Engineered gene transcription inhibition tool targeting hepatitis B virus gene and application thereof

Abstract

Provided is an epigenetic editor comprising a transcriptional activator-like effector (TALE) domain, at least one epigenetic modification domain, and at least one transcriptional regulatory domain; wherein the TALE domain can be specifically combined with a target nucleotide sequence on an HBV (Hepatitis B Virus) gene and / or a regulatory element of the HBV gene.

Description

Engineered gene transcription repression tools targeting hepatitis b virus genes and uses thereof TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to an epigenetic editing agent for regulating hepatitis B virus (HBV) gene expression and uses thereof. BACKGROUND

[0002] Hepatitis B virus (HBV) is a hepadnavirus that causes chronic hepatitis B infection, which can lead to persistent liver inflammation, significantly increasing the risk of liver cirrhosis and liver cancer in patients. According to the World Health Organization (WTO), there are currently more than 250 million cases of chronic hepatitis B caused by hepatitis B virus worldwide, resulting in more than 800,000 deaths per year, which is a great burden on global public health. As a populous country, the number of hepatitis B carriers in China accounts for about one-third of the world. Since the 1980s, with the increase of hepatitis B vaccination rate, the new cases of hepatitis B in China have been effectively controlled, but the current domestic hepatitis B infection is still a huge amount.

[0003] Current treatment methods for HBV such as nucleos(t)ide analogues and IFN can effectively inhibit viral replication, but it is difficult to achieve functional cure, i.e., reduce surface antigen (HBsAg) to undetectable levels, and long-term medication is required. Once treatment is stopped, HBV replication will rebound, and the main reason for this phenomenon is that after HBV infects hepatocytes, it forms covalently closed circular DNA (cccDNA), and the HBV genome can be integrated into the host hepatocyte genome. cccDNA and integrated DNA can be stably used as a template for viral replication and protein expression for a long time. Current conventional treatment does not eliminate both, therefore, eliminating or silencing cccDNA and integrated DNA can effectively achieve the treatment of hepatitis B infection. Since cccDNA can assemble with histones in the host hepatocyte nucleus to form a structure similar to a chromosome, its transcriptional regulation can also be affected by epigenetic modification. There are three CpG islands in the HBV genome, referred to as CG I, CG II and CGI II, respectively. CpG islands are key regions for achieving epigenetic regulation. Transcription of the HBV genome is regulated by four promoters (Xp, Cp, Sp1, Sp2) and two enhancers (Enh I and Enh II), wherein Xp, Cp, Enh I and Enh II are located within the CG II region, and Sp1 and Sp2 are located near CG I and CG III, therefore, epigenetic editing of CpG islands can affect multiple regulatory elements, thereby affecting the transcription of the viral genome.

[0004] Introducing epigenetic modifications in specific regulatory regions or specific sites of HBV genome can change chromatin structure, thereby adjusting target genes to a transcriptionally repressed state, achieving the silencing regulation of target genes, without cutting DNA, avoiding the possibility of generating double-strand breaks of genome, fundamentally eliminating the risk of activating unpredictable DNA repair mechanisms and possibly producing immunogenic truncated or mutant proteins. However, most of the research on specific epigenetic editing tools targeting HBV genome is still in the early stage, and the development of epigenetic editing technology capable of continuously curing or eliminating HBV virus still faces many unknown and limited challenges. SUMMARY

[0005] In one aspect, the present application provides an epigenetic editing agent comprising a transcription activator-like effector (TALE) domain, at least one epigenetic modification domain, and at least one transcription regulation domain, wherein: 1) the TALE domain, the at least one epigenetic modification domain, and the at least one transcription regulation domain are directly or indirectly linked; or 2) the TALE domain, the at least one epigenetic modification domain, and at least one recruiting domain A are directly or indirectly linked to form a first fusion, and the at least one transcription regulation domain and at least one recruiting domain A’ are directly or indirectly linked to form a second fusion; or 3) the TALE domain, the at least one transcription regulation domain, and at least one recruiting domain A are directly or indirectly linked to form a first fusion, and the at least one epigenetic modification domain and at least one recruiting domain A’ are directly or indirectly linked to form a second fusion; and: 2) and 3) the recruiting domain A and the recruiting domain A’ are capable of interacting to enable the fusion or a portion thereof of one of the first fusion and the second fusion to be recruited in the vicinity of the other fusion; the TALE domain is capable of specifically binding to a target nucleotide sequence on an HBV gene and / or a regulatory element of an HBV gene.

[0006] In some embodiments, the target nucleotide sequence is selected from the sequence set forth in any one of SEQ ID NOs: 61-1697 and 3336-3355.

[0007] In some embodiments, the TALE domain comprises an engineered RVD domain capable of recognizing and specifically binding to the target nucleotide sequence.

[0008] In some embodiments, the TALE domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1698-3334 and 3356-8266.

[0009] In some embodiments, the epigenetic modification domain is selected from the group consisting of DNA deaminase activity, DNA methyltransferase activity, DNA demethylase activity, DNA aminase activity, DNA oxidizing activity, DNA helicase activity, histone acetyltransferase activity, histone deacetylase activity, histone methyltransferase activity, histone demethylase activity, histone kinase activity, histone phosphatase activity, histone ubiquitin ligase activity, and histone deubiquitinating activity.

[0010] In some embodiments, the epigenetic modification domain comprises a DNA methyltransferase (DNMT) and / or a functionally active fragment thereof.

[0011] In some embodiments, the DNA methyltransferase is selected from the group consisting of DNMT3A, DNMT3B, DNMT3C, DNMT1, DNMT2, and DNMT3L.

[0012] In some embodiments, the epigenetic modification domain comprises a plurality of DNA methyltransferases and / or functionally active fragments thereof, and the plurality of DNA methyltransferases and / or functionally active fragments thereof are linked by a linker sequence.

[0013] In some embodiments, the epigenetic modification domain comprises at least one DNMT3A and at least one DNMT3L.

[0014] In some embodiments, the DNA methyltransferase comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-6.

[0015] In some embodiments, the epigenetic modification domain comprises DNMT3A and DNMT3L, and the C-terminus of the DNMT3A is linked to the N-terminus of the DNMT3L, or the C-terminus of the DNMT3L is linked to the N-terminus of the DNMT3A.

[0016] In some embodiments, the transcriptional regulation domain is a transcriptional activation domain or a transcriptional repressor domain.

[0017] In some embodiments, the transcription repressor domain is selected from the group consisting of: KRAB, ZIM3 KRAB, ZNF680, ZNF554, ZNF264, ZNF582, ZNF324, ZNF669, ZNF354A, ZNF82, ZNF595, ZNF419, ZNF566, ZIM2, EHMT2, SUV39H1, ZFPM1, TRIM28, EZH2, MXD1, SID, LSD1, HP1a, HDAC3, ZNF436, ZNF257, ZNF675, ZNF490, ZNF320, ZNF331, ZNF816, ZNF41, ZNF189, ZNF528, ZNF543, ZNF140, ZNF610, ZNF350, ZNF8, ZNF30, ZNF98, ZNF677, ZNF596, ZNF214, ZNF37A, ZNF34, ZNF250, ZNF547, ZNF273, ZFP82, ZNF224, ZNF33A, ZNF45, ZNF175, ZNF184, ZFP28-1, ZFP28-2, ZNF18, ZNF213, ZNF394, ZFP1, ZFP14, ZNF416, ZNF557, ZNF729, ZNF254, ZNF764, ZNF785, ZNF10, CBX5, RYBP, YAF2, MGA, CBX1, SCMH1, MPP8, SUMO3, HERC2, BIN1, PCGF2, TOX, FOXA1, FOXA2, IRF2BP1, IRF2BP2, IRF2BP1_2 N-terminal domain, HOXA13, HOXB13, HOXC13, HOXA11, HOXC11, HOXC10, HOXA10, HOXB9, HOXA9, ZFP28, ZN334, ZN568, ZN37A, ZN181, ZN510, ZN862, ZN140, ZN208, ZN248, ZN571, ZN699, ZN726, ZIK1, ZNF2, Z705F, ZNF14, ZN471, ZN624, ZNF84, ZNF7, ZN891, ZN337, Z705G, ZN529, ZN729, ZN419, Z705A, ZN302, ZN486, ZN621, ZN688, ZN33A, ZN554, ZN878, ZN772, ZN224, ZN184, ZN544, ZNF57, ZN283, ZN549, ZN211, ZN615, ZN253, ZN226, ZN730, Z585A, ZN732, ZN681, ZN667, ZN649, ZN470, ZN484, ZN431, ZN382,ZN254, ZN124, ZN607, ZN317, ZN620, ZN141, ZN584, ZN540, ZN75D, ZN555, ZN658, ZN684, RBAK, ZN829, ZN582, ZN112, ZN716, HKR1, ZN350, ZN480, ZN416, ZNF92, ZN100, ZN736, ZNF74, ZN443, ZN195, ZN530, ZN782, ZN791, ZN331, Z354C, ZN157, ZN727, ZN550, ZN793, ZN235, ZN724, ZN573, ZN577, ZN789, ZN718, ZN300, ZN383, ZN429, ZN677, ZN850, ZN454, ZN257, ZN264, ZN485, ZN737, ZNF44, ZN596, ZN565, ZN543, ZFP69, SUMO1, ZNF12, ZN169, ZN433, ZN175, ZN347, ZNF25, ZN519, Z585B, ZN517, ZN846, ZN230, ZNF66, ZN713, ZN816, ZN426, ZN674, ZN627, ZNF20, Z587B, ZN316, ZN233, ZN611, ZN556, ZN234, ZN560, ZNF77, ZN682, ZN614, ZN785, ZN445, ZFP30, ZN225, ZN551, ZN610, ZN528, ZN284, ZN418, ZN490, ZN805, Z780B, ZN763, ZN285, ZNF85, ZN223, ZNF90, ZN557, ZN425, ZN229, ZN606, ZN155, ZN222, ZN442, ZNF91, ZN135, ZN778, ZN534, ZN586, ZN567, ZN440, ZN583, ZN441, ZNF43, ZN589, ZN563, ZN561, ZN136, ZN630, ZN527, ZN333, Z324B, ZN786, ZN709, ZN792, ZN599, ZN613, ZF69B, ZN799, ZN569, ZN564, ZN546, ZFP92, ZN723, ZN439, ZFP57, ZNF19, ZN404, ZN274, CBX3, ZN250, ZN570, ZN675, ZN695, ZN548, ZN132, ZN738, ZN420, ZN626, ZN559, ZN460, ZN268, ZN304, ZN605, ZN844, SUMO5, ZN101, ZN783, ZN417, ZN182, ZN823,ZN177, ZN197, ZN717, ZN669, ZN256, ZN251, CBX4, CDY2, CDYL2, ZN562, ZN461, Z324A, ZN766, ID2, ZN214, CBX7, ID1, CREM, SCX, ASCL1, ZN764, SCML2, TWST1, CREB1, TERF1, ID3, CBX8, GSX1, NKX22, ATF1, TWST2, ZNF17, TOX3, TOX4, ZMYM3, I2BP1, RHXF1, SSX2, I2BPL, ZN680, TRI68, HXA13, PHC3, TCF24, HXB13, HEY1, PHC2, ZNF81, FIGLA, SAM11, KMT2B, HEY2, JDP2, HXC13, ASCL4, HHEX, GSX2, ETV7, ASCL3, PHC1, OTP, I2BP2, VGLL2, HXA11, PDLI4, ASCL2, CDX4, ZN860, LMBL4, PDIP3, NKX25, CEBPB, ISL1, CDX2, PROP1, SIN3B, SMBT1, HXC11, HXC10, PRS6A, VSX1, NKX23, MTG16, HMX3, HMX1, KIF22, CSTF2, CEBPE, DLX2, PPARG, PRIC1, UNC4, BARX2, ALX3, TCF15, TERA, VSX2, HXD12, CDX1, TCF23, ALX1, HXA10, RX, CXXC5, SCML1, NFIL3, DLX6, MTG8, CEBPD, SEC13, FIP1, ALX4, LHX3, PRIC2, MAGI3, NELL1, PRRX1, MTG8R, RAX2, DLX3, DLX1, NKX26, NAB1, SAMD7, PITX3, WDR5, MEOX2, NAB2, DHX8, CBX6, EMX2, CPSF6, HXC12, KDM4B, LMBL3, PHX2A, EMX1, NC2B, DLX4, SRY, ZN777, ZN398, GATA3, BSH, SF3B4, TEAD1, TEAD3, RGAP1, PHF1, GATA2, FOXO3, ZN212, IRX4, ZBED6, LHX4, SIN3A, RBBP7, NKX61, R51A1, MB3L1, DLX5, NOTC1, TERF2, ZN282, RGS12, ZN840, SPI2B, PAX7, NKX62, ASXL2, FOXO1, GATA1, ZMYM5, LRP1, MIXL1, SGT1, LMCD1, CEBPA, SOX14,WTIP, PRP19, NKX11, RBBP4, DMRT2, SMCA2, and functionally active fragments thereof.

[0018] In some embodiments, the transcription repressor domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 7-32.

[0019] In some embodiments, the transcription repressor domain comprises a zinc-finger protein-based transcription factor or a functionally active fragment thereof.

[0020] In some embodiments, the zinc-finger protein-based transcription factor is a Krüppel-associated box (KRAB) or a KRAB domain derived from ZIM3 (ZIM3 KRAB).

[0021] In some embodiments, the transcription regulation domain comprises two or more of the zinc-finger protein-based transcription factors or functionally active fragments thereof, which are of the same species or are of different species.

[0022] In some embodiments, the two or more zinc-finger protein-based transcription factors are connected by a linker sequence.

[0023] In some embodiments, the linker sequence is an XTEN linker sequence.

[0024] In some embodiments, the transcription repressor domain comprises a histone modification domain.

