Engineered gene transcription-suppression tool targeting PCSK9, and use thereof

Abstract

The present application relates to an epigenetic editing agent, which contains a transcription activator-like effector (TALE) domain, at least one epigenetic modification domain, and at least one transcriptional regulatory domain, wherein the TALE domain can specifically bind to a target nucleotide sequence on the PCSK9 gene and / or on a regulatory element of the PCSK9 gene.

Description

Engineered gene transcriptional repression tools targeting PCSK9 and their applications Technical Field

[0001] This application relates to the field of biomedicine, specifically to an epigenetic editing agent for regulating PCSK9 gene expression and its uses. Background Technology

[0002] Mixed hyperlipidemia is the most common type of lipoprotein metabolism or transport abnormality among people with dyslipidemia. A series of clinical studies both domestically and internationally have confirmed that mixed hyperlipidemia is a significant cause of atherosclerotic cardiovascular disease (ASCVD), including coronary heart disease, stroke, and peripheral vascular disease. Mixed hyperlipidemia is a lipid disorder that includes both hypercholesterolemia and hypertriglyceridemia. This classification is primarily based on the specific abnormalities in blood lipid levels; type II hyperlipidemia is the most common type, especially type IIb, which involves simultaneous elevations in both low-density lipoprotein cholesterol (LDL-C) and triglyceride levels, and is the most prevalent form of mixed hyperlipidemia. Therefore, addressing the simultaneous elevation of LDL-C and triglyceride levels is the primary target for managing mixed hyperlipidemia.

[0003] Currently, there are many therapeutic drugs targeting LDL-C. Among them, PCSK9, as a popular target for lipid-lowering drug development, has received considerable attention in recent years. After PCSK9 is produced in the body, it leads to the degradation of low-density lipoprotein receptors (LDL-R) on the surface of liver cells, thereby hindering the liver's effective removal of cholesterol from the blood, especially low-density lipoprotein cholesterol (LDL-C), ultimately leading to elevated blood lipid levels. Inhibiting PCSK9 expression provides a solution to this problem. By reducing PCSK9 expression, the binding of PCSK9 to LDL-R can be reduced, thereby preventing the degradation of LDL-R on the surface of liver cells. This results in more LDL-R on the liver surface, thus more effectively clearing LDL-C from the blood. Currently, drug strategies for reducing PCSK9 expression mainly include: inhibitors, monoclonal antibodies, siRNA, and gene-editing drugs. Among these, inhibitors, monoclonal antibodies, and siRNA all have the problem of long-term dosing, requiring patients to take medication for extended periods, and some patients are intolerant, while others with complications cannot receive treatment. Gene therapy drugs, such as Verve-101 developed by Verve Therapeutics in the United States, are base editors that are delivered to the liver via lipid nanoparticles. They replace one nucleotide in the PCSK9 gene with another, thereby inactivating the PCSK9 gene. However, the genetic risks associated with the genome-cutting activity of these drugs still need to be assessed. Therefore, there is an urgent need for a drug that can be administered once, is effective for life, and does not require genome cutting, thus avoiding genetic risks. Summary of the Invention

[0004] On one hand, this application provides an epigenetic editing agent comprising a transcription activator-like effector (TALE) domain, at least one epigenetic modification domain, and at least one transcriptional regulatory domain, wherein: 1) the TALE domain, the at least one epigenetic modification domain, and the at least one transcriptional regulatory domain are directly or indirectly connected; or 2) the TALE domain, the at least one epigenetic modification domain, and at least one recruitment domain A' are directly or indirectly connected to form a first fusion, and the at least one transcriptional regulatory domain is directly or indirectly connected to at least one recruitment domain A' to form a second fusion; or 3) The TALE domain, the at least one transcriptional regulatory domain, and the at least one recruitment domain A are directly or indirectly connected to form a first fusion, and the at least one epigenetic modification domain is directly or indirectly connected to the at least one recruitment domain A' to form a second fusion; and: 2) and 3) the recruitment domain A and the recruitment domain A' are able to interact so that a fusion of one of the first fusion and the second fusion, or a portion thereof, can be recruited to the vicinity of the other fusion; the TALE domain is able to specifically bind to target nucleotide sequences on the PCSK9 gene and / or the regulatory elements of the PCSK9 gene.

[0005] In some embodiments, the target nucleotide sequence is selected from any one of SEQ ID NO:116-660.

[0006] In some implementations, the TALE domain includes an engineered RVD domain that is capable of recognizing and specifically binding to the target nucleotide sequence.

[0007] In some embodiments, the TALE domain comprises the amino acid sequence of any one of SEQ ID NO:35-38 and 661-2840, 2865, 2867-2868.

[0008] In some embodiments, the epigenetic modification domain is selected from: DNA deamination activity, DNA methyltransferase activity, DNA demethylase activity, DNA amination activity, DNA oxidation 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 deubiquitination activity.

[0009] In some embodiments, the epigenetic modification domain comprises a DNA methyltransferase (DNMT) and / or a functionally active fragment thereof.

[0010] In some embodiments, the DNA methyltransferase is selected from DNMT3A, DNMT3B, DNMT3C, DNMT1, DNMT2, and DNMT3L.

[0011] In some embodiments, the epigenetic modification domain comprises multiple DNA methyltransferases and / or their functionally active fragments, and the multiple DNA methyltransferases and / or their functionally active fragments are linked by adapter sequences.

[0012] In some embodiments, the epigenetic modification domain comprises at least one DNMT3A and at least one DNMT3L.

[0013] In some embodiments, the DNA methyltransferase comprises the amino acid sequence shown in any one of SEQ ID NOs:39-44, 2860.

[0014] In some embodiments, the epigenetic modification domain includes DNMT3A and DNMT3L, and the C-terminus of DNMT3A is connected to the N-terminus of DNMT3L, or the C-terminus of DNMT3L is connected to the N-terminus of DNMT3A.

[0015] In some implementations, the transcriptional regulatory domain is a transcriptional activation domain or a transcriptional repressor domain.

[0016] In some embodiments, the transcriptional repressor domain is selected from: 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, IRF2BPL IRF-2BP1_2N-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 their functionally active fragments.

[0017] In some embodiments, the transcriptional repressor domain comprises the amino acid sequence shown in any one of SEQ ID NOs:45-70, 2861.

[0018] In some implementations, the transcriptional repressor domain comprises a zinc finger protein-based transcription factor or a functionally active fragment thereof.

[0019] In some embodiments, the zinc finger protein-based transcription factor is a Krüppel-associated repressor (KRAB) or a KRAB domain derived from ZIM3 (ZIM3 KRAB).

[0020] In some embodiments, the transcriptional regulatory domain comprises two or more zinc finger-based transcription factors or their functionally active fragments, wherein the two or more zinc finger-based transcription factors are of the same type or different types.

[0021] In some implementations, the two or more zinc finger protein-based transcription factors are linked by adapter sequences.

[0022] In some implementations, the connector sequence is an XTEN connector sequence.

[0023] In some implementations, the transcriptional repressor domain includes a histone modification domain.

[0024] In some embodiments, the histone modification domain is selected from: EZH2, HDAC3, HDAC1, EHMT2, PRMT1, PRMT5, SETDB1, hSIRT1, HP1a, LSD1, and their functionally active fragments.

[0025] In some implementations, the epigenetic modification domain and the transcriptional regulatory domain are both located at the N-terminus or C-terminus of the TALE domain.

[0026] In some implementations, the epigenetic modification domain and the transcriptional regulatory domain are located at the N-terminus and C-terminus of the TALE domain, respectively.

[0027] In some embodiments, the fusion peptide is sequentially connected from the N-terminus to the C-terminus to: 1) the epigenetic modification domain, the transcriptional regulatory domain, and the TALE domain; or 2) the transcriptional regulatory domain, the epigenetic modification domain, and the TALE domain; or 3) the TALE domain, the epigenetic modification domain, and the transcriptional regulatory domain; or 4) the TALE domain, the transcriptional regulatory domain, and the epigenetic modification domain; or 5) the epigenetic modification domain, the TALE domain, and the transcriptional regulatory domain; or 6) the transcriptional regulatory domain, the TALE domain, and the epigenetic modification domain.

[0028] In some embodiments, the fusion peptide is sequentially linked from the N-terminus to the C-terminus to: 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.

[0029] In some embodiments, the fusion peptide comprises the following domains: TALE-DNMT3A-DNMT3L-ZIM3KRAB, 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, where - indicates that the structural domains of the fusion are directly and / or indirectly connected, and the structural domains are arranged in order from the N end to the C end.

[0030] In some embodiments, the epigenetic editing agent is a complex peptide comprising the first fusion compound and the second fusion compound.

[0031] In some implementations, the first fusion comprises, from the N-terminus to the C-terminus, an epigenetic modification domain, a TALE domain, and a recruitment domain A.

[0032] In some embodiments, the second fusion compound contains a transcriptional repressor domain and a recruitment domain A' from the N-terminus to the C-terminus, or it contains a recruitment domain A' and a transcriptional repressor domain from the N-terminus to the C-terminus.

[0033] In some implementations, the first fusion compound comprises, from the N-terminus to the C-terminus, a recruitment domain A, a TALE domain, and a transcriptional repressor domain.

[0034] In some embodiments, the second fusion comprises, from the N-terminus to the C-terminus, an epigenetic modification domain and a recruitment domain A', or from the N-terminus to the C-terminus, a recruitment domain A' and an epigenetic modification domain.

[0035] In some embodiments, the complex peptide comprises the following features: 1) the first fusion compound comprises, from N-terminus to C-terminus, an epigenetic modification domain, a TALE domain, and a recruitment domain A, and the second fusion compound comprises, from N-terminus to C-terminus, a transcriptional repressor domain and a recruitment domain A'; or 2) the first fusion compound comprises, from N-terminus to C-terminus, an epigenetic modification domain, a TALE domain, and a recruitment domain A, and the second fusion compound comprises, from N-terminus to C-terminus, a recruitment domain A' and a transcriptional repressor domain; or 3) the first fusion compound comprises, from N-terminus to C-terminus, a recruitment domain A, a TALE domain, and a transcriptional repressor domain, and the second fusion compound comprises, from N-terminus to C-terminus, an epigenetic modification domain and a recruitment domain A'; or 4) the first fusion compound comprises, from N-terminus to C-terminus, a recruitment domain A, a TALE domain, and a transcriptional repressor domain, and the second fusion compound comprises, from N-terminus to C-terminus, a recruitment domain A' and an epigenetic modification domain.

[0036] In some embodiments, the recruitment domain A is selected from one of two groups of domains, and the recruitment domain A' is selected from the other of two groups of domains: 1) universal control non-derepressor 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.

[0037] In some implementations, wherein: 1) one of the recruitment domains A and A' is a GCN4 domain and the other is an scFv domain; or 2) one of the recruitment domains A and A' is a GFP11 fragment and the other is a GFP1-10 domain; or 3) one of the recruitment domains A and A' is a GVKESLV domain and the other is a PDZ protein domain.

[0038] In some embodiments, the complex peptide comprises: 1) a fusion of one of the first and second fusions comprising DNMT(3A-3L)-TALE-n×GCN4, and the other fusion comprising a transcriptional repressor domain -scFv; or 2) a fusion of one of the first and second fusions comprising DNMT(3A-3L)-TALE-scFv, and the other fusion comprising a transcriptional repressor domain -GCN4; or 3) a fusion of one of the first and second fusions comprising DNMT(3A-3L)-TALE-n×GFP11, and the other fusion comprising a transcriptional repressor domain -GCN4. The first fusion and the second fusion contain DNMT(3A-3L)-TALE-GFP1-10; or 4) one of the first fusions and the second fusion contains DNMT(3A-3L)-TALE-GFP1-10, and the other fusion contains a transcriptional repressor domain-GFP11; or 5) one of the first fusions and the second fusion contains DNMT(3A-3L)-TALE-n×GCN4, and the other fusion contains scFv-transcriptional repressor domain; or 6) one of the first fusions and the second fusion contains DNMT(3A-3L)-TALE-scFv, and the other fusion contains GCN4-transcriptional repressor domain. Domain; or 7) one of the first fusion and the second fusion comprises DNMT(3A-3L)-TALE-n×GFP11, and the other fusion comprises a GFP1-10-transcriptional repressor domain; or 8) one of the first fusion and the second fusion comprises DNMT(3A-3L)-TALE-GFP1-10, and the other fusion comprises a GFP11-transcriptional repressor domain; or 9) one of the first fusion and the second fusion comprises an n×GCN4-TALE-transcriptional repressor domain, and the other fusion comprises DNMT(3A-3L)-scFv ; or 10) one of the first fusion and the second fusion contains a scFv-TALE-transcriptional repressor domain, and the other fusion contains DNMT(3A-3L)-GCN4; or 11) one of the first fusion and the second fusion contains an n×GFP11-TALE-transcriptional repressor domain, and the other fusion contains DNMT(3A-3L)-GFP1-10; or 12) one of the first fusion and the second fusion contains a GFP1-10-TALE-transcriptional repressor domain, and the other fusion contains DNMT(3A-3L)-GFP11;Or 13) One of the first fusion and the second fusion contains an n×GCN4-TALE-transcriptional repressor domain, and the other fusion contains scFv-DNMT(3A-3L); or 14) One of the first fusion and the second fusion contains an scFv-TALE-transcriptional repressor domain, and the other fusion contains GCN4-DNMT(3A-3L); or 15) One of the first fusion and the second fusion contains an n×GFP11-TALE-transcriptional repressor domain, and the other fusion contains GFP1-10-DNMT(3A-3L). MT(3A-3L); or 16) a fusion of one of the first fusion and the second fusion contains a GFP1-10-TALE-transcriptional repressor domain, and the other fusion contains GFP11-DNMT(3A-3L); wherein DNMT(3A-3L) indicates that DNMT3A and DNMT3L are directly or indirectly linked in any order, and - indicates that the domains at both ends are directly or indirectly linked in order from the N-terminus to the C-terminus; n×GCN4 or n×GFP11 respectively represent n copies of GCN4 linked by adapter sequences or n copies of GFP11 linked by adapter sequences, where n is selected from any integer from 1 to 20.

