Compositions, systems, and methods for modulation of hepatitis B virus by targeted gene silencing

Abstract

Provided herein is an epigenetic modifying DNA targeting system, such as a CRISPR-Cas / guide RNA (gRNA) system, for transcriptional suppression of hepatitis B virus (HBV) genes to promote a cellular phenotype conducive to reduced HBV infection. In some embodiments, the epigenetic modifying DNA targeting system binds to or targets a target site of at least one gene or its regulatory element within the hepatitis B virus DNA sequence in a cell. In some aspects, the provided system relates to transcriptional suppression of one or more hepatitis B virus genes and / or their regulatory elements. In some aspects, methods and uses related to the provided compositions are also provided herein, for example, methods and uses in suppressing the replication and expression of hepatitis B virus associated with hepatitis B infection. TIFF2025527567000256.tif89170

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 63 / 399,634, filed August 19, 2022, entitled "COMPOSITIONS, SYSTEMS, AND METHODS FOR REGULATION OF HEPATITIS B VIRUS THROUGH TARGETED GENE REPRESSION," U.S. Provisional Patent Application No. 63 / 472,236, filed June 9, 2023, entitled "COMPOSITIONS, SYSTEMS, AND METHODS FOR REGULATION OF HEPATITIS B VIRUS THROUGH TARGETED GENE REPRESSION," and U.S. Provisional Patent Application No. 63 / 472,236, filed August 7, 2023, entitled "COMPOSITIONS, SYSTEMS, AND METHODS FOR REGULATION OF HEPATITIS B VIRUS THROUGH TARGETED GENE REPRESSION." This application claims priority to U.S. Provisional Patent Application No. 63 / 531,309, entitled "PATENT REPRESSION," the contents of which are incorporated by reference in their entirety.

[0002] INCORPORATION-BY-REFERENCE TO SEQUENCE LISTING This application is filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file with the name 224742002040SeqList.xml, created on August 18, 2023, and having a size of 1,595,298 bytes. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

[0003] Field In some aspects, the present disclosure relates to an epigenetic modifying DNA targeting system, such as a CRISPR-Cas / guide RNA (gRNA) system, for transcriptional suppression of hepatitis B virus (HBV) genes to promote a cellular phenotype conducive to reduced HBV infection. In some embodiments, the epigenetic modifying DNA targeting system binds to or targets a target site of at least one gene or its regulatory element within the hepatitis B virus DNA sequence in a cell. In some aspects, the system is a multiplexed system that binds to or targets target sites within at least two genes or their regulatory elements. In some aspects, the system of the present disclosure relates to transcriptional suppression of one or more hepatitis B virus genes. In some aspects, the present disclosure is directed to methods and uses related to the provided compositions, for example, in suppressing hepatitis B virus replication and expression associated with the treatment of hepatitis B infection. [Background technology]

[0004] background A large patient population, estimated to be 1 million in the United States alone and 250 million worldwide, is suffering from chronic hepatitis B infection. However, current standard treatments, including the administration of nucleoside analogues, PEGylated interferon, antisense oligonucleotides, and siRNA approaches to inhibit viral DNA transcription, 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 of the Invention

[0005] overview Provided herein is an epigenetic modifying DNA targeting system for suppressing transcription of one or more Hepatitis B Virus (HBV) genes, comprising at least one DNA targeting module, wherein each of the at least one DNA targeting module comprises a fusion protein comprising (a) a DNA-binding domain for targeting to a target site within a Hepatitis B Virus DNA sequence, and (b) at least one transcription repressor effector domain. In some embodiments, the at least one DNA-binding domain comprises a Cas-gRNA combination comprising (a) a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-associated (Cas) protein or a variant thereof and (b) at least one guide RNA (gRNA); a zinc finger protein (ZFP); a transcription activator-like effector (TALE); a meganuclease; a homing endonuclease; or an I-SceI enzyme or a variant thereof, and optionally, the DNA-binding domain comprises a catalytically inactive variant of any of the foregoing. In some embodiments, the Hepatitis B virus DNA sequence is an HBV gene or a regulatory element thereof. In some embodiments, the at least one DNA targeting module comprises multiple DNA targeting modules for targeting multiple target sites in one or more genes or regulatory elements thereof.In some embodiments, the plurality of DNA targeting modules comprises at least a first DNA targeting module and a second DNA targeting module, wherein (1) the first DNA targeting module represses transcription of a first HBV gene, the first DNA targeting module comprising a first fusion protein comprising (a) a DNA binding domain for targeting a target site of the first gene or its regulatory DNA element, and (b) at least one transcription repressor domain; and (2) the second DNA targeting module represses transcription of a second HBV gene, the second DNA targeting module comprising (a) a DNA binding domain for targeting a target site of the second gene or its regulatory DNA element. and (b) a second fusion protein comprising at least one transcriptional repressor domain, wherein optionally, the first DNA targeting module and the second DNA targeting module share the same fusion protein, such that the first and second fusion proteins are the same, and the DNA binding domain of the fusion protein is a CRISPR-associated (Cas) protein or a variant thereof; the first DNA targeting module comprises a first guide RNA (gRNA) that targets a target site in a first HBV gene or a regulatory element thereof, and the second DNA targeting module comprises a second gRNA that targets a target site in a second HBV gene or a regulatory element thereof.

[0006] Also provided herein is an epigenetic modifying DNA targeting system for suppressing transcription of one or more hepatitis B virus (HBV) genes, wherein the DNA targeting system comprises: (a) a fusion protein comprising a CRISPR-associated (Cas) protein or a variant thereof and at least one transcriptional repressor effector domain; and (b) a plurality of guide RNAs (gRNAs) comprising at least a first gRNA and a second gRNA, wherein the first gRNA targets a target site in a first HBV gene or its regulatory element, and the second gRNA targets a target site in a second HBV gene or its regulatory element, wherein the first and second genes or their regulatory elements regulate hepatitis B virus replication and / or HBV transcription. In some embodiments, the DNA targeting system further comprises a third gRNA that targets a target site in a third gene or its regulatory element that regulates hepatitis B virus replication and / or HBV transcription. In some embodiments, the system further includes a fourth gRNA targeting a target site in a fourth gene or its regulatory element, optionally a fifth gRNA targeting a fifth gene or its regulatory element, and / or optionally a sixth gRNA targeting a target site in a sixth gene or its regulatory element, wherein the gene or its regulatory element regulates Hepatitis B virus replication and / or HBV transcription. In some embodiments, the first, second, third, fourth, fifth, and / or sixth gene or its regulatory element are different.

[0007] Also provided herein is an epigenetic modifying DNA targeting system for suppressing transcription of one or more Hepatitis B Virus (HBV) genes, wherein the DNA targeting system comprises: (a) a fusion protein comprising a CRISPR-associated (Cas) protein or a variant thereof and at least one transcriptional repressor effector domain; and (b) multiple guide RNAs (gRNAs) that target multiple target sites in multiple genes or regulatory elements thereof, wherein the multiple genes or regulatory elements thereof regulate Hepatitis B Virus replication and / or HBV transcription.

[0008] Also provided herein is an epigenetic modifying DNA targeting system for suppressing transcription of more than one Hepatitis B Virus (HBV) gene, comprising a single DNA targeting module, wherein the DNA targeting module comprises: (a) a fusion protein comprising a CRISPR-associated (Cas) protein or a variant thereof and at least one transcriptional repressor effector domain; and (b) guide RNAs (gRNAs) that target multiple target sites in multiple genes or regulatory elements thereof, wherein the multiple genes or regulatory elements thereof regulate Hepatitis B Virus replication and / or HBV transcription.

[0009] In any of the embodiments herein, inhibiting transcription results in reduced replication of HBV and / or reduced levels of HBV proteins.

[0010] In any of the embodiments herein, the DNA targeting system does not introduce gene disruption or DNA breaks.

[0011] In any of the embodiments herein, at least one DNA binding module comprises multiple DNA binding modules that together target multiple target sites within the HBV DNA sequence, and optionally, each DNA binding module targets a different target site within the HBV DNA sequence.

[0012] In any of the embodiments herein, the multiple target sites are 2, 3, 4, 5, or 6 different target sites. In any of the embodiments herein, the multiple target sites are each within a different HBV gene or regulatory element thereof.

[0013] In any of the embodiments herein, each target site is within the same HBV gene or regulatory element thereof.

[0014] In any of the embodiments herein, the system comprises 2 to 10 DNA targeting modules.

[0015] In any of the embodiments herein, any two or more of the DNA targeting modules share the same fusion protein, or any two or more of the DNA targeting modules comprise different fusion proteins.

[0016] In any of the embodiments herein, the DNA-binding domain of each DNA-targeting module comprises a fusion protein comprising a CRISPR-associated (Cas) protein or a variant thereof and at least one transcriptional repressor effector domain, wherein each DNA-targeting module comprises a unique gRNA.

[0017] In any of the embodiments herein, the or each of the target sites is in HBV viral DNA that exists in the form of a covalently closed circular DNA (cccDNA), an incomplete double-stranded DNA (rcDNA), and / or is integrated into human genomic DNA. In any of the embodiments herein, the or each of the target sites is located in or near a gene or a regulatory element thereof that is involved in controlling HBV replication and / or HBV transcription.

[0018] In any of the embodiments herein, the gene involved in controlling HBV replication and / or transcription encodes a polymerase, an envelope protein, a capsid protein, a transcription factor, or a transcriptional transactivator. In any of the embodiments herein, the gene involved in controlling HBV replication and / or transcription is a polymerase gene, an S-family gene, an X-gene, or a core family gene.

[0019] In any of the embodiments herein, at least one target site is within the gene or regulatory elements of the X-gene encoding Hepatitis B virus protein X (HBx).

[0020] In any of the embodiments herein, the target site or each of the target sites is located at or near a regulatory element of an HBV gene involved in controlling HBV replication and / or HBV transcription. In some embodiments, the regulatory element is a promoter region. In some embodiments, the promoter region is a pre-S1 promoter, a pre-S2 promoter, an X promoter, or a basal core promoter. In some embodiments, the regulatory element is an enhancer region. In some embodiments, the enhancer region is an Enh1 or Enh2 enhancer region. In some embodiments, the regulatory element is a transcript processing control region.

[0021] In any of the embodiments herein, the target site or each of the target sites is within the coding region of the HBV gene. In any of the embodiments herein, the target site or each of the target sites is located within 500 base pairs (bp), 1000 bp, or 1500 bp of the transcription start site. In any of the embodiments herein, the target site or each of the target sites is located within a target region located between 0 and 3300 base pairs (bp) of the HBV genome, optionally between 0 and 3182 bp, corresponding to the positions relative to the HBV genome set forth in SEQ ID NO:650. In any of the embodiments herein, the target site or each of the target sites is located within a target region located between 43 bp and 490 bp, 1033 bp and 1749 bp, 1800 bp and 1950 bp, or 2953 bp and 3182 bp of the HBV genome, corresponding to the positions relative to the HBV genome set forth in SEQ ID NO:650. In any of the embodiments herein, the target site or each of the target sites is located within a target region located from 1 bp to 42 bp, 491 bp to 1032 bp, 1750 bp to 1799 bp, or 1951 bp to 2952 bp of the HBV genome, which correspond to the positions relative to the HBV genome set forth in SEQ ID NO:650. In any of the embodiments herein, the target site or each of the target sites is within a CpG island of the HBV genome. In any of the embodiments herein, the target site or each of the target sites is located within a target region located from 67 bp to 392 bp, 1033 bp to 1749 bp, or 2215 bp to 2490 bp of the HBV genome, which correspond to the positions relative to the HBV genome set forth in SEQ ID NO:650. In any of the embodiments herein, the or each of the target sites is located within a target region located between base pairs 1033 bp and 1749 bp in the Hepatitis B virus sequence, with reference to nucleotide positions in SEQ ID NO:650. In any of the embodiments herein, the or each of the target sites is within a target region located within 300 base pairs upstream of the Hepatitis B X protein (HBx) start codon.

[0022] Also provided herein is an epigenetic modifying DNA targeting system for suppressing transcription of one or more Hepatitis B Virus (HBV) genes, comprising at least one DNA targeting module, wherein each of the at least one DNA targeting module comprises a fusion protein comprising (a) a DNA binding domain for targeting to a target site within a target region spanning no more than 300 base pairs upstream of the Hepatitis B X protein (HBx) start codon, and (b) at least one transcriptional repressor effector domain.

[0023] In any of the embodiments herein, the or each of the target sites is located within the HBx basic core promoter region. In any of the embodiments herein, the or each of the target sites is located within the HBx promoter / enhancer region.

[0024] In any of the embodiments herein, the target site or each of the target sites is within a target region spanning 250 base pairs upstream from the Hepatitis B X protein (HBx) start codon. In any of the embodiments herein, with reference to the HBV genome set forth in SEQ ID NO:650, the target site or each of the target sites is within a target region having a sequence corresponding to a sequence located 1060-1480 bp base pairs of the HBV genome. In any of the embodiments herein, the target site or each of the target sites is within a target region spanning 150 base pairs upstream from the Hepatitis B X protein (HBx) start codon. In any of the embodiments herein, the target site or each of the target sites is within a target region spanning 120 base pairs upstream from the Hepatitis B X protein (HBx) start codon. In any of the embodiments herein, with reference to the HBV genome set forth in SEQ ID NO:650, the target site or each of the target sites is within a target region sequence corresponding to a sequence spanning 1250-1374 bp of the HBV genome. In any of the embodiments herein, with reference to the HBV genome set forth in SEQ ID NO:650, the target site or each of the target sites is within a target region sequence corresponding to a sequence spanning 1255-1302 bp of the HBV genome. In any of the embodiments herein, with reference to the HBV genome set forth in SEQ ID NO:650, the target site or each of the target sites is within a target region sequence corresponding to a sequence spanning 1260-1300 bp of the HBV genome.

[0025] In any of the embodiments herein, the target site or each of the target sites is at least 70% homologous to all hepatitis B virus genomes. In any of the embodiments herein, the target site or each of the target sites is at least 70% homologous to at least 1000 hepatitis B virus genomes. In any of the embodiments herein, the target site or each of the target sites is at least 70% homologous to at least 1000 hepatitis B virus genomes, and includes up to two mismatches.

[0026] In any of the embodiments herein, the target site, or each of the target sites, comprises a sequence set forth in any one of SEQ ID NOs: 1-195, a contiguous portion of at least 14 nucleotides (nt) thereof, or a complementary sequence of any of the foregoing. In any of the embodiments herein, the target site, or each of the target sites, comprises a sequence set forth in any one of SEQ ID NOs: 175, 138, 192, 152, 118, 125, 185, 63, 116, 124, 35, 82, a contiguous portion of at least 14 nucleotides (nt) thereof, or a complementary sequence of any of the foregoing. In any of the embodiments herein, the target site, or each of the target sites, comprises a sequence set forth in any one of SEQ ID NOs: 175, 138, 192, 152, 118, 125, 185, 63, 116, 124, 35, 82. In any of the embodiments herein, the target site, or each of the target sites, comprises a sequence set forth in any one of SEQ ID NOs:5, 6, 12, 18, 22, 26, 29, 38, 42, 43, 51, 56, 61, 63, 68, 72, 75, 79, 82, 84, 88, 89, 98, 99, 113, 116, 121, 124, 125, 118, 130, 133, 135, 138, 143, 150, 152, 155, 158, 164, 165, 175, 176, 182, 185, 189, 190, 192, a contiguous portion thereof of at least 14 nucleotides (nt), or the complementary sequence of any of the foregoing. In any of the embodiments herein, the target site or each of the target sites comprises a sequence set forth in any one of SEQ ID NOs:5, 6, 12, 18, 22, 26, 29, 38, 42, 43, 51, 56, 61, 63, 68, 72, 75, 79, 82, 84, 88, 89, 98, 99, 113, 116, 121, 124, 125, 118, 130, 133, 135, 138, 143, 150, 152, 155, 158, 164, 165, 175, 176, 182, 185, 189, 190, 192.In any of the embodiments herein, the target site, or each of the target sites, is set forth in any one of SEQ ID NOs: 12, 18, 20, 22, 26, 27, 46, 50, 63, 66, 73, 79, 185, 192, or a contiguous portion of at least 14 nucleotides thereof, or a complementary sequence of any of the foregoing. In any of the embodiments herein, the target site, or each of the target sites, is set forth in any one of SEQ ID NOs: 12, 18, 20, 22, 26, 27, 46, 50, 63, 66, 73, 79, 185, 192. In any of the embodiments herein, the target site, or each of the target sites, comprises the sequence set forth in SEQ ID NO: 22, or a contiguous portion of at least 14 nucleotides thereof, or a complementary sequence of any of the foregoing, optionally wherein the target site is set forth in SEQ ID NO: 22. In any of the embodiments herein, the target site or each of the target sites comprises the sequence set forth in SEQ ID NO:63, a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing, and optionally the target site is set forth in SEQ ID NO:63.

[0027] In any of the embodiments herein, the gRNA or each of the gRNAs comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 196-390. In any of the embodiments herein, the gRNA or each of the gRNAs further comprises a sequence set forth in SEQ ID NO: 587. In any of the embodiments herein, the gRNA or each of the gRNAs comprises a sequence set forth in any one of SEQ ID NOs: 196-390. In any of the embodiments herein, the gRNA or each of the gRNAs is set forth in any one of SEQ ID NOs: 391-585. In any of the embodiments herein, the gRNA or each of the gRNAs comprises a sequence set forth in any one of SEQ ID NOs: 370, 333, 387, 347, 313, 320, 380, 256, 258, 311, 319, 230, 272, a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing; and optionally, the gRNA or each of the gRNAs is set forth in any one of SEQ ID NOs: 565, 528, 542, 508, 515, 575, 515, 453, 506, 514, 425, or 472. In any of the embodiments herein, the gRNA or each of the gRNAs comprises a sequence set forth in any one of SEQ ID NOs: 370, 333, 387, 347, 313, 320, 380, 256, 258, 311, 319, 230, 272, and optionally, the gRNA or each of the gRNAs is set forth in any one of SEQ ID NOs: 565, 528, 542, 508, 515, 575, 515, 453, 506, 514, 425, or 472.In any of the embodiments herein, the or each of the gRNAs is selected from the group consisting of SEQ ID NOs. or a sequence set forth in any one of SEQ ID NOs: 200, 201, 207, 217, 221, 224, 233, 237, 238, 246, 251, 256, 258, 263, 267, 274, 270, 277, 279, 283, 284, 293, 294, 308, 311, 313, 316, 319, 320, 325, 328, 330, 333, 338, 345, 347, 350, 353, 359, 360, 370, 371, 377, 380, 384, 385, 387, or a contiguous portion thereof of at least 14 nucleotides, or the complementary sequence of any of the foregoing; and optionally, the gRNA or each of the gRNAs is selected from the group consisting of: Nos. 369, 395, 402, 408, 412, 416, 419, 428, 432, 433, 441, 446, 451, 453, 458, 462, 465, 469, 472, 474, 478, 479, 488, 489, 503, 506, 508, 511, 514, 515, 520, 523, 525, 575, 528, 533, 540, 542, 545, 548, 554, 555, 565, 566, 572, 579, 580, or 582.In any of the embodiments herein, the gRNA or each of the gRNAs comprises a sequence set forth in any one of SEQ ID NOs: 200, 201, 207, 217, 221, 224, 233, 237, 238, 246, 251, 256, 258, 263, 267, 274, 270, 277, 279, 283, 284, 293, 294, 308, 311, 313, 316, 319, 320, 325, 328, 330, 333, 338, 345, 347, 350, 353, 359, 360, 370, 371, 377, 380, 384, 385, or 387; and optionally, the gRNA or each of the gRNAs comprises a sequence set forth in any one of SEQ ID NOs: 200, 201, 207, 217, 221, 224, 233, 237, 238, 246, 251, 256, 258, 263, 267, 274, 270, 277, 279, 283, 284, 293, 294, 308, 311, 313, 316, 319, 320, 325, 328, 330, 333, 338, 345, 347, 350, 353, 359, 360, 370, 371, 377, Nos. 369, 395, 402, 408, 412, 416, 419, 428, 432, 433, 441, 446, 451, 453, 458, 462, 465, 469, 472, 474, 478, 479, 488, 489, 503, 506, 508, 511, 514, 515, 520, 523, 525, 575, 528, 533, 540, 542, 545, 548, 554, 555, 565, 566, 572, 579, 580, or 582. In any of the embodiments herein, the gRNA or each of the gRNAs comprises a sequence set forth in any one of SEQ ID NOs: 207, 213, 215, 217, 221, 222, 241, 245, 258, 261, 268, 274, 380, 387, a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing; and optionally, the gRNA or each of the gRNAs is set forth in any one of SEQ ID NOs: 402, 408, 410, 412, 416, 417, 436, 440, 453, 456, 463, 469, 575, 582. In any of the embodiments herein, the gRNA or each of the gRNAs comprises a sequence set forth in any one of SEQ ID NOs: 207, 213, 215, 217, 221, 222, 241, 245, 258, 261, 268, 274, 380, 387. In any of the embodiments herein, the gRNA or each of the gRNAs is set forth in any one of SEQ ID NOs: 402, 408, 410, 412, 416, 417, 436, 440, 453, 456, 463, 469, 575, 582.In any of the embodiments herein, the gRNA or each of the gRNAs comprises the sequence set forth in SEQ ID NO:217, a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence to any of the foregoing. In any of the embodiments herein, the gRNA or each of the gRNAs comprises the sequence set forth in SEQ ID NO:217. In any of the embodiments herein, the gRNA or each of the gRNAs is set forth in any one of SEQ ID NOs:412. In any of the embodiments herein, the gRNA or each of the gRNAs comprises the sequence set forth in SEQ ID NO:258, a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence to any of the foregoing. In any of the embodiments herein, the gRNA or each of the gRNAs comprises the sequence set forth in SEQ ID NO:258. In any of the embodiments herein, the gRNA or each of the gRNAs is set forth in any one of SEQ ID NOs:453.

[0028] In any of the embodiments herein, the target site or each of the target sites is at least 90% homologous to all hepatitis B virus genomes. In any of the embodiments herein, the target site or each of the target sites is at least 90% homologous to at least 1000 hepatitis B virus genomes. In any of the embodiments herein, the target site or each of the target sites is at least 90% homologous to at least 1000 hepatitis B virus genomes, and includes up to two mismatches, optionally one or two mismatches.

[0029] In any of the embodiments herein, the target site or each of the target sites comprises a sequence set forth in any one of SEQ ID NOs:35-100, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

[0030] In any of the embodiments herein, the gRNA or each of the gRNAs comprises a gRNA spacer sequence comprising a sequence set forth in any one of SEQ ID NOs: 230-295. In any of the embodiments herein, the gRNA or each of the gRNAs further comprises a sequence set forth in SEQ ID NO: 587. In any of the embodiments herein, the gRNA or each of the gRNAs comprises a sequence set forth in any one of SEQ ID NOs: 230-295, and optionally, the gRNA or each of the gRNAs is set forth in any one of SEQ ID NOs: 425-490.

[0031] In any of the embodiments herein, the maximum two mismatches are located in the first 12 nt of the 5' end of the protospacer.

[0032] In any of the embodiments herein, the or each of the target sites is at least 90% homologous to at least 1000 hepatitis B virus genomes and contains 0 mismatches.

[0033] In any of the embodiments herein, the target site comprises a sequence set forth in any one of SEQ ID NOs: 1-34, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence to any of the foregoing. In any of the embodiments herein, the or each of the gRNAs comprises a gRNA spacer sequence comprising a sequence set forth in SEQ ID NOs: 196-229.

[0034] In any of the embodiments herein, the gRNA spacer sequence is 14 nt to 24 nt, or 16 nt to 22 nt in length. In any of the embodiments herein, the gRNA spacer sequence is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length.

[0035] In any of the embodiments herein, the gRNA spacer sequence comprises modified nucleotides for increased stability.

[0036] In any of the embodiments herein, at least one gRNA further comprises the sequence set forth in SEQ ID NO: 587. In any of the embodiments herein, the gRNA, or each of the gRNAs, comprises the sequence set forth in any one of SEQ ID NOs: 196-229, and optionally, the gRNA, or each of the gRNAs, is set forth in any one of SEQ ID NOs: 391-424. In any of the embodiments herein, the gRNA or each of the gRNAs comprises a sequence set forth in any one of SEQ ID NOs: 207, 213, 215, 217, 221, 222, 241, 245, 258, 261, 268, 274, 380, 387, and optionally, the gRNA or each of the gRNAs is set forth in any one of SEQ ID NOs: 402, 408, 410, 412, 416, 417, 436, 440, 453, 456, 463, 469, 575, 582. In any of the embodiments herein, the gRNA comprises a sequence set forth in SEQ ID NO: 217, and optionally, the gRNA is set forth in SEQ ID NO: 412.

[0037] In any of the embodiments herein, the Cas protein or variant thereof is a Cas9 protein or variant thereof. In any of the embodiments herein, the Cas protein or variant thereof is a Cas12 protein or variant thereof. In any of the embodiments herein, the Cas protein or variant thereof is a variant Cas protein, wherein the variant Cas protein lacks nuclease activity or is an inactive Cas (dCas) protein. In any of the embodiments herein, the variant Cas protein is a variant Cas9 protein that lacks nuclease activity or is an inactive Cas9 (dCas9) protein. In any of the embodiments herein, the Cas9 protein or variant thereof is a Staphylococcus aureus Cas9 (SaCas9) protein or variant thereof. In any of the embodiments herein, the variant Cas9 is a Staphylococcus aureus dCas9 protein (dSaCas9) that includes at least one amino acid mutation selected from D10A and N580A, with reference to the numbering of positions in SEQ ID NO:596. In any of the embodiments herein, the variant Cas9 protein includes the sequence set forth in SEQ ID NO:597, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:597. In any of the embodiments herein, the Cas9 protein or variant thereof is a Streptococcus pyogenes Cas9 (SpCas9) protein or variant thereof. In any of the embodiments herein, the variant Cas9 is a Streptococcus pyogenes dCas9 (dSpCas9) protein that includes at least one amino acid mutation selected from D10A and H840A, with reference to the numbering of the positions in SEQ ID NO:598.In any of the embodiments herein, the variant Cas9 protein comprises the sequence set forth in SEQ ID NO:599, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0038] In any of the embodiments herein, at least one DNA-binding domain comprises an engineered zinc finger protein (eZFP). In any of the embodiments herein, at least one DNA-binding domain is an eZFP. In any of the embodiments herein, the target site comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 1045, 1046, 1052, at least a 12-nt contiguous portion thereof, or a complementary sequence of any of the foregoing. In any of the embodiments herein, the target site comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 1045, 1046, 1052.

[0039] In any of the embodiments herein, the zinc finger protein comprises six zinc fingers, designated F1 to F6 in order from the N-terminus to the C-terminus, and the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000002.tif17157TIFF2025527567000003.tif238164TIFF2025527567000004.tif131164.

[0040] In any of the embodiments herein, the zinc finger protein comprises six zinc fingers, designated F1 to F6 in order from the N-terminus to the C-terminus, and the amino acid sequence of each zinc finger recognition region is as follows: As shown in TIFF2025527567000005.tif17156.

[0041] In any of the embodiments herein, the zinc finger protein comprises six zinc fingers, designated F1 to F6 in order from the N-terminus to the C-terminus, and the amino acid sequence of each zinc finger recognition region is as follows: As shown in TIFF2025527567000006.tif17156.

[0042] In any of the embodiments herein, the zinc finger protein comprises six zinc fingers, designated F1 to F6 in order from the N-terminus to the C-terminus, and the amino acid sequence of each zinc finger recognition region is as follows: As shown in TIFF2025527567000007.tif17156.

[0043] a) an eZFP that binds to a target site within one or more HBV genes or regulatory elements thereof; b) at least one effector domain that inhibits transcription of one or more HBV genes; Also provided herein is an epigenetic modifying DNA targeting system comprising: wherein the zinc finger protein comprises six zinc fingers, designated F1 to F6 in N-terminal to C-terminal order, and the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000008.tif218164TIFF2025527567000009.tif151168.

[0044] In any of the embodiments herein, the engineered zinc finger protein comprises a sequence set forth in any one of SEQ ID NOs:692-719, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the engineered zinc finger protein is encoded by a nucleotide sequence set forth in any one of SEQ ID NOs:888-915, or a portion thereof, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0045] a) an engineered zinc finger protein that binds to a target site within one or more HBV genes or regulatory elements thereof; b) at least one effector domain that inhibits transcription of one or more HBV genes; Also provided herein is an epigenetic modifying DNA targeting system comprising: wherein the zinc finger protein comprises six zinc fingers, designated F1 to F6 in N-terminal to C-terminal order, and the amino acid sequence of each zinc finger recognition region is as follows: As shown in TIFF2025527567000010.tif17157.

