetic silencing of the cccDNA form and of the HBV integrated into the human genomic DNA. In embodiments of the provided methods, a lipid nanoparticle carrying a particular epigenetic-modifying DNA-targeting system as described herein is administered to a subject with CHB infection for delivery of the epigenetic-modifying DNA-targeting system to hepatocytes for treating the infection.24MF-36384587022474-20039.40

[0111] Provided embodiments relate to administration of particular dose amounts to a subject. The results and provided embodiments are based on the first therapy of its kind such that no dosing paradigm heretofore exists. Moreover, identifying a dose for humans for treating CHB is complicated because cynomolgus monkeys cannot contract HBV. Yet, cynomolgus monkeys are the most human relevant model utilized for determining dose of an LNP -based therapy, since they are genetically and physiologically more like humans than rodent species. Moreover, tissue distribution and biochemical components involved with the uptake of LNPs by the liver are expected to be better modeled in this species when compared to other traditional large animal-species used in toxicology studies. To solve the problem of lack of a CHB model in cynomolgus monkeys, pharmacodynamic (PD) assessment in another liver relevant, epigenetically regulated surrogate target gene was made, proprotein convertase subtilisin / kexin type 9 (PCSK9). These studies as described in Example 3 used the same LNP and mRNA episilencer fusion protein and differed only by replacing the HBV-specific gRNA with a cyno PCSK9 (cPCSK9)-targeting gRNA. Combined with results from preclinical studies and of toxicology studies of the HBV epi-silencer, a dose range for administration was determined as described in Example 4. The orthogonal approach to determining feasible dosing as provided herein contemplates a lower efficacious dose due to the higher potency of the provided HBV episilencer making it more potent at silencing its target.

[0112] In some embodiments, the results support dosing as low as 0.1 mg / kg or 10 mg, such as 0.2 mg / kg or 12 mg. As described herein, a dose of 0.20 mg / kg is expected to silence 74.8% - 89.9% of HBV genomes with higher levels of virus silencing possible with higher doses. In some embodiments, the dose is 0.1 mg / kg to 100 mg / kg, such as 0.2 mg / kg to 0.65 mg / kg, for example, at or about 0.2 mg / kg, about 0.45 mg / kg, about 0.65 mg / kg, or about 0.70 mg / kg. In some embodiments, the dose is greater than 0.65 mg / kg but is lower than 1.0 mg / kg. In some embodiments, flat or fixed doses (not weight-based dosing) are also contemplated and include administration of a dose as low as 10 mg, such as about 12 mg up to about 120 mg, for example about 12 mg to about 45 mg, about 20 mg to about 70 mg, or about 30 mg to about 65 mg. In some of any of the provide embodiments, the dose amount administered is no more than 100 mg. In some of any of the provide embodiments, the dose amount administered is no more than 120 mg. In some embodiments, the dose is administered as a single infusion. In some embodiments, one or more further infusion of the lipid nanoparticle may be administered to the subject. In some of any of the provided embodiments, the subject is a human subject.25MF-36384587022474-20039.40

[0113] The provided epi-silencer therapy for the treatment of CHB is able to directly and durably prevents intDNA and cccDNA activity by inducing local HBV sequence specific DNA methylation without cutting the DNA backbone to stop the production of new viral particles, potentially leading to a functional cure in patients with CHB. In some embodiments, functional cure is defined as sustained HBsAg loss in addition to undetectable HBV DNA 6 months posttreatment (Wong et al., 2022). Currently, reports indicate that spontaneous functional cure occurs in only ~1% of patients with CHB (Nguyen et al., 2020). Results herein, including preclinical data, indicate that the provided epi-silencer therapy can induce an epigenetically silent state in both intDNA and HBV cccDNA reservoirs to thereby prevent the transcription of all HBV RNA transcripts. This lack of transcription is proposed to prevent or reduce viremia, and thereby could enable patients to have stable control of viremia and viral gene expression, potentially without other or continuous AVT.

[0114] The approaches described herein demonstrate high efficacy, safety, and stability. In some embodiments, the approaches target all the forms of HBV in the same approach, utilize non-mutagenic platforms, and targeting the source of transcription rather than downstream transcripts. As methylation can be inherited by the cellular progeny, the durability of the epiediting approaches offers promise for treatment of HBV infection. In some embodiments, the epigenetic approaches result in silencing of HBV replication, HBV transcription, and production of proteins form the HBV DNA. The provided embodiments are not contingent on immune reboot nor on infected hepatocyte clearance but are based on a direct epigenetic silencing (e.g., HBV repression).

[0115] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0116] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.I. METHODS AND USES IN TREATING HEPATITIS B VIRAL INFECTIONS

[0117] Provided herein are methods of treating chronic hepatitis B (CHB) infections. The provided methods include methods for administering a dose of lipid nanoparticles comprising26MF-36384587022474-20039.40 the epigenetic modifying DNA-targeting systems described herein. In some embodiments, the LNP compositions are useful in treating a chronic hepatitis B infection in a human subject. Such methods and uses include therapeutic methods and uses, for example, involving administration of the compositions, to a subject having a disease, condition, or disorder, such as an HBV viral infection or is associated with an HBV viral infection. In certain embodiments, the subject has been diagnosed with liver disease caused by a Hepatitis B virus infection or a Hepatitis B virus infection. In some embodiments, the HBV viral infection or the associated HBV viral infection is selected from hepatitis D virus infection, delta hepatitis, acute hepatitis B, acute fulminant hepatitis B, chronic hepatitis B, liver fibrosis, end-stage liver disease, or cancer such as hepatocellular carcinoma. In some embodiments, the associated HBV viral infection is hepatitis D virus infection. In some embodiments, the HBV viral infection is chronic hepatitis B infection (CHB).

[0118] In some embodiments, the compositions disclosed herein are administered in an effective amount to effect treatment of subjects infected with HDV. More particularly, embodiments of the present disclosure relates to treatment of subjects identified in whom HDV infection arises in the presence of hepatitis B virus (HBV) infection. HDV is a defective, helper-virus-dependent pathogen that requires hepatitis B surface antigen (HBsAg) supplied by HBV to assemble, release, and spread. As a result, clinically relevant HDV infection occurs in individuals who are concurrently infected with HBV or who have an existing HBV infection. HDV infection typically arises by (i) co-infection, in which HDV and HBV are acquired simultaneously, or (ii) superinfection, in which HDV is acquired by a subject with chronic HBV infection. Superinfection is associated with higher rates of chronicity and accelerated liver disease progression relative to co-infection. The compositions disclosed herein effect treatment in subjects who are coninfected (wherein HBV infection and HDV infection occurred at the same time). The compositions disclosed herein effect treatment in subjects with a HDV superinfection (wherein the subject is infected with HDV after first having been infected with HBV). Uses include uses of the compositions in such methods and treatments, and in the preparation of a medicament in order to carry out such therapeutic methods. Such methods and uses include therapeutic methods and use, for example, involving administration of the compositions, to a subject having an HBV and HDV coinfection and / or a HDV superinfection. In some embodiments, the methods are carried out by administering the LNP compositions to the human subject having an HBV and HDV coinfection and / or a HDV superinfection.27MF-36384587022474-20039.40

[0119] The compositions are administered in an effective amount to effect treatment of HBV infection, and in particular CHB. Uses include uses of the compositions in such methods and treatments, and in the preparation of a medicament in order to carry out such therapeutic methods. Such methods and uses include therapeutic methods and use, for example, involving administration of the compositions, to a subject having a disease, condition, or disorder, such as an HBV viral infection or is associated with an HBV viral infection. In some embodiments, the HBV viral infection is a chronic hepatitis B (CHB) viral infection. In some embodiments, the methods are carried out by administering the LNP compositions to the human subject having CHB.

[0120] Provided herein are methods of treating hepatitis B viral infections, e.g., including administering to a subject in need thereof, a dose of lipid nanoparticles comprising an epigenetic-modifying DNA-targeting system. In some embodiments, the epigenetic-modifying DNA-targeting system modifying DNA-targeting system comprises a DNA-targeting module for repressing transcription of one or more Hepatitis B viral (HBV) genes. In some embodiments, the DNA-targeting module comprises a gRNA for targeting to a target site in a HBV DNA sequence and a polynucleotide encoding a fusion protein comprising a deactivated Streptococcus pyogenes Cas9 (dSpCas9) protein and at least one transcriptional repressor effector domain comprising a KRAB domain and a DNA methyltransferase 3 (DNMT3) domain. In some embodiments, the polynucleotide encoding the fusion protein is mRNA. In some embodiments, the dose of lipid nanoparticles administered to the subject comprises (a) between at or about 0.1 mg / kg and at or about 1 mg / kg, or (b) between at or about 10 mg to about 100 mg.

[0121] In some embodiments, the target site is present in a covalently closed circular DNA (cccDNA), relaxed circular DNA (rcDNA) and / or is integrated in the genomic DNA. In some embodiments, the target site is at or near a gene or regulatory element thereof involved in HBV replication and / or HBV transcription, such as a regulatory element or a coding region. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 27. In some embodiments, provided is a gRNA for targeting to a target site set forth in SEQ ID NO: 27.

[0122] Provided is a method of treating chronic hepatitis B (CHB) infection in a subject in need thereof, the method comprising administering to the subject a dose of lipid nanoparticles comprising an epigenetic-modifying DNA-targeting system comprising a DNA-targeting module for repressing total viral Hepatitis B Viral (HBV) mRNA transcripts. In some embodiments, the DNA-targeting module comprises: (a) a gRNA for targeting to a target site in a HBV DNA sequence, wherein the target site comprises the sequence set forth in SEQ ID NO:28MF-36384587022474-20039.4027; and (b) mRNA encoding a fusion protein comprising a a deactivated Streptococcus pyogenes Cas9 (dSpCas9) protein and at least one transcriptional repressor effector domain comprising a KRAB domain and a DNA methyltransferase 3 (DNMT3) domain.

[0123] In some of any of the embodiments, the DNMT3 domain includes a DNMT3 A domain and a DNM3TL domain, also referred to as DNMT3A / 3L. In some embodiments, the fusion protein contains a DNMT3 A / 3L domain, a KRAB domain and the spCas9. In some embodiments, the encoded fusion protein comprises in N- to C-terminal order a DNMT3A / 3L domain, SpCas9, and a KRAB. In some embodiments, the fusion protein is a DNTM3A / 3L- spCas9-KRAB fusion protein.

[0124] Exemplary fusion proteins and nucleotide sequences encoding the same are described herein.

[0125] In some embodiments, the dose of lipid nanoparticles administered to the subject is based on the weight of the human subject (weight-based dosing). In some embodiments, a dose of lipid nanoparticles is administered, and the dose is between at or about 0.1 mg / kg and at or about 1 mg / kg. However, in some such embodiments, the dose administered to a subject based on weight does not exceed 100 mg. By way of example, if the subject is over 100 kg (e.g., 105 kg) and the dose to be administered to the subject is 1 mg / kg, the subject is administered a dose of 100 mg. In some embodiments, the dose administered is not based on the weight of the human subject and is a flat dose (e.g., a fixed dose that is not based on the weight of the individual). In some embodiments, a dose of lipid nanoparticles is administered, and the dose is between at or about 10 mg to about 100 mg. In some embodiments, a dose of lipid nanoparticles is administered, and the dose is between at or about 10 mg to about 105 mg. In some embodiments, a dose of lipid nanoparticles is administered, and the dose is between at or about 10 mg to about 110 mg. In some embodiments, a dose of lipid nanoparticles is administered, and the dose is between at or about 10 mg to about 115 mg. In some embodiments, a dose of lipid nanoparticles is administered, and the dose is between at or about 10 mg to about 100 mg. In some embodiments, the dose of nanoparticles administered is at or at least about 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 115 mg, or 120 mg.

[0126] In some embodiments, a dose of lipid nanoparticles is administered in a dose of between 10-20 mg, 10-30 mg, 10-40 mg, 10-50 mg, 10-60 mg, 10-70 mg, 10-80 mg, 10-90 mg, 10-100 mg, 10-105 mg, 10-110 mg, 10-115 mg, 10-120 mg, 15-25 mg, 20-30 mg, 25-35 mg, 30- 40 mg, 35-45 mg, 40-50 mg, 45-55 mg, 50-60 mg, 55-65 mg, 60-70 mg, 65-75 mg, 70-80 mg,29MF-36384587022474-20039.4075-85 mg, 80-90 mg, 85-95 mg, 90-100 mg, 95-105 mg, 100-110 mg, 105-115 mg, or 110-120 mg. In some embodiments, a dose of lipid nanoparticles is administered in a dose of between 12 mg to about 45 mg, about 20 mg to about 70 mg, or about 30 mg to about 65 mg. In some embodiments, a dose of lipid nanoparticles is administered in a dose of between 12 mg to about 45 mg. In some embodiments, a dose of lipid nanoparticles is administered in a dose of between about 20 mg to about 70 mg. In some embodiments, a dose of lipid nanoparticles is administered in a dose of between or about 30 mg to about 65 mg.

[0127] In provided embodiments, the DNA-targeting systems comprise synthetic transcription factors that are able to modulate, such as reduce or repress, transcription of viral sequences in a targeted manner. In some embodiments, the DNA-targeting systems target repression of regulatory elements of HBV genes present in a covalently closed circular (cccDNA) form or in HBV DNA integrated into the human genomic DNA. In provided embodiments, the provided epigenetic-modifying DNA-targeting system reduces transcription of the viral sequences, including a plurality of genes present on both intDNA and cccDNA, and thereby promotes silencing of HBV replication and / or transcription. In some embodiments, delivery of the LNP comprising a DNA-targeting system to a subject represses transcription of total viral Hepatitis B Viral (HBV) mRNA transcripts, including one or more HBV genes, resulting in silencing of HBV replication and / or HBV transcription in a subject to thereby treat a disease or condition, including chronic Hepatitis B infection. The provided embodiments can be used to target multiple genetic mechanisms to treat HBV in infected patients, while avoiding the viral resistance, cost related to prolonged treatments, and poor efficacies of current combination therapies. This approach offers substantial clinical solutions to the treatment of HBV infections by reducing viral replication as well as transcription from both cccDNA and integrated HBV DNA, and circumventing the problems associated with current therapies.

[0128] In some embodiments, the methods of administering a dose of lipid nanoparticles comprising the DNA-targeting system (e.g., a gRNA for targeting to a target site and an mRNA encoding a fusion protein) to a subject as provided herein are carried out in vivo (i.e. in a subject). In some embodiments, the lipid nanoparticles are administered via intravenous administration to a human subject (e.g., human patient). In some embodiments, the dose is administered as a single infusion. In some embodiments, one or more further infusion of the lipid nanoparticle may be administered to the subject. In some embodiments, the subject is a human subject. In some embodiments, the subject is greater than or equal to 18 years of age. In30MF-36384587022474-20039.40 some embodiments, the subject is human. In some embodiments, the subject is greater than or equal to 12 years of age. In some embodiments, the subject has a weight of at least 40 kg.

[0129] In some embodiments, the methods and uses for administering the DNA-targeting systems result in delivery of the DNA-targeting system to liver cells (e.g. hepatocytes). In some embodiments, the methods of administering the lipid nanoparticles comprising the epigenetic- modifying DNA-targeting system to a subject contacts the DNA-targeting system with a liver cell or a population of liver cells. In some embodiments, the contacting introduces the lipid nanoparticles comprising the epigenome-modifying DNA-targeting system into the liver cell, such as where it is able to translocate or localize to the nucleus of the liver cell. In some embodiments, the methods treat an HBV infection in the liver cell or one or more liver cells in the population.

[0130] Also provided are methods of reducing transcription of one or more genes in a cell or total HBV mRNA transcripts comprising a Hepatitis B viral sequence. In some embodiments, the method comprises introducing into the cell an epigenetic-modifying DNA-targeting system that induces targeted CpG methylation of a CpG island (e.g., CpG island II) in a Hepatitis B viral sequence. In some embodiments, the method comprises introducing into the cell an epigenetic-modifying DNA-targeting system that induces targeted CpG methylation of CpG island II in a Hepatitis B viral sequence.

[0131] CpG islands are genomic regions that contain a high frequency of CG dinucleotides. Thus, these regions generally have a GC percentage that is greater than about 50% and with an observed / expected CpG ratio that is greater than about 60%. (Gardiner-Garden et al. “CpG islands in vertebrate genomes,” J Mol Biol 196: 261-282 (1987)). CpG islands are often located in the vicinity of genes. Methylation comprises epigenetic methylation of cytosine residues in DNA at sites where it is not typically present in normal cells. Detection of methylation of the CpG islands can be carried out according to methods of this invention, as well as any art-known method, including, but not limited to, e.g., methylation specific-polymerase chain reaction (MS- PCR), a method of nucleic acid amplification that is well known in the art. In this assay, bisulfite modification of the DNA sequence allows the detection of differences between methylated and unmethylated alleles. Reaction of the DNA with sodium bisulfite converts all unmethylated cytosines to uracil, which is recognized as thymine by Taq polymerase, but does not affect methylated cytosines. Amplification with primers specific for methylated or unmethylated DNA discriminates between methylated and unmethylated DNA. This assay provides a simple and fast way of surveying multiple samples to detect methylation of cytosines in the region of interest31MF-36384587022474-20039.40(Widschwendter et al., “Methylation and silencing of the retinoic acid receptor-beta2 gene in breast cancer” J Natl. Cancer Inst. 92(10):826-832 (2000)). Other methods known in the art for detection and / or quantitative analysis of DNA methylation include, but are not limited to, the chromatin immunoprecipitation assay (ChIP) (Mulero-Navarro et al., Carcinogenesis 27: 1099- 1104 (2006); Nakagawachi et al., Oncogene 22:8835-8844 (2003)); MethyLight®, a bisulfite modification-dependent fluorescence-based real time PCR assay (Eads et al. Nucleic Acids Res. 28(8) e32 (2000); Erhlich et al., Oncogene 21 :6694-6702 (2002)); pyrosequencing (Lee et al. Clinical Cancer Research 14:2664-2672 (2008); Dejeux et al., J. Mol. Diagn. 9:510-520 (2007)); and the Sequenom® MassARRAY® system (Sequenom, Inc., San Diego, Calif.), which utilizes MALDI-TOF mass spectrometry in combination with RNA base specific cleavage (MassCLEAVE™ kit) (Sequenom, Inc., San Diego, Calif.).

[0132] In some embodiments, the delivery to liver cells leads to an epigenetic change in the HB V gene or regulatory element, or a combination of genes or regulatory elements, which are targeted by the DNA-targeting system. In some embodiments, the epigenetic change comprises a change in at least one of: DNA accessibility, histone methylation, acetylation, phosphorylation, ubiquitylation, sumoylation, ribosylation, citrullination, and DNA methylation. In some embodiments, the epigenetic change is an altered DNA methylation of a target site in a target gene or a regulatory element thereof as described herein. In some embodiments, the epigenetic change is a histone modification of a target site in a target gene or a regulatory element thereof as described herein. In some embodiments, the delivery of the DNA-targeting system to a liver cell (e.g. hepatocyte) results in reduction of HBV mRNA transcripts or genes and / or regulatory element thereof in the liver of the subject.

[0133] In some embodiments, the modifications in the HBV epigenome is by targeting target sites within a gene and / or regulatory element thereof as described herein with a provided epigenetic-modifying DNA-targeting system to change the epigenome of HBV. In some embodiments, the modified HBV includes an epigenetic change in a target site at or near a gene or regulatory element thereof involved in controlling HBV replication and / or HBV transcription, comprising polymerase gene, S-family gene, X-gene, or core family gene, or a promoter, enhancer, or a transscript processing control region. In some embodiments, the epigenetic change of any of the above target sites is a change in at least one of: DNA accessibility, histone methylation, acetylation, phosphorylation, ubiquitylation, sumoylation, ribosylation, citrullination, and DNA methylation, compared to a comparable unmodified cell (e.g. liver cell) not subjected to the method, i.e. not contacted or introduced with the DNA-targeting system32MF-36384587022474-20039.40 described herein.

[0134] In some embodiments, the administration of the DNA-binding system modulates expression of a gene and / or regulatory element thereof or a combination of genes and / or regulatory element thereof in a liver cell, such as those described in Section LB. In some embodiments, the gene, transcript or combination of genes and transcripts, such as those described in Section I.B, is reduced in comparison to a comparable unmodified cell (e.g. liver) not subjected to the method, i.e. not contacted or introduced with the DNA-targeting system described herein. In some embodiments, reduced transcription of the combination of genes and / or regulatory elements thereof reduces HBV replication and protein levels, in a subject. In some embodiments, the liver cell in the subject has been modulated to have reduced transcription of the polymerase gene, S-family gene, X-gene, or core-family gene. In some embodiments, the expression of each gene of the combination of genes in the modified liver cell is reduced by at least 1.2-fold or more compared to the expression of the same gene in a comparable unmodified liver cell, such as reduced by at or about or greater than 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more.

[0135] In some aspects, reducing transcription of a Hepatitis B viral DNA sequence results in repression, such as silencing, of Hepatitis B viral replication and / or transcription. For instance, reduced transcription of the Hepatitis B viral DNA sequences results in a reduction of HBsAg transcripts and / or total HBV RNA. Reduced transcription of the Hepatitis B viral DNA sequences may also result in reduced HBsAg levels from integrated and cccDNA form and / or HBcrAg levels from cccDNA form.

[0136] In some embodiments, reducing transcription comprises a reduction in Hepatitis B surface antigen (HBsAg) and / or Hepatitis B viral core-related-antigen (HBcrAg) protein levels. In some embodiments, reducing transcription comprises a reduction in HBsAg transcript and / or protein levels by at least 90%. In some embodiments, reducing transcription comprises a reduction in HBcrAg transcript and / or protein levels by at least 50% from the cccDNA.

[0137] In some embodiments, reducing transcription comprises a reduction in Hepatitis B pre-core (“preC”), pre-genomic (“pgRNA”), preSl, preS2 / S, and HBx levels.

[0138] With reference to the provided disclosure, it is understood that a cell that is positive (+) for HBV (e.g., HBV infected cell) means that the cell expresses any of the HBV markers (e.g., HBV RNA transcripts and / or proteins) described herein. Likewise, it is understood that a cell that is negative (-) for a particular marker is a cell that does not express the marker at a level that is detectable. Antibodies and other binding entities can be used to detect expression levels33MF-36384587022474-20039.40 of marker proteins to identify or detect a given cell surface marker. Suitable antibodies may include polyclonal, monoclonal, fragments (such as Fab fragments), single chain antibodies and other forms of specific binding molecules. Antibody reagents for cell surface markers above are readily known to a skilled artisan. A number of well-known methods for assessing expression level of surface markers or proteins may be used, such as detection by affinity -based methods, e.g., immunoaffinity-based methods, e.g., in the context of surface markers, such as by flow cytometry. In some embodiments, the label is a fluorophore and the method for detection or identification of cell surface markers on cells (e.g. hepatocytes) is by flow cytometry. In some embodiments, different labels are used for each of the different markers by multicolor flow cytometry. In some embodiments, surface expression can be determined by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting the binding of the antibody to the marker.

[0139] In some embodiments, a cell (e.g. hepatocyte) is positive (pos or +) for a particular marker if there is detectable presence on or in the cell of a particular marker, which can be an intracellular marker or a surface marker (e.g., HBeAg, HBsAg). In some embodiments, surface expression is positive if staining by flow cytometry is detectable at a level substantially above the staining detected carrying out the same procedures with an isotype-matched control under otherwise identical conditions and / or at a level substantially similar to, or in some cases higher than, a cell known to be positive for the marker and / or at a level higher than that for a cell known to be negative for the marker. In some embodiments, a cell (e.g. a hepatocyte) contacted by a DNA-targeting system described herein, has decreased expression for a particular marker (e.g. HBeAg) if the staining is substantially lower than a similar cell that was not contacted by the DNA-targeting system.

[0140] In some embodiments, a cell (e.g. hepatocyte) is negative (neg or -) for a particular marker if there is an absence of detectable presence on or in the cell of a particular marker, which can be an intracellular marker or a surface marker. In some embodiments, surface expression is negative if staining is not detectable by flow cytometry at a level substantially above the staining detected carrying out the same procedures with an isotype-matched control under otherwise identical conditions and / or at a level substantially lower than a cell known to be positive for the marker and / or at a level substantially similar to a cell known to be negative for the marker.

[0141] In some embodiments, the LNP composition comprising a provided DNA-targeting system reduces the total level of HBV RNA in a cell, including viral RNA species transcribed34MF-36384587022474-20039.40 from cccDNA and integrated HBV DNA templates. In some embodiments, total HBV RNA is reduced by at least about 70%, 80%, 90%, 95%, or 99% compared to that in a control cell or subject. Reduction of total HBV RNA may correspond to decreased expression of one or more viral proteins, including HBsAg, HBeAg, and HBcrAg.

[0142] In provided embodiments, epigenetic repression of HBV gene transcription from covalently closed circular DNA (cccDNA) and / or integrated HBV DNA templates results in decreased production of viral RNA transcripts and corresponding viral proteins. Such repression may be evidenced by decreased levels of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and / or hepatitis B core-related antigen (HBcrAg) in a cell or biological sample infected with HBV. For example, repression of transcription of the HBV surface (S) gene leads to reduced levels of hepatitis B surface antigen (HBsAg) in the cell or in the circulation of the subject. Reductions in HBsAg may reflect silencing of transcripts derived from both cccDNA and intDNA, whereas decreases in HBeAg and HBcrAg predominantly indicate repression of transcription from cccDNA templates. Accordingly, measurement of HBsAg, HBeAg, and HBcrAg levels provides quantitative readouts of the extent of HBV gene transcriptional silencing achieved by the epigenetic-modifying DNA-targeting system. In some embodiments, a decrease in HBsAg protein levels may serve as an indicator or readout of reduced HBV gene transcription and overall viral gene silencing from both cccDNA and intDNA.

[0143] In some embodiments, the LNP composition comprising a provided DNA-targeting system reduces the total level of HBsAg transcript or protein levels in an HBV infected cell. In some embodiments, the HBsAg transcript or protein levels is reduced by at least about 70%, 80%, 90%, 95%, or 99% compared to that in a control cell or subject.

[0144] In some embodiments, the phenotype of infected cells and / or individuals is characterized functionally. In some aspects, the phenotype can be characterized by the presence of HBV RNA transcripts in infected cells. In some aspects, the phenotype can be characterized by the presence of any one or combination of the HBV proteins in infected cells. In some aspects, the phenotype can be characterized by the presence of antibodies to any of the markers described herein. In some aspects, the antibodies include but are not limited to anti-HBc-IgM, anti-HBc total, and antibodies to HBeAg. In some embodiments, the RNA transcripts, proteins and / or antibodies are measured, detected, and / or quantified by any suitable technique known in the art. For instance, the RNA transcripts may be measured, detected and / or quantified using35MF-36384587022474-20039.40 real-time PCR techniques. The HBV proteins (e.g., HBsAg, HBeAg and / or HBcrAg) may be measured, detected and / or quantified using enzyme-linked immunosorbent assays (ELISAs).

[0145] Various methods may be utilized to characterize the transcription or expression levels of a gene or viral transcript sequence in a cell (e.g. hepatocyte) such as after the cell has been contacted or introduced with a provided DNA-targeting system. In some embodiments, analyzing the transcription activity or expression of a sequence or gene may be by RNA analysis. In some embodiments, the RNA analysis includes RNA quantification. In some embodiments, the RNA quantification occurs by reverse transcription quantitative PCR (RT- qPCR), multiplexed qRT-PCR, fluorescence in situ hybridization (FISH), RNA-sequencing (RNA-seq) or combinations thereof.

[0146] In some embodiments, the gene or transcript is one in which expression of the gene or presence of the transcript in the cell (e.g. HBV infected cell, such as a hepatocyte), is reduced after having been contacted or introduced with a provided DNA-targeting system.

[0147] In some embodiments, the reduction in gene expression or the change in the level of transcripts in a cell (e.g. HBV infected cell, such as a hepatocyte) is about a log2 fold change of at least 1.25-fold, 1.5-fold, 1.75-fold, 2.0-fold, 2.5-fold, 2.75-fold, 3.0-fold, 3.25-fold, 3.5-fold,3.75-fold, 4.0 fold, 4.25-fold, 4.5-fold, 4.75-fold, 5.0-fold, 5.25-fold, 5.5-fold, 5.75-fold, 6.25- fold, 6.50-fold, 6.75-fold, 7.0-fold. 7.25-fold, 7.50-fold, 7.75-fold, 8.0-fold, 8.25-fold, 8.5-fold,8.75-fold, 9.0-fold or any value between any of the foregoing compared to that in a control cell. In some embodiments, the reduction in gene expression or the change in the level of transcripts in an HBV infected cell, such as a hepatocyte, is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100% or any value between any of the foregoing compared to that in a control cell. In some embodiments, the reduction in gene expression or the change in the level of transcripts in a HBV infected cell is greater than 75% compared to that in a control cell. In some embodiments, the reduction in gene expression or the change in the level of transcripts in a HBV infected cell is greater than 80% compared to that in a control cell. In some embodiments, the reduction in gene expression or the change in the level of transcripts in a HBV infected cell is greater than 85% compared that in a control cell. In some embodiments, the reduction in gene expression or the change in the level of transcripts in a HBV infected cell is greater than 90% compared that in a control cell. In some of any such embodiments, the control cell is a HBV infected cell similarly infected with HBV or in which HBV viral sequences are present as cccDNA and intDNA, but in which such cell has not36MF-36384587022474-20039.40 been contacted with or delivered a provided LNP carrying an epigenetic-modifying DNA targeting system.A. Lipid nanoparticles

[0148] In some embodiments, the epigenetic-modifying DNA-targeting systems, including gRNAs and polynucleotides (e.g., mRNA) encoding the fusion proteins, or components thereof described herein, are incorporated in lipid nanoparticles (LNPs), such as for delivery. In some embodiments, the lipid nanoparticle is a vector for delivery. Provided herein is a lipid nanoparticle comprising an epigenetic-modifying DNA-targeting system comprising a DNA- targeting module for repressing total viral Hepatitis B Viral (HBV) mRNA transcripts.