[0025] In some embodiments, the histone modification domain is selected from the group consisting of EZH2, HDAC3, HDAC1, EHMT2, PRMT1, PRMT5, SETDB1, hSIRT1, HP1a, LSD1, and functionally active fragments thereof.

[0026] In some embodiments, the epigenetic modification domain and the transcription regulation domain are co-located at either the N-terminus or the C-terminus of the TALE domain.

[0027] In some embodiments, the epigenetic modification domain and the transcription regulation domain are located at the N-terminus and the C-terminus of the TALE domain, respectively.

[0028] In some embodiments, the fusion peptide has, in order from N-terminus to C- terminus: 1) the epigenetic modification domain, the transcriptional regulation domain, and the TALE domain; or 2) the transcriptional regulation domain, the epigenetic modification domain, and the TALE domain; or 3) the TALE domain, the epigenetic modification domain, and the transcriptional regulation domain; or 4) the TALE domain, the transcriptional regulation domain, and the epigenetic modification domain; or 5) the epigenetic modification domain, the TALE domain, and the transcriptional regulation domain; or 6) the transcriptional regulation domain, the TALE domain, and the epigenetic modification domain.

[0029] In some embodiments, the fusion peptide has, in order from N-terminus to C- terminus: 1) one or a combination of DNMT3A and DNMT3L, one or more zinc finger protein-based transcription factors, and a TALE domain; or 2) one or more zinc finger protein-based transcription factors, one or a combination of DNMT3A and DNMT3L, and a TALE domain; or 3) a TALE domain, one or a combination of DNMT3A and DNMT3L, and one or more zinc finger protein-based transcription factors; or 4) a TALE domain, one or more zinc finger protein-based transcription factors, and one or a combination of DNMT3A and DNMT3L; or 5) one or a combination of DNMT3A and DNMT3L, a TALE domain, and one or more zinc finger protein-based transcription factors; or 6) one or more zinc finger protein-based transcription factors, a TALE domain, and one or a combination of DNMT3A and DNMT3L.

[0030] In some embodiments, the fusion peptide comprises the following domains: TALE-DNMT3A-DNMT3L-ZIM3 KRAB, TALE-ZIM3 KRAB-DNMT3L-DNMT3A, TALE-ZIM3 KRAB-DNMT3A-DNMT3L, ZIM3 KRAB-DNMT3A-DNMT3L-TALE, DNMT3A-DNMT3L-ZIM3 KRAB-TALE, DNMT3A-DNMT3L-ZNF324-TALE, DNMT3A-DNMT3L-ZNF419-TALE, DNMT3A-DNMT3L-TALE-EZH2, DNMT3A-DNMT3L-TALE-HDAC3, DNMT3A-DNMT3L-TALE-HP1a, DNMT3A-DNMT3L-TALE-KRAB, DNMT3A-DNMT3L-TALE-ZIM3 KRAB, wherein - indicates that each part domain of the fusion is directly and / or indirectly connected, and the each part domain is in the order from N-terminus to C-terminus.

[0031] In some embodiments, the epigenetic editing agent is a complex peptide comprising the first fusion and the second fusion.

[0032] In some embodiments, the first fusion comprises, in order from N-terminus to C-terminus: 1) an epigenetic modification domain, a TALE domain, and a recruiting domain A, or 2) an epigenetic modification domain, a recruiting domain A, and a TALE domain, or 3) a TALE domain, a recruiting domain A, and an epigenetic modification domain, or 4) a TALE domain, an epigenetic modification domain, and a recruiting domain A, or 5) a recruiting domain A, an epigenetic modification domain, and a TALE domain, or 6) a recruiting domain A, a TALE domain, and an epigenetic modification domain.

[0033] In some embodiments, the second fusion comprises, in order from N-terminus to C-terminus: a transcriptional repressor domain and a recruiting domain A’, or in order from N-terminus to C-terminus: a recruiting domain A’ and a transcriptional repressor domain.

[0034] In some embodiments, the first fusion comprises, in order from N-terminus to C-terminus: 1) a recruiting domain A, a TALE domain, and a transcriptional repressor domain, or 2) a recruiting domain A, a transcriptional repressor domain, and a TALE domain, or 3) a TALE domain, a recruiting domain A, and a transcriptional repressor domain, or 4) a TALE domain, a transcriptional repressor domain, and a recruiting domain A, or 5) a transcriptional repressor domain, a TALE domain, and a recruiting domain A, or 6) a transcriptional repressor domain, a recruiting domain A, and a TALE domain.

[0035] In some embodiments, the second fusion comprises, in order from N- to C-terminus, an epigenetic modification domain and a recruiting domain A', or in order from N- to C-terminus, a recruiting domain A' and an epigenetic modification domain.

[0036] In some embodiments, the complex peptide comprises the feature: 1) the first fusion comprises, in order from N- to C-terminus, an epigenetic modification domain, a TALE domain, and a recruiting domain A, and the second fusion comprises, in order from N- to C-terminus, a transcriptional repressor domain and a recruiting domain A'; or 2) the first fusion comprises, in order from N- to C-terminus, an epigenetic modification domain, a TALE domain, and a recruiting domain A, and the second fusion comprises, in order from N- to C-terminus, a recruiting domain A' and a transcriptional repressor domain; or 3) the first fusion comprises, in order from N- to C-terminus, a recruiting domain A, a TALE domain, and a transcriptional repressor domain, and the second fusion comprises, in order from N- to C-terminus, an epigenetic modification domain and a recruiting domain A'; or 4) the first fusion comprises, in order from N- to C-terminus, a recruiting domain A, a TALE domain, and a transcriptional repressor domain, and the second fusion comprises, in order from N- to C-terminus, a recruiting domain A' and an epigenetic modification domain; or 5) the first fusion comprises, in order from N- to C-terminus, an epigenetic modification domain, a recruiting domain A, and a TALE domain, and the second fusion comprises, in order from N- to C-terminus, a recruiting domain A' and a transcriptional repressor domain; or 6) the first fusion comprises, in order from N- to C-terminus, a TALE domain, an epigenetic modification domain, and a recruiting domain A, and the second fusion comprises, in order from N- to C-terminus, a recruiting domain A' and a transcriptional repressor domain; or 7) the first fusion comprises, in order from N- to C-terminus, a TALE domain, a recruiting domain A, and an epigenetic modification domain, and the second fusion comprises, in order from N- to C-terminus, a recruiting domain A' and a transcriptional repressor domain.

[0037] In some embodiments, the recruiting domain A is selected from any one of the first group of domains and the recruiting domain A' is selected from any one of the second group of domains: 1) a general control non-depressible 4 (GCN4), a GFP 11 fragment derived from split green fluorescent protein (GFP), or a GVKESLV polypeptide; and 2) a single-chain antibody (scFv), a GFP 1-10 fragment derived from split green fluorescent protein (GFP), or a PDZ protein domain.

[0038] In some embodiments, wherein: 1) one of the recruiting domain A and the recruiting domain A' is a domain of GCN4, and wherein the other is a scFv; or 2) one of the recruiting domain A and the recruiting domain A' is a domain of GFP 11 fragment, and wherein the other is GFP 1-10; or 3) one of the recruiting domain A and the recruiting domain A' is a domain of GVKESLV, and wherein the other is a PDZ protein domain.

[0039] In some embodiments, the complex peptide comprises the features: 1) one of the first fusion and the second fusion comprises DNMT(3A-3L)-TALE-n x GCN4, and the other comprises a transcription repressor domain-scFv; or 2) one of the first fusion and the second fusion comprises DNMT(3A-3L)-TALE-scFv, and the other comprises a transcription repressor domain-GCN4; or 3) one of the first fusion and the second fusion comprises DNMT(3A-3L)-TALE-n x GFP11, and the other comprises a transcription repressor domain-GFP1-10; or 4) one of the first fusion and the second fusion comprises DNMT(3A-3L)-TALE-GFP1-10, and the other comprises a transcription repressor domain-GFP11; or 5) one of the first fusion and the second fusion comprises DNMT(3A-3L)-TALE-n x GCN4, and the other comprises scFv-transcription repressor domain; or 6) one of the first fusion and the second fusion comprises DNMT(3A-3L)-TALE-scFv, and the other comprises GCN4-transcription repressor domain; or 7) one of the first fusion and the second fusion comprises DNMT(3A-3L)-TALE-n x GFP11, and the other comprises GFP1-10-transcription repressor domain; or 8) one of the first fusion and the second fusion comprises DNMT(3A-3L)-TALE-GFP1-10, and the other comprises GFP11-transcription repressor domain; or 9) one of the first fusion and the second fusion comprises n x GCN4-TALE-transcription repressor domain, and the other comprises DNMT(3A-3L)-scFv; or 10) one of the first fusion and the second fusion comprises scFv-TALE-transcription repressor domain, and the other comprises DNMT(3A-3L)-GCN4; or 11) one of the first fusion and the second fusion comprises n x GFP11-TALE-transcription repressor domain, and the other comprises DNMT(3A-3L)-GFP1-10; or 12) one of the first fusion and the second fusion comprises GFP1-10-TALE-transcription repressor domain, and the other comprises DNMT(3A-3L)-GFP11.or 17) the fusion of one of the first fusion and the second fusion comprises a scFv- transcription repressor domain, and the other fusion comprises DNMT(3A-3L)-n x GCN4-TALE; or 18) the fusion of one of the first fusion and the second fusion comprises a scFv- transcription repressor domain, and the other fusion comprises TALE-DNMT(3A-3L)-n x GCN4; or 19) the fusion of one of the first fusion and the second fusion comprises a scFv- transcription repressor domain, and the other fusion comprises TALE-n x GCN4-DNMT(3A-3L); wherein DNMT(3A-3L) represents DNMT3A and DNMT3L linked directly or indirectly in any order, - represents the domains at its two ends are linked directly or indirectly in order from N-terminus to C-terminus; n x GCN4 or n x GFP11 represents n copies of GCN4 or n copies of GFP11, respectively, linked by a linker sequence, n is selected from any integer from 1 to 20.

[0040] In some embodiments, the epigenetic editing agent further comprises a nuclear localization signal and / or a marker domain.

[0041] In some embodiments, the epigenetic editing agent is capable of providing a modification of at least one nucleotide in the vicinity of the HBV gene and / or within a regulatory element of the HBV gene.

[0042] In another aspect, the present application provides a nucleic acid encoding the epigenetic editing agent described herein.

[0043] In some embodiments, the nucleic acid comprises a first nucleic acid segment encoding the first fusion, and a second nucleic acid segment encoding the second fusion, the first nucleic acid segment linked to the second nucleic acid segment by a nucleic acid segment encoding a cleavage peptide.

[0044] In some embodiments, the cleavage peptide is a 2A peptide and / or an IRES.

[0045] In some embodiments, the 2A peptide is selected from P2A, T2A, E2A, and F2A.

[0046] In some embodiments, the nucleic acid is a recombinant vector.

[0047] In some embodiments, the recombinant vector further comprises a non-coding region.

[0048] In some embodiments, the non-coding region is selected from an intron, a regulatory element, a promoter, an enhancer, a termination sequence, and a 5’ and 3’ untranslated region.

[0049] In another aspect, the present application provides a delivery vehicle comprising the epigenetic editing agent described herein and / or the nucleic acid described herein, and optionally comprising a liposome and / or a lipid nanoparticle.

[0050] In another aspect, the present application provides a composition comprising the epigenetic editing agent described herein, the nucleic acid described herein, and / or the delivery vehicle described herein.

[0051] In another aspect, the present application provides a cell comprising the epigenetic editing agent described herein, the nucleic acid described herein, the delivery vehicle described herein, and / or the composition described herein.

[0052] In another aspect, the present application provides a kit comprising the epigenetic editing agent described herein, the nucleic acid described herein, the delivery vehicle described herein, the composition described herein, and / or the cell described herein.

[0053] In another aspect, the present application provides a method of modulating expression of an HBV gene product, the method comprising administering the epigenetic editing agent described herein, the nucleic acid described herein, the delivery vehicle described herein, the composition described herein, the cell described herein, and / or the kit described herein.

[0054] In some embodiments, the method comprises introducing the epigenetic modifier, the nucleic acid, the delivery vehicle, the composition, the cell, and / or the kit into a cell containing the HBV gene.

[0055] In some embodiments, the method comprises contacting the epigenetic modifier, the nucleic acid, the delivery vehicle, and / or the composition with the HBV gene and / or a regulatory element of the HBV gene.

[0056] In some embodiments, the regulatory element comprises a core promoter, a proximal promoter, a distal enhancer, a silencer, an insulator element, a boundary element, and / or a locus control region.

[0057] In another aspect, the present application provides a method of treating or alleviating a disease or a condition associated with hepatitis B virus (HBV) infection, the method comprising administering to a subject in need thereof an effective amount of the epigenetic editing agent described herein, the nucleic acid described herein, the delivery vehicle described herein, the composition described herein, the cell described herein, and / or the kit described herein.

[0058] In another aspect, the present application provides use of the epigenetic editing agent described herein, the nucleic acid described herein, the delivery vehicle described herein, the composition described herein, the cell described herein, and / or the kit described herein for the manufacture of a medicament for treating or alleviating a disease or a condition associated with hepatitis B virus (HBV) infection.

[0059] In some embodiments, the disease or the condition associated with hepatitis B virus (HBV) infection comprises hepatitis, cirrhosis, liver fibrosis, and hepatocellular carcinoma caused by HBV infection.