[0046] In any of the embodiments herein, the eZFP comprises a sequence set forth in SEQ ID NO:709, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the engineered zinc finger protein comprises a sequence set forth in any one of SEQ ID NO:709.

[0047] In any of the embodiments herein, the engineered zinc finger protein is encoded by a nucleotide sequence set forth in SEQ ID NO:905, or a portion thereof, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In any of the embodiments herein, the engineered zinc finger protein is encoded by a sequence set forth in any one of SEQ ID NO:905.

[0048] a) an engineered zinc finger protein that binds to a target site within one or more HBV genes or regulatory elements thereof; b) at least one effector domain that inhibits transcription of one or more HBV genes; Also provided herein is an epigenetic modifying DNA targeting system comprising: a zinc finger protein comprising six zinc fingers, designated F1 to F6 in N-terminal to C-terminal order, and the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000011.tif17157. In some embodiments, the engineered zinc finger protein comprises the sequence set forth in SEQ ID NO:710, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the engineered zinc finger protein comprises the sequence set forth in any one of SEQ ID NO:710. In any of the embodiments herein, the engineered zinc finger protein is encoded by the sequence set forth in SEQ ID NO:906, or a portion thereof, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In any of the embodiments herein, the engineered zinc finger protein is encoded by a sequence set forth in any one of SEQ ID NO:906.

[0049] a) an engineered zinc finger protein that binds to a target site within one or more HBV genes or regulatory elements thereof; b) at least one effector domain that inhibits transcription of one or more HBV genes; Also provided herein is an epigenetic modifying DNA targeting system comprising: wherein the zinc finger protein comprises six zinc fingers, designated F1 to F6 in N-terminal to C-terminal order, and the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000012.tif17156. In some embodiments, the engineered zinc finger protein comprises the sequence set forth in SEQ ID NO:716, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the engineered zinc finger protein comprises the sequence set forth in any one of SEQ ID NO:716. In some embodiments, the engineered zinc finger protein is encoded by the sequence set forth in SEQ ID NO:912, or a portion thereof, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the engineered zinc finger protein is encoded by a sequence set forth in any one of SEQ ID NO:912.

[0050] In any of the embodiments herein, at least one effector domain induces transcriptional repression. In any of the embodiments herein, at least one effector domain is a DNA methyltransferase. In any of the embodiments herein, at least one effector domain comprises a DNA methyltransferase and a repressor domain capable of recruiting a heterochromatin inducer, or optionally, the heterochromatin inducer comprises a histone methyltransferase. In any of the embodiments herein, at least one effector domain comprises a DNA methyltransferase and a histone methyltransferase. In any of the embodiments herein, at least one effector domain is selected from a KRAB repressor domain, an ERF repressor domain, an Mxi1 repressor domain, a SID4X repressor domain, a Mad-SID repressor domain, an LSD1 repressor domain, or a DNMT3A, DNMT3A-3L, a DNMT3A / L-KRAB fusion repressor domain, a DNMT3B domain binding protein, an EZH2 repressor domain, or an LSD1 repressor domain, or a variant of any of the foregoing. In any of the embodiments herein, at least one effector domain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence selected from any one of SEQ ID NO:590, or 600-608, 651, 661, 664, 665, 666, 668, and 669, or a domain thereof, a portion thereof, or any of the foregoing. In any of the embodiments herein, at least one effector domain comprises a KRAB domain or a variant thereof. In any of the embodiments herein, at least one effector domain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence set forth in SEQ ID NO:590, a portion thereof, or any of the foregoing.In any of the embodiments herein, at least one effector domain comprises a DNMT3A / L domain or a variant thereof. In any of the embodiments herein, at least one effector domain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:604 and 607, a portion thereof, or any of the foregoing; or at least one effector domain comprises a sequence set forth in SEQ ID NO:651, a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:651.

[0051] In some embodiments, the fusion protein comprises a DNMT3A / 3L-dSpCas9-KRAB fusion protein. In some embodiments, the fusion protein comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:645, a portion thereof, or any of the foregoing.

[0052] In any of the embodiments herein, at least one effector domain is fused to the N-terminus, C-terminus, or both the N-terminus and C-terminus of the DNA-binding domain or component thereof.

[0053] In any of the embodiments herein, the fusion protein is encoded by a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:680, a portion thereof, or any of the foregoing. In any of the embodiments herein, the fusion protein is encoded by the sequence set forth in SEQ ID NO:680.

[0054] In any of the embodiments herein, the fusion protein is encoded by a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in any one of SEQ ID NOs:916-943, a portion thereof, or any of the foregoing. In any of the embodiments herein, the fusion protein comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in any one of SEQ ID NOs:944-971, a portion thereof, or any of the foregoing. In any of the embodiments herein, the fusion protein comprises a sequence set forth in any one of SEQ ID NOs:961, 962, or 968.

[0055] In any of the embodiments herein, the fusion protein comprises a DNMT3A / 3L-eZFP-KRAB fusion protein.

[0056] In any of the embodiments herein, the fusion protein is encoded by a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in any one of SEQ ID NOs:972-999, a portion thereof, or any of the foregoing. In any of the embodiments herein, the fusion protein is encoded by a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in any one of SEQ ID NOs:933, 934, or 940, a portion thereof, or any of the foregoing. In any of the embodiments herein, the fusion protein comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in any one of SEQ ID NOs: 1000-1027, a portion thereof, or any of the foregoing. In any of the embodiments herein, the fusion protein comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in any one of SEQ ID NOs: 1017, 1018, or 1024, a portion thereof, or any of the foregoing.

[0057] In any of the embodiments herein, the fusion protein further comprises one or more nuclear localization signals (NLS).

[0058] In any of the embodiments herein, the fusion protein further comprises one or more linkers connecting two or more of the DNA-binding domain, the at least one effector domain, and the one or more nuclear localization signals.

[0059] In any of the embodiments herein, the DNA targeting system targets the entire hepatitis B virus genome.

[0060] In any of the embodiments herein, the DNA targeting system targets at least 70% of all hepatitis B virus genomes. In any of the embodiments herein, the DNA targeting system targets at least 60% of all hepatitis B virus genomes. In any of the embodiments herein, the DNA targeting system targets at least 50% of all hepatitis B virus genomes.

[0061] In any of the embodiments herein, the DNA targeting system is unable to introduce a gene disruption or a DNA break at or near the target site.

[0062] In any of the embodiments herein, inhibiting transcription of one or more HBV genes results in a reduction in RNA and / or protein levels from the HBV DNA sequence. In any of the embodiments herein, inhibiting transcription comprises a reduction in total hepatitis B virus RNA transcript levels. In any of the embodiments herein, inhibiting transcription comprises a reduction in hepatitis B pre-core ("pre-C"), pre-genome ("pgRNA"), pre-S1, pre-S2 / S, and HBx levels. In any of the embodiments herein, inhibiting transcription comprises a reduction in HBx levels. In any of the embodiments herein, inhibiting transcription comprises a reduction in hepatitis B surface antigen (HBsAg) and / or hepatitis B virus core-related antigen (HbcrAg) protein levels. In any of the embodiments herein, inhibiting transcription comprises at least a 90% reduction in HbsAg transcript and / or protein levels. In any of the embodiments herein, inhibiting transcription comprises at least a 50% reduction in HbcrAg transcript and / or protein levels from cccDNA.

[0063] Also provided herein is a guide RNA (gRNA) that binds to a target site within a hepatitis B virus DNA sequence. In some embodiments, the hepatitis B virus DNA sequence is a hepatitis B virus (HBV) gene or a regulatory element thereof. In some embodiments, the target site is present in a covalently closed circular DNA (cccDNA) form, an incomplete double-stranded DNA (rcDNA) form, and / or is integrated into human genomic DNA. In any of the embodiments herein, the target site is located in or near a gene or its regulatory element involved in controlling HBV replication and / or HBV transcription. In any of the embodiments herein, the gene involved in controlling HBV replication and / or HBV transcription encodes a polymerase, an envelope protein, a capsid protein, a transcription factor, or a transcriptional transactivator. In any of the embodiments herein, the gene involved in controlling HBV replication and / or HBV transcription is a polymerase gene, an S-family gene, an X-gene, or a core-family gene. In any of the embodiments herein, the target site is within the gene or regulatory elements of the X-gene encoding Hepatitis B virus protein X (HBx). In any of the embodiments herein, the target site is at or near regulatory elements involved in controlling HBV replication and / or HBV transcription.

[0064] In any of the embodiments herein, the regulatory element is a promoter region.In any of the embodiments herein, the promoter region is a pre-S1 promoter, a pre-S2 promoter, an X promoter, or a basic core promoter.In any of the embodiments herein, the regulatory element is an enhancer region.In any of the embodiments herein, the enhancer region is an Enh1 or Enh2 enhancer region.In any of the embodiments herein, the regulatory element is a transcript processing control region.

[0065] In any of the embodiments herein, the target site is a coding region. In any of the embodiments herein, the target site is located within 500 bp, 1000 bp, or 1500 bp of the transcription start site. In any of the embodiments herein, the target site is located within a target region located between 0 and 3300 base pairs (bp) of the HBV genome, optionally between 0 and 3189 bp, corresponding to the position relative to the HBV genome set forth in SEQ ID NO:650. In any of the embodiments herein, the target site is located within a target region located between 43 bp and 490 bp, 1033 bp and 1749 bp, 1800 bp and 1950 bp, or 2953 bp and 3182 bp of the HBV genome, corresponding to the position relative to the HBV genome set forth in SEQ ID NO:650. In any of the embodiments herein, the target site is located within a target region located at base pairs between 1 bp and 42 bp, between 491 bp and 1032 bp, between 1750 bp and 1799 bp, or between 1951 bp and 2952 bp of the HBV genome, corresponding to the positions relative to the HBV genome set forth in SEQ ID NO:650.

[0066] In any of the embodiments herein, the or each of the target sites is within a CpG island of the HBV genome. In any of the embodiments herein, the target site is located within a target region located between 67 bp and 392 bp, between 1033 bp and 1749 bp, or between 2215 bp and 2490 bp of the HBV genome, corresponding to the position relative to the HBV genome set forth in SEQ ID NO:650. In any of the embodiments herein, the target site is located within a target region located between 1033 bp and 1749 bp of the HBV genome, corresponding to the position relative to the HBV genome set forth in SEQ ID NO:650. In any of the embodiments herein, the or each of the target sites is within a target region extending within 300 base pairs upstream of the Hepatitis B X protein (HBx) start codon.

[0067] Also provided herein is a gRNA (gRNA) that binds to a target site within a target region extending within 300 base pairs upstream of the Hepatitis B X protein (HBx) start codon. In any of the embodiments herein, the target site is located within the HBx basic core promoter region. In any of the embodiments herein, the target site is located within the HBx promoter / enhancer region. In any of the embodiments herein, the target site is within a target region extending within 250 base pairs upstream of the Hepatitis B X protein (HBx) start codon. In any of the embodiments herein, the target site is within a target region extending 1060-1480 bp of the HBV genome, corresponding to the position relative to the HBV genome set forth in SEQ ID NO:650. In any of the embodiments herein, the target site is within a target region extending within 150 base pairs upstream of the Hepatitis B X protein (HBx) start codon. In any of the embodiments herein, the target site is within a target region extending within 120 base pairs upstream of the Hepatitis B X protein (HBx) start codon. In any of the embodiments herein, with reference to the HBV genome set forth in SEQ ID NO:650, the target site is within a target region sequence corresponding to a sequence spanning 1250-1374 bp of the HBV genome. In any of the embodiments herein, with reference to the HBV genome set forth in SEQ ID NO:650, the target site is within a target region sequence corresponding to a sequence spanning 1255-1302 bp of the HBV genome. In any of the embodiments herein, with reference to the HBV genome set forth in SEQ ID NO:650, the target site is within a target region sequence corresponding to a sequence spanning 1260-1300 bp of the HBV genome.In any of the embodiments herein, the gRNA is selected from the group consisting of SEQ ID or 387, a contiguous portion thereof of at least 14 nucleotides, or the complementary sequence of any of the foregoing; and optionally, the gRNA or each of the gRNAs comprises a sequence set forth in any one of SEQ ID NOs:200, 201, 207, 217, 221, 224, 233, 237, 238, 246, 251, 256, 258, 263, 267, 274, 270, 277, 279, 283, 284, 293, 294, 308, 311, 313, 316, 319, 320, 325, 328, 330, 333, 338, 345, 347, 350, 353, 359, 360, 369, 370, 371, 377, 380, 384, 385, or 387; Nos. 395, 402, 408, 412, 416, 419, 428, 432, 433, 441, 446, 451, 453, 458, 462, 465, 469, 472, 474, 478, 479, 488, 489, 503, 506, 508, 511, 514, 515, 520, 523, 525, 575, 528, 533, 540, 542, 545, 548, 554, 555, 565, 566, 572, 579, 580, or 582. In any of the embodiments herein, the gRNA is set forth in any one of SEQ ID NOs:395, 402, 408, 412, 416, 419, 428, 432, 433, 441, 446, 451, 453, 458, 462, 465, 469, 472, 474, 478, 479, 488, 489, 503, 506, 508, 511, 514, 515, 520, 523, 525, 575, 528, 533, 540, 542, 545, 548, 554, 555, 565, 566, 572, 579, 580, or 582.In any of the embodiments herein, the gRNA comprises a sequence set forth in any one of SEQ ID NOs: 207, 213, 215, 217, 221, 222, 241, 245, 258, 261, 268, 274, 380, 387, a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing, and optionally, the gRNA, or each of the gRNAs, is set forth in any one of SEQ ID NOs: 402, 408, 410, 412, 416, 417, 436, 440, 453, 456, 463, 469, 575, or 582. In any of the embodiments herein, the gRNA comprises the sequence set forth in any one of SEQ ID NOs:402, 408, 410, 412, 416, 417, 436, 440, 453, 456, 463, 469, 575, or 582. In any of the embodiments herein, the gRNA comprises the sequence set forth in SEQ ID NO:217, a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing, and optionally the gRNA, or each of the gRNAs, is set forth in any one of SEQ ID NOs:412. In any of the embodiments herein, the gRNA comprises the sequence set forth in SEQ ID NO:412.

[0068] Also provided herein is a CRISPR-associated (Cas)-gRNA combination comprising: (a) a CRISPR-associated (Cas) protein or variant thereof; and (b) at least one guide RNA (gRNA) of any one of claims 165-202, which targets the Cas protein or variant thereof to a target site in a Hepatitis B virus DNA sequence. In some embodiments, the Cas protein or variant thereof is a Cas9 protein or variant thereof. In any embodiment herein, the Cas protein or variant thereof is a variant Cas protein, wherein the variant Cas protein lacks nuclease activity or is an inactive Cas9 (dCas) protein. In any embodiment herein, the variant Cas protein is a variant Cas9 protein that lacks nuclease activity or is an inactive Cas9 (dCas9) protein. In any embodiment herein, the Cas9 protein or variant thereof is a Staphylococcus aureus Cas9 (SaCas9) protein or variant thereof. In some embodiments, the variant Cas9 is a Staphylococcus aureus dCas9 protein (dSaCas9) comprising at least one amino acid mutation selected from D10A and N580A, with reference to the position numbering of SEQ ID NO:596. In some embodiments, the variant Cas9 protein comprises the sequence set forth in SEQ ID NO:597, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the Cas9 protein or variant thereof is a Streptococcus pyogenes Cas9 (SpCas9) protein or variant thereof. In some embodiments, the variant Cas9 is a Streptococcus pyogenes dCas9 (dSpCas9) protein comprising at least one amino acid mutation selected from D10A and H840A, with reference to the position numbering of SEQ ID NO:598.In some embodiments, the variant Cas9 protein comprises the sequence set forth in SEQ ID NO:599, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0069] Also provided are polynucleotides encoding the epigenetic modifying DNA targeting systems disclosed herein or fusion proteins of the DNA targeting systems disclosed herein, the gRNAs disclosed herein, the CRISPR Cas-gRNA combinations disclosed herein, or portions or components of any of the foregoing.

[0070] Also provided are multiple polynucleotides encoding the epigenetic modifying DNA targeting system disclosed herein or a fusion protein of the DNA targeting system disclosed herein, the gRNA disclosed herein, the CRISPR Cas-gRNA combination disclosed herein, or portions or components of any of the foregoing.

[0071] Also provided are vectors comprising the polynucleotides disclosed herein.Also provided are vectors comprising a plurality of the polynucleotides disclosed herein.

[0072] In any of the embodiments herein, the vector is a viral vector. In some embodiments herein, the vector is an adeno-associated virus (AAV) vector. In some embodiments herein, the vector is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9. In some embodiments herein, the vector is a lentiviral vector. In some embodiments herein, the vector is a non-viral vector. In some embodiments herein, the non-viral vector is selected from lipid nanoparticles, liposomes, exosomes, or cell-penetrating peptides. In any of the embodiments herein, the vector exhibits tropism for hepatitis B virus-infected cells. In some embodiments herein, the vector comprises one vector or two or more vectors.

[0073] Also provided herein is a method for promoting epigenetic modification within a target region in a hepatitis B virus sequence, the method comprising introducing an epigenetic modifying DNA targeting system that targets a target site within the target region into an HBV-infected cell containing the hepatitis virus sequence.

[0074] Also provided herein is a method for increasing CpG methylation within a target region in a hepatitis B virus sequence, the method comprising introducing an epigenetic modifying DNA targeting system that targets a target site within the target region into an HBV-infected cell containing the hepatitis virus sequence.

[0075] Also provided herein is a method for promoting epigenetic modification of a target region in a Hepatitis B virus sequence, the method comprising introducing an epigenetic modifying DNA targeting system disclosed herein, a gRNA disclosed herein, a CRISPR Cas-gRNA combination disclosed herein, a polynucleotide disclosed herein, a plurality of polynucleotides disclosed herein, a vector disclosed herein, or a portion or component of any of the foregoing, into an HBV-infected cell containing a Hepatitis B virus sequence.

[0076] Also provided herein is a method for increasing CpG methylation of a target region in a Hepatitis B virus sequence, the method comprising introducing an epigenetic modifying DNA targeting system disclosed herein, a gRNA disclosed herein, a CRISPR Cas-gRNA combination disclosed herein, a polynucleotide disclosed herein, a plurality of polynucleotides disclosed herein, a vector disclosed herein, or a portion or component of any of the foregoing, into an HBV-infected cell containing a Hepatitis B virus sequence.

[0077] In any of the embodiments herein, the target region comprises a contiguous sequence of nucleotides within the sequence corresponding to 1033 bp to 1749 bp in the Hepatitis B virus sequence, with reference to the nucleotide positions of SEQ ID NO:650.

[0078] Also provided herein is a method for reducing transcription of one or more genes in HBV-infected cells containing a hepatitis B virus sequence, the method comprising introducing into the cell an epigenetic modifying DNA targeting system that induces targeted CpG methylation within the hepatitis B virus sequence, with reference to the nucleotide positions of SEQ ID NO:650.

[0079] Also provided herein is a method for reducing hepatitis B virus infection in HBV-infected cells, comprising introducing into cells containing hepatitis B virus sequences an epigenetic modifying DNA targeting system that induces targeted CpG methylation within a target region in the hepatitis B virus sequence, with reference to the nucleotide position of SEQ ID NO:650.

[0080] In any of the embodiments herein, the epigenetic modifying DNA targeting system includes at least one DNA targeting module comprising a fusion protein including (a) a DNA binding domain for targeting a target site in a Hepatitis B virus DNA sequence, and (b) at least one effector domain including a DNA methyltransferase effector domain.

[0081] In any of the embodiments herein, the region of CpG methylation is within 500 base pairs of the target region. In any of the embodiments herein, the introducing occurs in vivo or ex vivo in a subject.

[0082] In any of the embodiments herein, the cell is a mammalian cell. In any of the embodiments herein, the cell is a human cell. In any of the embodiments herein, the cell contains integrated HBV DNA. In any of the embodiments herein, the cell is a hepatocyte containing a pool of episomal HBV cccDNA. In some embodiments, the hepatocyte expresses an HBV protein, wherein the HBV protein is HBsAg, HBeAg, or HBcrAg, or a combination thereof.

[0083] Also provided herein is a method for reducing hepatitis virus infection in a subject, comprising administering to a subject infected with hepatitis B an epigenetic modifying DNA targeting system that increases CpG methylation within a target region in a hepatitis B virus sequence, wherein the epigenetic modifying DNA targeting system comprises: (a) a DNA binding domain for targeting to a target site in the hepatitis B virus DNA sequence; and (b) at least one effector domain comprising a DNA methyltransferase effector domain.

[0084] In any of the embodiments herein, the target region is a CpG-containing region in the HBV genome. In any of the embodiments herein, with reference to the nucleotide positions of SEQ ID NO:650, the target region comprises a contiguous sequence of nucleotides within a sequence corresponding to 67 bp to 392 bp, 1033 bp to 1749 bp, or 2215 bp to 2490 bp within the hepatitis B virus sequence. In any of the embodiments herein, with reference to the nucleotide positions of SEQ ID NO:650, the target region comprises a contiguous sequence of nucleotides within a sequence corresponding to 1033 bp to 1749 bp within the hepatitis B virus sequence. In any of the embodiments herein, the target region is located within 300 base pairs upstream of the hepatitis B X protein (HBx) start codon. In any of the embodiments herein, the target region is within the HBx basic core promoter region. In any of the embodiments herein, the target region is within the HBx promoter / enhancer region. In any of the embodiments herein, the target region is within 250 base pairs upstream of the Hepatitis B X protein (HBx) start codon. In any of the embodiments herein, with reference to the nucleotide positions of SEQ ID NO:650, the target region comprises a contiguous sequence of nucleotides within the sequence corresponding to 1060 bp to 1480 bp in the Hepatitis B virus sequence. In any of the embodiments herein, the target region is within 150 base pairs upstream of the Hepatitis B X protein (HBx) start codon. In any of the embodiments herein, the target region is within 120 base pairs upstream of the Hepatitis B X protein (HBx) start codon. In any of the embodiments herein, with reference to the nucleotide positions of SEQ ID NO:650, the target region comprises a contiguous sequence of nucleotides within the sequence corresponding to 1250 bp to 1374 bp in the Hepatitis B virus sequence. In some embodiments, the target region has the sequence set forth in SEQ ID NO:1068. In any of the embodiments herein, the target region comprises a contiguous sequence of nucleotides within the sequence corresponding to 1260 bp to 1300 bp in the Hepatitis B virus sequence, with reference to the nucleotide positions of SEQ ID NO:650.In some embodiments, the target region has the sequence shown in SEQ ID NO:1070.

[0085] In any of the embodiments herein, the DNA-binding domain comprises a Cas-gRNA combination comprising (a) a CRISPR-associated (Cas) protein or a variant thereof, and (b) at least one guide RNA (gRNA); a zinc finger protein (ZFP); a transcription activator-like effector (TALE); a meganuclease; a homing endonuclease; or an I-SceI enzyme or a variant thereof, and optionally, the DNA-binding domain comprises a catalytically inactive variant of any of the foregoing.

[0086] In any of the embodiments herein, the method comprises a CRISPR-associated (Cas) protein or variant thereof, and (b) at least one guide RNA (gRNA) of any one of claims 165-202 that targets the Cas protein or variant thereof to a target site at a target site within a Hepatitis B virus DNA sequence.

[0087] In any of the embodiments herein, the target site, or each of the target sites, comprises a sequence set forth in any one of SEQ ID NOs:5, 6, 12, 18, 22, 26, 29, 38, 42, 43, 51, 56, 61, 63, 68, 72, 75, 79, 82, 84, 88, 89, 98, 99, 113, 116, 121, 124, 125, 118, 130, 133, 135, 138, 143, 150, 152, 155, 158, 164, 165, 175, 176, 182, 185, 189, 190, 192, a contiguous portion thereof of at least 14 nucleotides (nt), or the complementary sequence of any of the foregoing. 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: 12, 18, 20, 22, 26, 27, 46, 50, 63, 66, 73, 79, 185, 192, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In any of the embodiments herein, the target site, or each of the target sites, comprises the sequence set forth in SEQ ID NO: 22, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.In any of the embodiments herein, the or each of the gRNAs is selected from the group consisting of SEQ ID NOs. or a sequence set forth in any one of SEQ ID NOs: 200, 201, 207, 217, 221, 224, 233, 237, 238, 246, 251, 256, 258, 263, 267, 274, 270, 277, 279, 283, 284, 293, 294, 308, 311, 313, 316, 319, 320, 325, 328, 330, 333, 338, 345, 347, 350, 353, 359, 360, 370, 371, 377, 380, 384, 385, 387, or a contiguous portion thereof of at least 14 nucleotides, or the complementary sequence of any of the foregoing; and optionally, the gRNA or each of the gRNAs is selected from the group consisting of: Nos. 395, 402, 408, 412, 416, 419, 428, 432, 433, 441, 446, 451, 453, 458, 462, 465, 469, 472, 474, 478, 479, 488, 489, 503, 506, 508, 511, 514, 515, 520, 523, 525, 575, 528, 533, 540, 542, 545, 548, 554, 555, 565, 566, 572, 579, 580, or 582. In any of the embodiments herein, the gRNA or each of the gRNAs comprises a sequence set forth in any one of SEQ ID NOs: 207, 213, 215, 217, 221, 222, 241, 245, 258, 261, 268, 274, 380, 387, a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing; and optionally, the gRNA or each of the gRNAs is set forth in any one of SEQ ID NOs: 402, 408, 410, 412, 416, 417, 436, 440, 453, 456, 463, 469, 575, 582. In any of the embodiments herein, the gRNA or each of the gRNAs comprises a sequence set forth in any one of SEQ ID NOs:217, a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing, and optionally, the gRNA or each of the gRNAs is set forth in any one of SEQ ID NOs:412.

[0088] In any of the embodiments herein, at least one DNA-binding domain comprises an engineered zinc finger protein (eZFP). In any of the embodiments herein, the target site comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 1045, 1046, 1052, at least a 12-nt contiguous portion thereof, or a complementary sequence of any of the foregoing. In any of the embodiments herein, the target site comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 1045, 1046, 1052.

[0089] In any of the embodiments herein, at least one effector domain is a DNA methyltransferase. In any of the embodiments herein, at least one effector domain comprises a DNA methyltransferase and a repressor domain capable of recruiting a heterochromatin inducer, or optionally, the heterochromatin inducer comprises a histone methyltransferase. In any of the embodiments herein, at least one effector domain comprises a DNA methyltransferase and a histone methyltransferase. In any of the embodiments herein, at least one effector domain comprises a DNMT3A / L domain or a variant thereof. In any of the embodiments herein, at least one effector domain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NOs: 604 and 607, a portion thereof, or any of the foregoing. In any of the embodiments herein, at least one effector domain further comprises a KRAB domain or a variant thereof. In any of the embodiments herein, at least one effector domain further comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence set forth in SEQ ID NO:590, a portion thereof, or any of the foregoing. In any of the embodiments herein, the DNA targeting system comprises a DNMT3A / 3L-dSpCas9-KRAB domain or a variant thereof. In any of the embodiments herein, the DNA targeting system comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence set forth in SEQ ID NO:645, a portion thereof, or any of the foregoing.

[0090] In any of the embodiments herein, the DNA targeting system comprises the sequence set forth in SEQ ID NO:680, a portion thereof, or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In any of the embodiments herein, the DNA targeting system comprises the sequence set forth in SEQ ID NO:680.

[0091] Also provided herein is a method for suppressing the transcription of one or more genes in hepatitis B virus-infected cells, comprising introducing into hepatitis B virus-infected cells an epigenetic modifying DNA targeting system disclosed herein, a gRNA disclosed herein, a CRISPR Cas-gRNA combination disclosed herein, a polynucleotide disclosed herein, a plurality of polynucleotides disclosed herein, a vector disclosed herein, or any part or component thereof.In some embodiments, one or more genes are epigenetically modified by the DNA targeting system.In some embodiments, the transcription of one or more genes is reduced compared to comparable cells that have not been subjected to the method.

[0092] In any of the embodiments herein, transcription of one or more genes is reduced by at least about 1.25-fold, 1.5-fold, 1.75-fold, 2.0-fold, 2.5-fold, 2.75-fold, 3.0-fold, 3.5-fold, 3.75-fold, 4.0-fold, 4.5-fold, 4.75-fold, 5.0-fold, 5.25-fold, 5.5-fold, 5.75-fold, or 6-fold. In any of the embodiments herein, inhibiting transcription of one or more genes results in reduced HBV replication and / or HBV transcription. In any of the embodiments herein, the HBV-infected cell is a mammalian cell.