[0149] The lipid nanoparticle refers to particles having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) which include specified lipids. The lipid nanoparticle may also include the polynucleotides for the epi-silencer, such as a nucleic acid (e.g., mRNA) encoding the fusion protein and a gRNA. In some embodiments, lipid nanoparticles are included in a formulation that can be used to deliver the epi-silencer to hepatocytes. In some embodiments, the epi-silencer as the active agent, such as a nucleic acid (e.g., mRNA encoding the fusion protein) and gRNA, may be encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells e.g. an adverse immune response.

[0150] In some embodiments, the lipid nanoparticles include charged lipids (e.g., cationic lipids), neutral lipids, steroids and polymer conjugated lipids. In some embodiments, the LNP comprises a cationic lipid, a PEGylated lipid, a phosphatidylcholine, and a steroid such as a sterols and their derivatives.

[0151] The cationic lipid is a lipid that is capable of being positively charged. Among exemplary cationic lipids are lipids that include one or more amine group(s) which bear the positive charge. Exemplary cationic lipids are ionizable such that they can exist in a positively charged or neutral form depending on pH. The ionication of the cationic lip may affect the surface charge of the lipid nanoparticle under different pH conditions. This charge state can influence plasma protein absorption, blood clearance and tissue distribution (Semple, S. C., et al., Adv. Drug Deliv Rev 32:3-17 (1998)), as well as the ability to form endosomolytic non- bilayer structures (Hafez, I. M., et 35 al., Gene Ther 8: 1188-1196 (2001)) for the intracellular delivery of nucleic acids.

[0152] In one embodiment, the cationic lipids for use in the disclosed LNPs have the37MF-36384587022474-20039.40 following structure (I):or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein:R1is optionally substituted C1-C24 alkyl or optionally substituted C2-C24 alkenyl;R2and R3are each independently optionally substituted C1-C36 alkyl;R4and R5are each independently optionally substituted Ci-Ce alkyl, or R4and R5join, along with the N to which they are attached, to form a heterocyclyl or heteroaryl;L1, L2, and L3are each independently optionally substituted Ci-Cis alkylene;G1is a direct bond, -(CH2)nO(C=O)-, -(CH2)n(C=O)O-, or -(C=O)-;G2and G3are each independently -(C=O)O- or -O(C=O)-; and n is an integer greater than 0.

[0153] In some embodiments, the cationic lipid has the following structure (IA):

[0154] In some embodiments, the cationic lipid has the following structure (IB):

[0155] In some embodiments, R1is optionally substituted Ce-Ci8 alkyl or C14-C18 alkenyl.MF-36384587022474-20039.40In certain embodiments, R1is Cs alkyl, C9 alkyl, C10 alkyl, C12 alkyl, C14 alkyl, or Ci6 alkyl. In some more specific embodiments, R1is Ci6 alkenyl. In certain more specific embodiments, R1is unbranched. In some embodiments, R1is branched. In certain embodiments, R1is unsubstituted.

[0156] In some embodiments, G1is a direct bond, -(CH2)nO(C=O)-, or -(CH2)n(C=O)O-. In certain embodiments, G1is a direct bond. In some more specific embodiments, G1is - (CH2)n(C=O)O- and n is greater than 1. In some embodiments, n is 1-20. In some embodiments n is 1-10. In some embodiments n is 5-11. In some embodiments, n is 6-10. In certain more specific embodiments, n is 5, 6, 7, 8, 9, or 10. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In certain embodiments, n is 8. In some embodiments, n is 9. In some embodiments, n is 10.

[0157] In some embodiments, L1is Ci-Ce alkylene. In certain embodiments, L1is C2 alkylene, C3 alkylene, or C4 alkylene. In some more specific embodiments, L1is unbranched. In certain more specific embodiments, L1is unsubstituted.

[0158] In some embodiments, R2is C8-C24 alkyl. In some embodiments, R3is C8-C24 alkyl. In some more specific embodiments, R2and R3are both C8-C24 alkyl. In some embodiments, R2and R3are each independently C11 alkyl, C12 alkyl, C13 alkyl, C14 alkyl, C15 alkyl, Ci6 alkyl, Cis alkyl, or C20 alkyl. In certain embodiments, R2is branched. In more specific embodiments, R3is branched. In some more specific embodiments, R2and R3each independently have one of the following structures:wherein:R6and R7are each independently C2-C12 alkyl.

[0159] In some embodiments, R2and R3each independently have one of the following structures:

[0160] In some embodiments, L2and L3are each independently C4-C10 alkylene. In certain embodiments, L2and L3are both C5 alkylene. In some more specific embodiments, L2and L339MF-36384587022474-20039.40 are both Ce alkylene. In certain embodiments, L2and L3are both Cx alkylene. In some more specific embodiments, L2and L3are both C9 alkylene. In some embodiments, L2is unbranched. In some embodiments, L3is unbranched. In more specific embodiments, L2is unsubstituted. In some embodiments, L2is unsubstituted.

[0161] In some embodiments, R4and R5are each independently Ci-Ce alkyl. In more specific embodiments, R4and R5are both methyl. In certain embodiments, R4and R5are both ethyl. In certain embodiments, R4is methyl and R5is n-butyl. In some embodiments, R4and R5are both n-butyl. In different embodiments, R4is methyl and R5is n-hexyl.

[0162] In some embodiments, R4and R5join, along with the N to which they are attached, to form a heterocyclyl. In certain embodiments, the heterocyclyl is a 5-membered heterocyclyl. In some embodiments, the heterocyclyl has the following structure:

[0163] In various different embodiments, the cationic lipid has one of the structures set forth in Table 1 below.MF-36384587022474-20039.4041MF-36384587022474-20039.4042MF-36384587022474-20039.40MF-36384587022474-20039.40MF-36384587022474-20039.4045MF-36384587022474-20039.40

[0164] The cationic lipids, and LNPs comprising the same, can be prepared according to procedures known in the art, including those set forth in WO 2020 / 146805, which is incorporated herein by reference.

[0165] In some embodiments, the lipid nanoparticles include a neutral lipid. The neutral lipid is a lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, these include phosphotidylcholines such as 1,2-Distearoyl-sn-glycero- 3 -phosphocholine (DSPC), l,2-Dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 1,2- Dimyristoyl-sn-glycero-3 -phosphocholine (DMPC), 1 -Palmitoyl-2-oleoy 1 -sn-glycero-3 - phosphocholine (POPC), l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), phophatidyl ethanol an lines such as l,2-Dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), sphingomy-elins (SM), ceramides, steroids such as sterols and their derivatives. Neutral lipids may be synthetic or naturally derived. In various embodiments, the molar ratio of the cationic lipid to the neutral lipid ranges from about 2: 1 to about 8: 1.

[0166] In some embodiments, the lipid nanoparticles includes a phosphotidylcholine. In some embodiments, the phosphotidylcholine is l,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0167] In some embodiments, the lipid nanoparticles includes a steroid, such as a sterol.46MF-36384587022474-20039.40

[0168] In some embodiments, the non-cationic lipids comprised by the lipid nanoparticles include one or more steroids. Steroids useful for the lipid nanoparticles described herein include, but are not limited to, cholestanes such as cholesterol, cholanes such as cholic acid, pregnanes such as progesterone, androstanes such as testosterone, and estranes such as estradiol. Further steroids include, but are not limited to, cholesterol (ovine), cholesterol sulfate, desmosterol-d6, cholesterol-d7, lathosterol-d7, desmosterol, stigmasterol, lanosterol, dehydrocholesterol, dihydrolanosterol, zymosterol, lathosterol, zymosterol-d5, 14-demethyl-lanosterol, 14-demethyl- lanosterol-d6, 8(9)- dehydrocholesterol, 8(14)-dehydrocholesterol, diosgenin, DHEA sulfate, DHEA, lanosterol- d6, dihydrolanosterol-d7, campesterol-d6, sitosterol, lanosterol-95, Dihydro FF-MAS-d6, zymostenol-d7, zymostenol, sitostanol, campestanol, campesterol, 7- dehydrodesmosterol, pregnenolone, sitosterol-d7, Dihydro T-MAS, Delta 5 -avenaster ol, Brassicasterol, Dihydro FF-MAS, 24-methylene cholesterol, cholic acid derivatives, cholesteryl esters, and glycosylated sterols. In particular embodiments, the lipid nanoparticles comprise cholesterol. In some of these embodiments, the molar ratio of the cationic lipid to cholesterol ranges from about 2: 1 to 1 : 1.

[0169] In some embodiments, the lipid nanoparticles comprise a polymer conjugated lipid. Such lipid conjugates include, but are not limited to, ceramide PEG derivatives such as C8 PEG2000 ceramide, C16 PEG2000 ceramide, C8 PEG5000 ceramide, C16 PEG5000 ceramide, C8 PEG750 ceramide, and C16 PEG750 ceramide, phosphoethanolamine PEG derivatives such as 16:0 PEG5000PE, 14:0 PEG5000 PE, 18:0 PEG5000 PE, 18: 1 PEG5000 PE, 16:0 PEG3000 PE, 14:0 PEG3000 PE, 18:0 PEG3000 PE, 18: 1 PEG3000 PE, 16:0 PEG2000 PE, 14:0 PEG2000 PE, 18:0 PEG2000 PE, 18: 1 PEG2000 PE 16:0 PEG1000 PE, 14:0 PEG1000 PE, 18:0 PEG1000 PE, 18: 1 PEG 1000 PE, 16:0 PEG750 PE, 14:0 PEG750 PE, 18:0 PEG750 PE, 18: 1 PEG750 PE, 16:0 PEG550 PE, 14:0 PEG550 PE, 18:0 PEG550 PE, 18: 1 PEG550 PE, 16:0 PEG350 PE, 14:0 PEG350 PE, 18:0 PEG350 PE, and 18: 1 PEG350, sterol PEG derivatives such as Chol-PEG600, and glycerol PEG derivatives such as DMG-PEG5000, DSG-PEG5000, DPG- PEG5000, DMG-PEG3000, DSG-PEG3000, DPG-PEG3000, DMG-PEG2000, DSG- PEG2000, DPG-PEG2000, DMG-PEG1000, DSG-PEG1000, DPG-PEG1000, DMG- PEG750, DSG- PEG750, DPG-PEG750, DMG-PEG550, DSG-PEG550, DPG-PEG550, DMG-PEG350, DSG- PEG350, and DPG-PEG350. In some embodiments, the lipid conjugate is a DMG-PEG. In some particular embodiments, the lipid conjugate is DMG- PEG2000. In some particular embodiments, the lipid conjugate is DMG-PEG5000.47MF-36384587022474-20039.40

[0170] Other exemplary polyme-conjugated lipids for us in the LNPs include a pegylated diacylglycerol (PEG-DAG) a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-0-(2’,3’-di(tetradecanoyloxy)propyl-l-0-(co- methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as co-methoxy(polyethoxy)ethyl-N-(2,3- di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(co - methoxy(polyethoxy)ethyl)carbamate. In various embodiments, the molar ratio of the cationic lipid to the pegylated lipid ranges from about 100: 1 to about 25: 1.

[0171] In some embodiments, the LNPs comprise a pegylated lipid having the following structure (II):or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and z has a mean value ranging from 30 to 60.

[0172] In some embodiments, R10and R11are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms. In other embodiments, the average z is about 45.

[0173] In some of the foregoing embodiments of the pegylated lipid (II), R10and R11are not both n-octadecyl when z is 42. In some embodiments, R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 18 carbon atoms. In some embodiments, R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 12 to 16 carbon atoms. In some embodiments, R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing 12 carbon atoms. In some embodiments, R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing 14 carbon atoms. In other embodiments, R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing 16 carbon atoms. In still more embodiments, R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing 1848MF-36384587022474-20039.40 carbon atoms. In still other embodiments, R10is a straight or branched, saturated or unsaturated alkyl chain containing 12 carbon atoms and R11is a straight or branched, saturated or unsaturated alkyl chain containing 14 carbon atoms.

[0174] In various embodiments, z spans a range that is selected such that the PEG portion of (II) has an average molecular weight of about 400 to about 6000 g / mol. In some embodiments, the average z is about 45.

[0175] In other embodiments, the pegylated lipid has one of the following structures:

[0176] wherein n spans a range such that the average molecular weight of the pegylated lipid is about 2500 g / mol.

[0177] In certain embodimens, the LNPs include a pegylated lipid having structure Ila, i.e., 2-[2-(co-methoxy (polyethyleneglycol2000) ethoxy]-N,N-ditetradecylacetamide

[0178] The pegylated lipids can be prepared according to procedures known in the art, including those set forth in WO 2015 / 199952, which is incorporated herein by reference.

[0179] In some embodiments, the cationic lipid is a bis(2-butyloctyl) 10-(N-decyl-4- (dimethylamino)butanamido)nonadecanedioate (1-18). In some embodiments, the cationic lipid has the structure:49MF-36384587022474-20039.40

[0180] In some embodiments, the neutral lipid is l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC). In some embodiments, DSPC has the structure:

[0181] In some embodiments, the PEGylated lipid is 2-[2-(co-methoxy(polyethyleneglycol2000) ethoxy]-N,N-ditetradecylacetamide (Ila). In some embodiments, the PEGylated lipid has the structure:

[0182] In some embodiments, the lipid nanoparticle comprises a cholesterol. In some embodiments, cholesterol has the structure:50MF-36384587022474-20039.40

[0183] In some embodiments, the lipid nanoparticles comprise: a cationic lipid that is bis(2- butyloctyl) 10-(N-decyl-4-(dimethylamino)butanamido)nonadecanedioate, 1 ,2-distearoyl-sn- glycero-3 -phosphocholine (DSPC), a PEGylated lipid that is 2-[2-(co-methoxy (polyethyleneglycol2000) ethoxy]-N,N-ditetradecylacetamide, and cholesterol.

[0184] In some embodiments, the molar concentration of the cationic lipid is from about 20% to about 80%, from about 30% to about 70%, from about 40% to about 60%, from about 45% to about 55%, or about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or about 80% of the total lipid molar concentration, wherein the total lipid molar concentration is the sum of the cationic lipid, the neutral lipid, and the polymer-conjugated lipid molar concentrations.

[0185] In certain aspects, the lipid nanoparticle comprises from 41 to 49 mol percent, from 41 to 48 mol percent, from 42 to 48 mol percent, from 43 to 48 mol percent, from 44 to 48 mol percent, from 45 to 48 mol percent, from 46 to 48 mol percent, or from 47.2 to 47.8 mol percent of the cationic lipid. In certain specific embodiments, the lipid nanoparticle comprises about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9 or 48.0 mol percent of the cationic lipid.

[0186] In certain embodiments, the lipid nanoparticles comprise a molar ratio of cationic lipid to any of the polynucleotides of from about 1 to about 20, from about 2 to about 16, from about 4 to about 12, from about 6 to about 10, or about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20.

[0187] In some embodiments, the lipid nanoparticles can comprise at least one non-cationic lipid. In particular embodiments, the molar concentration of the non-cationic lipids is from about 20% to about 80%, from about 30% to about 70%, from about 40% to about 70%, from about51MF-36384587022474-20039.4040% to about 60%, from about 46% to about 50%, or about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 48.5%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or about 80% of the total lipid molar concentration. Non-cationic lipids include, in some embodiments, phospholipids and steroids.

[0188] In certain other embodiments, the the LNPs include a neutral lipid that is present in a concentration ranging from 5 to 15 mol percent, 7 to 13 mol percent, or 9 to 11 mol percent. In certain specific embodiments, the neutral lipid is present in a concentration of about 9.5, 10 or 10.5 mol percent. In some embodiments, the molar ratio of the cationic lipid to the neutral lipid ranges from about 4.1 : 1.0 to about 4.9: 1.0, from about 4.5: 1.0 to about 4.8:1.0, or from about 4.7: 1.0 to 4.8: 1.0.

[0189] In different embodiments, the steroid is cholesterol. In some embodiments, the steroid is present in a concentration ranging from 39 to 49 molar percent, 40 to 46 molar percent, from 40 to 44 molar percent, from 40 to 42 molar percent, from 42 to 44 molar percent, or from 44 to 46 molar percent. In certain specific embodiments, the steroid is present in a concentration of 40, 41, 42, 43, 44, 45, or 46 molar percent. In certain embodiments, the molar ratio of cationic lipid to the steroid ranges from 1.0:0.9 to 1.0: 1.2, or from 1.0: 1.0 to 1.0: 1.2. In different embodiments, the steroid is cholesterol.

[0190] In some embodiments, the cationic lipid / DSPC / PEGylated lipid / cholesterol are formulated in a ratio of 47.5 / 10 / 2.5 / 40 mol %.

[0191] In other embodiments, the ratio of cationic lipid to mRNA in the LNP (i.e., N / P, were N represents the moles of cationic lipid and P represents the moles of phosphate present as part of the nucleic acid backbone) range from 2: 1 to 30: 1, for example 3 : 1 to 22: 1. In other embodiments, N / P ranges from 6: 1 to 20: 1 or 2: 1 to 12: 1. Exemplary N / P ranges include about 3: 1, about 6: l, about 12: 1 and about 22: 1. In some embodiments, the N / P range is about 6: 1.

[0192] In some embodiments, the ratio of cationic lipid to mRNA (N / P) is 6.0.

[0193] In some embodiments, the total RNA in the LNP is about 0.5 mg, about 0.75 mg, about 1 mg, about 1.25 mg, about 1.50mg, about 1.75 mg, about 2.0 mg, about 2.25 mg, about 2.50 mg, or any of the foregoing. In some embodiments, the total RNA in the LNP is 1 mg.

[0194] In some embodiments, the ratio of the mRNA encoding the fusion protein (e.g., the mRNA comprising the sequence set forth in SEQ ID NO: 1) and the sgRNA (e.g., SEQ ID NO: 38) is about 4: 1, about 3: 1, about 2: 1, about 1 : 1, about 1 :2, about 1 :3, about 1 :4, or a value between any of the foregoing. For example, the ratio of the mRNA encoding the fusion protein and the sgRNA is about 4: 1 to about 1 :4. In some embodiments, the ratio of the mRNA52MF-36384587022474-20039.40 encoding the fusion protein and the sgRNA is about 4: 1 to about 1 : 1. In some embodiments, the ratio of the mRNA encoding the fusion protein and the sgRNA is 2: 1.

[0195] In some embodiments, the lipid nanoparticle is formulated in 300 mM sucrose in phosphate buffered saline, about pH 7.0.

[0196] In some embodiments, provided herein is an LNP HBV epi-silencer therapy in which encapsulated in the lipid nanoparticle is (a) a gRNA for targeting to a target site in a HBV DNA sequence, wherein the target site comprises the sequence set forth in SEQ ID NO: 27; and (b) an mRNA encoding a fusion protein comprising a deactivated Streptococcus pyogenes Cas9 (dSpCas9) protein and at least one transcriptional repressor effector domain comprising a KRAB domain and a DNA methyltransferase 3 (DNMT3) domain that has DNA methyltransferase activity, such as the fusion DNMT3A / 3L-SpCas9-KRAB, for example an mRNA comprising a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1 or 100% sequence identity to SEQ ID NO: 1 . In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 26, such as the sgRNA set forth in SEQ ID NO: 38. In some embodiments, the lipid nanoparticle comprises: a cationic lipid that is bis(2 -butyloctyl) 10-(N-decyl-4-(dimethylamino)butanamido)nonadecanedioate, 1,2-distearoyl- sn-glycero-3 -phosphocholine (DSPC), a PEGylated lipid that is 2-[2-(co-methoxy (polyethyleneglycol2000) ethoxy]-N,N-ditetradecylacetamide, and cholesterol. In some embodiments, the cationic lipid / DSPC / PEGylated lipid / cholesterol are formulated in a ratio of 47.5 / 10 / 2.5 / 40 mol %.

[0197] The lipid nanoparticles for use in the method can be prepared by various techniques which are known to a skilled artisan. Nucleic acid-lipid particles and methods of preparation are disclosed in, for example, U.S. Patent Publication Nos. 20040142025 and 20070042031.

[0198] In some embodiments, the lipid nanoparticles will have a size within the range of about 25 to about 500 nm. In some embodiments, the lipid nanoparticles have a size from about 50 nm to about 300 nm, or from about 60 nm to about 120 nm. The size of the lipid nanoparticles may be determined by quasi-electric light scattering (QELS) as described in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421A150 (1981). A variety of methods are known in the art for producing a population of lipid nanoparticles of particular size ranges, for example, sonication or homogenization. One such method is described in U.S. Pat. No. 4,737,323.

[0199] In some embodiments, the lipid nanoparticles comprise a cell targeting molecule such as, for example, a targeting ligand (e.g., antibodies, scFv proteins, DART molecules, peptides, aptamers, and the like) anchored on the surface of the lipid nanoparticle that53MF-36384587022474-20039.40 selectively binds the lipid nanoparticles to the targeted cell, such as any cell described herein, e.g. a hepatocyte.

[0200] In some embodiments, the vector (e.g., lipid nanoparticle) exhibits liver cell and / or hepatocyte tropism.B. Epigenetic-modifying DNA targeting system (epi-silencer)

[0201] In provided embodiments, for the target gene or regulatory element thereof that is targeted, the DNA-targeting systems include a DNA- binding domain (e.g., Streptococcus pyogenes Cas9 (dSpCas9)) that binds to a target site in a gene or regulatory element thereof. In some embodiments, the DNA-targeting systems are composed of a DNA-targeting module that includes the DNA-binding domain and additionally include at least one effector domain that is able to epigenetically modify one or more DNA bases of the gene or regulatory element thereof, in which the epigenetic modification results in a reduction in transcription of the gene (e.g. inhibits transcription or reduces transcription of the gene compared to the absence of the DNA- targeting system). Hence, the terms DNA-targeting system and epigenetic-modifying DNA targeting system may be used herein interchangeably. In some embodiments, the DNA-targeting systems include a polynucleotide encoding a fusion protein comprising a deactivated Streptococcus pyogenes Cas9 (dSpCas9) protein capable of being targeted to the target site; and (b) at least one effector domain capable of reducing transcription of the gene. For instance, the at least one effector domain is a transcription repressor domain.

[0202] In some embodiments, the DNA-targeting system contains a DNA-targeting module, where the DNA-targeting module is capable of targeting a target site in an HBV viral sequence, such as target gene or regulatory element thereof as provided. In some embodiments, the DNA- targeting module includes (a) a gRNA for targeting to a target site in a HBV DNA sequence and (b) a fusion protein comprising a deactivated Cas9 (dCas9), such as dSpCas9 protein, and at least one transcriptional repressor effector domain capable of reducing transcription of a viral HBV transcript or gene.

[0203] In some embodiments, the DNA- binding domain comprises or is derived from a Cas protein or variant thereof such as a nuclease-inactive Cas or dCas (e.g. dCas9), and the DNA- targeting system comprises one or more guide RNAs (gRNAs), such as a combination of gRNAs (e.g. two gRNAs or three gRNAs). In some embodiments, the gRNA comprises a spacer sequence that is capable of targeting and / or hybridizing to the target site. In some embodiments, the gRNA is capable of complexing with the Cas protein or variant thereof. In some aspects, the gRNA directs or recruits the Cas protein or variant thereof to the target site. In some54MF-36384587022474-20039.40 embodiments, the effector domain comprises a transcription repressor effector domain (such as those described in Section I B.2(b), and / or is capable of reducing transcription of the gene.

[0204] In some embodiments, the epigenetic-modifying DNA targeting system provided as part of the LNP HBV epi-silencer therapy herein is composed of a fusion protein of a deactivated Cas9 (dCas9) linked to a DNA methyltransferase and KRAB effector domains, and also includes a gRNA for targeting a target site of the viral HBV sequence. In some of any of the embodiments, the DNMT3 domain includes a DNMT3 A domain and a DNM3TL domain, also referred to as DNMT3A / 3L. In some embodiments, the fusion protein contains a DNMT3A / 3L domain, a KRAB domain and the spCas9. In some embodiments, the encoded fusion protein comprises in N- to C-terminal order a DNMT3A / 3L domain, SpCas9, and a KRAB. In some embodiments, the fusion protein is a DNTM3A / 3L-spCas9-KRAB fusion protein.

[0205] Exemplary target sites of a viral HBV sequence and gRNA for targeting the same are described in Section I.B. l. Exemplary fusion proteins and nucleotide sequences encoding the same are described below in Section I.B.2.

[0206] In some embodiments, the gRNA is provided as RNA and a polynucleotide encoding the fusion protein is mRNA. The mRNA can be 5' capped and / or 3' polyadenylated.1. Target sites and Guide RNAs

[0207] Provided herein as part of the epigenetic-modifying DNA targeting system and LNP HBV epi-silencer therapy are guide RNAs for targeting to a target site in a HBV DNA sequence. In some embodiments, the guide RNAs (gRNA) are for use with the LNP HBV epi-silencer therapy in a method of treating chronic hepatitis B (CHB) infection in a subject in need thereof such as described herein. a. Target sites

[0208] In some embodiments, the target site is in a Hepatitis B viral DNA sequence. In some embodiments, the epigenetic-modifying DNA-targeting system comprises a DNA-targeting module for repressing transcription of total viral Hepatitis B Viral (HBV) mRNA transcripts by targeting to the target site. In some aspects, repressing transcription of the HBV gene, such as reduced gene expression, results in silencing of HBV replication (e.g., reduced HBV replication) and / or HBV transcription.

[0209] In any of the embodiments herein, the target site is a gene and / or regulatory element thereof in the Hepatitis B viral (HBV) genome. In some embodiments, the target site is present55MF-36384587022474-20039.40 in a covalently closed circular DNA (cccDNA) and is an HBV viral sequence integrated in the human genomic DNA. In some embodiments, the target site is at or near a gene involved in HBV replication and / or HBV transcription. In some aspects, the target site is at or near a promoter. In some aspects, the target site is near an enhancer region. In some aspects, the target site is at or near transcript processing control region. In some aspects, the target site may be any sequence optimal for depositing DNA methylation for silencing HBV transcription.

[0210] In provided embodiments, the cccDNA transcribes five HBV RNAs (0.7 kb, 2.1 kb, 2.4 kb, longer and shorter 3.5 kb RNAs) under the host RNA polymerase. Transcription of cccDNA is controlled by four promoters- the basal core, preSl, preS2, and X promoters and two enhancers- enhancers I and II. The 0.7-kb RNA can be translated to HBV X protein (HBx) which acts as a transcriptional regulator. The 2.1 -kb RNA can be translated to HBV small surface protein (S) and middle surface protein (M). The 2.4-kb RNA can be translated to HBV large surface protein (L). L, M, and S can self-assemble to form empty subviral particles (SVPs) (including spherical SVPs and filamentous SVPs) that are secreted with only filamentous SVPs and virions containing significant amounts of L protein. The spherical SVPs are secreted through the constitutive secretory pathway. The filamentous SVPs are secreted by the endosomal sorting complex required for transport (ESCRT) machinery through multivesicular bodies (MVB). The longer 3.5-kb RNA is termed pre-core RNA (preC RNA) and can be translated to pre-Core protein, better known as HBV e antigen (HBeAg). The shorter 3.5-kb RNA is pre-genomic RNA (pgRNA) that has two roles, as the translation template for HBV polymerase (Pol) and Core proteins and as the replication template for intra-capsid (formed by Core protein polymerization) reverse transcription by Pol to form HBV rcDNA. These nucleocapsids can then be enveloped by HBV surface proteins (L, M, and S) to form mature virions and secreted through the ESCRT / MVB pathway. Alternatively, these nucleocapsids can also be transported to the nucleus to form cccDNA. In some embodiments, repressing transcription and / or translation of the HBV gene, such as reduced gene expression, results in silencing of any of the following HBV markers: HBV HBV X protein (HBx), Hepatitis B surface antigens (HBsAg) such as small surface protein (S), middle surface protein (M), or HBV large surface protein (L), HBV e antigen (HBeAg). In some embodiments, repressing transcription and / or translation of the HBV gene, such as reduced gene expression, results in silencing of HB core-related antigens (HBcrAg). HBcrAg includes 3 precore / core protein products, including hepatitis B core antigen (HBeAg), HBeAg, and a 22-kDA precore protein (p22cr). In some aspects, cccDNA, HBV total DNA, serum HBcrAg, HBsAg, HBeAg, hepatitis56MF-36384587022474-20039.40B core antibody (anti-HBc), HBV DNA, HBV RNA are quantified as readouts for measuring reduced HBV transcription and / or translation. In some embodiments, the target site is in a gene that encodes any of the HBV proteins. In some embodiments, the target site is in a regulatory element (e.g. promoter or enhancer) of a gene that encodes any of the HBV proteins.