[0060] The epigenetic editing agent and its encoding nucleic acid, vector, composition, cell, and the like products provided by the present application have at least one of the following advantages: significant transcriptional regulation efficiency of HBV gene, rich modification forms of HBV gene (such as DNA methylation modification, histone acetylation modification, etc.), flexible and diverse connection modes between various regulatory elements, and significantly improved recruitment effect of the complex peptide based on the SunTag recruitment strategy. BRIEF DESCRIPTION OF DRAWINGS

[0061] The specific features of the invention involved in the present application are shown in the appended claims. The features and advantages of the invention involved in the present application can be better understood by referring to the exemplary embodiments and the accompanying drawings described in detail below. The drawings are briefly described as follows:

[0062] FIGS. 1-10 show the inhibition efficiency of the epigenetic editing agent described herein (comprising different tool structures and TALE domains) on HBsAg and HBeAg in the cell line HepG2.2.15.

[0063] FIGS. 11A-11B show the inhibition of HBV marker levels in a HBV infected human primary hepatocyte (PHH) line by the epigenetic editing agents described herein, and the relationship between the inhibition and the dose administered.

[0064] FIGS. 12A-12B show the knockdown of HBV markers (HBV marker levels are log base 10) in a transgenic HBV mouse by the epigenetic editing agents described herein. DETAILED DESCRIPTION

[0065] The present application will be described by specific example, and other advantages and effects of the present application will be easily understood by those skilled in the art from the disclosure.

[0066] TERMINOLOGY

[0067] In the present application, the term "epigenetic modification domain" generally refers to a domain that is capable of altering gene expression or cellular phenotype of a cell population upon administration. It is understood that such alteration refers to one or more functionally relevant modifications to the genome that do not involve changes to the nucleic acid sequence. Examples of such modifications are DNA methylation and histone modification, both of which are important for the regulation of gene expression without changing the underlying DNA sequence.

[0068] In the present application, the term "TALE" is a polypeptide comprising one or more TALE repeat domains / units. Naturally occurring TALEs or "wild-type TALEs" are nucleic acid binding proteins secreted by numerous species of the genus Xanthomonas. TALE polypeptides contain a nucleic acid binding domain composed of tandem repeats of a highly conserved monomer polypeptide, which is primarily 33, 34, or 35 amino acids in length, and differs from one another primarily in amino acid positions 12 and 13. In preferred embodiments, the nucleic acid is DNA. As used herein, the polypeptide monomer of a TALE is used to refer to the highly conserved repeating polypeptide sequence within the TALE nucleic acid binding domain, and the term "repeat variable diresidue" or "RVD" is used to refer to the highly variable amino acids at positions 12 and 13 of the polypeptide monomer. A general representation of a TALE monomer contained within a DNA binding domain is X 1-11 -(X 12 X 13 )-X 14-33或34或或35 where the subscript indicates the amino acid position, and X represents any amino acid. X 12 X 13 indicates the RVD. In some TALE polypeptide monomers, the variable amino acid at position 13 is absent or not present, and in such monomers the RVD consists of a single amino acid. In such cases, the RVD can alternatively be represented as X*, where X represents X12 and (*) indicates X 13 is absent. The DNA binding domain comprises several repeats of TALE monomers and this can be represented as (X 1-11 -(X 12 X 13 )-X 14-33或34或或35 ) z wherein in preferred embodiments z is at least 5-40. In further preferred embodiments z is at least 10.-26.

[0069] TALE monomers have nucleotide binding affinities determined by the type of amino acid within their RVD. For example, polypeptide monomers with RVDs of NI preferentially bind to adenine (A), polypeptide monomers with RVDs of NG preferentially bind to thymine (T), polypeptide monomers with RVDs of HI) preferentially bind to cytosine (C), and monomers with RVDs of NN preferentially bind to both adenine (A) and guanine (G). In other embodiments, monomers with RVDs of IG preferentially bind to T. Thus, the number and order of polypeptide monomer repeats in the nucleic acid binding domain of a TALE determines its nucleic acid target specificity. In further embodiments of the application, monomers with RVDs of NS recognize all four base pairs and can bind to A, T, G, or C. The structure and function of TALEs are further described, for example, in Moscou et al., Science 326: 1501 (2009); Boch et al., Science 326: 1509-1512 (2009); and Zhang et al., Nature Biotechnology 29: 149-153 (2011), each of which is incorporated by reference in its entirety. The repeat domains of TALEs are involved in the binding of the TALE to its cognate target DNA sequence. These repeat units (or "repeat sequences") exhibit at least some sequence homology to other TALE repeat sequences within naturally occurring TALE proteins. See, e.g., U.S. Patent Publication No. 20110301073. The TALE binding domains involved in the present application can be "engineered" to bind to a predetermined nucleotide sequence, for example, via engineering (altering one or more amino acids) of the recognition helix region of naturally occurring TALE proteins. Thus, engineered DNA binding proteins (TALEs) are non-naturally occurring proteins. Non-limiting examples of methods for engineering DNA binding proteins are design and selection. Designed DNA binding proteins are non-naturally occurring proteins whose design and / or composition is derived primarily from rational criteria. Rational design criteria include the application of substitution rules and computational algorithms for processing information in information databases that store existing TALE designs and binding data. See, e.g., U.S. Patents 6,140,081; 6,453,242; and 6,534,261; see also WO 98 / 53058; WO 98 / 53059; WO 98 / 53060; WO 02 / 016536 and WO 03 / 016496 and U.S. Publication No. 20110301073.

[0070] In the present application, the term “recruitment” generally refers to the recruitment between protein molecules, which specifically refers to the recruitment of proteins to other molecules to perform specific biological functions. Such recruitment mainly relies on the affinity of intermolecular interaction, and the affinity is generally considered to be related to the spatial structure of the protein molecule, which is relatively complex. The interaction mechanism may exemplarily include, but is not limited to, hydrogen bond, ionic interaction, hydrophobic interaction, van der Waals force, etc. non-covalent bond interaction. For example, some proteins can recruit enzymes to catalyze chemical reactions, or recruit other proteins to form complexes. These recruitment is essential for many cellular processes, such as signal transduction, DNA replication and gene expression, etc.

[0071] In the present application, the term “DNA methyltransferase” generally refers to an enzyme that catalyzes the transfer of a methyl group to DNA. Non-limiting examples of DNA methyltransferases include DNMT1, DNMT 3A, DNMT 3B, and DNMT 3L. For example, by DNA methylation, DNA methyltransferases can modify the activity of a DNA fragment (e.g., regulate gene expression) without altering the DNA sequence. As described herein, an epigenetic editing agent can include one or more (e.g., two) DNA methyltransferases. When a DNA methyltransferase is included as part of an epigenetic editing agent (fusion peptide or complex peptide), the DNA methyltransferase can be referred to as a “DNA methyltransferase domain”. In aspects, the DNA methyltransferase domain comprises a variant or homolog of an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to DNMT3A. In aspects, the DNA methyltransferase domain comprises a variant or homolog of an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to DNMT3L.

[0072] In the present application, the term “functionally active fragment” generally refers to a fragment having a partial region of a full-length protein or nucleic acid, but retains or partially retains the biological activity or function of the full-length protein or nucleic acid. For example, a functionally active fragment can retain or partially retain the ability of a full-length protein to bind to another molecule. For example, a functionally active fragment of a DNA methyltransferase can retain or partially retain the biological activity function of a full-length DNA methyltransferase to catalyze the transfer of a methyl group to DNA.

[0073] In the present application, the terms "inhibit," "repress," "silence," and the like generally refer to a decrease in gene expression and / or activity. For example, administration of a substance of the present application can negatively affect (e.g., decrease) the activity of a nucleic acid sequence relative to the activity of the nucleic acid sequence in the absence of the substance (e.g., fusion protein, complex, nucleic acid, vector) (control). For example, inhibition can refer to a reduction in a disease or a symptom of a disease. For example, inhibition includes at least partially, partially, or completely blocking activation (e.g., transcription) of a nucleic acid sequence, or decreasing, preventing, or delaying activation of a nucleic acid sequence. For example, the inhibition activity can be 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less of the control.

[0074] In the present application, the term "transcriptional repressor" generally refers to a substance and / or agent, such as a protein (e.g., a transcription factor or fragment thereof), that binds to a target nucleic acid sequence and results in a decrease in the expression level of a gene product associated with the target nucleic acid sequence. For example, the gene product can be an RNA (e.g., mRNA) transcribed from a gene or a polypeptide translated from an mRNA transcribed from a gene. Generally, an increase or decrease in mRNA levels results in an increase or decrease in the level of a polypeptide translated therefrom. Expression levels can be determined using standard techniques for measuring mRNA or protein. Non-limiting examples of transcriptional repressors include: mSin3 interaction domain (SID) protein, methyl-CpG-binding domain 2 (MBD2), MBD3, DNA methyltransferase (DNMT) 1 (DNMT1), DNMT2A, DNMT3A, DNMT3B, DNMT3L, retinoblastoma protein (Rb), methyl CpG binding protein 2 (Mecp2), GATA-1 and its cofactor Fog1, MAT2 regulator (ROM2), Arabidopsis HD2A protein (AtHID2A), lysine-specific demethylase 1 (LSD1), and / or Krüppel-associated box (KRAB).

[0075] In the present application, the term "KRAB" also referred to as "Krüppel-associated box domain" or "Krüppel-associated box domain," generally refers to a transcriptional repression domain of about 45 to about 75 amino acid residues present in the transcription factor of human zinc finger protein. In aspects, the KRAB domain can include a variant or homolog of an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a ZIM3 KRAB domain or a KOX1 KRAB domain.

[0076] In the present application, the term "split green fluorescent protein" generally refers to a polypeptide that is capable of splitting and upon recombination immediately forms an active green fluorescent protein.

[0077] In the present application, the term "GCN4" is a transcription factor in S. cerevisiae, a "master regulator" in the yeast genome, regulating nearly one-tenth of the yeast genome, which is a highly conserved protein, whose homolog in mammals is Activating Transcription factor-4 (ATF4).

[0078] In the present application, the term "PDZ protein" generally refers to a naturally occurring protein containing a PDZ domain. Exemplary PDZ proteins include CASK, MPP1, DLG1, DLG2, PSD95, NeDLG, TIP-33, SYNla, TIP-43, LDP, LIM, LIMK1, LIMK2, MPP2, NOS1, AF6, PTN_4, prIL16, 41.8kD, KIAA0559, RGS12, KIAA0316, DVL1, TIP-40, TIAM1, MINT1, MAGI-I, MAGI-2, MAGI-3, KIAA0303, CBP, MINT3, TIP-2, KIAA0561, and / or TIP-I.

[0079] In the present application, the term "single chain antibody" or "scFv (Single Chain Antibody)" generally refers to a single chain polypeptide containing one or more antigen binding sites. In addition, although the H and L chains of Fv fragments are encoded by different genes, they can be joined, directly or through a peptide, such as by recombinant means, to produce a single protein chain (called single chain antibody, sAb; Bird et al. 1988 Science 242:423-426; and Huston et al. 1988 PNAS 85:5879-5883) in which the H and L chains are connected. The single chain antibody is also included in the term "antibody", can be used as a binding determinant in the design and manufacture of multispecific binding molecules, and can be prepared by recombinant techniques or enzymatic or chemical cleavage of intact antibodies.

[0080] In the present application, the term "directly or indirectly connected" generally refers to either "directly connected" or "indirectly connected." "Directly connected" generally refers to a direct linkage between the connected substances (e.g., amino acid sequence segments) without a spacer component (e.g., an amino acid residue or derivative thereof) in between. For example, an amino acid sequence segment X is directly connected to another amino acid sequence segment Y via an amide bond between the C-terminal amino acid of the amino acid sequence segment X and the N-terminal amino acid of the amino acid sequence segment Y. "Indirectly connected" generally refers to a linkage between the connected substances (e.g., amino acid sequence segments) with a spacer component (e.g., an amino acid residue or derivative thereof) in between. For example, as used herein, the spacer component is a "linker" or "linker sequence," which generally refers to a linker that connects two or more moieties. In various embodiments, the linker is connected to the amino acid sequence of the remainder of the compound (e.g., the first or second fusion of a fusion peptide or a composite peptide provided herein) at the N-terminus and the C-terminus. For example, the term "XTEN," "XTEN linker," or "XTEN polypeptide" as used herein refers to a recombinant polypeptide (e.g., an unstructured recombinant peptide) that lacks hydrophobic amino acid residues. In some embodiments, the XTEN linker sequence comprises one or more fragment sequences taken from the amino acid sequence set forth in SEQ ID NO: 60 (XTEN full-length polypeptide) that comprises no less than 16 contiguous amino acids. The development and use of XTEN can be found, for example, in Schellenberger et al., Nature Biotechnology 27, 1186-1190 (2009), which is incorporated by reference herein in its entirety. For example, in some embodiments of the present application, the linker sequence comprises a GS linker peptide comprising the sequence: (GS) a (GGS)b(GGGS)c(GGGGS)d, wherein G represents a glycine residue (Gly), S represents a serine residue (Ser), and a, b, c, and d represent integers greater than or equal to 0. For example, the spacer component (linker sequence) used in the present application is selected from the amino acid sequence set forth in any one of SEQ ID NOs: 42-59 (SEQ ID NO: 43 is GSG).

[0081] In the present application, the term "nuclear localization sequence" or "nuclear localization signal" or "NLS" generally refers to a peptide that directs a protein to the nucleus. For example, the NLS includes five basic positively charged amino acids. For example, the NLS can be located at any position on the peptide chain.