[0093] In any of the embodiments herein, the HBV-infected cells are human cells. In any of the embodiments herein, the cells contain integrated HBV DNA. In any of the embodiments herein, the cells are hepatocytes containing a pool of episomal HBV cccDNA. In any of the embodiments herein, the hepatocytes express HBV proteins, wherein the HBV proteins are HBsAg and / or HBeAg. In any of the embodiments herein, the HBV-infected cells are present in a subject.

[0094] In any of the embodiments herein, the subject is a human. In any of the embodiments herein, the subject has an HBV viral infection. In any of the embodiments herein, the subject has hepatocytes containing integrated HBV DNA. In any of the embodiments herein, the subject has hepatocytes containing a pool of episomal HBV cccDNA. In any of the embodiments herein, the subject has hepatocytes expressing HBV proteins, wherein the HBV proteins are HBsAg, HBeAg, or HBcrAg, and combinations thereof.

[0095] In any of the embodiments herein, the subject has a disease, condition or disorder associated with HBV virus infection.In any of the embodiments herein, the disease, condition or disorder is liver disease or cancer.In any of the embodiments herein, the disease, condition or disorder is acute hepatitis, chronic hepatitis, liver failure or liver cirrhosis.In any of the embodiments herein, the disease, condition or disorder is cancer, and optionally, the cancer is hepatocellular carcinoma.

[0096] Also provided herein is a pharmaceutical composition comprising the vector disclosed herein. In any of the embodiments herein, the vector is conjugated with an amino sugar derivative of galactose, and optionally, the vector is conjugated with an N-acetylgalactosamine (GalNAc) moiety.

[0097] Also provided herein are pharmaceutical compositions comprising an epigenetic modifying DNA targeting system disclosed herein or a fusion protein disclosed herein, a gRNA disclosed herein, a CRISPR Cas-gRNA combination disclosed herein, a polynucleotide disclosed herein, a plurality of polynucleotides disclosed herein, a vector disclosed herein, or a portion or component of any of the foregoing.

[0098] Also provided herein is a pharmaceutical composition for use in treating an HBV viral infection in a subject. In any of the embodiments herein, the subject has a disease, condition, or disorder associated with an HBV viral infection.

[0099] Also provided herein are pharmaceutical compositions for use in treating a disease, disorder, or condition in a subject associated with HBV viral infection.

[0100] Also provided herein are pharmaceutical compositions for use in the manufacture of a medicament for treating an HBV viral infection in a subject. In some embodiments, the HBV viral infection is associated with a disease, disorder, or condition.

[0101] Also provided herein are pharmaceutical compositions for use in the manufacture of a medicament for treating a disease, condition, or disorder in a subject associated with HBV viral infection.

[0102] In any of the embodiments herein, the disease, condition, or disorder is liver disease or cancer.In any of the embodiments herein, the disease, condition, or disorder is acute hepatitis, chronic hepatitis, liver failure, or liver cirrhosis.In any of the embodiments herein, the disease, condition, or disorder is cancer, optionally hepatocellular carcinoma.In any of the embodiments herein, the pharmaceutical composition is for in vivo administration to a subject.

[0103] In any of the embodiments herein, after administration of the pharmaceutical composition, transcription of one or more HBV genes is suppressed in the cells of the subject. In any of the embodiments herein, the one or more HBV genes are involved in controlling HBV replication and / or HBV transcription. In any of the embodiments herein, the one or more genes are polymerase genes, S-family genes, X-genes, or core family genes.

[0104] Also provided herein is a method for treating a disease, condition, or disorder in a subject in need thereof, comprising administering to the subject an epigenetic modifying DNA targeting system disclosed herein, a gRNA disclosed herein, a CRISPR Cas-gRNA combination disclosed herein, a polynucleotide disclosed herein, a plurality of polynucleotides disclosed herein, a vector disclosed herein, a pharmaceutical composition disclosed herein, or a portion or component of any of the foregoing.

[0105] Also provided herein is a method of reducing Hepatitis B virus infection in a subject, comprising administering to a subject having Hepatitis B virus infection an epigenetic modifying DNA targeting system disclosed herein, a gRNA disclosed herein, a CRISPR Cas-gRNA combination disclosed herein, a polynucleotide disclosed herein, a plurality of polynucleotides disclosed herein, a vector disclosed herein, a pharmaceutical composition disclosed herein, or a portion or component of any of the foregoing.

[0106] Also provided herein are engineered zinc finger proteins (eZFPs) that bind to target sites in one or more HBV genes or regulatory elements thereof, wherein the target sites are within a target region spanning 1033 bp to 1749 bp of the HBV genome, corresponding to the location relative to the HBV genome set forth in SEQ ID NO:650. In any of the embodiments herein, the target site is within a target region spanning 300 base pairs upstream of the Hepatitis B X protein (HBx) start codon. In any of the embodiments herein, the target site is located within the HBx basic core promoter region. In any of the embodiments herein, the target site is located within the HBx promoter / enhancer region. In any of the embodiments herein, the target site is within a target region spanning 250 base pairs upstream of the Hepatitis B X protein (HBx) start codon. In any of the embodiments herein, the target site is within a target region spanning 1060 to 1480 bp of the HBV genome, corresponding to the location relative to the HBV genome set forth in SEQ ID NO:650. In any of the embodiments herein, the target site is within a target region spanning 150 base pairs upstream from the Hepatitis B X protein (HBx) start codon. In any of the embodiments herein, the target site is within a target region spanning 120 base pairs upstream from the Hepatitis B X protein (HBx) start codon. In any of the embodiments herein, with reference to the HBV genome set forth in SEQ ID NO:650, the target site is within a target region sequence corresponding to a sequence spanning 1250-1374 bp of the HBV genome. In some embodiments, the target region has the sequence set forth in SEQ ID NO:1068. In any of the embodiments herein, with reference to the HBV genome set forth in SEQ ID NO:650, the target site is within a target region sequence corresponding to a sequence spanning 1255-1302 bp of the HBV genome. In some embodiments, the target region has the sequence set forth in SEQ ID NO:1069.In any of the embodiments herein, with reference to the HBV genome set forth in SEQ ID NO:650, the target site is within a target region sequence corresponding to a sequence spanning 1260-1300 bp of the HBV genome. In some embodiments, the target region has a sequence set forth in SEQ ID NO:1070. In any of the embodiments herein, with reference to the HBV genome set forth in SEQ ID NO:650, the target site is within a target region sequence corresponding to a sequence spanning 1255 bp to 1290 bp of the HBV genome. In any of the embodiments herein, the target site comprises a nucleotide sequence set forth in any one of SEQ ID NOs:1028-1055, at least a 12 nt contiguous portion thereof, or a complementary sequence to any of the foregoing. In any of the embodiments herein, the target site comprises a nucleotide sequence set forth in any one of SEQ ID NOs:1028-1055.

[0107] In any of the embodiments herein, the target site comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 1045, 1046, or 1052, a contiguous portion thereof of at least 12 nt, or a complementary sequence of any of the foregoing. In any of the embodiments herein, the target site comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 1045, 1046, or 1052.

[0108] In any of the embodiments herein, the zinc finger protein comprises six zinc fingers, designated F1 to F6 in order from the N-terminus to the C-terminus, and the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000013.tif171164TIFF2025527567000014.tif211170.

[0109] In any of the embodiments herein, the zinc finger protein comprises six zinc fingers, designated F1 to F6 in order from the N-terminus to the C-terminus, and the amino acid sequence of each zinc finger recognition region is as follows: As shown in TIFF2025527567000015.tif17161.

[0110] In any of the embodiments herein, the zinc finger protein comprises six zinc fingers, designated F1 to F6 in order from the N-terminus to the C-terminus, and the amino acid sequence of each zinc finger recognition region is as follows: As shown in TIFF2025527567000016.tif17161.

[0111] In any of the embodiments herein, the zinc finger protein comprises six zinc fingers, designated F1 to F6 in order from the N-terminus to the C-terminus, and the amino acid sequence of each zinc finger recognition region is as follows: As shown in TIFF2025527567000017.tif17162.

[0112] Also provided herein are eZFPs that bind to target sites in one or more HBV genes or regulatory elements thereof, wherein the zinc finger protein comprises six zinc fingers, designated F1 to F6 in N-terminal to C-terminal order, and the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000018.tif151162TIFF2025527567000019.tif231164.

[0113] In any of the embodiments herein, the engineered zinc finger protein comprises a sequence set forth in any one of SEQ ID NOs:692-719, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In any of the embodiments herein, the engineered zinc finger protein is encoded by a sequence set forth in any one of SEQ ID NOs:888-915, or a portion thereof, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0114] Also provided herein are engineered zinc finger proteins that bind to target sites in one or more HBV genes or regulatory elements thereof, wherein the zinc finger proteins are comprised of, from N- to C-terminal order, F1 to F6 ( TIFF2025527567000020.tif17157). In some embodiments, the engineered zinc finger protein comprises the sequence set forth in SEQ ID NO:709, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the engineered zinc finger protein comprises the sequence set forth in any one of SEQ ID NO:709. In some embodiments, the engineered zinc finger protein is encoded by the sequence set forth in SEQ ID NO:905, or a portion thereof, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the engineered zinc finger protein is encoded by a sequence set forth in any one of SEQ ID NO:905.

[0115] Also provided herein is an engineered zinc finger protein that binds to a target site in one or more HBV genes or regulatory elements thereof, wherein the zinc finger protein comprises six zinc fingers, designated F1 to F6 in N-terminal to C-terminal order, and the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000021.tif17155. In some embodiments, the engineered zinc finger protein comprises the sequence set forth in SEQ ID NO:710, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the engineered zinc finger protein comprises the sequence set forth in any one of SEQ ID NO:710. In some embodiments, the engineered zinc finger protein is encoded by the sequence set forth in SEQ ID NO:906, or a portion thereof, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the engineered zinc finger protein is encoded by a sequence set forth in any one of SEQ ID NO:906.

[0116] Also provided herein is an engineered zinc finger protein that binds to a target site in one or more HBV genes or regulatory elements thereof, wherein the zinc finger protein comprises six zinc fingers, designated F1 to F6 in N-terminal to C-terminal order, and the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000022.tif17157. In some embodiments, the engineered zinc finger protein comprises the sequence set forth in SEQ ID NO:716, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the engineered zinc finger protein comprises the sequence set forth in any one of SEQ ID NO:716. In some embodiments, the engineered zinc finger protein is encoded by the sequence set forth in SEQ ID NO:912, or a portion thereof, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the engineered zinc finger protein is encoded by a sequence set forth in any one of SEQ ID NO:912. [Brief explanation of the drawings]

[0117] [Figure 1] Figure 1 shows the fold change in total HBV RNA mediated by each guide RNA and dCas9-KRAB effector fusion protein. Fold change is shown relative to the central targeting position of each gRNA across all HBV genotypes. [Figure 2] Shows the conservation of guide RNA across HBV genome types (HBV0 to HBV12). [Figure 3] Figures 3A-3B show the suppression of total HBV RNA (Figure 3A) and HBsAg (Figure 3B) mediated by the top gRNA candidates from the selection (HBVg_192 (SEQ ID NO: 582), HBVg_17 (SEQ ID NO: 407), HBVg_63 (SEQ ID NO: 453)) at day 5 post-transfection. [Figure 4]Figure 1 shows the fold change in total HBV RNA mediated by each guide RNA and DNMT3A / L-dCas9-KRAB-effector fusion protein. The fold change is shown relative to the central targeting position of each gRNA across the HBV genome. [Figure 5] Shown is the suppression of total HBV RNA mediated by the top gRNA candidates (HBVg_142 (SEQ ID NO:532), HBVg_138 (SEQ ID NO:528), HBVg_185 (SEQ ID NO:575), HBVg_152 (SEQ ID NO:542)) from the screen at 41 and 61 days post-transfection. [Figure 6A] Figures 6A-6B show the sustained and stable suppression of total HBV RNA mediated by gRNAs (HBVg_63 (SEQ ID NO:453), HBVg_185 (SEQ ID NO:575), and HBVg_56 (SEQ ID NO:446)). [Figure 6B] Figures 6A-6B show the sustained and stable suppression of total HBV RNA mediated by gRNAs (HBVg_63 (SEQ ID NO:453), HBVg_185 (SEQ ID NO:575), and HBVg_56 (SEQ ID NO:446)). [Figure 7] Normalized total HBV RNA expression after re-administration of exemplary gRNA and epi-editor combinations is shown. [Figure 8] Figure 1 shows the fold change in 3.5 kilobases (kb) of HBV RNA mediated by each guide RNA and the DNMT3A / L-dSpCas9-KRAB effector fusion protein in a true infection model. The fold change is shown relative to the central targeting position of each gRNA across the HBV genome. [Figure 9] Figure 1 shows the inter-infection consistency using a correlation plot between the two cccDNA selections. [Figure 10] gRNAs that suppressed either integrated HBV, cccDNA, or both integrated and cccDNA are shown. [Figure 11]We list single gRNAs that targeted HBV cccDNA and integrated DNA and achieved multiplexed HBV inhibition at multiple genes and their regulatory elements. [Figure 12] We list sequences targeted by a single gRNA that achieved multiplexed HBV suppression at multiple genes and their regulatory elements. [Figure 13] Figure 1 shows the fold change in total HBV RNA mediated by each guide RNA and an exemplary epi-editor. The fold change is shown relative to the central targeting position of each gRNA across the HBV genome. [Figure 14] Figure 1 shows the suppression of target HBV cccDNA transcripts by individual gRNAs. [Figure 15A] Figures 15A-15B show suppression of cccDNA in a primary human hepatocyte (PHH) infection model. Figure 15A shows suppression of HBV RNA mediated by HBVg_22 (SEQ ID NO: 412) from two PHH donors. Figure 15B shows suppression mediated by HBVg_22 (SEQ ID NO: 412) in PHH cells infected with two doses of HBV. [Figure 15B] See legend to Figure 15A. [Figure 16] Figures 16A-16B show a comparison between suppression mediated by HBVg_22 (SEQ ID NO:412) in combination with either dSpCas9-KRAB alone (SEQ ID NO:595) or DNMT3A / L-dSpCas9-KRAB ("D3AL-K"; SEQ ID NO:645) fusion in the HepG2.NTCP (Figure 16A) and PxB PHH (Figure 16B) models. [Figure 17] Figure 1 shows multiplexed targeted transcriptional repression of different regions within HBV RNA in Hep3B cells. [Figure 18] Figure 1 shows repression mediated by either individual gRNAs or multiple gRNAs in a PLC / PRF / 5 (Alexander) cell model. [Figure 19]We demonstrate a multiplexed approach using a combination of two gRNAs and an exemplary dSpCas9-effector in the PXB primary human hepatocyte (PHH) cell model. [Figure 20A] Figures 20A and 20B show methyl capture sequencing analysis of cccDNA and integrated HBV DNA. [Figure 20B] Figures 20A and 20B show methyl capture sequencing analysis of cccDNA and integrated HBV DNA. [Figure 21] Figure 1 shows the increase in methylation patterns after delivery of mRNA encoding DNMT3A / L-dSpCas9-KRAB together with various gRNAs. [Figure 22] 1 shows persistent CpG island 2 methylation patterns after delivery of mRNA encoding DNMT3A / L-dCas9-KRAB and HBVg_22 (SEQ ID NO:412) into an HBV-infected PHH donor. [Figure 23A] Figure 23A and Figure 23B reveal RNA sequencing analysis of gRNA- and epi-editor-dependent changes to gene expression. [Figure 23B] Figure 23A and Figure 23B reveal RNA sequencing analysis of gRNA- and epi-editor-dependent changes to gene expression. [Figure 23C] Figure 23C shows minimal changes in differentially expressed genes mediated by gRNA compared to the lipid-only control. [Figure 23D] Figure 23D shows that there are no differentially expressed genes between non-targeting gRNA and HBVg_22 (SEQ ID NO:412) at any dose or time point. [Figure 24] 1 shows a schematic representation of an in vivo study in human chimeric liver mice. [Figure 25]Figure 1 shows suppression mediated by an exemplary DNMT3A / L-dCas9-KRAB fusion protein in combination with gRNA HBVg_22 (SEQ ID NO:412) 5 days (D5) after administration. Suppression was measured by monitoring HBsAg protein levels and HBV DNA 2 days before (pre-donation) and 5 days after (D5) lipid nanoparticle administration. [Figure 26] Pre- vs. post-treatment fold change in metric is shown. [Figure 27A] Figures 27A-27D show suppression in FRG mice after refeeding with HBVg_22 (SEQ ID NO:412) alone or multiplexed with HBVg_185 (SEQ ID NO:575), or delivered by GalNAc-conjugated LNPs, in combination with mRNA encoding either dCas9-KRAB (Figures 27A-27C) or DNMT3A / L-dCas9-KRAB (Figure 27D). [Figure 27B] See legend to Figure 27A. [Figure 27C] See legend to Figure 27A. [Figure 27D] See legend to Figure 27A. [Figure 28A] Figures 28A-28C show human chimeric FRG mice after delivery with GalNAc-conjugated PEG LNPs. Figure 28A shows stable suppression after a single dose of LNPs containing mRNA encoding DNMT3A / 3L-dSpCas9-KRAB and HBVg_22 (SEQ ID NO: 412) at day 33. Figure 28B shows tracking of the suppression level in one mouse. Figure 28C shows tissue samples with a significant reduction in pgRNA signal in HBVg_22 (SEQ ID NO: 412)-delivered mice compared to mice administered a non-targeting gRNA. [Figure 28B] See legend to Figure 28A. [Figure 28C] See legend to Figure 28A. [Figure 29A]Figures 29A-29B show the fold change in total HBV RNA mediated by each fusion protein containing a KRAB epi-editor and a zinc finger protein (ZFP). Two batches of ZFP-KRAB fusion proteins were screened for HBV suppression in Hep3B cells (Figures 29A and 29B). [Figure 29B] See legend to Figure 29A. [Figure 30] 1 shows a preliminary comparison between the repression induced by ZFP-KRAB fusion proteins and dCas9-KRAB fusion proteins in combination with gRNA in Hep3B cells. [Figure 31A] Figures 31A-31B show the targeting location of each ZFP along the X-promoter (HBx) region. The highlighted regions in Figure 31A represent the regions within the HBx promoter targeted by the most effective gRNAs (HBVg_22 and HBVg_63) as well as the most effective ZFP-KRAB fusion proteins (eZFP_18, eZFP_19, and eZFP_25). Figure 31B shows the sites within the HBx region targeted by gRNAs (HBVg_22 and HBVg_63) and ZFP-KRAB fusion proteins (eZFP_18, eZFP_19, and eZFP_25). [Figure 31B] See legend to Figure 31A. [Figure 32] Figures 32A-32B show the fold change in total HBV RNA mediated by each eZFP-KRAB fusion protein in HepG2.NTCP cells. [Figure 33] The fold change in total HBV RNA mediated by each eZFP-KRAB fusion protein is shown along with the conservation of the fusion protein across HBV subtypes. [Figure 34] The fold change in total HBV RNA mediated by each DNMT3A / L-eZFP-KRAB fusion protein at days 4 and 15 post-transfection is shown. [Figure 35]Shows minimal changes in differentially expressed genes mediated by eZFP-KRAB fusion proteins compared to lipid-only controls. [Figure 36] Figures 36A-36B reveal RNA sequencing analysis of eZFP-KRAB fusion protein-dependent changes to gene expression compared to either GFP (Figure 36A) or a non-targeting (NT) control (Figure 36B). DETAILED DESCRIPTION OF THE INVENTION

[0118] Detailed Description Hepatitis B is a potentially life-threatening liver infection caused by the hepatitis B virus (HBV). HBV infection is a global public health problem that leads to chronic liver infection and increases the risk of cirrhosis and liver cancer. The WHO estimated that 296 million people worldwide were living with chronic hepatitis B infection in 2019, including 1 million in the United States, with 1.5 million new infections each year. In 2019, hepatitis B resulted in an estimated 820,000 deaths, primarily from cirrhosis and hepatocellular carcinoma (primary liver cancer).

[0119] 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)). HBV virions contain a compact, partially double-stranded, approximately 3.2 kb incomplete double-stranded DNA (rcDNA) genome. The genome contains four lesions: a covalently bound HBV polymerase and a 10-nucleotide (nt) DNA flap at the 5' end on the minus strand; and a 5'-capped RNA primer and a single-stranded DNA (ssDNA) gap on the plus strand. The HBV genome is an approximately 3.2 kilobase double-stranded DNA molecule, but can be longer or shorter (e.g., up to 3300 bp or more in size) depending on the specific HBV strain. An exemplary HBV genome is the hepatitis B virus genome (hepatitis B virus subtype ayw, complete genome, GenBank: U95551.1), SEQ ID NO: 650. At least 10 genotypes (A-J) have been identified, with divergence between genotypes of 8% or less. Genotype subtypes also exist, including those classified as HBV genotypes A (A1-A7), genotypes B (B1-B9), genotypes C (C1-C16), genotypes D (D1-D8), and genotypes F (F1-F4) (Zhang et al. World J Gastroenterol., 2015, 22:126-144). Within a genotype, the divergence in sequence identity is only about 4%. It is understood that the provided systems and methods are applicable to multiple HBV genomes, particularly given the high sequence similarity. For purposes herein, reference to the numbering of nucleotide positions is the nucleotide (base pair) numbering of the HBV DNA sequence set forth in GenBank Accession No. U95551.1, as shown in SEQ ID NO:650.Those skilled in the art understand that, for example, the target sites or base pair positions described herein in another HBV genome may not be at the same position, but may still be homologous or substantially homologous (e.g., one, two, or three mismatches), such as when determined by aligning the HBV genome sequence with the sequence set forth in SEQ ID NO:650. Thus, the corresponding position or positions can be readily identified by aligning the HBV genome sequence with the reference sequence set forth in SEQ ID NO:650. Hepatitis B virus comprises a circular genome; therefore, for purposes herein, references to the numbering of nucleotide positions in a linear HBV DNA sequence may be shifted by a small number of nucleotides, for example, depending on the start of the linear sequence. For example, the sequences set forth in SEQ ID NO:650 and SEQ ID NO:1071 are the same sequence, but the start of the linear sequence is shifted by two nucleotides due to differences in the start of the linear sequence. Identifying corresponding sequence regions between different sequences of an HBV genome sequence is well within the skill of one of ordinary skill in the art.

[0120] The HBV life cycle involves processes such as viral entry, cccDNA formation, transcription, replication, assembly, secretion, and integration. After viral entry into hepatocytes via the bile acid transporter NTCP11, the viral nucleocapsid containing the HBV rcDNA is transported to the nucleus. The rcDNA is released, and four lesions on the rcDNA are completely repaired to form a supercoiled cccDNA molecule (also known as a minichromosome). Viral repair factors are not required for repair, and cccDNA often relies on host DNA repair mechanisms, including TDP2, DNA polymerase (POL) κ, POL α, DNA ligases 1 and 3, and flap endonuclease 1. HBV hijacks host ubiquitous and liver-enriched transcription factors for cccDNA transcriptional regulation. cccDNA is the primary viral reservoir driving chronic HBV infection and serves as the template for all HBV viral transcripts. Another form of HBV DNA in the host is stably integrated HBV DNA into the host genome (Zhao K., et al., Cell Press-The Innovation 1(2): 1-10 (2020)). Double-stranded linear DNA (dslDNA) is the preferred substrate for integration into the host genome. Because there is little sequence homology between viral and cellular DNA, the NHEJ DNA repair pathway has been proposed as the mechanism for HBV DNA integration. HBV DNA integration occurs at double-strand breaks throughout the host genome, and terminal deletions of up to 200 bp from the integrated HBV DNA are common. Neither specific chromosomal hotspots nor common recurrence sites have been observed among patients. There is some evidence of enrichment at specific genomic sites within tumor tissues (Sung W., et al., Nature Genetics 44(7):765-9 (2012)). Although progeny virus is not produced, integrated HBV DNA can produce viral RNA and proteins. HBV DNA integration occurs more frequently in hepatoma cells (84%) than in normal liver tissue (30%).

[0121] Current standard treatments include nucleoside analogs (e.g., lamivudine) and pegylated interferon therapy. Nucleoside analogs act by inhibiting HBV polymerase activity, resulting in a decrease in viral replication. However, prolonged treatment duration, increased viral resistance, and the emergence of mutant strains have limited the effectiveness of nucleoside therapy (Papatheodoridis GV et al., Am. J. Gastroenterol 97(7):1618-28 (2002). PEGylated interferon therapy, alone or in combination with nucleoside analogs (e.g., lamivudine), has been tested to suppress viral DNA transcription. PEGylated interferon therapy has been shown to mediate distinct effects on the innate and adaptive arms of the immune system, having a significant depleting effect 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 of Infectious Disease 212(7):1042-51 (2015) marcellin P., et al., New England Journal of Medicine 351(12):1206-17 (2004)). Neither nucleotide analogs nor PEGylation therapy can eliminate or suppress the production of HBV surface antigen (HBsAg), which is associated with a poor prognosis for HBV infection. Other therapies, including antisense oligonucleotide (ASO) and siRNA approaches focused on reducing HBsAg to achieve a functional cure (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 promise in inhibiting the synthesis of HBsAg, HBeAg, and HBV DNA.However, the functional benefit of any of these treatments on liver tissue regeneration is unclear.

[0122] Current antiviral therapies rarely achieve cure because they inhibit replication of the cytoplasmic HBV genome and do not directly target cccDNA, a form that serves as an HBV replication intermediate and residual viral reservoir (Yang G., et al., Theranostics 9(24):7345-58 (2019)). Genome manipulation approaches, such as nucleases or base editors, target the removal or mutagenesis of the cccDNA pool to functionally cure the infection. However, such nuclease-based therapies have the potential to generate chromosomal abnormalities and are therefore undesirable, highlighting the need for better HBV therapies.

[0123] The persistence of episomal cccDNA pools in infected hepatocytes remains a significant obstacle to their complete elimination by anti-HBV therapy. cccDNA accumulates in the nucleus as chromatin-like cccDNA minichromosomes assembled by histones and non-histones. Due to its non-native state, cccDNA exhibits aberrant chromatin regulation. For example, changes in the epigenetic state of cccDNA have been found to direct 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 sites H4K5 and H4K12, contributing to the assembly of cccDNA. Acetylation marks on the histones of cccDNA in turn promote HBV replication and cccDNA accumulation. This transcriptional activity is primarily driven by the presence or absence of activating epigenetic marks on cccDNA; repressive histone marks (e.g., H3K27me3 and H3K9me3) are scarce, suggesting limited repression in cccDNA (Tropberger P. et al., PNAS, 112(42):E5715-E5724 (2015), Riviere L., et al., J Hepatol 15(00450):S0168-8278 (2015)).

[0124] Favorable clinical outcomes are associated with key epigenetic features within the cccDNA minichromosome. Studies have found that cccDNA contains methylation-prone CpG islands that are associated with HBV behavior (Zhang Y., et al., PlosOne 9(10):e110442 (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 correlated with low levels of serum HBV DNA and HBsAg titers in patients (Zhang Y., et al., PlosOne 9(10):e110442 (2014)). HBV genotype, HBeAg positivity, patient age, and stage of liver fibrosis were found to correlate with the CpG methylation status of cccDNA. In vitro methylation studies further confirmed that methylation of CpG island II could significantly reduce cccDNA transcription and subsequent viral core DNA replication (Zhang Y., et al., PlosOne 9(10):e110442 (2014)), demonstrating the importance of chromatin as a potential target for regulating cccDNA and for the treatment of chronic HBV infection.Antiviral drugs and broad-spectrum epigenetic modifiers, such as IFNα, have been implicated in reducing post-translational modifications of active histones, thereby transcriptionally downregulating cccDNA transcription (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)).

[0125] The provided embodiments are based on the recognition that epigenetically silencing one or more HBV viral genes, including those present in cccDNA, may be a viable therapeutic approach for curing HBV infection. Disclosed herein are approaches for achieving amelioration of infection and potentially functional cure from HBV through precise epigenetic silencing of cccDNA forms, incomplete double-stranded DNA (rcDNA) forms, and HBV integrated into human genomic DNA. The approaches described herein demonstrate high efficacy, safety, and stability. In some embodiments, these approaches target all forms of HBV in the same approach, utilize a non-mutagenetic platform, and target the transcription source rather than downstream transcripts. Because methylation can be inherited by cellular progeny, the persistence of epi-editing approaches holds promise for treating HBV infection. In some embodiments, the approaches described herein target multiple locations on the viral genome to ensure a deep and sustained response across HBV variants. In some embodiments, epigenetic approaches result in silencing of HBV replication, HBV transcription, and protein production from HBV DNA. Provided embodiments are based on direct epigenetic silencing (e.g., HBV suppression) without the requirement of immune restart or elimination of infected hepatocytes.