[0211] In some embodiments, the target site for an epigenetic-modifying DNA-targeting system is in a gene involved in HBV replication and / or HBV transcription. In some aspects, the target site for an epigenetic-modifying DNA-targeting system is in or near a gene or a regulatory element thereof involved in controlling HBV replication and / or HBV transcription. In some embodiments, the gene involved in HBV replication and / or HBV transcription is a X-gene. In some embodiments, the gene involved in HBV replication and / or transcription encodes a transcription factor or transcriptional transactivator. In some embodiments, the regulatory element thereof involved in HBV replication and / or HBV transcription is an enhancer region. In some embodiments, the promoter region is a X promoter or a basal core promoter. In some embodiments, the enhancer region is an Enhl enhancer and / or an Enh2 enhancer region.

[0212] In some embodiments, the target site is a sequence within a target region that has a sequence corresponding to the sequence positioned between base pair (bp) positions: 1033 bp - 1749 bp (CpG Island 2) of the HBV genome with reference to the Hepatitis B Virus genome (Hepatitis B virus subtype ayw, complete genome, GenBank: U95551.1), SEQ ID NO 21.

[0213] In some embodiments, the gRNA targets a site positioned between 1033 bp - 1749 bp (CpG Island 2) of the HBV genome. In some embodiments, the gRNA targets a site positioned between 1255-1302 bp of the HBV genome corresponding to positions with reference to the Hepatitis B Virus genome (Hepatitis B virus subtype ayw, complete genome, GenBank: U95551.1), SEQ ID NO: 21. In some embodiments, the gRNA targets a site positioned between 1260-1300 bp bp of the HBV genome corresponding to positions with reference to the Hepatitis B Virus genome (Hepatitis B virus subtype ayw, complete genome, GenBank: U95551.1), SEQ ID NO: 21. In some embodiments, the gRNA targets a site at or near a regulatory element involved in HBV replication and / or transcription. In some embodiments, the gRNA targets the X-gene. In some embodiments, the gRNA targets the X promoter regions. In some embodiments, the gRNA targets Enhl or an Enh2 enhancer region. In some embodiments, the gRNA targets an HBV coding region.

[0214] In some embodiments, the target site is in a regulatory element of the X-gene. The X- gene (also known as HBx, HBVgp3, peptide X, pX) is a gene that encodes a multifunctional protein that modulates transcriptional regulation, protein degradation pathways, apoptosis, signal57MF-36384587022474-20039.40 transduction, cell cycle progress, and genetic stability by directly or indirectly interacting with host factors. The X-gene protein modulates protein degradation pathways, apoptosis, transcription, signal transduction, cell cycle progress, and genetic stability by directly or indirectly interacting with host factors. In some embodiments, the target site is a sequence within a target region that has a sequence corresponding to the sequence positioned between 1376 bp - 1749 bp of the HBV genome with reference to the Hepatitis B Virus genome (Hepatitis B virus subtype ayw, complete genome, GenBank: U95551.1), SEQ ID NO: 21. In some embodiments, the target site is within a target region that has a sequence corresponding to the sequence located at base pairs between 1255-1302 bp with reference to the HBV genome set forth in SEQ ID NO: 21. In some embodiments, the target site is within a target region that has a sequence corresponding to the sequence located at base pairs between 1260-1300 bp with reference to the HBV genome set forth in SEQ ID NO: 21.

[0215] In some embodiments, the start codon for encoding the HBx protein (HBx start codon) is at residue base pair 1376 of the HBV genome corresponding to positions with reference to the HBV genome set forth in SEQ ID NO: 21. It is found herein that targeting a target site in the X-gene in this region upstream of the start codon using a provided epigenetic- modifying DNA targeting system exhibits high activity for repressing viral replication and transcription of HBV infected cells. In some embodiments, the target region is in a CpG island of the HBV genome. In some embodiments, the target site is a sequence within a target region that has a sequence corresponding to the sequence positioned between 1033-1749 bp with reference to the HBV genome set forth in SEQ ID NO:21. In some embodiments, the target site is in the Enhancer II region, such as within a target region that has a sequence corresponding to the sequence positioned between between 1636-1744 bp with reference to the HBV genome set forth in SEQ ID NO: 21. In some embodiments, the target site is in the HBx promoter / Enhancer I region, such as within a target region that has a sequence corresponding to the sequence positioned between between 1100-1300 bp with reference to the HBV genome set forth in SEQ ID NO: 21. In some embodiments, the target site is within a target region that has a sequence corresponding to the sequence positioned between between 1100-1300 bp with reference to the HBV genome set forth in SEQ ID NO: 21. In some embodiments, the target site is in the basal core promoter region, such as within a target region that has a sequence corresponding to the sequence positioned between 1600-1749 bp with reference to the HBV genome set forth in SEQ ID NO: 21.

[0216] In some embodiments, the target site is located within about 50 base pairs (bp),58MF-36384587022474-20039.40 about 100 bp, about 150 bp, about 200 bp, about 250 bp, about 300 bp, about 350 bp, about 400 bp, about 450 bp, about 500 bp, about 600b p, about 650 bp, about 700 bp, about 750 bp, about 800 bp, about 850 bp, about 900 bp, about 1000 bp, about 1050 bp, about 1100 bp, about 1200 bp, about 1250 bp, about 1300 bp, about 1350 bp about 1400 bp, about 1450 bp, about 1500 bp, of a transcription start site. In some embodiments, the transcription start site is a start site for the HBx gene. In some embodiments, the target site is located within about 300 bp of a transcription start site of an HBx gene. In some embodiments, the target site is located within about 350 bp of a transcription start site of an HBx gene.

[0217] According to phylogenetic analyses and sequence divergence, HBV can be classified into 10 genotypes (A to J) based upon an inter-group divergence of 8 percent or more in the complete nucleotide sequence (Norder H, et.a., Complete genomes, phylogenetic relatedness, and structural proteins of six strains of the hepatitis B virus, four of which represent two new genotypes. Virology. 1994 Feb;198(2):489-503; Stuyver L, et.al., A new genotype of hepatitis B virus: complete genome and phylogenetic relatedness. J Gen Virol. 2000 Jan;81(Pt l):67-74, Arauz-Ruiz P, et.al., Genotype H: a new Amerindian genotype of hepatitis B virus revealed in Central America. J Gen Virol. 2002 Aug;83(Pt 8):2059-2073)). There is evidence suggesting that HBV genotypes influence clinical outcomes, mutational patterns in the precore and core promoter regions, HbeAg seroconversion rates, and response to interferon therapy. Most genotypes have specific geographic distributions; genotypes A and D are prevalent in Western Europe and North America, and genotypes B and C are prevalent in East Asia and Oceania.

[0218] In some embodiments, the target site is at least 70% homologous to all Hepatitis B viral genotypes (e.g., genomes). In some embodiments, the target site is at least 70% homologous to at least 500, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, at least 5000, at least 5500, at least 6000, at least 6500, at least 7000 Hepatitis B viral genomes. In some embodiments, the target site is at least 70% homologous to at least 1000 Hepatitis B viral genomes and comprises up to two mismatches.

[0219] In some embodiments, the target site is at least 90% homologous to all Hepatitis B viral genomes. In some embodiments, target site is at least 90% homologous to at least 500, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, at least 5000, at least 5500, at least 6000, at least 6500, at least 7000 Hepatitis B viral genomes.

[0220] In some embodiments, the target site is at least 90% homologous to at least 1000 Hepatitis B viral genomes and comprises one or two mismatches. In some embodiments, the59MF-36384587022474-20039.40 mismatches are located in the first 12 nt on the 5’ end of the protospacer adjacent motif (PAM) as represented by ‘n’ in ‘nnnnnnnnnnnnNNNNNNNN-NGG’. In some embodiments, the target site is at least 90% homology to at least 1000 Hepatitis B viral genomes and comprises zero mismatches.

[0221] In some of any embodiments, that target site is a sequence of 14 to 22 nucleotides. In some of any embodiments, that target site is a sequence of 14 to 19 nucleotides. In some of any embodiments, that target site is a sequence of 14 nucleotides. In some of any embodiments, that target site is a sequence of 15 nucleotides. In some of any embodiments, that target site is a sequence of 16 nucleotides. In some of any embodiments, that target site is a sequence of 17 nucleotides. In some of any embodiments, that target site is a sequence of 18 nucleotides. In some of any embodiments, that target site is a sequence of 19 nucleotides.

[0222] In provided embodiments, reference to a target site or complement thereof (interchangeably, “complementary sequence”) refers to the region of DNA to which the spacer sequence hybridizes within and can be designated by a sequence that is complementary to the spacer (including the reverse complement when accounting for the 5’ to 3’ directionality of a sequence) or the sequence of the other strand of the paired DNA in the region. For instance, for purposes herein, the target site is typically designated by the same sequence as the spacer sequence (except that the target site sequence is DNA instead of RNA), which is the sequence of the other strand of the paired DNA that is in the region that the spacer hybridizes to. It is understood that reference to a target site or complement thereof (or variations thereof, such as “complementary”) can be a complement of the target site sequence, including the reverse complement (e.g., taking into account 5’ to 3’ directionality of a sequence).

[0223] Thus, it is understood that reference to a target site may represent the target site sequence or a complement sequence. Also, depiction of a spacer sequence (RNA), it is understood that the uracil (“U”) in the sequence can alternatively be depicted as a “T”, such as in accordance with WIPO Standard ST. 26 sequence rules.

[0224] In any of the embodiments provided herein, the target site is complementary to a referenced sequence (i.e. particular sequence set forth by SEQ ID NO with reference to the Sequence Listing). In some of any embodiments, a complementary sequence is a reverse complement of the referenced sequence.

[0225] In any of the embodiments provided herein, the target site comprises the referenced sequence (i.e. particular sequence set forth by SEQ ID NO with reference to the Sequence Listing). In any of the embodiments provided herein, the target site is the sequence set forth by60MF-36384587022474-20039.40 the referenced sequence (i.e. particular sequence set forth by SEQ ID NO with reference to the Sequence Listing).

[0226] In any of the embodiments provided herein, the target site is a contiguous portion of at least 14 nucleotides (14 nt) of a referenced sequence (i.e. particular sequence set forth by SEQ ID NO with reference to the Sequence Listing). In some embodiments, the contiguous portion is 15 nucleotides. In some embodiments, the contiguous portion is 16 nucleotides. In some embodiments, the contiguous portion is 17 nucleotides. In some embodiments, the contiguous portion is 18 nucleotides. In some embodiments, the contiguous portion is 19 nucleotides.

[0227] In any of the embodiments herein, the target site, or each of the target sites, comprises the sequence set forth in any one of SEQ ID NOS: 27, 63-113, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some embodiments, the target site is a contiguous portion of any one of SEQ ID NOS: 27, 63- 113 that is 15, 16, 17, 18 or 19 nucleotides in length, or a complementary sequence of any of the foregoing. In some embodiments, the target site is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a target site sequence described herein above. In some embodiments, the target site is the sequence set forth in any one of SEQ ID NO: 27, 63-113 In some embodiments, the reduction in gene expression or the change in the level of transcripts in a cell is greater than 90% compared to the level of gene in a control cell.

[0228] In some embodiments, the target site, or each of the target sites, comprises the sequence set forth in SEQ ID NO: 27, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In any of the embodiments herein, the target site, or each of the target sites, comprises a contiguous portion of the sequence set forth in SEQ ID NO: 27 that is 14-19 nucleotides (nt) in length (such as 14, 15, 16, 17, 18 or 19 nucleotides in length), or a complementary sequence of the foregoing.

[0229] In some embodiments, the gRNA targets a target site that comprises the sequence set forth in SEQ ID NO: 27. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 27. In some embodiments, the gRNA targets a target site set forth in SEQ ID NO: 27. b. Guide RNAs

[0230] Provided herein is a guide RNA for targeting to a target site in a HBV DNA sequence. In some embodiments, the guide RNA is for a method of treating chronic hepatitis B61MF-36384587022474-20039.40(CHB) infection in a subject in need thereof.

[0231] In some aspects, the gRNA is a nucleic acid that promotes the specific targeting or homing of the gRNA / Cas RNP complex to the target site of the target gene and / or regulatory element thereof, such as any described herein. In some embodiments, a target site of a gRNA may be referred to as a protospacer.

[0232] Provided herein are gRNAs, such as gRNAs that target or bind to a target site or DNA regulatory element thereof. In some embodiments, the gRNA is capable of complexing with the Cas protein (e.g., dSpCas9) or variant thereof. In some embodiments, the gRNA comprises a gRNA spacer sequence (i.e. a spacer sequence or a guide sequence) that is capable of hybridizing to the target site, or that is complementary to the target site, such as any target site described herein. In some embodiments, the gRNA comprises a scaffold sequence that complexes with or binds to the Cas protein.

[0233] In some embodiments, the gRNAs provided herein are chimeric gRNAs. In general, gRNAs can be unimolecular (i.e. consisting of a single RNA molecule), or modular (comprising more than one, and typically two, separate RNA molecules). Modular gRNAs can be engineered to be unimolecular, wherein sequences from the separate modular RNA molecules are comprised in a single gRNA molecule, sometimes referred to as a chimeric gRNA, synthetic gRNA, or single gRNA. In some embodiments, the chimeric gRNA is a fusion of two noncoding RNA sequences: a crRNA sequence and a tracrRNA sequence, for example as described in WO 2013 / 176772 Al, or Jinek, M. et al. Science 337(6096):816-21 (2012). In some embodiments, the chimeric gRNA mimics the naturally occurring crRNA:tracrRNA duplex involved in the Type II Effector system, wherein the naturally occurring crRNA:tracrRNA duplex acts as a guide for the Cas9 protein.

[0234] In some aspects, the spacer sequence of a gRNA is a polynucleotide sequence comprising at least a portion that has sufficient complementarity with the target site or DNA regulatory element thereof (e.g. any described herein) to hybridize with a target site in the target gene and / or regulatory element thereof and direct sequence-specific binding of a CRISPR complex to the sequence of the target site. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. In some embodiments, the gRNA comprises a spacer sequence that is complementary, e.g., at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% (e.g., fully complementary), to the target site. The strand of the target nucleic acid comprising the target site sequence may be referred to as the “complementary strand” of the target nucleic acid.62MF-36384587022474-20039.40

[0235] In some embodiments, the gRNA spacer sequence is between about 14 nucleotides (nt) and about 26 nt, or between 16 nt and 22 nt in length. In some embodiments, the gRNA spacer sequence is 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt or 22 nt, 23 nt, 24 nt, 25 nt, or 26 nt in length. In some embodiments, the gRNA spacer sequence is 18 nt, 19 nt, 20 nt, 21 nt or 22 nt in length. In some embodiments, the gRNA spacer sequence is 19 nt in length.

[0236] A target site of a gRNA may be referred to as a protospacer. In some aspects, the spacer is designed to target a protospacer with a specific protospacer-adjacent motif (PAM), i.e. a sequence immediately adjacent to the protospacer that contributes to and / or is required for Cas binding specificity. Different CRISPR / Cas systems have different PAM requirements for targeting. For example, in some embodiments, S. pyogenes Cas9 uses the PAM 5 ’-NOGS’, where N is any nucleotide.

[0237] Cas proteins may use or be engineered to use different PAMs from those listed above. For example, mutated SpCas9 proteins may use the PAMs 5’-NGG-3’, 5’-NGAN-3’, 5’- NGNG-3’, 5’-NGAG-3’, or 5’-NGCG-3’. In some embodiments, the protospacer of a gRNA for complexing with S. pyogenes Cas9 or variant thereof is set forth in SEQ ID NO: 26.

[0238] A spacer sequence may be selected to reduce the degree of secondary structure within the spacer sequence. Secondary structure may be determined by any suitable polynucleotide folding algorithm.

[0239] In some embodiments, the gRNA (including the guide sequence) will comprise the base uracil (U), whereas DNA encoding the gRNA molecule will comprise the base thymine (T). While not wishing to be bound by theory, in some embodiments, it is believed that the complementarity of the guide sequence with the target sequence contributes to specificity of the interaction of the gRNA molecule / Cas molecule complex with a target nucleic acid. It is understood that in a guide sequence and target sequence pair, the uracil bases in the guide sequence will pair with the adenine bases in the target sequence.

[0240] In some embodiments, one, more than one, or all of the nucleotides of a gRNA can have a modification, e.g., to render the gRNA less susceptible to degradation and / or improve bio-compatibility. By way of non-limiting example, the backbone of the gRNA can be modified with a phosphorothioate, or other modification(s). In some cases, a nucleotide of the gRNA can comprise a 2’ modification, e.g., a 2-acetylation, e.g., a 2’ methylation, or other modification(s).

[0241] Methods for designing gRNAs and exemplary targeting domains can include those described in, e.g., International PCT Pub. Nos. WO 2014 / 197748 A2, WO 2016 / 130600 A2 , WO 2017 / 180915 A2 , WO 2021 / 226555 A2 , WO 2013 / 176772 Al , WO 2014 / 152432 A2 ,63MF-36384587022474-20039.40WO 2014 / 093661 A2 , WO 2014 / 093655 A2 , WO 2015 / 089427 Al , WO 2016 / 049258 A2 , WO 2016 / 123578 Al , WO 2021 / 076744 Al , WO 2014 / 191128 Al , WO 2015 / 161276 A2 , WO 2017 / 193107 A2, and WO 2017 / 093969 Al.

[0242] In some embodiments, the gRNA targets a target site as described in Section I.B.1. In some embodiments, the gRNA targets a target site that comprises a sequence selected from any one of SEQ ID NOS: 27, 63-113 as shown in Table 2, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the gRNA for targeting to the target site comprises the sequence set forth in SEQ ID NOS: 26, 114-164, a contiguous portion thereof of at least 14 nucleotides (e.g. 14, 15, 16, 17, 18 or 19 nucleotides), or a complementary sequence of any of the foregoing or a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the gRNA for targeting to the target site comprises the sequence set forth in SEQ ID NO: 26, 114-164. In some embodiments, the gRNA for targeting to the target site is set forth in SEQ ID NO: 26, 114-164.

[0243] In some embodiments, the gRNA for targeting to the target site comprises the sequence set forth in SEQ ID NO: 26, a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing or a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the gRNA for targeting to the target site comprises the sequence set forth in SEQ ID NO: 26. In some embodiments, the gRNA for targeting to the target site is set forth in SEQ ID NO: 26.

[0244] In some embodiments, the gRNA further comprises a scaffold sequence. In some embodiments, the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 39. In some embodiments, the scaffold sequence comprises the sequence set forth in SEQ ID NO: 39 (GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC), or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 39. In some embodiments, the scaffold sequence is set forth in SEQ ID NO: 39.64MF-36384587022474-20039.40

[0245] In some embodiments, the gRNA comprises the sequence selected from any one of SEQ ID NOS: 38, 165-215 (e.g, as shown in Table 3), or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of SEQ ID NO: 38, 165-215. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 38, 165-215. In some embodiments, the gRNA is set forth in SEQ ID NO: 38, 165-215.

[0246] In some embodiments, the gRNA comprises the sequence selected from any one of SEQ ID NOS: 38 (e.g., as shown in Table 3), or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of SEQ ID NO: 38. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 38. In some embodiments, the gRNA is set forth in SEQ ID NO: 38 (HBVg l).

[0247] In some embodiments, any of the provided gRNA sequences is complexed with a Cas9. In some embodiments, the Cas9 is a dCas9. In some embodiments, the dCas9 is a dSpCas9, such as a dSpCas9 set forth in SEQ ID NO: 25.Table 2. Target site sequences and gRNA spacers65MF-36384587022474-20039.4066MF-36384587022474-20039.40Table 3. Gene-targeting gRNAs67MF-36384587022474-20039.4068MF-36384587022474-20039.402. mRNA and Fusion proteins

[0248] In some aspects, the DNA-targeting systems provided herein comprise a DNA- targeting module that is a fusion protein directed to the target site by the gRNA and that contains one or more transcriptional repression domains. In some embodiments, a provided lipid69MF-36384587022474-20039.40 nanoparticle carries such as encapsulates a polynucleotide encoding the fusion protein and the gRNA. In some embodiments, the polynucleotide encoding the fusion protein is an mRNA. In some embodiments, all of the components of the fusion protein provided herein are encoded in one polynucleotide (e.g., mRNA).

[0249] In some embodiments, the encoded fusion protein comprises: (a) a DNA- binding domain (e.g., a deactivated Streptococcus pyogenes Cas9 (dSpCas9) protein) for targeting to a target site in a Hepatitis B viral DNA sequence, such as a gene or regulatory element thereof, for example any as described herein, and (b) at least one transcriptional repressor effector domain (e.g., DNMT3A / L and KRAB). In some embodiments, the fusion protein contains a a deactivated Streptococcus pyogenes Cas9 (dSpCas9) protein or variant thereof, such as described in Section I B.2 (a), and at least one transcriptional repressor effector domain, such as any described in Section I B.2(b). In some embodiments, the at least one transcriptional repressor effector domain compres a KRAB domain and a DNA methyltransferase 3 (DNMT3) domain that has DNA methyltransferase activity. In some aspects, the fusion protein is targeted to a target site of a HBV viral sequence, such as a regulatory element of an HB V gene, such as any described in Section I.B.1, and leads to reduced or repressed transcription of the gene.

[0250] In some embodiments, the fusion protein is encoded from an mRNA eapsulated in the lipid nanoparticle. In some embodiments, the mRNA further comprises one or more untranslated regions (UTR) near the 5’ end and / or the 3’ end. In some embodiments, the mRNA further comprises a UTR near the 5’ end and near the 3’ end. In some embodiments, the mRNA further comprises a UTR at the 5’ end and at the 3’ end. In some embodiments, the mRNA further comprises a polyA sequence at the 3’ end.

[0251] In some embodiments, the mRNA comprises one or more UTRs, a nucleotide sequence encoding the fusion protein, and a polyA sequence. In some embodiments, the mRNA comprises from 5’ end to the 3’ end: a UTR, a nucleotide sequence encoding the fusion protein, a UTR, and a polyA sequence. In some embodiments, the mRNA comprises from the 5’ end to the 3’ end: the UTR comprising the sequence set forth in SEQ ID NO: 9, a nucleotide sequence encoding the fusion protein, the UTR comprising the sequence set forth in SEQ ID NO: 10, and a polyA sequence comprising the sequence set forth in SEQ ID NO: 19.

[0252] In some embodiments, the nucleotide sequence encoding the fusion protein is one tht encodes a fusion protein comprising a DNMT3 A domain, a DNMT3L domain, a dSpCas9, a KRAB domain, one or more linkers, and one or more nuclear localization signals (NLS). In some embodiments, the encoded fusion protein comprises a DNMT3 A domain, a DNMT3L70MF-36384587022474-20039.40 domain, a dSpCas9, a KRAB domain. In some embodiments, the encoded fusion protein further comprises one or more linkers, and one or more NLS. In some embodiments, the encoded fusion protein comprises from N-terminus to C-terminus: a DNMT3A domain, a linker, the DNMT3L domain, a linker, a NLS, a linker, the dSpCas9, a linker, a NLS, a linker, the KRAB, and a NLS. In some embodiments, the fusion protein is encoded by a nucleotide sequence that includes, from the 5’ end to the 3’ end: a nucleotide sequence encoding the DNMT3A domain comprising the sequence set forth in SEQ ID NO: 2, a nucleotide sequence encoding a linker comprising the sequence set forth in SEQ ID NO: 11, a nucleotide sequence encoding the DNMT3L domain comprising the sequence set forth in SEQ ID NO: 3, a nucleotide sequence encoding the linker comprising the sequence set forth in SEQ ID NO: 12, a nucleotide sequence encoding an NLS comprising the sequence set forth in SEQ ID NO: 13, a nucleotide sequence encoding a linker comprising the sequence set forth in SEQ ID NO: 14, a nucleotide sequence encoding the dSpCas9 comprising the sequence set forth in SEQ ID NO: 4, a nucleotide sequence encoding a linker comprising the sequence set forth in SEQ ID NO: 15, a nucleotide sequence encoding an NLS comprising the sequence set forth in SEQ ID NO: 16, a nucleotide sequence encoding the linker comprising the sequence set forth in SEQ ID NO: 17, a nucleotide sequence encoding the KRAB comprising the sequence set forth in SEQ ID NO: 5, and a nucleotide sequence encoding the NLS comprising the sequence set forth in SEQ ID NO: 18.

[0253] In some embodiments, the encoded fusion protein comprises from the N-terminus to the C-terminus: the DNMT3A domain comprising the sequence set forth in SEQ ID NO: 22, the linker comprising the sequence set forth in SEQ ID NO: 34, the DNMT3L domain comprising the sequence set forth in SEQ ID NO: 23, the linker comprising the sequence set forth in SEQ ID NO: 35, the NLS comprising the sequence set forth in SEQ ID NO: 32, the linker comprising the sequence set forth in SEQ ID NO: 36, the dSpCas9 comprising the sequence set forth in SEQ ID NO: 25, the linker comprising the sequence set forth in SEQ ID NO: 37, the NLS comprising the sequence set forth in SEQ ID NO: 16, the linker comprising the sequence set forth in SEQ ID NO: 33, the KRAB comprising the sequence set forth in SEQ ID NO: 28, the NLS comprising the sequence set forth in SEQ ID NO: 32.

[0254] In some embodiments, the polynucleotide is an mRNA molecule that comprises a nucleotide sequence encoding a DNMT3A / L-dSpCas9-KRAB fusion protein, such as the sequence set forth in SEQ ID NO:41, or a sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto. In some embodiments, the mRNA comprises the sequence set forth in SEQ ID NO: 1, or a sequence71MF-36384587022474-20039.40 having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto. In some embodiments, the mRNA is set forth in SEQ ID NO: 1.

[0255] In some embodiments, the mRNA comprises the sequence set forth in SEQ ID NO: 1, or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the foregoing. In some embodiments, the mRNA comprises the sequence set forth in SEQ ID NO: 1.

[0256] Provided herein is a method of treating chronic hepatitis B (CHB) infection in a subject in need thereof, the method comprising administering to the subject a dose of lipid nanoparticles comprising an epigenetic-modifying DNA-targeting system comprising a DNA- targeting module for repressing total viral Hepatitis B Viral (HBV) mRNA transcripts. In some embodiments, the DNA-targeting module comprises: (a) a gRNA for targeting to a target site in a HBV DNA sequence, wherein the target site comprises the sequence set forth in SEQ ID NO: 27; and (b) a mRNA comprising from the N-terminus to the C-terminus: the UTR comprising the sequence set forth in SEQ ID NO: 9, a nucleotide encoding a fusion protein, the UTR comprising the sequence set forth in SEQ ID NO: 10, and a polyA sequence comprising the sequence set forth in SEQ ID NO: 19. In some embodiments, the fusion protein comprises from the N-terminus to the C-terminus: the DNMT3 A domain comprising the sequence set forth in SEQ ID NO: 22, the linker comprising the sequence set forth in SEQ ID NO: 34, the DNMT3L domain comprising the sequence set forth in SEQ ID NO: 23, the linker comprising the sequence set forth in SEQ ID NO: 35, the NLS comprising the sequence set forth in SEQ ID NO: 32, the linker comprising the sequence set forth in SEQ ID NO: 36, the dSpCas9 comprising the sequence set forth in SEQ ID NO: 25, the linker comprising the sequence set forth in SEQ ID NO: 37, the NLS comprising the sequence set forth in SEQ ID NO: 16, the linker comprising the sequence set forth in SEQ ID NO: 33, the KRAB comprising the sequence set forth in SEQ ID NO: 28, the NLS comprising the sequence set forth in SEQ ID NO: 32. In some embodiments, the fusion protein is encoded by a nucleotide sequence that includes, from the 5’ end to the 3’ end: a nucleotide sequence encoding the DNMT3A domain comprising the sequence set forth in SEQ ID NO: 2, a nucleotide sequence encoding a linker comprising the sequence set forth in SEQ ID NO: 11, a nucleotide sequence encoding the DNMT3L domain comprising the sequence set forth in SEQ ID NO: 3, a nucleotide sequence encoding the linker comprising the sequence set forth in SEQ ID NO: 12, a nucleotide sequence encoding an NLS comprising the sequence set forth in SEQ ID NO: 13, a nucleotide sequence encoding a linker comprising the sequence set forth in SEQ ID NO: 14, a nucleotide sequence encoding the72MF-36384587022474-20039.40 dSpCas9 comprising the sequence set forth in SEQ ID NO: 4, a nucleotide sequence encoding a linker comprising the sequence set forth in SEQ ID NO: 15, a nucleotide sequence encoding an NLS comprising the sequence set forth in SEQ ID NO: 16, a nucleotide sequence encoding the linker comprising the sequence set forth in SEQ ID NO: 17, a nucleotide sequence encoding the KRAB comprising the sequence set forth in SEQ ID NO: 5, and a nucleotide sequence encoding the NLS comprising the sequence set forth in SEQ ID NO: 18.