[0082] In the present application, the term "marker" refers to a peptide that can be introduced into an expression vector that can be used to allow for the deletion and / or purification of the expression product of one or more vector insert fragments. Such markers are well known in the art and include radiolabeled amino acids or polypeptides to which biotin moieties can be attached that can be detected by labeled avidin (e.g., streptavidin containing a fluorescent label or enzymatic activity that can be detected by optical or colorimetric methods). Affinity markers such as FLAG, glutathione-S-transferase, maltose binding protein, cellulose binding domain, thioredoxin, NusA, mistin, chitin binding domain, cutinase, AGT, GFP and other markers in wide use such as those used in the ProBond® protein expression and purification system. Further non-limiting examples for polypeptides include, but are not limited to, the following: a histidine tag, a radioisotope or radionuclide (e.g.,3H,14C,35S,90Y,99Tc,111In,125I,177Lu,166Ho or153Sm); a fluorescent marker (e.g., FITC, rhodamine, lanthanide phosphors), an enzymatic marker (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); a chemiluminescent marker; a biotin moiety; an overhanging polypeptide epitope recognized by a second reporter (e.g., leucine zipper pair sequence, binding site for a secondary antibody, metal binding domain, epitope tag); and a magnetic reagent such as a gadolinium chelate.

[0083] In the present application, the specific proteins (e.g., KRAB, TALE, Dnmt3A, Dnmt3L) can include any native form of the protein or a variant or homolog that maintains the activity of the protein (e.g., has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the activity compared to the native protein). In aspects, the variant or homolog has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity across the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 continuous amino acid portion) compared to the naturally occurring form.

[0084] In the present application, the term "modification of nucleotides" can mean that the nucleic acids described in the present application are synthesized or modified by methods well established in the art, for example, the methods described in "Current protocols in nucleic acid chemistry" Beaucage, S.L. et al., (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated by reference herein. The modification can include, but is not limited to, end modification such as 5'-end modification (e.g., phosphorylation, conjugation, inverted linkage) or 3'-end modification (e.g., conjugation, DNA nucleotide, inverted linkage, etc.); base modification such as substitution with a stabilized base, destabilized base, or base pairing with an extended pairing group library base, removal of base (abasic nucleotide), or conjugated base; sugar modification (e.g., sugar modification at 2'-position or 4'-position) or substitution of sugar; or backbone modification including modification or substitution of phosphodiester linkage.

[0085] In the present application, the terms "nucleic acid" and "polynucleotide", "nucleotide", "nucleotide sequence", and "oligonucleotide" are used interchangeably and generally refer to a polymer of nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and its complement in single-, double-, or multi-stranded form. For example, a nucleotide can be a ribonucleotide, a deoxyribonucleotide, or a modified version thereof. For example, a nucleotide can be single- and double-stranded DNA, single- and double-stranded RNA, and hybrid molecules having a mixture of single- and double-stranded DNA and RNA. For example, a nucleotide can include, but is not limited to, any type of RNA, such as mRNA, siRNA, miRNA, sgRNA, and guide RNA, and any type of DNA, genomic DNA, plasmid DNA, and minicircle DNA, and any fragment thereof. The term also encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring.

[0086] In the present application, the term "sequence encoding for" or "nucleic acid encoding for" generally refers to a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence that encodes a protein. The coding sequence can also include initiation and termination signals operably linked to regulatory elements comprising a promoter and polyadenylation signal capable of directing expression in the cells of the individual or mammal to which the nucleic acid is administered. The coding sequence can be codon-optimized.

[0087] In the present application, the term "delivery vehicle" generally refers to a transfer vehicle capable of delivering an agent (e.g., a nucleic acid molecule) to a target cell. The delivery vehicle can deliver the agent to a specific subset of cells. For example, the delivery vehicle is targeted to certain types of cells by virtue of the inherent properties of the delivery vehicle or by virtue of moieties coupled to the vehicle, contained within (or bound to the vehicle such that the moiety and the delivery vehicle remain together, and such that the moiety is sufficient to target the delivery vehicle). The delivery vehicle can also increase the in vivo half-life of the agent to be delivered and / or the bioavailability of the agent to be delivered. The delivery vehicle can include a viral vector, a virus-like particle, a polycationic vector, a peptide vector, a liposome, and / or a hybrid vector. For example, if the target cell is a hepatocyte, the properties of the delivery vehicle (e.g., size, charge, and / or pH) can be effective to deliver the delivery vehicle and / or the molecule encapsulated therein to the target cell, reduce immune clearance, and / or promote residence in the target cell.

[0088] In the present application, the term "liposome" generally refers to a vesicle with an internal space that is isolated from the external medium by one or more bilayered membranes. In some embodiments, the bilayered membrane can be formed by amphiphilic molecules, such as synthetic or naturally derived lipids that contain spatially segregated hydrophilic and hydrophobic domains; in other embodiments, the bilayered membrane can be formed by amphiphilic polymers and surfactants. In some embodiments, the liposome is a spherical vesicular structure consisting of a single or multiple lipid bilayers surrounding an internal aqueous compartment, and an outer, relatively impermeable, lipophilic phospholipid bilayer. In some embodiments, liposomes are biocompatible, non-toxic, can deliver hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their load across biological membranes and the blood-brain barrier (BBB). Liposomes can be made from several different types of lipids, e.g., phospholipids. Liposomes can comprise natural phospholipids and lipids such as 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), sphingomyelin, egg phosphatidylcholine, monosialic ganglioside, or any combination thereof. To alter the structure and properties of the liposome, several other additives can be added to the liposome. For example, the liposome can also comprise cholesterol, sphingomyelin, and / or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), e.g., to increase stability and / or prevent leakage of the internal cargo of the liposome.

[0089] The term "lipid nanoparticle (LNP)" generally refers to a particle comprising a plurality (i.e., more than one) of lipid molecules physically associated (e.g., covalently or non-covalently) with one another through intermolecular forces. An LNP can be, for example, a microsphere (including unilamellar and multilamellar vesicles, e.g., liposomes), a dispersed phase in an emulsion, a micelle, or an internal phase in a suspension. An LNP can encapsulate a nucleic acid within a cationic lipid particle (e.g., a liposome), and can be relatively easily delivered to cells. In some examples, a lipid nanoparticle does not contain any viral components, which helps to minimize safety and immunogenicity issues. The lipid particles can be used for in vitro, ex vivo, and in vivo delivery. The lipid particles can also be used for cell populations of various scales. LNP of the present application can be readily prepared by various methods known in the art, for example, by mixing an organic phase with an aqueous phase. Mixing of the two phases can be achieved by microfluidic devices and impinging stream reactors. The more thoroughly the organic and aqueous phases are mixed, the better the entrapment efficiency and particle size distribution of the LNP obtained. Preferably, the particle size of the LNP can be adjusted by varying the speed of mixing of the organic and aqueous phases. The faster the speed of mixing, the smaller the particle size of the LNP prepared will be. The entrapment efficiency can be optimized by adjusting the N / P (ionizable lipid / nucleic acid) ratio of the LNP system. In some examples, LNP can be used to deliver DNA molecules and / or RNA molecules (such as mRNA). In certain instances, LNP can be used to deliver RNP complexes.

[0090] In the present application, the term "recombinant vector" generally refers to a nucleic acid molecule capable of transporting another nucleic acid with it. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Alternatively, the vector can be linearized. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell

[0091] In the present application, the term "regulatory element" refers to genetic elements that control the expression of nucleic acid sequences. For example, splicing signals, promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for replication, transcription, and translation of a coding sequence in a recipient cell. Not all of these control sequences need to be present. Transcription control signals in eukaryotes generally include "promoter" and "enhancer" elements. Promoters and enhancers are comprised of short arrays of DNA sequences that are regulatory elements that promote the initiation of transcription of operably linked coding regions, and enhancers are regulatory elements that increase the rate of genetic transcription by increasing the activity of the promoter located closest in space on the same DNA molecule, which of these sequences specifically interact with cellular proteins involved in transcription (Maniatis et al., Science 236: 1237 (1987), incorporated by reference in its entirety). Promoter and enhancer elements have been isolated from a variety of eukaryotic sources, including yeast, insect and mammalian cells, and viruses (similar control sequences, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer depends on the recipient cell type. Some eukaryotic promoters and enhancers have a wide host range, while other promoters and enhancers are functional within a limited subset of cell types (for reviews, see, e.g., Voss et al., Trends Biochem. Sci., 11: 287 (1986); and Maniatis et al. (ibid.), incorporated by reference in their entireties). For example, the SV40 early gene enhancer is very active in a wide variety of cell types from many mammalian species and has been used to express proteins in many mammalian cells (Dijkema et al., EMBO J. 4: 761 (1985), incorporated by reference in its entirety). Promoter and enhancer elements derived from the human elongation factor 1-alpha gene (Uetsuki et al., J. Biol. Chem., 264: 5791 (1989); Kim et al., Gene 91: 217 (1990); and Mizushima and Nagata, Nucl. Acids. Res., 18: 5322 (1990)), the long terminal repeat of the Rous Sarcoma Virus (Gorman et al., Proc. Natl. Acad. Sci. U.S.A. 79: 6777 (1982)), and the human cytomegalovirus (Boshart et al., Cell 41: 521 (1985)) are also useful for expressing proteins in different mammalian cell types, which references are incorporated by reference in their entireties. Promoters and enhancers can exist naturally or together. For example, the retroviral long terminal repeat contains both promoter and enhancer elements.In general, the action of a promoter and enhancer is independent of the gene being transcribed or translated. Thus, the enhancer and promoter used can be "endogenous," "exogenous," or "heterologous" with respect to the gene to which they are operably linked. An "endogenous" enhancer / promoter is one that is naturally associated with a given gene in the genome. An "exogenous" or "heterologous" enhancer or promoter is one that is placed in juxtaposition to a gene by genetic manipulation (i.e., molecular biology techniques), which manipulation is such that transcription of the gene is directed by the linked enhancer / promoter. The presence of a "splicing signal" on an expression vector generally results in high level expression of the recombinant transcript. In certain embodiments, the "splicing signal" mediates the removal of introns from the primary RNA transcript, consisting of a splice donor and acceptor site (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nded., Cold Spring Harbor Laboratory Press, New York (1989), pp. 16.7-16.8, incorporated by reference in its entirety). A commonly used splice donor and acceptor site is that of the splice junction of the 16S RNA from SV40. In certain embodiments, a "transcription termination signal" is generally present downstream of the polyadenylation signal, and is several hundred nucleotides in length. For example, the term "poly A signal" or "poly A sequence" denotes a DNA sequence that directs the termination and polyadenylation of the nascent RNA transcript. Efficient polyadenylation of the recombinant transcript is often necessary because transcripts lacking a poly A signal are unstable and are rapidly degraded. The poly A signal used in an expression vector can be "heterologous" or "endogenous." An endogenous poly A signal is one that is naturally present at the 3' end of the coding region of a given gene in the genome. A heterologous poly A signal is one that is isolated from one gene and operably linked to the 3' end of another gene. A commonly used heterologous poly A signal is the SV40 poly A signal. The SV40 poly A signal is contained on a 237 bp BamHI / BcII restriction fragment and directs termination and polyadenylation (Sambrook et al., supra, 16.6-16.7, incorporated by reference in its entirety).

[0092] In the present application, the term "subject" generally refers to an animal, typically a mammal, such as a human, a non-human primate (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), a domestic animal (dogs and cats), a farm animal (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs), and a laboratory animal (mice, rats, rabbits, guinea pigs). Human subjects include fetal, neonatal, infant, juvenile, and adult subjects. Subjects include animal disease models, for example mice and other animal models of blood coagulation diseases (such as HemA), and other animal models known to those skilled in the art.

[0093] In the present application, the term "comprising" generally means including the recited elements but not excluding other elements.

[0094] In the present application, the term "consisting of generally means including only the recited elements and no other elements.

[0095] In the present application, the term "about" generally means a range of variation of 0.5-10% above or below the specified numerical value, for example, a range of variation of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified numerical value.

[0096] DETAILED DESCRIPTION

[0097] In one aspect, the present application provides an epigenetic editing agent comprising a transcription activator-like effector (TALE) domain, at least one epigenetic modification domain, and at least one transcription regulation domain, wherein: 1) the TALE domain, the at least one epigenetic modification domain, and the at least one transcription regulation domain are directly or indirectly linked; or 2) the TALE domain, the at least one epigenetic modification domain, and at least one recruiting domain A are directly or indirectly linked to form a first fusion, and the at least one transcription regulation domain and at least one recruiting domain A’ are directly or indirectly linked to form a second fusion; or 3) the TALE domain, the at least one transcription regulation domain, and at least one recruiting domain A are directly or indirectly linked to form a first fusion, and the at least one epigenetic modification domain and at least one recruiting domain A’ are directly or indirectly linked to form a second fusion; and: 2) and 3) the recruiting domain A and the recruiting domain A’ are capable of interacting to enable the fusion or a portion thereof of one of the first fusion and the second fusion to be recruited in the vicinity of the other fusion; the TALE domain is capable of specifically binding to a target nucleotide sequence on an HBV gene and / or a regulatory element of an HBV gene.

[0098] In another aspect, the present application provides a nucleic acid encoding the epigenetic editing agent described herein. For example, the nucleic acid comprises DNA and / or mRNA. For example, the nucleic acid can be used to treat or alleviate a disease or a condition thereof associated with abnormal expression of a target gene and / or abnormal activity of a target gene. In some embodiments, the nucleic acid is mRNA; one or more modification techniques can be used to generate a more stable mRNA. Known mRNA modification techniques can be broadly classified into three categories: synthesizing mRNA with artificially synthesized non-natural ribonucleic acid instead of natural ribonucleic acid; adding 5' caps, 3' poly(A) "tails", and UTR (untranslated region) sequences; and using special new formulation techniques to effectively protect mRNA. Among them, the preferred mRNA modification technique can be to synthesize mRNA with artificially synthesized non-natural ribonucleic acid instead of natural ribonucleic acid. Chemical modifications on eukaryotic mRNA can be broadly classified into three categories: methylation, pseudouridine (Ψ), and hypoxanthine. For example, the chemical modification can be selected from the group consisting of: pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine. For example, the nucleic acid is a recombinant vector comprising a nucleic acid encoding the complex described herein. For example, a recombinant vector can refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. A recombinant vector can include a single-stranded, double-stranded, or partially double-stranded nucleic acid molecule; a nucleic acid molecule comprising one or more free ends, no free ends (e.g., circular); a nucleic acid molecule comprising DNA, RNA, or both; and other kinds of polynucleotides known in the art. For example, a viral vector can be used. A viral vector can comprise a viral-derived DNA or RNA sequence for packaging into a virus (e.g., retrovirus, replication-defective retrovirus, adenovirus, replication-defective adenovirus, and adeno-associated virus AAV). Viruses and viral vectors can be used for delivery in vitro, ex vivo, and / or in vivo.