[0126] The embodiments provided herein include an epigenetic modifying DNA targeting system comprising at least one DNA targeting module for inhibiting the transcription of one or more hepatitis B virus (HBV) genes and / or their regulatory elements; wherein each of the at least one DNA targeting module comprises a fusion protein comprising (a) a DNA binding domain for targeting a target site within the hepatitis B virus DNA sequence, and (b) at least one transcriptional repressor effector domain. In some embodiments, the provided epigenetic modifying DNA targeting system is for multiplexed targeted inhibition of multiple different genes or their regulatory elements that regulate hepatitis B virus (HBV) replication and / or HBV transcription. In some embodiments, the epigenetic modifying DNA targeting system comprises multiple DNA targeting modules for inhibiting the transcription of multiple genes or their regulatory elements that regulate hepatitis B virus (HBV) replication and / or HBV transcription. In some embodiments, the DNA targeting module comprises: (a) a fusion protein comprising a CRISPR-associated (Cas) protein or a variant thereof and at least one transcriptional repressor effector domain; and (b) a plurality of guide RNAs (gRNAs), including at least a first gRNA and a second gRNA. In some embodiments, the first gRNA targets a target site in a first gene or its regulatory element, and the second gRNA targets a target site in a second gene or its regulatory element. The first and second genes or their regulatory elements regulate hepatitis B virus replication and / or HBV transcription. Polynucleotides, vectors, and compositions containing the DNA targeting system or fusion proteins of the DNA targeting system are also provided herein.

[0127] In some embodiments of the provided epigenetic modifying DNA targeting systems, the DNA binding domain is a nuclease-inactive CRISPR-associated (Cas) protein or a variant thereof, such as dead Cas (dCas, e.g., dCas9), and the DNA targeting system further comprises at least one gRNA capable of forming a complex with Cas. In some embodiments, the DNA binding domain is a nuclease-inactive CRISPR-associated (Cas) protein or a variant thereof complexed with a guide RNA (gRNA). In such systems, the gRNA has a spacer sequence capable of hybridizing to a target site of a gene or its regulatory element. Cas / gRNA combinations, polynucleotides, associated gRNA containing compositions, and methods involving or related to the epigenetic modifying DNA targeting system are also provided herein.

[0128] In some embodiments of the provided epigenetic modifying DNA targeting system, the DNA binding domain is a protein domain engineered for sequence-specific binding to target site.For example, in some embodiments, the DNA binding domain is a zinc finger (ZFN)-based DNA binding domain as described herein, or a transcription activator-like effector DNA binding domain.

[0129] Also provided herein is the method of using epigenetic modifying DNA targeting system to modulate the transcription or phenotype of liver cell.Also provided herein is the method of using epigenetic modifying DNA targeting system to suppress HBV replication and / or protein level.In some embodiments, the method can be used in the therapy of treating HBV infection, such as hepatitis.

[0130] In some embodiments, the target site is present in covalently closed circular DNA (cccDNA), incomplete double-stranded DNA (rcDNA), and / or integrated into genomic DNA. In some embodiments, the target site is located in or near a gene or its regulatory element, e.g., a regulatory element or coding region, involved in HBV replication and / or HBV transcription. Also provided herein is an epigenetic modification DNA targeting system that is multiplexed with multiple DNA targeting modules, thereby enabling the system to target a combination of such genes or their regulatory elements. In some embodiments, each module of the DNA targeting system suppresses the transcription of a different gene. Also provided herein is a method of using the epigenetic modification DNA targeting system to reduce HBV replication and / or transcription. In some embodiments, the method can be used to treat liver disease (e.g., hepatitis), cancer (e.g., hepatocellular carcinoma), or HBV infection (acute or chronic hepatitis).

[0131] Thus, in some embodiments, the DNA targeting system comprises a synthetic transcription factor capable of modulating, for example, reducing or suppressing, gene transcription in a targeted manner. In provided embodiments, the provided epigenetic modifying DNA targeting system reduces the transcription of a gene and / or its regulatory element or multiple genes and / or their regulatory elements, thereby promoting the silencing of HBV replication and / or transcription. The provided embodiments can be used to target multiple genetic mechanisms to treat HBV in infected patients while avoiding viral resistance, the costs associated with long-term treatment, and the lack of efficacy of current combination therapies. This approach reduces not only viral replication but also transcription from both cccDNA and integrated HBV DNA, thereby providing a substantial clinical solution to the treatment of HBV infection by avoiding the problems associated with current therapies.

[0132] All publications, including patent documents, scientific articles, and databases, referenced 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. To the extent that a definition set forth herein contradicts or otherwise conflicts with a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth herein shall take precedence over the definition incorporated herein by reference.

[0133] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0134] I. DNA Targeting Systems In some embodiments, a DNA targeting system is provided, which can specifically target a target site in at least one gene (also referred to herein as target gene) or its DNA regulatory element (e.g., regulatory element) to reduce the transcription of at least one gene. In provided embodiments, for each of the target genes or their regulatory elements to be targeted, the DNA targeting system comprises a DNA binding domain that binds to the target site in the gene or its regulatory element. In some embodiments, the DNA targeting system additionally comprises at least one effector domain that can epigenetically modify one or more DNA bases of the gene or its regulatory element, and this epigenetic modification leads to reduced transcription of the gene (e.g., inhibit or reduce transcription of the gene compared to the absence of the DNA targeting system). Thus, the terms DNA targeting system and epigenetic modifying DNA targeting system can be used interchangeably herein. In some embodiments, the DNA targeting system comprises a fusion protein comprising (a) at least one DNA binding domain that can be targeted to a target site, and (b) at least one effector domain that can reduce transcription of the gene. By way of example, at least one effector domain is a transcriptional repressor domain.

[0135] In some embodiments, the DNA targeting system contains at least one DNA targeting module, and each DNA targeting module of the system is a component of the DNA targeting system that can independently target one target site in a target gene or its regulatory element as provided. In some embodiments, each DNA targeting module includes (a) a DNA binding domain that can be targeted to a target site in a target gene or its regulatory element that regulates HBV replication and / or HBV transcription, and (b) an effector domain that can reduce the transcription of the gene.

[0136] In some embodiments, the DNA targeting system comprises a single DNA targeting module for targeting the repression of a single gene. In some embodiments, the DNA targeting module comprises (a) a DNA binding domain capable of being targeted to a target site of a target gene or its regulatory element that regulates HBV replication and / or HBV transcription, and (b) an effector domain capable of reducing transcription of the gene.

[0137] In some embodiments, the DNA targeting system comprises a single DNA targeting module for targeting the suppression of more than one gene or its regulatory elements. Thus, in some embodiments, a single DNA targeting module provides a multiplexed epigenetic modification DNA targeting system that targets more than one gene or its regulatory elements for modulation (e.g., suppression). In some embodiments, the DNA targeting module comprises (a) a DNA binding domain that can be targeted to the target site of more than one target gene or its regulatory elements that regulates HBV replication and / or HBV transcription, and (b) an effector domain that can reduce the transcription of the gene. In some embodiments, the DNA targeting system comprises a single DNA targeting module for targeting the suppression of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, or at least 30 genes or their regulatory elements. In certain embodiments, the DNA targeting module is cross-reactive with each of the target sites of more than one gene. In some embodiments, a single DNA targeting module provides a multiplexed epigenetic modifying DNA targeting system that suppresses the transcription of more than one gene or its regulatory elements. In some embodiments, the DNA targeting module suppresses the transcription of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, or at least 30 genes or their regulatory elements.

[0138] In some embodiments, the DNA targeting system is a multiplexed DNA targeting system, comprising a plurality of DNA targeting modules, each DNA targeting module targets different target sites of one or more genes or their regulatory elements.In some embodiments, different target sites are in the same region of gene or its regulatory elements.In some embodiments, different target sites are present in regulatory elements, such as promoters.In some embodiments, target sites overlap, so that any two or more DNA targeting modules bind to overlapping target sites.

[0139] In some embodiments, DNA targeting system comprises multiple DNA targeting modules, wherein each DNA targeting module is for targeting the suppression of different genes.In some embodiments, DNA targeting system is a multiplexed DNA targeting system, that is, it is targeted to the target site in more than one gene or its regulatory element.The term DNA targeting system can include the multiplexed epigenetic modifying DNA targeting system that comprises more than one DNA targeting module.In some embodiments, each DNA targeting module in the multiplexed epigenetic modifying DNA targeting system targets the target site in a gene or its regulatory element that is different from the other DNA targeting modules in the system, and suppresses different genes.In some embodiments, each DNA targeting module in the multiplexed epigenetic modifying DNA targeting system targets the target site in more than one gene or its regulatory element, and suppresses the transcription of more than one gene. In some embodiments, each DNA targeting module represses transcription of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30 genes.

[0140] The multiplexed epigenetic modifying DNA targeting system comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30 DNA targeting modules, or any value between any of the foregoing. In some embodiments, the multiplexed epigenetic modifying DNA targeting system represses transcription of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, or at least 30 genes.

[0141] In some embodiments, any two DNA targeting modules of a DNA targeting system comprise separate (i.e., non-overlapping) components. In some embodiments, each DNA targeting module of a DNA targeting system comprises separate (i.e., non-overlapping) components. For example, a DNA targeting system may comprise a first DNA targeting module comprising a first fusion protein comprising a DNA binding domain (e.g., a ZFN- or TALE-based DNA binding domain) that targets a first target site, and a second DNA targeting module comprising a second fusion protein comprising a second DNA binding domain (e.g., a ZFN- or TALE-based DNA binding domain) that targets a second target site.

[0142] In some embodiments, any two DNA targeting modules of a DNA targeting system may contain shared (i.e., overlapping) components. In some embodiments, each DNA targeting module of a DNA targeting system contains shared (i.e., overlapping) components. For example, a DNA targeting system may contain: a first DNA targeting module comprising (a) a fusion protein comprising a Cas protein and a transcriptional repressor domain, and (b) a first gRNA complexed with the Cas protein to target a first target site in a first HBV gene or its regulatory element; and a second DNA targeting module comprising (a) a fusion protein of the first DNA targeting module, and (b) a second gRNA complexed with the Cas protein to target a second target site in a second HBV gene or its regulatory element. It will be understood that providing two or more different gRNAs for a given Cas protein allows different molecules of the same Cas protein to be targeted to the target sites of the two or more gRNAs. Conversely, different Cas protein variants (e.g., SpCas9 and SaCas9) are compatible with different gRNA scaffold sequences and PAMs, as described herein. Thus, it is possible to engineer a single DNA targeting system that contains multiple non-overlapping CRISPR / Cas-based DNA targeting modules.

[0143] In some aspects, the present invention provides an epigenetic modifying DNA targeting system, comprising a plurality of DNA targeting modules for suppressing the transcription of a plurality of genes that regulate HBV replication and / or HBV transcription. In some embodiments, the plurality of DNA targeting modules comprises a first DNA targeting module for suppressing the transcription of a first gene of the plurality of genes or their regulatory elements, and a second DNA targeting module for suppressing the transcription of a second gene of the plurality of genes. In some embodiments, each DNA targeting module comprises a fusion protein comprising (a) a DNA binding domain for targeting a target site of one of the plurality of genes, and (b) at least one transcriptional repressor domain. In some embodiments, the target site is located in or near an HBV gene or its regulatory element. In some embodiments, the HBV gene or its regulatory element is involved in the control of HBV replication and / or HBV transcription. The regulatory element may be a promoter region (e.g., a pre-S1 promoter, a pre-S2 promoter, an X promoter, or a basal core promoter), an enhancer region (e.g., an Enh1 or Enh2 enhancer region), or any other transcript processing control region (e.g., a region involved in 5' capping, splicing, and / or 3' polyadenylation).

[0144] In some aspects, provided herein is an epigenetic modifying DNA targeting system for suppressing transcription of one or more Hepatitis B Virus (HBV) genes, the epigenetic modifying DNA targeting system comprising at least one DNA targeting module; each of the at least one DNA targeting module comprising a fusion protein comprising (a) a DNA binding domain for targeting to a target site in a Hepatitis B virus DNA sequence, e.g., an HBV gene or a regulatory element thereof, and (b) at least one transcriptional repressor effector domain.

[0145] In some aspects, the present disclosure provides an epigenetic modifying DNA targeting system for suppressing the transcription of one or more hepatitis B virus (HBV) genes, comprising at least one DNA targeting module, wherein each of the at least one DNA targeting module comprises: (a) a fusion protein comprising a clustered regularly interspaced short palindromic repeats-associated (Cas) protein or a variant thereof and at least one transcriptional repressor effector domain; and (b) a plurality of guide RNAs (gRNAs) targeting a plurality of target sites in a plurality of genes or their regulatory elements, wherein the plurality of genes or their regulatory elements regulate hepatitis B virus replication and / or HBV transcription. In some aspects of the provided embodiments, the plurality of target sites are 2, 3, 4, 5, or 6 different target sites. In some aspects of the provided embodiments, each of the plurality of target sites is in a different HBV gene or its regulatory element.

[0146] In aspects of provided embodiments, the DNA targeting system provided herein targets a gene or its regulatory elements to reduce transcription of one or more Hepatitis B virus (HBV) genes in HBV-infected cells, wherein the reduced transcription modulates one or more activities or functions of the HBV-infected cells, such as the expression of HBV RNA and / or HBV proteins. In some embodiments, the reduced transcription of the gene results in reduced expression of the gene, i.e., reduced gene expression, in the infected cells. In some embodiments, the reduced transcription of the gene, e.g., reduced gene expression, results in reduced expression of a protein, i.e., reduced protein expression, in the infected cells.

[0147] In some aspects, the cell is a liver cell, for example, hepatocyte, hepatic stellate cell (HSC), Kupffer cell, and hepatic sinusoidal endothelial cell.For example, the present invention provides a DNA targeting system that targets a gene or its regulatory element to reduce the transcription of HBV gene in target cell, and the reduced transcription modulates one or more activities or functions of HBV, for example, the transcription and protein expression of HBV.In some embodiments, the reduced transcription of gene results in the reduced expression of gene in target cell, i.e., reduced gene expression.In some aspects, the cell is a liver cell.

[0148] In some aspects, the cells are from a human subject. In some aspects, the cells are cells in a subject (i.e., cells in vivo).

[0149] In some embodiments, the DNA-binding domain comprises or is derived from a CRISPR-associated (Cas) protein, a zinc finger protein (ZFP), a transcription activator-like effector (TALE), a meganuclease, a homing endonuclease, an I-SceI enzyme, or a variant thereof. In some embodiments, the DNA-binding domain comprises a catalytically inactive (e.g., nuclease-inactive or nuclease-inactivated) variant of any of the foregoing. In some embodiments, the DNA-binding domain comprises an inactive Cas9 (dCas9) protein or a variant thereof that is catalytically inactivated and therefore inactive in nuclease activity and unable to cleave DNA.

[0150] 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), e.g., a combination of gRNAs (e.g., two gRNAs or three gRNAs). In some embodiments, the gRNA comprises a spacer sequence capable of targeting and / or hybridizing to a target site. In some embodiments, the gRNA is capable of complexing with a Cas protein or variant thereof. In some aspects, the gRNA directs or recruits a Cas protein or variant thereof to a target site. In some embodiments, the effector domain comprises a transcriptional repressor domain and / or is capable of reducing transcription of a gene. In some embodiments, the effector domain directly or indirectly induces reduced transcription of a gene. In some embodiments, the effector domain induces, catalyzes, or induces transcriptional repression. In some embodiments, the effector domain induces transcriptional repression. In some aspects, the effector domain is selected from a KRAB domain, an ERF repressor domain, an MXI1 domain, a SID4X domain, a MAD-SID domain, a DNMT family protein domain (e.g., DNMT3A or DNMT3B), a fusion of one or more DNMT family proteins or domains thereof (e.g., DNMT3A / L, which includes a fusion of the DNMT3A and DNMT3L domains), an LSD1, a SunTag domain, an EZH2 domain, a partial 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.

[0151] In some embodiments, the fusion protein of the DNA targeting system comprises a dCas9-KRAB fusion protein. In some embodiments, the fusion protein of the DNA targeting system comprises a DNMT3A / L-dCas9-KRAB fusion protein. In some embodiments, the fusion protein of the DNA targeting system comprises a KRAB-dCas9-DNMT3A / L fusion protein.

[0152] Exemplary components and characteristics of DNA targeting systems are provided in the following subsections below.

[0153] A. Target Site and Location In some embodiments herein, the target site is a gene and / or its regulatory element in the hepatitis B virus (HBV) genome. In some embodiments, the target site is present in covalently closed circular DNA (cccDNA), incompletely relaxed circular DNA (rcDNA), and / or integrated into human genomic DNA. In some embodiments, the target site is in a hepatitis B virus DNA sequence. In some embodiments, the hepatitis B virus DNA sequence is an HBV gene or its regulatory element. In some embodiments, the target site is in or near a gene involved in HBV replication and / or HBV transcription. In some embodiments, the epigenetic modifying DNA targeting system includes at least one DNA targeting module for targeting to the target site to suppress one or more HBV transcriptions. In some aspects, suppressing HBV gene transcription, e.g., reduced gene expression, results in silencing of HBV replication (e.g., reduced HBV replication) and / or silencing of HBV transcription.

[0154] In the context of the provided disclosure, an HBV-positive (+) cell (e.g., an HBV-infected cell) is understood to mean that the cell expresses any of the HBV markers described herein (e.g., HBV RNA transcripts and / or proteins). Similarly, a cell negative (-) for a particular marker is understood to be a cell that does not express that marker at undetectable levels. Antibodies and other binding entities can be used to detect the expression level of marker proteins to identify or detect a given cell surface marker. Suitable antibodies can include polyclonal, monoclonal, fragments (such as Fab fragments), single-chain antibodies, and other forms of specific binding molecules. Antibody reagents for the above-mentioned cell surface markers are readily known to those of skill in the art. Numerous well-known methods can be used to assess the expression level of a surface marker or protein, such as affinity-based methods, e.g., immunoaffinity-based methods, e.g., in the case of surface markers, by flow cytometry. In some embodiments, the label is a fluorophore, and the method for detecting or identifying a cell surface marker on cells (e.g., hepatocytes) is by flow cytometry. In some embodiments, different labels are used for 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 the marker and detecting binding of the antibody to the marker.

[0155] In some embodiments, a cell (e.g., hepatocyte) is positive (pos or +) for a particular marker (e.g., HBeAg, HBsAg) if the particular marker, which can be an intracellular or surface marker, is detectably present on or within the cell. In some embodiments, surface expression is positive if staining by flow cytometry is detectable at a level substantially greater than that detected using an isotype-matched control and the same procedure under otherwise identical conditions, and / or at a level substantially similar to, or in some cases higher than, that for cells known to be positive for the marker, and / or at a level higher than that for cells known to be negative for the marker. In some embodiments, cells (e.g., hepatocytes) contacted with a DNA targeting system described herein have reduced expression of a particular marker (e.g., HBeAg) if they stain substantially less than similar cells not contacted with a DNA targeting system.

[0156] In some embodiments, a cell (e.g., a hepatocyte) is negative (neg or -) for a particular marker if the particular marker, which can be an intracellular or surface marker, is not detectably present on or in the cell. In some embodiments, surface expression is negative if staining is not detectable by flow cytometry at a level substantially greater than that detected using an isotype-matched control under otherwise identical conditions and performing the same procedure, and / or at a level substantially less than that detected in cells known to be positive for that marker, and / or at a level substantially similar to that detected in cells known to be negative for that marker.

[0157] In some embodiments, the phenotype of infected cells and / or individuals is functionally characterized. 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 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, antibodies include, but are not limited to, antibodies to anti-HBc-IgM, total anti-HBc, and HBeAg. In some embodiments, RNA transcripts, proteins, and / or antibodies are measured, detected, and / or quantified by any suitable technique known in the art. By way of example, RNA transcripts can be measured, detected, and / or quantified using real-time PCR techniques. HBV proteins (e.g., HBsAg, HBeAg, and / or HBcrAg) can be measured, detected, and / or quantified using enzyme-linked immunosorbent assay (ELISA).

[0158] The target genes and / or their regulatory elements for modulation by the provided DNA targeting systems, including the multiplexed epigenetic modifying DNA targeting systems herein, include any whose transcription and expression is reduced in cells (e.g., HBV-infected cells). Various methods can be used to characterize the transcription or expression level of genes in cells (e.g., hepatocytes), such as after contacting or introducing the provided DNA targeting systems into cells. In some embodiments, analyzing the transcription activity or expression of genes can be done by RNA analysis. In some embodiments, RNA analysis includes RNA quantification. In some embodiments, RNA quantification is performed by reverse transcription quantitative PCR (RT-qPCR), multiplexed qRT-PCR, fluorescent in situ hybridization (FISH), RNA sequencing (RNA-seq), or a combination thereof.

[0159] In some embodiments, the expression or presence of a gene or transcript in a cell (e.g., HBV-infected cell, e.g., hepatocyte) is reduced after contacting or introducing a provided DNA targeting system, such as a multiplexed epigenetic DNA targeting system.In some aspects, multiple genes or transcripts are targeted by the multiplexed epigenetic DNA targeting system, for example, by one or more DNA targeting modules thereof.In such a system, each gene or transcript of the multiplexed DNA targeting system is reduced after contacting or introducing a provided multiplexed epigenetic DNA targeting system. In some embodiments, the reduction in gene expression or change in transcript level in a cell (e.g., an HBV-infected cell, e.g., a hepatocyte) is about a log2 fold change of at least 1.25x, 1.5x, 1.75x, 2.0x, 2.5x, 2.75x, 3.0x, 3.25x, 3.5x, 3.75x, 4.0x, 4.25x, 4.5x, 4.75x, 5.0x, 5.25x, 5.5x, 5.75x, 6.25x, 6.50x, 6.75x, 7.0x, 7.25x, 7.50x, 7.75x, 8.0x, 8.25x, 8.5x, 8.75x, 9.0x, or any value in between, relative to the level of the gene in a control cell. In some embodiments, the reduction in gene expression or change in transcript level in cells (e.g., HBV-infected cells, e.g., hepatocytes) 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 above, compared to the level of the gene in control cells. In some embodiments, the reduction in gene expression or change in transcript level in cells is more than 90% compared to the level of the gene in control cells. In some embodiments, the guide RNA is set forth in SEQ ID NO:565, 528, 542, 508, 515, 575, 515, 453, 506, 514, 425, or 472.In some embodiments, the reduction in gene expression or change in transcript level in the cell is greater than 75% compared to the level of the gene in a control cell. In some embodiments, the guide RNA is selected from the group consisting of SEQ ID NOs: 565 (HBVg_175), 528 (HBVg_138), 582 (HBVg_192), 542 (HBVg_152), 508 (HBVg_118), 515 (HBVg_125), 575 (HBVg_185), 453 (HBVg_63), 506 (HBVg_116), 514 (HBVg_124), 395 (HBVg_5), 472 (HBVg_8 2), 451(HBVg_61), 488(HBVg_98), 540(HBVg_150), 533(HBVg_143), 572(HBVg_182), 566(HBVg_17 6), 489(HBVg_99), 469(HBVg_79), 408(HBVg_18), 465(HBVg_75), 402(HBVg_12), 474(HBVg_84), 5 25(HBVg_135), 416(HBVg_26), 396(HBVg_6), 554(HBVg_164), 419(HBVg_29), 545(HBVg_155), 446 (HBVg_56), 580(HBVg_190), 555(HBVg_165), 412(HBVg_22), 428(HBVg_38), 458(HBVg_68), 548(H BVg_158), 511(HBVg_121), 432(HBVg_42), 441(HBVg_51), 433(HBVg_43), 579(HBVg_189), 479(HBVg_89), 478(HBVg_88), 520(HBVg_130), 462(HBVg_72), 523(HBVg_133), and 503(HBVg_113).

[0160] In a provided embodiment, cccDNA transcribes five HBV RNAs (0.7 kb, 2.1 kb, 2.4 kb, long, and short 3.5 kb RNAs) under the influence of host RNA polymerase. Transcription of cccDNA is controlled by four promoters, the basic core, pre-S1, pre-S2, and X promoters, and two enhancers, enhancer I and II (Figure 1). The 0.7 kb RNA can be translated into the HBV X protein (HBx), which acts as a transcriptional regulator. The 2.1 kb RNA can be translated into the HBV small surface protein (S) and middle surface protein (M). The 2.4 kb RNA can be translated into the HBV large surface protein (L). L, M, and S can self-assemble to form hollow subviral particles (SVPs) (including spherical and filamentous SVPs), and only filamentous SVPs are secreted in virions containing large amounts of L protein. Globular SVPs are secreted via the constitutive secretory pathway. Filamentous SVPs are secreted via the endosomal sorting complex transport (ESCRT) machinery through multivesicular bodies (MVBs). The long 3.5 kb RNA, called pre-core RNA (pre-C RNA), can be translated into the pre-core protein commonly known as HBV e antigen (HBeAg). The short 3.5 kb RNA is a pregenomic RNA (pgRNA) that serves two roles: as a translation template for HBV polymerase (Pol) and core protein, and as a replication template for reverse transcription within the capsid (formed by core protein polymerization) by Pol to form HBV rcDNA. These nucleocapsids are then enveloped by HBV surface proteins (L, M, and S) to form mature virions, which can be secreted via the ESCRT / MVB pathway. Alternatively, these nucleocapsids can be transported to the nucleus to form cccDNA. In some embodiments, suppressing the transcription and / or translation of HBV genes, e.g., reduced gene expression, results in silencing of any of the following HBV markers: HBV HBV X protein (HBx), hepatitis B surface antigen (HBsAg), HBV e antigen (HBeAg), such as the small surface protein (S), middle surface protein (M), or HBV large surface protein (L).In some embodiments, suppressing HBV gene transcription and / or translation, e.g., reduced gene expression, results in silencing of HB core-related antigen (HBcrAg). HBcrAg comprises three precore / core protein products, including hepatitis B core antigen (HBcAg), HBeAg, and 22 kDA precore protein (p22cr). In some aspects, cccDNA, HBV total DNA, serum HBcrAg, HBsAg, HBeAg, hepatitis B core antibody (anti-HBc), HBV DNA, and HBV RNA are quantified as readouts to measure reduced HBV transcription and / or translation. In some embodiments, the target site is in a gene encoding any of the HBV proteins. In some embodiments, the target site is in a regulatory element (e.g., a promoter or enhancer) of a gene encoding any of the HBV proteins.

[0161] In some embodiments, the target site of the epigenetic modifying DNA targeting system is in a gene involved in HBV replication and / or HBV transcription. In some aspects, the target site of the epigenetic modifying DNA targeting system is in or near a gene or its regulatory element involved in the control of HBV replication and / or HBV transcription. In some embodiments, the gene involved in HBV replication and / or HBV transcription is a polymerase gene, an S-family gene, an X-gene, and / or a core-family gene. In some embodiments, the gene involved in HBV replication and / or transcription encodes a polymerase, an envelope protein, a capsid protein, a transcription factor, or a transcription transactivator. In some embodiments, the regulatory element involved in HBV replication and / or HBV transcription is a promoter region, an enhancer region, and / or any transcript processing control region. In some embodiments, the promoter region is a pre-S1 promoter, a pre-a S2 promoter, an X promoter, or a basal core promoter. In some embodiments, the enhancer region is an Enh1 enhancer and / or an Enh2 enhancer region. In some embodiments, the transcript processing control region is a region encoding signals for 5'-end capping, splicing, and / or 3'-end polyadenylation.

[0162] In some embodiments, the target site is a sequence within a target region having a sequence corresponding to a sequence located between base pair (bp) positions 1 bp to 42 bp, 491 bp to 1032 bp, 1750 bp to 1799 bp, or 1951 bp to 2952 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 650. In some embodiments, the target site is within a target region of the HBV genome having the sequence set forth in SEQ ID NO: 1056, or a complementary sequence thereof. In some embodiments, the target site is within a target region of the HBV genome having the sequence set forth in SEQ ID NO: 1058, or a complementary sequence thereof. In some embodiments, the target site is within a target region of the HBV genome having the sequence set forth in SEQ ID NO: 1060, or a complementary sequence thereof. In some embodiments, the target site is within a target region of the HBV genome having the sequence set forth in SEQ ID NO:1062 or its complementary sequence.

[0163] In some embodiments, the target site is a sequence within a target region having a sequence corresponding to a sequence located between base pair (bp) positions 43 bp to 490 bp, 1033 bp to 1749 bp, 1800 bp to 1950 bp, or 2953 bp to 3182 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:650. In some embodiments, the target site is within a target region of the HBV genome having the sequence set forth in SEQ ID NO:1057, or a complementary sequence thereof. In some embodiments, the target site is within a target region of the HBV genome having the sequence set forth in SEQ ID NO:1059, or a complementary sequence thereof. In some embodiments, the target site is within a target region of the HBV genome having the sequence set forth in SEQ ID NO:1061, or a complementary sequence thereof. In some embodiments, the target site is within a target region of the HBV genome having the sequence set forth in SEQ ID NO:1063 or its complementary sequence.