[0039] In some implementations, the epigenetic editing agent further comprises nuclear localization signal and / or marker domains.

[0040] In some embodiments, the epigenetic editing agent is capable of providing modification of at least one nucleotide in the vicinity of the PCSK9 gene and / or within the regulatory elements of the PCSK9 gene.

[0041] On the other hand, this application provides nucleic acids encoding the epigenetic editing agents described in this application.

[0042] In some embodiments, the nucleic acid comprises a first nucleic acid fragment encoding the first fusion compound and a second nucleic acid fragment encoding the second fusion compound, the first and second nucleic acid fragments being linked by a nucleic acid fragment encoding a cleavage peptide.

[0043] In some embodiments, the cleavage peptide is a 2A peptide and / or IRES.

[0044] In some embodiments, the 2A peptide is selected from P2A, T2A, E2A, and F2A.

[0045] In some implementations, the nucleic acid is a recombinant vector.

[0046] In some implementations, the recombinant vector also includes a non-coding region.

[0047] In some implementations, the non-coding region is selected from introns, modulating elements, promoters, enhancers, termination sequences, and 5' and 3' untranslated regions.

[0048] On the other hand, this application provides a delivery vector comprising the epigenetic editing agent and / or the nucleic acid described in this application, and optionally comprising liposomes and / or lipid nanoparticles.

[0049] On the other hand, this application provides a composition comprising the epigenetic editing agent described in this application, the nucleic acid described in this application, and / or the delivery vector described in this application.

[0050] On the other hand, this application provides a cell comprising the epigenetic editing agent, the nucleic acid, the delivery vector, and / or the composition described in this application.

[0051] On the other hand, this application provides a kit comprising the epigenetic editing agent described in this application, the nucleic acid described in this application, the delivery vector described in this application, the composition described in this application, and / or the cells described in this application.

[0052] On the other hand, this application provides a method for regulating the expression of the PCSK9 gene product, the method comprising administering the epigenetic editing agent described in this application, the nucleic acid described in this application, the delivery vector described in this application, the composition described in this application, the cells described in this application, and / or the kit described in this application.

[0053] In some embodiments, the method includes introducing the epigenetic modifier, the nucleic acid, the delivery vector, the composition, the cell, and / or the kit into cells containing the PCSK9 gene.

[0054] In some embodiments, the method includes contacting the epigenetic modifier, the nucleic acid, the delivery vector, and / or the composition with the PCSK9 gene and / or the regulatory elements of the PCSK9 gene.

[0055] In some implementations, the regulatory element includes a core promoter, a proximal promoter, a distal enhancer, a silencer, an insulator element, a boundary element, and / or a locus control region.

[0056] On the other hand, this application provides a method for treating or alleviating a disease or condition associated with abnormal PCSK9 gene expression and / or abnormal PCSK9 gene activity, the method comprising administering to a subject in need an effective amount of the epigenetic editing agent described in this application, the nucleic acid described in this application, the delivery vector described in this application, the composition described in this application, the cells described in this application, and / or the kit described in this application.

[0057] On the other hand, this application provides the use of the epigenetic editing agent, nucleic acid, delivery vector, composition, cell, and / or kit described in this application for preparing a drug for treating or alleviating diseases or symptoms associated with abnormal PCSK9 gene expression and / or abnormal PCSK9 gene activity.

[0058] The epigenetic editing agents and their encoded nucleic acids, vectors, compositions, cells, and other products provided in this application have at least one of the following advantages: significant PCSK9 gene transcriptional regulation efficiency, rich PCSK9 gene modification forms (such as DNA methylation modification, histone acetylation modification, etc.) and regulatory range, relatively flexible and diverse connection methods between various regulatory elements, and the significantly improved recruitment effect of the complex peptide based on the SunTag recruitment strategy. Attached Figure Description

[0059] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0060] Figures 1-6 show the effect of the epigenetic editing agent described in this application on inhibiting the expression of the target gene PCSK9 in mice;

[0061] Figure 7 shows the PCSK9 level in primate blood after administration of V35-223;

[0062] Figure 8 shows the level of LDL-c in primate blood after administration of V35-223. Detailed Implementation

[0063] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0064] Terminology Definition

[0065] In this application, the term "epigenetic modification domain" generally refers to a domain capable of altering gene expression or cell phenotype in a cell population upon administration. It is understood that such alteration refers to one or more function-related modifications to the genome without involving changes to the nucleic acid sequence. Examples of such modifications are DNA methylation and histone modifications, both of which are important for the regulation of gene expression without altering the basic DNA sequence.

[0066] In this application, the term "TALE" refers to a polypeptide containing one or more TALE repeating domains / units. Naturally occurring TALEs, or "wild-type TALEs," are nucleic acid-binding proteins secreted by numerous species of Proteobacteria. TALE polypeptides contain a nucleic acid-binding domain consisting of tandem repeats of highly conserved monomeric polypeptides, which are primarily 33, 34, or 35 amino acids in length and differ primarily from each other at amino acid positions 12 and 13. In a preferred embodiment, the nucleic acid is DNA. As used herein, a polypeptide monomer of TALE is used to refer to a highly conserved repeating polypeptide sequence within the TALE nucleic acid-binding domain, and the terms "repeated variable diresidue" or "RVD" are used to refer to a highly variable amino acid 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35 The subscript indicates the position of the amino acid, and X represents any amino acid. 12 X 13 Indicating RVD. In some TALE polypeptide monomers, the variable amino acid at position 13 is missing or absent, and in such monomers, RVD consists of a single amino acid. In such cases, RVD can alternatively be represented as X*, where X represents X. 12 And (*) indicates X 13 No. The DNA-binding domain contains several repeats of the TALE monomer, and this can be represented as (X 1-11 -(X 12 X 13 )-X 14-33或34或35 ) z In a preferred embodiment, z is at least 5-40. In a further preferred embodiment, z is at least 10-26.

[0067] TALE monomers possess nucleotide binding affinity determined by the amino acid types within their RVDs. For example, polypeptide monomers with RVDs containing NI preferentially bind to adenine (A), polypeptide monomers with RVDs containing NG preferentially bind to thymine (T), polypeptide monomers with RVDs containing HD preferentially bind to cytosine (C), and monomers with RVDs containing NN preferentially bind to both adenine (A) and guanine (G). In other embodiments, monomers with RVDs containing IG preferentially bind to T. Therefore, the number and order of repeats of polypeptide monomers within the nucleotide-binding domain of a TALE determine its nucleic acid target specificity. In a further embodiment of this application, monomers with RVDs containing NS recognize all four base pairs and can bind to A, T, G, or C. The structure and function of TALE 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 herein by reference in its entirety. The repeating domains of TALE are involved in the binding of TALE to its homologous target DNA sequences. These repeating units (or “repetitive sequences”) exhibit at least some sequence homology with other TALE repeating sequences within naturally occurring TALE proteins. See, for example, U.S. Patent Publication No. 20110301073. The TALE binding domains involved in this application can be “engineered” to bind to predetermined nucleotide sequences, for example via engineering (changing one or more amino acids) of the recognition helical 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 include design and selection. The designed DNA-binding protein is a non-naturally occurring protein, and its design and / or composition are primarily derived from rational design criteria. Rational design criteria include the application of substitution rules and computational algorithms used to process information in an information database storing existing TALE designs and binding data. See, for example, U.S. Patents 6,140,081; 6,453,242; and 6,534,261; also see WO 98 / 53058; WO 98 / 53059; WO 98 / 53060; WO02 / 016536 and WO 03 / 016496 and U.S. Publication No. 20110301073.

[0068] In this application, the term "recruitment" generally refers to recruitment between protein molecules, specifically the recruitment of other molecules by a protein to perform a particular biological function. This recruitment relies primarily on the affinity of intermolecular interactions, which is often considered complex and related to the spatial structure of the protein molecule. Interaction mechanisms may include, but are not limited to, non-covalent interactions such as hydrogen bonds, ionic interactions, hydrophobic interactions, and van der Waals forces. For example, some proteins can recruit enzymes to catalyze chemical reactions or recruit other proteins to form complexes. These recruitment processes are crucial for many cellular processes, such as signal transduction, DNA replication, and gene expression.

[0069] In this application, the term "DNA methyltransferase" generally refers to an enzyme that catalyzes the transfer of methyl groups to DNA. Non-limiting examples of DNA methyltransferases include DNMT1, DNMT 3A, DNMT 3B, and DNMT 3L. For example, through DNA methylation, DNA methyltransferases can modify the activity of DNA fragments (e.g., regulate gene expression) without altering the DNA sequence. As described herein, epigenetic editing agents may 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 may be referred to as a "DNA methyltransferase domain". In all respects, the DNA methyltransferase domain comprises a variant or homolog of DNMT 3A having an amino acid sequence that is 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 identical. In all respects, the DNA methyltransferase domain comprises a variant or homolog of DNMT3L having an amino acid sequence that is 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 identical.

[0070] In this application, the term "functionally active fragment" generally refers to a fragment that has 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 may 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 may retain or partially retain the biological activity of a full-length DNA methyltransferase in catalyzing the transfer of methyl groups to DNA.

[0071] In this application, the terms "inhibition," "repression," "silencing," etc., generally refer to a reduction in gene expression and / or activity. For example, the application of the substance of this application may have a negative impact (e.g., reduction) on the activity of a nucleic acid sequence relative to the activity in the absence of a substance (e.g., fusion protein, complex, nucleic acid, vector) (control). For example, inhibition may refer to a reduction in disease or disease symptoms. For example, inhibition includes at least partially, partially, or completely blocking the activation (e.g., transcription) of a nucleic acid sequence, or reducing, preventing, or delaying the activation of a nucleic acid sequence. For example, the inhibitory activity may be 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or lower than that in the control.

[0072] In this 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 causes a decrease in the expression level of a gene product associated with the target nucleic acid sequence. For example, the gene product may be RNA (e.g., mRNA) transcribed from a gene or a polypeptide translated from mRNA transcribed from a gene. Typically, an increase or decrease in mRNA levels leads to an increase or decrease in the level of the polypeptide translated from it. Expression levels can be determined using standard techniques for measuring mRNA or protein. Examples of non-restrictive transcriptional repressors include: mSin3-interacting domain (SID) proteins, 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 (AtHD2A), lysine-specific demethylase 1 (LSD1), and / or Krüppel-related box (KRAB).

[0073] In this application, the term "KRAB" is also referred to as "Krüppel-associated box domain" or "Krüppel-associated box domain," which generally refers to about 45 to about 75 amino acid residues of the transcriptional repressor domain present in transcription factors of human zinc finger proteins. In various aspects, the KRAB domain may include variants or homologs having an amino acid sequence that is 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 identical to the ZIM3 KRAB domain or the KOX1 KRAB domain.

[0074] In this application, the term "split green fluorescent protein" generally refers to a polypeptide that is capable of splitting and immediately forming an active green fluorescent protein upon recombination.

[0075] In this application, the term "GCN4" refers to a transcription factor in Saccharomyces cerevisiae, which is a "master regulator" in the yeast genome that regulates nearly one-tenth of the yeast genome. It is a highly conserved protein, and its mammalian homologue is Activating Transcription Factor-4 (ATF4).

[0076] In this 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, SYN1a, TIP-43, LDP, LIM, LIMK1, LIMK2, MPP2, NOS1, AF6, PTN_4, prIL16, 41.8kD, KIAA0559, RGS12, KIAA0316, DVL1, TIP-40, TIAM1, MINT1, MAGI-1, MAGI-2, MAGI-3, KIAA0303, CBP, MINT3, TIP-2, KIAA0561, and / or TIP-1.