[0099] In another aspect, the present application provides a delivery vehicle comprising an epigenetic editing agent described herein and / or a nucleic acid described herein, and optionally comprising a liposome and / or a lipid nanoparticle. For example, the delivery vehicle can be introduced into a cell by a physical delivery method. Examples of physical methods include microinjection, electroporation, and hydrodynamic delivery. For example, LNPs can encapsulate nucleic acids in cationic lipid particles (e.g., liposomes) and can be relatively easily delivered to cells. In some examples, the lipid nanoparticles are free of any viral components, which helps to minimize safety and immunogenicity concerns. The lipid particles can be used for in vitro, ex vivo, and in vivo delivery. The components of the LNP can include a cationic lipid, an ionizable lipid, a PEGylated lipid, and / or a supporting lipid, and optionally a cholesterol component.

[0100] In another aspect, the present application provides a composition comprising an epigenetic editing agent described herein, a nucleic acid described herein, and / or a delivery vehicle described herein. For example, the epigenetic editing agent, the nucleic acid (or recombinant vector) encoding the epigenetic editing agent, and the delivery vehicle in the composition can be included in one composition, or separately included in different compositions. For example, the epigenetic editing agent, the nucleic acid (or recombinant vector) encoding the epigenetic editing agent, and / or the delivery vehicle in the composition can be used simultaneously, or separately.

[0101] In another aspect, the present application provides a cell comprising an epigenetic editing agent described herein, a nucleic acid described herein, a delivery vehicle described herein, and / or a composition described herein.

[0102] In another aspect, the present application provides a kit comprising an epigenetic editing agent described herein, a nucleic acid described herein, a delivery vehicle described herein, a composition described herein, and / or a cell described herein. For example, the kit further comprises at least one container holding the above components. For example, the kit comprises more than one of the above components, which further comprises a second, third, and / or other container(s) other than the container(s) holding the above components, in which the more than one of the above components can be separately placed. For example, the kit can place the above components in various combinations in the container(s). For example, the kit further comprises a buffer reagent, a device for mixing, a device for measuring, a device for sorting, and / or a device for labeling. For example, the kit further comprises a package for holding the various containers. For example, the kit further comprises an instruction manual for using the components of the kit. For example, the instruction manual comprises a physical form of paper and / or an electronically readable form.

[0103] In another aspect, the present application provides a method of modulating expression of a HBV gene product, the method comprising administering the epigenetic editing agent described herein, the nucleic acid described herein, the delivery vehicle described herein, the composition described herein, the cell described herein, and / or the kit described herein. For example, the method of inhibiting expression of a HBV gene is introducing the epigenetic editing agent, the nucleic acid, the delivery vehicle, the composition, the cell, and / or the kit into a cell containing a HBV gene. For example, the introducing into a cell can be introducing into a cell using a non-viral or viral-based transfection method. For example, the non-viral transfection method includes any suitable method of introducing into a cell without using viral DNA or viral particles as a delivery system, non-limiting examples of non-viral transfection methods include nanoparticle encapsulation (e.g., lipid nanoparticle, gold nanoparticle, etc.), calcium phosphate transfection, liposome transfection, nucleofection, sonoporation, transfection by heat shock, magnetic transfection, and electroporation of a nucleic acid encoding the epigenetic editing agent. For example, the viral-based transfection method includes any viral vector suitable for use in the methods described herein, non-limiting examples of which include, but are not limited to, retroviral, adenoviral, lentiviral, and / or adeno-associated viral vectors. For example, the method of inhibiting expression of a HBV gene further comprises introducing the epigenetic editing agent, the nucleic acid, the delivery vehicle, the composition, the cell, and / or the kit into a cell from an external environment. For another example, the method of inhibiting expression of a HBV gene comprises contacting the epigenetic editing agent, the nucleic acid, the delivery vehicle, and / or the composition with a HBV gene and / or a transcriptional regulatory element of the HBV gene. For example, the epigenetic editing agent is a complex peptide comprising a first fusion and a second fusion, the contacting refers to the TALE domain comprised by the epigenetic editing agent specifically recognizing and hybridizing to a specific region in the HBV gene, while the first fusion and the second fusion are recruited to the vicinity of the HBV gene or within the transcriptional regulatory region thereof through the direct or indirect interaction of the recruiting domain A and the recruiting domain A’ thereof, thereby modulating the expression of the nucleic acid thereof.

[0104] In another aspect, the present application provides a method of treating or ameliorating a disease or a condition associated with hepatitis B virus (HBV) infection, comprising administering to a subject in need thereof an effective amount of the epigenetic editing agent described herein, the nucleic acid described herein, the delivery vehicle described herein, the composition described herein, the cell described herein, and / or the kit described herein. For example, the method of treatment comprises mixing the epigenetic editing agent, the nucleic acid, the delivery vehicle, the composition, the cell, and / or the kit with a therapeutic agent and performing systemic delivery to a subject in need thereof such that it is widely exposed to a substantial portion of the body, which can be performed by any means known in the art, including but not limited to intravenous, intra-arterial, subcutaneous, intracavitary, and intraperitoneal delivery. For example, the method of treatment comprises mixing the epigenetic editing agent, the nucleic acid, the delivery vehicle, the composition, the cell, and / or the kit with a therapeutic agent and performing local delivery to a subject in need thereof such that it is directed to a target site within an organism, which can be performed by, for example, direct injection into a disease site (e.g., a tumor or an inflammation site) or a target organ (e.g., liver, heart, pancreas, kidney, etc.). For example, the local delivery includes local administration or local injection techniques, including but not limited to intramuscular, subcutaneous, or intradermal injection. For example, the local delivery does not exclude systemic pharmacological effects.

[0105] In another aspect, the present application provides the use of the epigenetic editing agent described herein, the nucleic acid described herein, the delivery vehicle described herein, the composition described herein, the cell described herein, and / or the kit described herein for the manufacture of a medicament for treating or ameliorating a disease or a condition associated with hepatitis B virus (HBV) infection.

[0106] In another aspect, the present application provides the epigenetic editing agent described herein, the nucleic acid described herein, the delivery vehicle described herein, the composition described herein, the cell described herein, or the kit described herein for use in treating or ameliorating a disease or a condition associated with hepatitis B virus (HBV) infection.

[0107] Fusion peptide

[0108] In some embodiments, the epigenetic modification domain and the transcriptional regulation domain are located at the N-terminus or the C-terminus of the TALE domain. For example, the fusion peptide has, from N-terminus to C-terminus, the epigenetic modification domain, the transcriptional regulation domain, and the TALE domain. For example, the fusion peptide has, from N-terminus to C-terminus, the transcriptional regulation domain, the epigenetic modification domain, and the TALE domain. For example, the fusion peptide has, from N-terminus to C-terminus, the TALE domain, the epigenetic modification domain, and the transcriptional regulation domain. For example, the fusion peptide has, from N-terminus to C-terminus, the TALE domain, the transcriptional regulation domain, and the epigenetic modification domain.

[0109] In some embodiments, the epigenetic modification domain and the transcriptional regulation domain are located at the N-terminus and the C-terminus of the TALE domain, respectively. For example, the fusion peptide has, from N-terminus to C-terminus, the epigenetic modification domain, the TALE domain, and the transcriptional regulation domain.

[0110] In some specific embodiments, the fusion peptide has, from N-terminus to C-terminus: 1) one or a combination of DNMT3A and DNMT3L, one or more than one zinc-finger protein-based transcription factor, and the TALE domain; or 2) one or more than one zinc-finger protein-based transcription factor, one or a combination of DNMT3A and DNMT3L, and the TALE domain; or 3) the TALE domain, one or a combination of DNMT3A and DNMT3L, and one or more than one zinc-finger protein-based transcription factor; or 4) the TALE domain, one or more than one zinc-finger protein-based transcription factor, and one or a combination of DNMT3A and DNMT3L; or 5) one or a combination of DNMT3A and DNMT3L, the TALE domain, and one or more than one zinc-finger protein-based transcription factor.

[0111] In some embodiments of the above cases, the epigenetic modification domain can comprise DNMT3A and DNMT3L, the C-terminus of the DNMT3A is connected to the N-terminus of the DNMT3L, or the C-terminus of the DNMT3L is connected to the N-terminus of the DNMT3A. Also in these cases, the transcriptional regulation domain can comprise a transcriptional repressor domain. For example, the transcriptional regulation domain comprises a zinc-finger protein-based transcription factor or a functionally active fragment thereof. Specifically, the zinc-finger protein-based transcription factor can comprise KRAB. For example, in some embodiments of the present application, the zinc-finger protein-based transcription factor can be selected from the group consisting of ZIM3 KRAB and KOX1 KRAB.

[0112] For example, the epigenetic editing agent provided in this application may be selected from the example fusion peptides shown in Table 1. For example, the structure of the epigenetic editing agent provided in this application may also be selected from (but not limited to) the fusion tools involved in patent application publication numbers WO2024 / 131917A1, TW202440930A1, WO2024 / 131940A1, TW202440931A, WO2023 / 165597A1 and their family applications AU2023228989A1 and TW202346588A (wherein, for example, "nucleic acid binding domain", "DNA binding domain", "CasN", "dCas9", etc., may be replaced with the TALE domain of the amino acid sequence contained in any one of SEQ ID NO: 1698-3334 and 3356-8266 provided in this application), the disclosures of the above-mentioned patent applications are hereby incorporated by reference.

[0113] Table 1. Example structures and sequences of fusion peptide tools

[0114] Taking tools V1 and V3 as examples respectively, when X TALE When the sequence is the amino acid sequence shown in SEQ ID NO: 5663: the amino acid sequence of the V1 tool is shown in SEQ ID NO: 8269, and the mRNA sequence encoding the V1 tool is shown in SEQ ID NO: 8270. The V1 tool targets the target nucleic acid sequence on the HBV gene as shown in SEQ ID NO: 731; the amino acid sequence of the V3 tool is shown in SEQ ID NO: 8273, and the mRNA sequence encoding the V3 tool is shown in SEQ ID NO: 8274. The V3 tool targets the target nucleic acid sequence on the HBV gene as shown in SEQ ID NO: 731.

[0115] Complex peptides and their first and second fusion compounds

[0116] In some implementations, the first and second fusions of the epigenetic editing agent (complex peptide) of this application can generally be divided into two cases: (1) one of the two fusions contains a TALE domain, an epigenetic modification domain and a recruitment domain A, and the other fusion contains a transcriptional repressor domain and a recruitment domain A', or (2) one of the two fusions contains a TALE domain, a transcriptional repressor and a recruitment domain A, and the other fusion contains an epigenetic modification domain and a recruitment domain A'.

[0117] Specifically, in some embodiments under the above (1), one of the two fusions can comprise, in order from N- to C-terminus, an epigenetic modification domain, a TALE domain, and a recruiting domain A. For example, in some embodiments under the above (2), one of the two fusions can comprise, in order from N- to C-terminus, a recruiting domain A, a TALE domain, and a transcription repressor domain. For example, in some embodiments under the above (1), the other of the two fusions can comprise, in order from N- to C-terminus, a transcription repressor domain and a recruiting domain A', or a recruiting domain A' and a transcription repressor domain, i.e., the transcription repressor domain and the recruiting domain A' can be connected in an order interchangeable. For example, in some embodiments under the above (2), the other of the two fusions can comprise, in order from N- to C-terminus, an epigenetic modification domain and a recruiting domain A', or a recruiting domain A' and an epigenetic modification domain, i.e., the epigenetic modification domain and the recruiting domain A' can be connected in an order interchangeable.

[0118] In some more specific embodiments, the TALE domain can comprise, but is not limited to, an amino acid sequence set forth in any one of SEQ ID NOs: 1698-3334 and 3356-8266.