[0257] Provided herein is a method of treating chronic hepatitis B (CHB) infection in a subject in need thereof, the method comprising administering to the subject a dose of lipid nanoparticles comprising (a) a gRNA for targeting to a target site in a HBV DNA sequence, wherein the target site comprises the sequence set forth in SEQ ID NO: 27; and (b) a mRNA comprising the sequence set forth in SEQ ID NO: 1 or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the foregoing.

[0258] In some embodiments, the polynucleotide as provided herein can be codon optimized for efficient translation into protein in the eukaryotic cell or animal of interest. For example, codons can be optimized for expression in humans, mice, rats, hamsters, cows, pigs, cats, dogs, fish, amphibians, plants, yeast, insects, and so forth. Programs for codon optimization are available as freeware. Commercial codon optimization programs are also available. a. CRISPR-Based DNA Binding domains

[0259] The provided epigenetic DNA-targeting systems include fusion proteins based on CRISPR / Cas systems, i.e., CRISPR / Cas-based DNA-targeting systems, that are able to guide the transcriptional repression domain of the fusion protein to a target site in a target gene or regulatory element thereof via a Cas-gRNA complex. Hence, the DNA-targeting system also include the gRNA, provided in combination or as a complex with the dCas9 protein or variant thereof, for targeting of the DNA-targeting system to the target site of the target gene or regulatory element thereof. In some embodiments, the fusion protein is guided to a specific target site sequence of the target gene or regulatory element thereof by the guide RNA, wherein the effector domain mediates targeted epigenetic modification to reduce or repress transcription of the target gene.

[0260] In some embodiments, the DNA- binding domain comprises or is derived from a CRISPR associated (Cas) protein or variants thereof. In some embodiments, the DNA- binding domain comprises a catalytically inactive (e.g. nuclease-inactive or nuclease-inactivated) variant of the foregoing. In some embodiments, the DNA- binding domain comprises a deactivated Cas9 (dCas9) protein or variant thereof that is a catalytically inactivated so that it is inactive for73MF-36384587022474-20039.40 nuclease activity and is not able to cleave the DNA.

[0261] In some aspects, CRISPR / Cas-based DNA-targeting systems comprises a Cas protein that is a variant that lacks nuclease activity (i.e. is a dCas protein). In some embodiments, the Cas protein is mutated so that nuclease activity is reduced or eliminated. Such Cas proteins are referred to as deactivated Cas or dead Cas (dCas) or nuclease-inactive Cas (iCas) proteins, as referred to interchangeably herein. In some embodiments, the variant Cas protein is a variant Cas9 protein that lacks nuclease activity or that is a deactivated Cas9 (dCas9, or iCas9) protein.

[0262] The CRISPR system (also known as CRISPR / Cas system, or CRISPR-Cas system) refers to a conserved microbial nuclease system, found in the genomes of bacteria and archaea, that provides a form of acquired immunity against invading phages and plasmids. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), refers to loci containing multiple repeating DNA elements that are separated by non-repeating DNA sequences called spacers. Spacers are short sequences of foreign DNA that are incorporated into the genome between CRISPR repeats, serving as a 'memory' of past exposures. Spacers encode the DNA-targeting portion of RNA molecules that confer specificity for nucleic acid cleavage by the CRISPR system. CRISPR loci contain or are adjacent to one or more CRISPR-associated (Cas) genes, which can act as RNA-guided nucleases for mediating the cleavage, as well as non-protein coding DNA elements that encode RNA molecules capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage.

[0263] In Type II CRISPR / Cas systems with the Cas protein Cas9, two RNA molecules and the Cas9 protein form a ribonucleoprotein (RNP) complex to direct Cas9 nuclease activity. The CRISPR RNA (crRNA) contains a spacer sequence that is complementary to a target nucleic acid sequence (target site), and that encodes the sequence specificity of the complex. The transactivating crRNA (tracrRNA) base-pairs to a portion of the crRNA and forms a structure that complexes with the Cas9 protein, forming a Cas / RNA RNP complex.

[0264] Naturally occurring CRISPR / Cas systems, such as those with Cas9, have been engineered to allow efficient programming of Cas / RNA RNPs to target desired sequences in cells of interest, both for gene-editing and modulation of gene expression. The tracrRNA and crRNA have been engineered to form a single chimeric guide RNA molecule, commonly referred to as a guide RNA (gRNA), for example as described in WO 2013 / 176772 Al, WO 2014 / 093661 A2, WO 2014 / 093655 A2, Jinek, M. et al. Science 337(6096): 816-21 (2012), or Cong, L. et al. Science 339(6121): 819-23 (2013). The spacer sequence of the gRNA can be chosen by a user to target the Cas / gRNA RNP complex to a desired locus, e.g. a desired target74MF-36384587022474-20039.40 site in the target gene and / or regulatory element thereof.

[0265] Cas proteins have also been engineered to allow targeting of Cas / gRNA RNPs without inducing cleavage at the target site. Mutations in Cas proteins can reduce or abolish nuclease activity of the Cas protein, rendering the Cas protein catalytically inactive. Cas proteins with reduced or abolished nuclease activity are referred to as deactivated Cas (dCas), or nuclease-inactive Cas (iCas) proteins, as referred to interchangeably herein. Exemplary deactivated Cas9 (dCas9) derived from S. pyogenes contains silencing mutations of the RuvC and HNH nuclease domains (D10A and H840A), for example as described in WO 2013 / 176772 Al, WO 2014 / 093661 A2, Jinek, M. et al. Science 337(6096):816-21 (2012), and Qi, L. et al. Cell 152(5): 1173-83 (2013). Exemplary dCas variants derived from the Casl2 system (i.e. Cpfl) are described, for example in WO 2017 / 189308 Al and Zetsche, B. et al. Cell 163(3):759-71 (2015). Conserved domains that mediate nucleic acid cleavage, such as RuvC and HNH endonuclease domains, are readily identifiable in Cas orthologues, and can be mutated to produce inactive variants, for example as described in Zetsche, B. et al. Cell 163(3):759-71 (2015).

[0266] In some embodiments, the dCas9 protein can comprise a sequence derived from a naturally occurring Cas9 molecule, or variant thereof. In some embodiments, the dCas9 protein can comprise a sequence derived from a naturally occurring Cas9 molecule of S. pyogenes, or variant thereof. In some embodiments, the dCas9 protein comprises a sequence derived from a naturally occurring Cas9 molecule of S. pyogenes.

[0267] In some embodiments, the dCas9 protein can comprise a sequence derived from a naturally occurring Cas9 molecule of S. thermophilus, S. aureus, C. jejuni, N. meningitidis, F. novicida, S. canis, S. auricularis. Non-limiting examples of Cas9 orthologs from other bacterial strains include but are not limited to: Cas proteins identified in Acaryochloris marina MBIC1 1017; Acetohalobium arabaticum DSM 5501; Acidithiobacillus caldus,' Acidithiobacillus ferrooxidans ATCC 23270; Alicyclobacillus acidocaldarius LAA1; Alicyclobacillus acidocaldarius subsp. acidocaldarius DSM 446; Allochromatium vinosum DSM 180; Ammonifex degensii KC4; Anabaena variabilis ATCC 29413; Arthrospira maxima CS-328; Arthrospira platensis str. Paraca; Arthrospira sp. PCC 8005; Bacillus pseudomycoides DSM 12442; Bacillus selenitireducens MLS10; Burkholderiales bacterium 1 1 47; Caldicelulosiruptor becscii DSM 6725; Candidatus Desulforudis awt / axvzator MP104C; Caldicellulosiruptor hydrothermalis 108; Clostridium phage c-st; Clostridium botulinum A3 str. Loch Maree; Clostridium botulinum Ba4 str. 657; Clostridium difficile QCD-63q42; Crocosphaera watsonii WH 8501; Cyanothece sp.75MF-36384587022474-20039.40ATCC 51142; Cyanothece sp. CCY0110; Cyanothece sp. PCC 7424; Cyanothece sp. PCC 7822; Exiguobacterium sibiricum 255-15; Finegoldia magna ATCC 29328; Ktedonobacter racemifer DSM 44963; Lactobacdlus delbrueckii subsp. bulgaricus PB2003 / 044-T3-4; Lactobacdlus salivarius ATCC 11741; Listeria innocua: Lyngbya sp. PCC 8106; Marinobacter sp. ELB17; Methanohalobium evestigatum Z-7303; Microcystis phage Ma-LMMOl; Microcystis aeruginosa NIES-843; Microscilla marina ATCC 23134; Microcoleus chthonoplastes PCC 7420; Neisseria meningitidis,' Nitrosococcus halophilus Nc4; Nocardiopsis dassonvillei subsp. dassonvillei DSM 43111; Nodularia spumigena CCY9414; Nostoc sp. PCC 7120; Oscillatoria sp. PCC 6506; Pelotomaculum thermopropionicum SI; Petrotoga mobilis SJ95; Polar omonas naphthalenivorans CJ2; Polaromonas sp. JS666; Pseudoalter omonas haloplanktis TAC125; Streptomyces pristinaespiralis ATCC 25486; Streptomyces pristinaespiralis ATCC 25486; Streptococcus thermophilus,' Streptomyces viridochromogenes DSM 40736; Streptosporangium roseum DSM 43021; Synechococcus sp. PCC 7335; and Thermosipho africanus TCF52B (Chylinski et al., RNA Biol., 2013; 10(5): 726-737).

[0268] Dead Cas-fusion proteins with transcriptional and / or epigenetic regulators have been used as a versatile platform for ectopically regulating gene expression in target cells. These include fusion of a Cas with an effector domain, such as a transcriptional activator or transcriptional repressor. For example, fusing dCas9 with a transcriptional repressor such as KRAB (Kriippel associated box) can result in robust repression of gene expression. A variety of dCas-fusion proteins with transcriptional and epigenetic regulators can be engineered for regulation of gene expression, for example as described in WO 2014 / 197748, WO 2016 / 130600, WO 2017 / 180915, WO 2021 / 226555, WO 2013 / 176772, WO 2014 / 152432, WO 2014 / 093661, WO 2021 / 247570, Adli, M. Nat. Commun. 9, 1911 (2018), Perez-Pinera, P. et al. Nat. Methods 10, 973-976 (2013), Mali, P. et al. Nat. Biotechnol. 31, 833-838 (2013), Maeder, M. L. et al. Nat. Methods 10, 977-979 (2013), Gilbert, L. A. et al. Cell 154(2):442-451 (2013), and Nunez, J.K. et al. Cell 184(9):2503-2519 (2021).

[0269] In some embodiments, the dCas9 protein or variant thereof is derived from a Streptococcus pyogenes Cas9 (SpCas9) protein or a variant thereof. In some embodiments, the variant Cas9 is a Streptococcus pyogenes dCas9 (dSpCas9) protein that comprises at least one amino acid mutation selected from D10A and H840A, with reference to numbering of positions of SEQ ID NO: 24. In some embodiments, the variant Cas9 protein comprises the sequence set forth in SEQ ID NO:25, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.76MF-36384587022474-20039.40

[0270] In some embodiments, the dSpCas9 protein binds to the target site in a target gene or regulatory element thereof without mediating nucleic acid cleavage at the target site. In some embodiments, the DNA-targeting system does not introduce a genetic disruption or a DNA break. The CRISPR / Cas-based DNA-targeting systems may be used to modulate expression of a target gene in a cell, such as a hepatocyte. b. Transcriptional Repressor Effector Domains

[0271] In some aspects, the DNA-targeting systems provided herein include a polynucleotide (e.g. mRNA) encoding a fusion protein comprising one or more effector domains. In some embodiments, the effector domains include at least one transcriptional repressor effector domain. In some embodiments, the fusion protein comprises: (a) a dSpCas9 DNA binding domain for targeting to a target site in a Hepatitis B viral DNA sequence, such as a gene or regulatory element thereof, for example any described above, and (b) at least one transcriptional repressor effector domain. In some aspects, the transcriptional repressor effector domain (e.g., a KRAB and a DNMT3 domain) is capable of reducing transcription of a viral HBV transcript, such as a gene.

[0272] In some embodiments, the effector domain directly or indirectly leads to reduced transcription of one or more HBV mRNA transcripts or genes. In some embodiments, the effector domain induces, catalyzes or leads to transcription repression. In some embodiments, the effector domain induces transcription repression. In some aspects, the effector domain is selected from a KRAB domain, a DNMT family protein domain (e.g. DNMT3 A or DNMT3B), a fusion of one or more DNMT family proteins or domains thereof (e.g. DNMT3A / L, which comprises a fusion of DNMT3A and DNMT3L domains), a partially or fully functional fragment or domain of any of the foregoing, or a combination of any of the foregoing. In some embodiments, the effector domain is KRAB. In some embodiments, the effector domain is DNMT3A / L.

[0273] In some embodiments, the effector domain comprises a KRAB domain, or a variant thereof. The KRAB-containing zinc finger proteins make up the largest family of transcriptional repressors in mammals. The Kriippel associated box (KRAB) domain is a transcriptional repressor domain present in many zinc finger protein-based transcription factors. The KRAB domain comprises charged amino acids and can be divided into sub-domains A and B. The KRAB domain recruits corepressors KAP1 (KRAB-associated protein- 1), epigenetic readers such as heterochromatin protein 1 (HP1), and other chromatin modulators to induce transcriptional repression through heterochromatin formation. KRAB-mediated gene repression77MF-36384587022474-20039.40 is associated with loss of histone H3 -acetylation and an increase in H3 lysine 9 trimethylation (H3K9me3) at the repressed gene promoters. KRAB domains, including in dCas fusion proteins, have been described, for example, in WO 2017 / 180915, WO 2014 / 197748, US 2019 / 0127713, WO 2013 / 176772, Urrutia R. et al. Genome Biol. 4, 231 (2003), Groner A. C. et al. PLoS Genet. 6, el000869 (2010). In some embodiments, the effector domain comprises at least one KRAB domain or a variant thereof. In some embodiments, the KRAB domain comprises a ZNF10 domain, KOX1 domain, ZIM3 domain, ZNF93 domain, or variant thereof.

[0274] In some embodiments, the KRAB domain comprises at least one ZNF10 domain. In some embodiments, the KRAB domain comprises any of the sequences set forth in SEQ ID NOS: 28-31, or a portion thereof, or a combination thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing. In some embodiments, the KRAB domain is encoded by any of the sequences set forth in SEQ ID NOS: 5-8, or a portion thereof, or a combination thereof, or any sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

[0275] In some embodiments, the KRAB domain comprises the sequence set forth in SEQ ID NOS: 28 or a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing. In some embodiments, the KRAB domain comprises the sequence set forth in SEQ ID NOS: 28. In some embodiments, the KRAB domain is set forth in SEQ ID NO: 28.

[0276] In some embodiments, the KRAB domain is encoded by SEQ ID NO: 5, or a portion thereof, or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing. In some embodiments, the KRAB domain is encoded by the sequence set forth in SEQ ID NO: 5.

[0277] In some embodiments, the effector domain is from a DNMT3 or is a portion or a functionally active variant thereof with DNA methyltransferase activity. The DNMT3 A and DNMT3B are two DNA methyltransferases that catalyze de novo methylation, which depending on the site may be associated with transcriptional repression. DNMTs, such as DNMT3s, mediate transfer of a methyl group from the universal methyl donor, S-adenosyl-L-methionine (SAM), to the 5-position of cytosine residues. In some aspects, these DNMT3 DNA methyltransferases induce de novo methylation of a cytosine base to methylated 5- methylcytosine. DNMT3, including in dCas fusion proteins, have been described, for example, in US20190127713, Liu, X. S. et al. Cell 167, 233-247.el7 (2016), Lei, Y. et al. Nat.78MF-36384587022474-20039.40Commun. 8, 16026 (2017). DNMT3 proteins, such as DNMT3A and DNMT3B, contain an N- terminal part that is naturally involved in regulatory activity and targeting, and a C-terminal catalytic domain termed the MTase C5-type domain. In some embodiments, an effector domain in embodiments provided herein includes a catalytically active portion of a DNMT3 A or a DNMT3B that contains a catalytically active C-terminal domain. In particular, isolated catalytic domains of DNMT3a and DNMT3b are catalytically active (see e.g. Gowher and Jeltsch (2002) J. Biol. Chem., 277:20409).

[0278] In any of the embodiments herein, the region of CpG methylation is within about 1000 bp, about 750 bp, about 600 bp, about 500 bp, about 450 bp, about 400 bp, about 350 bp, about 300 bp, about 250 bp, about 200 bp, about 150 bp, about 100 bp, about 50 bp of the target region.

[0279] In some embodiments, the DNMT3 domain may be an effector domain of DNMT3A that is catalytically active. In some embodiments, the effector domain may be the full-length of DNMT3 A or a catalytically active portion thereof. In some embodiments, the effector domain is a catalytically active portion that is less than the full-length sequence of DNMT3A. In some embodiments, a catalytically active portion is a contiguous sequence of amino acids that confers DNA methyltransferase activity, such as by mediating methylation of a cytosine base to methylated 5-methylcytosine. In some embodiments, the contiguous sequence of amino acids is a contiguous C-terminal portion of a DNMT3 protein, such as DNMT3 A, that is from 280 amino acids to 330 amino acids in length. In some embodiments, the contiguous portion is 280 amino acids, 290 amino acids, 300 amino acids, 310 amino acids, 320 amino acids, or 330 amino acids in length, or is a length of any value between any of the foregoing. In some embodiments, a catalytically active portion of a DNMT, such as a DNMT3, includes a SAM-dependent MTase C5-type domain. In some embodiments, the DNMT3 domain, such as a domain of DNMT3A or DNMT3B, is of human origin.

[0280] In some embodiments, the effector domain is from DNMT3 A or a catalytically active portion or variant thereof. An exemplary DNMT3A domain is set forth in SEQ ID NO: 22, 218, or is a catalytically active portion thereof, or is an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 22, 218 or the catalytically active portion thereof that exhibits DNA methyltransferase activity. In some embodiments, the DNMT3A domain is set forth in SEQ ID NO: 22, or is a catalytically active portion thereof, or is an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 22 or the catalytically active portion79MF-36384587022474-20039.40 thereof that exhibits DNA methyltransferase activity. In some embodiments, the catalytically active portion is a contiguous portion of amino acids of SEQ ID NO: 22 that includes the SAM- dependent MTase C5-type domain.

[0281] In some embodiments, the DNMT3L or portion thereof is of animal origin. In some embodiments, the domain from DNMT3A is of human origin.

[0282] In some embodiments, the DNMT3A domain is set forth in SEQ ID NO: 22, or is a catalytically active portion thereof, or is an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:22 or the catalytically active portion thereof that exhibits DNA methyltransferase activity. In some embodiments, the DNMT3L or portion thereof is of animal origin. In some embodiments, the domain from DNMT3L is of murine origin. In some embodiments, the domain from DNMT3L is of human origin. In some embodiments, the DNMT3A domain is encoded by a sequence set forth in SEQ ID NO: 2 or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2. In some embodiments, the DNMT3A domain is encoded by a sequence set forth in SEQ ID NO: 2.

[0283] In some embodiments, the effector domain includes a DNMT3L, or a portion or a variant of DNMT3L or the portion thereof. DNMT3L (DNA (cytosine-5)-methyltransf erase 3- like) is a catalytically inactive regulatory factor of DNA methyltransferases that can either promote or inhibit DNA methylation depending on the context. DNMT3L is essential for the function of DNMT3A and DNMT3B; DNMT3L interacts with DNMT3A and DNMT3B and enhances their catalytic activity. For instance, DNMT3L interacts with the catalytic domain of DNMT3 A or DNMT3B to form a heterodimer, demonstrating that DNMT3L has dual functions of binding an unmethylated histone tail and activating DNA methyltransferase. In some embodiments, reference to a portion or variant of a DNMT3L for purposes herein refers to a sufficient C-terminal sequence portion of DNMT3L that interacts with the catalytic domain of DNMT3 A or DNMT3B and is able to stimulate or promote DNA methyltransferase activity of DNMT3A or DNMT3B (see e.g. Jia et al. Nature, 2007, 449:248-251; Gowher et al. J. Biol. Chem., 2005, 280: 13341-13348). In some embodiments, the DNMT3L or portion thereof is of animal origin. In some embodiments, the domain from DNMT3L is of murine origin. In some embodiments, the domain from DNMT3L is of human origin.

[0284] In some embodiments, the DNMT3L domain is a DNMT3L, or a C-terminal portion or variant thereof, that interacts with the catalytic domain of DNMT3A to form a heterodimer to provide for a more active DNA methyltransferase. In some embodiments, the effector domain is80MF-36384587022474-20039.40 a fusion domain of a DNMT3 A domain and the DNMT3L domain (DNMT3A / 3L). In some embodiments, the fusion of a DNMT3 A domain and the DNMT3L domain (DNMT3A / 3L) comprises SEQ ID NO: 42, or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 42. In some embodiments, the DNMT3 A domain and DNMT3L domain are connected by a linker.

[0285] In some embodiments, the DNMT3L domain is a C-terminal portion of DNMT3L composed of a contiguous C-terminal portion of the full-length DNMT3L that does not include the N-terminal cysteine-rich ATRX-Dnmt3-Dnmt3L (ADD) domain. In some embodiments, the DNMT3L domain is a contiguous C-terminal portion of DNMT3L that is less than 220 amino acids in length, such as between 100 and 215 amino acids, such as at or about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210 or 215 amino acids in length, or a length between a value of any of the foregoing. In some embodiments, the DNMT3L domain is a contiguous C- terminal portion of DNMT3L that is 205, 206, 207, 208, 209, 210, 211, 212, 213, 214 or 215 amino acids in length.

[0286] An exemplary DNMT3L domain is set forth in SEQ ID NO:43, or is a portion thereof, or is an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 43 or the portion thereof. In some embodiments, the DNMT3L domain is a contiguous C-terminal portion of the full-length DNMT3L set forth in SEQ ID NO: 43 that does not include the N-terminal cysteine-rich ATRX- Dnmt3-Dnmt3L (ADD) domain (corresponding to residues 75-207 of the sequence set forth in SEQ ID NO: 43). In some embodiments, the DNMT3L domain is a contiguous C-terminal portion of the full-length DNMT3L set forth in SEQ ID NO: 43 that is less than 220 amino acids in length, such as between 100 and 215 amino acids, such as at or about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210 or 215 amino acids in length, or a length between a value of any of the foregoing. In some embodiments, the DNMT3L domain is a contiguous C-terminal portion of the full-length DNMT3L set forth in SEQ ID NO: 43 that is 205, 206, 207, 208, 209, 210, 211, 212, 213, 214 or 215 amino acids in length.

[0287] In some embodiments, the DNMT3L domain is set forth in SEQ ID NO: 23, or is a portion thereof, or is an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 23. In some embodiments, the DNMT3L domain is set forth in SEQ ID NO: 23. In some embodiments, the DNMT3L domain does not contain an N-terminal methionine, such as set forth in SEQ ID NO: 23. In some embodiments, the DNMT3L domain is encoded by a nucleotide sequence comprising SEQ ID81MF-36384587022474-20039.40NO: 3 or is a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 3. In some embodiments, the DNMT3L domain is set forth in SEQ ID NO: 3.

[0288] In some embodiments, the DNMT3L domain is a human or humanized DNMT3L. Corresponding sequences of human are highly homologous to the Dnmt3L derived from mouse and have a sequence identity of at least 90% with the murine sequence. It is within the level of a skilled artisan to humanize a non-human sequence of a DNMT3L domain, such as a domain of a murine DNMT3L. In some embodiments, the effector domain includes a DNMT3L domain that is a humanized variant of the murine DMT3L set forth in SEQ ID NO: 43 or a portion thereof that is able to interact with DNMT3 A or DNMT3 A. In some embodiments, the effector domain includes a DNMT3L domain that is a humanized variant of the murine C-terminal portion of DNMT3L set forth in SEQ ID NO: 23.

[0289] An exemplary DNMT3L domain of human origin is set forth in SEQ ID NO: 44, or is a portion thereof, or is an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 44 or the portion thereof. In some embodiments, the DNMT3L domain is a contiguous C-terminal portion of the full-length DNMT3L set forth in SEQ ID NO: 44 that does not include the N-terminal cysteine-rich ATRX- Dnmt3-Dnmt3L (ADD) domain (corresponding to residues 41-73 of the sequence set forth in SEQ ID NO: 44). In some embodiments, the DNMT3L domain is a contiguous C-terminal portion of the full-length DNMT3L set forth in SEQ ID NO: 44 that is less than 220 amino acids in length, such as between 100 and 215 amino acids, such as at or about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210 or 215 amino acids in length, or a length between a value of any of the foregoing. In some embodiments, the DNMT3L domain is a contiguous C-terminal portion of the full-length DNMT3L set forth in SEQ ID NO: 44 that is 205, 206, 207, 208, 209, 210, 211, 212, 213, 214 or 215 amino acids in length.

[0290] In some embodiments, the DNMT3L domain comprises the sequence set forth in SEQ ID NO: 45, or is a portion thereof, or is an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:45. In some embodiments, the DNMT3L domain is set forth in SEQ ID NO: 45. In some embodiments, the DNMT3L domain contains an N-terminal methionine.

[0291] In some embodiments, the effector domain comprises a fusion of DNMT3A and DNMT3L (DNMT3A / L). The fusion protein contains DNMT3A and DNMT3L domains that can be any as described above. In some embodiments, the fusion protein contains the DNMT3 A82MF-36384587022474-20039.40 domain set forth in SEQ ID NO: 22 and the DNMT3L domain set forth in SEQ ID NO: 23, arranged in any order. In some embodiments, the DNMT3 A and DNMT3L domains present in a provided fusion protein are separated from each other in the fusion protein by an intervening sequence, such as the DNA-binding domain, another effector domain or a linker. In some embodiments, the domains are either directly linked to each other or they are linked via a linker, such as a peptide linker. In some embodiments, the DNMT3 A and DNMT3L domains are connected as a fusion domain via a linker that connects the DNMT3 A domain and the DNMT3L domain. Exemplary linkers are described herein. In some embodiments, the linker is the linker set forth in SEQ ID NO: 34.

[0292] An exemplary DNMT3A / L fusion domain is set forth in SEQ ID NO: 42. In some embodiments, the effector domain comprises the sequence set forth in SEQ ID NO: 42, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 42, and exhibits DNA methyltransferase activity. In

[0293] Any of a variety of assays are known to assess or monitor methyltransferase (MTase) ativity. In some embodiments, exemplary assays to assess DNA methyltransferase activity include, but are not limited to, radio DNA MTase assays, colorimetric DNA MTase activity assays, fluorescent DNA MTase activity assays, chemiluminescent / bioluminescent DNA MTase activity assays, electrochemical DNA MTase activity assays, and elctrogenerated chemiluminescence (ECL) DNA MTase activity assays. Exemplary assays are described in Poh et al. Theranostics, 2016, 6:369-391; Li et al., Methods Appl. Fluoresc., 2017, 5:012002; Deng et al., Anal Chem., 2014, 86:2117-23; and Ma et al. J Mater Chem B., 2020, 8:3488-3501.

[0294] In some embodiments, the at least one effector domain is fused to the N-terminus, the C-terminus, or both the N-terminus and the C-terminus, of the DNA- binding domain (e.g., dCas). In some embodiments, the at least one effector domain is fused to the DNA- binding domain directly, or via any intervening amino acid sequence, such as a linker sequence or a nuclear localization sequence (NLS). In some embodiments, the at least one effector domain of the fusion protein includes more than one effector domains. In some embodiments, at least two effector domains of the fusion protein are different. In some embodiments, the effector domains and the DNA-binding domain are arranged in any order. The effector domains and the DNA- binding domain may be arranged in any order.

[0295] In some embodiments, the at least one effector domain of the fusion protein includes two different effector domains. The two different effector domains and the DNA-binding domain can be arranged in any order. In some embodiments, each of the effector domains are83MF-36384587022474-20039.40N-terminal to the DNA-binding domain in which a first effector domain is fused to the N- terminus of the second effector domain and the second effector domain is fused to the N- terminus of the DNA-binding domain. In some embodiments, the fusion protein of a provided DNA-binding system, or a DNA-targeting module thereof, comprises from N- to C-terminal order: a first transcriptional repressor effector domain, a second transcriptional repressor effector domain and the DNA binding domain. In some embodiments, each of the effector domains are C-terminal to the DNA-binding domain in which a first effector domain is fused to the C- terminus of the DNA-binding domain and the second effector domain is fused to the C-terminus of the first effector domain. In some embodiments, the fusion protein of a provided DNA- binding system, or a DNA-targeting module thereof, comprises from N- to C-terminal order: a DNA-binding domain, a first transcriptional repressor effector domain, and a second transcriptional repressor effector domain. In some embodiments, the DNA-binding domain is between the effector domains, in which one effector domain is fused to the N-terminus of the DNA-binding domain and the other effector domain is fused to the C-terminus of the DNA- binding domain. In some embodiments, the fusion protein of a provided DNA-binding system, or a DNA-targeting module thereof, comprises from N- to C-terminal order: a first transcriptional effector domain, a DNA-binding domain, and a second transcriptional repressor effector domain. In some embodiments, one or more of the components may be fused to eachother directly, or via any intervening amino acid sequence, such as via a linker sequence or a nuclear localization sequence (NLS).