[0164] In some embodiments, the target site is a sequence within a target region having a sequence corresponding to a sequence located between base pair (bp) positions 67 bp to 392 bp (CpG island 1), 1033 bp to 1749 bp (CpG island 2), or 2215 bp to 2490 bp (CpG island 3) 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:650. In some embodiments, the target site is within a target region of the HBV genome having the sequence set forth in SEQ ID NO:1064, or a complementary sequence thereof. In some embodiments, the target site is within a target region of the HBV genome having the sequence set forth in SEQ ID NO:1059, or a complementary sequence thereof. In some embodiments, the target site is within a target region of the HBV genome having the sequence set forth in SEQ ID NO:1066 or its complementary sequence.

[0165] In some embodiments, the target site is in the polymerase gene or its regulatory elements. The polymerase gene (also known as the P gene) encodes a multifunctional enzyme (P polymerase, also known as HBVgp1, DNA-directed DNA polymerase) that converts the viral RNA genome to dsDNA within the viral cytoplasmic capsid. The polymerase exhibits DNA polymerase activity, which can copy either DNA or RNA templates, and RNase H activity, which cleaves the RNA strand of an RNA-DNA heteroduplex in a partially processive 3'- to 5'-endonuclease mode. The polymerase gene ORF completely overlaps with the pre-S / S ORF and partially overlaps with the core family and X gene ORFs. In some embodiments, the target site is a sequence within a target region having a sequence corresponding to a sequence located between 1 bp and 1621 bp, 1374 bp and 1838 bp, or 2307 bp and 3182 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:650.

[0166] In some embodiments, the target site is in an S-family gene or its regulatory element. In some embodiments, the target site is in the S gene, pre-S1 promoter, and / or pre-S2 promoter region. S-family genes encode three distinct, structurally related envelope proteins synthesized from alternative start codons, called large (L), medium (M), and small (S) hepatitis B (HB) proteins (also referred to as L-HBs, M-HBs, and s-HBs, respectively). The three proteins share the same carboxy terminus but have different amino-terminal extensions. In some embodiments, the target site is a sequence within a target region having a sequence corresponding to a sequence located between 1 bp and 837 bp, 1 bp and 155 bp, or 2854 bp and 3182 bp of the hepatitis B virus genome (hepatitis B virus subtype ayw, complete genome, GenBank: U95551.1) SEQ ID NO:650.

[0167] In some embodiments, the target site is in the X-gene or its regulatory elements. The X-gene (also known as HBx, HBVgp3, peptide X, pX) is a gene encoding a multifunctional protein that modulates transcriptional regulation, proteolytic pathways, apoptosis, signal transduction, cell cycle progression, and genetic stability by directly or indirectly interacting with host factors. The X-gene protein modulates proteolytic pathways, apoptosis, transcription, signal transduction, cell cycle progression, and genetic stability by directly or indirectly interacting with host factors. In some embodiments, the target site is a sequence within a target region having a sequence corresponding to a sequence located between 1374 bp and 1838 bp of the HBV genome, with reference to SEQ ID NO:650 of the hepatitis B virus genome (hepatitis B virus subtype ayw, complete genome, GenBank: U95551.1). In some embodiments, the start codon encoding the HBx protein (HBx start codon) is at base pair 1376 of the HBV genome, corresponding to the position with reference to the HBV genome set forth in SEQ ID NO:650. In some embodiments, the start codon encoding the HBx protein (HBx start codon) is located at base pair 1374 of the HBV genome, corresponding to the position in reference to the HBV genome set forth in SEQ ID NO:1071. Targeting a target site in the X-gene in this region upstream of the start codon using the epigenetic modifying DNA targeting system provided herein has been found to exhibit high activity in suppressing viral replication and transcription in HBV-infected cells. In some embodiments, the target region is located in a CpG island of the HBV genome. In some embodiments, the target site is a sequence within a target region having a sequence corresponding to a sequence located between 1033 and 1749 bp in reference to the HBV genome set forth in SEQ ID NO:650. In some embodiments, the target site is located in the HBx promoter / enhancer #1 region, e.g., within a target region having a sequence corresponding to a sequence located between 1100 and 1350 bp in reference to the HBV genome set forth in SEQ ID NO:650.In some embodiments, the target site is in the basic core promoter region, within a target region having a sequence corresponding to a sequence located between 1600 and 1750 bp with reference to the HBV genome, e.g., as set forth in SEQ ID NO:650.

[0168] In some embodiments, the target site is within a target region spanning within 300 base pairs (bp), 250 bp, 200 bp, 150 bp, 140 bp, 130 bp, 120 bp, 110 bp, or 100 bp upstream of the HBx start codon. In some embodiments, the target site is within a target region having a sequence corresponding to a sequence located between 1250 and 1374 bp with reference to the HBV genome set forth in SEQ ID NO:650. In some embodiments, the target site is within a target region of the HBV genome having a sequence set forth in SEQ ID NO:1068 or a complementary sequence thereof. In some embodiments, the target site is a sequence within a target region having a sequence corresponding to a sequence located between 1255 and 1302 bp with reference to the HBV genome set forth in SEQ ID NO:650. In some embodiments, the target site is within a target region of the HBV genome, the target site being within a target region having the sequence set forth in SEQ ID NO:1069 or a complementary sequence thereof. In some embodiments, the target site is a sequence within a target region having a sequence corresponding to a sequence located between 1260 and 1300 bp with reference to the HBV genome set forth in SEQ ID NO:650. In some embodiments, the target site is within a target region of the HBV genome, the target site being within a target region having the sequence set forth in SEQ ID NO:1070 or a complementary sequence thereof. In some embodiments, the or each of the target sites is within a target region located between 1060 and 1480 bp of the HBV genome corresponding to a position with reference to the HBV genome set forth in SEQ ID NO:650. In some embodiments, the target site is within a target region of the HBV genome, the target region having the sequence set forth in SEQ ID NO:1067. Exemplary DNA binding systems for targeting target sites within such regions are provided herein, including CRISPR / Cas systems and systems with various DNA binding domains, including ZFPs.

[0169] In some embodiments, the target site is in a core family gene or its regulatory element. In some embodiments, the regulatory element is the Enh2 promoter. In some embodiments, the regulatory element is a basic core promoter (BCP). The core promoter (CP) region of a viral genome plays a central role in viral replication and morphogenesis (Quarleri J, World Journal of Gastroenterology 20(2): 425-435 (2014)). The core promoter region directs the initiation of transcription for the synthesis of both pre-core mRNA and pre-genomic RNA (pgRNA). The CP region consists of a basic core promoter (BCP) that initiates pre-core mRNA (also known as pre-C, C gene, HBVgp4) and pgRNA transcription, and an upstream regulatory region (URR) that contains positive and negative regulatory elements that modulate promoter activity. Several transcription factors bind to regulatory sequence elements of the CP, such as C / EBP, HNF1, HNF3 / 4, and COUP-TF1, to differentially regulate the synthesis of pre-C mRNA and pgRNA. The presence of AT-rich regions or TATA-like boxes within the CP also contributes to the transcription of pgRNA. Pre-core mRNA encodes the outer core antigen (also known as capsid protein, pre-capsid protein, HBeAg, pre-core protein, or p25), which self-assembles to form an icosahedral capsid that packages the viral genome. pgRNA is translated to form the polymerase, nucleocapsid protein HBcAg, and soluble secreted HBeAg protein. pgRNA is then incorporated into progeny nucleocapsids and reverse-transcribed into DNA by the co-assembled viral polymerase, resulting in new HBV virions.These mature, incompletely double-stranded DNA (rcDNA)-containing nucleocapsids can either redeploy their genome to the nucleus of the same cell to assemble a pool of 10-100 copies of cccDNA molecules or interact with envelope proteins in the ER / Golgi and be secreted as new infectious virions (Pollicino T., et al., Journal of Hepatology, 61(2):P408-417 (2014)). In some embodiments, the target site is in a core family gene or its regulatory element. In some aspects, targeting one or more sites within a core family gene or its regulatory element comprises suppression of pgRNA transcripts. In some aspects, suppression of pgRNA transcripts comprises silencing of HBV replication. In some embodiments, the target site is a sequence within a target region having a sequence corresponding to sequences located between 1590 bp to 1815 bp, 1636 bp to 1744, 1751 bp to 1769, 1814 bp to 1900 bp, 1816 bp to 2455, and 1800 bp to 1950 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: 650. In some embodiments, reduced transcription comprises a reduction in total Hepatitis B virus RNA transcript levels. In some embodiments, reduced transcription comprises a reduction in Hepatitis B precore ("pre-C") and / or pregenomic ("pgRNA") RNA levels.

[0170] In some aspects, the target site is a coding region. In some embodiments, the gene involved in HBV replication and / or HBV transcription encodes an S family protein (HBsAg) such as HBV X protein (HBx), small surface protein (S-HBs), middle surface protein (M-HBs), or HBV large surface protein (L-HBs), precore protein (HBeAg), HBV core-related antigen (HBcrAg), polymerase, core, and precore proteins. In some aspects, the target site is a sequence within a target region having a sequence corresponding to a sequence located between 1 bp and 42 bp, 43 bp and 1090 bp, 1091 bp and 1849 bp, or 1850 bp and 2455 bp, or 2455 bp and 3182 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: 650.

[0171] In some embodiments, inhibiting transcription comprises reducing hepatitis B surface antigen (HBsAg) and / or hepatitis B virus core-related antigen (HBcrAg) protein levels. In some embodiments, inhibiting transcription comprises reducing HBsAg transcript and / or protein levels by at least 90%. In some embodiments, inhibiting transcription comprises reducing HBcrAg transcript and / or protein levels by at least 50% from cccDNA.

[0172] In some embodiments, suppressing transcription includes reducing Hepatitis B precore ("preC"), pregenome ("pgRNA"), preS1, preS2 / S, and HBx levels.

[0173] In some embodiments, the multiplexed epigenetic modifying DNA targeting system targets or binds to a target site in a gene, such as any of those described above. In some embodiments, the target site is located in a regulatory DNA element of a gene in a cell (e.g., a hepatocyte). In some embodiments, the regulatory DNA element is a sequence to which a gene regulatory protein can bind and affect the transcription of the gene. In some embodiments, the regulatory DNA element is a cis, trans, distal, proximal, upstream, or downstream regulatory DNA element of a gene. In some embodiments, the regulatory DNA element is a promoter or enhancer of a gene. In some embodiments, the target site is located within a promoter, enhancer, exon, intron, untranslated region (UTR), 5'UTR, or 3'UTR of a gene. In some embodiments, a promoter is a nucleotide sequence to which RNA polymerase binds to initiate transcription of a gene. In some embodiments, a promoter is a nucleotide sequence typically located between 100 bp and 1000 bp from the transcription start site of a gene, e.g., within about 100 bp, about 500 bp, or about 1000 bp from the transcription start site of a gene. In some embodiments, a target site is located within a sequence of unknown or known function suspected to be capable of controlling expression of a gene.

[0174] In some embodiments, the target site is located within about 50 base pairs (bp), 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 600 bp, 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, or about 1500 bp of the transcription start site.

[0175] In some embodiments, the target site is located within a target region located between 1 bp and 3300 bp of the HBV genome. In some embodiments, the target site is a sequence within a target region having a sequence corresponding to a sequence located between 43 bp and 490 bp, 1033 bp and 1749 bp, 1800 bp and 1950 bp, or 2953 bp and 3182 of the HBV genome, with reference to Hepatitis B virus genome (Hepatitis B virus subtype ayw, complete genome, GenBank: U95551.1) SEQ ID NO 650. In some embodiments, the target site is a sequence within a target region having a sequence corresponding to a sequence located between 1 bp and 42 bp, 491 bp and 1032 bp, 1750 bp and 1799 bp, or 1951 bp and 2952 bp, or 3198 bp and 3182 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 650. Based on phylogenetic analysis and sequence divergence, HBV can be classified into 10 genotypes (A-J) based on intergroup divergence of 8% or more in the complete nucleotide sequence (Norder H, et al., 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 1):67-74; Arauz-Ruiz P, et al., Genotype H: a new American genotype of hepatitis B virus revealed in Central America. J Gen Virol. 2002 Aug;83(Pt 8):2059-2073).Evidence suggests that HBV genotype influences clinical outcomes, mutation 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 common in Western Europe and North America, while genotypes B and C are common in East Asia and Oceania.

[0176] In some embodiments, the target site is at least 70% homologous to all Hepatitis B virus genotypes (e.g., genomes), hi 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, or at least 7000 Hepatitis B virus genomes.

[0177] In some embodiments, the target site is at least 70% homologous to at least 1000 hepatitis B virus genomes and contains up to two mismatches. In some embodiments, the target site comprises the sequence set forth in any one of SEQ ID NOs: 1-195, a contiguous portion thereof of at least 14 nucleotides (nt) of any one of SEQ ID NOs: 1-195, or the complementary sequence of any of the foregoing. In some embodiments, the target site is a contiguous portion of any one of SEQ ID NOs: 1-195 that is 15, 16, 17, 18, or 19 nucleotides in length, or the complementary sequence of any of the foregoing. In some embodiments, the target site is a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, 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 set forth herein above. In some embodiments, the target site is a sequence set forth in any one of SEQ ID NOs: 1-195.

[0178] In some of the embodiments, the target site is a sequence of 14-22 nucleotides. In some of the embodiments, the target site is a sequence of 14-19 nucleotides. In some of the embodiments, the target site is a sequence of 14 nucleotides. In some of the embodiments, the target site is a sequence of 15 nucleotides. In some of the embodiments, the target site is a sequence of 16 nucleotides. In some of the embodiments, the target site is a sequence of 17 nucleotides. In some of the embodiments, the target site is a sequence of 18 nucleotides. In some of the embodiments, the target site is a sequence of 19 nucleotides.

[0179] In any of the embodiments provided herein, the target site is complementary to a referenced sequence (i.e., a particular sequence designated by a SEQ ID NO with reference to a sequence listing). In some of any of the embodiments, the complementary sequence is the reverse complement of the referenced sequence.

[0180] In any of the embodiments provided herein, the target site comprises a referenced sequence (i.e., a specific sequence designated by a SEQ ID NO with reference to a sequence listing). In any of the embodiments provided herein, the target site is a sequence designated by a referenced sequence (i.e., a specific sequence designated by a SEQ ID NO with reference to a sequence listing).

[0181] 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., a particular sequence designated by a SEQ ID NO with reference to a 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.

[0182] In some embodiments, the target site is at least 90% homologous to all Hepatitis B virus genomes, hi some embodiments, the 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 virus genomes.

[0183] In some embodiments, the target site is at least 90% homologous to at least 1000 hepatitis B virus genomes and contains one or two mismatches. In some embodiments, the target site comprises the sequence set forth in any one of SEQ ID NOs:35-100, a contiguous portion thereof of at least 14 nt of any one of SEQ ID NOs:35-100, or the complementary sequence of any of the foregoing. In some embodiments, the target site is a contiguous portion of any one of SEQ ID NOs:35-100 that is 15, 16, 17, 18, or 19 nucleotides in length, or the complementary sequence of any of the foregoing. In some embodiments, the target site is a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, 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 set forth herein above. In some embodiments, the target site is a sequence set forth in any one of SEQ ID NOs:35-100.

[0184] In some embodiments, the mismatch is located in the first 12 nt on the 5' end of the protospacer adjacent motif (PAM), as represented by the "n" in "nnnnnnnnnnnnNNNNNNNN-NGG."

[0185] In some embodiments, the target site is at least 90% homologous to at least 1000 hepatitis B virus genomes and contains zero mismatches. In some embodiments, the target site comprises the sequence set forth in any one of SEQ ID NOs: 1-34, a contiguous portion thereof of at least 14 nt of any one of SEQ ID NOs: 1-34, or the complementary sequence of any of the foregoing. In some embodiments, the target site is a contiguous portion of any one of SEQ ID NOs: 1-34 that is 15, 16, 17, 18, or 19 nucleotides in length, or the complementary sequence of any of the foregoing. In some embodiments, the target site is a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, 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 set forth herein above. In some embodiments, the target site is a sequence set forth in any one of SEQ ID NOs: 1-34.

[0186] In any of the embodiments herein, the target site, or each of the target sites, comprises a sequence set forth in any one of SEQ ID NOs: 175, 138, 192, 152, 118, 125, 185, 63, 116, 124, 35, 82, 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: 175, 138, 192, 152, 118, 125, 185, 63, 116, 124, 35, 82 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 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, 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 a sequence set forth in any one of SEQ ID NOs: 175, 138, 192, 152, 118, 125, 185, 63, 116, 124, 35, 82. In some embodiments, the reduction in gene expression or change in transcript levels in the cells is greater than 90% compared to the levels of said gene in control cells.

[0187] In any of the embodiments herein, the target site, or each of the target sites, comprises a sequence set forth in any one of SEQ ID NOs:5, 10, 12, 18, 22, 26, 29, 38, 56, 61, 62, 63, 68, 72, 79, 80, 82, 84, 98, 99, 116, 118, 121, 124, 125, 135, 138, 143, 150, 152, 158, 164, 175, 176, 182, 185, 189, 190, 192, a contiguous portion thereof of at least 14 nucleotides (nt), or the complementary sequence of any of the foregoing. In some embodiments, the target site is a contiguous portion of any one of SEQ ID NOs:5, 10, 12, 18, 22, 26, 29, 38, 56, 61, 62, 63, 68, 72, 75, 79, 80, 82, 84, 98, 99, 116, 118, 121, 124, 125, 135, 138, 143, 150, 152, 158, 164, 175, 176, 182, 185, 189, 190, 192 that is 15, 16, 17, 18 or 19 nucleotides in length, or the complementary sequence of any of the foregoing. In some embodiments, the target site is a sequence having 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 a sequence set forth in any one of SEQ ID NOs:5, 10, 12, 18, 22, 26, 29, 38, 56, 61, 62, 63, 68, 72, 75, 79, 80, 82, 84, 98, 99, 116, 118, 121, 124, 125, 135, 138, 143, 150, 152, 158, 164, 175, 176, 182, 185, 189, 190, or 192. In some embodiments, the reduction in gene expression or change in transcript level in the cell is greater than 80% compared to the level of the gene in a control cell.

[0188] In any of the embodiments herein, the target site, or each of the target sites, comprises a sequence set forth in any one of SEQ ID NOs:5, 6, 12, 18, 22, 26, 29, 38, 42, 43, 51, 56, 61, 63, 68, 72, 75, 79, 82, 84, 88, 89, 98, 99, 113, 116, 121, 124, 125, 118, 130, 133, 135, 138, 143, 150, 152, 155, 158, 164, 165, 175, 176, 182, 185, 189, 190, 192, a contiguous portion thereof of at least 14 nucleotides (nt), or the complementary sequence of any of the foregoing. In some embodiments, the target site is a contiguous portion of any one of SEQ ID NOs:5, 6, 12, 18, 22, 26, 29, 38, 42, 43, 51, 56, 61, 63, 68, 72, 75, 79, 82, 84, 88, 89, 98, 99, 113, 116, 121, 124, 125, 118, 130, 133, 135, 138, 143, 150, 152, 155, 158, 164, 165, 175, 176, 182, 185, 189, 190, 192 that is 15, 16, 17, 18, or 19 nucleotides in length, or the complementary sequence of any of the foregoing. In some embodiments, the target site is a sequence having 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 a sequence set forth in any one of SEQ ID NOs:5, 6, 12, 18, 22, 26, 29, 38, 42, 43, 51, 56, 61, 63,68, 72, 75, 79, 82, 84, 88, 89, 98, 99, 113, 116, 121, 124, 125, 118, 130, 133, 135, 138, 143, 150, 152, 155, 158, 164, 165, 175, 176, 182, 185, 189, 190, or 192.In some embodiments, the reduction in gene expression or change in transcript levels in the cell is greater than 75% compared to the level of said gene in a control cell.

[0189] 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: 22, 63, 75, 99, 116, 124, 138, 143, 150, 152, 175, 176, 192, 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 comprises the sequence of SEQ ID NOs: 22, 63, 75, 99, 116, 124, 138, 143, 150, 152, 175, 176, 192, or a complementary sequence of any of the foregoing that is 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the target site is a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, 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 the target site sequences described herein above. In some embodiments, the target site is a sequence set forth in any one of SEQ ID NOs: 22, 63, 75, 99, 116, 124, 138, 143, 150, 152, 175, 176, or 192.

[0190] In any of the embodiments herein, the target site, or each of the target sites, comprises a sequence set forth in any one of SEQ ID NOs: 12, 18, 20, 22, 26, 27, 46, 50, 63, 66, 73, 79, 185, 192, 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: 12, 18, 20, 22, 26, 27, 46, 50, 63, 66, 73, 79, 185, 192 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 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, 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 a sequence set forth in any one of SEQ ID NOs: 12, 18, 20, 22, 26, 27, 46, 50, 63, 66, 73, 79, 185, or 192.

[0191] In some embodiments, the or each of the target sites comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 1028-1055, a contiguous portion thereof of at least 12 nt, or a complementary sequence of any of the foregoing. In some embodiments, the target site is a contiguous portion of any of SEQ ID NOs: 1028-1055 that is 13, 14, 16, 16, 17, or 18 nucleotides in length, or a complementary sequence of any of the foregoing. In some embodiments, the target site is the sequence set forth in any one of SEQ ID NOs: 1028-1055.

[0192] In some embodiments, the target site or each of the target sites comprises the sequence set forth in SEQ ID NO:22, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence thereof. 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:22 that is 14-19 nucleotides (nt) in length, or a complementary sequence thereof. In some embodiments, the target site is the sequence set forth in SEQ ID NO:22. In some embodiments, the target site can be targeted by a DNA targeting system provided herein. In some embodiments, the DNA binding domain is dSpCas9, which is used in combination with a complementary gRNA for targeting to the target site. In some embodiments, the gRNA has a spacer sequence set forth in SEQ ID NO:217 or a contiguous portion thereof that is complementary to the target site. In some embodiments, the gRNA further comprises a scaffold sequence for dSpCas9 set forth in SEQ ID NO:587. In some embodiments, the DNA targeting system comprises a dSpCas9 fusion protein with an effector domain described herein and a gRNA shown in SEQ ID NO:22 (e.g., HBVg_22).

[0193] In some embodiments, the target site or each of the target sites comprises the sequence set forth in SEQ ID NO:63, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence thereof. 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:63 that is 14-20 nucleotides (nt) in length, or a complementary sequence thereof. In some embodiments, the target site is the sequence set forth in SEQ ID NO:63. In some embodiments, the target site can be targeted by a DNA targeting system provided herein. In some embodiments, the DNA binding domain is dSpCas9, which is used in combination with a complementary gRNA for targeting to the target site. In some embodiments, the gRNA has a spacer sequence set forth in SEQ ID NO:217 or a contiguous portion thereof that is complementary to the target site. In some embodiments, the gRNA further comprises a scaffold sequence for SpCas9 set forth in SEQ ID NO:587. In some embodiments, the DNA targeting system comprises a dSpCas9 fusion protein with an effector domain described herein and a gRNA shown in SEQ ID NO:63 (e.g., HBVg_63).

[0194] In any of the embodiments herein, the target site or each of the target sites comprises the sequence set forth in SEQ ID NO: 1045, a contiguous portion thereof of at least 12 nucleotides (nt), or a complementary sequence thereof. 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: 1045 that is 12-18 nucleotides (nt) in length, or a complementary sequence thereof. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 1045. In some embodiments, the DNA-binding domain is an eZFP for targeting to the target site. In some embodiments, the ZFP comprises a recognition motif set forth in SEQ ID NOs: 822, 823, 824, 825, 826, and 827. In some embodiments, the eZFP has a sequence set forth in SEQ ID NO: 709. In some embodiments, the eZFP is an eZFP designated eZFP_18.

[0195] In any of the embodiments herein, the target site or each of the target sites comprises the sequence set forth in SEQ ID NO: 1046, a contiguous portion thereof of at least 12 nucleotides (nt), or a complementary sequence thereof. 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: 1046 that is 12-18 nucleotides (nt) in length, or a complementary sequence thereof. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 1046. In some embodiments, the DNA-binding domain is an eZFP for targeting to the target site. In some embodiments, the ZFP comprises a recognition motif set forth in SEQ ID NOs: 828, 829, 830, 831, 832, and 833. In some embodiments, the eZFP has a sequence set forth in SEQ ID NO: 710. In some embodiments, the eZFP is an eZFP designated eZFP_19.

[0196] In any of the embodiments herein, the target site or each of the target sites comprises the sequence set forth in SEQ ID NO: 1052, a contiguous portion thereof of at least 12 nucleotides (nt), or a complementary sequence thereof. 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: 1052 that is 12-18 nucleotides (nt) in length, or a complementary sequence thereof. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 1052. In some embodiments, the DNA-binding domain is an eZFP for targeting to the target site. In some embodiments, the ZFP comprises a recognition motif set forth in SEQ ID NOs: 864, 865, 866, 867, 868, and 869. In some embodiments, the eZFP has a sequence set forth in SEQ ID NO: 716. In some embodiments, the eZFP is an eZFP designated eZFP_25.

[0197] In some embodiments, the target site is present in covalently closed circular DNA (cccDNA), imperfect double-stranded DNA (rcDNA), and / or integrated into human genomic DNA. In some embodiments, targeting the target site results in silencing of HBV replication (e.g., reduced HBV replication) and / or silencing of HBV transcription.

[0198] In some embodiments, the present invention provides a multiplexed epigenetic modifying DNA targeting system, which targets a combination of at least two target genes or their regulatory DNA elements as described herein.In some embodiments, the multiplexed epigenetic modifying DNA targeting system targets two, three, four, five, six or more target genes or their regulatory DNA elements as described herein.

[0199] In some embodiments, in the multiplexed epigenetic modifying DNA targeting system provided, each of the target sites is in a different HBV gene. In some embodiments, each of the target sites is in the same HBV gene.

[0200] In some embodiments, provided herein is a multiplexed epigenetic modifying DNA targeting system that targets any combination of the genes and / or their regulatory elements described herein.

[0201] In some embodiments, a multiplexed epigenetic modifying DNA targeting system is provided herein that targets a first gene or its regulatory element and a second gene or its regulatory element. In some embodiments, the first gene or its regulatory element is selected from the list consisting of a polymerase gene, an S-family gene, an X-gene, a core family gene, a pre-S1 promoter, a pre-S2 promoter, an X promoter, a basal core promoter, an Enh1 enhancer, an Enh2 enhancer, a transcript processing control region, and any coding region within the HBV genome; the second gene or its regulatory element is selected from the list consisting of a polymerase gene, an S-family gene, an X-gene, a core family gene, a pre-S1 promoter, a pre-S2 promoter, an X promoter, a basal core promoter, an Enh1 enhancer, an Enh2 enhancer, a transcript processing control region, and any coding region within the HBV genome; and the first gene or its regulatory element is different from the second gene or its regulatory element. The first and second target sites can be any of those described above.

[0202] In some embodiments, the present disclosure provides a multiplexed epigenetic modifying DNA targeting system, which targets a first regulatory element and a second regulatory element.In some embodiments, the first regulatory element and the second regulatory element are selected from the combinations listed in Table 1.

[0203] Table 1. Combinations of first and second regulatory elements targeted by the multiplexed epigenetic modifying DNA targeting system provided herein. TIFF2025527567000023.tif61147

[0204] In some embodiments, the present invention provides a multiplexed epigenetic modifying DNA targeting system that targets a first gene or its regulatory element, a second gene or its regulatory element, and a third gene or its regulatory element.In some embodiments, the first gene or its regulatory element is selected from the list consisting of a polymerase gene, an S-family gene, an X-gene, a core family gene, a pre-S1 promoter, a pre-S2 promoter, an X promoter, a basal core promoter, an Enh1 enhancer, an Enh2 enhancer, a transcript processing control region, and any coding region in the HBV genome, and the second gene or its regulatory element is selected from the list consisting of a polymerase gene, an S-family gene, an X-gene, a core family gene, a pre-S1 promoter, a pre-S2 promoter, an X promoter, a basal core promoter, an Enh1 enhancer, an Enh2 enhancer, a transcript processing control region, and any coding region in the HBV genome. the first gene or its regulatory element is selected from the list consisting of a polymerase gene, an S-family gene, an X-gene, a core-family gene, a pre-S1 promoter, a pre-S2 promoter, an X promoter, a basal core promoter, an Enh1 enhancer, an Enh2 enhancer, and a transcript processing control region, and the first gene or its regulatory element, the second gene or its regulatory element, and the third gene or its regulatory element are different from each other. The first, second, and third target sites can be any of those described above.