[0119] In some more specific embodiments, the transcriptional repressor is selected from one or more of the following domains: KRAB, ZIM3 KRAB, ZNF680, ZNF554, ZNF264, ZNF582, ZNF324, ZNF669, ZNF354A, ZNF82, ZNF595, ZNF419, ZNF566, ZIM2, EHMT2, SUV39H1, ZFPM1, TRIM28, EZH2, MXD1, SID, LSD1, HP1a, HDAC3, HDAC1, PRMT1, SETDB1, hSIRT1, ZNF436, ZNF257, ZNF675, ZNF490, ZNF320, ZNF331, ZNF816, ZNF41, ZNF189, ZNF528, ZNF543, ZNF140, ZNF610, ZNF350, ZNF8, ZNF30, ZNF98, ZNF677, ZNF596, ZNF214, ZNF37A, ZNF34, ZNF250, ZNF547, ZNF273, ZFP82, ZNF224, ZNF33A, ZNF45, ZNF175, ZNF184, ZFP28-1, ZFP28-2, ZNF18, ZNF213, ZNF394, ZFP1, ZFP14, ZNF416, ZNF557, ZNF729, ZNF254, ZNF764, ZNF785, ZNF10, CBX5, RYBP, YAF2, MGA, CBX1, SCMH1, MPP8, SUMO3, HERC2, BIN1, PCGF2, TOX, FOXA1, FOXA2, IRF2BP1, IRF2BP2, IRF2BP LIRF-2BP1_2 N-terminal domain, HOXA13, HOXB13, HOXC13, HOXA11, HOXC11, HOXC10, HOXA10, HOXB9, HOXA9, ZFP28, ZN334, ZN568, ZN37A, ZN181, ZN510, ZN862, ZN140, ZN208, ZN248, ZN571, ZN699, ZN726, ZIK1, ZNF2, Z705F, ZNF14, ZN471, ZN624, ZNF84, ZNF7, ZN891, ZN337, Z705G, ZN529, ZN729, ZN419, Z705A, ZN302, ZN486, ZN621, ZN688, ZN33A, ZN554, ZN878, ZN772, ZN224, ZN184, ZN544, ZNF57, ZN283, ZN549, ZN211, ZN615, ZN253, ZN226, ZN730, Z585A, ZN732,ZN681, ZN667, ZN649, ZN470, ZN484, ZN431, ZN382, ZN254, ZN124, ZN607, ZN317, ZN620, ZN141, ZN584, ZN540, ZN75D, ZN555, ZN658, ZN684, RBAK, ZN829, ZN582, ZN112, ZN716, HKR1, ZN350, ZN480, ZN416, ZNF92, ZN100, ZN736, ZNF74, ZN443, ZN195, ZN530, ZN782, ZN791, ZN331, Z354C, ZN157, ZN727, ZN550, ZN793, ZN235, ZN724, ZN573, ZN577, ZN789, ZN718, ZN300, ZN383, ZN429, ZN677, ZN850, ZN454, ZN257, ZN264, ZN485, ZN737, ZNF44, ZN596, ZN565, ZN543, ZFP69, SUMO1, ZNF12, ZN169, ZN433, ZN175, ZN347, ZNF25, ZN519, Z585B, ZN517, ZN846, ZN230, ZNF66, ZN713, ZN816, ZN426, ZN674, ZN627, ZNF20, Z587B, ZN316, ZN233, ZN611, ZN556, ZN234, ZN560, ZNF77, ZN682, ZN614, ZN785, ZN445, ZFP30, ZN225, ZN551, ZN610, ZN528, ZN284, ZN418, ZN490, ZN805, Z780B, ZN763, ZN285, ZNF85, ZN223, ZNF90, ZN557, ZN425, ZN229, ZN606, ZN155, ZN222, ZN442, ZNF91, ZN135, ZN778, ZN534, ZN586, ZN567, ZN440, ZN583, ZN441, ZNF43, ZN589, ZN563, ZN561, ZN136, ZN630, ZN527, ZN333, Z324B, ZN786, ZN709, ZN792, ZN599, ZN613, ZF69B, ZN799, ZN569, ZN564, ZN546, ZFP92, ZN723, ZN439, ZFP57, ZNF19, ZN404, ZN274, CBX3, ZN250, ZN570, ZN675, ZN695, ZN548, ZN132, ZN738, ZN420, ZN626, ZN559, ZN460, ZN268, ZN304, ZN605,ZN844, SUMO5, ZN101, ZN783, ZN417, ZN182, ZN823, ZN177, ZN197, ZN717, ZN669, ZN256, ZN251, CBX4, CDY2, CDYL2, ZN562, ZN461, Z324A, ZN766, ID2, ZN214, CBX7, ID1, CREM, SCX, ASCL1, ZN764, SCML2, TWST1, CREB1, TERF1, ID3, CBX8, GSX1, NKX22, ATF1, TWST2, ZNF17, TOX3, TOX4, ZMYM3, I2BP1, RHXF1, SSX2, I2BPL, ZN680, TRI68, HXA13, PHC3, TCF24, HXB13, HEY1, PHC2, ZNF81, FIGLA, SAM11, KMT2B, HEY2, JDP2, HXC13, ASCL4, HHEX, GSX2, ETV7, ASCL3, PHCl, OTP, I2BP2, VGLL2, HXA11, PDLI4, ASCL2, CDX4, ZN860, LMBL4, PDIP3, NKX25, CEBPB, ISL1, CDX2, PROP1, SIN3B, SMBT1, HXC11, HXC10, PRS6A, VSX1, NKX23, MTG16, HMX3, HMX1, KIF22, CSTF2, CEBPE, DLX2, PPARG, PRIC1, UNC4, BARX2, ALX3, TCF15, TERA, VSX2, HXD12, CDX1, TCF23, ALX1, HXA10, RX, CXXC5, SCML1, NFIL3, DLX6, MTG8, CEBPD, SEC13, FIP1, ALX4, LHX3, PRIC2, MAGI3, NELL1, PRRX1, MTG8R, RAX2, DLX3, DLX1, NKX26, NAB1, SAMD7, PITX3, WDR5, MEOX2, NAB2, DHX8, CBX6, EMX2, CPSF6, HXC12, KDM4B, LMBL3, PHX2A, EMX1, NC2B, DLX4, SRY, ZN777, ZN398, GATA3, BSH, SF3B4, TEAD1, TEAD3, RGAP1, PHF1, GATA2, FOXO3, ZN212, IRX4, ZBED6, LHX4, SIN3A, RBBP7, NKX61, R51A1, MB3L1, DLX5, NOTC1, TERF2, ZN282, RGS12, ZN840, SPI2B, PAX7, NKX62, ASXL2, FOXO1,GATA1, ZMYM5, LRP1, MIXL1, SGT1, LMCD1, CEBPA, SOX14, WTIP, PRP19, NKX11, RBBP4, DMRT2, SMCA2, and functionally active fragments thereof.

[0120] In some more specific embodiments, the epigenetic modification domain comprises at least one DNA methyltransferase or a functionally active fragment thereof. For example, the DNA methyltransferase is selected from the group consisting of DNMT3A, DNMT3B, DNMT3c, DNMT1, DNMT2, and DNMT3L. For example, the DNA methylation domain comprises at least one DNMT3A and at least one DNMT3L. For example, the at least one DNMT3A and the at least one DNMT3L are linked in an interchangeable order. For example, the DNA methylation domain comprises one DNMT3A and one DNMT3L, and they are linked in an interchangeable order. For example, the DNA methyltransferase comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-6.

[0121] The first fusion and the second fusion of the complex peptides described herein are formed into an aggregated complex through the interaction between the recruiting domains comprised in each. Accordingly, the present disclosure provides non-limiting examples of combinations of the recruiting domain A and the recruiting domain A': (1) one of the domains of the recruiting domain A and the recruiting domain A' is GCN4, and the other domain is scFv; or (2) one of the domains of the recruiting domain A and the recruiting domain A' is GFP11 fragment, and the other domain is GFP1-10; or (3) one of the domains of the recruiting domain A and the recruiting domain A' is GVKESLV, and the other domain is PDZ protein domain. The same logic applies to the cases where GFP11 and GFP1-10 are derived from split GFP (SEQ ID NO: 38) to form the recruiting domain A and the recruiting domain A' respectively, and can be applied to other classes of fluorescent proteins, such as mCherry (SEQ ID NO: 80), eYFP (SEQ ID NO: 82), eCFP (SEQ ID NO: 81), etc., i.e., different sets of recruiting domain A and recruiting domain A' can be obtained by splitting mCherry, splitting eYFP, or splitting eCFP, respectively, for use in the complex peptides provided herein. In some embodiments, one of the first fusion and the second fusion of the complex peptides of the present disclosure can comprise two or more recruiting domains, and they are linked by a linker sequence. Exemplary amino acid sequences of the recruiting domains can comprise any one of SEQ ID NOs: 33-37.

[0122] For example, the epigenetic editing agent provided in this application may be selected from the example complex peptides shown in Table 2. For example, the structure of the epigenetic editing agent provided in this application may also be selected from (but not limited to) the first fusion, second fusion, and complex tool involved in patent application publication numbers WO2024 / 131917A1, TW202440930A1, WO2024 / 131940A1, TW202440931A, WO2023 / 165597A1 and their family applications AU2023228989A1 and TW202346588A (wherein, for example, "nucleic acid binding domain", "DNA binding domain", "CasN", "dCas9", etc., may be replaced with the TALE domain of the amino acid sequence contained in any one of SEQ ID NO: 1698-3334 and 3356-8266 provided in this application), the disclosures of the above-mentioned patent applications are hereby incorporated by reference.

[0123] Table 2. Example structures and sequences of complex peptide tools

[0124] Taking the V2 tool as an example, when X TALE When the sequence is the amino acid sequence shown in SEQ ID NO: 5663: the amino acid sequence of the V2 tool is shown in SEQ ID NO: 8271, and the mRNA sequence encoding the V1 tool is shown in SEQ ID NO: 8272. The V2 tool targets the target nucleic acid sequence on the HBV gene as shown in SEQ ID NO: 731.

[0125] Without being limited by any theory, the embodiments described below are merely for illustrating the epigenetic editing agent, preparation method, and use of this application, and are not intended to limit the scope of the invention.

[0126] Example

[0127] Example 1

[0128] The epigenetic editing agent of this application exhibits high inhibition efficiency of HBV-D gene expression in the HepG2.2.15 cell line.

[0129] The cell line HepG2.2.15 integrated with HBV-D type genome (SEQ ID NO: 3335) was used as a research model in this example (model preparation method referred from: Mol Ther Nucleic Acids. 2020 Jun 5: 20: 480-490. doi: 10.1016 / j.omtn.2020.03.005.). The epigenetic editing of the conserved region can lead to the decrease of HBV viral marker parameters, i.e., the decrease of the amount or level of HBsAg and HBeAg markers.

[0130] Experimental method: Different versions of tools were transcribed into mRNA in vitro, and LNP was prepared according to the reference (Musunuru, K., Chadwick, A. C., Mizoguchi, T. et al. In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates. Nature 593, 429-434 (2021.)).

[0131] On day 0, HepG2.2.15 was plated in 24-well plates at a density of 5x10 5 The density of cells per well was 5x10 5 The density of cells per well was 5x10

[0132] Table 3 List of epigenetic editing tool information (Figure 1, detection time: day 5)

[0133] Table 4 List of epigenetic editing tool information (Figure 2, detection time: day 3)

[0134] Table 5 List of epigenetic editing tool information (Figure 3, detection time: day 4)

[0135] Table 6 List of epigenetic editing tool information (Figure 4, detection time: day 4)

[0136] Table 7 List of epigenetic editing tool information (Figure 5, detection time: day 6)

[0137] Table 8 List of epigenetic editing tool information (Figure 6, detection time: day 3)

[0138] Table 9 List of epigenetic editing tool information (Figure 7, detection time: day 16)

[0139] Table 10 List of epigenetic editing tool information (Figure 8, detection time: day 4)

[0140] Table 11 List of epigenetic editing tool information (Figure 9, detection time: day 6)

[0141] Table 12 List of epigenetic editing tool information (Figure 10, detection time: day 4)

[0142] Example 2

[0143] Inhibition efficiency of the epigenetic editing agent of the present application on HBV-D gene expression in human primary hepatocytes

[0144] This example uses human primary hepatocytes (PHH) infected with HBV-D type (SEQ ID NO: 3335) as a research model. Epigenetic editing against the conserved region can lead to a decrease in HBV viral marker parameters, i.e., a decrease in the amount or level of HBsAg, HBeAg marker production.

[0145] Experimental method: Different versions of tools were transcribed into mRNA in vitro, and prepared into LNP according to the reference (Musunuru, K., Cnadwick, A. C., Mizoguchi, T. et al. In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates. Nature 593, 429-434 (2021.)).

[0146] The cell experiment part of this embodiment was commissioned to WuXi AppTec, and the cells and viruses used during the experiment were provided by WuXi AppTec. This experiment used D hepatitis B virus (GenBank: U95551) to infect the PHH cell line. The experimental scheme is roughly as follows: after the primary hepatocytes were infected with HBV for two days, mRNA of different versions of the tool was delivered to the primary hepatocytes using LNP, and the culture supernatant was collected every 3 days after administration to detect the expression levels of HBV surface antigen (HBsAg), core antigen (HBcAg) and HBV DNA in the supernatant. The HBV-infected PHH cell line was treated with three tools (experimental group numbers 18, 28, 37 corresponding to the experimental groups in the table of Example 1) at two mRNA delivery concentrations of 0.78 ug / ml and 1.56 ug / ml, respectively, and the experimental results are shown in FIG. 11A. On the 12th day after administration, the three tools significantly inhibited the expression of HBV antigens and DNA.

[0147] In addition, the mRNA of two groups of tools (experimental group numbers 85 and 71 corresponding to the experimental groups in the table of Example 1, wherein the amino acid sequence and the encoding mRNA sequence of the tool of experimental group 85 are shown as SEQ ID NO: 8269 and 8270, respectively) was delivered to the above-mentioned HBV-infected primary hepatocytes according to a concentration gradient, and the concentration gradient was set to 0.0025 ug / ml, 0.0078 ug / ml, 0.025 ug / ml, 0.078 ug / ml, 0.25 ug / ml, 0.78 ug / ml and 2.5 ug / ml. The culture supernatant was collected every two days after administration, and the expression levels of HBV surface antigen (HBsAg), core antigen (HBcAg) and HBV DNA in the supernatant were detected. The experimental results are shown in FIG. 11B. On the eighth day after administration, the inhibition effect of the two tools on HBV antigens and DNA showed a dose-dependent effect.