[0077] In this 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. Additionally, although the H and L chains of the Fv fragment are encoded by different genes, they can be linked together directly or via peptides. For example, through recombinant methods, the H and L chains can be linked into a single protein chain (called a single-chain antibody, sAb; Bird et al. 1988 Science 242: 423-426; and Huston et al. 1988 PNAS 85: 5879-5883) using synthetic linkers. This single-chain antibody is also included in the term "antibody," which can be used as a binding determinant in the design and manufacture of multispecific binding molecules, and can be prepared by recombinant techniques or by enzymatic or chemical cleavage of intact antibodies.

[0078] In this application, the term "directly or indirectly linked" generally refers to the opposite of "directly linked" or "indirectly linked." "Directly linked" generally refers to a direct connection. For example, a direct connection can be a situation where linked substances (e.g., amino acid sequence segments) are directly linked without a spacer component (e.g., amino acid residues or derivatives thereof); for example, amino acid sequence segment X is directly linked to another amino acid sequence segment Y via an amide bond formed by the C-terminal amino acid of amino acid sequence segment X and the N-terminal amino acid of amino acid sequence segment Y. "Indirectly linked" generally refers to a situation where linked substances (e.g., amino acid sequence segments) are indirectly linked with a spacer component (e.g., amino acid residues or derivatives thereof). For example, as used herein, the spacer component is a "linker" or "linker sequence," which generally refers to a linker comprising two or more parts. In various embodiments, the linker is connected at the N-terminus and C-terminus to the amino acid sequence of the remaining portion of the compound (e.g., the first or second fusion of the fusion peptide or complex peptide provided herein). For example, as used herein, the terms "XTEN," "XTEN linker," or "XTEN peptide" refer to a recombinant peptide lacking hydrophobic amino acid residues (e.g., an unstructured recombinant peptide). In some embodiments, the XTEN adapter sequence comprises one or more fragment sequences truncated from the amino acid sequence (full-length XTEN polypeptide) shown in SEQ ID NO:98, wherein the one or more fragment sequences comprise at least 16 consecutive amino acids. In this application, XTEN 80 refers to a full-length 80-amino acid XTEN adapter and fusion proteins derived from a full-length 80-amino acid XTEN adapter. In some embodiments, XTEN 80 may be the sequence shown in SEQ ID NO 83. Adapters derived from XTEN 80 also fall within the scope of XTEN 80. For example, as shown in the table below, linker 15-fused XTEN80 linker 1, where the last 46 amino acids are derived from XTEN80. Another example is linker 22-fused XTEN80 linker 2 or linker 12-XTEN 80 linker 2, which are derived by inserting the XTEN80 sequence into XTEN16.

[0079] In this application, XTEN 16 refers to the full-length 16-amino acid XTEN linker and fusion proteins derived from the full-length 16-amino acid XTEN linker. In some embodiments, XTEN 16 can be the sequence shown in SEQ ID NO 90. Linkers derived from XTEN 16 also fall within the scope of XTEN 16. For example, as shown in the table below, linker11-XTEN 16linker 1 is a full-length 16-amino acid XTEN linker. linker10-XTEN 16linker 2 is based on linker11-XTEN 16linker 1 with the addition of DG at the N segment, and linker9-XTEN 16linker 3 is based on linker11-XTEN 16linker 1 with the addition of G at the N segment. Therefore, linker10-XTEN 16linker 2 and linker9-XTEN 16linker 3 should be considered as a type of XTEN 16.

[0080] The development and use of XTEN can be found, for example, in Schellenberger et al., *Nature Biotechnology*, 27, 1186-1190 (2009), which is incorporated herein by reference in its entirety. For instance, in some embodiments of this application, the adapter sequence comprises a GS linker peptide, which comprises the sequence: (GS) a (GGS) b (GGGS) c (GGGGS) d In this context, 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 this application is selected from the amino acid sequences shown in any one of SEQ ID NO:80-97, 2848-2856, and 2869-2871 (SEQ ID NO:81 is GSG).

[0081] The connectors used in this application are shown in Table 1 below.

[0082] Table 1 - Connector List

[0083] In this application, the terms "nuclear localization sequence" or "nuclear localization signal" or "NLS" generally refer to a peptide that directs a protein to the cell nucleus. For example, an NLS comprises five basic, positively charged amino acids. For example, an NLS can be located at any position on the peptide chain. For example, the NLS used in this application is selected from the amino acid sequences shown in any one of SEQ ID NO:2857-2859.

[0084] NLS can include sv40 NLS, c-myc NLS, and BPSV40 NLS. Unless otherwise specified, NLS can be any one of the three types of NLS mentioned above.

[0085] In this application, the term "marker" refers to a peptide that can be introduced into an expression vector, which can be used to allow 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 comprise radiolabeled amino acids or polypeptides linked to a biotinylated moiety detectable by a labeled avidin (e.g., streptomycin containing a fluorescent label or enzymatic activity detectable 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, keratinase, AGT, GFP, and other widely used markers, such as those used in protein expression and purification systems, are also included. Further non-limiting examples of peptides include, but are not limited to, the following: histidine labels, radioisotopes or radionuclides (e.g., 3H, 14C, 35S, 90Y, 99Tc, 111In, 125I, 177Lu, 166Ho, or 153Sm); fluorescent labels (e.g., FITC, rhodamine, lanthanides); enzyme labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent labels; biotin groups; pendant peptide antigenic determinants recognized by a second reporter (e.g., leucine zipper pairs, binding sites for secondary antibodies, metal-binding domains, antigenic determinant markers); and magnetic agents, such as gadolinium chelates.

[0086] In this application, the specific protein (e.g., KRAB, TALE, Dnmt3A, Dnmt3L) may include any native form of the protein or a variant or homolog that maintains the activity of the protein (e.g., having at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity compared to the native protein). In each aspect, the variant or homolog has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., 50, 100, 150, or 200 consecutive amino acid segments) compared to the native form.

[0087] In this application, the term "nucleotide modification" may refer to the synthesis or modification of the nucleic acid described in this invention by methods well-established in the art, such as those described in "Current protocols in nucleic acid chemistry" Beaucage, SL et al., (Edrs.), John Wiley & Sons, Inc., New York, NY, USA (which are incorporated herein by reference). Such modifications may include, but are not limited to: terminal modifications, such as 5'-terminal modifications (e.g., phosphorylation, conjugation, inverted linkage) or 3'-terminal modifications (e.g., conjugation, DNA nucleotides, inverted linkage, etc.); base modifications, such as substitution with a stable base, a destabilized base, or a base paired with an expanded library of bases, base removal (base-free nucleotide), or conjugated bases; sugar modifications (e.g., sugar modification at the 2' or 4' position) or sugar substitution; or backbone modifications, including modification or substitution of phosphodiester bonds.

[0088] In this application, the term "nucleic acid" is used interchangeably with "polynucleotide," "nucleotide," "nucleotide sequence," and "oligonucleotide," and generally refers to a nucleotide (e.g., deoxyribonucleotide or ribonucleotide) and polymers thereof in single-stranded, double-stranded, or multi-stranded form, or complementary forms thereof. For example, a nucleotide can be a ribonucleotide, a deoxyribonucleotide, or a modified version thereof. For example, a nucleotide can be a single-stranded and double-stranded DNA, a single-stranded and double-stranded RNA, or a hybrid molecule having a mixture of single-stranded and double-stranded DNA and RNA. For example, a nucleotide can be, 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 microcircular DNA, and any fragment thereof. The term also covers nucleic acids containing known nucleotide analogs or modified backbone residues or bonds, said nucleic acids being synthetic, naturally occurring, or non-natural.

[0089] In this application, the terms "sequence encoding..." or "nucleic acid encoding..." generally refer to a nucleic acid (RNA or DNA molecule) containing a nucleotide sequence encoding a protein. The coding sequence may also include start and stop signals operatively linked to regulatory elements comprising promoters and polyadenylation signals capable of directing expression in the cells of an individual or mammal to which the nucleic acid has been administered. Codon optimization of the coding sequence is possible.

[0090] In this application, the term "delivery carrier" generally refers to a transfer medium capable of delivering a reagent (e.g., a nucleic acid molecule) to target cells. A delivery carrier can deliver a reagent to a specific cell subclass. For example, the delivery carrier can target certain cell types by means of inherent characteristics of the delivery carrier or by a portion coupled to the carrier, a portion contained therein (or a portion bound to the carrier such that the portion and the delivery carrier remain together, thereby making the portion sufficient to target the delivery carrier). Delivery carriers can also improve the in vivo half-life and / or bioavailability of the reagent to be delivered. Delivery carriers may include viral vectors, virus-like particles, polycationic carriers, peptide carriers, liposomes, and / or hybridization carriers. For example, if the target cells are hepatocytes, the properties of the delivery carrier (e.g., size, charge, and / or pH) can effectively deliver the delivery carrier and / or the molecules encapsulated therein to the target cells, reduce immune clearance, and / or promote residence in the target cells.

[0091] In this application, the term "liposome" generally refers to a vesicle with an internal space that is isolated from an external medium by one or more bilayer membranes. In some embodiments, the bilayer membrane can be formed from amphiphilic molecules, such as synthetic or naturally derived lipids comprising spatially isolated hydrophilic and hydrophobic domains; in other embodiments, the bilayer membrane can be formed from amphiphilic polymers and surfactants. In some embodiments, the liposome is a spherical vesicle structure consisting of a single or multiple lipid bilayer surrounding an internal aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. In some embodiments, the liposome is biocompatible, non-toxic, capable of delivering hydrophilic and lipophilic drug molecules, protecting their carriers from degradation by plasma enzymes, and transporting their load across biological membranes and the blood-brain barrier (BBB). Liposomes can be made from several different types of lipids, such as phospholipids. Liposomes may contain natural phospholipids and lipids such as 1,2-distearate-sn-glycerol-3-phosphatidylcholine (DSPC), sphingomyelin, lecithin, monosialotetrahexosylganglioside, or any combination thereof. Several other additives may be added to liposomes to modify their structure and properties. For example, liposomes may also contain cholesterol, sphingomyelin, and / or 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), for example, to increase stability and / or prevent leakage of the internal carriers of the liposomes.

[0092] The term "lipid nanoparticle (LNP)" generally refers to a particle containing multiple (i.e., more than one) lipid molecules physically bound together by intermolecular forces (e.g., covalent or non-covalent). LNPs can be, for example, microspheres (including monolayer and multilayer vesicles, such as liposomes), dispersed phases in emulsions, micelles, or internal phases in suspensions. LNPs can encapsulate nucleic acids within cationic lipid particles (e.g., liposomes) and can be delivered to cells relatively easily. In some instances, lipid nanoparticles are free of any viral components, which helps 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 sizes. The LNPs of this application can be readily prepared by various methods known in the art, such as by mixing an organic phase with an aqueous phase. Mixing of the two phases can be achieved using microfluidic devices and impinging flow reactors. The more thoroughly the organic and aqueous phases are mixed, the better the encapsulation efficiency and particle size distribution of the obtained LNPs. Preferably, the particle size of the LNPs can be adjusted by varying the mixing rate of the organic and aqueous phases. The faster the mixing rate, the smaller the particle size of the prepared LNPs. Encapsulation efficiency can be optimized by adjusting the N / P (ionizable lipid / nucleic acid) ratio of the LNP system. In some instances, LNPs can be used to deliver DNA molecules and / or RNA molecules (such as mRNA). In certain cases, LNPs can be used to deliver RNP complexes.

[0093] In this application, the term "recombinant vector" generally refers to a nucleic acid molecule capable of transporting itself and another nucleic acid linked to it. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which an additional DNA segment can be linked. Alternatively, the vector can be linear. Another type of vector is a viral vector, in which an additional DNA segment can be linked to the viral genome. Certain vectors are capable of autonomous replication within the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication and augmented mammalian vectors). Other vectors (e.g., non-augmented mammalian vectors) can integrate into the host cell's genome after introduction and thus replicate along with the host genome.