[0205] In some embodiments, the present invention provides a multiplexed epigenetic modifying DNA targeting system that targets the first regulatory element, the second regulatory element, and the third regulatory element. In some embodiments, the first regulatory element is selected from the list consisting of L-HBs promoter, M-HBs promoter, S-HBs promoter, X-promoter, basal core promoter, Enh1 enhancer, and Enh2 enhancer, the second regulatory element is selected from the list consisting of L-HBs promoter, M-HBs promoter, S-HBs promoter, X-promoter, basal core promoter, Enh1 enhancer, and Enh2 enhancer, and the third regulatory element is selected from the list consisting of L-HBs promoter, M-HBs promoter, S-HBs promoter, X-promoter, basal core promoter, Enh1 enhancer, and Enh2 enhancer, and the first, second, and third regulatory elements are different. In some embodiments, the first regulatory element is an Enh1 enhancer, the second regulatory element is selected from the list consisting of an L-HBs promoter, an M-HBs promoter, an S-HBs promoter, an X promoter, a basal core promoter, and an Enh2 enhancer, and the third regulatory element is selected from the list consisting of an L-HBs promoter, an M-HBs promoter, an S-HBs promoter, an X promoter, a basal core promoter, and an Enh2 enhancer, and the second regulatory element and the third regulatory element are different. In some embodiments, the first regulatory element is an Enh1 enhancer, the second regulatory element is an L-HBs, and the third regulatory element is selected from the list consisting of an M-HBs promoter, an S-HBs promoter, an X promoter, a basal core promoter, and an Enh2 enhancer.

[0206] In some embodiments, the first regulatory element, the second regulatory element, and the third regulatory element are selected from the combinations listed in Table 2.

[0207] Table 2: Combinations of first, second, and third regulatory elements targeted by the multiplexed epigenetic modifying DNA targeting system provided herein TIFF2025527567000024.tif61159

[0208] In some embodiments, the present invention provides a multiplexed epigenetic modifying DNA targeting system, which targets a first gene or its regulatory element, a second gene or its regulatory element, a third gene or its regulatory element, and a fourth gene or its regulatory element.In some embodiments, the first gene or its regulatory element is selected from the list consisting of polymerase gene, S-family gene, X-gene, core family gene, pre-S1 promoter, pre-S2 promoter, X promoter, basal core promoter, Enh1 enhancer, Enh2 enhancer, transcript processing control region, and any coding region in the HBV genome; the second gene or its regulatory element is selected from the list consisting of polymerase gene, S-family gene, X-gene, core family gene, pre-S1 promoter, pre-S2 promoter, X promoter, basal core promoter, Enh1 enhancer, Enh2 enhancer, transcript processing control region, and any coding region in the HBV genome; and the third gene or its regulatory element is the first gene or its regulatory element, the second gene or its regulatory element, the third gene or its regulatory element, and the fourth gene or its regulatory element are selected from the list consisting of a polymerase gene, an S-family gene, an X-gene, a core family gene, a pre-S1 promoter, a pre-S2 promoter, an X promoter, a basal core promoter, an Enh1 enhancer, an Enh2 enhancer, and a transcript processing control region; and the first gene or its regulatory element, the second gene or its regulatory element, the third gene or its regulatory element, and the fourth gene or its regulatory element are different from one another. The first, second, third, and fourth target sites can be any of those described above.

[0209] In some embodiments, the present invention provides a multiplexed epigenetic modifying DNA targeting system that targets the same gene or its regulatory element.For example, two or more multiplexed epigenetic modifying DNA targeting systems target the same or common gene or its regulatory element.In some embodiments, the gene or its regulatory element is selected from the list consisting of polymerase gene, S-family gene, X-gene, core family gene, pre-S1 promoter, pre-S2 promoter, X promoter, basic core promoter, Enh1 enhancer, Enh2 enhancer, transcript processing control region, and any coding region in HBV genome, and the second gene or its regulatory element is selected from the list consisting of polymerase gene, S-family gene, X-gene, core family gene, pre-S1 promoter, pre-S2 promoter, X promoter, basic core promoter, Enh1 enhancer, Enh2 enhancer, transcript processing control region, and any coding region in HBV genome. In some embodiments, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30 multiplexed epigenetic modifying DNA targeting systems target the same gene or its regulatory elements.

[0210] In some embodiments, the target site for multiplexed editing (e.g., by the multiplexed epigenetic modifying DNA targeting system described herein) is the sequence set forth in any one of SEQ ID NOs: 12, 18, 20, 22, 26, 27, 46, 50, 63, 66, 73, 79, 185, 192. In some embodiments, the target site for multiplexed editing (e.g., by the multiplexed epigenetic modifying DNA targeting system described herein) is the sequence set forth in SEQ ID NO: 22. The target site can be any as described above.

[0211] B. CRISPR-Based DNA Targeting System Provided herein is an epigenetic DNA targeting system that is based on CRISPR / Cas system, that is, the DNA targeting system that is based on CRISPR / Cas can be combined with the target site in target gene or its regulatory element.In some embodiments, provided epigenetic DNA targeting system is a multiplexed epigenetic DNA targeting system that is based on CRISPR / Cas system, that is, the DNA targeting system that is based on CRISPR / Cas can target the site in the combination of target gene or its regulatory element.

[0212] In some embodiments, the CRISPR / Cas DNA binding domain is nuclease-inactive, e.g., comprises dCas (e.g., dCas9), whereby the system binds to a target site in a target gene or its regulatory element without mediating nucleic acid cleavage at the target site. In some embodiments, the DNA targeting system does not introduce gene disruption or DNA cleavage. A CRISPR / Cas-based DNA targeting system can be used to modulate the expression of a target gene in a cell, e.g., a hepatocyte. In some embodiments, the target gene or its regulatory element can comprise any of the enzymes described herein, including any of those described in Section IA above. In some embodiments, the target site in the target gene or its regulatory element can comprise any of the enzymes described herein, including any of those described in Section IA above. In some embodiments, a CRISPR / Cas-based DNA targeting system can comprise any known Cas enzyme, generally a nuclease-inactive Cas or dCas. In some embodiments, a CRISPR / Cas-based DNA targeting system comprises a fusion protein of a nuclease-inactive Cas protein or a variant thereof with an effector domain (e.g., a transcriptional repressor) that reduces gene transcription, and at least one gRNA.

[0213] The CRISPR system (also known as the CRISPR / Cas system or CRISPR-Cas system) refers to a conserved microbial nuclease system found in bacterial and archaeal genomes that provides a form of adaptive immunity against invading phages and plasmids. Clustered regularly interspaced short palindromic repeats (CRISPR) refers to a locus containing multiple repetitive DNA elements separated by non-repetitive DNA sequences called spacers. Spacers are short sequences of foreign DNA that are integrated between CRISPR repeats in the genome and serve as a "memory" of past exposure. Spacers encode the DNA-targeting portion of an RNA molecule that confers specificity for nucleic acid cleavage by the CRISPR system. CRISPR loci contain or are adjacent to one or more CRISPR-associated (Cas) genes that can act as RNA-guided nucleases to mediate cleavage, as well as non-protein-coding DNA elements that encode RNA molecules capable of programming the specificity of CRISPR-mediated nucleic acid cleavage.

[0214] In the type II CRISPR / Cas system, which contains 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 the target nucleic acid sequence (target site) and encodes the sequence specificity of the complex. The trans-activating crRNA (tracrRNA) base-pairs with a portion of the crRNA and forms a structure that complexes with the Cas9 protein to form the Cas / RNA RNP complex.

[0215] Naturally occurring CRISPR / Cas system, for example, CRISPR / Cas system with Cas9, has been engineered to enable efficient programming of Cas / RNA RNP to target desired sequence in cells of interest for both gene editing and gene expression modulation.TracrRNA and crRNA have been engineered to form a single chimeric guide RNA molecule, generally referred to as guide RNA (gRNA), as described in, for example, WO 2013 / 176772 A1, 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 selected by the user to target the Cas / gRNA RNP complex to a desired locus, e.g., a desired target site in a target gene and / or its regulatory elements.

[0216] Cas proteins have also been engineered to enable targeting of Cas / gRNA RNPs without inducing cleavage at the target site. Mutations in Cas proteins can reduce or abolish the nuclease activity of the Cas protein, resulting in the Cas protein being catalytically inactive. Cas proteins with reduced or abolished nuclease activity are referred to as inactive Cas (dCas) or nuclease-inactive Cas (iCas) proteins, and are referred to interchangeably herein. Exemplary inactive Cas9 (dCas9) derived from S. pyogenes contains silencing mutations (D10A and H840A) in the RuvC and HNH nuclease domains, as described, for example, in WO 2013 / 176772 A1, 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 Cas12 system (i.e., Cpf1) are described, for example, in WO 2017 / 189308 A1 and Zetsche, B. et al. Cell 163(3):759-71 (2015). Conserved domains that mediate nucleic acid cleavage, such as the RuvC and HNH endonuclease domains, are readily identifiable in Cas orthologs and can be mutated to produce inactive variants, as described, for example, in Zetsche, B. et al. Cell 163(3):759-71 (2015).

[0217] dCas-fusion proteins with transcriptional and / or epigenetic regulators have been used as a versatile platform for ectopically modulating gene expression in target cells. These include fusions of Cas with effector domains, such as transcriptional activators or repressors. For example, dCas9 can be fused to transcriptional activators such as VP64 (a polypeptide composed of four tandem copies of VP16, the 16-amino acid transactivation domain of herpes simplex virus) to robustly induce gene expression. Alternatively, dCas9 can be fused to transcriptional repressors such as KRAB (Kruppel-associated box) to robustly inhibit gene expression. For example, 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, ML et al. Nat. Methods 10, 977-979 (2013), Gilbert, LA et al. Cell 154(2):442-451 (2013), and Nunez, JK et al. Cell 184(9):2503-2519 (2021), various dCas-fusion proteins with transcriptional and epigenetic regulators can be engineered to regulate gene expression.

[0218] In some aspects, a DNA targeting system is provided, comprising a fusion protein comprising a DNA-binding domain comprising a nuclease-inactive Cas protein or a variant thereof and an effector domain (i.e., a transcriptional repressor) for reducing transcription or inducing transcriptional repression when targeted to a target gene or its regulatory element in a cell (e.g., a hepatocyte). In such embodiments, the DNA targeting system also comprises one or more gRNAs provided in combination or complex with a dCas protein or a variant thereof for targeting the DNA targeting system to a target site in the target gene or its regulatory element. In some embodiments, the fusion protein is guided by a guide RNA to a specific target site sequence in the target gene or its regulatory element, where the effector domain mediates targeted epigenetic modification to reduce or repress transcription of the target gene. In some embodiments, a combination of gRNAs guides the fusion protein to a combination of target site sequences in a combination of genes or their regulatory elements, where the effector domain mediates targeted epigenetic modification to reduce or repress transcription of the combination of target genes. As described further below, any of a variety of effector domains that reduce or suppress transcription can be used.

[0219] 1. CRISPR-based DNA-binding domains In some aspects, the DNA binding domain comprises or is derived from a CRISPR-associated (Cas) protein or variant thereof and is nuclease inactive (i.e., a dCas protein).

[0220] In some embodiments, the Cas protein is derived from a Class 1 CRISPR system (i.e., a multi-Cas protein system), such as a Type I, Type III, or Type IV CRISPR system. In some embodiments, the Cas protein is derived from a Class 2 CRISPR system (i.e., a single Cas protein system), such as a Type II, Type V, or Type VI CRISPR system. In some embodiments, the Cas protein is derived from a Type V CRISPR system. In some embodiments, the Cas protein is derived from a Cas12 protein (i.e., Cpfl) or a variant thereof, as described, for example, in WO 2017 / 189308 A1 and Zetsche, B. et al. Cell. 163(3):759-71 (2015). In some embodiments, the Cas protein is derived from a Type II CRISPR system. In some embodiments, the Cas protein is a Cas protein described in, e.g., WO 2013 / 176772 A1, WO 2014 / 152432 A2, WO 2014 / 093661 A2, WO 2014 / 093655 A2, Jinek, M. et al. Science 337(6096):816-21 (2012), Mali, P. et al. Science 339(6121):823-6 (2013), Cong, L. et al. Science 339(6121):819-23 (2013), Perez-Pinera, P. et al. Nat. Methods 10, 973-976 (2013), or Mali, P. et al. Nat. Biotechnol. 31, 833-838 (2013), derived from the Cas9 protein or a variant thereof. Various CRISPR / Cas systems and related Cas proteins for use in gene editing and regulation are described, for example, in Moon, SB et al. Exp. Mol. Med. 51, 1-11 (2019), Zhang, FQ Rev. Biophys. 52, E6 (2019), and Makarova KS et al. Methods Mol. Biol. 1311:47-75 (2015).

[0221] In some embodiments, the dCas9 protein can comprise a sequence derived from a naturally occurring Cas9 molecule or a variant thereof. In some embodiments, the dCas9 protein can comprise a sequence derived from a naturally occurring Cas9 molecule of S. pyogenes, S. thermophilus, S. aureus, C. jejuni, N. meningitidis, F. novicida, S. canis, or S. auricularis, or a variant thereof. In some embodiments, the dCas9 protein can comprise a sequence derived from a naturally occurring Cas9 molecule of S. aureus. In some embodiments, the dCas9 protein can comprise a sequence derived from a naturally occurring Cas9 molecule of S. pyogenes.

[0222] Non-limiting examples of Cas9 orthologs from other bacterial strains include Acaryochloris marina MBIC11017; Acetohalobium arabaticum DSM 5501; Acidithiobacillus caldus; Acidithiobacillus ferrooxidans ATCC 23270; Alicyclobacillus acidocaldarius LAA1; Alicyclobacillus acidocaldarius subsp. acidocaldarius DSM 446; Allochromatium vinosum DSM 180; and Ammonifex degenzii. 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 audaxviator 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. ATCC 51142; Cyanothece sp. CCY0110; Cyanothece sp. PCC 7424; Cyanothece sp. PCC 7822; Exiguobacterium sibiricum 255-15; Finegoldia magna ATCC 29328; Ktedonobacter racemifer DSM 44963; Lactobacillus delbrueckii subsp. bulgaricus PB2003 / 044-T3-4; Lactobacillus salivarius ATCC 11741; Listeria innocua; Lyngbya sp. PCC 8106; Marinobacter sp. ELB17; Methanohalobium evestigatum Z-7303; Microcystis phage Ma-LMM01; 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; Polaromonas naphthalenivorans CJ2; Polaromonas sp. JS666; Pseudoalteromonas haloplanktis TAC125; Streptomyces pristinaespiralis Cas proteins identified in 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).

[0223] In some aspects, the Cas protein is a variant lacking nuclease activity (i.e., a dCas protein). In some embodiments, the Cas protein is mutated to reduce or eliminate nuclease activity. Such Cas proteins are referred to as inactive Cas or dead Cas (dCas) or nuclease-inactive Cas (iCas) proteins, and are referred to interchangeably herein. In some embodiments, the variant Cas protein is a variant Cas9 protein that lacks nuclease activity or is an inactive Cas9 (dCas9 or iCas9) protein.

[0224] In some embodiments, the Cas9 protein or variant thereof is derived from a Staphylococcus aureus Cas9 (SaCas9) protein or variant thereof. In some embodiments, the variant Cas9 is a Staphylococcus aureus dCas9 protein (dSaCas9) comprising at least one amino acid mutation selected from D10A and N580A with reference to the numbering of positions in SEQ ID NO:596. In some embodiments, the variant Cas9 protein comprises the sequence set forth in SEQ ID NO:597, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0225] In some embodiments, the Cas9 protein or variant thereof is derived from a Streptococcus pyogenes Cas9 (SpCas9) protein or variant thereof. In some embodiments, the variant Cas9 is a Streptococcus pyogenes dCas9 (dSpCas9) protein comprising at least one amino acid mutation selected from D10A and H840A with reference to the numbering of positions in SEQ ID NO:598. In some embodiments, the variant Cas9 protein comprises the sequence set forth in SEQ ID NO:599, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0226] 2. Guide RNA In some embodiments, a Cas protein (e.g., dCas9) is provided in combination with or complexed with one or more guide RNAs (gRNAs). In some aspects, the gRNA is a nucleic acid that facilitates specific targeting or homing of the gRNA / Cas RNP complex to a target site in a target gene and / or its regulatory elements, such as any of those described above. In some embodiments, the target site of the gRNA may be referred to as a protospacer.

[0227] Provided herein are gRNAs, e.g., gRNAs that target or bind to a target site or its DNA regulatory element, e.g., any of those described in Section IA above. In some embodiments, the gRNA is capable of complexing with a Cas protein or a variant thereof. In some embodiments, the gRNA comprises a gRNA spacer sequence (i.e., a spacer sequence or guide sequence) that is capable of hybridizing to or complementary to a target site, such as any of the target sites described in Section IA or further below. In some embodiments, the gRNA comprises a scaffold sequence that complexes with or binds to a Cas protein.

[0228] In some embodiments, the gRNAs provided herein are chimeric gRNAs. Generally, gRNAs can be unimolecular (i.e., consisting of a single RNA molecule) or modular (comprising more than one, typically two, separate RNA molecules). Modular gRNAs can be engineered to be unimolecular, in which sequences from separate modular RNA molecules are included in a single gRNA molecule (sometimes referred to as chimeric gRNAs, synthetic gRNAs, or single gRNAs). In some embodiments, chimeric gRNAs are fusions of two non-coding RNA sequences: a crRNA sequence and a tracrRNA sequence, as described, for example, in WO 2013 / 176772 A1 or Jinek, M. et al. Science 337(6096):816-21 (2012). In some embodiments, chimeric gRNAs mimic the naturally occurring crRNA:tracrRNA duplex involved in type II effector systems, where the naturally occurring crRNA:tracrRNA duplex acts as a guide for the Cas9 protein.

[0229] In some aspects, the spacer sequence of the gRNA is a polynucleotide sequence comprising at least a portion having sufficient complementarity with the target site or its DNA regulatory elements (e.g., any of those described in Section IA) to hybridize with the target site in the target gene and / or its regulatory elements and direct sequence-specific binding of the CRISPR complex to the sequence of the target site. Full complementarity is not necessary, as long as there is sufficient complementarity to cause hybridization and promote CRISPR complex formation. In some embodiments, the gRNA comprises a spacer sequence that is complementary to the target site, e.g., at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% complementary (e.g., fully complementary). The strand of the target nucleic acid that comprises the target site sequence may be referred to as the "complementary strand" of the target nucleic acid.

[0230] In some embodiments, the gRNA spacer sequence is about 14 nucleotides (nt) to about 26 nt, or 16 nt to 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.

[0231] The target site of the gRNA may be referred to as a protospacer. In some aspects, the spacer is designed to target the 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'-NGG-3' (SEQ ID NO: 629), where N is any nucleotide. S. aureus Cas9 uses the PAM 5'-NNGRRT-3' (SEQ ID NO: 630), where N is any nucleotide and R is G or A. N. meningitidis Cas9 uses the PAM 5'-NNNNGATT-3' (SEQ ID NO: 631), where N is any nucleotide. C. jejuni Cas9 uses PAM 5'-NNNNRYAC-3' (SEQ ID NO:632), where N is any nucleotide, R is G or A, and Y is C or T. S. thermophilus uses PAM 5'-NNAGAAW-3' (SEQ ID NO:633), where N is any nucleotide, and W is A or T. F. novicida Cas9 uses PAM 5'-NGG-3' (SEQ ID NO:634), where N is any nucleotide. T. denticola Cas9 uses PAM 5'-NAAAAC-3' (SEQ ID NO:635), where N is any nucleotide. Cas12a (also known as Cpf1) from various species uses the PAM 5'-TTTV-3' (SEQ ID NO:636). Cas proteins may use or be engineered to use different PAMs from those listed above.For example, a mutated SpCas9 protein may use the PAM 5'-NGG-3' (SEQ ID NO:629), 5'-NGAN-3' (SEQ ID NO:637), 5'-NGNG-3' (SEQ ID NO:638), 5'-NGAG-3' (SEQ ID NO:639), or 5'-NGCG-3' (SEQ ID NO:640). In some embodiments, the protospacer of a gRNA for complexing with S. pyogenes Cas9 or a variant thereof is set forth in SEQ ID NO:588. In some embodiments, the protospacer of a gRNA for complexing with S. aureus Cas9 or a variant thereof is set forth in SEQ ID NO:589.

[0232] The 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.

[0233] In some embodiments, the gRNA (including the guide sequence) will contain the base uracil (U), while the DNA encoding the gRNA molecule will contain the base thymine (T). Without wishing to be bound by theory, it is believed that in some embodiments, the complementarity between the guide sequence and the target sequence contributes to the specificity of the interaction between the gRNA molecule / Cas molecule complex and the target nucleic acid. It is understood that in a pair of a guide sequence and a target sequence, the uracil base in the guide sequence pairs with the adenine base in the target sequence.

[0234] In some embodiments, one, more than one, or all of the nucleotides of the gRNA can have a modification, e.g., to make the gRNA less susceptible to degradation and / or improve biocompatibility. As a non-limiting example, the backbone of the gRNA can be modified with phosphorothioates or other modifications. In some cases, the nucleotides of the gRNA can include a 2' modification, e.g., 2-acetylation, e.g., 2' methylation, or other modification.

[0235] Methods for designing gRNAs and exemplary targeting domains are described, for example, in International PCT Publication Nos. WO 2014 / 197748 A2, WO 2016 / 130600 A2, WO 2017 / 180915 A2, WO 2021 / 226555 A2, WO 2013 / 176772 A1, WO 2014 / 152432 A2, WO 2014 / 093661 A2, WO 2014 / 093655 A2, WO 2015 / 089427 A1, WO 2016 / 049258 A2, WO 2016 / 123578 A1, WO The present invention may include those described in WO 2021 / 076744 A1, WO 2014 / 191128 A1, WO 2015 / 161276 A2, WO 2017 / 193107 A2, and WO 2017 / 093969 A1.

[0236] In some embodiments, the gRNAs provided herein target target sites present in covalently closed circular DNA (cccDNA), incomplete double-stranded DNA (rcDNA). In some aspects, the target site can be any HBV genomic sequence that is optimal for inserting DNA methylation that results in silencing of HBV RNA transcription.

[0237] In some embodiments, the target site is located in or near a gene or its regulatory element involved in the control of HBV replication and / or HBV transcription. In some aspects, the target site is located in or near a promoter. In some aspects, the target site is located near an enhancer region. In some aspects, the target site is located in or near a transcript processing control region. In some aspects, the target site can be any gene that is suitable for inserting DNA methylation to silence HBV transcription.

[0238] In some embodiments, the gRNA targets a target site comprising a sequence selected from any one of SEQ ID NOs: 1-34 as set forth in Table 3, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence to any of the foregoing, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100%, 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.

[0239] In some embodiments, the gRNA targets a target site comprising a sequence selected from any one of SEQ ID NOs: 35-100 as set forth in Table 4, or a contiguous portion thereof of at least 14 nt, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100%, 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.

[0240] In some embodiments, the gRNA targets a target site comprising a sequence selected from any one of SEQ ID NOs: 101-195 as set forth in Table 5, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100%, 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.

[0241] In some embodiments, the gRNA further comprises a scaffold sequence as set forth in SEQ ID NO:587. In some embodiments, the gRNA further comprises a scaffold sequence. In some embodiments, the scaffold sequence is In some embodiments, the scaffold sequence is set forth in SEQ ID NO:587.

[0242] In some embodiments, the gRNA comprises a sequence selected from any one of SEQ ID NOs: 391-585 as set forth in Table 6, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100%, 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 NOs: 391-585. In some embodiments, the gRNA is set forth in any one of SEQ ID NOs: 391-585.

[0243] In some embodiments, the gRNA comprises a sequence selected from any one of SEQ ID NOs:391-424, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100%, 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.

[0244] In some embodiments, the gRNA comprises a sequence selected from any one of SEQ ID NOs:425-490, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100%, 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.

[0245] In some embodiments, the gRNA comprises a sequence selected from any one of SEQ ID NOs:490-585, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100%, 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.

[0246] In some embodiments, any of the provided gRNA sequences is provided complexed or in combination with Cas9. In some embodiments, the Cas9 is dCas9. In some embodiments, the dCas9 is dSpCas9, such as the dSpCas9 set forth in SEQ ID NO:599.

[0247] Table 3. Target site sequences and gRNA spacers for >90% HBV genome conservation and no mismatches (HBV0). TIFF2025527567000026.tif137165TIFF2025527567000027.tif166165

[0248] Table 4. Target site sequences and gRNA spacers for >90% HBV genome conservation and 1-2 mismatches (HBV1). TIFF2025527567000028.tif54170TIFF2025527567000029.tif241170TIFF2025527567000030.tif241170TIFF2025527567000031.tif34170

[0249] Table 5. Target site sequences and gRNA spacers for 70-90% HBV genome conservation and up to two mismatches (HBV2). TIFF2025527567000032.tif179170TIFF2025527567000033.tif241170TIFF2025527567000034.tif241170TIFF2025527567000035.tif150170

[0250] Table 6. Gene-targeting gRNAs TIFF2025527567000036.tif63170TIFF2025527567000037.tif235170TIFF2025527567000038.tif235170 TIFF2025527567000039.tif235170TIFF2025527567000040.tif235170TIFF2025527567000041.tif235170 TIFF2025527567000042.tif235170TIFF2025527567000043.tif235170TIFF2025527567000044.tif23517 0TIFF2025527567000045.tif235170TIFF2025527567000046.tif235170TIFF2025527567000047.tif13170

[0251] In some embodiments, the gRNAs provided herein target target sites in the Hepatitis B virus genome. In some embodiments, the gRNA targets a site located between 0 and 3300 bp of the HBV genome. In some embodiments, the gRNA targets a site located between 43 bp and 490 bp, 1033 bp and 1749 bp, 1800 bp and 1950 bp, or 2953 bp and 3182 of the HBV genome, corresponding to positions referenced in Hepatitis B virus genome (Hepatitis B virus subtype ayw, complete genome, GenBank: U95551.1) SEQ ID NO: 650. In some embodiments, the gRNA targets a site located between 1 bp and 42 bp, 491 bp and 1032 bp, 1750 bp and 1799 bp, or 1951 bp and 2952 bp of the HBV genome corresponding to positions referenced in the Hepatitis B virus genome (Hepatitis B virus subtype ayw, complete genome, GenBank: U95551.1) SEQ ID NO:650. 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 a polymerase gene, an S-family gene, an X-gene, or a core-family gene. In some embodiments, the gRNA targets the M / S-HBs, X, basic core, or L-HBs promoter region. In some embodiments, the gRNA targets the Enh1 or Enh2 enhancer region. In some embodiments, the gRNA targets the HBV coding region.

[0252] In some embodiments, a gRNA provided herein comprises a sequence selected from any one of SEQ ID NOs: 196-229, a contiguous portion thereof of at least 14 nucleotides (e.g., 14, 15, 16, 17, 18, or 19 nucleotides), a complementary sequence to any of the foregoing, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, 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 comprises a spacer sequence comprising a sequence selected from any one of SEQ ID NOs:230-295, a contiguous portion thereof of at least 14 nt (e.g., 14, 15, 16, 17, 18, or 19 nucleotides), or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, 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 comprises a spacer sequence comprising a sequence selected from any one of SEQ ID NOs:296-390, a contiguous portion thereof of at least 14 nt (e.g., 14, 15, 16, 17, 18, or 19 nucleotides), or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, 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 further comprises a scaffold sequence.In some embodiments, the scaffold sequence comprises the sequence set forth in SEQ ID NO:587, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100%, 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:587. In some embodiments, a gRNA comprising a spacer sequence and a scaffold sequence comprises a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100%, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% sequence identity to a sequence selected from any one of SEQ ID NOs: 391-585, or all or a portion thereof.

[0253] In some embodiments, the gRNA comprises a sequence selected from any one of SEQ ID NOs: 370, 333, 387, 347, 313, 320, 380, 256, 258, 311, 319, 230, 272, a contiguous portion thereof of at least 14 nucleotides (e.g., 14, 15, 16, 17, 18, or 19 nucleotides), a complementary sequence of any of the foregoing, or a sequence 80% complementary to any of the foregoing. , 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, or a spacer sequence comprising a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity. In some embodiments, the gRNA further comprises a scaffold sequence. In some embodiments, the scaffold sequence comprises the sequence set forth in SEQ ID NO:587, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100%, 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:587. In some embodiments, a gRNA comprising a spacer sequence and a scaffold sequence comprises a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100%, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% sequence identity to a sequence selected from any one of SEQ ID NOs: 565, 528, 542, 508, 515, 575, 515, 453, 506, 514, 425, or 472, or all or a portion thereof. In some embodiments, the gRNA is set forth in SEQ ID NO:565, 528, 542, 508, 515, 575, 515, 453, 506, 514, 425, or 472.