[0296] In some embodiments, the fusion protein contains the DNA-binding domain, and the KRAB domain and DNMT3A / 3L fusion domain as first and second effector domains. In some embodiments, a first effector domain is fused to the N-terminus of the second effector domain and the second effector domain is fused to the N-terminus of the DNA-binding domain. In some embodiments, a fusion protein provided herein comprises in order: DNMT3A / L-KRAB- dSpCas9. In some embodiments, a fusion protein provided herein comprises in order: KRAB- DNMT3A / L-dSpCas9. In some embodiments, each of the KRAB domain and DNMT3A / 3L domain are C-terminal to the DNA-binding domain in which a first effector domain is fused to the C-terminus of the DNA-binding domain and the second effector domain is fused to the C- terminus of the first effector domain. In some embodiments, a fusion protein provided herein comprises in order: dSpCas9- DNMT3A / L-KRAB. In some embodiments, a fusion protein provided herein comprises in order: dSpCas9- KRAB- DNMT3A / L. In some embodiments, the DNA-binding domain is between the KRAB domain and DNMT3A / 3L domain, in which one84MF-36384587022474-20039.40 effector domain is fused to the N-terminus of the DNA-binding domain and the other effector domain is fused to the C-terminus of the DNA-binding domain. In some embodiments, a fusion protein provided herein comprises in order: KRAB-dSpCas9- DNMT3A / L. In some embodiments, a fusion protein provided herein comprises in order: DNMT3A / L-dSpCas9- KRAB.

[0297] In some embodiments, a fusion protein named herein comprises elements of the named fusion protein in any configuration or order. For example, a dSpCas9-KRAB- DNMT3A / L fusion protein may comprise dSpCas9, KRAB, and DNMT3A / L in any order. For example, a dSpCas9-KRAB-DNMT3A / L fusion protein may comprise from N-terminal to C- terminal, DNMT3A / L, dSpCas9, and KRAB. A fusion protein named herein may comprise additional elements. For example, a DNMT3A / L-dSpCas9-KRAB- fusion protein may comprise one or more linkers, NLS sequences, or other sequences in any combination or order. In some embodiments, the fusion protein of the DNA-targeting system comprises a DNMT3A / L- dSpCas9-KRAB- fusion protein. In some embodiments, the fusion protein of the DNA-targeting system comprises a KRAB-dSpCas9- DNMT3A / L- fusion protein. In some embodiments, the fusion protein of the DNA-targeting system comprises a KRAB-dSpCas9- DNMT3A / L- fusion protein. In some embodiments, the fusion protein is a DNMT3A / L-dSpCas9-KRAB protein. In some embodiments, the fusion protein is a KRAB- DNMT3A / L- dSpCas9 protein. In some embodiments, the fusion protein is a DNMT3A / L- KRAB-dSpCas9 protein.

[0298] In some embodiments, the fusion protein of the DNA-targeting system, or a DNA- targeting module thereof, comprises the sequence set forth in SEQ ID NO: 41, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the fusion protein comprises the sequence set forth in SEQ ID NO: 41.

[0299] In some embodiments, the polynucleotide is an mRNA molecule that comprises a sequence encoding a DNMT3A / L-dCas9-KRAB fusion protein. In some embodiments, the mRNA encoding the fusion protein comprises the sequence set forth in SEQ ID NO: 1, or a sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto. In some embodiments, the mRNA is set forth in SEQ ID NO: 1.

[0300] In some embodiments, the polynucloeotide encoding the fusion protein may include one or more linker or nuclear localization signals (NLS), such as at the N- or C-terminus of the fusion protein or between the Cas and the KRAB domain or between the Cas and the85MF-36384587022474-20039.40DNMT3A / 3L domains or between the DNMT3 A and the DNMT3L, or one or a combination of the foregoing. The linker or NLS can be any as described herein. In some embodiments, the one or more linkers connect the DNA- binding domain or a component thereof to at least one effector domain of the fusion protein. A linker may be included anywhere in the polypeptide sequence of the fusion protein, for example, between the effector domain and the DNA- binding domain or a component thereof. In some embodiments, the one or more linkers connect two effector domains of the fusion protein. Depending on the number of effector domains, the fusion protein can include 1, 2, 3, 4 or more linkers. In some embodiments, the linkers may be the same or they may be different.

[0301] In some embodiments, the NLS is at the N- or C-terminus, between N- or C-terminus and the KRAB domain, between the N- or C-terminus and the DNMT3A / L domain, between the dSpCas9 and the DNMT3A / 3L domains, between the DNMT3 A and the DNMT3L domains, and / or between the dSpCas9 and the KRAB domain.

[0302] In some embodiments, the NLS is between the DNMT3 A and the DNMT3L, between the DNMT3L and the first NLS, between the first NLS and the dSpCas9, between the dSpCas9 and a second NLS, between the second NLS and the KRAB, and between the KRAB and a 3 ’ UTR.

[0303] In some embodiments, a polynucleotide encoding the fusion protein of the DNA- targeting system, or a DNA-targeting module thereof, comprises one or more nuclear localization signals (NLS). In some embodiments, the polynucleotide encoding the fusion protein described herein comprises one or more nuclear localization sequences (NLSs), such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. When more than one NLS is present, each may be selected independently of the others, such that a single NLS may be present in more than one copy and / or in combination with one or more other NLSs present in one or more copies. Non-limiting examples of NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV(SEQ ID NO: 32); the NLS from nucleoplasmin (e.g. the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 33); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 46) or RQRRNELKRSP (SEQ ID NO: 47); the hRNPAl M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 48); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 49) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 50) and PPKKARED (SEQ ID NO: 51) of the myoma T protein; the sequence PQPKKKPL86MF-36384587022474-20039.40(SEQ ID NO: 52) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 53) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 54) and PKQKKRK (SEQ ID NO: 55) of the influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO: 56) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO: 57) of the mouse Mxl protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 58) of the human poly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 60) of the steroid hormone receptors (human) glucocorticoid.

[0304] In some embodiments, the NLS comprises the sequence set forth in any one of SEQ ID NOS: 32 or 33, a portion thereof, or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the foregoing. In some embodiments, the NLS comprises the sequence set forth in any one of SEQ ID NOS: 32 or 33. In some embodiments, the NLS comprises the sequences set forth in any one of SEQ ID NOS: 32 and 33.

[0305] In some embodiments, the NLS comprises the sequence set forth in any one of SEQ ID NOS: 13, 16, 18, a portion thereof, or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the foregoing. In some embodiments, the NLS comprises the sequence set forth in any one of SEQ ID NOS: 13, 16, or 18.

[0306] In general, the one or more NLSs are of sufficient strength to drive accumulation of the fusion protein in a detectable amount in the nucleus of a eukaryotic cell. In general, strength of nuclear localization activity may derive from the number of NLSs in the fusion protein, the particular NLS(s) used, or a combination of these factors. Detection of accumulation in the nucleus may be performed by any suitable technique. For example, a detectable marker may be fused to the fusion protein, such that location within a cell may be visualized, such as in combination with a means for detecting the location of the nucleus (e.g. a stain specific for the nucleus such as DAPI). Cell nuclei may also be isolated from cells, the contents of which may then be analyzed by any suitable process for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay. Accumulation in the nucleus may also be determined indirectly, such as by an assay for the effect of the fusion protein (e.g. an assay for altered gene expression activity in a cell transformed with the DNA-targeting system comprising the fusion protein), as compared to a control condition (e.g. an untransformed cell).

[0307] In some embodiments, the polynucleotide further comprises or encodes one or more linkers connecting two or more of: the dSpCas9, the KRAB domain, the DNMT3 A domain, the DNMT3L domain, and the one or more nuclear localization signals. A linker may be of any length and designed to promote or restrict the mobility of components in the fusion protein. In87MF-36384587022474-20039.40 some embodiments, the linker is a peptide linker. A linker may comprise any amino acid sequence of about 2 to about 100, about 5 to about 80, about 10 to about 60, or about 20 to about 50 amino acids. A linker may comprise an amino acid sequence of at least about 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or 85 amino acids. A linker may comprise an amino acid sequence of less than about 100, 90, 80, 70, 60, 50, or 40 amino acids. A skilled artisan can readily choose an appropriate linker for the connection of two domains. In some embodiments, the linker is a flexible linker. Flexible linkers are generally composed of small, non-polar or polar residues such as glycine, serine or threonine. In some embodiments, the linker is the Gly4Ser(n) linker, whereby n is an integer of 1 to 10. A linker may include sequential or tandem repeats of an amino acid sequence that is 2 to 20 amino acids in length. Linkers may be rich in amino acids glycine (G), serine (S), and / or alanine (A). Linkers may include, for example, a GS linker. An exemplary GS linker is represented by the sequence GGGGS or the formula (GGGGS)n, wherein n is an integer that represents the number of times the GGGGS sequence is repeated (e.g. between 1 and 10 times). The number of times a linker sequence is repeated can be adjusted to optimize the linker length and achieve appropriate separation of the functional domains.

[0308] In some embodiments, the one or more linkers is independently selected from a linker comprising the sequence set forth in any one of SEQ ID NO: 11, 12, 14, 15, or 17.

[0309] Other examples of linkers may include, for example, GGGGG, GGAGG, GGGGS SS, or GGGGAAA.

[0310] In some embodiments, artificial linker sequences can be used. In some embodiments, the linker is EASGSGRASPGIPGSTR (SEQ ID NO: 216). In some embodiments, the linker is KRPAATKKAGQAKKKKAS (SEQ ID NO: 217). In some embodiments, the linker is linker is GH4GVPAA (SEQ ID NO: 36). In some embodiments, the linker is SSGNSNANSRGPSFSSGLVPLSLRGSH (SEQ ID NO: 34). In some embodiments, the linker is EASGSGRASPGIPGSTRN (SEQ ID NO: 35). In some embodiments, the linker is SGG. In some embodiments, the linker is AS.

[0311] In some embodiments, the linker comprises the sequence set forth in any one of SEQ ID NOS: 34, 35, 36, 37, a portion thereof, or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the foregoing. In some embodiments, the linker comprises the sequence set forth in any one of SEQ ID NOS: 34, 35, 36, 37. In some embodiments, the linker is encoded by the sequence set forth in any one of SEQ ID NOS: 11, 12, 14, 15 a portion thereof, or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%,88MF-36384587022474-20039.4096%, 97%, 98%, or 99% sequence identity to the foregoing. In some embodiments, the linker comprises the sequence set forth in any one of SEQ ID NOS: 11, 12, 14, 15.

[0312] In some embodiments, the linker is linker is GIHGVPAA (SEQ ID NO: 36). In some embodiments, the linker is SSGNSNANSRGPSFSSGLVPLSLRGSH (SEQ ID NO: 34). In some embodiments, the linker is EASGSGRASPGIPGSTRN (SEQ ID NO: 35). In some embodiments, the linker is SGG. In some embodiments, the linker is AS.

[0313] In some embodiments, the mRNA comprises a nucleotide sequence encoding the fusion protein. In some embodiments, the nucleotide sequence comprises the linker sequences set forth in SEQ ID NOS: 34, 35, 36, a portion thereof, or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the foregoing. In some embodiments, the nucleotide sequence comprises the linker sequences set forth in SEQ ID NOS: 34, 35, 36. In some embodiments, the nucleotide sequence further comprises the sequences SGG and AS.

[0314] In some embodiments of the method, the mRNA comprises a 5' cap. In some embodiments, the 5’ cap is an altered nucleotide on the 5’ end of primary transcripts such as messenger RNA. In some aspects, the 5’ caps of the mRNA improves one or more of RNA stability and processing, mRNA metabolism, the processing and maturation of an RNA transcript in the nucleus, transport of mRNA from the nucleus to the cytoplasm, mRNA stability, and efficient translation of mRNA to protein. In some embodiments, a 5’ cap can be a naturally- occurring 5’ cap or one that differs from a naturally-occurring cap of an mRNA. A 5’ cap may be any 5' cap known to a skilled artisan. In certain embodiments, the 5' cap is selected from the group consisting of an Anti-Reverse Cap Analog (ARCA) cap, a 7-methyl-guanosine (7mG) cap, a CleanCap® analog, a vaccinia cap, and analogs thereof. For instance, the 5’ cap may include, without limitation, an anti-reverse cap analogs (ARCA) (US7074596), 7-methyl- guanosine, CleanCap® analogs, such as Cap 1 analogs (Trilink; San Diego, CA), or enzymatically capped using, for example, a vaccinia capping enzyme or the like.

[0315] In some embodiments, the mRNA may be polyadenylated. In some embodiments, the polynucleotide encoding the fusion protein further comprises a polyA sequence. In some embodiments, the polyA sequence comprises a sequence set forth in SEQ ID NO: 19, or a sequence that has at least 90% sequence identity to the foregoing. In some embodiments, the polyA sequence comprises a sequence set forth in SEQ ID NO: 19. In some embodiments, the polynucleotide comprises a polyA sequence at the 3’ end. The mRNA may contain various 5’ and 3’ untranslated sequence elements to enhance expression of the encoded protein and / or89MF-36384587022474-20039.40 stability of the mRNA itself. In some embodiments, the polynucleotide encoding the fusion protein further comprises one or more untranslated regions (UTR) near the 5’ end and / or the 3’ end, optionally a UTR near the 5’ end and near the 3’ end. In some aspects, different transcriptional control sequences may be selected for use in the polynucleotide, for example to achieve the appropriate level of expression. For example, in some embodiments, UTRs can be selected that facilitate expression in a specific tissue or cell type (e.g. liver or hepatocyte).

[0316] In some embodiments, the UTR near the 5’ end comprises a sequence set forth in SEQ ID NO: 9 or a sequence that has at least 90% sequence identity to the foregoing and the UTR near the 3’ end comprises a sequence set forth in SEQ ID NO: 10 or a sequence that has at least 90% sequence identity to the foregoing. In some embodiments, the UTR at or near the 5’ end is set forth in SEQ ID NO: 9. In some embodiments, the UTR at or near the 3’ end is set forth in SEQ ID NO: 10.

[0317] In some embodiments, the polynucleotide (i.e., the mRNA described herein) comprises at least one nucleoside modification. The mRNA may contain modifications of naturally-occurring nucleosides to nucleoside analogs. Any nucleoside analogs known in the art are envisioned. Such nucleoside analogs can include, for example, those described in US 8,278,036. In certain embodiments of the method, the nucleoside modification is selected from the group consisting of a modification from uridine to pseudouridine and uridine to Nl- methyl pseudouridine. In particular embodiments of the method the nucleoside modification is from uridine to pseudouridine.

[0318] In some embodiments, the mRNA (Table 4) comprises from 5’ end to the 3’ end: a UTR, a nucleotide sequence encoding the fusion protein, a UTR, and a polyA sequence. In some embodiments, the nucleotide sequence encoding the fusion protein comprises, from 5’ end to the 3’ end, nucleotide sequences encoding: a DNMT3A domain, a linker, the DNMT3L domain, a linker, a NLS, a linker, the dSpCas9, a linker, a NLS, a linker, the KRAB, and a NLS.

[0319] In some embodiments, the mRNA comprises from the 5’ end to the 3’ end: the UTR comprising the sequence set forth in SEQ ID NO: 9, a nucleotide sequence encoding the fusion protein, the UTR comprising the sequence set forth in SEQ ID NO: 10, and a polyA sequence comprising the sequence set forth in SEQ ID NO: 19. In some embodiments, the DNMT3A domain of the fusion protein is encoded by the sequence set forth in SEQ ID NO: 2, the linker of the fusion protein is encoded by the sequence set forth in SEQ ID NO: 11, the DNMT3L domain of the fusion protein is encoded by the sequence set forth in SEQ ID NO: 3, the linker of the fusion protein is encoded by the sequence set forth in SEQ ID NO: 12, the NLS of the fusion90MF-36384587022474-20039.40 protein is encoded by the sequence set forth in SEQ ID NO: 13, the linker of the fusion protein is encoded by the sequence set forth in SEQ ID NO: 14, the dSpCas9 of the fusion protein is encoded by the sequence set forth in SEQ ID NO: 4, the linker of the fusion protein is encoded by the sequence set forth in SEQ ID NO: 15, the NLS of the fusion protein is encoded by the sequence set forth in SEQ ID NO: 16, the linker of the fusion protein is encoded by the sequence set forth in SEQ ID NO: 17, the KRAB of the fusion protein is encoded by the sequence set forth in SEQ ID NO: 5, the NLS of the fusion protein is encoded by the sequence set forth in SEQ ID NO: 18. In some embodiments, the fusion protein comprises from the N- terminus to the C-terminus: the DNMT3 A domain comprising the sequence set forth in SEQ ID NO: 22, the linker comprising the sequence set forth in SEQ ID NO: 34, the DNMT3L domain comprising the sequence set forth in SEQ ID NO: 23, the linker comprising the sequence set forth in SEQ ID NO: 35, the NLS comprising the sequence set forth in SEQ ID NO: 32, the linker comprising the sequence set forth in SEQ ID NO: 36, the dSpCas9 comprising the sequence set forth in SEQ ID NO: 25, the linker comprising the sequence set forth in SEQ ID NO: 37, the NLS comprising the sequence set forth in SEQ ID NO: 16, the linker comprising the sequence set forth in SEQ ID NO: 33, the KRAB comprising the sequence set forth in SEQ ID NO: 28, the NLS comprising the sequence set forth in SEQ ID NO: 32.

[0320] An exemplary mRNA encoding the fusion protein has features set forth in Table 4.91MF-36384587022474-20039.40

[0321] In some embodiments, the polynucleotide encoding the fusion protein is an mRNA. In some embodiments, the mRNA can be produced using methods known in the art such as in vitro transcription. In some embodiments, the polynucleotide (i.e., mRNA) comprises the sequence set forth in SEQ ID NO: 1, a portion thereof, or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the foregoing. In some embodiments, the polynucleotide (i.e., mRNA) comprises the sequence set forth in SEQ ID NO: 1, a portion thereof, or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the foregoing. In some embodiments, the mRNA comprises the sequence set forth in SEQ ID NO: 1, a portion thereof, or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the foregoing. In some embodiments, the mRNA comprises the sequence set forth in SEQ ID NO: 1, a portion thereof, or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the foregoing.

[0322] In some embodiments, the polynucleotide is an mRNA that comprises the sequence set forth in SEQ ID NO: 1. In some embodiments, the mRNA comprises the sequence set forth in SEQ ID NO: 1.

[0323] Provided herein is a method of treating chronic hepatitis B (CHB) infection in a subject in need thereof, the method comprising administering to the subject a dose of lipid nanoparticles comprising an epigenetic-modifying DNA-targeting system comprising a DNA- targeting module for repressing total viral Hepatitis B Viral (HBV) mRNA transcripts. In some embodiments, the DNA-targeting module comprises: (a) a gRNA for targeting to a target site in a HBV DNA sequence. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 27; and (b) a mRNA comprising from the 5’ end to the 3’ end: the UTR comprising the sequence set forth in SEQ ID NO: 9, a nucleotide encoding a fusion protein, the UTR comprising the sequence set forth in SEQ ID NO: 10, and a polyA sequence comprising the sequence set forth in SEQ ID NO: 19. In some embodiments, the fusion protein comprises from the N-terminus to the C-terminus: the DNMT3 A domain comprising the sequence set forth in SEQ ID NO: 22, the linker comprising the sequence set forth in SEQ ID NO: 34, the DNMT3L domain comprising the sequence set forth in SEQ ID NO: 23, the linker comprising92MF-36384587022474-20039.40 the sequence set forth in SEQ ID NO: 35, the NLS comprising the sequence set forth in SEQ ID NO: 32, the linker comprising the sequence set forth in SEQ ID NO: 36, the dSpCas9 comprising the sequence set forth in SEQ ID NO: 25, the linker comprising the sequence set forth in SEQ ID NO: 37, the NLS comprising the sequence set forth in SEQ ID NO: 16, the linker comprising the sequence set forth in SEQ ID NO: 33, the KRAB comprising the sequence set forth in SEQ ID NO: 28, the NLS comprising the sequence set forth in SEQ ID NO: 32. In some embodiments, the DNMT3 A domain of the fusion protein is encoded by the sequence set forth in SEQ ID NO: 2, the linker of the fusion protein is encoded by the sequence set forth in SEQ ID NO: 11, the DNMT3L domain of the fusion protein is encoded by the sequence set forth in SEQ ID NO: 3, the linker of the fusion protein is encoded by the sequence set forth in SEQ ID NO: 12, the NLS of the fusion protein is encoded by the sequence set forth in SEQ ID NO: 13, the linker of the fusion protein is encoded by the sequence set forth in SEQ ID NO: 14, the dSpCas9 of the fusion protein is encoded by the sequence set forth in SEQ ID NO: 4, the linker of the fusion protein is encoded by the sequence set forth in SEQ ID NO: 15, the NLS of the fusion protein is encoded by the sequence set forth in SEQ ID NO: 16, the linker of the fusion protein is encoded by the sequence set forth in SEQ ID NO: 17, the KRAB of the fusion protein is encoded by the sequence set forth in SEQ ID NO: 5, the NLS of the fusion protein is encoded by the sequence set forth in SEQ ID NO: 18.

[0324] Provided herein is a method of treating chronic hepatitis B (CHB) infection in a subject in need thereof, the method comprising administering to the subject a dose of lipid nanoparticles comprising (a) a gRNA for targeting to a target site in a HBV DNA sequence. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 27; and (b) a mRNA comprising the sequence set forth in SEQ ID NO: 1 or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the foregoing.C. Dosing and Administration

[0325] Provided herein is a method of treating chronic hepatitis B (CHB) infection in a subject in need thereof, the method comprising administering to the subject a dose of lipid nanoparticles comprising an epigenetic-modifying DNA-targeting system comprising a DNA- targeting module for repressing transcription of total viral Hepatitis B Viral (HBV) mRNA transcripts. In some embodiments, the dose comprises (a) between at or about 0.1 mg / kg and at or about 1 mg / kg, or (b) between at or about 10 mg to about 100 mg.

[0326] In some embodiments, the subject is human. In some embodiments, the subject has hepatitis B viral infection. In some embodiments, the Hepatitis B viral infection is a chronic93MF-36384587022474-20039.40 infection. In some embodiments, the infection is an acute infection.

[0327] In some embodiments, the human subject is an adult human. In some embodiments, the subject is greater than or equal to 18 years of age. In some embodiments, the subject is greater than or equal to 12 years of age.

[0328] In some aspects, the subject is selected for treatment based on, or is characterized by, one or more of the following: 1) 18 to 75 years of age (inclusive); 2) diagnosed with CHB defined as HBsAg (+) for > 12 months and baseline titers of HBsAg > 500 lU / mL; 3) has received treatment with a nucleoside analogues (NA; entecavir, tenofovir disoproxil fumarate or tenofovir alafenamide) for > 12 months with a stable dose for > 6 months; 4) HBV DNA <90 HJ / mL for >6 months.

[0329] In some embodiments, the human subject has cells (e.g., hepatocytes) comprising cccDNA and / or integrated HBV DNA. In some embodiments, the subject has hepatocytes comprising cccDNA and integrated HBV DNA.

[0330] In some aspects, the cell is a liver cell, such as a hepatocyte, hepatic stellate cells (HSCs), kupffer cells, and liver sinusoidal endothelial cells. In some aspects, the cell is a hepatocyte.

[0331] In some embodiments, the subject has hepatocytes expressing HBV proteins, wherein the HBV proteins are HBsAg, HBeAg, or HBcrAg and combinations thereof.

[0332] In some embodiments, the subject has been diagnosed as positive for HBsAg for at least about 12 months. In some embodiments, the subject has an HBsAg titer (e.g., HBsAg baseline titre) of at least about 250 HJ / mL, 300 HJ / mL, 400 HJ / mL, 500 HJ / mL, 600 HJ / mL, 700, 800, 900, or any of the foregoing. In some embodiments, the subject has an HBsAg titer of at least about 500 HJ / mL.

[0333] In some embodiments, the subject has or has been diagnosed as being positive for HBV DNA levels of less than about 50 U / mL, about 60 U / mL, about 70 U / mL, about 80 U / mL, about 90 U / mL, about 100 U / mL, about 110 U / mL or any of the foregoing. In some embodiments, the subject has or has been diagnosed as being positive for HBV DNA levels for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, atleast about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, or any of the foregoing. In some embodiments, the subject has or has been diagnosed as being positive for HBV DNA levels of less than about 90 U / mL for at least 6 months. In some embodiments, the subject has HBV DNA of less than 90 lU / ml for 6 months. In some embodiments, the subject has HBV DNA of less than 90 lU / ml for94MF-36384587022474-20039.406 months.

[0334] In some embodiments, has or has been diagnosed with chronic Hepatitis B (CHB) defined as HBsAg positive for about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 24 months, or any of the foregoing. In some embodiments, has or has been diagnosed with CHB defined as HBsAg positive for 12 months. In some embodiments, has or has been diagnosed with CHB defined as HBsAg positive for greater than 12 months. In some embodiments, has or has been diagnosed with CHB defined as HBsAg positive for greater than or equal to 12 months.

[0335] In some embodiments, the subject has not received treatment with a nucleoside analog. In some embodiments, the subject has received treatment with a nucleoside analog for at least about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, or any of the foregoing. In some embodiments, the subject has received treatment with one nucleoside analog. In some embodiments, the subject has received treatment with at least one nucleoside analog. In some embodiments, the subject has received treatment with two or more nucleoside analogs. In some embodiments, the subject has received treatment with three or more nucleoside analogs. In some embodiments, the subject has received treatment with four or more nucleoside analogs, such as four, five, six, seven, eight, nine, ten analogs.

[0336] The subject may have received or is receiving any suitable nucleoside analogs. In some embodiments, the nucleoside analog comprises one or more of entecavir, tenofovir disoproxil fumarate, or tenofovir alafenamide, or any suitable nucleoside analog known in the art. In some embodiments, the subject has received treatment with a nucleoside analog. In some embodiments, the nucleoside analog is entecavir (ETV), tenofovir disoproxil fumarate (TDF) or tenofovir alafenamide (TAF) for 12 months of more than 12 months with a stable dose for 6 months or more than 6 months. In some embodiments, the subject has received the nucleoside analog prior to the start of administration of the lipid nanoparticle described herein. In some embodiments, the subject is receiving the nucleoside analog at the time of administration of the lipid nanoparticle and continues to receive the nucleoside analog. In some embodiments, the treatment with the nucleoside analog is discontinued at the time of administration of the lipid nanoparticle.

[0337] In some embodiments, the subject (a) has or has been diagnosed with chronic95MF-36384587022474-20039.40Hepatitis B (CHB) defined as HBsAg positive for 12 months or more than 12 months; (b) has baseline titers of HBsAg of more than 500 lU / ml; (c) has received treatment with a nucleoside analog, wherein the nucleoside analog is entecavir (ETV), tenofovir disoproxil fumarate (TDF) or tenofovir alafenamide (TAF) for 12 months of more than 12 months with a stable dose for 6 months or more than 6 months; or (d) has HBV DNA of less than 90 lU / ml for 6 months or less than 6 months. In some embodiments, the subject (a) has or has been diagnosed with chronic Hepatitis B (CHB) defined as HBsAg positive for 12 months or more than 12 months; (b) has baseline titers of HBsAg of more than 500 lU / ml; (c) has received treatment with a nucleoside analog, wherein the nucleoside analog is entecavir (ETV), tenofovir disoproxil fumarate (TDF) or tenofovir alafenamide (TAF) for 12 months of more than 12 months with a stable dose for 6 months or more than 6 months; and (d) has HBV DNA of less than 90 lU / ml for 6 months or less than 6 months.