[0094] In this application, the term "regulatory element" refers to a genetic element that controls the expression of a nucleic acid sequence. Examples include splicing signals, promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, etc., which together enable the replication, transcription, and translation of coding sequences in recipient cells. Not all of these control sequences are required. Transcriptional control signals in eukaryotes typically include "promoter" and "enhancer" elements. Promoters and enhancers consist of short arrays of DNA sequences; promoters are regulatory elements that promote the initiation of transcription in operatively linked coding regions, while enhancers are regulatory elements that increase the rate of genetic transcription by increasing the activity of the nearest promoter on the same DNA molecule. These sequences specifically interact with cellular proteins involved in transcription (Maniatis et al., Science 236:1237 (1987), incorporated herein by reference in its entirety). Promoter and enhancer elements have been isolated from a wide range of eukaryotic sources, including genes in yeast, insect and mammalian cells, and viruses (similar control sequences, i.e., promoters, have also been found in prokaryotes). The choice of specific promoters and enhancers depends on the recipient cell type. Some eukaryotic promoters and enhancers have a broad host range, while others function within a limited subgroup of cell types (for reviews, see, e.g., Voss et al., Trends Biochem. Sci., 11:287 (1986); and Maniatis et al. (ibid.), incorporated herein by reference in their entirety). For example, the SV40 early gene enhancer is highly active in a wide range of cell types from many mammalian species and has been used to express proteins in a variety of mammalian cells (Dijkema et al., EMBO J. 4:761 (1985), incorporated herein by reference in its entirety). Promoter and enhancer elements derived from the human elongation factor 1-α 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)), long terminal repeat sequences of Rous sarcoma virus (Gorman et al., Proc. Natl. Acad. Sci. USA 79:6777 (1982)), and human cytomegalovirus (Boshart et al., Cell 41:521 (1985)) can also be used to express proteins in different mammalian cell types, and the aforementioned references are incorporated herein by reference in their entirety. Promoters and enhancers can exist naturally, alone or together. For example, long terminal repeat sequences of retroviruses contain promoter and enhancer elements.Generally, the function of promoters and enhancers is independent of the gene being transcribed or translated. Therefore, the enhancers and promoters used can be “endogenous,” “exogenous,” or “heterogeneous” relative to the gene to which they are operatively linked. An “endogenous” enhancer / promoter is one that is naturally linked to a given gene in the genome. An “exogenous” or “heterogeneous” enhancer or promoter is one that is juxtaposed with a gene through genetic manipulation (i.e., molecular biology techniques), such that transcription of that gene is directed by the linked enhancer / promoter. The presence of a “splicing signal” on the expression vector typically leads to high levels of expression of the recombinant transcript. In some embodiments, the “splicing signal” mediates the removal of introns from the primary RNA transcript, consisting of a splicing donor and recipient site (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York (1989), pp. 16.7–16.8, incorporated herein by reference in its entirety). The commonly used splicing donor and acceptor sites are splice sites of 16S RNA derived from SV40. In some implementations, the "transcription termination signal" is typically located downstream of the polyadenylation signal and is several hundred nucleotides in length. For example, the term "poly A signal" or "poly A sequence" refers to the DNA sequence that directs the termination and polyadenylation of nascent RNA transcripts. Efficient polyadenylation of recombinant transcripts is often necessary because transcripts lacking a poly A signal are unstable and rapidly degraded. The poly A signal used in expression vectors can be "heterologous" or "endogenous." An endogenous poly A signal is a signal naturally present at the 3' end of the coding region of a given gene in the genome. A heterologous poly A signal is a signal isolated from one gene and operatively 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 / BclI-restricting fragment and directs termination and polyadenylation (Sambrook et al., ibid., 16.6–16.7, incorporated herein by reference in its entirety).

[0095] In this application, the term "subject" generally refers to an animal, typically a mammal such as a human, non-human primates (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), livestock (dogs and cats), farm animals (poultry such as chickens and ducks, horses, cattle, goats, sheep, pigs), and laboratory animals (mice, rats, rabbits, guinea pigs). Human subjects include fetuses, newborns, infants, adolescents, and adult subjects. Subjects include animal disease models, such as mice and other animal models of blood clotting disorders (such as HemA), and other animal models known to those skilled in the art.

[0096] In this application, the term "comprising" generally means including the explicitly specified features, but does not exclude other elements.

[0097] In this application, the term “selected from” generally refers to the selection of objects and all combinations thereof. For example, “selected from (:) A, B and C” means all combinations of A, B and C, such as A, B, C, A+B, A+C, B+C or A+B+C.

[0098] In this application, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range 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 a specified value.

[0099] Invention Details

[0100] On one hand, this application provides an epigenetic editing agent comprising a transcription activator-like effector (TALE) domain, at least one epigenetic modification domain, and at least one transcriptional regulatory domain, wherein: 1) the TALE domain, the at least one epigenetic modification domain, and the at least one transcriptional regulatory domain are directly or indirectly connected; or 2) the TALE domain, the at least one epigenetic modification domain, and at least one recruitment domain A' are directly or indirectly connected to form a first fusion, and the at least one transcriptional regulatory domain is directly or indirectly connected to at least one recruitment domain A' to form a second fusion; or 3) The TALE domain, the at least one transcriptional regulatory domain, and the at least one recruitment domain A are directly or indirectly connected to form a first fusion, and the at least one epigenetic modification domain is directly or indirectly connected to the at least one recruitment domain A' to form a second fusion; and: 2) and 3) the recruitment domain A and the recruitment domain A' are able to interact so that a fusion of one of the first fusion and the second fusion, or a portion thereof, can be recruited to the vicinity of the other fusion; the TALE domain is able to specifically bind to target nucleotide sequences on the PCSK9 gene and / or the regulatory elements of the PCSK9 gene.

[0101] On the other hand, this application provides nucleic acids encoding the epigenetic editing agents described in this application. For example, the nucleic acid comprises DNA and / or mRNA. For example, the nucleic acid can be used to treat or alleviate diseases or symptoms associated with abnormal target gene expression and / or abnormal target gene activity. In some embodiments, the nucleic acid is mRNA; one or more modification techniques can be used to produce more stable mRNA. Known mRNA modification techniques can be broadly classified into three categories: synthesizing mRNA by replacing natural ribonucleic acid with artificially synthesized non-natural ribonucleic acid; adding 5' caps, 3' poly(A) tails, and UTR (untranslated region) sequences; and employing special novel formulation techniques to effectively protect mRNA. Among these, preferred mRNA modification techniques can be used to synthesize mRNA by replacing natural ribonucleic acid with artificially synthesized non-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 may be selected from: pseudouridine, N1-methylpseuuridine, N1-ethylpseuuridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deazo-pseuuridine, 2-thio-1-methylpseuuridine, 2-thio-5-aza-uridine, 2-thio-dihydropseuuridine, 2-thio-dihydrouridine, 2-thio-pseuuridine, 4-methoxy-2-thio-pseuuridine, 4-methoxy-pseuuridine, 4-thio-1-methylpseuuridine, 4-thio-pseuuridine, 5-aza-uridine, dihydropseuuridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine. For example, the nucleic acid is a recombinant vector containing a nucleic acid encoding the complex described in this application. For example, a recombinant vector may refer to a nucleic acid molecule capable of transporting another nucleic acid linked thereto. Recombinant vectors may include single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules containing one or more free ends, or without free ends (e.g., circular); nucleic acid molecules containing DNA, RNA, or both; and other types of polynucleotides known in the art. For example, viral vectors may be used. Viral vectors may contain virus-derived DNA or RNA sequences for packaging into viruses (e.g., retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses, and adeno-associated virus (AAV)). Viruses and viral vectors may be used for in vitro, ex vivo, and / or in vivo delivery.

[0102] On the other hand, this application provides a delivery vector comprising the epigenetic editing agent and / or nucleic acid described in this application, and optionally liposomes and / or lipid nanoparticles. For example, the delivery vector can be introduced into cells by physical delivery methods. 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 delivered to cells relatively easily. In some examples, the lipid nanoparticles do not contain any viral components, which helps to minimize safety and immunogenicity issues. Lipid particles can be used for in vitro, ex vivo, and in vivo delivery. The components of LNPs may include cationic lipids, ionizable lipids, polyethylene glycol-modified lipids and / or supporting lipids, and optionally a cholesterol component.

[0103] On the other hand, this application provides a composition comprising the epigenetic editing agent, the nucleic acid, and / or the delivery vector described in this application. For example, the epigenetic editing agent, the nucleic acid (or recombinant vector) encoding the epigenetic editing agent, and the delivery vector in the composition may be contained simultaneously in one composition or separately in different compositions. For example, when using the epigenetic editing agent, the nucleic acid (or recombinant vector) encoding the epigenetic editing agent, and / or the delivery vector in the composition, they may be used simultaneously or separately.

[0104] On the other hand, this application provides a cell comprising the epigenetic editing agent, the nucleic acid, the delivery vector, and / or the composition described in this application.

[0105] On the other hand, this application provides a kit comprising the epigenetic editing agent, nucleic acid, delivery vector, composition, and / or cells described in this application. For example, the kit further comprises at least one container for holding the aforementioned components. For example, the kit comprises more than one of the aforementioned components and further comprises a second, third, and / or other container besides the container, in which the one or more of the aforementioned components can be separately placed. For example, the kit can hold various combinations of the aforementioned components in the containers. For example, the kit further comprises buffer reagents, mixing devices, measuring devices, sorting devices, and / or labeling devices. For example, the kit further comprises packaging for accommodating various containers. For example, the kit further comprises instructions for using the kit components. For example, the instructions may be in physical paper form and / or machine-readable electronic form.

[0106] On the other hand, this application provides a method for regulating the expression of the PCSK9 gene product, the method comprising administering the epigenetic editing agent described in this application, the nucleic acid described in this application, the delivery vector described in this application, the composition described in this application, the cells described in this application, and / or the kit described in this application. For example, the method for inhibiting PCSK9 gene expression is to introduce the epigenetic editing agent, the nucleic acid, the delivery vector, the composition, the cells, and / or the kit into cells containing the PCSK9 gene. For example, the introduction into cells may be done using non-viral or virus-based transfection methods. For example, the non-viral transfection method includes any suitable method for introducing cells without using viral DNA or viral particles as a delivery system. Examples of non-limiting non-viral transfection methods include nanoparticle encapsulation of nucleic acids encoding the epigenetic editing agent (e.g., lipid nanoparticles, gold nanoparticles, etc.), calcium phosphate transfection, liposome transfection, nuclear transfection, acoustic perforation, transfection by heat shock, magnetic transfection, and electroporation. For example, virus-based transfection methods include any viral vector suitable for the methods described in this application, with non-limiting examples including but not limited to retroviruses, adenoviruses, lentiviruses, and / or adeno-associated virus vectors. For example, the method for inhibiting PCSK9 gene expression further includes introducing the epigenetic editing agent, the nucleic acid, the delivery vector, the composition, the cells, and / or the kit from the external environment into the cells. As another example, the method for inhibiting PCSK9 gene expression includes contacting the epigenetic editing agent, the nucleic acid, the delivery vector, and / or the composition with the PCSK9 gene and / or the transcriptional regulatory elements of the PCSK9 gene. For example, the epigenetic editing agent is a complex peptide comprising a first fusion and a second fusion, wherein the contact refers to the TALE domain contained in the epigenetic editing agent specifically recognizing and hybridizing with a specific region in the PCSK9 gene, while the first and second fusions are recruited to the vicinity of the PCSK9 gene or its transcriptional regulatory region through direct or indirect interactions of their recruitment domains A and A', thereby regulating the expression of its nucleic acid.

[0107] On the other hand, this application provides a method for treating or alleviating a disease or symptom associated with abnormal PCSK9 gene expression and / or abnormal PCSK9 gene activity, the method comprising administering an effective amount of the epigenetic editing agent, the nucleic acid, the delivery vector, the composition, the cells, and / or the kit described in this application to a subject in need. For example, the treatment method comprises mixing the epigenetic editing agent, the nucleic acid, the delivery vector, the composition, the cells, and / or the kit with a therapeutic agent and delivering it systemically to a subject in need, exposing them extensively to a large 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 treatment method comprises mixing the epigenetic editing agent, the nucleic acid, the delivery vector, the composition, the cells, and / or the kit with a therapeutic agent and delivering it locally to a subject in need, allowing it to directly reach a target site within the organism, which can be achieved, for example, by direct injection into a disease site (e.g., a tumor or site of inflammation) or a target organ (e.g., the liver, heart, pancreas, kidney, etc.). For example, the local delivery includes local application or injection techniques, including but not limited to intramuscular, subcutaneous, or intradermal injection. For example, the local delivery does not exclude systemic pharmacological effects.

[0108] On the other hand, this application provides the use of the epigenetic editing agent, nucleic acid, delivery vector, composition, cell, and / or kit described in this application for preparing a drug for treating or alleviating diseases or symptoms associated with abnormal PCSK9 gene expression and / or abnormal PCSK9 gene activity.

[0109] On the other hand, this application provides the epigenetic editing agent, nucleic acid, delivery vector, composition, cell, or kit described in this application for the treatment or relief of diseases or symptoms associated with abnormal PCSK9 gene expression and / or abnormal PCSK9 gene activity.

[0110] Fusion peptide

[0111] In some embodiments, the epigenetic modification domain and the transcriptional regulatory domain are both located at the N-terminus or C-terminus of the TALE domain. For example, the fusion peptide sequentially connects the epigenetic modification domain, the transcriptional regulatory domain, and the TALE domain from the N-terminus to the C-terminus. (This is repeated three times in the original text.)

[0112] In some embodiments, the epigenetic modification domain and the transcriptional regulatory domain are located at the N-terminus and C-terminus of the TALE domain, respectively. For example, the fusion peptide is sequentially linked from the N-terminus to the C-terminus with the epigenetic modification domain, the TALE domain, and the transcriptional regulatory domain.

[0113] In some specific embodiments, the fusion peptide is sequentially linked from the N-terminus to the C-terminus to: 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.