[0254] In some embodiments, the gRNA comprises a sequence selected from any one of SEQ ID NOs: 200, 205, 207, 213, 217, 221, 224, 233, 251, 256, 257, 258, 263, 267, 274, 275, 277, 279, 293, 294, 311, 313, 316, 319, 320, 330, 333, 338, 345, 347, 353, 359, 370, 371, 377, 380, 384, 385, 387, at least 14 nucleotides (e.g., 14, 15, 16, 17, 18, or 19 nucleotides) a spacer sequence comprising a contiguous portion thereof of any of the foregoing, the complement of any of the foregoing, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, 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 further comprises a scaffold sequence. In some embodiments, the scaffold sequence comprises the sequence set forth in SEQ ID NO:587, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100%, 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:587.In some embodiments, the gRNA comprising the spacer sequence and the scaffold sequence is selected from any one of SEQ ID NOs: 395, 400, 402, 408, 412, 416, 419, 428, 446, 451, 452, 453, 458, 462, 465, 469, 470, 472, 474, 488, 489, 506, 508, 511, 514, 515, 525, 528, 533, 540, 542, 548, 554, 565, 566, 572, 575, 579, 580, 582. In some embodiments, the gRNA comprises a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% sequence identity to the sequence, or all or a portion thereof. NO: A sequence selected from any one of 200, 201, 207, 217, 221, 224, 233, 237, 238, 246, 251, 256, 258, 263, 267, 274, 270, 277, 279, 283, 284, 293, 294, 308, 311, 313, 316, 319, 320, 325, 328, 330, 333, 338, 345, 347, 350, 353, 359, 360, 370, 371, 377, 380, 384, 385, 387, at least 14 nucleotides (e.g., 14, 15, 16 99.5%, 99.9% or 100%, or a sequence having 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 further comprises a scaffold sequence.In some embodiments, the scaffold sequence comprises the sequence set forth in SEQ ID NO:587, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100%, 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:587. In some embodiments, a gRNA comprising a spacer sequence and a scaffold sequence is NO:369, 395, 402, 408, 412, 416, 419, 428, 432, 433, 441, 446, 451, 453, 458, 462, 465, 469, 472, 474, 478, 479, 488, 4 89, 503, 506, 508, 511, 514, 515, 520, 523, 525, 575, 528, 533, 540, 542, 545, 548, 554, 555, 565, 566, 572, 579, 580, 58 2, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100%, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% sequence identity to a sequence selected from any one of In some embodiments, the gRNA is set forth in SEQ ID NO:369, 395, 402, 408, 412, 416, 419, 428, 432, 433, 441, 446, 451, 453, 458, 462, 465, 469, 472, 474, 478, 479, 488, 489, 503, 506, 508, 511, 514, 515, 520, 523, 525, 575, 528, 533, 540, 542, 545, 548, 554, 555, 565, 566, 572, 579, 580, or 582.

[0255] In some embodiments, the gRNA is a sequence selected from any one of SEQ ID NOs: 207, 213, 215, 217, 221, 222, 241, 245, 258, 261, 268, 274, 380, 387, a contiguous portion thereof of at least 14 nucleotides (e.g., 14, 15, 16, 17, 18, or 19 nucleotides), a complementary sequence of any of the foregoing, or an 8-nucleotide sequence of any of the foregoing. and a spacer sequence comprising a sequence having 0%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100%, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% sequence identity. In some embodiments, the gRNA comprises a spacer sequence comprising the sequence of SEQ ID NO:217, a contiguous portion thereof of at least 14 nucleotides (e.g., 14, 15, 16, 17, 18, or 19 nucleotides), the complement of any of the foregoing, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, 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:217. In some embodiments, the gRNA further comprises a scaffold sequence. In some embodiments, the scaffold sequence comprises the sequence set forth in SEQ ID NO:587, or a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100%, 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:587.In some embodiments, a gRNA comprising a spacer sequence and a scaffold sequence comprises a sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100%, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% sequence identity to a sequence selected from any one of SEQ ID NOs: 402, 408, 410, 412, 416, 417, 436, 440, 453, 456, 463, 469, 575, 582, or all or a portion thereof. In some embodiments, the provided multiplexed epigenetic modifying DNA targeting system for epigenetic modification of at least two genes and / or their regulatory elements comprises any of the above-mentioned gRNAs complexed with a Cas protein, for example, a Cas9 protein. In some embodiments, the Cas9 is dCas9. In some embodiments, the dCas9 is dSpCas9, for example, dSpCas9 set forth in SEQ ID NO:599, or variants and / or fusions thereof.

[0256] In some embodiments, provided herein are combinations of gRNAs. In some embodiments, provided herein are multiplexed epigenetic modifying DNA targeting systems comprising combinations of gRNAs.

[0257] In some embodiments, the gRNA combination comprises at least two gRNAs that target at least two different genes or their regulatory elements. In some embodiments, the gRNA combination comprises a first gRNA targeted to a first gene or its regulatory element and a second gRNA targeted to a second gene or its regulatory element. In some embodiments, the first gRNA targets a gene or regulatory element thereof selected from the list consisting of a polymerase gene, an S-family gene, an X-gene, a core-family gene, a pre-S1 promoter, a pre-S2 promoter, an X promoter, a basal core promoter, an Enh1 enhancer, an Enh2 enhancer, a transcript processing control region, and any coding region within the HBV genome; the second gRNA targets a gene or regulatory element thereof selected from the list consisting of a polymerase gene, an S-family gene, an X-gene, a core-family gene, a pre-S1 promoter, a pre-S2 promoter, an X promoter, a basal core promoter, an Enh1 enhancer, an Enh2 enhancer, a transcript processing control region, and any coding region within the HBV genome; and the first and second gRNAs target different genes or regulatory elements thereof. In some embodiments, the first gRNA targets the Enh1 enhancer, and the second gRNA targets a gene selected from the list consisting of the L-HBs promoter, the M-HBs promoter, the S-HBs promoter, the X promoter, the basal core promoter, the S-gene promoter, and the Enh2 enhancer. In some embodiments, the first gRNA targets the Enh1 enhancer, and the second gRNA targets the L-HBs. In some embodiments, the first gRNA and the second gRNA target a combination of two genes selected from the combinations of genes listed in Table 1. In some embodiments, the first gRNA and the second gRNA are each independently selected from any of the gRNAs described herein.

[0258] In some embodiments, a gRNA combination comprises at least three gRNAs that target at least three different genes or regulatory elements thereof. In some embodiments, a gRNA combination comprises a first gRNA targeted to a first gene, a second gRNA targeted to a second gene, and a third gRNA targeted to a third gene. In some embodiments, a gRNA combination comprises at least three gRNAs that target at least three different genes or regulatory elements thereof. In some embodiments, a gRNA combination comprises a first gRNA targeted to a first gene or regulatory element thereof, a second gRNA targeted to a second gene or regulatory element thereof, and a third gRNA targeted to a third gene or regulatory element thereof. In some embodiments, the first gRNA targets a gene or a regulatory element thereof selected from the list consisting of a polymerase gene, an S-family gene, an X-gene, a core family gene, a pre-S1 promoter, a pre-S2 promoter, an X promoter, a basal core promoter, an Enh1 enhancer, an Enh2 enhancer, a transcript processing control region, and any coding region within the HBV genome, and the second gRNA targets a gene or a regulatory element thereof selected from the list consisting of a polymerase gene, an S-family gene, an X-gene, a core family gene, a pre-S1 promoter, a pre-S2 promoter, an X promoter, a basal core promoter, an Enh1 enhancer, an Enh2 enhancer, and any coding region within the HBV genome. the first gRNA targets a gene or its regulatory element selected from the list consisting of a polymerase gene, an S-family gene, an X-gene, a core family gene, a pre-S1 promoter, a pre-S2 promoter, an X promoter, a basal core promoter, an Enh1 enhancer, an Enh2 enhancer, a transcript processing control region, and any coding region within the HBV genome; the third gRNA targets a gene or its regulatory element selected from the list consisting of a polymerase gene, an S-family gene, an X-gene, a core family gene, a pre-S1 promoter, a pre-S2 promoter, an X promoter, a basal core promoter, an Enh1 enhancer, an Enh2 enhancer, a transcript processing control region, and any coding region within the HBV genome; and the first, second, and third gRNAs target different genes or their regulatory elements.In some embodiments, the first gRNA targets the Enh1 enhancer, the second gRNA targets a gene selected from the list consisting of the L-HBs promoter, the M-HBs promoter, the S-HBs promoter, the X-promoter, the basal core promoter, the S-gene promoter, the Enh1 enhancer, and the Enh2 enhancer, the third gRNA targets a gene selected from the list consisting of the L-HBs promoter, the M-HBs promoter, the S-HBs promoter, the X-promoter, the basal core promoter, the S-gene promoter, the Enh1 enhancer, and the Enh2 enhancer, and the second gRNA and the third gRNA target different genes. In some embodiments, the first gRNA targets the Enh1 enhancer, the second gRNA targets the L-HBs promoter, and the third gRNA targets a gene selected from the list consisting of the M-HBs promoter, the S-HBs promoter, the X promoter, the basal core promoter, the S-gene promoter, and the Enh2 enhancer. In some embodiments, the first gRNA, the second gRNA, and the third gRNA target a combination of three genes selected from the combinations of genes listed in Table 2. In some embodiments, the first gRNA, the second gRNA, and the third gRNA are each independently selected from any of the gRNAs described herein.

[0259] In some embodiments, a gRNA combination comprises at least four gRNAs targeting at least four different genes or regulatory elements thereof. In some embodiments, a gRNA combination comprises a first gRNA targeted to a first gene, a second gRNA targeted to a second gene, a third gRNA targeted to a third gene, and a fourth gRNA targeted to a fourth gene. In some embodiments, a gRNA combination comprises at least four gRNAs targeting at least four different genes or regulatory elements thereof. In some embodiments, a gRNA combination comprises a first gRNA targeted to a first gene or regulatory element thereof, a second gRNA targeted to a second gene or regulatory element thereof, a third gRNA targeted to a third gene or regulatory element thereof, and a fourth gRNA targeted to a third gene or regulatory element thereof.In some embodiments, the first gRNA targets a gene or a regulatory element thereof selected from the list consisting of a polymerase gene, an S-family gene, an X-gene, a core family gene, a pre-S1 promoter, a pre-S2 promoter, an X promoter, a basal core promoter, an Enh1 enhancer, an Enh2 enhancer, a transcript processing control region, and any coding region within the HBV genome; the second gRNA targets a gene or a regulatory element thereof selected from the list consisting of a polymerase gene, an S-family gene, an X-gene, a core family gene, a pre-S1 promoter, a pre-S2 promoter, an X promoter, a basal core promoter, an Enh1 enhancer, an Enh2 enhancer, a transcript processing control region, and any coding region within the HBV genome; and the third gRNA targets a gene or a regulatory element thereof selected from the list consisting of a polymerase gene, an S-family gene, an X-gene, a core family gene, a pre-S1 promoter, a pre-S2 promoter, an X promoter, a basal core promoter, an Enh1 enhancer, an Enh2 enhancer, a transcript processing control region, and any coding region within the HBV genome. the first gRNA targets a gene or regulatory element thereof selected from the list consisting of an S-family gene, an X-gene, a core family gene, a pre-S1 promoter, a pre-S2 promoter, an X promoter, a basal core promoter, an Enh1 enhancer, an Enh2 enhancer, a transcript processing control region, and any coding region within the HBV genome; the fourth gRNA targets a gene or regulatory element thereof selected from the list consisting of a polymerase gene, an S-family gene, an X-gene, a core family gene, a pre-S1 promoter, a pre-S2 promoter, an X promoter, a basal core promoter, an Enh1 enhancer, an Enh2 enhancer, a transcript processing control region, and any coding region within the HBV genome; and the first, second, third, and fourth gRNAs target different genes or regulatory elements thereof.In some embodiments, the first gRNA targets the Enh1 enhancer, the second gRNA targets a gene selected from the list consisting of the L-HBs promoter, the M-HBs promoter, the S-HBs promoter, the X-promoter, the basal core promoter, the S-gene promoter, the Enh1 enhancer, and the Enh2 enhancer, the third gRNA targets a gene selected from the list consisting of the L-HBs promoter, the M-HBs promoter, the S-HBs promoter, the X-promoter, the basal core promoter, the S-gene promoter, the Enh1 enhancer, and the Enh2 enhancer, the fourth gRNA targets a gene selected from the list consisting of the L-HBs promoter, the M-HBs promoter, the S-HBs promoter, the X-promoter, the basal core promoter, the S-gene promoter, the Enh1 enhancer, and the Enh2 enhancer, and the first, second, third, and fourth gRNAs target different genes or regulatory elements thereof. In some embodiments, the first gRNA, the second gRNA, the third gRNA, and the fourth target four genes, or combinations of regulatory elements thereof, selected from the genes, or combinations of regulatory elements thereof, listed in Table 2. In some embodiments, the first gRNA, the second gRNA, the third gRNA, and the fourth gRNA are each independently selected from any of the gRNAs described herein.

[0260] In some embodiments, the gRNA combination comprises at least five gRNAs that target at least five different genes or their regulatory elements. In some embodiments, the gRNA combination comprises at least six gRNAs that target at least six different genes and / or their regulatory elements. In some embodiments, the first, second, third, fourth, fifth, and / or sixth genes or their regulatory elements are different.

[0261] C. Engineered Zinc Finger Proteins (eZFPs) In some aspects, provided herein are zinc finger proteins (ZFPs), such as engineered zinc finger proteins (eZFPs). In some embodiments, the eZFPs are capable of binding to or bind to target sites in HBV genes or regulatory elements of genes in hepatitis B virus sequences. In some aspects, the eZFPs can facilitate specific targeting of effector domains for transcriptional repression of genes or regulatory elements. In some embodiments, provided herein are epigenetic modifying DNA targeting systems, including fusion proteins comprising eZFPs and one or more other elements, such as effector domains for transcriptional repression. Thus, in some aspects, the eZFPs facilitate reduced expression of HBV genes or regulatory elements, for example, in the context of compositions and methods for treating HBV-related diseases or disorders, such as HBV virus infection, liver disease, or cancer.

[0262] In some embodiments, a zinc finger protein (ZFP), zinc finger DNA-binding protein, or zinc finger DNA-binding domain is a protein, or a domain within a larger protein, that binds to DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through the coordination of zinc ions. Among these, ZFPs are artificial or engineered ZFPs (eZFPs), which contain ZFP domains, typically 9-18 nucleotides long, that target specific DNA sequences and are generated by the assembly of individual zinc fingers. ZFPs include those with two, three, four, five, or six fingers, where a single finger domain is approximately 30 amino acids long and contains an alpha helix containing two invariant histidine residues coordinated through zinc with two cysteines in a single beta turn. Generally, the sequence specificity of a ZFP can be altered by making amino acid substitutions at the four helix positions (-1, 2, 3, and 6) of the zinc finger's recognition helix, also known as the zinc finger recognition region. Thus, for example, a ZFP or ZFP-containing molecule, eg, a fusion protein, can be non-naturally occurring, eg, engineered to bind to an optimal target site.

[0263] In some embodiments, zinc fingers can be custom-designed (i.e., designed by the user) and / or obtained from commercial sources.Various methods are available for designing zinc finger proteins.For example, the method for designing zinc finger proteins to bind to target DNA sequences of interest is described in, for example, Liu, Q. et al., PNAS, 94(11):5525-30 (1997);Wright, DA et al., Nat. Protoc., 1(3):1637-52 (2006);Gersbach, CA et al., Acc. Chem. Res., 47(8):2309-18 (2014);Bhakta MS et al., Methods Mol. Biol., 649:3-30 (2010);And Gaj et al., Trends Biotechnol, 31(7):397-405 (2013). In addition, various web-based tools are publicly available for designing zinc finger proteins to bind to a DNA target sequence of interest (see, for example, the Zinc Finger Tools design website from Scripps, available on the World Wide Web at scripps.edu / barbas / zfdesign / zfdesignhome.php). Various commercial services are also available for designing zinc finger proteins to bind to a DNA target sequence of interest.See, for example, commercially available services or kits provided by Creative Biolabs (World Wide Web, creative-biolabs.com / Design-and-Synthesis-of-Artificial-Zinc-Finger-Proteins.html), the Zinc Finger Consortium Modular Assembly Kit available from Addgene (World Wide Web, addgene.org / kits / zfc-modular-assembly / ), or the CompoZr Custom ZFN Service from Sigma Aldrich (World Wide Web, sigmaaldrich.com / life-science / zinc-finger-nuclease-technology / custom-zfn.html).

[0264] In some embodiments, the present invention provides an epigenetic modifying DNA targeting system, comprising a fusion protein comprising an eZFP and one or more other elements, such as an effector domain for transcriptional repression. In some embodiments, at least one DNA binding domain of the epigenetic modifying DNA targeting system comprises an engineered zinc finger protein (eZFP). In some embodiments, the epigenetic modifying DNA targeting system comprises an engineered zinc finger protein (eZFP) that binds to a target site in one or more HBV genes or their regulatory elements. The target site targeted by any of the provided eZFPs can be any of those described herein, for example, any of the target sites described in Section IA.

[0265] In some embodiments, the target site of an eZFP provided herein (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system) is located in one or more HBV genes or regulatory elements thereof. In some embodiments, the target site is located in a CpG island (e.g., CpG island 1, CpG island 2, CpG island 3) of the HBV genome. In some embodiments, the target site is located in CpG island 2 of the HBV genome. In some embodiments, the target site is located within a target region spanning 1033 bp to 1749 bp of the HBV genome, corresponding to the position with reference to the HBV genome set forth in SEQ ID NO:650. In some embodiments, the target site is located within a target region spanning within 300 base pairs (bp), 250 bp, 200 bp, 150 bp, 140 bp, 130 bp, 120 bp, 110 bp, or 100 bp upstream of the HBx start codon. In some embodiments, the target site is within a target region sequence corresponding to a sequence spanning 1250-1374 bp with reference to the HBV genome set forth in SEQ ID NO:650. In some embodiments, the target region has the sequence set forth in SEQ ID NO:1068. In some embodiments, the target site is within a target region sequence corresponding to a sequence spanning 1255-1302 bp with reference to the HBV genome set forth in SEQ ID NO:650. In some embodiments, the target region has the sequence set forth in SEQ ID NO:1069. In some embodiments, the target site is within a target region sequence corresponding to a sequence spanning 1260-1300 bp with reference to the HBV genome set forth in SEQ ID NO:650. In some embodiments, the target region has the sequence set forth in SEQ ID NO:1070.

[0266] In some embodiments, the target site of an eZFP provided herein (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system) comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 1028-1055, at least a 12-nt contiguous portion thereof, or a complementary sequence of any of the foregoing. In some embodiments, the target site of an eZFP provided herein comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 1028-1055. In some embodiments, the target site of an eZFP provided herein comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 1045, 1046, or 1052, at least a 12-nt contiguous portion thereof, or a complementary sequence of any of the foregoing. In some embodiments, the target site comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 1045, 1046, or 1052.

[0267] In some embodiments, the target site is contained in a double-stranded DNA, e.g., an HBV sequence integrated into human genomic DNA. In some embodiments, the target site is contained in a covalently closed circular (cccDNA) HBV sequence. In some embodiments, the target site is contained in an incomplete double-stranded DNA (rcNDA) HBV sequence. In some embodiments, an eZFP is capable of binding to a target site. In some embodiments, an eZFP binds to a target site. In some embodiments, the binding is target-specific. For example, in some embodiments, an eZFP binds to a target site and does not bind to other sites comprising different sequences. For example, in some embodiments, an individual eZFP disclosed herein binds to a target site set forth in any one of SEQ ID NOs: 1028-1055 and does not bind to a different target site. In some embodiments, an individual eZFP disclosed herein binds to a target site set forth in any one of SEQ ID NOs: 1045, 1046, or 1052 and does not bind to a different target site. In some embodiments, the target sites of the eZFPs provided herein comprise the sequences shown in Table 7.

[0268] Table 7. eZFP target sequences TIFF2025527567000048.tif164128

[0269] In some embodiments, the target site of an eZFP provided herein (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system) comprises the nucleotide sequence set forth in SEQ ID NO: 1045. In some embodiments, at least a 12-nt contiguous portion thereof, or a complementary sequence of any of the foregoing. In some embodiments, the target site of an eZFP provided herein comprises the sequence set forth in SEQ ID NO: 1045.

[0270] In some embodiments, the target site of an eZFP provided herein (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system) comprises the nucleotide sequence set forth in SEQ ID NO: 1046. In some embodiments, at least a 12-nt contiguous portion thereof, or a complementary sequence of any of the foregoing. In some embodiments, the target site of an eZFP provided herein comprises the sequence set forth in SEQ ID NO: 1046.

[0271] In some embodiments, the target site of an eZFP provided herein (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system) comprises the nucleotide sequence set forth in SEQ ID NO: 1052. In some embodiments, at least a 12-nt contiguous portion thereof, or a complementary sequence of any of the foregoing. In some embodiments, the target site of an eZFP provided herein comprises the sequence set forth in SEQ ID NO: 1052.

[0272] In some embodiments, an eZFP comprises multiple zinc fingers. In some embodiments, each zinc finger comprises a recognition region. In some embodiments, the recognition regions together facilitate sequence-specific binding of the eZFP, e.g., to a specific target site. In some embodiments, an eZFP comprises six zinc fingers, designated F1-F6 from N-terminus to C-terminus, each of which comprises a corresponding recognition region F1-F6 that facilitates sequence-specific binding to a specific target site.

[0273] In some embodiments, the characteristics of eZFPs that target specific target sites provided herein are set forth in Table E4. In some embodiments, the eZFPs comprise six zinc fingers, designated F1-F6 in N- to C-terminal order, each containing a corresponding recognition region F1-F6 as set forth in Table E4. In some embodiments, the recognition regions F1-F6 facilitate specific binding to a designated target site sequence in Table E4. In some embodiments, the eZFPs comprise an amino acid sequence comprising a recognition region as set forth in Table E4. In some embodiments, the eZFPs can be encoded by a DNA sequence as set forth in Table 8.

[0274] Table 8. eZFP DNA sequences TIFF2025527567000049.tif143165TIFF2025527567000050.tif237165TIFF2025527567000051.tif24116 5TIFF2025527567000052.tif241165TIFF2025527567000053.tif241165TIFF2025527567000054.tif62165

[0275] In some embodiments, an eZFP (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system) comprises a zinc finger protein that includes six zinc fingers, designated F1 to F6 from the N-terminus to the C-terminus. In some embodiments, the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000055.tif151164TIFF2025527567000056.tif231170

[0276] In some embodiments, the eZFP (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system) comprises a sequence set forth in any one of SEQ ID NOs: 692-719, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP is encoded by a nucleotide sequence set forth in any one of SEQ ID NOs: 888-915, or a portion thereof, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0277] In some embodiments, provided herein is an eZFP (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system), e.g., eZFP_1 as described herein. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1028, at least a 12-nt contiguous portion thereof, or the complementary sequence of any of the foregoing. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1028. In some embodiments, the eZFP comprises six zinc fingers, designated F1-F6 in order from N-terminus to C-terminus, each comprising a corresponding zinc finger recognition region F1-F6, wherein the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000057.tif17159: In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:692, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:692. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:888, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:888.

[0278] In some embodiments, provided herein is an eZFP (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system), e.g., eZFP_2 as described herein. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1029, at least a 12-nt contiguous portion thereof, or the complementary sequence of any of the foregoing. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1029. In some embodiments, the eZFP comprises six zinc fingers, designated F1-F6 in order from N-terminus to C-terminus, each comprising a corresponding zinc finger recognition region F1-F6, wherein the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000058.tif17156. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:693, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:693. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:889, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:889.

[0279] In some embodiments, provided herein is an eZFP (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system), e.g., eZFP_3 as described herein. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1030, at least a 12-nt contiguous portion thereof, or the complementary sequence of any of the foregoing. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1030. In some embodiments, the eZFP comprises six zinc fingers, designated F1-F6 in order from N-terminus to C-terminus, each comprising a corresponding zinc finger recognition region F1-F6, wherein the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000059.tif17156. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:694, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:694. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:890, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:890.

[0280] In some embodiments, provided herein is an eZFP (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system), e.g., eZFP_4 as described herein. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1031, at least a 12-nt contiguous portion thereof, or the complementary sequence of any of the foregoing. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1031. In some embodiments, the eZFP comprises six zinc fingers, designated F1-F6 in order from N-terminus to C-terminus, each comprising a corresponding zinc finger recognition region F1-F6, wherein the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000060.tif17156. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:695, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:695. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:891, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:891.

[0281] In some embodiments, provided herein is an eZFP (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system), e.g., eZFP_5 as described herein. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1032, at least a 12-nt contiguous portion thereof, or the complementary sequence of any of the foregoing. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1032. In some embodiments, the eZFP comprises six zinc fingers, designated F1-F6 in order from N-terminus to C-terminus, each comprising a corresponding zinc finger recognition region F1-F6, wherein the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000061.tif17157. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:696, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:696. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:892, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:892.

[0282] In some embodiments, provided herein is an eZFP (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system), e.g., eZFP_6 as described herein. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1033, at least a 12-nt contiguous portion thereof, or the complementary sequence of any of the foregoing. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1033. In some embodiments, the eZFP comprises six zinc fingers, designated F1-F6 in order from N-terminus to C-terminus, each comprising a corresponding zinc finger recognition region F1-F6, wherein the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000062.tif17156. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:697, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:697. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:893, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:893.

[0283] In some embodiments, provided herein is an eZFP (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system), e.g., eZFP_7 as described herein. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1034, at least a 12-nt contiguous portion thereof, or the complementary sequence of any of the foregoing. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1034. In some embodiments, the eZFP comprises six zinc fingers, designated F1-F6 in order from N-terminus to C-terminus, each comprising a corresponding zinc finger recognition region F1-F6, wherein the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000063.tif17157. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:698, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:698. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:894, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:894.

[0284] In some embodiments, provided herein is an eZFP (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system), e.g., eZFP_8 as described herein. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1035, at least a 12-nt contiguous portion thereof, or the complementary sequence of any of the foregoing. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1035. In some embodiments, the eZFP comprises six zinc fingers, designated F1-F6 in order from N-terminus to C-terminus, each comprising a corresponding zinc finger recognition region F1-F6, wherein the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000064.tif17158. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:699, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:699. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:895, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:895.

[0285] In some embodiments, provided herein is an eZFP (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system), e.g., eZFP_9 as described herein. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1036, at least a 12-nt contiguous portion thereof, or the complementary sequence of any of the foregoing. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1036. In some embodiments, the eZFP comprises six zinc fingers, designated F1-F6 in order from N-terminus to C-terminus, each comprising a corresponding zinc finger recognition region F1-F6, wherein the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000065.tif17157. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:700, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:700. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:896, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:896.

[0286] In some embodiments, provided herein is an eZFP (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system), e.g., eZFP_10, as described herein. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1037, at least a 12-nt contiguous portion thereof, or the complementary sequence of any of the foregoing. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1037. In some embodiments, the eZFP comprises six zinc fingers, designated F1-F6 in order from N-terminus to C-terminus, each comprising a corresponding zinc finger recognition region F1-F6, wherein the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000066.tif17156. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:701, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:701. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:897, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:897.

[0287] In some embodiments, provided herein is an eZFP (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system), e.g., eZFP_11, as described herein. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1038, at least a 12-nt contiguous portion thereof, or the complementary sequence of any of the foregoing. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1038. In some embodiments, the eZFP comprises six zinc fingers, designated F1-F6 in order from N-terminus to C-terminus, each comprising a corresponding zinc finger recognition region F1-F6, wherein the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000067.tif17156. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:702, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:702. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:898, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:898.

[0288] In some embodiments, provided herein is an eZFP (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system), e.g., eZFP_12 as described herein. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1039, at least a 12-nt contiguous portion thereof, or the complementary sequence of any of the foregoing. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1039. In some embodiments, the eZFP comprises six zinc fingers, designated F1-F6 in order from N-terminus to C-terminus, each comprising a corresponding zinc finger recognition region F1-F6, wherein the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000068.tif17156. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:703, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:703. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:899, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:899.

[0289] In some embodiments, provided herein is an eZFP (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system), e.g., eZFP_13 as described herein. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1040, at least a 12-nt contiguous portion thereof, or the complementary sequence of any of the foregoing. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1040. In some embodiments, the eZFP comprises six zinc fingers, designated F1-F6 in order from N-terminus to C-terminus, each comprising a corresponding zinc finger recognition region F1-F6, wherein the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000069.tif17158. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:704, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:704. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:900, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:900.