[0338] In some aspects, a subject is excluded from treatment or is not selected for treatment do to one or more features that may include: 1) Participants with any evidence or history of liver disease including but not limited to cirrhosis, drug- or alcohol -related liver disease, autoimmune hepatitis, hemochromatosis, Wilson's disease, a-1 antitrypsin deficiency, primary biliary cirrhosis, primary sclerosing cholangitis, portal hypertension, hepatic encephalopathy, nonalcoholic steatohepatitis or any other non-HBV liver disease considered clinically significant; 2) Diagnosed or suspected hepatocellular carcinoma (alpha-fetoprotein concentration >200 ng / mL or liver ultrasound or other imaging with findings suggestive of HCC at any time). A liver ultrasound to assess for HCC or other abnormalities will be performed if the participant has not had one performed (with documentation of results available) within 6 months before the Day 1 Visit; 3) History of Hepatitis C or D infection. Participants who have no evidence of cirrhosis, have completed a curative intent regimen for Hepatitis C, and are deemed by the PI to have no active Hepatitis C infection and no increased risk for hepatotoxicity may be considered for inclusion; 4) Positive testing for HIV-1, HIV-2, HAV, HCV, or HDV at the Screening Visit that suggests an ongoing infection; 5) Liver testing showing the following: i) Alanine aminotransferase (ALT) / aspartate aminotransferase (AST) > 1.5 x upper limit of normal (ULN) and total bilirubin > 1. 5 x ULN at the Screening and Day -1 Visits; ii) For participants with a history of Gilbert’s Syndrome, total bilirubin > 2 x ULN at the Screening Visit; iii) Serum albumin <3.5 g / dL at the Screening and Day -1 Visits; iv) Out of reference range unless a clinician determined clinically nonsignificant activated partial thromboplastin time (aPTT) and international normalized ratio (INR) at the Screening and Day -1 Visits; v) Transient96MF-36384587022474-20039.40Elastography (FibroScan) score >8.8 kPa at the Screening Visit; vi) Liver biopsy (if available and performed with 6 months prior to the Screening Visit): fibrosis of 3 or higher on Batts- Ludwig or METAVIR or stage > 3 by Ishak scoring system; 6) Participants with any of the following laboratory values at the Screening or Day -1 Visits: i) Hemoglobin <10 g / dL, ii) Absolute neutrophil count <1.5 x 103 / pL, iii) Platelets <150,000 cells / mm3, or iv) Estimated glomerular filtration rate (eGFR) <60 mL / min / 1.73 m2 by the CKD-EPI (2021) equation; 7) Known or suspected acute febrile illness, symptomatic viral including COVID-19, bacterial (including upper respiratory tract infection), or fungal (non-cutaneous) infection within approximately 2 weeks before the Screening Visit. Screening is allowed when it’s unlikely there will be any potential impact on screening assessment due to the infection; 8) History of receiving HBV vaccine (licensed or experimental) within the 6 months before the Screening Visit or planned to have this vaccine during the study or have received any other vaccination during screening; 9) Previous treatment with an HBV-targeted treatment other than NAs within the 6 months before the Screening Visit or planned use during the study; 10) Use of corticosteroids above 5 mg / day of prednisone (or equivalent) or other immunosuppressive medications within 4 weeks prior to Screening (topical, intra-articular and inhaled steroids allowed; 11) Received during the 6-month period before the Screening Visit any medications or other treatments that may adversely affect the immune system such as allergy injections, immunoglobulins, blood products, interferons, cytotoxic drugs, or other drugs that are known to be frequently associated with significant major organ toxicity; 12) Unable or unwilling to take the required pre-investigational product infusion treatment medication regimen; 13) History of anaphylaxis or known hypersensitivity to any LNP component (or its excipients) or contraindication to high-dose steroids; 14) History of active malignancy within 5 years prior to the Screening Visit or during the screening period, except curatively resected basal cell or squamous cell carcinoma of skin (past medical history of cancer is not exclusionary if the participant has been disease free for at least 5 years since the time of diagnosis and treatment); 15) Uncontrolled hypertension (systolic blood pressure [BP] >160 mmHg, diastolic BP >100 mmHg) despite maximal medical treatment; 16) Prior liver, heart, or other solid organ transplant or bone marrow transplant; 17) Any of the following within the last 12 months prior to the Screening Visit: myocardial infarction (MI), transient ischemic attack (TIA), cerebrovascular accident, pulmonary embolism or New York Health Association Class III or IV heart failure; 18) Prior participation in any gene therapy, approved gene therapy or clinical study, (eg, AAV, CRISPR, etc); however, if the study was unblinded or documentation otherwise exists that the97MF-36384587022474-20039.40 participant was randomized to the placebo control group and did not receive active gene transfer agent, the participant may be considered for this study; 19) Receiving an investigational intervention or participating in another clinical study within 30 days or within 5 half-lives of the drug prior to the Screening Visit. Note: Observational, non-interventional registry trials (studies with no procedural assessments) are acceptable; 20) Participant has a history of alcohol or drug abuse as determined by the clinician within 3 years prior to the Screening Visit; or 21) Any medical or psychiatric condition, laboratory abnormality, or other reason that, in the investigator’s opinion, could adversely affect the safety of the participant, impair the assessment of study results, or preclude compliance with the study.

[0339] In some embodiments, a therapeutic amount of the pharmaceutical composition (e.g., lipid nanoparticles comprising the epigenetic-modifying DNA-targeting system) is administered. Typically, precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, extent of infection, and condition of the patient (subject). Generally, dosages and routes of administration of the pharmaceutical composition are determined according to the size and condition of the subject, according to standard pharmaceutical practice. For example, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models such as mice, rats, rabbits, dogs, pigs, or monkeys. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. The exact dosage can be determined in light of factors related to the subject requiring treatment. Dosage and administration can be adjusted to provide sufficient levels of the active compound or to maintain the desired effect. Factors that may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy.

[0340] In some embodiments, modeling and simulation of pharmacokinetic (PK) and pharmacodynamic (PD) profiles observed in control animals and animal models of disease (e.g., mouse or cynomolgus models) can be used to predict or determine patient dosing. For example, PK data from non-human primates (e.g., cynomolgus monkeys) can be used to estimate human PK. Similarly, mouse or rat PK and PD data can be used to predict human dosing. The observed animal data can be used to inform computational models which can be used to simulate human dose response.98MF-36384587022474-20039.40

[0341] In some embodiments, methods provided herein include administering a pharmaceutical composition (e.g., LNP comprising epigenetic-modifying DNA-targeting system) in an amount in which a dose is known or predicted to repressing transcription of total viral Hepatitis B Viral (HBV) mRNA transcripts. The particular amount can be determined experimentally or empirically. In some embodiments, the amount can be empirically determined from in vitro binding data or from animal models.

[0342] In some embodiments, the HBV targeted epi-silencer disclosed herein (e.g., LNP formulated epigenetic-modifying DNA-targeting system) is administered as a single infusion (e.g., single dose). In some embodiments, one or more further infusion of the LNP formulated epigenetic-modifying DNA-targeting system may be administered to the subject. Further refinement of the appropriate dosage is routinely made. Appropriate dosages may be ascertained through use of appropriate dose-response data.

[0343] It is understood that reference to the “dose” or “dose amount” of a lipid nanoparticle (LNP) composition is the mass of the nucleic acid active ingredient, such as the RNA, contained within the LNP formulation, unless otherwise specified. The total nucleic acid dose may represent the combined mass of all such RNA species formulated within the administered LNP composition, including the mRNA encoding an effector fusion protein and one or more guide RNAs. Accordingly, dosing amounts are expressed in terms of the quantity of nucleic acid administered, such as in milligrams (mg) or milligrams per kilogram of body weight (mg / kg).

[0344] Typically, precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). In some embodiments, when referencing dosage based on mg / kg of the subject, an average human subject is considered to have a mass of about 60 kg-75 kg, such as 60 kg and / or a body surface area (BSA) of 1.73 m2

[0345] In some embodiments, the results support dosing as low as 0.1 mg / kg, such as 0.2 mg / kg. As described herein, a dose of 0.20 mg / kg is expected to silence 74.8% - 89.9% of HBV genomes with higher levels of virus silencing possible with higher doses.

[0346] Among the provided methods are methods of reducing Hepatitis virus infection in a subject, the method comprising administering to the subject a dose of lipid nanoparticles comprising an epigenetic-modifying DNA-targeting system comprising a DNA-targeting module for repressing transcription of total viral Hepatitis B Viral (HBV) mRNA transcripts, wherein the DNA-targeting module comprises: (a) a gRNA for targeting to a target site in a99MF-36384587022474-20039.40HBV DNA sequence, wherein the target site comprises the sequence set forth in SEQ ID NO: 27, a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing; and (b) an mRNA encoding a fusion protein comprising a deactivated Streptococcus pyogenes Cas9 (dSpCas9) protein and at least one transcriptional repressor effector domain comprising a KRAB domain and a DNA methyltransferase 3 (DNMT3) domain that has DNA methyltransferase activity, and wherein the dose comprises (a) between at or about 0.1 mg / kg and at or about 1 mg / kg, or (b) between at or about 10 mg to about 100 mg.

[0347] Also provided herein are methods of reducing Hepatitis virus infection in a subject comprising administering to a subject in need thereof, the method comprising administering to the subject a dose of lipid nanoparticles comprising an epigenetic-modifying DNA-targeting system that increases CpG methylation of a region within a target region in a Hepatitis B viral sequence. In some embodiments, the target region comprises CpG island 2 in a Hepatitis B viral sequence. In some embodiments, the target region comprises a contiguous sequence of nucleotides within the sequence corresponding to 1033 bp -1749 bp in a Hepatitis B viral sequence with reference to nucleotide positions of SEQ ID NO: 21. In some embodiments, the target region comprises a contiguous sequence of nucleotides within the sequence corresponding to 1255 bp -1290 bp in a Hepatitis B viral sequence with reference to nucleotide positions of SEQ ID NO: 21. In some embodiments, the epigenetic modifying DNA-targeting system comprises: (a) a gRNA for targeting to a target site in a HBV DNA sequence, wherein the target site comprises the sequence set forth in SEQ ID NO: 27, a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing; and (b) an mRNA encoding a fusion protein comprising a deactivated Streptococcus pyogenes Cas9 (dSpCas9) protein and at least one transcriptional repressor effector domain comprising a KRAB domain and a DNA methyltransferase 3 (DNMT3) domain that has DNA methyltransferase activity. In some embodiments, the dose comprises (a) between at or about 0.1 mg / kg and at or about 1 mg / kg, or (b) between at or about 10 mg to about 100 mg.

[0348] In some embodiments, the dose is 0.1 mg / kg to 100 mg / kg, such as 0.2 mg / kg to 0.65 mg / kg, for example, at or about 0.2 mg / kg, about 0.45 mg / kg or about 0.65 mg / kg. In some embodiments, the dose is greater than 0.65 mg / kg but is lower than 1.0 mg / kg. In some embodiments, flat or fixed doses (not weight-based dosing) are also contemplated and include administration of a dose as low as 10 mg up to as high as 120mg. In some embodiments, flat or fixed doses (not weight-based dosing) are also contemplated and include administration of a100MF-36384587022474-20039.40 dose as low as 10 mg up to as high as lOOmg, such as about 12 mg to to 65 mg, for example about 12 mg to about 45 mg.

[0349] In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA- targeting system is administered in an amount between at or about 0.1 mg / kg and and at or about 1 mg / kg, inclusive. In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA-targeting system is administered in an amount between at or about 0.1 mg / kg and at or about 1 mg / kg, between at or about 0.1 mg / kg and at or about 0.8 mg / kg, between at or about 0.1 mg / kg and at or about 7 mg / kg, between at or about 0.1 mg / kg and at or about 5, between at or about 0.1 mg / kg and at or about 4 mg / kg, between at or about 0.1 mg / kg and at or about 3 mg / kg mg / kg, between at or about 0.15 mg / kg and at or about 0.8 mg / kg, between at or about 0.15 mg / kg and at or about 0.7 mg / kg, between at or about 0.15 mg / kg and at or about 0.4 mg / kg, between at or about 0.15 mg / kg and at or about 0.2 mg / kg, between at or about 0.3 mg / kg and at or about 0.65 mg / kg, between at or about 0.3 mg / kg and at or about 0.5 mg / kg, between at or about 0.3 mg / kg and at or about 0.4 mg / kg, between at or about 0.2 mg / kg and at or about 0.45 mg / kg, between at or about 0.2 mg / kg and at or about 0.4 mg / kg, between at or about 0.2 mg / kg and at or about 0.3 mg / kg, between at or about 0.2 mg / kg and at or about 0.25 mg / kg, each inclusive.

[0350] In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA- targeting system is or is about 0.1 mg / kg, about 0.2 mg / kg, about 0.25 mg / kg, about 0.3 mg / kg, about 0.35 mg / kg, about 0.4 mg / kg, about 0.45 mg / kg, about 0.5 mg / kg, about 0.55 mg / kg, about 0.6 mg / kg, about 0.65 mg / kg, about 0.7 mg / kg, about 0.75 mg / kg, about 0.8 mg / kg, about 0.85 mg / kg, about 0.95 mg / kg, about 1 mg / kg, or a value between any of the foregoing.

[0351] In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA- targeting system comprises about 0.2 mg / kg. In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA-targeting system is 0.2 mg / kg.

[0352] In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA- targeting system comprises about 0.45mg / kg. In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA-targeting system is 0.45 mg / kg.

[0353] In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA- targeting system comprises about 0.65 mg / kg. In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA-targeting system is 0.65 mg / kg.

[0354] In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA- targeting system is greater than 0.65 mg / kg and less than 1 mg / kg.101MF-36384587022474-20039.40

[0355] In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA- targeting system is administered in an amount between at or about 10 mg to about 100 mg. In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA-targeting system is administered in an amount between at or about 10 to between at or about 100 mg, between at or about 10 to between at or about 90 mg, between at or about 10 to between at or about 80 mg, between at or about 20 to between at or about 100 mg, between at or about 20 to between at or about 80 mg, between at or about 20 to between at or about 60 mg, between at or about 20 to between at or about 40 mg, between at or about 27 mg to between at or about about 39 mg, between at or about 25 mg to between at or about about 100 mg, between at or about 25 mg to between at or about about 80 mg, between at or about 25 mg to between at or about about 60 mg, between at or about 25 mg to between at or about about 40 mg, between at or about 70 mg to between at or about 100 mg, between at or about 80 mg to between at or about 100 mg, between at or about 90 mg to between at or about 100 mg, between at or about 70 mg to between at or about 80 mg, or between any of the foregoing.

[0356] In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA- targeting system comprises between about 10 to about 100 mg.

[0357] In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA- targeting system comprises between about 12 mg to about 20 mg.

[0358] In some embodiments, if the subject is 100 kg or greater the dose of the LNP formulated epigenetic-modifying DNA-targeting system comprises no more than 20 mg. In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA-targeting system comprises no more than 20 mg. In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA-targeting system is no more than 20 mg.

[0359] In some embodiments, if the subject is 100 kg or greater the dose comprises no more than 45 mg. In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA- targeting system comprises no more than 45 mg. In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA-targeting system is no more than 45 mg.

[0360] In some embodiments, if the subject is 100 kg or greater the dose is no more than 100 mg. In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA- targeting system comprises no more than 100 mg. In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA-targeting system is no more than 100 mg.

[0361] In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA- targeting system comprises no more than 12 mg. In some embodiments, the dose of the LNP102MF-36384587022474-20039.40 formulated epigenetic-modifying DNA-targeting system is no more than 12 mg.

[0362] In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA- targeting system comprises no more than 27 mg. In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA-targeting system is no more than 27 mg.

[0363] In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA- targeting system comprises no more than 39 mg. In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA-targeting system is no more than 39 mg.

[0364] In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA- targeting system comprises no more than 100 mg. In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA-targeting system is no more than 100 mg.

[0365] The administration of the subject compositions may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one embodiment, the therapeutic composition is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the therapeutic composition is administered by i.v. injection.

[0366] In some embodiments, the pharmaceutical composition is administered to a subject through any route, including orally, transdermally, by inhalation, intravenously, intra-arterially, intramuscularly, direct application to a wound site, application to a surgical site, intraperitoneally, by suppository, subcutaneously, intradermally, transcutaneously, by nebulization, intrapleurally, intraventricularly, intra-articularly, intraocularly, intraspinally, intratumorally or systemically.

[0367] In some embodiments, the pharmaceutical composition (e.g., the lipid nanoparticle comprising the epigenetic-modifying DNA-targeting systems) are administered to the subject intraveneously. In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA-targeting system to the subject via intravenous administration is about 0.2 mg / kg. In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA-targeting system to the subject via intravenous administration is about 0.45 mg / kg. In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA-targeting system to the subject via intravenous administration is about 0.465 mg / kg. In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA-targeting system to the subject via intravenous administration is greater than 0.65 mg / kg and less than 1 mg / kg.103MF-36384587022474-20039.40

[0368] In some embodiments, the pharmaceutical composition (e.g., the lipid nanoparticle comprising the epigenetic-modifying DNA-targeting systems) are administered to the subject intraveneously. In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA-targeting system to the subject via intravenous administration is no more than 12 mg. In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA-targeting system to the subject via intravenous administration is no more than 27 mg. In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA-targeting system to the subject via intravenous administration is no more than 39 mg. In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA-targeting system to the subject via intravenous administration is no more than 70 mg. In some embodiments, the dose of the LNP formulated epigenetic-modifying DNA-targeting system to the subject via intravenous administration is no more than 100 mg.In some embodiments, the subject has a weight of at least about 40 kg, about 45 kg, about 50 kg, about 55 kg, about 60 kg, about 65 kg, of any of the foregoing. In some embodiments, the subject has a weight of at least about 40 kg.

[0369] In some embodiments, the pharmaceutical composition (including LNP comprising the epigenetic modyifying DNA-targeting systems described herein) is administered parenterally. In some embodiments, the pharmaceutical composition is in a form suitable for infusion injection, for example by intravenous injection. In some embodiments, the infusion duration is, is at least, or is about 30 minutes, 40 minutes, 50 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11, hours, or 12 hours. In some embodiments the infusion duration is between about 30 minutes and 6 hours. In some embodiments, the infusion duration is between about 30 minutes and 5 hours. In some embodiments, the infusion duration is between about 30 minutes and 4 hours. In some embodiments, the infusion duration is between about 30 minutes and 3 hours. In some embodiments, the infusion duration is between about 30 minutes and 2 hours. In some embodiments, the infusion duration is between about 30 minutes and 1 hour. In some embodiments, the infusion duration is or is about 30 minutes. In some embodiments the infusion duration is or up to about 12 hours.

[0370] In some embodiments, the subject is administered a single dose of the LNP formulated epigenetic-modifying DNA-targeting system. In embodiments in which a single dose of the LNP composition is administered, the dose may be any of the dosage amounts or ranges described herein, including but not limited to those expressed in terms of mass (e.g., mg or104MF-36384587022474-20039.40 mg / kg). For example, each dose may be about 0.1 to 1 mg / kg, such as about 0.2 mg / kg, 0.45 mg / kg, or 0.65 mg / kg, or a value between any of the foregoing, or may be a fixed amount of about 10 to 100 mg, such as about 12 mg, 27 mg, 39 mg, 52 mg, or 100 mg, or a value between any of the foregoing. In certain embodiments, a single administration of the LNP composition provides sufficient epigenetic-modifying activity to induce durable transcriptional silencing of HBV genes. Such a single administration may be sufficient to reduce HBV total RNA, including transcripts derived from cccDNA and / or intDNA, or to reduce HBV transcriptional activity, as evidenced by decreased HBsAg transcript or protein levels. In some embodiments, the reduction in HBV total RNA or HBsAg transcript or protein levels is at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% or more, relative to the level in the subject prior to treatment, or relative to a control cell or subject not exposed to the LNP composition. In some embodiments, such reduction persists for 1 year or more, such as 2, 3, 4, 5 or more years, following administration.

[0371] In some embodiments, the subject is administered more than one dose of the LNP formulated epigenetic-modifying DNA-targeting system. The number of doses may vary depending on the desired level and duration of epigenetic modification and may include, for example, two, three, four, five, or more administrations. In some embodiments, the subject is administered two doses of the LNP formulated epigenetic-modifying DNA-targeting system. In some embodiments, the subject is administered three doses of the LNP formulated epigenetic- modifying DNA-targeting system. In some embodiments, the subject is administered four doses of the LNP formulated epigenetic-modifying DNA-targeting system. In some embodiments, the subject is administered five doses of the LNP formulated epigenetic-modifying DNA-targeting system. In some embodiments, the subject is administered greater than five doses of the LNP formulated epigenetic-modifying DNA-targeting system. In provided embodiments, a multipledose administration regimen is sufficient to reduce HBV total RNA, including transcripts derived from cccDNA and / or intDNA, or to reduce HBV transcriptional activity, as evidenced by decreased HBsAg transcript or protein levels. In some embodiments, the reduction in HBV total RNA or HBsAg transcript or protein levels is at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% or more, relative to the level in the subject prior to treatment, or relative to a control cell or subject not exposed to the LNP composition. In some embodiments, such reduction persists for 1 year or more, such as 2, 3, 4, 5 or more years, following administration.

[0372] In some embodiments in which more than one dose of the LNP formulated105MF-36384587022474-20039.40 epigenetic-modifying DNA-targeting system is administered, the administration is a finite multiple dosing regimen, wherein a finite number of discrete doses are administered over a defined treatment period, rather than continuous or indefinite dosing. Finite multiple dosing may include administration of two or more doses, typically three or more, each comprising a fixed or predetermined quantity of the LNP formulation and administered at defined intervals. The total number of doses in such regimens is limited and predetermined, e.g., two, three, four, five, or six doses, after which no further administration occurs unless a retreatment cycle is initiated. The dosing interval in a finite multiple dosing regimen may be an interval as described. Such finite multiple dosing can achieve durable and sustained epigenetic reprogramming while reducing the risk of cumulative toxicity, immune sensitization, and / or undesired prolonged activity of the epigenetic-modifying DNA-targeting system.

[0373] In some embodiments, the multiple doses may be administered at regular or variable intervals. In certain embodiments, successive doses are separated by a period ranging from about one hour to about 24 months, such as from about one hour to about one day, from about one day to about one week, from about one week to about one month, or from about one month to about six months. In some embodiments, the interval between doses is about 1, 3, 7, 14, 21, or 28 days, or about 1, 2, 3, 6, or 12 months, or a value between any of the foregoing. In some embodiments, the dosing interval may be fixed (i.e. each dose of the multiple-dose administration is separated by the same interval) or may be variable (i.e. at least one interval between two doses differs from another interval within the dosing regimen). The specific dosing interval may be selected to achieve sustained or periodic exposure to the LNP -formulated epigenetic-modifying DNA-targeting system, taking into account factors such as the desired persistence of the epi-editor effect, the therapeutic indication being treated or other factors within the skill and knowledge of a clinician or treating physician. In certain embodiments, the multiple doses are administered according to a finite multiple dosing regimen, as described herein.

[0374] In some embodiments, each dose of the multiple-dose administration independently comprises any of the dosage amounts or ranges described herein, and the doses may be the same or different. For example, each dose may be about 0.1 to 1 mg / kg, such as about 0.2 mg / kg, 0.45 mg / kg, or 0.65 mg / kg, or a value between any of the foregoing, or may be a fixed amount of about 10 to 100 mg, such as about 12 mg, 27 mg, 39 mg, 52 mg, or 100 mg, or a value between any of the foregoing. In some embodiments, each dose may contain an equivalent (same) amount of the LNP formulated epigenetic-modifying DNA-targeting system, or successive doses106MF-36384587022474-20039.40 may be increased or decreased relative to a prior dose. The total cumulative amount administered across the multiple doses may therefore represent a sum of identical or nonidentical individual doses, delivered according to the multiple dosing schedule.

[0375] In some embodiments, in a multipe-dose administration regimen, each dose (which may be an equivalent amount for each dose administered) is delivered at consistent intervals within a 6 month period, such as within a 3 month period, for example over approximately 8 to 12 weeks. In some embodiments, the multiple dosing is a finite multiple dosing regimen that may include 2 to 6 doses administered once every 14 to 60 days, such as once every 21 to 42 days. In some embodiments, the finite dosing schedule may include 3 to 5 doses administered every two weeks (Q2W), once every 28 days ( i.e. every 4 weeks (Q4W)) or once every 6 weeks (Q6W). In some embodiments, three doses are administered once every 21 days (Q3Wx3). In some embodiments, four doses are administered once every 21 days (Q3Wx4). In some embodiments, three doses are administered once every 28 days (Q4Wx3). In some embodiments, four doses are administered once every 28 days (Q4Wx4). In some embodiments, three doses are administered once every 6 weeks (Q6Wx3). In some embodiments, four doses are administered once every 6 weeks (Q6Wx4).

[0376] In some embodiments, the pharmaceutical composition is administered in combination with an infusion prophylaxis regimen. In some embodiments, the prophylaxis regimen may be administered 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more hours after the administration of the pharmaceutical composition. In some embodiments, the prophylaxis regimen may be administered within 1 to 2 hours of administration of the pharmaceutical composition. In some embodiments, the prophylaxis regimen may comprise one or more of the following: acetaminophen / paracetamol, steroid, Hl blocker, or an H2 blocker. In some embodiments, the prophylaxis regimen may comprise one or more of the following: oral acetaminophen / paracetamol, IV steroid, IV Hl blocker, oral Hl blocker, IV H2 blocker, or oral H2 blocker. In some embodiments, the prophylaxis regimen may comprise one or more of the following: oral acetaminophen / paracetamol (approximately 500 mg to 650 mg); IV steroid (eg, dexamethasone 10 mg or equivalent); IV Hl blocker (eg, diphenhydramine 50 mg or equivalent); oral Hl blocker (eg, cetirizine 10 mg or equivalent), or IV or oral H2 blocker (eg, famotidine 20 mg or equivalent).

[0377] In some embodiments, the pharmaceutical composition is administered as a monotherapy (i.e., as a single agent) or as a combination therapy (i.e., in combination with one or more additional agents). In some embodiments, the additional agent is a nucleoside analog.107MF-36384587022474-20039.40In some embodiments, the nucleoside analog is entecavir (ETV), tenofovir disoproxil fumarate (TDF) or tenofovir alafenamide (TAF).

[0378] In aspects of the provided embodiments, a DNA-targeting system provided herein targets a gene or regulatory element thereof to reduce transcription of total viral Hepatitis B Viral (HBV) mRNA transcripts in an Hepatitis B Virus (HBV) infected cell, in which the reduced transcription modulates one or more activities or functions of the HBV infected cells, such as expression of HBV RNA and / or HBV proteins. In some embodiments, reduced transcription of the gene results in a reduction in expression of the gene, i.e. reduced gene expression, in the infected cell. In some embodiments, reduced transcription of the gene, such as reduced gene expression, results in a reduction in expression of the protein, i.e. reduced protein expression, in the infected cell.

[0379] Once the epigenetic-modifying DNA-targeting system is administered to the subject (e.g., human), the biological activity of the modified cell populations in some aspects is measured by any of a number of known methods. Parameters to assess include reduced levels of HBV transcripts and / or HBV DNA levels in the liver cells (e.g. by qRT-PCR / qPCR), reduced levels of of HBV proteins (e.g., HBsAg, HBeAg) inside and on the surface of the liver cells (e.g., assessed by fluorescence staining and flow cytometry), and / or HBeAg, measured by analysis of blood serum using ELISA). In some aspects the biological activity is measured by assessing clinical outcome. Specific thresholds for the parameters can be set to determine the efficacy of the methods of therapy provided herein.

[0380] In some embodiments, the method reduces the levels of Hepatitis B virus in the subject. In some embodiments, the method reduces Hepatitis B virus RNA and / or protein levels in the subject. In some embodiments, the method reduces Hepatitis B virus RNA and / or protein levels in the subject. In some embodiments, the expression of HBV DNA or pre-genomic (pgRNA) is reduced. In some embodiments, the levels of HBsAg, HBeAg, HBcrAg, and / or HBeAg and p-HBcAg are reduced. In some embodiments, the levels of HBV DNA or pgRNA are reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, the levels of HBV DNA or pgRNA are reduced by at least 10%. In some embodiments, the levels of HBV DNA or pgRNA are reduced by at least 20%. In some embodiments, the levels of HBV DNA or pgRNA are reduced by at least 30%. In some embodiments, the levels of HBV DNA or pgRNA are reduced by at least 40%. In some embodiments, the levels of HBV DNA or pgRNA are reduced by at least 50%. In some embodiments, the levels of HBV DNA or pgRNA are reduced by at least 60%. In108MF-36384587022474-20039.40 some embodiments, the levels of HBV DNA or pgRNA are reduced by at least 70%. In some embodiments, the levels of HBV DNA or pgRNA are reduced by at least 80%. In some embodiments, the levels of HBV DNA or pgRNA are reduced by at least 90%.

[0381] In some embodiments, the levels of HBsAg, HBeAg, HBcrAg, and / or HBcAg and p- HBcAg are reduced by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, the levels of HBsAg, HBeAg, HBcrAg, and / or HBcAg and p-HBcAg are reduced by at least 10%. In some embodiments, the levels of HBsAg, HBeAg, HBcrAg, and / or HBcAg and p-HBcAg are reduced by at least 20%. In some embodiments, the levels of HBsAg, HBeAg, HBcrAg, and / or HBcAg and p-HBcAg are reduced by at least 30%. In some embodiments, the levels of HBsAg, HBeAg, HBcrAg, and / or HBcAg and p-HBcAg are reduced by at least 40%. In some embodiments, the levels of HBsAg, HBeAg, HBcrAg, and / or HBcAg and p-HBcAg are reduced by at least 50%.

[0382] In some embodiments, the method results in a change in the levels of stiffness and / or fat content.

[0383] In some embodiments, the levels of anti-HBsAg, anti-HBcAg, and / or anti-HBeAg antibodies are reduced. In some embodiments, the levels of HBV-specific peripheral blood mononuclear cell immune markers are reduced.

[0384] In some aspects, dose limiting toxicities include the following (event occurring within the first 4 weeks after dosing): 1) any Common Terminology Criteria for Adverse Events (CTCAE) Grade 3 (except lab event) or higher AE that is possibly, probably, or definitely related to investigational product (IP); 2) any CTCAE Grade 3 laboratory abnormality that persists for > 2 weeks and is possibly, probably, or definitely related to IP; 3) any adverse event (AE) that meets protocol-defined stopping criteria; 4) any other AE or laboratory abnormality that would preclude further dosing. In some embodiments, dose limiting toxicities may be graded according to the CTCAE Version 5.0. In some embodiments, a toxicity -treatment agent can be administered at first signs or symptoms of a toxicity (e.g., paracetamol and / or coricosteroids).II. PHARMACEUTICAL COMPOSITIONS AND FORMULATIONS

[0385] In some aspects, provided herein are compositions, such as pharmaceutical compositions and formulations for administration, that include any of the DNA-targeting systems described herein, or any of the polynucleotides or vectors encoding the same. In some aspects, the pharmaceutical composition contains one or more DNA-targeting systems provided herein or a component thereof. In some aspects, the pharmaceutical composition comprises one109MF-36384587022474-20039.40 or more vectors, e.g., viral vectors that contain polynucleotides that encode one or more components of the DNA-targeting systems provided herein. Such compositions can be used in accord with the provided methods, and / or with the provided articles of manufacture or compositions, such as in the prevention or treatment of diseases, conditions, and disorders, or in detection, diagnostic, and prognostic methods.