[0114] In some embodiments described above, the epigenetic modification domain may comprise DNMT3A and DNMT3L, with the C-terminus of DNMT3A connected to the N-terminus of DNMT3L, or vice versa. Similarly, in these embodiments, the transcriptional regulatory domain may comprise a transcriptional repressor domain. For example, the transcriptional regulatory domain may comprise a zinc finger protein-based transcription factor or a functionally active fragment thereof. Specifically, the zinc finger protein-based transcription factor may comprise KRAB; for example, in some embodiments of this application, the zinc finger protein-based transcription factor may be selected from ZIM3 KRAB and KOX1 KRAB.

[0115] The following structures may include the TALE-DNMT3A-DNMT3L-ZIM3 KRAB, TALE-ZIM3KRAB-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, or DNMT3A-DNMT3L-TALE-ZIM3 KRAB structures as described above.

[0116] Furthermore, an exemplary fusion structure is described in the following scheme: V1 scheme: NLS-TALE HPT15-2×NLS-XTEN80-DNMT3A-DNMT3L-XTEN16-ZIM3, which may include the following components from the N end to the C end, and the following components may be directly or indirectly connected to each other:

[0117] NLS (e.g., SEQ ID NO:2858), TALE HPT15 (e.g., SEQ ID NO:883), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857), DNMT3A (e.g., SEQ ID NO:2860), DNMT3L-CD (e.g., SEQ ID NO:41), Zim3 Krab (e.g., SEQ ID NO:2861).

[0118] Furthermore, the V1 solution may further include an XTEN connector, for example, the N-terminal to the C-terminal may include the following components, which may be directly or indirectly connected to each other:

[0119] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0120] TALE HPT15, for example, the sequence shown in SEQ ID NO:883;

[0121] One or more (e.g., two) sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0122] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0123] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0124] DNMT3L-CD, for example, a sequence as shown in SEQ ID NO:41;

[0125] Linker10-XTEN 16 linker 2, for example, the sequence shown in SEQ ID NO:89;

[0126] Zim3 Krab, for example, the sequence shown in SEQ ID NO:2861.

[0127] V2 Solution: NLS-TALE HPT15-2×NLS-XTEN80-ZIM3-XTEN80-hDNMT3L-DNMT3A, which can include the following components from the N-terminal to the C-terminal, and these components can be directly or indirectly connected to each other:

[0128] NLS (e.g., SEQ ID NO:2858), TALE HPT15 (e.g., SEQ ID NO:883), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857), Zim3 Krab (e.g., SEQ ID NO:2861), hDNMT3L (e.g., SEQ ID NO:42), DNMT3A (e.g., SEQ ID NO:2860).

[0129] Furthermore, the V2 solution may further include XTEN connectors, for example, the N-terminal to the C-terminal may include the following components, which may be directly or indirectly connected to each other:

[0130] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0131] TALE HPT15, for example, the sequence shown in SEQ ID NO:883;

[0132] One or more (e.g., two) sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0133] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0134] Zim3 Krab, for example, the sequence shown in SEQ ID NO:2861;

[0135] linker12-XTEN 80linker 2, for example, the sequence shown in SEQ ID NO:91;

[0136] Human DNMT3L, for example, the sequence shown in SEQ ID NO:42;

[0137] Linker3-flexible peptide linker, for example, the sequence shown in SEQ ID NO:82;

[0138] DNMT3A, for example, the sequence shown in SEQ ID NO:2860.

[0139] V3 solution: NLS-TALE HPT15-2×NLS-XTEN80-ZIM3-XTEN80-DNMT3A-hDNMT3L, which can include the following components from the N-terminal to the C-terminal, and these components can be directly or indirectly connected to each other:

[0140] NLS (e.g., SEQ ID NO:2857), HPT15 (e.g., SEQ ID NO:883), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857), Zim3 Krab (e.g., SEQ ID NO:2861), DNMT3A (e.g., SEQ ID NO:2860), hDNMT3L (e.g., SEQ ID NO:42).

[0141] Furthermore, the V3 solution may further include XTEN connectors, for example, the N-terminal to the C-terminal may include the following components, which may be directly or indirectly connected to each other:

[0142] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0143] HPT15, for example, the sequence shown in SEQ ID NO:883;

[0144] One or more (e.g., two) sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0145] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0146] Zim3 Krab, for example, the sequence shown in SEQ ID NO:2861;

[0147] Linker 22-fused XTEN80 linker 2, for example, the sequence shown in SEQ ID NO:2855;

[0148] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0149] hDNMT3L, for example, the sequence shown in SEQ ID NO:42.

[0150] V4 solution: ZIM3-BFP-DNMT3A-hDNMT3L-XTEN80-NLS-TALE HPT15-2×NLS, which can include the following components from the N-terminal to the C-terminal, and these components can be directly or indirectly connected to each other:

[0151] Zim3 Krab (e.g., SEQ ID NO:2861), BFP (e.g., SEQ ID NO:2866), DNMT3A (e.g., SEQ ID NO:2860), hDNMT3L (e.g., SEQ ID NO:42), NLS (e.g., SEQ ID NO:2857), HPT15 (e.g., SEQ ID NO:883), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857).

[0152] Furthermore, the V4 solution may further include XTEN connectors, for example, the N-terminal to the C-terminal may include the following components, which may be directly or indirectly connected to each other:

[0153] Zim3 Krab, for example, the sequence shown in SEQ ID NO:2861;

[0154] linker11-XTEN 16linker 1, for example, the sequence shown in SEQ ID NO:90;

[0155] BFP (blue fluorescent protein), for example, the sequence shown in SEQ ID NO:2866;

[0156] linker11-XTEN 16linker 1, for example, the sequence shown in SEQ ID NO:90;

[0157] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0158] hDNMT3L, for example, a sequence as shown in SEQ ID NO:42;

[0159] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0160] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0161] HPT15, for example, the sequence shown in SEQ ID NO:883;

[0162] One or more (e.g., two) sv40 NLS, such as the sequence shown in SEQ ID NO:2857.

[0163] V5 Solution: NLS-DNMT3A-hDNMT3L-XTEN80-ZIM3-XTEN80-NLS-TALE HPT15-2×NLS, which can include the following components from the N-terminal to the C-terminal, and these components can be directly or indirectly connected to each other:

[0164] NLS (e.g., SEQ ID NO: 2858), DNMT3A (e.g., SEQ ID NO: 2860), hDNMT3L (e.g., SEQ ID NO: 42), Zim3 Krab (e.g., SEQ ID NO: 2861), NLS (e.g., SEQ ID NO: 2857), TALE HPT15 (e.g., SEQ ID NO: 883), NLS (e.g., SEQ ID NO: 2857), NLS (e.g., SEQ ID NO: 2857).

[0165] Furthermore, the V5 solution may further include XTEN connectors, for example, the N-terminal to the C-terminal may include the following components, which may be directly or indirectly connected to each other:

[0166] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0167] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0168] hDNMT3L, for example, a sequence as shown in SEQ ID NO:42;

[0169] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0170] Zim3 Krab, for example, the sequence shown in SEQ ID NO:2861;

[0171] Linker 15-fused XTEN80 linker 1, for example, the sequence shown in SEQ ID NO:2848;

[0172] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0173] TALE HPT15, for example, the sequence shown in SEQ ID NO:883;

[0174] Linker5-GS linker, for example, the sequence shown in SEQ ID NO:84;

[0175] One or more (e.g., two) sv40 NLS, such as the sequence shown in SEQ ID NO:2857.

[0176] V6 Solution: NLS-DNMT3A-hDNMT3L-XTEN80-ZNF324-XTEN80-NLS-TALE HPT15-2×NLS, which can include the following components from the N-terminal to the C-terminal, and these components can be directly or indirectly connected to each other:

[0177] NLS (e.g., SEQ ID NO:2858), DNMT3A (e.g., SEQ ID NO:2860), hDNMT3L (e.g., SEQ ID NO:42), Znf324 (e.g., SEQ ID NO:51), NLS (e.g., SEQ ID NO:2857), HPT15 (e.g., SEQ ID NO:883), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857).

[0178] Furthermore, the V6 solution may further include XTEN connectors, for example, the N-terminal to the C-terminal may include the following components, which may be directly or indirectly connected to each other:

[0179] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0180] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0181] hDNMT3L, for example, a sequence as shown in SEQ ID NO:42;

[0182] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0183] Znf324, for example, the sequence shown in SEQ ID NO:51;

[0184] Linker 15-fused XTEN linker 1, for example, the sequence shown in SEQ ID NO:2848;

[0185] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0186] HPT15, for example, the sequence shown in SEQ ID NO:883;

[0187] One or more (e.g., two) sv40 NLS, such as the sequence shown in SEQ ID NO:2857.

[0188] V7 Solution: NLS-DNMT3A-hDNMT3L-XTEN80-ZNF419-GS-XTEN80-NLS-TALE HPT15-2×NLS, which can include the following components from the N-terminal to the C-terminal, and these components can be directly or indirectly connected to each other:

[0189] NLS (e.g., SEQ ID NO:2858), DNMT3A (e.g., SEQ ID NO:2860), hDNMT3L (e.g., SEQ ID NO:42), Znf419 (e.g., SEQ ID NO:56), NLS (e.g., SEQ ID NO:2857), HPT15 (e.g., SEQ ID NO:883), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857).

[0190] Furthermore, the V7 solution may further include XTEN connectors, for example, the N-terminal to the C-terminal may include the following components, which may be directly or indirectly connected to each other:

[0191] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0192] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0193] hDNMT3L, for example, a sequence as shown in SEQ ID NO:42;

[0194] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0195] Znf419, for example, the sequence shown in SEQ ID NO:56;

[0196] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0197] HPT15, for example, the sequence shown in SEQ ID NO:883;

[0198] One or more (e.g., two) sv40 NLS, such as the sequence shown in SEQ ID NO:2857.

[0199] V8 solution: NLS-DNMT3A-DNMT3L-XTEN80-NLS-TALE HPT15-2×NLS-XTEN16-EZH2, which can include the following components from the N-terminal to the C-terminal, and these components can be directly or indirectly connected to each other:

[0200] NLS (e.g., SEQ ID NO:2858), DNMT3A (e.g., SEQ ID NO:2860), DNMT3L-CD (e.g., SEQ ID NO:41), NLS (e.g., SEQ ID NO:2857), HPT15 (e.g., SEQ ID NO:883), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857), EZH2 (e.g., SEQ ID NO:63).

[0201] Furthermore, the V8 solution may further include XTEN connectors, for example, the N-terminal to the C-terminal may include the following components, which may be directly or indirectly connected to each other:

[0202] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0203] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0204] DNMT3L-CD, for example, a sequence as shown in SEQ ID NO:41;

[0205] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0206] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0207] HPT15, for example, the sequence shown in SEQ ID NO:883;

[0208] One or more (e.g., two) sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0209] linker11-XTEN 16linker 1, for example, the sequence shown in SEQ ID NO:90;

[0210] EZH2, for example, the sequence shown in SEQ ID NO:63.

[0211] The V9 solution, NLS-DNMT3A-DNMT3L-XTEN80-NLS-TALE HPT15-2×NLS-XTEN16-HDAC3, can include the following components from its N-terminal to its C-terminal. These components can be directly or indirectly connected to each other:

[0212] NLS (e.g., SEQ ID NO:2858), DNMT3A (e.g., SEQ ID NO:2860), DNMT3L-CD (e.g., SEQ ID NO:41), NLS (e.g., SEQ ID NO:2857), HPT15 (e.g., SEQ ID NO:883), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857), HA tag (e.g., SEQ ID NO:2862), HDAC3 (e.g., SEQ ID NO:69).

[0213] Furthermore, the V9 solution may further include an XTEN connector, for example, the N-terminal to the C-terminal may include the following components, which may be directly or indirectly connected to each other:

[0214] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0215] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0216] DNMT3L-CD, for example, a sequence as shown in SEQ ID NO:41;

[0217] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0218] HPT15, for example, the sequence shown in SEQ ID NO:883;

[0219] One or more (e.g., two) sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0220] linker11-XTEN 16linker 1, for example, the sequence shown in SEQ ID NO:90;

[0221] HDAC3, for example, the sequence shown in SEQ ID NO:69.

[0222] V10 solution: NLS-DNMT3A-DNMT3L-XTEN80-NLS-TALE HPT15-2×NLS-XTEN16-HP1a, which can include the following components from the N-terminal to the C-terminal, and the following components can be directly or indirectly connected to each other:

[0223] NLS (e.g., SEQ ID NO:2858), DNMT3A (e.g., SEQ ID NO:2860), DNMT3L-CD (e.g., SEQ ID NO:41), NLS (e.g., SEQ ID NO:2857), HPT15 (e.g., SEQ ID NO:883), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857), HP1a (e.g., SEQ ID NO:67).

[0224] Furthermore, the V10 solution may further include an XTEN connector, for example, the N-terminal to the C-terminal may include the following components, which may be directly or indirectly connected to each other:

[0225] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0226] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0227] DNMT3L-CD, for example, a sequence as shown in SEQ ID NO:41;

[0228] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0229] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0230] HPT15, for example, the sequence shown in SEQ ID NO:883;

[0231] One or more (e.g., two) sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0232] linker11-XTEN 16linker 1, for example, the sequence shown in SEQ ID NO:90;

[0233] HP1a, for example, the sequence shown in SEQ ID NO:67.