[0148] Example 3

[0149] Inhibition efficiency of the epigenetic editing agent of the present application on HBV-A gene expression in mice

[0150] The transgenic HBV mice (C57BL / 6-HBV) used in this example have a 1.28-length HBV genome (type A, GenBank: AF305422.1) inserted into their genome. The experimental protocol is to transcribe different versions of the tool into mRNA in vitro, and prepare LNP according to the reference (Musunuru, K., Chadwick, A.C., Mizoguchi, T. et al. In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates. Nature 593, 429-434 (2021).). The mRNA of different versions of the tool is delivered into transgenic HBV mice by tail vein injection using lipid nanoparticles (LNP) (dose 0.25 mg / kg), and the negative control is injected with PBS. Blood samples were collected periodically after administration to detect the levels of secreted HBV surface antigen (HBsAg) and core antigen (HBcAg) in serum, and the level of HBV DNA was detected by qPCR. (Hepatitis B virus e antigen quantitative kit: Maike Biological, item number IM4403003; Hepatitis B virus surface antigen quantitative kit: Maike Biological, item number IM4403001; Hepatitis B virus nucleic acid quantitative kit: Shengxiang Biological, 2015340008; Detection method according to kit instructions). The experimental results after administration are shown in Figure 12A. After delivering different tools (experimental group numbers 18, 28, 37 correspond to the experimental groups in the table of Example 1) into transgenic HBV mice, the expression levels of antigens HBsAg / HBeAg and HBV DNA in serum were significantly reduced, and the stable inhibition effect was still maintained as the days after administration increased.

[0151] In addition, the mRNA of two groups of tools (corresponding to experimental groups 85 and 71 in the table of Example 1, wherein the amino acid sequence and the encoding mRNA sequence of the tool of experimental group 85 are SEQ ID NO: 8269 and 8270, respectively) were delivered into the transgenic HBV mice by tail vein injection using the above-mentioned lipid nanoparticles (LNP) at different doses (0.25 mg / kg, 0.75 mg / kg, 1.5 mg / kg, respectively), and the negative control was injected with PBS. Blood samples were collected periodically after administration, and the levels of secreted HBV surface antigen (HBsAg), core antigen (HBcAg) and HBV DNA in the serum were detected in the same manner as described above. The experimental results after administration are shown in FIG. 12B. After delivering different doses of tools into the transgenic HBV mice, the expression levels of antigens HBsAg / HBeAg and HBV DNA in the serum were significantly reduced, and the stable inhibitory effect was still maintained as the days after administration increased, and the drug efficacy showed a dose-dependent effect.

Claims

1. An epigenetic editing agent comprising a transcription activator-like effector (TALE) domain, at least one epigenetic modification domain, and at least one transcription regulation domain, wherein: 1) the TALE domain, the at least one epigenetic modification domain, and the at least one transcription regulation domain are directly or indirectly linked; or 2) the TALE domain, the at least one epigenetic modification domain, and at least one recruiting domain A are directly or indirectly linked to form a first fusion, and the at least one transcription regulation domain and at least one recruiting domain A’ are directly or indirectly linked to form a second fusion; or 3) the TALE domain, the at least one transcription regulation domain, and at least one recruiting domain A are directly or indirectly linked to form a first fusion, and the at least one epigenetic modification domain and at least one recruiting domain A’ are directly or indirectly linked to form a second fusion; and: 2) and 3) the recruiting domain A and the recruiting domain A’ are capable of interacting to enable the fusion or a portion thereof of one of the first fusion and the second fusion to be recruited in proximity to the other fusion; the TALE domain is capable of specifically binding to a target nucleotide sequence on an HBV gene and / or a regulatory element of an HBV gene.

2. The epigenetic editing agent of claim 1, the target nucleotide sequence is selected from the group consisting of the sequence of any one of SEQ ID NOs: 61-1697 and 3336-3355.

3. The epigenetic editing agent of claim 1 or 2, the TALE domain comprises an engineered RVD domain capable of recognizing and specifically binding to the target nucleotide sequence.

4. The epigenetic editing agent of any one of claims 1-3, the TALE domain comprises the amino acid sequence of any one of SEQ ID NOs: 1698-3334 and 3356-8266.

5. The epigenetic editing agent of any one of claims 1-4, the epigenetic modification domain is selected from the group consisting of: DNA deaminase activity, DNA methyltransferase activity, DNA demethylase activity, DNA aminating activity, DNA oxidizing activity, DNA helicase activity, histone acetyltransferase activity, histone deacetylase activity, histone methyltransferase activity, histone demethylase activity, histone kinase activity, histone phosphatase activity, histone ubiquitin ligase activity, and histone deubiquitinating activity.

6. The epigenetic editing agent of any one of claims 1-5, the epigenetic modification domain comprises a DNA methyltransferase (DNMT) and / or a functionally active fragment thereof.

7. The epigenetic editing agent of claim 6, the DNA methyltransferase is selected from the group consisting of DNMT3A, DNMT3B, DNMT3C, DNMT1, DNMT2, and DNMT3L. ​ ​ ​ ​ ​ ​ 8. The epigenetic editing agent of any one of claims 1-7, wherein the epigenetic modification domain comprises a plurality of DNA methyltransferases and / or functionally active fragments thereof, and the plurality of DNA methyltransferases and / or functionally active fragments thereof are connected by a linker sequence.

9. The epigenetic editing agent of any one of claims 1-8, wherein the epigenetic modification domain comprises at least one DNMT3A and at least one DNMT3L.

10. The epigenetic editing agent of any one of claims 6-8, wherein the DNA methyltransferase comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-6.

11. The epigenetic editing agent of any one of claims 1-10, wherein the epigenetic modification domain comprises DNMT3A and DNMT3L, and a C-terminus of the DNMT3A is connected to an N-terminus of the DNMT3L or a C-terminus of the DNMT3L is connected to an N-terminus of the DNMT3A.

12. The epigenetic editing agent of any one of claims 1-11, wherein the transcriptional regulation domain is a transcriptional activation domain or a transcriptional repressor domain.

13. The epigenetic editing agent of claim 12, the transcriptional repressor domain is selected from the group consisting of: KRAB, ZIM3KRAB, ZNF680, ZNF554, ZNF264, ZNF582, ZNF324, ZNF669, ZNF354A, ZNF82, ZNF595, ZNF419, ZNF566, ZIM2, EHMT2, SUV39H1, ZFPM1, TRIM28, EZH2, MXD1, SID, LSD1, HP1a, HDAC3, ZNF436, ZNF257, ZNF675, ZNF490, ZNF320, ZNF331, ZNF816, ZNF41, ZNF189, ZNF528, ZNF543, ZNF140, ZNF610, ZNF350, ZNF8, ZNF30, ZNF98, ZNF677, ZNF596, ZNF214, ZNF37A, ZNF34, ZNF250, ZNF547, ZNF273, ZFP82, ZNF224, ZNF33A, ZNF45, ZNF175, ZNF184, ZFP28-1, ZFP28-2, ZNF18, ZNF213, ZNF394, ZFP1, ZFP14, ZNF416, ZNF557, ZNF729, ZNF254, ZNF764, ZNF785, ZNF10, CBX5, RYBP, YAF2, MGA, CBX1, SCMH1, MPP8, SUMO3, HERC2, BIN1, PCGF2, TOX, FOXA1, FOXA2, IRF2BP1, IRF2BP2, IRF2BP1_2 N-terminal domain, HOXA13, HOXB13, HOXC13, HOXA11, HOXC11, HOXC10, HOXA10, HOXB9, HOXA9, ZFP28, ZN334, ZN568, ZN37A, ZN181, ZN510, ZN862, ZN140, ZN208, ZN248, ZN571, ZN699, ZN726, ZIK1, ZNF2, Z705F, ZNF14, ZN471, ZN624, ZNF84, ZNF7, ZN891, ZN337, Z705G, ZN529, ZN729, ZN419, Z705A, ZN302, ZN486, ZN621, ZN688, ZN33A, ZN554, ZN878, ZN772, ZN224, ZN184, ZN544, ZNF57, ZN283, ZN549, ZN211, ZN615, ZN253, ZN226, ZN730, Z585A, ZN732, ZN681, ZN667, ZN649, ZN470, ZN484,ZN431, ZN382, ZN254, ZN124, ZN607, ZN317, ZN620, ZN141, ZN584, ZN540, ZN75D, ZN555, ZN658, ZN684, RBAK, ZN829, ZN582, ZN112, ZN716, HKR1, ZN350, ZN480, ZN416, ZNF92, ZN100, ZN736, ZNF74, ZN443, ZN195, ZN530, ZN782, ZN791, ZN331, Z354C, ZN157, ZN727, ZN550, ZN793, ZN235, ZN724, ZN573, ZN577, ZN789, ZN718, ZN300, ZN383, ZN429, ZN677, ZN850, ZN454, ZN257, ZN264, ZN485, ZN737, ZNF44, ZN596, ZN565, ZN543, ZFP69, SUMO1, ZNF12, ZN169, ZN433, ZN175, ZN347, ZNF25, ZN519, Z585B, ZN517, ZN846, ZN230, ZNF66, ZN713, ZN816, ZN426, ZN674, ZN627, ZNF20, Z587B, ZN316, ZN233, ZN611, ZN556, ZN234, ZN560, ZNF77, ZN682, ZN614, ZN785, ZN445, ZFP30, ZN225, ZN551, ZN610, ZN528, ZN284, ZN418, ZN490, ZN805, Z780B, ZN763, ZN285, ZNF85, ZN223, ZNF90, ZN557, ZN425, ZN229, ZN606, ZN155, ZN222, ZN442, ZNF91, ZN135, ZN778, ZN534, ZN586, ZN567, ZN440, ZN583, ZN441, ZNF43, ZN589, ZN563, ZN561, ZN136, ZN630, ZN527, ZN333, Z324B, ZN786, ZN709, ZN792, ZN599, ZN613, ZF69B, ZN799, ZN569, ZN564, ZN546, ZFP92, ZN723, ZN439, ZFP57, ZNF19, ZN404, ZN274, CBX3, ZN250, ZN570, ZN675, ZN695, ZN548, ZN132, ZN738, ZN420, ZN626, ZN559, ZN460, ZN268, ZN304, ZN605, ZN844, SUMO5, ZN101, ZN783, ZN417,ZN182, ZN823, ZN177, ZN197, ZN717, ZN669, ZN256, ZN251, CBX4, CDY2, CDYL2, ZN562, ZN461, Z324A, ZN766, ID2, ZN214, CBX7, ID1, CREM, SCX, ASCL1, ZN764, SCML2, TWST1, CREB1, TERF1, ID3, CBX8, GSX1, NKX22, AIF1, TWST2, ZNF17, TOX3, TOX4, ZMYM3, I2BP1, RHXF1, SSX2, I2BPL, ZN680, TRI68, HXA13, PHC3, TCF24, HXB13, HEY1, PHC2, ZNF81, FIGLA, SAM11, KMT2B, HEY2, JDP2, HXC13, ASCL4, HHEX, GSX2, ETV7, ASCL3, PHC1, OTP, I2BP2, VGLL2, HXA11, PDLI4, ASCL2, CDX4, ZN860, LMBL4, PDIP3, NKX25, CEBPB, ISL1, CDX2, PROP1, SIN3B, SMBT1, HXC11, HXC10, PRS6A, VSX1, NKX23, MTG16, HMX3, HMX1, KIF22, CSTF2, CEBPE, DLX2, PPARG, PRIC1, UNC4, BARX2, ALX3, TCF15, TERA, VSX2, HXD12, CDX1, TCF23, ALX1, HXA10, RX, CXXC5, SCML1, NFIL3, DLX6, MTG8, CEBPD, SEC13, FIP1, ALX4, LHX3, PRIC2, MAGI3, NELL1, PRRX1, MTG8R, RAX2, DLX3, DLX1, NKX26, NAB1, SAMD7, PITX3, WDR5, MEOX2, NAB2, DHX8, CBX6, EMX2, CPSF6, HXC12, KDM4B, LMBL3, PHX2A, EMX1, NC2B, DLX4, SRY, ZN777, ZN398, GAIA3, BSH, SF3B4, TEAD1, TEAD3, RGAP1, PHF1, GATA2, FOXO3, ZN212, IRX4, ZBED6, LHX4, SIN3A, RBBP7, NKX61, R51A1, MB3L1, DLX5, NOTC1, TERF2, ZN282, RGS12, ZN840, SPI2B, PAX7, NKX62, ASXL2, FOXO1, GATA1, ZMYM5, LRP1, MIXL1, SGT1, LMCD1,CEBPA, SOX14, WTIP, PRP19, NKX11, RBBP4, DMRT2, SMCA2, and functionally active fragments thereof.

14. The epigenetic editing agent of claim 12 or 13, wherein the transcriptional repressor domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 7-32.

15. The epigenetic editing agent of any one of claims 12-14, wherein the transcriptional repressor domain comprises a zinc-finger protein-based transcription factor or a functionally active fragment thereof.

16. The epigenetic editing agent of claim 15, wherein the zinc-finger protein-based transcription factor is Krüppel-associated box (KRAB) or a KRAB domain derived from ZIM3 (ZIM3 KRAB).

17. The epigenetic editing agent of any one of claims 1-16, wherein the transcriptional regulation domain comprises two or more of the zinc-finger protein-based transcription factors or functionally active fragments thereof, which are the same species or different species.

18. The epigenetic editing agent of claim 17, wherein the two or more zinc-finger protein-based transcription factors are connected by a linker sequence.

19. The epigenetic editing agent of claim 18, wherein the linker sequence is an XTEN linker sequence.

20. The epigenetic editing agent of claim 12, wherein the transcriptional repressor domain comprises a histone modification domain.

21. The epigenetic editing agent of claim 20, wherein the histone modification domain is selected from the group consisting of EZH2, HDAC3, HDAC1, EHMT2, PRMT1, PRMT5, SETDB1, hSIRT1, HP1a, LSD1, and functionally active fragments thereof.

22. The epigenetic editing agent of any one of claims 1-21, which is a fusion peptide.

23. The epigenetic editing agent of claim 22, wherein the epigenetic modification domain and the transcription regulation domain are co-located at either N-terminus or C-terminus of the TALE domain.

24. The epigenetic editing agent of claim 22, wherein the epigenetic modification domain and the transcription regulation domain are located at N-terminus and C-terminus of the TALE domain, respectively.