[0386] The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject or a cell to which the formulation would be administered.

[0387] In some embodiments, the pharmaceutical composition may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be functional molecules as vehicles, adjuvants, carriers, or diluents.

[0388] A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0389] In some aspects, the choice of carrier is determined in part by the particular agent and / or by the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars110MF-36384587022474-20039.40 such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0390] In some embodiments, the pharmaceutically acceptable excipient may be a transfection facilitating agent, which may include surface active agents, such as immune- stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents.

[0391] In some embodiments, the transfection facilitating agent is a polyanion, polycation, including poly-L-glutamate (LGS), or lipid. In some embodiments, the transfection facilitating agent is poly-L-glutamate. In some embodiments, the transfection facilitating agent may also include surface active agents such as immune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs and vesicles such as squalene and squalene, and hyaluronic acid may also be used administered in conjunction with the genetic construct. In some embodiments, the DNA vector encoding the DNA-targeting system may also include a transfection facilitating agent such as lipids, liposomes, including lecithin liposomes or other liposomes known in the art, as a DNA- liposome mixture (see for example WO9324640), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents. In some embodiments, the transfection facilitating agent is a polyanion, polycation, including poly-L- glutamate (LGS), or lipid.

[0392] Compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.

[0393] In some embodiments, the lipid nanoparticle carrying the epigenetic DNA-targeting111MF-36384587022474-20039.40 system provided herein is formulated in 100- 500 mM sucrose in phosphate buffered saline. In some embodiments, the lipid nanoparticle carrying the epigenetic DNA-targeting system herein is formulated in sucrose in an amount that is at or about 100 mN, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM, 500 mM, or any amount between any of the foregoing. In some embodiments, the lipid nanoparticle carrying the epigenetic DNA-targeting system herein is formulated in about 300 mM sucrose. In some embodiments, the pH of the formulation is neutral pH. In some embodiments, the pH of the formulation is about pH 7.0 to pH. 7.4, such as at or about pH 7.1, pH 7.2, pH 7.3, or pH 7.4. In some embodiments, the lipid nanopartitcle carrying the epigenetic DNA-targeting system herein is formulated in about 300 mM sucrose in phosphate buffered saline at pH about 7.0 to about 7.4. In some embodiments, the pH is about pH 7.0. In some embodiments, the pH is about pH 7.4.

[0394] Sterile injectable solutions can be prepared by incorporating the agent in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The formulations to be used for in vivo or ex vivo administration or use are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.

[0395] The pharmaceutical composition in some embodiments contains components in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.

[0396] In some embodiments, the composition can be administered to a subject by any suitable means, for example, by bolus infusion or by injection, e.g., by intravenous or subcutaneous injection. In some embodiments, a given dose is administered by a single bolus administration of the composition. In some embodiments, the composition is administered by multiple bolus administrations of the composition, for example, over a period of no more than 3 days, or by continuous infusion administration of the composition. In some embodiments, the composition is administered parenterally, for example by intravenous, intramuscular,112MF-36384587022474-20039.40 subcutaneous, or intraperitoneal administration. In some embodiments, the composition is administered to a subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

[0397] For the prevention or treatment of disease, the appropriate dosage may depend on the type of disease to be treated, the type of agent or agents, the type of cells or recombinant receptors, the severity and course of the disease, whether the agent or cells are administered for preventive or therapeutic purposes, previous therapy, the subject’s clinical history and response to the agent or the cells, and the discretion of the attending physician. The compositions are in some embodiments suitably administered to the subject at one time or over a series of treatments.V. KITS AND ARTICLES OF MANUFACTURE

[0398] Also provided are articles of manufacture, systems, apparatuses, and kits useful in performing the provided embodiments. In some embodiments, the provided articles of manufacture or kits contain any of the DNA-targeting systems described herein, any of the gRNAs described herein, any of the fusion proteins described herein, any of the polynucleotides described herein, any of the pluralities of polynucleotides described herein, any of the vectors described herein, any of the pluralities of vectors described herein, or a portion or a component of any of the foregoing, or any combination thereof. In some embodiments, the articles of manufacture or kits include polypeptides, polynucleotides, nucleic acids, and / or vectors useful in performing the provided methods.

[0399] . In some embodiments, the articles of manufacture or kits include lipid nanoparticles comprising a gRNA for targeting to a target site in the HB V DNA sequence and an mRNA encoding a fusion protein, as described herein.

[0400] In some embodiments, the articles of manufacture or kits include one or more containers, typically a plurality of containers, packaging material, and a label or package insert on or associated with the container or containers and / or packaging, generally including instructions for use, e.g., instructions for introducing or administering.

[0401] Also provided are articles of manufacture, systems, apparatuses, and kits useful in administering the provided compositions, e.g., pharmaceutical compositions, e.g., for use in therapy or treatment. In some embodiments, the articles of manufacture or kits provided herein contain vectors and / or plurality of vectors, such as any vectors and / or plurality of vectors described herein. In some aspects, the articles of manufacture or kits provided herein can be used for administration of the vectors and / or plurality of vectors, and can include instructions for113MF-36384587022474-20039.40 use.

[0402] The articles of manufacture and / or kits containing cells or cell compositions for therapy, may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container in some embodiments holds a composition which is by itself or combined with another composition effective for treating, preventing and / or diagnosing the condition. In some embodiments, the container has a sterile access port. Exemplary containers include an intravenous solution bags, vials, including those with stoppers pierceable by a needle for injection, or bottles or vials for orally administered agents. The label or package insert may indicate that the composition is used for treating a disease or condition. The article of manufacture may further include a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further include another or the same container comprising a pharmaceutically-acceptable buffer. It may further include other materials such as other buffers, diluents, filters, needles, and / or syringes.

[0403] In some aspects, the pharmaceutical composition is packaged in vials. In some embodiments, the vials contain about 1 ml, about 1.25 ml, about 1.5 ml, about 2 ml, about 2.25ml, about 3 ml, about 3.5 ml, about 4 ml, about 4.5 ml, about 5 ml, or between any of the foregoing. In some embodiments, the vials contain about 2 ml of the pharmaceutical composition.

[0404] In some aspects, the pharmaceutical formulation is no more than about 1 mg / ml, about 1.5 mg / ml, about 2 mg / ml, about 2.5 mg / ml, about 3 mg / ml. In some embodiments, the pharmaceutical formulation is no more than about 2 mg / ml.

[0405] In some embodiments, the pharmaceutical composition is packaged in vials. In some embodiments, the containers (e.g., vials) are single use vials. In some embodiments, the containers (e.g., vials) are multi use vials. For example, if the dose mg / kg is higher more vials may be needed. In another example, if the subject is 100 kg or more up to 10 vials may be needed.

[0406] In some embodiments, the pharmaceutical composition is formulated in a multiuse via and comprises no more than about 2 mg / ml.VI. DEFINITIONS114MF-36384587022474-20039.40

[0407] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0408] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or more.” It is understood that aspects and variations described herein include “consisting” and / or “consisting essentially of’ aspects and variations.

[0409] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.

[0410] The term “about” as used herein refers to the usual error range for the respective value readily known. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”. In some embodiments, “about” may refer to ±25%, ±20%, ±15%, ±10%, ±5%, or ±l%.

[0411] As used herein, the term, “corresponding to” with reference to positions of a nucleotide sequence (or base pair) or protein sequence (amino acid), such as recitation that nucleotides or amino acid positions “correspond to” base pair or amino acid positions in a disclosed sequence, such as set forth in the Sequence listing, refers to nucleotides or amino acid positions identified upon alignment with the disclosed sequence to maximize identity using a115MF-36384587022474-20039.40 standard alignment algorithm, such as the GAP algorithm. By aligning the sequences, corresponding residues can be identified, for example, using conserved and identical amino acid residues as guides. In general, to identify corresponding positions, the sequences of amino acids are aligned so that the highest order match is obtained (see, e.g. : Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; Carrillo et al. (1988) SIAM J Applied Math 48: 1073).

[0412] A “gene,” includes a DNA region encoding a gene product, as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and / or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions. The sequence of a gene is typically present at a fixed chromosomal position or locus on a chromosome in the cell.

[0413] A “regulatory element” or “DNA regulatory element,” which terms are used interchangeably herein, in reference to a gene refers to DNA regions which regulate the production of a gene product, whether or not such regulatory sequences are adjacent to coding and / or transcribed sequences. Accordingly, a regulatory element includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions.

[0414] As used herein, a “target site” or “target nucleic acid sequence” is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule (e.g. a DNA- binding domain disclosed herein) will bind, provided sufficient conditions for binding exist.

[0415] The term “expression” with reference to a gene or “gene expression” refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or can be a protein produced by translation of an mRNA. Gene products also include RNAs which are modified, by processes such as116MF-36384587022474-20039.40 capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristoylation, and glycosylation. Hence, reference to expression or gene expression includes protein (or polypeptide) expression or expression of a transcribable product of or a gene such as mRNA. The protein expression may include intracellular expression or surface expression of a protein. Typically, expression of a gene product, such as mRNA or protein, is at a level that is detectable in the cell.

[0416] As used herein, a “detectable” expression level, means a level that is detectable by standard techniques known to a skilled artisan, and include for example, differential display, RT (reverse transcriptase)-coupled polymerase chain reaction (PCR), Northern Blot, and / or RNase protection analyses as well as immunoaffinity -based methods for protein detection, such as flow cytometry, ELISA, or western blot. The degree of expression levels need only be large enough to be visualized or measured via standard characterization techniques.

[0417] As used herein, the term “DNA-targeting system” or “epigenetic-modifying DNA- targeting system” refers to a composition comprising a DNA- binding domain (such as any Cas, ZFN, or TALE-based DNA- binding domain described herein) that targets a target site of a target gene and / or regulatory element thereof. In some embodiments, the DNA-targeting system is engineered to target the target site. In some embodiments, the DNA-targeting system comprises one or more effector domains that modify transcription of the target gene and / or regulatory element thereof when recruited to the target site by the DNA-targeting system. In some embodiments, the DNA-targeting system is capable of targeting more than one target site (i.e. a plurality of target sites), such as 2, 3, 4, 5, 6, or more target sites.

[0418] As used herein, the term “DNA-targeting module” refers to any composition or portion of a DNA-targeting system described herein that targets one target site. For example, an individual DNA-targeting module may comprise a gRNA and a fusion protein comprising a DNA-binding domain and a transcriptional repressor domain (e.g. DNMT3A / 3L-dSpCas9- KRAB). A DNA-targeting system provided herein may comprise 2 to 10 DNA-targeting modules.

[0419] As used herein, the term “reduced expression” or “decreased expression” means any form of expression that is lower than the expression in an original or source cell that does not contain the modification for modulating a particular gene expression by a DNA-targeting system, for instance a wild-type expression level (which can be absence of expression or immeasurable expression as well). Reference herein to “reduced expression,” or “decreased117MF-36384587022474-20039.40 expression” is taken to mean a decrease in gene expression relative to the level in a cell that does not contain the modification, such as the original source cell prior to contacting with, or engineering to introduce, the DNA-binding system into the T cell, such as an unmodified cell or a wild-type T cell. The decrease in expression can be at least 5%, 10%, 20%, 30%, 40% or 50%, 60%, 70%, 80%, 85%, 90%, or 100% or even more. In some cases, the decrease in expression can be at least 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80- fold, 90-fold, 100-fold, 200-fold or more.

[0420] As used herein, the term “reduced transcription” or “decreased transcription” refers to the level of transcription of a gene that is lower than the transcription of the gene in an original or source cell that does not contain the modification for modulating transcription by a DNA-targeting system, for instance a wild-type transcription level of a gene. Reference to reduced transcription or decreased transcription can refer to reduction in the levels of a transcribable product of a gene such as mRNA. Any of a variety of methods can be used to monitor or quantitate a level of a transcribable product such as mRNA, including but not limited to, real-time quantitative RT (reverse transcriptase)- polymerase chain reaction (qRT-PCR), Northern Blot, microarray analysis, or RNA sequencing (RNA-Seq). The reduction in transcription can be at least 5%, 10%, 20%, 30%, 40% or 50%, 60%, 70%, 80%, 85%, 90%, or 100% or even more. In some cases, the reduction in transcription can be at least 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold or more.

[0421] As used herein, an “epigenetic modification” refers to changes in the gene expression that are not caused by changes in the DNA sequences but are due to events like DNA methylations, histone modifications, miRNA expression modulation.

[0422] As used herein, the term “modification” or “modified” with reference to a T cell refers to any change or alteration in a cell that impacts gene expression in the cell. In some embodiments, the modification is an epigenetic modification that directly changes the epigenetic state of a gene or regulatory elements thereof to alter (e.g. decrease) expression of a gene product. In some embodiments, a modification described herein results in decreased expression of a target gene or selected polynucleotide sequence.

[0423] As used herein, a “fusion” molecule is a molecule in which two or more subunit molecules are linked, such as covalently. Examples of a fusion molecule include, but are not limited to, fusion proteins (for example, a fusion between a DNA-binding domain such as a ZFP, TALE DNA-binding domain or CRISPR-Cas protein and one or more effector domains,118MF-36384587022474-20039.40 such as a transactivation domain). The fusion molecule also may be part of a system in which a polynucleotide component associates with a polypeptide component to form a functional molecule (e.g., a CRISPR / Cas system in which a single guide RNA associates with a functional domain to modulate gene expression). Fusion molecules also include fusion nucleic acids, for example, a nucleic acid encoding the fusion protein. Expression of a fusion protein in a cell can result from delivery of the fusion protein to the cell or by delivery of a polynucleotide encoding the fusion protein to a cell, where the polynucleotide is transcribed, and the transcript is translated, to generate the fusion protein.

[0424] The term “methylation,” as used herein, refers to the presence of epigenetic methylation of cytosine residues in DNA at sites where it is not typically present in normal cells.

[0425] The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a selfreplicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” Among the vectors are viral vectors, such as adenoviral vectors or lentiviral vectors.

[0426] The term “expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include, but are not limited to, cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0427] The term “isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0428] The term "polynucleotide" refers to a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomelic "nucleotides." The monomelic119MF-36384587022474-20039.40 nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means.

[0429] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0430] As used herein, “percent (%) amino acid sequence identity” and “percent identity” when used with respect to an amino acid sequence (reference polypeptide sequence) is defined as the percentage of amino acid residues in a candidate sequence (e.g., the subject antibody or fragment) that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various known ways, in some embodiments, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences can be determined, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0431] In some embodiments, “operably linked” may include the association of components, such as a DNA sequence, (e.g. a heterologous nucleic acid) and a regulatory sequence(s), in such a way as to permit gene expression when the appropriate molecules (e.g. transcriptional repressor proteins) are bound to the regulatory sequence. Hence, it means that the components120MF-36384587022474-20039.40 described are in a relationship permitting them to function in their intended manner.

[0432] An amino acid substitution may include replacement of one amino acid in a polypeptide with another amino acid. The substitution may be a conservative amino acid substitution or a non-conservative amino acid substitution. Amino acid substitutions may be introduced into a binding molecule, e.g., antibody, of interest and the products screened for a desired activity, e.g., retained / improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

[0433] Amino acids generally can be grouped according to the following common sidechain properties:

[0434] (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He;

[0435] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

[0436] (3) acidic: Asp, Glu;

[0437] (4) basic: His, Lys, Arg;

[0438] (5) residues that influence chain orientation: Gly, Pro;

[0439] (6) aromatic: Trp, Tyr, Phe.

[0440] In some embodiments, conservative substitutions can involve the exchange of a member of one of these classes for another member of the same class. In some embodiments, non-conservative amino acid substitutions can involve exchanging a member of one of these classes for another class.

[0441] As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.

[0442] As used herein, a “subject” or an “individual,” which are terms that are used interchangeably, is a mammal. In some embodiments, a “mammal” includes humans, nonhuman primates, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, monkeys, etc. In some embodiments, the subject or individual is human. In some embodiments, the subject is a patient that is known or suspected of having a disease, disorder or condition.

[0443] As used herein, the term “treating” and “treatment” includes administering to a subject an effective amount of cells (e.g. T cells), such as such cells that have been modified by a DNA-targeting system or polynucleotide(s) encoding the DNA-targeting system described herein, so that the subject has a reduction in at least one symptom of the disease or an improvement in the disease, for example, beneficial or desired clinical results. For purposes of121MF-36384587022474-20039.40 this technology, beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Treating can refer to prolonging survival as compared to expected survival if not receiving treatment. Thus, one of skill in the art realizes that a treatment may improve the disease condition, but may not be a complete cure for the disease. In some embodiments, one or more symptoms of a disease or disorder are alleviated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% upon treatment of the disease.

[0444] The term “therapeutically effective amount” refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term "therapeutically effective amount" includes that amount of a biological molecule, s...

Claims

22474-20039.40CLAIMSWHAT IS CLAIMED:

1. A method of treating chronic hepatitis B (CHB) infection in a subject in need thereof, the method comprising administering to the subject a dose of lipid nanoparticles comprising an epigenetic-modifying DNA-targeting system comprising a DNA-targeting module for repressing transcription of total viral Hepatitis B Viral (HBV) mRNA transcripts, wherein the DNA- targeting module comprises:(a) a gRNA for targeting to a target site in a HBV DNA sequence, wherein the target site comprises the sequence set forth in SEQ ID NO: 27, a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing; and(b) an mRNA encoding a fusion protein comprising a deactivated Streptococcus pyogenes Cas9 (dSpCas9) protein and at least one transcriptional repressor effector domain comprising a KRAB domain and a DNA methyltransferase 3 (DNMT3) domain that has DNA methyltransferase activity, and wherein the dose comprises (a) between at or about 0.1 mg / kg body weight of the subject and at or about 1.5 mg / kg body weight of the subject, or (b) between at or about 10 mg to about 100 mg.

2. The method of claim 1, wherein the subject has an HBV-associated viral infection.

3. The method of claim 2, wherein the HBV-associated viral infection is hepatitis D virus infection.

4. A method of treating hepatitis D virus (HDV) infection in a subject in need thereof, the method comprising administering to the subject a dose of lipid nanoparticles comprising an epigenetic-modifying DNA-targeting system comprising a DNA-targeting module for repressing transcription of total viral Hepatitis B Viral (HBV) mRNA transcripts, wherein the DNA- targeting module comprises:(a) a gRNA for targeting to a target site in a HBV DNA sequence, wherein the target site comprises the sequence set forth in SEQ ID NO: 27, a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing; and193MF-36384587022474-20039.40(b) an mRNA encoding a fusion protein comprising a deactivated Streptococcus pyogenes Cas9 (dSpCas9) protein and at least one transcriptional repressor effector domain comprising a KRAB domain and a DNA methyltransferase 3 (DNMT3) domain that has DNA methyltransferase activity, and wherein the dose comprises (a) between at or about 0.1 mg / kg body weight of the subject and at or about 1.5 mg / kg body weight of the subject, or (b) between at or about 10 mg to about 100 mg.

5. The method of any one of claims 1-4, wherein the dose comprises (a) between at or about 0.1 mg / kg body weight of the subject and at or about 1 mg / kg body weight of the subject, or (b) between at or about 10 mg to about 100 mg.

6. The method of any one of claims 1-5 wherein the target site comprises the sequence set forth in SEQ ID NO: 27.

7. The method of any one of claims 1-6, wherein the fusion protein comprising the KRAB domain comprises a K0X1 domain, a ZNF10 domain, or a ZIM3 domain.

8. The method of any one of claims 1-7, wherein the KRAB domain comprises a ZNF10 domain.

9. The method of any one of claims 1-8, wherein the KRAB domain comprises the sequence set forth in SEQ ID NO: 28, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 28.

10. The method of any one of claims 1-9, wherein the DNMT3 domain comprises a DNMT3A domain.

11. The method of claim 10, wherein the DNMT3 A comprises the sequence set forth in SEQ ID NO: 22, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 22.194MF-36384587022474-20039.4012. The method of claim 10 or claim 11, wherein the DNMT3A comprises the sequence set forth in SEQ ID NO: 22.

13. The method of any one of claims 1-12, wherein the DNMT3 domain comprises a DNMT3L domain or a variant thereof.

14. The method of claim 12, wherein the DNMT3 domain comprises the sequence set forth in SEQ ID NO: 23, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 23.

15. The method of claim 13 or claim 14, wherein the DNMT3 domain comprises the sequence set forth in SEQ ID NO: 23.

16. The method of any one of claims 1-15, wherein the DNMT3 domain comprises a DNMT3 A domain and a DNMT3L domain (DNMT3A / L domain).

17. The method of any one of claims 1-16, wherein the fusion protein comprises from the N-terminus to the C-terminus: a DNMT3 A domain and a DNMT3L domain (DNMT3A / L domain) , a dSpCas9 domain, and a KRAB domain.

18. The method of claim 16 or claim 17, wherein the DNMT3 domain is a DNMT3A / L domain and the DNMT3 A domain and the DNMT3L domain are separated by a linker, wherein the linker comprises the sequence set forth in SEQ ID NO: 34.

19. The method of any of claims 1-18, wherein the DNMT3 domain is a DNMT3A / L domain and comprises the sequence set forth in SEQ ID NO: 42 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 42.

20. The method of any one of claims 1-19, wherein the fusion protein comprises a sequence comprising at least 90% sequence identity to SEQ ID NO: 42 and, a sequence comprising at least 90% sequence identity to SEQ ID NO: 28.195MF-36384587022474-20039.4021. The method of any one of claims 1-20, wherein the fusion protein comprising the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO: 24.

22. The method of any one of claims 1-21, wherein the dSpCas9 protein comprises the sequence set forth in SEQ ID NO: 25, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 25.

23. The method of any one of claims 1-22, wherein the dSpCas9 protein comprises the sequence set forth in SEQ ID NO: 25.

24. The method of any one of claims 1-23, wherein the fusion protein further comprises one or more nuclear localization signals (NLS).

25. The method of claim 24, wherein the NLS is at the N- or C-terminus, between N- or C-terminus and the KRAB domain, between the N- or C-terminus and the DNMT3A / L domain, between the dSpCas9 and the DNMT3A / 3L domains, or between the dSpCas9 and the KRAB domain.

26. The method of claim 24 or claim 25, wherein the NLS comprises the sequence set forth in any one of SEQ ID NOS: 13, 16, 18, a portion thereof, or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the foregoing.

27. The method of any one of claims 24-26, wherein the NLS comprises the sequence set forth in any one of SEQ ID NOS: 13, 16, and 18.

28. The method of any one of claims 1-27, wherein fusion protein further comprises one or more linkers connecting two or more of: the DNMT3 A domain, the DNMT3L domain, the dSpCas9, the KRAB domain, and the one or more nuclear localization signals (NLS).

29. The method of claim 28, wherein the one or more linkers is independently selected from a linker comprising the sequence set forth in any one of SEQ ID NO: 11, 12, 14, 15, or 17.196MF-36384587022474-20039.4030. The method of any one of claims 1-29, wherein the mRNA further comprises one or more untranslated regions (UTR) near the 5’ end and / or the 3’ end, optionally a UTR near the 5’ end and near the 3’ end.

31. The method of claim 30, wherein the UTR near the 5’ end comprises a sequence set forth in SEQ ID NO: 9 or a sequence that has at least 90% sequence identity to SEQ ID NO:9 and the UTR near the 3’ end comprises a sequence set forth in SEQ ID NO: 10 or a sequence that has at least 90% sequence identity to SEQ ID NO: 10.

32. The method of any one of claims 1-31, wherein the mRNA further comprises a polyA sequence at the 3’ end.

33. The method of any of claims 1-32, wherein the mRNA comprises from 5’ end to the 3’ end: a UTR, a nucleotide sequence encoding the fusion proteina UTR, and a polyA sequence.

34. The method of any one of claims 1-33, wherein the mRNA encoding the fusion protein comprises from the 5’ end to the 3’ end: the UTR comprising the sequence set forth in SEQ ID NO: 9, a nucleotide sequence encoding the fusion protein, the UTR comprising the sequence set forth in SEQ ID NO: 10, and a polyA sequence comprising the sequence set forth in SEQ ID NO: 19.

35. The method of claim 33 or claim 34, wherein the nucleotide sequence encoding the fusion protein comprises, in order from the 5’ end to the 3’ end, nucleotides encoding: a DNMT3 A domain, a linker, the DNMT3L domain, a linker, a NLS, a linker, the dSpCas9, a linker, a NLS, a linker, the KRAB, and a NLS.

36. The method of any of claims 33-35, wherein the nucleotide sequence encoding the fusion protein comprises, in order from the 5’ end to the 3’ end: a nucleotide sequence encoding the DNMT3A domain comprising the sequence set forth in SEQ ID NO: 2, a nucleotide sequence encoding a linker comprising the sequence set forth in SEQ ID NO: 11, a nucleotide sequence encoding the DNMT3L domain comprising the sequence set forth in SEQ197MF-36384587022474-20039.40ID NO: 3, a nucleotide sequence encoding the linker comprising the sequence set forth in SEQ ID NO: 12, a nucleotide sequence encoding an NLS comprising the sequence set forth in SEQID NO: 13, a nucleotide sequence encoding a linker comprising the sequence set forth in SEQID NO: 14, a nucleotide sequence encoding the dSpCas9 comprising the sequence set forth inSEQ ID NO: 4, a nucleotide sequence encoding a linker comprising the sequence set forth inSEQ ID NO: 15, a nucleotide sequence encoding an NLS comprising the sequence set forth in SEQ ID NO: 16, a nucleotide sequence encoding the linker comprising the sequence set forth in SEQ ID NO: 17, a nucleotide sequence encoding the KRAB comprising the sequence set forth in SEQ ID NO: 5, and a nucleotide sequence encoding the NLS comprising the sequence set forth in SEQ ID NO: 18.

37. The method of any one of claims 1-36, wherein the mRNA comprises the sequence set forth in SEQ ID NO: 1, or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1.

38. A method of treating chronic hepatitis B (CHB) infection in a subject in need thereof, the method comprising administering to the subject a dose of lipid nanoparticles comprising an epigenetic-modifying DNA-targeting system comprising a DNA-targeting module for repressing total viral Hepatitis B Viral (HBV) mRNA transcripts, wherein the DNA- targeting module comprises:(a) a gRNA for targeting to a target site in a HBV DNA sequence, wherein the target site comprises the sequence set forth in SEQ ID NO: 27; and(b) an mRNA comprising from the 5’ end to the 3’ end a UTR comprising the sequence set forth in SEQ ID NO: 9, a nucleotide encoding a fusion protein, a UTR comprising the sequence set forth in SEQ ID NO: 10, and a polyA sequence comprising the sequence set forth in SEQ ID NO: 19, wherein the nucleotide sequence encoding the fusion protein comprises , in order from the 5’ end to the 3’ end: a nucleotide sequence encoding the DNMT3A domain comprising the sequence set forth in SEQ ID NO: 2, a nucleotide sequence encoding a linker comprising the sequence set forth in SEQ ID NO: 11, a nucleotide sequence encoding the DNMT3L domain comprising the sequence set forth in SEQ ID NO: 3, a nucleotide sequence encoding the linker comprising the sequence set forth in SEQ ID NO: 12, a nucleotide sequence encoding an NLS comprising the sequence set forth in SEQ ID NO: 13, a nucleotide sequence encoding a linker198MF-36384587022474-20039.40 comprising the sequence set forth in SEQ ID NO: 14, a nucleotide sequence encoding the dSpCas9 comprising the sequence set forth in SEQ ID NO: 4, a nucleotide sequence encoding a linker comprising the sequence set forth in SEQ ID NO: 15, a nucleotide sequence encoding an NLS comprising the sequence set forth in SEQ ID NO: 16, a nucleotide sequence encoding the linker comprising the sequence set forth in SEQ ID NO: 17, a nucleotide sequence encoding the KRAB comprising the sequence set forth in SEQ ID NO: 5, and a nucleotide sequence encoding the NLS comprising the sequence set forth in SEQ ID NO: 18, wherein the dose comprises (a) between at or about 0.1 mg / kg and at or about 1.5 mg / kg, or (b) between at or about 10 mg to about 100 mg.

39. A method of treating chronic hepatitis B (CHB) infection in a subject in need thereof, the method comprising administering to the subject a dose of lipid nanoparticles comprising (a) a gRNA for targeting to a target site in a HBV DNA sequence, wherein the target site comprises the sequence set forth in SEQ ID NO: 27; and (b) a mRNA comprising the sequence set forth in SEQ ID NO: 1 or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1, wherein the dose comprises (a) between at or about 0.1 mg / kg and at or about 1.5 mg / kg, or (b) between at or about 10 mg to about 100 mg.