[0234] The V25 solution, NLS-DNMT3A-DNMT3L-XTEN80-NLS-TALE HPT15-2×NLS-XTEN16-Krab, can include the following components from the N-terminal to the C-terminal, and these components can be directly or indirectly connected to each other:

[0235] NLS (e.g., SEQ ID NO:2858), DNMT3A (e.g., SEQ ID NO:2860), DNMT3L-CD (e.g., SEQ ID NO:41), NLS (e.g., SEQ ID NO:2857), HPT15-2 (e.g., SEQ ID NO:2865), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857), KRAB (e.g., SEQ ID NO:45).

[0236] Furthermore, the V25 solution may further include XTEN connectors, for example, the N-terminal to the C-terminal may include the following components, which may be directly or indirectly connected to each other:

[0237] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0238] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0239] DNMT3L-CD, for example, a sequence as shown in SEQ ID NO:41;

[0240] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0241] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0242] HPT15, for example, the sequence shown in SEQ ID NO:883;

[0243] One or more (e.g., two) sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0244] linker11-XTEN 16linker 1, for example, the sequence shown in SEQ ID NO:90;

[0245] KRAB, for example, the sequence shown in SEQ ID NO:45.

[0246] The V26 solution, NLS-DNMT3A-hDNMT3L-XTEN80-NLS-TALE HPT15-2×NLS-XTEN16-Zim3, can include the following components from the N-terminal to the C-terminal, and these components can be directly or indirectly connected to each other:

[0247] NLS (e.g., SEQ ID NO: 2858), DNMT3A (e.g., SEQ ID NO: 2860), hDNMT3L (e.g., SEQ ID NO: 42), NLS (e.g., SEQ ID NO: 2857), HPT15 (e.g., SEQ ID NO: 883), NLS (e.g., SEQ ID NO: 2857), NLS (e.g., SEQ ID NO: 2857), Zim3 Krab (e.g., SEQ ID NO: 2861).

[0248] Furthermore, the V26 solution may further include an XTEN connector, for example, the N-terminal to the C-terminal may include the following components, which may be directly or indirectly connected to each other:

[0249] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0250] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0251] hDNMT3L, for example, a sequence as shown in SEQ ID NO:42;

[0252] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0253] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0254] HPT15, for example, the sequence shown in SEQ ID NO:883;

[0255] One or more (e.g., two) sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0256] linker11-XTEN 16linker 1, for example, the sequence shown in SEQ ID NO:90;

[0257] Zim3 Krab, for example, the sequence shown in SEQ ID NO:2861.

[0258] V27 Solution: NLS-DNMT3A-hDNMT3L-XTEN80-ZIM3-XTEN80-NLS-TALE HPT21-2×NLS, which can include the following components from the N-terminal to the C-terminal, and these components can be directly or indirectly connected to each other:

[0259] NLS (e.g., SEQ ID NO:2858), DNMT3A (e.g., SEQ ID NO:2860), hDNMT3L (e.g., SEQ ID NO:42), Zim3 Krab (e.g., SEQ ID NO:2861), NLS (e.g., SEQ ID NO:2857), TALE HPT21 (e.g., SEQ ID NO:819), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857).

[0260] Furthermore, the V27 solution may further include XTEN connectors, for example, the N-terminal to the C-terminal may include the following components, which may be directly or indirectly connected to each other:

[0261] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0262] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0263] hDNMT3L, for example, a sequence as shown in SEQ ID NO:42;

[0264] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0265] Zim3 Krab, for example, the sequence shown in SEQ ID NO:2861;

[0266] Linker 15-fused XTEN80 linker 1, for example, the sequence shown in SEQ ID NO:2848;

[0267] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0268] TALE HPT21, for example, the sequence shown in SEQ ID NO:819;

[0269] One or more (e.g., two) sv40 NLS, such as the sequence shown in SEQ ID NO:2857.

[0270] The V29 solution, NLS-DNMT3A-DNMT3L-XTEN80-NLS-TALE HPT21-2×NLS-XTEN16-Krab, can include the following components from the N-terminal to the C-terminal, and these components can be directly or indirectly connected to each other:

[0271] NLS (e.g., SEQ ID NO:2858), DNMT3A (e.g., SEQ ID NO:2860), DNMT3L-CD (e.g., SEQ ID NO:41), NLS (e.g., SEQ ID NO:2857), HPT21 (e.g., SEQ ID NO:819), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857), KRAB (e.g., SEQ ID NO:45).

[0272] Furthermore, the V29 solution may further include an XTEN connector, for example, the N-terminal to the C-terminal may include the following components, which may be directly or indirectly connected to each other:

[0273] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0274] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0275] DNMT3L-CD, for example, a sequence as shown in SEQ ID NO:41;

[0276] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0277] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0278] HPT21, for example, the sequence shown in SEQ ID NO:819;

[0279] One or more (e.g., two) sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0280] linker11-XTEN 16linker 1, for example, the sequence shown in SEQ ID NO:90;

[0281] KRAB, for example, the sequence shown in SEQ ID NO:45.

[0282] V30 Solution: NLS-DNMT3A-hDNMT3L-XTEN80-ZIM3-XTEN80-NLS-TALE HPT25-2×NLS, which can include the following components from the N-terminal to the C-terminal, and these components can be directly or indirectly connected to each other:

[0283] NLS (e.g., SEQ ID NO: 2858), DNMT3A (e.g., SEQ ID NO: 2860), hDNMT3L (e.g., SEQ ID NO: 42), Zim3 Krab (e.g., SEQ ID NO: 2861), NLS (e.g., SEQ ID NO: 2857), TALE HPT25 (e.g., SEQ ID NO: 958), NLS (e.g., SEQ ID NO: 2857), NLS (e.g., SEQ ID NO: 2857).

[0284] Furthermore, the V30 solution may further include an XTEN connector, for example, the N-terminal to the C-terminal may include the following components, which may be directly or indirectly connected to each other:

[0285] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0286] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0287] hDNMT3L, for example, a sequence as shown in SEQ ID NO:42;

[0288] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0289] Zim3 Krab, for example, the sequence shown in SEQ ID NO:2861;

[0290] Linker 15-fused XTEN80 linker 1, for example, the sequence shown in SEQ ID NO:2848;

[0291] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0292] TALE HPT25, for example, the sequence shown in SEQ ID NO:958;

[0293] Linker5-GS linker, for example, the sequence shown in SEQ ID NO:84;

[0294] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0295] sv40 NLS, for example, the sequence shown in SEQ ID NO:2857.

[0296] The V32 solution, NLS-DNMT3A-DNMT3L-XTEN80-NLS-TALE HPT25-2×NLS-XTEN16-Krab, can include the following components from the N-terminal to the C-terminal, and these components can be directly or indirectly connected to each other:

[0297] NLS (e.g., SEQ ID NO:2858), DNMT3A (e.g., SEQ ID NO:2860), DNMT3L-CD (e.g., SEQ ID NO:41), NLS (e.g., SEQ ID NO:2857), HPT25 (e.g., SEQ ID NO:958), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857), KRAB (e.g., SEQ ID NO:45).

[0298] Furthermore, the V32 solution may further include XTEN connectors, for example, the N-terminal to the C-terminal may include the following components, which may be directly or indirectly connected to each other:

[0299] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0300] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0301] DNMT3L-CD, for example, a sequence as shown in SEQ ID NO:41;

[0302] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0303] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0304] HPT25, for example, the sequence shown in SEQ ID NO:958;

[0305] One or more (e.g., two) sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0306] linker11-XTEN 16linker 1, for example, the sequence shown in SEQ ID NO:90;

[0307] KRAB, for example, the sequence shown in SEQ ID NO:45.

[0308] The V33 solution, NLS-DNMT3A-DNMT3L-XTEN80-NLS-TALE HPT11-2×NLS-XTEN16-Krab, can include the following components from the N-terminal to the C-terminal, and these components can be directly or indirectly connected to each other:

[0309] NLS (e.g., SEQ ID NO:2858), DNMT3A (e.g., SEQ ID NO:2860), DNMT3L-CD (e.g., SEQ ID NO:41), NLS (e.g., SEQ ID NO:2857), TALE HPT11 (e.g., SEQ ID NO:947), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857), KRAB (e.g., SEQ ID NO:45).

[0310] The V34 solution, NLS-DNMT3A-DNMT3L-XTEN80-NLS-TALE HPT13-2×NLS-XTEN16-Krab, can include the following components from the N-terminal to the C-terminal, and these components can be directly or indirectly connected to each other:

[0311] NLS (e.g., SEQ ID NO:2858), DNMT3A (e.g., SEQ ID NO:2860), DNMT3L-CD (e.g., SEQ ID NO:41), NLS (e.g., SEQ ID NO:2857), TALE HPT13 (e.g., SEQ ID NO:961), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857), KRAB (e.g., SEQ ID NO:45).

[0312] Furthermore, the V34 solution may further include an XTEN connector, for example, the N-terminal to the C-terminal may include the following components, which may be directly or indirectly connected to each other:

[0313] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0314] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0315] DNMT3L-CD, for example, a sequence as shown in SEQ ID NO:41;

[0316] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0317] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0318] TALE HPT13, for example, the sequence shown in SEQ ID NO:961;

[0319] HA tag, for example, a sequence as shown in SEQ ID NO:2862;

[0320] Linker5-GS linker, for example, the sequence shown in SEQ ID NO:84;

[0321] One or more (e.g., two) sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0322] linker11-XTEN 16linker 1, for example, the sequence shown in SEQ ID NO:90;

[0323] KRAB, for example, the sequence shown in SEQ ID NO:45.

[0324] The sequence composition of the above scheme is as follows:

[0325] The following are example fusion products of this application, whose amino acid sequences are shown below: bold—DNMT3A, underlined bold—DNMT3L, Italics—Connector sequence, <Bold underline>—Transcriptional regulatory domain, Bold underline—TALE.

[0326] 1) NLS-TALE HPT15-2×NLS-XTEN80-DNMT3A-DNMT3L-XTEN16-ZIM3 KRAB (V1 scheme, SEQ ID NO:1; TALE field sequence is SEQ ID NO:883):

[0327] 2) NLS-TALE HPT15-2×NLS-XTEN80-ZIM3 KRAB-XTEN80-(human)DNMT3L-DNMT3A (V2 scheme, SEQ ID NO:2; TALE field sequence is SEQ ID NO:883):

[0328] 3) NLS-TALE HPT15-2×NLS-XTEN80-ZIM3 KRAB-XTEN80-DNMT3A-(human)DNMT3L (V3 scheme, SEQ ID NO:3; TALE field sequence is SEQ ID NO:883):

[0329] 4) ZIM3 KRAB-BFP-DNMT3A-(human)DNMT3L-XTEN80-NLS-TALE HPT15-2×NLS (V4 scheme, SEQ ID NO:4; TALE field sequence is SEQ ID NO:883):

[0330] 5) NLS-DNMT3A-(human)DNMT3L-XTEN80-ZIM3 KRAB-XTEN80-NLS-TALE HPT15-2×NLS (V5 scheme, SEQ ID NO:5; TALE field sequence is SEQ ID NO:883):

[0331] 6) NLS-DNMT3A-(human)DNMT3L-XTEN80-ZNF324-XTEN80-NLS-TALE HPT15-2×NLS (V6 scheme, SEQ ID NO:6; TALE field sequence is SEQ ID NO:883):

[0332] 7) NLS-DNMT3A-(human)DNMT3L-XTEN80-ZNF419-GS-XTEN80-NLS-TALE HPT15-2×NLS (V7 scheme, SEQ ID NO:7; TALE field sequence is SEQ ID NO:883):

[0333] 8) NLS-DNMT3A-DNMT3L-XTEN80-NLS-TALE HPT15-2×NLS-XTEN16-EZH2 (V8 scheme, SEQ ID NO:8; TALE field sequence is SEQ ID NO:883):

[0334] 9) NLS-DNMT3A-DNMT3L-XTEN80-NLS-TALE HPT15-2×NLS-XTEN16-HDAC3 (V9 scheme, SEQ ID NO:9; TALE field sequence is SEQ ID NO:883):

[0335] 10) NLS-DNMT3A-DNMT3L-XTEN80-NLS-TALE HPT15-2×NLS-XTEN16-HP1a (V10 scheme, SEQ ID NO:10; TALE field sequence is SEQ ID NO:883):

[0336] 11) NLS-DNMT3A-DNMT3L-XTEN80-NLS-TALE HPT15-2×NLS-XTEN16-KRAB (V25 scheme, SEQ ID NO:25; TALE field sequence is SEQ ID NO:883):

[0337] 12) NLS-DNMT3A-(human)DNMT3L-XTEN80-NLS-TALE HPT15-2×NLS-XTEN16-ZIM3KRAB (V26 scheme, SEQ ID NO:26; TALE field sequence is SEQ ID NO:883):

[0338] 13) NLS-DNMT3A-(human)DNMT3L-XTEN80-ZIM3 KRAB-XTEN80-NLS-TALE HPT21-2×NLS (V27 scheme, SEQ ID NO:27; TALE field sequence is SEQ ID NO:819):

[0339] 14) NLS-DNMT3A-DNMT3L-XTEN80-NLS-TALE HPT21-2×NLS-XTEN16-KRAB (V29 scheme, SEQ ID NO:29; TALE field sequence is SEQ ID NO:819):

[0340] 15) NLS-DNMT3A-(human)DNMT3L-XTEN80-ZIM3 KRAB-XTEN80-NLS-TALE HPT25-2×NLS (V30 scheme, SEQ ID NO:30; TALE field sequence is SEQ ID NO:958):

[0341] 16) NLS-DNMT3A-DNMT3L-XTEN80-NLS-TALE HPT25-2×NLS-XTEN16-KRAB (V32 scheme, SEQ ID NO:32; TALE field sequence is SEQ ID NO:958):

[0342] 17) NLS-DNMT3A-DNMT3L-XTEN80-NLS-TALE HPT11-2×NLS-XTEN16-KRAB (V33 scheme, SEQ ID NO:33; TALE field sequence is SEQ ID NO:35):

[0343] 18) NLS-DNMT3A-DNMT3L-XTEN80-NLS-TALE HPT13-2×NLS-XTEN16-KRAB (V34 scheme, SEQ ID NO:34; TALE field sequence is SEQ ID NO:36):

[0344] In some specific implementations, the TALE sequence (underlined bold italic portion) in the above example fusions 1)-18) can also be replaced with an amino acid sequence selected from any one of SEQ ID NOs:35-38 and 661-2840, 2865, 2867-2868.