25. The epigenetic editing agent of any one of claims 22-24, wherein the fusion peptide comprises, in order from N-terminus to C-terminus: 1) the epigenetic modification domain, the transcription regulation domain, and the TALE domain; or 2) the transcription regulation domain, the epigenetic modification domain, and the TALE domain; or 3) the TALE domain, the epigenetic modification domain, and the transcription regulation domain; or 4) the TALE domain, the transcription regulation domain, and the epigenetic modification domain; or 5) the epigenetic modification domain, the TALE domain, and the transcription regulation domain; or 6) the transcription regulation domain, the TALE domain, and the epigenetic modification domain.

26. The epigenetic editing agent of any one of claims 22-25, wherein the fusion peptide comprises, in order from N-terminus to C-terminus: 1) one or a combination of DNMT3A and DNMT3L, one or more zinc-finger protein-based transcription factors, and a TALE domain; or 2) one or more zinc-finger protein-based transcription factors, one or a combination of DNMT3A and DNMT3L, and a TALE domain; or 3) a TALE domain, one or a combination of DNMT3A and DNMT3L, and one or more zinc-finger protein-based transcription factors; or 4) a TALE domain, one or more zinc-finger protein-based transcription factors, and one or a combination of DNMT3A and DNMT3L; or 5) one or a combination of DNMT3A and DNMT3L, a TALE domain, and one or more zinc-finger protein-based transcription factors; or 6) one or more zinc-finger protein-based transcription factors, a TALE domain, and one or a combination of DNMT3A and DNMT3L.

27. The epigenetic editing agent of any one of claims 22-26, the fusion peptide comprising the following domains: TALE-DNMT3A-DNMT3L-ZIM3 KRAB, TALE-ZIM3 KRAB-DNMT3L-DNMT3A, TALE-ZIM3 KRAB-DNMT3A-DNMT3L, ZIM3 KRAB-DNMT3A-DNMT3L-TALE, DNMT3A-DNMT3L-ZIM3 KRAB-TALE, DNMT3A-DNMT3L-ZNF324-TALE, DNMT3A-DNMT3L-ZNF419-TALE, DNMT3A-DNMT3L-TALE-EZH2, DNMT3A-DNMT3L-TALE-HDAC3, DNMT3A-DNMT3L-TALE-HP1a, DNMT3A-DNMT3L-TALE-KRAB, DNMT3A-DNMT3L-TALE-ZIM3 KRAB, wherein, - indicates that the respective domains of the fusion are directly and / or indirectly linked, and in order from N-terminus to C-terminus.

28. The epigenetic editing agent of any one of claims 1-21, which is a composite peptide comprising the first fusion and the second fusion.

29. The epigenetic editing agent of claim 28, wherein the first fusion comprises, in order from N-terminus to C-terminus: 1) an epigenetic modification domain, a TALE domain, and a recruitment domain A, or 2) an epigenetic modification domain, a recruitment domain A, and a TALE domain, or 3) a TALE domain, a recruitment domain A, and an epigenetic modification domain, or 4) a TALE domain, an epigenetic modification domain, and a recruitment domain A, or 5) recruiting domain A, epigenetic modification domain and TALE domain, or 6) recruiting domain A, TALE domain and epigenetic modification domain.

30. The epigenetic editing agent of claim 28 or 29, the second fusion comprising, in order from N- to C-terminus, a transcription repressor domain and recruiting domain A’, or a recruiting domain A’ and a transcription repressor domain.

31. The epigenetic editing agent of claim 28, the first fusion comprising, in order from N- to C-terminus: 1) recruiting domain A, TALE domain and transcription repressor domain, or 2) recruiting domain A, transcription repressor domain and TALE domain, or 3) TALE domain, recruiting domain A and transcription repressor domain, or 4) TALE domain, transcription repressor domain and recruiting domain A, or 5) transcription repressor domain, TALE domain and recruiting domain A, or 6) transcription repressor domain, recruiting domain A and TALE domain.

32. The epigenetic editing agent of claim 28 or 31, the second fusion comprising, in order from N- to C-terminus, an epigenetic modification domain and recruiting domain A’, or a recruiting domain A’ and an epigenetic modification domain.

33. The epigenetic editing agent of any one of claims 28-32, the complex peptide comprising the features: 1) the first fusion comprising, in order from N- to C-terminus, an epigenetic modification domain, TALE domain and recruiting domain A, the second fusion comprising, in order from N- to C-terminus, a transcription repressor domain and recruiting domain A’; or 2) the first fusion comprising, in order from N- to C-terminus, an epigenetic modification domain, TALE domain and recruiting domain A, the second fusion comprising, in order from N- to C-terminus, a recruiting domain A’ and a transcription repressor domain; or 3) the first fusion comprising, in order from N- to C-terminus, a recruiting domain A, TALE domain and transcription repressor domain, the second fusion comprising, in order from N- to C-terminus, an epigenetic modification domain and recruiting domain A’; or 4) the first fusion comprising, in order from N- to C-terminus, a recruiting domain A, TALE domain and transcription repressor domain, the second fusion comprising, in order from N- to C-terminus, a recruiting domain A’ and an epigenetic modification domain; or 5) the first fusion comprising, in order from N- to C-terminus, an epigenetic modification domain, recruiting domain A and TALE domain, the second fusion comprising, in order from N- to C-terminus, a recruiting domain A’ and a transcription repressor domain; or 6) the first fusion comprising, in order from N- to C-terminus, a TALE domain, epigenetic modification domain and recruiting domain A, the second fusion comprising, in order from N- to C-terminus, a recruiting domain A’ and a transcription repressor domain; or 7) the first fusion comprises, in order from N- to C-terminus, a TALE domain, a recruitment domain A, and an epigenetic modification domain, and the second fusion comprises, in order from N- to C-terminus, a recruitment domain A’ and a transcription repressor domain.

34. The epigenetic editing agent of any one of claims 1-33, wherein the recruitment domain A is selected from any one of one of the following two groups of domains, and the recruitment domain A’ is selected from any one of the other of the following two groups of domains: 1) a general control non-derepressible protein 4 (GCN4), a GFP11 fragment derived from split green fluorescent protein (GFP), or a GVKESLV polypeptide; and 2) a single chain antibody (scFv), a GFP1-10 fragment derived from split green fluorescent protein (GFP), or a PDZ protein domain.

35. The epigenetic editing agent of any one of claims 1-34, wherein: 1) the domain of one of the recruitment domain A and the recruitment domain A’ is GCN4, and the domain of the other is scFv; or 2) the domain of one of the recruitment domain A and the recruitment domain A’ is GFP11 fragment, and the domain of the other is GFP1-10; or 3) the domain of one of the recruitment domain A and the recruitment domain A’ is GVKESLV, and the domain of the other is PDZ protein domain.

36. The epigenetic editing agent of any one of claims 28-35, wherein the complex peptide comprises the features: 1) the fusion of one of the first fusion and the second fusion comprises DNMT(3A-3L)-TALE-n x GCN4, and the fusion of the other comprises a transcription repressor domain-scFv; or 2) the fusion of one of the first fusion and the second fusion comprises DNMT(3A-3L)-TALE-scFv, and the fusion of the other comprises a transcription repressor domain-GCN4; or 3) the fusion of one of the first fusion and the second fusion comprises DNMT(3A-3L)-TALE-n x GFP11, and the fusion of the other comprises a transcription repressor domain-GFP1-10; or 4) the fusion of one of the first fusion and the second fusion comprises DNMT(3A-3L)-TALE-GFP1-10, and the fusion of the other comprises a transcription repressor domain-GFP11; or 5) the fusion of one of the first fusion and the second fusion comprises DNMT(3A-3L)-TALE-n x GCN4, and the fusion of the other comprises scFv-transcription repressor domain; or 6) the fusion of one of the first fusion and the second fusion comprises DNMT(3A-3L)-TALE-scFv, and the fusion of the other comprises GCN4-transcription repressor domain. 7) one of the first fusion and the second fusion comprises DNMT(3A-3L)-TALE-n x GFP11, and the other comprises GFP1-10-transcription repressor domain; or 8) one of the first fusion and the second fusion comprises DNMT(3A-3L)-TALE-GFP1-10, and the other comprises GFP11-transcription repressor domain; or 9) one of the first fusion and the second fusion comprises n x GCN4-TALE-transcription repressor domain, and the other comprises DNMT(3A-3L)-scFv; or 10) one of the first fusion and the second fusion comprises scFv-TALE-transcription repressor domain, and the other comprises DNMT(3A-3L)-GCN4; or 11) one of the first fusion and the second fusion comprises n x GFP11-TALE-transcription repressor domain, and the other comprises DNMT(3A-3L)-GFP1-10; or 12) one of the first fusion and the second fusion comprises GFP1-10-TALE-transcription repressor domain, and the other comprises DNMT(3A-3L)-GFP11; or 13) one of the first fusion and the second fusion comprises n x GCN4-TALE-transcription repressor domain, and the other comprises scFv-DNMT(3A-3L); or 14) one of the first fusion and the second fusion comprises scFv-TALE-transcription repressor domain, and the other comprises GCN4-DNMT(3A-3L); or 15) one of the first fusion and the second fusion comprises n x GFP11-TALE-transcription repressor domain, and the other comprises GFP1-10-DNMT(3A-3L); or 16) one of the first fusion and the second fusion comprises GFP1-10-TALE-transcription repressor domain, and the other comprises GFP11-DNMT(3A-3L); or 17) one of the first fusion and the second fusion comprises scFv-transcription repressor domain, and the other comprises DNMT(3A-3L)-n x GCN4-TALE; or 18) one of the first fusion and the second fusion comprises scFv-transcription repressor domain, and the other comprises TALE-DNMT(3A-3L)-n x GCN4; or 19) the fusion of one of said first fusion and said second fusion comprises a scFv- transcription repressor domain, and the other fusion comprises a TALE-n x GCN4-DNMT(3A-3L); wherein DNMT(3A-3L) means that DNMT3A and DNMT3L are directly or indirectly linked in any order, - means that the domains at its two ends are directly or indirectly linked in order from N-terminus to C-terminus; n x GCN4 or n x GFP11 means n copies of GCN4 or n copies of GFP11 respectively linked by a linker sequence, n being selected from any integer from 1 to 20.

37. The epigenetic editing agent of any one of claims 1-36, further comprising a nuclear localization signal and / or a marker domain.

38. The epigenetic editing agent of any one of claims 1-37, capable of providing a modification of at least one nucleotide in the vicinity of the HBV gene and / or within a regulatory element of the HBV gene.

39. A nucleic acid encoding the epigenetic editing agent of any one of claims 1-38.

40. The nucleic acid of claim 39, comprising a first nucleic acid segment encoding the first fusion, and a second nucleic acid segment encoding the second fusion, the first and second nucleic acid segments being linked by a nucleic acid segment encoding a cleavage peptide.

41. The nucleic acid of claim 40, the cleavage peptide being a 2A peptide and / or an IRES.

42. The nucleic acid of claim 41, the 2A peptide being selected from P2A, T2A, E2A and F2A.

43. The nucleic acid of any one of claims 39-42, being a recombinant vector.

44. The nucleic acid of claim 43, the recombinant vector further comprising a non-coding region.

45. The nucleic acid of claim 44, the non-coding region being selected from an intron, a regulatory element, a promoter, an enhancer, a termination sequence, and a 5’ and 3’ untranslated region.

46. A delivery vehicle comprising the epigenetic editing agent of any one of claims 1-38 and / or the nucleic acid of any one of claims 40-46, and optionally a liposome and / or a lipid nanoparticle.

47. A composition comprising the epigenetic editing agent of any one of claims 1-38, the nucleic acid of any one of claims 39-45, and / or the delivery vehicle of claim 46.

48. A cell comprising the epigenetic editing agent of any one of claims 1-38, the nucleic acid of any one of claims 39-45, the delivery vehicle of claim 46, and / or the composition of claim 47.

49. A kit comprising the epigenetic editing agent of any one of claims 1-38, the nucleic acid of any one of claims 39-45, the delivery vehicle of claim 46, the composition of claim 47, and / or the cell of claim 48.

50. A method of modulating expression of an HBV gene product, the method comprising administering the epigenetic editing agent of any one of claims 1-38, the nucleic acid of any one of claims 39-45, the delivery vehicle of claim 46, the composition of claim 47, the cell of claim 48, and / or the kit of claim 49.

51. The method of claim 50, the method comprising introducing the epigenetic modifying agent, the nucleic acid, the delivery vehicle, the composition, the cell, and / or the kit to a cell containing the HBV gene.

52. The method of claim 51, the method comprising contacting the epigenetic modifying agent, the nucleic acid, the delivery vehicle, and / or the composition to the HBV gene and / or a regulatory element of the HBV gene.

53. The method of claim 52, the regulatory element comprising a core promoter, a proximal promoter, a distal enhancer, a silencer, an insulator element, a boundary element, and / or a locus control region.

54. A method of treating or ameliorating a disease or condition associated with a hepatitis B virus (HBV) infection, the method comprising administering to a subject in need thereof an effective amount of the epigenetic editing agent of any one of claims 1-38, the nucleic acid of any one of claims 39-45, the delivery vehicle of claim 46, the composition of claim 47, the cell of claim 48, and / or the kit of claim 49.

55. Use of the epigenetic editing agent of any one of claims 1-38, the nucleic acid of any one of claims 39-45, the delivery vehicle of claim 46, the composition of claim 47, the cell of claim 48, and / or the kit of claim 49 for the manufacture of a medicament for treating or ameliorating a disease or condition associated with a hepatitis B virus (HBV) infection.

56. The method of claim 54, the disease or condition associated with a hepatitis B virus (HBV) infection comprising hepatitis, cirrhosis, liver fibrosis, and hepatocellular carcinoma caused by HBV infection.

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