[0290] In some embodiments, provided herein is an eZFP (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system), e.g., eZFP_14 as described herein. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1041, at least a 12-nt contiguous portion thereof, or the complementary sequence of any of the foregoing. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1041. In some embodiments, the eZFP comprises six zinc fingers, designated F1-F6 in order from N-terminus to C-terminus, each comprising a corresponding zinc finger recognition region F1-F6, wherein the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000070.tif17157. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:705, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:705. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:901, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:901.

[0291] In some embodiments, provided herein is an eZFP (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system), e.g., eZFP_15 as described herein. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1042, at least a 12-nt contiguous portion thereof, or the complementary sequence of any of the foregoing. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1042. In some embodiments, the eZFP comprises six zinc fingers, designated F1-F6 in order from N-terminus to C-terminus, each comprising a corresponding zinc finger recognition region F1-F6, wherein the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000071.tif17156. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:706, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:706. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:902, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:902.

[0292] In some embodiments, provided herein is an eZFP (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system), e.g., eZFP_16 as described herein. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1043, at least a 12-nt contiguous portion thereof, or the complementary sequence of any of the foregoing. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1043. In some embodiments, the eZFP comprises six zinc fingers, designated F1-F6 in order from N-terminus to C-terminus, each comprising a corresponding zinc finger recognition region F1-F6, wherein the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000072.tif17157. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:707, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:707. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:903, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:903.

[0293] In some embodiments, provided herein is an eZFP (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system), e.g., eZFP_17, as described herein. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1044, at least a 12-nt contiguous portion thereof, or the complementary sequence of any of the foregoing. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1044. In some embodiments, the eZFP comprises six zinc fingers, designated F1-F6 in order from N-terminus to C-terminus, each comprising a corresponding zinc finger recognition region F1-F6, wherein the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000073.tif17158. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:708, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:708. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:904, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:904.

[0294] In some embodiments, provided herein is an eZFP (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system), e.g., eZFP_18 as described herein. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1045, at least a 12-nt contiguous portion thereof, or the complementary sequence of any of the foregoing. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1045. In some embodiments, the eZFP comprises six zinc fingers, designated F1-F6 in order from N-terminus to C-terminus, each comprising a corresponding zinc finger recognition region F1-F6, wherein the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000074.tif17157. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:709, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:709. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:905, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:905.

[0295] In some embodiments, provided herein is an eZFP (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system), e.g., eZFP_19, as described herein. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1046, at least a 12-nt contiguous portion thereof, or the complementary sequence of any of the foregoing. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1046. In some embodiments, the eZFP comprises six zinc fingers, designated F1-F6 in order from N-terminus to C-terminus, each comprising a corresponding zinc finger recognition region F1-F6, wherein the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000075.tif17157. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:710, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:710. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:906, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:906.

[0296] In some embodiments, provided herein is an eZFP (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system), e.g., eZFP_20 as described herein. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1047, at least a 12-nt contiguous portion thereof, or the complementary sequence of any of the foregoing. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1047. In some embodiments, the eZFP comprises six zinc fingers, designated F1-F6 in order from N-terminus to C-terminus, each comprising a corresponding zinc finger recognition region F1-F6, wherein the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000076.tif17158. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:711, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:711. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:907, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:907.

[0297] In some embodiments, provided herein is an eZFP (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system), e.g., eZFP_21 as described herein. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1048, at least a 12-nt contiguous portion thereof, or the complementary sequence of any of the foregoing. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1048. In some embodiments, the eZFP comprises six zinc fingers, designated F1-F6 in order from N-terminus to C-terminus, each comprising a corresponding zinc finger recognition region F1-F6, wherein the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000077.tif17156. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:712, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:712. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:908, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:908.

[0298] In some embodiments, provided herein is an eZFP (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system), e.g., eZFP_22 as described herein. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1049, at least a 12-nt contiguous portion thereof, or the complementary sequence of any of the foregoing. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1049. In some embodiments, the eZFP comprises six zinc fingers, designated F1-F6 in order from N-terminus to C-terminus, each comprising a corresponding zinc finger recognition region F1-F6, wherein the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000078.tif17155. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:713, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:713. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:909, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:909.

[0299] In some embodiments, provided herein is an eZFP (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system), e.g., eZFP_23 as described herein. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1050, at least a 12-nt contiguous portion thereof, or the complementary sequence of any of the foregoing. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1050. In some embodiments, the eZFP comprises six zinc fingers, designated F1-F6 in order from N-terminus to C-terminus, each comprising a corresponding zinc finger recognition region F1-F6, wherein the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000079.tif17156. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:714, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:714. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:910, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:910.

[0300] In some embodiments, provided herein is an eZFP (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system), e.g., eZFP_24 as described herein. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1051, at least a 12-nt contiguous portion thereof, or the complementary sequence of any of the foregoing. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1051. In some embodiments, the eZFP comprises six zinc fingers, designated F1-F6 in order from N-terminus to C-terminus, each comprising a corresponding zinc finger recognition region F1-F6, wherein the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000080.tif17156. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:715, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:715. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:911, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:911.

[0301] In some embodiments, provided herein is an eZFP (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system), e.g., eZFP_25 as described herein. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1052, at least a 12-nt contiguous portion thereof, or the complementary sequence of any of the foregoing. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1052. In some embodiments, the eZFP comprises six zinc fingers, designated F1-F6 in order from N-terminus to C-terminus, each comprising a corresponding zinc finger recognition region F1-F6, wherein the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000081.tif17156. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:716, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:716. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:912, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:912.

[0302] In some embodiments, provided herein is an eZFP (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system), e.g., eZFP_26 as described herein. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1053, at least a 12-nt contiguous portion thereof, or the complementary sequence of any of the foregoing. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1053. In some embodiments, the eZFP comprises six zinc fingers, designated F1-F6 in order from N-terminus to C-terminus, each comprising a corresponding zinc finger recognition region F1-F6, wherein the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000082.tif17156. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:717, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:717. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:913, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:913.

[0303] In some embodiments, provided herein is an eZFP (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system), e.g., eZFP_27 as described herein. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1054, at least a 12-nt contiguous portion thereof, or the complementary sequence of any of the foregoing. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1054. In some embodiments, the eZFP comprises six zinc fingers, designated F1-F6 in order from N-terminus to C-terminus, each comprising a corresponding zinc finger recognition region F1-F6, wherein the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000083.tif17155. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:718, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:718. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:914, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:914.

[0304] In some embodiments, provided herein is an eZFP (e.g., an eZFP included in a fusion protein of an epigenetic modifying DNA targeting system), e.g., eZFP_28 as described herein. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1055, at least a 12-nt contiguous portion thereof, or the complementary sequence of any of the foregoing. In some embodiments, the eZFP targets a target site comprising the nucleotide sequence set forth in SEQ ID NO: 1055. In some embodiments, the eZFP comprises six zinc fingers, designated F1-F6 in order from N-terminus to C-terminus, each comprising a corresponding zinc finger recognition region F1-F6, wherein the amino acid sequence of each zinc finger recognition region is as follows: TIFF2025527567000084.tif17156. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:719, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP comprises the amino acid sequence set forth in SEQ ID NO:719. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:915, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the eZFP is encoded by the nucleotide sequence set forth in SEQ ID NO:915.

[0305] D. Other DNA-binding domains In some of any provided embodiments, the DNA-binding domain comprises a transcription activator-like effector (TALE); a meganuclease; a homing endonuclease; or an I-SceI enzyme or variant thereof. In some embodiments, the DNA-binding domain comprises a catalytically inactive variant of any of the foregoing.

[0306] Transcription activator-like effectors (TALEs) are proteins naturally found in Xanthomonas bacteria. TALEs contain multiple repeat amino acid sequences, each with binding specificity for a single base in the target sequence. Each repeat contains a pair of variable residues (repeat variable dinucleotides; RVDs) at positions 12 and 13 that determine the nucleotide specificity of that repeat. In some embodiments, the RVDs involved in recognizing different nucleotides are HD for C, NG for T, NI for A, NN for G or A, NS for A, C, G, or T, HG for T, IG for T, NK for G, HA for C, ND for C, HI for C, HN for G, NA for G, SN for G or A, and YG for T, TL for A, VT for A or G, and SW for A. In some embodiments, the RVD can be mutated to other amino acid residues to modulate its specificity for the nucleotides A, T, C, and G, and in particular to enhance this specificity. Binding domains with similar modular base-pair-base nucleic acid binding properties can also be derived from different bacterial species. These alternative modular proteins can exhibit greater sequence diversity than TALE repeats.

[0307] In some embodiments, a "TALE DNA binding domain" or "TALE" is a polypeptide comprising one or more TALE repeat domains / units. The repeat domains, each containing a repeat variable di-residue (RVD), are responsible for binding of the TALE to its cognate target DNA sequence. A single "repeat unit" (also referred to as a "repeat") is typically 33-35 amino acids in length and exhibits at least some sequence homology to other TALE repeat sequences within naturally occurring TALE proteins. TALE proteins can be designed to bind to target sites using canonical or non-canonical RVDs within the repeat unit. See, e.g., U.S. Patent Nos. 8,586,526 and 9,458,205.

[0308] In some embodiments, the TALE is a fusion protein comprising a nucleic acid binding domain and an effector domain derived from the TALE. In some embodiments, one or more sites in the FXN locus can be targeted by the engineered TALE.

[0309] Zinc finger and TALE DNA-binding domains can be engineered to bind to a given nucleotide sequence, for example, through manipulation of the recognition helix region of naturally occurring zinc finger proteins (changing one or more amino acids), by manipulation of amino acids in the TALE repeats (repeated variable di-residue or RVD regions) involved in DNA binding, or by systematic ordering of modular DNA-binding domains, such as TALE repeats or ZFP domains. Therefore, engineered zinc finger proteins or TALE proteins are non-naturally occurring proteins. A non-limiting example of a method for engineering zinc finger proteins and TALEs is design and selection. Designed proteins are non-naturally occurring proteins whose design / composition arises primarily from rational criteria. Rational criteria for design include substitution rules and the application of computer algorithms to process information in databases storing information on existing ZFP or TALE designs (canonical and non-canonical RVDs) and binding data. See, e.g., U.S. Patent Nos. 9,458,205; 8,586,526; 6,140,081; 6,453,242; and 6,534,261; also WO 98 / 53058; WO 98 / 53059; WO 98 / 53060; WO 02 / 016536; and WO 03 / 016496.

[0310] E. Effector Domain In some aspects, the DNA targeting system provided herein comprises one or more effector domains.In some embodiments, the DNA targeting system provided herein comprises a fusion protein comprising (a) a DNA binding domain for targeting a target site in a Hepatitis B virus DNA sequence, for example, a gene or its regulatory element, for example, any of those described above, and (b) at least one effector domain.In some aspects, the effector domain can reduce the transcription of a gene, i.e., comprises a transcription repressor effector domain.In some aspects, the effector domain comprises a transcription repressor effector domain.

[0311] In some aspects, the effector domain, when ectopically recruited to a gene or its DNA regulatory elements, represses, induces, catalyzes, or leads to reduced transcription of the gene and / or its regulatory elements.

[0312] In some embodiments, the effector domain induces, catalyzes, or leads to transcriptional repression, transcriptional co-repression, transcriptional repression, transcription factor release, polymerization, histone modification, histone acetylation, histone deacetylation, nucleosome remodeling, chromatin remodeling, heterochromatin formation, protein degradation, ubiquitination, deubiquitination, phosphorylation, dephosphorylation, splicing, nucleic acid association, DNA methylation, DNA demethylation, histone methylation, histone demethylation, or DNA base oxidation. In some embodiments, the effector domain inhibits, induces, catalyzes, or leads to transcriptional repression or transcriptional co-repression. In some embodiments, the effector domain induces transcriptional repression. In some embodiments, the effector domain itself possesses one of the aforementioned activities (i.e., acts directly). In some embodiments, the effector domain recruits and / or interacts with a polypeptide domain having one of the aforementioned activities (i.e., acts indirectly).

[0313] Gene expression of endogenous mammalian genes, e.g., human genes, can be achieved by targeting a fusion protein comprising a DNA-binding domain (e.g., dCas9) and an effector domain to the mammalian gene or its regulatory DNA elements (e.g., promoters or enhancers) via one or more gRNAs. Any of a variety of effector domains are known and can be used in accordance with the provided embodiments. Repression of target genes and / or their regulatory elements by such effector domains as Cas fusion proteins with various Cas molecules and transcriptional repressor domains is described, for example, in WO2021226077, WO2017180915, WO2014197748, WO2014093655, US20190127713, WO2013176772, Adli, M. Nat. Commun. 9, 1911 (2018), Urrutia, R. Genome Biol. 4, 231 (2003), Groner, AC et al. PLoS Genet. 6, e1000869 (2010), Liu, XS et al. Cell 167, 233-247.e17 (2016), and Lei, Y. et al. Nat. Commun. 8, 16026 (2017).

[0314] In some embodiments, the effector domain can comprise a KRAB domain, an ERF repressor domain, an MXI1 domain, a SID4X domain, a MAD-SID domain, a DNMT family protein domain (e.g., DNMT3A or DNMT3B), a fusion of one or more DNMT family proteins or domains thereof (e.g., DNMT3A / L, which comprises a fusion of the DNMT3A and DNMT3L domains), an EZH2 domain, an LSD1, a SunTag domain, a partial or fully functional fragment or domain of any of the foregoing, or a combination of any of the foregoing. In some embodiments, the fusion protein can be dCas9-KRAB. In some embodiments, the fusion protein can be DNMT3A / L-dCas9-KRAB. In some embodiments, the fusion protein can be KRAB-dCas9-DNMT3A / L.

[0315] In some embodiments, the effector domain comprises a transcriptional repressor domain described in WO 2021 / 226077.

[0316] In some embodiments, the effector domain comprises a KRAB domain or a variant thereof. KRAB-containing zinc finger proteins constitute the largest transcriptional repressor family in mammals. The Kruppel-associated box (KRAB) domain is a transcriptional repressor domain present in many zinc finger protein-based transcription factors. The KRAB domain contains charged amino acids and can be divided into subdomains A and B. The KRAB domain recruits epigenetic readers such as the corepressor KAP1 (KRAB-associated protein-1), heterochromatin protein 1 (HP1), and other chromatin modulators, inducing transcriptional repression through heterochromatin formation. KRAB-mediated gene repression is accompanied by a loss of histone H3 acetylation and an increase in H3 lysine 9 trimethylation (H3K9me3) in the promoters of repressed genes. KRAB domains, including those in dCas fusion proteins, are 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), and Groner AC et al. PLoS Genet. 6, e1000869 (2010). In some embodiments, the effector domain comprises at least one KRAB domain or a variant thereof.

[0317] In some embodiments, the KRAB domain is set forth in SEQ ID NO: 590. In some embodiments, the effector domain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 590, or a portion thereof, or any of the foregoing.

[0318] In some embodiments, the KRAB domain is set forth in SEQ ID NO: 669. In some embodiments, the effector domain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 669, or a portion thereof, or any of the foregoing.

[0319] In some embodiments, the effector domain comprises at least one ERF repressor domain or a variant thereof. ERF (ETS2 repressor factor) is a potent transcriptional repressor containing a conserved ets-DNA binding domain and represses transcription through a distinct domain at the carboxyl terminus of the protein. ERF repressor domains, including those in dCas fusion proteins, are described, for example, in WO2017180915, WO2014197748, WO2013176772, and Mavrothalassitis, G., Ghysdael, J. Proteins of the ETS family with transcriptional repressor activity. Oncogene 19, 6524-6532 (2000). In some embodiments, the effector domain comprises at least one ERF repressor domain or a variant thereof. An exemplary ERF repressor domain is set forth in SEQ ID NO: 600. In some embodiments, the effector domain comprises the sequence set forth in SEQ ID NO:600, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

[0320] In some embodiments, the effector domain comprises at least one MXI1 domain or a variant thereof. The MXI1 domain functions by antagonizing myc transcriptional activity by competing for binding to myc-associated factor x (MAX). MXI1 domains, including those in dCas fusion proteins, are described, for example, in WO2017180915, WO2014197748, and US20190127713. In some embodiments, the effector domain comprises at least one MXI1 domain or a variant thereof. An exemplary MXI1 domain is set forth in SEQ ID NO:601. In some embodiments, the effector domain comprises the sequence set forth in SEQ ID NO:601, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing.

[0321] In some embodiments, the effector domain comprises at least one SID4X domain or a variant thereof. The mSin3-interacting domain (SID) is present on different transcriptional repressor proteins. SID interacts with the paired amphipathic alpha helix 2 (PAH2) domain of mSin3, a transcriptional repressor domain that binds to transcriptional repressor proteins such as the mSin3 A corepressor. A dCas9 molecule can be fused to four linked mSin3-interacting domains (SID4X). SID domains, including those in dCas fusion proteins, are described, for example, in WO2017180915, WO2014197748, and WO2014093655. In some embodiments, the effector domain comprises at least one SID domain or a variant thereof. An exemplary SID domain is set forth in SEQ ID NO:602. In some embodiments, the effector domain comprises the sequence set forth in SEQ ID NO:602, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of the foregoing.

[0322] In some embodiments, the effector domain comprises at least one MAD domain or a variant thereof. The MAD family proteins Mad1, Mxi1, Mad3, and Mad4 belong to the basic helix-loop-helix-zipper class and contain a conserved N-terminal region (called the Sin3 interaction domain (SID)) required for repression activity. MAD-SID domains, including those in dCas fusion proteins, are described, for example, in WO2017180915, WO2014197748, and WO2013176772. In some embodiments, the effector domain comprises at least one MAD-SID domain or a variant thereof. An exemplary MAD-SID domain is set forth in SEQ ID NO:603. In some embodiments, the effector domain comprises the sequence set forth in SEQ ID NO:603, or a portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of the foregoing.

[0323] In some embodiments, the effector domain is derived from DNMT3, or a portion or functionally active variant thereof with DNA methyltransferase activity.DNMT3A and DNMT3B are two DNA methyltransferases that catalyze de novo methylation, and depending on the site, may be involved in transcriptional repression.DNMTs, such as DNMT3, mediate the 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 cytosine bases to methylated 5-methylcytosine. DNMT3, including in dCas fusion proteins, is described, for example, in US20190127713, Liu, XS et al. Cell 167, 233-247.e17 (2016), and Lei, Y. et al. Nat. Commun. 8, 16026 (2017). DNMT3 proteins, such as DNMT3A and DNMT3B, contain an N-terminal portion naturally involved in regulatory activity and targeting, and a C-terminal catalytic domain called an MTase C5-type domain. In some embodiments, the effector domain in the embodiments provided herein comprises a catalytically active portion of DNMT3A or DNMT3B containing the catalytically active C-terminal domain. In particular, the isolated catalytic domains of DNMT3a and DNMT3b are catalytically active (see, e.g., Gowher and Jeltsch (2002) J. Biol. Chem., 277:20409).

[0324] In some embodiments, the DNMT3 domain can be a catalytically active effector domain of DNMT3A or DNMT3B. In some embodiments, the effector domain can be full-length DNMT3A or DNMT3B or a catalytically active portion thereof. In some embodiments, the effector domain is a catalytically active portion that is shorter than the full-length sequence of DNMT3A or DNMT3B. In some embodiments, the catalytically active portion is a contiguous sequence of amino acids that confers DNA methyltransferase activity, for example, by mediating the methylation of cytosine bases to methylated 5-methylcytosine. In some embodiments, the contiguous sequence of amino acids is a contiguous C-terminal portion of a DNMT3 protein, e.g., DNMT3A or DNMT3B, that is 280 to 330 amino acids in length. In some embodiments, the contiguous portion is 280, 290, 300, 310, 320, or 330 amino acids in length, or any value between any of the foregoing. In some embodiments, the catalytically active portion of a DNMT, e.g., DNMT3, comprises a SAM-dependent MTase C5-type domain. In some embodiments, the DNMT3 domain, e.g., the domain of DNMT3A or DNMT3B, is of human origin.

[0325] In some embodiments, the effector domain is derived from DNMT3A or a catalytically active portion or variant thereof. An exemplary DNMT3A domain is set forth in SEQ ID NO:604, or a catalytically active portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:604 or a catalytically active portion thereof that exhibits DNA methyltransferase activity. In some embodiments, the catalytically active portion is a contiguous portion of amino acids of SEQ ID NO:604 (e.g., corresponding to amino acids 634-912 of SEQ ID NO:604) that includes a SAM-dependent MTase C5-type domain. In some embodiments, the contiguous sequence of amino acids of SEQ ID NO:604 includes at least 250 amino acids, 275 amino acids, 300 amino acids, or 325 amino acids, or any value between any of the foregoing. In some embodiments, the contiguous sequence of amino acids is a contiguous portion of SEQ ID NO:604 that includes amino acids 634 to 912 and is between 280 amino acids and 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 any value in between any of the foregoing.

[0326] In some embodiments, the DNMT3A domain is set forth in SEQ ID NO:661, or a catalytically active portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:661 or a catalytically active portion thereof that exhibits DNA methyltransferase activity. In some embodiments, the DNMT3A domain is set forth in SEQ ID NO:661.

[0327] In some embodiments, the DNMT3A domain is set forth in SEQ ID NO:665, or a catalytically active portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:665 or a catalytically active portion thereof that exhibits DNA methyltransferase activity. In some embodiments, the DNMT3A domain is set forth in SEQ ID NO:665.

[0328] In some embodiments, the effector domain is derived from DNMT3B or a catalytically active portion or variant thereof that exhibits DNA methyltransferase activity. An exemplary DNMT3B domain is set forth in SEQ ID NO:605, or a catalytically active portion thereof, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:605 or a catalytically active portion thereof that exhibits DNA methyltransferase activity. In some embodiments, the catalytically active portion is a contiguous portion of amino acids of SEQ ID NO:605 (e.g., corresponding to amino acids 575-853 of SEQ ID NO:605) that includes a SAM-dependent MTase C5-type domain. In some embodiments, the contiguous sequence of amino acids of SEQ ID NO:605 includes at least 250 amino acids, 275 amino acids, 300 amino acids, or 325 amino acids, or any value between any of the foregoing. In some embodiments, the contiguous sequence of amino acids is a contiguous por...

Claims

1. Multiple polynucleotides, including the following: (a) A gRNA for targeting a target site in the hepatitis B virus DNA sequence, wherein the target site includes the sequence shown in SEQ ID NO: 22, and (b) mRNA encoding a fusion protein comprising an inactive Streptococcus pyogenes dCas9 (dSpCas9) protein and a transcription repressor effector domain comprising a KRAB domain and a DNMT3A / L domain, wherein the KRAB domain comprises the sequence shown in SEQ ID NO: 590 and the DNMT3A / L domain comprises the sequence shown in SEQ ID NO:

651.

2. A vector comprising a plurality of polynucleotides as described in claim 1.

3. The vector according to claim 2, wherein the vector is a lipid nanoparticle.

4. The plurality of polynucleotides according to claim 1, wherein the KRAB domain comprises the sequence shown in SEQ ID NO:

669.

5. The plurality of polynucleotides according to claim 1, wherein the gRNA comprises the sequence shown in SEQ ID NO:

217.

6. The plurality of polynucleotides according to claim 1, wherein the dSpCas9 comprises at least one amino acid mutation selected from D10A and H840A, with reference to the numbering of the position SEQ ID NO:

598.

7. The plurality of polynucleotides according to claim 1, wherein the dSpCas9 has at least 95% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 599, and contains alanine (A) at amino acid position 9 and alanine at amino acid position 839, corresponding to the amino acid residue in SEQ ID NO:

599.

8. The plurality of polynucleotides according to claim 1, wherein the fusion protein comprises, in order from the N-terminus to the C-terminus, the DNMT3A / L domain indicated by SEQ ID NO: 651, the dSpCas9 indicated by SEQ ID NO: 599, and the KRAB domain indicated by SEQ ID NO:

590.

9. The plurality of polynucleotides according to claim 8, wherein the gRNA comprises the sequence shown in SEQ ID NO:

412.

10. The plurality of polynucleotides according to claim 8, wherein the fusion protein further comprises one or more nuclear localization signals (NLS), the one or more NLS being located at the N-terminus or C-terminus of the fusion protein, between the dSpCas9 and the DNMT3A / L domain, or between the dSpCas9 and the KRAB domain.

11. The plurality of polynucleotides according to claim 10, wherein one or more NLSs are selected from the group consisting of the sequences PKKKRKV (SEQ ID NO: 593), KRPAATKKAGQAKKKK (SEQ ID NO: 592), and PKQKKRK (SEQ ID NO: 624).

12. The plurality of polynucleotides according to claim 11, wherein the fusion protein comprises, in order from the N-terminus to the C-terminus, the DNMT3A / L domain shown at SEQ ID NO: 651, the sequence shown at SEQ ID NO: 672, the sequence shown at SEQ ID NO: 670, the dSpCas9 shown at SEQ ID NO: 599, the sequence shown at SEQ ID NO: 677, the KRAB domain shown at SEQ ID NO: 669, and the sequence shown at SEQ ID NO:

593.

13. A vector comprising a plurality of polynucleotides as described in claim 12.

14. The vector according to claim 13, wherein the vector is a lipid nanoparticle.

15. The plurality of polynucleotides according to claim 12, wherein the gRNA comprises the sequence shown in SEQ ID NO:

412.

16. A vector comprising a plurality of polynucleotides as described in claim 15.

17. The vector according to claim 16, wherein the vector is a lipid nanoparticle.

18. Multiple polynucleotides, including the following: (a) A gRNA for targeting a target site in the hepatitis B virus DNA sequence, comprising the sequence shown in SEQ ID NO: 217, and (b) mRNA encoding a fusion protein comprising an inactive Streptococcus pyogenes dCas9 (dSpCas9) protein and a transcription repressor effector domain comprising a KRAB domain and a DNMT3A / L domain, wherein the KRAB domain comprises the amino acid sequence shown in SEQ ID NO: 669 and the DNMT3A / L domain comprises the amino acid sequence shown in SEQ ID NO:

651.

19. A vector comprising a plurality of polynucleotides according to claim 18.

20. The vector according to claim 19, wherein the vector is a lipid nanoparticle.

21. The plurality of polynucleotides according to claim 18, wherein the dSpCas9 has at least 95% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 599, and contains alanine (A) at amino acid position 9 and alanine at amino acid position 839, corresponding to the amino acid residue in SEQ ID NO:

599.

22. The plurality of polynucleotides according to claim 18, wherein the fusion protein comprises, in order from the N-terminus to the C-terminus, the DNMT3A / L domain indicated by SEQ ID NO: 651, the dSpCas9 indicated by SEQ ID NO: 599, and the KRAB domain indicated by SEQ ID NO:

669.

23. A vector comprising a plurality of polynucleotides as described in claim 22.

24. The vector according to claim 23, wherein the vector is a lipid nanoparticle.

25. The plurality of polynucleotides according to claim 22, wherein the gRNA comprises the sequence shown in SEQ ID NO:

412.

26. The plurality of polynucleotides according to claim 22, wherein the fusion protein further comprises one or more nuclear localization signals (NLS), and the one or more NLS are located at the N-terminus or C-terminus of the fusion protein, between the dSpCas9 and the DNMT3A / L domain, or between the dSpCas9 and the KRAB domain.

27. The plurality of polynucleotides according to claim 26, wherein one or more NLSs are selected from the group consisting of the sequences PKKKRKV (SEQ ID NO: 593), KRPAATKKAGQAKKKK (SEQ ID NO: 592), and PKQKKRK (SEQ ID NO: 624).

28. Multiple polynucleotides, including the following: (a) mRNA encoding a fusion protein including the following: (i) The Streptococcus pyogenes dCas9 (dSpCas9) protein shown in SEQ ID NO: 599, and (ii) A transcription repressor effector domain comprising a KRAB domain and a DNMT3A / L domain, wherein the KRAB domain comprises the amino acid sequence shown in SEQ ID NO: 669 and the DNMT3A / L domain comprises the amino acid sequence shown in SEQ ID NO: 651, and (b) A gRNA for targeting a target site in the hepatitis B virus DNA sequence, comprising the sequence shown in SEQ ID NO:

217.

29. The plurality of polynucleotides according to claim 28, wherein the gRNA comprises the sequence shown in SEQ ID NO:

412.

30. The plurality of polynucleotides according to claim 28, wherein the fusion protein comprises, in order from the N-terminus to the C-terminus, the DNMT3A / L domain shown at SEQ ID NO: 651, the sequence shown at SEQ ID NO: 672, the sequence shown at SEQ ID NO: 670, the dSpCas9 shown at SEQ ID NO: 599, the sequence shown at SEQ ID NO: 677, the KRAB domain shown at SEQ ID NO: 669, and the sequence shown at SEQ ID NO: 593.

Images