[0345] The structure of the epigenetic editing agent provided in this application may also be selected from (but is 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., can be replaced with the TALE domain of the amino acid sequence provided in this application containing any one of SEQ ID NO:35-38 and 661-2840, 2865, 2867-2868), the disclosures of the above-mentioned patent applications are hereby incorporated by reference.

[0346] Complex peptides and their first and second fusion compounds

[0347] 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'.

[0348] Specifically, in some implementations of scenario (1) above, one of the two fusions may contain, from N-terminus to C-terminus, an epigenetic modification domain, a TALE domain, and a recruitment domain A. For example, in some implementations of scenario (2) above, one of the two fusions may contain, from N-terminus to C-terminus, a recruitment domain A, a TALE domain, and a transcriptional repressor domain. For example, in some implementations of scenario (1) above, the other fusion may contain, from N-terminus to C-terminus, a transcriptional repressor domain and a recruitment domain A', or a recruitment domain A' and a transcriptional repressor domain, i.e., the transcriptional repressor domain and the recruitment domain A' may be connected in an interchangeable order. For example, in some implementations of scenario (2) above, the other fusion may contain, from N-terminus to C-terminus, an epigenetic modification domain and a recruitment domain A', or a recruitment domain A' and an epigenetic modification domain, i.e., the epigenetic modification domain and the recruitment domain A' may be connected in an interchangeable order.

[0349] In some more specific embodiments, the TALE field may include, but is not limited to, the amino acid sequences shown in any one of SEQ ID NO:35-38 and 661-2840, 2865, 2867-2868.

[0350] 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, ZNF 419, ZNF566, ZIM2, EHMT2, SUV39H1, ZFPM1, TRIM28, EZH2, MXD1, SID, LSD1, HP1a, HDAC 3. HDAC1, PRMT1, SETDB1, hSIRT1, ZNF436, ZNF257, ZNF675, ZNF490, ZNF320, ZNF331, Z NF816, ZNF41, ZNF189, ZNF528, ZNF543, ZNF140, ZNF610, ZNF350, ZNF8, ZNF30, ZNF98, Z NF677, ZNF596, ZNF214, ZNF37A, ZNF34, ZNF250, ZNF547, ZNF273, ZFP82, ZNF224, ZNF3 3A, ZNF45, ZNF175, ZNF184, ZFP28-1, ZFP28-2, ZNF18, ZNF213, ZNF394, ZFP1, ZFP14, ZN F416, ZNF557, ZNF729, ZNF254, ZNF764, ZNF785, ZNF10, CBX5, RYBP, YAF2, MGA, CBX1, SCMH1, MPP8, SUMO3, HERC2, BIN1, PCGF2, TOX, FOXA1, FOXA2, IRF2BP1, IRF2BP2, IRF2BPL IRF-2BP1_2N-terminal domain, HOXA13, HOXB13, HOXC13, HOXA11, HOXC11, HOXC10, HOXA10, HOXB9, HOXA9, ZFP28, ZN334, ZN568, ZN3 7A, 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 their functionally active fragments.

[0351] 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 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 interchangeably linked in sequence. For example, the DNA methylation domain comprises one DNMT3A and one DNMT3L, and they are interchangeably linked in sequence. For example, the DNA methyltransferase comprises the amino acid sequence shown in any one of SEQ ID NO: 39-44, 2860.

[0352] The first and second fusions of the complex peptides described in this application form aggregated complexes through the interaction between their respective recruitment domains. Therefore, this application provides non-limiting examples of combinations of recruitment domain A and recruitment domain A': (1) one of recruitment domain A and recruitment domain A' has a domain of GCN4, and the other domain is scFv; or (2) one of recruitment domain A and recruitment domain A' has a domain of a GFP11 fragment, and the other domain is GFP1-10; or (3) one of recruitment domain A and recruitment domain A' has a domain of GVKESLV, and the other domain is a PDZ protein domain. Similarly, the situation where GFP11 and GFP1-10 are derived from splitting GFP (SEQ ID NO:76) to form recruitment domain A and recruitment domain A', respectively, can also be applied to other classes of fluorescent proteins, such as mCherry (SEQ ID NO:77), eYFP (SEQ ID NO:79), eCFP (SEQ ID NO:78), etc. Different sets of recruitment domain A and recruitment domain A' can be obtained by splitting mCherry, eYFP, or eCFP for use in the composite peptide provided in this application. In some embodiments, one of the first fusion and the second fusion of the composite peptide of this application may contain two or more recruitment domains, which are linked by a linker sequence. The amino acid sequence of an exemplary recruitment domain may include any one of SEQ ID NO:71-75.

[0353] The following structures may include TALE-DNMT3A-DNMT3L-ZIM3 KRAB, TALE-ZIM3KRAB-DNMT3L-DNMT3A, TALE-ZIM3 KRAB-DNMT3A-DNMT3L, ZIM3 KRAB-DNMT3A-DNMT3L-TALE, DNMT3A-DNMT3L-ZIM3 as described above KRAB-TALE, DNMT3A-DNMT3L-ZNF324-TALE, DNMT3A-DNMT3L-ZNF419-TALE, DNMT3A-DNMT3L-TALE-EZH2, DNMT3 A-DNMT3L-TALE-HDAC3, DNMT3A-DNMT3L-TALE-HP1a, DNMT3A-DNMT3L-TALE-KRAB, or DNMT3A-DNMT3L-TALE-ZIM3 KRAB, KRAB-DNMT3A-DNMT3L-TALE, DNMT3L-DNMT3A-TALE-KRAB structure.

[0354] Furthermore, an exemplary fusion structure is described in the following scheme:

[0355] The V11 solution, NLS-scFv-KRAB, can include the following components from the N-terminal to the C-terminal, and these components can be directly or indirectly connected to each other:

[0356] NLS (e.g., SEQ ID NO:2858), scFv (e.g., SEQ ID NO:2863), KRAB (e.g., SEQ ID NO:45).

[0357] Furthermore, the V11 solution from the N-end to the C-end can include the following components, which can be directly or indirectly connected to each other:

[0358] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0359] scFv, for example, a sequence as shown in SEQ ID NO:2863;

[0360] KRAB, for example, the sequence shown in SEQ ID NO:45.

[0361] V12 Solution: NLS-DNMT3A-DNMT3L-NLS-TALE HPT15-2×NLS-4×GCN4-NLS, which can include the following components from the N-terminal to the C-terminal, and these components can be directly or indirectly connected to each other:

[0362] NLS (e.g., SEQ ID NO:2858), DNMT3L-CD (e.g., SEQ ID NO:41), NLS (e.g., SEQ ID NO:2857), TALE HPT15 (e.g., SEQ ID NO:883), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857), multiple GCN4s (e.g., SEQ ID NO:71), NLS (e.g., SEQ ID NO:2859), for example, scheme V12 may have 2, 3, or 4 GCN4s, each GCN4 being directly or indirectly connected to each other.

[0363] Furthermore, the V12 solution from the N-end to the C-end can include the following components, which can be directly or indirectly connected to each other:

[0364] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0365] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0366] DNMT3L-CD, for example, a sequence as shown in SEQ ID NO:41;

[0367] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0368] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0369] TALE HPT15, for example, the sequence shown in SEQ ID NO:883;

[0370] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0371] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0372] Multiple (e.g., 4) GCN4s, such as SEQ ID NO:71, are connected to each other via a connector (e.g., SEQ ID NO:86);

[0373] BPSV40 NLS, for example, the sequence shown in SEQ ID NO:2859.

[0374] The V13 solution, NLS-DNMT3A-DNMT3L-scFv, can include the following components from the N-terminal to the C-terminal, and these components can be directly or indirectly connected to each other:

[0375] NLS (e.g., SEQ ID NO:2858), DNMT3A (e.g., SEQ ID NO:2860), DNMT3L-CD (e.g., SEQ ID NO:41), scFv (e.g., SEQ ID NO:2863).

[0376] Furthermore, the V13 solution can include the following components from the N-end to the C-end, and these components can be directly or indirectly connected to each other:

[0377] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0378] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0379] DNMT3L-CD, for example, a sequence as shown in SEQ ID NO:41;

[0380] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0381] scFv, for example, the sequence shown in SEQ ID NO:2863.

[0382] The V14 solution, NLS-3×GCN4-NLS-TALE and HPT15-2×NLS-KRAB, can include the following components from the N-terminal to the C-terminal, and these components can be directly or indirectly connected to each other:

[0383] NLS (e.g., SEQ ID NO:2858), multiple GCN4s (e.g., SEQ ID NO:71), NLS (e.g., SEQ ID NO:2857), TALE HPT15 (e.g., SEQ ID NO:883), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857), KRAB (e.g., SEQ ID NO:45), for example, scheme V14 may have 2, 3, or 4 GCN4s, each GCN4 being directly or indirectly connected to each other.

[0384] Furthermore, the V14 solution can include the following components from the N-end to the C-end, and these components can be directly or indirectly connected to each other:

[0385] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0386] Multiple (e.g., 3) GCN4s, such as SEQ ID NO:71, are connected to each other via a connector (e.g., SEQ ID NO:86);

[0387] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0388] TALE HPT15, for example, the sequence shown in SEQ ID NO:883;

[0389] Multiple (e.g., two) sv40 NLS, for example, the sequence shown in SEQ ID NO:2857;

[0390] Linker10-XTEN 16 linker 2, for example, the sequence shown in SEQ ID NO:89;

[0391] KRAB, for example, the sequence shown in SEQ ID NO:45.

[0392] V15 scheme: NLS-scFv-ZIM3-2a-NLS-DNMT3A-hDNMT3L-NLS-TALE HPT15-2×NLS-10×GCN4-NLS (SEQ ID NO:15), which may include the following components from the N end to the C end, and the following components may be directly or indirectly connected to each other:

[0393] NLS (e.g., SEQ ID NO: 2858), scFv (e.g., SEQ ID NO: 2863), Zim3 Krab (e.g., SEQ ID NO: 2861), NLS (e.g., SEQ ID NO: 2858), DNMT3A (e.g., SEQ ID NO: 2860), hDNMT3L (e.g., SEQ ID NO: 42), NLS (e.g., SEQ ID NO: 2857), TALE HPT15 (e.g., SEQ ID NO: 883), NLS (e.g., SEQ ID NO: 2857), NLS (e.g., SEQ ID NO: 2857), GCN4 (e.g., SEQ ID NO: 71), multiple GCN4s (e.g., SEQ ID NO: 71), NLS (e.g., SEQ ID NO: 2859), for example, scheme V15 may have 2, 3, 4, 5, 6, 7, 8, 9 or 10 GCN4s, each GCN4 being directly or indirectly connected to each other.

[0394] Furthermore, the V15 solution, from the N-end to the C-end, can include the following components, which can be directly or indirectly connected to each other:

[0395] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0396] scFv, for example, a sequence as shown in SEQ ID NO:2863;

[0397] Zim3 Krab, for example, the sequence shown in SEQ ID NO:2861;

[0398] P2A, for example, a sequence as shown in SEQ ID NO:2864;

[0399] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0400] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0401] hDNMT3L, for example, a sequence as shown in SEQ ID NO:42;

[0402] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0403] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0404] TALE HPT15, for example, the sequence shown in SEQ ID NO:883;

[0405] Multiple (e.g., two) sv40 NLS, for example, the sequence shown in SEQ ID NO:2857;

[0406] Multiple (e.g., 10) GCN4s, such as SEQ ID NO:71, are connected to each other via a connector (e.g., SEQ ID NO:86);

[0407] BPSV40 NLS, for example, the sequence shown in SEQ ID NO:2859.