40. The method of claim 38 or claim 39, wherein the subject has an HBV-associated viral infection.

41. The method of claim 40, wherein the HBV-associated viral infection is hepatitis D virus infection.

42. A method of treating a hepatitis D virus infection in a subject in need thereof, the method comprising administering to the subject a dose of lipid nanoparticles comprising an epigenetic-modifying DNA-targeting system comprising a DNA-targeting module for repressing total viral Hepatitis B Viral (HBV) mRNA transcripts, wherein the DNA-targeting module comprises:(a) a gRNA for targeting to a target site in a HBV DNA sequence, wherein the target site comprises the sequence set forth in SEQ ID NO: 27; and199MF-36384587022474-20039.40(b) an mRNA comprising from the 5’ end to the 3’ end a UTR comprising the sequence set forth in SEQ ID NO: 9, a nucleotide encoding a fusion protein, a UTR comprising the sequence set forth in SEQ ID NO: 10, and a polyA sequence comprising the sequence set forth in SEQ ID NO: 19, wherein the nucleotide sequence encoding the fusion protein comprises , in order from the 5’ end to the 3’ end: a nucleotide sequence encoding the DNMT3A domain comprising the sequence set forth in SEQ ID NO: 2, a nucleotide sequence encoding a linker comprising the sequence set forth in SEQ ID NO: 11, a nucleotide sequence encoding the DNMT3L domain comprising the sequence set forth in SEQ ID NO: 3, a nucleotide sequence encoding the linker comprising the sequence set forth in SEQ ID NO: 12, a nucleotide sequence encoding an NLS comprising the sequence set forth in SEQ ID NO: 13, a nucleotide sequence encoding a linker comprising the sequence set forth in SEQ ID NO: 14, a nucleotide sequence encoding the dSpCas9 comprising the sequence set forth in SEQ ID NO: 4, a nucleotide sequence encoding a linker comprising the sequence set forth in SEQ ID NO: 15, a nucleotide sequence encoding an NLS comprising the sequence set forth in SEQ ID NO: 16, a nucleotide sequence encoding the linker comprising the sequence set forth in SEQ ID NO: 17, a nucleotide sequence encoding the KRAB comprising the sequence set forth in SEQ ID NO: 5, and a nucleotide sequence encoding the NLS comprising the sequence set forth in SEQ ID NO: 18, wherein the dose comprises (a) between at or about 0.1 mg / kg and at or about 1.5 mg / kg, or (b) between at or about 10 mg to about 100 mg.

43. A method of treating a hepatitis D virus infection in a subject in need thereof, the method comprising administering to the subject a dose of lipid nanoparticles comprising (a) a gRNA for targeting to a target site in a HBV DNA sequence, wherein the target site comprises the sequence set forth in SEQ ID NO: 27; and (b) a mRNA comprising the sequence set forth in SEQ ID NO: 1 or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1, wherein the dose comprises (a) between at or about 0.1 mg / kg and at or about 1.5 mg / kg, or (b) between at or about 10 mg to about 100 mg.

44. The method of any one of claims 38-43, wherein the dose comprises (a) between at or about 0.1 mg / kg and at or about 1.0 mg / kg, or (b) between at or about 10 mg to about 100 mg.200MF-36384587022474-20039.4045. The method of any of claims 1-44, wherein the mRNA comprises the sequence set forth in SEQ ID NO: 1.

46. The method of any one of claims 1-45, wherein the gRNA for targeting to the target site comprises a spacer sequence comprising the sequence set forth in SEQ ID NO: 26, a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing.

47. The method of claim 46, wherein the spacer sequence is 17 to 24 nucleotides in length, optionally at or about 20 nucleotides in length.

48. The method of any one of claims 1-47, wherein the gRNA comprises a spacer sequence for targeting to the target site that comprises the sequence set forth in SEQ ID NO: 26.

49. The method of any one of claims 1-48, wherein the gRNA comprises a spacer sequence for targeting to the target site that is set forth in SEQ ID NO: 26.

50. The method of any one of claims 1-49, wherein the gRNA comprises a scaffold sequence for dSpCas9.

51. The method of claim 50, wherein the scaffold sequence comprises a sequence set forth in SEQ ID NO: 39 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 39.

52. The method of claim 50 or claim 51, wherein the scaffold sequence is set forth in SEQ ID NO: 39.

53. The method of any of claims 1-52, wherein the gRNA for targeting to the target site comprises the sequence set forth in SEQ ID NO: 38 or a complementary sequence of the foregoing.

54. The method of any one of claims 1-53, wherein the gRNA for targeting to the target site is set forth in SEQ ID NO: 38.201MF-36384587022474-20039.4055. The method of any one of claims 1-54, wherein the lipid nanoparticles comprise a cationic lipid having the following structure (I):or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein:R1is optionally substituted C1-C24 alkyl or optionally substituted C2-C24 alkenyl;R2and R3are each independently optionally substituted C1-C36 alkyl;R4and R5are each independently optionally substituted Ci-Ce alkyl, or R4and R5join, along with the N to which they are attached, to form a heterocyclyl or heteroaryl;L1, L2, and L3are each independently optionally substituted Ci-Cis alkylene;G1is a direct bond, -(CH2)nO(C=O)-, -(CH2)n(C=O)O-, or -(C=O)-;G2and G3are each independently -(C=O)O- or -O(C=O)-; and n is an integer greater than 0.

56. The method of claim 55, wherein the cationic lipid is selected from a compound in Table 1.

57. The method of claim 55 or claim 56, wherein the cationic lipid is 1-18.

58. The method of any one of claims 1-57, wherein the lipid nanoparticles comprise a pegylated lipid having the following structure (II):or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted202MF-36384587022474-20039.40 by one or more ester bonds; and z has a mean value ranging from 30 to 60.

59. The method of claim 58, wherein the pegylated lipid is Ila.

60. The method of any one of claims 1-59, wherein the lipid nanoparticles comprises: a cationic lipid that is bis(2 -butyloctyl) 10-(N-decyl-4- (dimethylamino)butanamido)nonadecanedioate, l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), a PEGylated lipid that is 2-[2-(co-methoxy (polyethyleneglycol2000) ethoxy]-N,N-ditetradecylacetamide, and cholesterol, wherein the cationic lipid / DSPC / PEGylated lipid / cholesterol are formulated in a ratio of 47.5 / 10 / 2.5 / 40 mol %.

61. The method of any of claims 55-60, wherein the ratio of cationic lipid to mRNA (N / P) is 6.0.

62. The method of claim 1-61, wherein the total RNA in the lipid nanoparticle is 1 mg.

63. The method of any of claims 1-62, wherein the ratio of the mRNA encoding the fusion protein and the gRNA is 2: 1.

64. The method of any of claims 1-63, wherein the lipid nanoparticle is formulated in 300 mM sucrose in phosphate buffered saline at pH about 7.0 to about 7.4.

65. The method of any one of claims 1-64, wherein the dose is an amount between at or about 0.1 mg / kg body weight of the subject and about 1 mg / kg body weight of the subject.

66. The method of any one of claims 1-65, wherein the dose is an amount between at or about 0.15 mg / kg body weight of the subject and about 0.8 mg / kg body weight of the subject, between at or about 0.3 mg / kg body weight of the subject and at or about 0.65 mg / kg body weight of the subject, or between at or about 0.2 mg / kg body weight of the subject and at or about 0.45 mg / kg body weight of the subject.203MF-36384587022474-20039.4067. The method of any one of claims 1- 66, wherein the dose is at or about 0.2 mg / kg body weight of the subject.

68. The method of any one of claims 1-66, wherein the dose is at or about 0.45 mg / kg body weight of the subject.

69. The method of any one of claims 1-66, wherein the dose is at or about 0.65 mg / kg body weight of the subject.

70. The method of any one of claims 1-66, wherein the dose is greater than 0.65 mg / kg body weight of the subject and less than 100 mg / kg body weight of the subject.

71. The method of any one of claims 1-70, wherein if the subject is 100 kg or greater then the dose is no more than 100 mg.

72. The method of any one of claims 1-64, wherein the dose is an amount between about 10 to about 100 mg.

73. The method any one of claims 1-64 and 72, wherein the dose is an amount from at or about 12 mg to about 52 mg, from at or about 12 mg to about 39 mg, from at or about 12 mg to about 36 mg, from at or about 12 mg to about 27 mg, from at or about 12 mg to at or about 16 mg, from at or about 27 mg to at or about 100 mg, from at or about 27 mg to about 39 mg or from at or about 52 mg to at our about 100 mg.

74. The method of any one of claims 1-73, wherein the dose is from about 75 mg to about 90 mg.

75. The method of any one of claims 1-74, wherein a single dose of the lipid nanoparticles is administered to the subject.

76. The method of any one of claims 1-74, comprising administering two or more doses of the lipid nanoparticles to the subject as part of a multiple-dose regimen.204MF-36384587022474-20039.4077. The method of claim 76, wherein two, three, four, five, or six doses of the lipid nanoparticles are administered to the subject.

78. The method of claim 76 or claim 77, wherein the multipe-dose regimen is a finite multiple-dosing regimen in which a finite number of discrete doses are administered over a defined treatment period.

79. The method of claim 78, wherein the finite multiple-dosing regimen comprises two, three, four, five, or six total doses.

80. The method of any one of claims 76-79, wherein each dose of the multiple-dose regimen independently comprises a dosage amount or range as recited in any one of claims 56- 64.

81. The method of any one of claims 76-80, wherein each dose of the multiple-dose regimen independently is from about 0.1 to about 1 mg / kg, optionally about 0.2 mg / kg, 0.45 mg / kg, or 0.65 mg / kg.

82. The method of any one of claims 76-80, wherein each dose of the multiple-dose regimen is a fixed amount from about 10 to 100 mg, optionally about 12 mg, 27 mg, 39 mg, 52 mg, or 100 mg, or a value between any of the foregoing.

83. The method of any one of claims 76-81, wherein the multiple-dose regimen comprises 2 to 6 doses administered once every 14 to 60 days.

84. The method of any one of claims 76-83, wherein each dose of the multiple-dose regimen is administered once every 21 to 42 days.

85. The method of any one of claims 76-84, wherein the each dose of the multiple-dose regimen is administered every two weeks (Q2W), once every 28 days (Q4W), or once every six weeks (Q6W).205MF-36384587022474-20039.4086. The method of any one of claims 76-85, wherein the multiple-dose regimen is selected from three doses once every 28 days (Q4Wx3), four doses once every 28 days (Q4Wx4), three doses once every 21 days (Q3Wx3), or four doses once every six weeks (Q6Wx4).

87. The method of any one of claims 76-86, wherein the multiple-dose regimen comprises three doses once every 28 days (Q4Wx3).

88. The method of any one of claims 1-87, wherein the lipid nanoparticles are administered via intravenous administration.

89. The method of any one of claims 1-88, wherein the subject is a human.

90. The method of any one of claims 1-89, wherein the subject is an adult.

91. The method of any one of claims 1-89, wherein the subject is 12 years or older.

92. The method of any one of claims 1-91, wherein the subject has a weight of at least 40 kg.

93. The method of any one of claims 1-92, wherein the subject has hepatocytes comprising cccDNA and / or integrated HBV DNA.

94. The method of any one of claims 1-93, wherein the subject has hepatocytes comprising cccDNA and integrated HBV DNA.

95. The method of any one of claims 1-94, wherein the subject has hepatocytes expressing HBV proteins, wherein the HBV proteins are HBsAg, HBeAg, or HBcrAg and combinations thereof.

96. The method of any one of claims 1-95, wherein the subject has been diagnosed as positive for HBsAg for at least about 12 months.206MF-36384587022474-20039.4097. The method of any one of claims 1-96, wherein the subject has an HBsAg titer of at least about 500 lU / mL.

98. The method of any one of claims 1-97, wherein the subject has received a treatment with a nucleoside analog for at least about 12 months.

99. The method of claim 98, wherein the nucleoside analog comprises one or more of entecavir, tenofovir disoproxil fumarate, or tenofovir alafenamide100. The method of claim 98 or claim 99, wherein the subject is receiving the nucleoside analog at the time of administration of the lipid nanoparticle and continues to receive the nucleoside analog.

101. The method of claim 98 or claim 99, wherein the treatment with the nucleoside analog is discontinued at the time of administration of the lipid nanoparticle.

102. The method of any one of claims 1-101, wherein the subject has or has been diagnosed as being positive for HBV DNA levels of less than about 90 U / mL for at least 6 months.

103. The method of any one of claims 1-102 wherein, the subject(e) has or has been diagnosed with chronic Hepatitis B (CHB) defined as HBsAg positive for 12 months or more than 12 months;(f) has baseline titers of HBsAg of more than 500 lU / ml;(g) has received treatment with a nucleoside analog, wherein the nucleoside analog is entecavir (ETV), tenofovir disoproxil fumarate (TDF) or tenofovir alafenamide (TAF) for 12 months of more than 12 months with a stable dose for 6 months or more than 6 months; and(h) has HBV DNA of less than 90 lU / ml for 6 months or less than 6 months.

104. The method of any one of claims 1-103, wherein the method reduces the levels of Hepatitis B virus in the subject.207MF-36384587022474-20039.40105. The method of any one of claims 1-104, wherein the method reduces Hepatitis B virus RNA and / or protein levels in the subject.

106. The method of any one of claims 1-105, wherein the expression of HBV DNA and / or pre-genomic (pgRNA) is reduced.

107. The method of any one of claims 1-106, wherein the levels of HBsAg, HBeAg, HBcrAg, and / or HBeAg and / or p-HBcAg are reduced.

108. The method of any one of claims 1-107, wherein the levels of stiffness and / or fat are changed.

109. The method of any one of claims 1-108, wherein the levels of anti-HBsAg, anti- HBcAg, and / or anti-HBeAg antibodies and / or HBV-specific PBMC immune markers are reduced.

110. A lipid nanoparticle comprising an epigenetic-modifying DNA-targeting system comprising a DNA-targeting module for repressing total viral Hepatitis B Viral (HBV) mRNA transcripts, wherein: the DNA-targeting module comprises:(a) a gRNA for targeting to a target site in a HBV DNA sequence, wherein the target site comprises the sequence set forth in SEQ ID NO: 27, a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing; and(b) an mRNA encoding a fusion protein comprising a deactivated Streptococcus pyogenes Cas9 (dSpCas9) protein and at least one transcriptional repressor effector domain comprising a KRAB domain and a DNA methyltransferase 3 (DNMT3) domain that has DNA methyltransferase activity; and wherein the lipid nanoparticles comprise a cationic lipid having the following structure (I):208MF-36384587022474-20039.40or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein:R1is optionally substituted C1-C24 alkyl or optionally substituted C2-C24 alkenyl;R2and R3are each independently optionally substituted C1-C36 alkyl;R4and R5are each independently optionally substituted Ci-Ce alkyl, or R4and R5join, along with the N to which they are attached, to form a heterocyclyl or heteroaryl;L1, L2, and L3are each independently optionally substituted Ci-Cis alkylene;G1is a direct bond, -(CH2)nO(C=O)-, -(CH2)n(C=O)O-, or -(C=O)-;G2and G3are each independently -(C=O)O- or -O(C=O)-; and n is an integer greater than 0.

111. The lipid nanoparticle of claim 110 wherein the target site comprises the sequence set forth in SEQ ID NO: 27.

112. The lipid nanoparticle of claim 110 or claim 111, wherein the fusion protein comprising the KRAB domain comprises a KOX1 domain, a ZNF10 domain, or a ZIM3 domain.

113. The lipid nanoparticle of any one of claims 110-112, wherein the KRAB domain comprises a ZNF10 domain.

114. The lipid nanoparticle of any one of claims 110-113, wherein the KRAB domain comprises the sequence set forth in SEQ ID NO: 28, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO :28.

115. The lipid nanoparticle of any one of claims 110-114, wherein the DNMT3 domain comprises a DNMT3 A domain.209MF-36384587022474-20039.40116. The lipid nanoparticle of claim 115, wherein the DNMT3A comprises the sequence set forth in SEQ ID NO: 22, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 22.

117. The lipid nanoparticle of claim 113 or claim 116, wherein the DNMT3A comprises the sequence set forth in SEQ ID NO: 22.

118. The lipid nanoparticle of any one of claims 110-117, wherein the DNMT3 domain comprises a DNMT3L domain or a variant thereof.

119. The lipid nanoparticle of claim 118, wherein the DNMT3 domain comprises the sequence set forth in SEQ ID NO: 23, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 23.

120. The lipid nanoparticle of claim 118 or claim 119, wherein the DNMT3 domain comprises the sequence set forth in SEQ ID NO: 23.

121. The lipid nanoparticle of any one of claims 110-120, wherein the DNMT3 domaincomprises a DNMT3 A domain and a DNMT3L domain (DNMT3 A / L domain).

122. The lipid nanoparticle of any one of claims 110-121, wherein the fusion protein comprises from the N-terminus to the C-terminus: a DNMT3 A domain and a DNMT3L domain (DNMT3A / L domain), a dSpCas9 domain, and a KRAB domain.

123. The lipid nanoparticle of claim 121 or claim 122, wherein the DNMT3 domain is a DNMT3A / L domain and the DNMT3 A domain and the DNMT3L domain are separated by a linker, wherein the linker comprises the sequence set forth in SEQ ID NO: 34.

124. The lipid nanoparticle of any of claims 110-123, wherein the DNMT3 domain is a DNMT3A / L domain and comprises the sequence set forth in SEQ ID NO: 42 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 42.210MF-36384587022474-20039.40125. The lipid nanoparticle of any one of claims 110-124, wherein the fusion protein comprises a sequence comprising at least 90% sequence identity to SEQ ID NO: 42 and, a sequence comprising at least 90% sequence identity to SEQ ID NO: 28.

126. The lipid nanoparticle of any one of claims 110-125, wherein the fusion protein comprising the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO: 24.

127. The lipid nanoparticle of any one of claims 110-126, wherein the dSpCas9 protein comprises the sequence set forth in SEQ ID NO: 25, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 25.

128. The lipid nanoparticle of any one of claims 110-127, wherein the dSpCas9 protein comprises the sequence set forth in SEQ ID NO: 25.

129. The lipid nanoparticle of any one of claims 110-128, wherein fusion protein further comprises one or more nuclear localization signals (NLS).

130. The lipid nanoparticle of claim 129, wherein the NLS is at the N- or C-terminus, between N- or C-terminus and the KRAB domain, between the N- or C-terminus and the DNMT3A / L domain, between the dSpCas9 and the DNMT3A / 3L domains, or between the dSpCas9 and the KRAB domain.

131. The lipid nanoparticle of claim 129 or claim 130, wherein the NLS comprises the sequence set forth in any one of SEQ ID NOS: 13, 16, 18, a portion thereof, or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the foregoing.

132. The lipid nanoparticle of any one of claims 129-131, wherein the NLS comprises the sequence set forth in any one of SEQ ID NOS: 13, 16, and 18.211MF-36384587022474-20039.40133. The lipid nanoparticle of any one of claims 110-132, wherein fusion protein further comprises one or more linkers connecting two or more of: the DNMT3A domain, the DNMT3L domain, the dSpCas9, the KRAB domain, and the one or more nuclear localization signals.

134. The lipid nanoparticle of claim 133, wherein the one or more linkers is independently selected from a linker comprising the sequence set forth in any one of SEQ ID NO: 11, 12, 14, 15, or 17.

135. The lipid nanoparticle of any one of claims 110-134, wherein the mRNA further comprises one or more untranslated regions (UTR) near the 5’ end and / or the 3’ end, optionally a UTR near the 5’ end and near the 3 ’ end.

136. The lipid nanoparticle of claim 135, wherein the UTR near the 5’ end comprises a sequence set forth in SEQ ID NO: 9 or a sequence that has at least 90% sequence identity to SEQ ID NO: 9 and the UTR near the 3’ end comprises a sequence set forth in SEQ ID NO: 10 or a sequence that has at least 90% sequence identity to SEQ ID NO: 10.

137. The lipid nanoparticle of any one of claims 110-136, wherein the mRNA further comprises a polyA sequence at the 3’ end.

138. The lipid nanoparticle of any of claims 110-137, wherein the mRNA comprises from 5’ end to the 3’ end: a UTR, a nucleotide sequence encoding the fusion protein a UTR, and a polyA sequence.

139. The lipid nanoparticle of any one of claims 110-138, wherein the mRNA encoding the fusion protein comprises from the 5’ end to the 3’ end: the UTR comprising the sequence set forth in SEQ ID NO: 9, a nucleotide sequence encoding the fusion protein, the UTR comprising the sequence set forth in SEQ ID NO: 10, and a polyA sequence comprising the sequence set forth in SEQ ID NO: 19.

140. The lipid nanoparticle of claim 138 or claim 139, wherein the nucleotide sequence encoding the fusion protein comprises, in order from the 5’ end to the 3’ end,212MF-36384587022474-20039.40 nucleotides encoding: a DNMT3 A domain, a linker, the DNMT3L domain, a linker, a NLS, a linker, the dSpCas9, a linker, a NLS, a linker, the KRAB, and a NLS.

141. The lipid nanoparticle of any of claims 138-140, wherein the nucleotide sequence encoding the fusion protein comprises, in order from the 5’ end to the 3’ end: a nucleotide sequence encoding the DNMT3A domain comprising the sequence set forth in SEQ ID NO: 2, a nucleotide sequence encoding a linker comprising the sequence set forth in SEQ ID NO: 11, a nucleotide sequence encoding the DNMT3L domain comprising the sequence set forth in SEQ ID NO: 3, a nucleotide sequence encoding the linker comprising the sequence set forth in SEQ ID NO: 12, a nucleotide sequence encoding an NLS comprising the sequence set forth in SEQID NO: 13, a nucleotide sequence encoding a linker comprising the sequence set forth in SEQID NO: 14, a nucleotide sequence encoding the dSpCas9 comprising the sequence set forth inSEQ ID NO: 4, a nucleotide sequence encoding a linker comprising the sequence set forth inSEQ ID NO: 15, a nucleotide sequence encoding an NLS comprising the sequence set forth in SEQ ID NO: 16, a nucleotide sequence encoding the linker comprising the sequence set forth in SEQ ID NO: 17, a nucleotide sequence encoding the KRAB comprising the sequence set forth in SEQ ID NO: 5, and a nucleotide sequence encoding the NLS comprising the sequence set forth in SEQ ID NO: 18.

142. The lipid nanoparticle of any one of claims 110-141, wherein the mRNA comprises the sequence set forth in SEQ ID NO: 1, or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1.

143. A lipid nanoparticle comprising an epigenetic-modifying DNA-targeting system comprising a DNA-targeting module for repressing total viral Hepatitis B Viral (HBV) mRNA transcripts, wherein: the DNA-targeting module comprises:(a) a gRNA for targeting to a target site in a HBV DNA sequence, wherein the target site comprises the sequence set forth in SEQ ID NO: 27; and(b) an mRNA comprising from the 5’ end to the 3’ end: a UTR comprising the sequence set forth in SEQ ID NO: 9, a nucleotide encoding a fusion protein, a UTR comprising the sequence set forth in SEQ ID NO: 10, and a polyA sequence comprising the sequence set forth in SEQ ID NO: 19, wherein the nucleotide sequence encoding the fusion protein comprises , in213MF-36384587022474-20039.40 order from the 5’ end to the 3’ end: a nucleotide sequence encoding the DNMT3A domain comprising the sequence set forth in SEQ ID NO: 2, a nucleotide sequence encoding a linker comprising the sequence set forth in SEQ ID NO: 11, a nucleotide sequence encoding the DNMT3L domain comprising the sequence set forth in SEQ ID NO: 3, a nucleotide sequence encoding the linker comprising the sequence set forth in SEQ ID NO: 12, a nucleotide sequence encoding an NLS comprising the sequence set forth in SEQ ID NO: 13, a nucleotide sequence encoding a linker comprising the sequence set forth in SEQ ID NO: 14, a nucleotide sequence encoding the dSpCas9 comprising the sequence set forth in SEQ ID NO: 4, a nucleotide sequence encoding a linker comprising the sequence set forth in SEQ ID NO: 15, a nucleotide sequence encoding an NLS comprising the sequence set forth in SEQ ID NO: 16, a nucleotide sequence encoding the linker comprising the sequence set forth in SEQ ID NO: 17, a nucleotide sequence encoding the KRAB comprising the sequence set forth in SEQ ID NO: 5, and a nucleotide sequence encoding the NLS comprising the sequence set forth in SEQ ID NO: 18, and wherein the lipid nanoparticles comprise a cationic lipid having the following structure (I):or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein:R1is optionally substituted C1-C24 alkyl or optionally substituted C2-C24 alkenyl;R2and R3are each independently optionally substituted C1-C36 alkyl;R4and R5are each independently optionally substituted Ci-Ce alkyl, or R4and R5join, along with the N to which they are attached, to form a heterocyclyl or heteroaryl;L1, L2, and L3are each independently optionally substituted Ci-Cis alkylene;G1is a direct bond, -(CH2)nO(C=O)-, -(CH2)n(C=O)O-, or -(C=O)-;G2and G3are each independently -(C=O)O- or -O(C=O)-; and n is an integer greater than 0.214MF-36384587022474-20039.40144. A lipid nanoparticle comprising an epigenetic-modifying DNA-targeting system comprising a DNA-targeting module for repressing total viral Hepatitis B Viral (HBV) mRNA transcripts, wherein the DNA-targeting module comprises (a) a gRNA for targeting to a target site in a HBV DNA sequence, wherein the target site comprises the sequence set forth in SEQ ID NO: 27; and (b) a mRNA comprising the sequence set forth in SEQ ID NO: 1 or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1, and wherein the lipid nanoparticles comprise a cationic lipid having the following structure (I):or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein:R1is optionally substituted C1-C24 alkyl or optionally substituted C2-C24 alkenyl;R2and R3are each independently optionally substituted C1-C36 alkyl;R4and R5are each independently optionally substituted Ci-Ce alkyl, or R4and R5join, along with the N to which they are attached, to form a heterocyclyl or heteroaryl;L1, L2, and L3are each independently optionally substituted Ci-Cis alkylene;G1is a direct bond, -(CH2)nO(C=O)-, -(CH2)n(C=O)O-, or -(C=O)-;G2and G3are each independently -(C=O)O- or -O(C=O)-; and n is an integer greater than 0.

145. The lipid nanoparticle of any of claims 110-144, wherein the mRNA comprises the sequence set forth in SEQ ID NO: 1.

146. The lipid nanoparticle of any one of claims 110-145, wherein the gRNA for targeting to the target site comprises a spacer sequence comprising the sequence set forth in SEQ ID NO: 26, a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing.215MF-36384587022474-20039.40147. The lipid nanoparticle of claim 146, wherein the spacer sequence is 17 to 24 nucleotides in length, optionally at or about 20 nucleotides in length.

148. The lipid nanoparticle of any one of claims 110-147, wherein the gRNA comprises a spacer sequence for targeting to the target site that comprises the sequence set forth in SEQ ID NO: 26.

149. The lipid nanoparticle of any one of claims 110-148, wherein the gRNA comprises a spacer sequence for targeting to the target site that is set forth in SEQ ID NO: 26.

150. The lipid nanoparticle of any of claims 110-149, wherein the gRNA comprises a scaffold sequence for dSpCas9.

151. The lipid nanoparticle of claim 150, wherein the scaffold sequence comprises a sequence set forth in SEQ ID NO: 39 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 39.

152. The lipid nanoparticle of claim 150 or claim 151, wherein the scaffold sequence is set forth in SEQ ID NO: 39.

153. The lipid nanoparticle of any of claims 110-152, wherein the gRNA for targeting to the target site comprises the sequence set forth in SEQ ID NO: 38 or a complementary sequence of the foregoing.

154. The lipid nanoparticle of any one of claims 110-153, wherein the gRNA for targeting to the target site is set forth in SEQ ID NO: 38.

155. The lipid nanoparticle of any of claims 110-154, wherein the cationic lipid is selected from a compound in Table 1.

156. The lipid nanoparticle of any of claims 110-155, wherein the cationic lipid is I- 18.216MF-36384587022474-20039.40157. The lipid nanoparticle of any one of claims 110-156, wherein the lipid nanoparticles comprise a pegylated lipid having the following structure (II):or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:R10and R11are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and z has a mean value ranging from 30 to 60.

158. The lipid nanoparticle of claim 157, wherein the pegylated lipid is Ila.

159. The lipid nanoparticle of any of claims 110-158, comprising a cationic lipid that is bis(2 -butyloctyl) 10-(N-decyl-4-(dimethylamino)butanamido)nonadecanedioate, l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), a PEGylated lipid that is 2-[2-(co-methoxy (polyethyleneglycol2000) ethoxy]-N,N-ditetradecylacetamide, and cholesterol, wherein the cationic lipid / DSPC / PEGylated lipid / cholesterol are formulated in a ratio of 47.5 / 10 / 2.5 / 40 mol %.

160. The lipid nanoparticle of any of claims 110-159, wherein ratio of cationic lipid to mRNA (N / P) is 6.0.

161. The lipid nanoparticle of any of claims 110-160, wherein the total RNA in the lipid nanoparticle is 1 mg.

162. The lipid nanoparticle of any of claims 110-161, wherein the ratio of the mRNA encoding the fusion protein and the sgRNA is 2: 1.

163. The lipid nanoparticle of any of claims 110-162, wherein the lipid nanoparticle is formulated in 300 mM sucrose in phosphate buffered saline at pH about 7.0 to about 7.4.217MF-363845870

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