[0408] The first fusion of the V15 scheme (SEQ ID NO:102) may include the following components from the N end to the C end, which may be directly or indirectly connected to each other: NLS (e.g., SEQ ID NO:2858), scFv (e.g., SEQ ID NO:2863), and Zim3 Krab (e.g., SEQ ID NO:2861).

[0409] Furthermore, the first fusion of the V15 scheme (SEQ ID NO:102) may include the following components from the N-terminus to the C-terminus, and these components may be directly or indirectly connected to each other:

[0410] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0411] scFv, for example, a sequence as shown in SEQ ID NO:2863;

[0412] Zim3 Krab, for example, the sequence shown in SEQ ID NO:2861.

[0413] The second fusion of the V15 scheme (SEQ ID NO:103) may include the following components from the N end to the C end, which may be directly or indirectly connected to each other: NLS (e.g., SEQ ID NO:2858), DNMT3A (e.g., SEQ ID NO:2860), hDNMT3L (e.g., SEQ ID NO:42), NLS (e.g., SEQ ID NO:2857), TALE HPT15 (e.g., SEQ ID NO:883), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857), GCN4 (e.g., SEQ ID NO:71), and multiple GCN4s (e.g., SEQ ID NO:71).

[0414] Furthermore, the second fusion of the V15 scheme (SEQ ID NO:103) may include the following components from the N-terminus to the C-terminus, and these components may be directly or indirectly connected to each other:

[0415] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0416] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0417] hDNMT3L, for example, a sequence as shown in SEQ ID NO:42;

[0418] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0419] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0420] TALE HPT15, for example, the sequence shown in SEQ ID NO:883;

[0421] Multiple (e.g., two) sv40 NLS, for example, the sequence shown in SEQ ID NO:2857;

[0422] Multiple (e.g., 10) GCN4s, such as SEQ ID NO:71, are connected to each other via a connector (e.g., SEQ ID NO:86);

[0423] BPSV40 NLS, for example, the sequence shown in SEQ ID NO:2859.

[0424] V16 scheme: NLS-KRAB-scFv-2a-NLS-DNMT3A-DNMT3L-NLS-TALE HPT15-2×NLS-2×GCN4-NLS (SEQ ID NO:16), which may include the following components from the N end to the C end, and the following components may be directly or indirectly connected to each other:

[0425] NLS (e.g., SEQ ID NO:2858), KRAB (e.g., SEQ ID NO:45), scFv (e.g., SEQ ID NO:2863), NLS (e.g., SEQ ID NO:2858), DNMT3A (e.g., SEQ ID NO:2860), hDNMT3L (e.g., SEQ ID NO:42), NLS (e.g., SEQ ID NO:2857), TALE HPT15 (e.g., SEQ ID NO:883), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857), GCN4 (e.g., SEQ ID NO:71), GCN4 (e.g., SEQ ID NO:71), NLS (e.g., SEQ ID NO:2859).

[0426] Furthermore, the V16 solution from the N-end to the C-end can include the following components, which can be directly or indirectly connected to each other:

[0427] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0428] KRAB, for example, a sequence as shown in SEQ ID NO:45;

[0429] scFv, for example, a sequence as shown in SEQ ID NO:2863;

[0430] P2A, for example, a sequence as shown in SEQ ID NO:2864;

[0431] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0432] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0433] hDNMT3L, for example, a sequence as shown in SEQ ID NO:42;

[0434] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0435] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0436] TALE HPT15, for example, the sequence shown in SEQ ID NO:883;

[0437] Multiple (e.g., two) sv40 NLS, for example, the sequence shown in SEQ ID NO:2857;

[0438] Multiple (e.g., two) GCN4s, such as the sequence shown in SEQ ID NO:71, are connected to each other by a connector (e.g., SEQ ID NO:86);

[0439] BPSV40 NLS, for example, the sequence shown in SEQ ID NO:2859.

[0440] The first fusion of the V16 scheme (SEQ ID NO: 104) may include the following components from the N end to the C end, which may be directly or indirectly connected to each other: NLS (e.g., SEQ ID NO: 2858), KRAB (e.g., SEQ ID NO: 45), and scFv (e.g., SEQ ID NO: 2863).

[0441] Furthermore, the first fusion component of the V16 solution, from the N-end to the C-end, may include the following components, which may be directly or indirectly connected to each other:

[0442] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0443] KRAB, for example, a sequence as shown in SEQ ID NO:45;

[0444] scFv, for example, the sequence shown in SEQ ID NO:2863.

[0445] The second fusion of the V16 scheme (SEQ ID NO:105) may include the following components from the N end to the C end, which may be directly or indirectly connected to each other: NLS (e.g., SEQ ID NO:2858), DNMT3A (e.g., SEQ ID NO:2860), hDNMT3L (e.g., SEQ ID NO:42), NLS (e.g., SEQ ID NO:2857), TALE HPT15 (e.g., SEQ ID NO:883), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857), GCN4 (e.g., SEQ ID NO:71), GCN4 (e.g., SEQ ID NO:71), NLS (e.g., SEQ ID NO:2859).

[0446] Furthermore, the second fusion component of the V16 scheme, from the N-end to the C-end, may include the following components, which may be directly or indirectly connected to each other:

[0447] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0448] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0449] hDNMT3L, for example, a sequence as shown in SEQ ID NO:42;

[0450] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0451] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0452] TALE HPT15, for example, the sequence shown in SEQ ID NO:883;

[0453] Multiple (e.g., two) sv40 NLS, for example, the sequence shown in SEQ ID NO:2857;

[0454] Multiple (e.g., two) GCN4s, such as the sequence shown in SEQ ID NO:71, are connected to each other by a connector (e.g., SEQ ID NO:86);

[0455] BPSV40 NLS, for example, the sequence shown in SEQ ID NO:2859.

[0456] V17 Solution: NLS-DNMT3A-DNMT3L-scFv-2a-NLS-1×GCN4-NLS-TALE HPT15-2×NLS-KRAB, which can include the following components from the N-terminal to the C-terminal, and these components can be directly or indirectly connected to each other:

[0457] NLS (e.g., SEQ ID NO:2858), DNMT3L-CD (e.g., SEQ ID NO:41), scFv (e.g., SEQ ID NO:2863), NLS (e.g., SEQ ID NO:2858), GCN4 (e.g., SEQ ID NO:71), NLS (e.g., SEQ ID NO:2857), TALE HPT15 (e.g., SEQ ID NO:883), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857), KRAB (e.g., SEQ ID NO:45).

[0458] Furthermore, the V17 solution from the N-end to the C-end can include the following components, which can be directly or indirectly connected to each other:

[0459] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0460] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0461] DNMT3L-CD, for example, a sequence as shown in SEQ ID NO:41;

[0462] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0463] scFv, for example, a sequence as shown in SEQ ID NO:2863;

[0464] P2A, for example, a sequence as shown in SEQ ID NO:2864;

[0465] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0466] GCN4, for example, the sequence shown in SEQ ID NO:71;

[0467] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0468] TALE HPT15, for example, the sequence shown in SEQ ID NO:883;

[0469] Multiple (e.g., 2) sv40 NLS, for example, the sequence shown in SEQ ID NO:2857;

[0470] Linker10-XTEN 16 linker 2, for example, the sequence shown in SEQ ID NO:89;

[0471] KRAB, for example, the sequence shown in SEQ ID NO:45.

[0472] The first fusion of the V17 scheme may include the following components from the N end to the C end, which may be directly or indirectly connected to each other: NLS (e.g., SEQ ID NO:2858), DNMT3A (e.g., SEQ ID NO:2860), hDNMT3L-CD (e.g., SEQ ID NO:41), and scFv (e.g., SEQ ID NO:2863).

[0473] Furthermore, the first fusion component of the V17 solution, from the N-end to the C-end, may include the following components, which may be directly or indirectly connected to each other:

[0474] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0475] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0476] DNMT3L-CD, for example, a sequence as shown in SEQ ID NO:41;

[0477] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0478] scFv, for example, the sequence shown in SEQ ID NO:2863.

[0479] The second fusion of the V17 scheme may include the following components from the N end to the C end, which may be directly or indirectly connected to each other: NLS (e.g., SEQ ID NO:2858), GCN4 (e.g., SEQ ID NO:71), NLS (e.g., SEQ ID NO:2857), TALE HPT15 (e.g., SEQ ID NO:883), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857), and KRAB (e.g., SEQ ID NO:45).

[0480] Furthermore, the second fusion component of the V17 scheme, from the N-end to the C-end, may include the following components, which may be directly or indirectly connected to each other:

[0481] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0482] GCN4, for example, the sequence shown in SEQ ID NO:71;

[0483] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0484] TALE HPT15, for example, the sequence shown in SEQ ID NO:883;

[0485] Multiple (e.g., 2) sv40 NLS, for example, the sequence shown in SEQ ID NO:2857;

[0486] Linker10-XTEN 16 linker 2, for example, the sequence shown in SEQ ID NO:89;

[0487] KRAB, for example, the sequence shown in SEQ ID NO:45.

[0488] V18 solution: NLS-DNMT3A-hDNMT3L-NLS-scFv-2a-NLS-3×GCN4-NLS-TALE HPT15-2×NLS-ZIM3, which can include the following components from the N-terminal to the C-terminal, and the following components can be directly or indirectly connected to each other:

[0489] NLS (e.g., SEQ ID NO:2858), DNMT3A (e.g., SEQ ID NO:2860), hDNMT3L (e.g., SEQ ID NO:42), NLS (e.g., SEQ ID NO:2857), scFv (e.g., SEQ ID NO:2863), NLS (e.g., SEQ ID NO:2858), multiple GCN4s (e.g., SEQ ID NO:71) (e.g., 3, 4, 5, 6, 7 GCN4s), NLS (e.g., SEQ ID NO:2857), TALE HPT15 (e.g., SEQ ID NO:883), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857), Zim3 Krab (e.g., SEQ ID NO:2861).

[0490] Furthermore, the V18 solution, from the N-end to the C-end, can include the following components, which can be directly or indirectly connected to each other:

[0491] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0492] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0493] hDNMT3L, for example, a sequence as shown in SEQ ID NO:42;

[0494] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0495] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0496] scFv, for example, a sequence as shown in SEQ ID NO:2863;

[0497] P2A, for example, a sequence as shown in SEQ ID NO:2864;

[0498] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0499] Multiple (e.g., 3) GCN4s, such as the sequence shown in SEQ ID NO:71, are connected to each other by a connector (e.g., SEQ ID NO:86);

[0500] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0501] TALE HPT15, for example, the sequence shown in SEQ ID NO:883;

[0502] Multiple (e.g., 2) sv40 NLS, for example, the sequence shown in SEQ ID NO:2857;

[0503] Linker10-XTEN 16 linker 2, for example, the sequence shown in SEQ ID NO:89;

[0504] Zim3 Krab, for example, the sequence shown in SEQ ID NO:2861.

[0505] The first fusion of the V18 scheme from the N end to the C end may include the following components, which may be directly or indirectly connected to each other: NLS (e.g., SEQ ID NO:2858), DNMT3A (e.g., SEQ ID NO:2860), hDNMT3L (e.g., SEQ ID NO:42), NLS (e.g., SEQ ID NO:2857), and scFv (e.g., SEQ ID NO:2863).

[0506] Furthermore, the first fusion component of the V18 solution, from the N-end to the C-end, may include the following components, which may be directly or indirectly connected to each other:

[0507] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0508] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0509] hDNMT3L, for example, a sequence as shown in SEQ ID NO:42;

[0510] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0511] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0512] scFv, for example, a sequence as shown in SEQ ID NO:2863;

[0513] The second fusion of the V18 scheme may include the following components from the N end to the C end, which may be directly or indirectly connected to each other: multiple GCN4 (e.g., SEQ ID NO:71) (e.g., 3, 4, 5, 6, 7 GCN4), TALE HPT15 (e.g., SEQ ID NO:883), and Zim3 Krab (e.g., SEQ ID NO:2861).

[0514] Furthermore, the second fusion component of the V18 solution, from the N-end to the C-end, may include the following components, which may be directly or indirectly connected to each other:

[0515] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0516] Multiple (e.g., 3) GCN4s, such as the sequence shown in SEQ ID NO:71, are connected to each other by a connector (e.g., SEQ ID NO:86);

[0517] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0518] TALE HPT15, for example, the sequence shown in SEQ ID NO:883;

[0519] Multiple (e.g., 2) sv40 NLS, for example, the sequence shown in SEQ ID NO:2857;

[0520] Linker10-XTEN 16 linker 2, for example, the sequence shown in SEQ ID NO:89;

[0521] Zim3 Krab, for example, the sequence shown in SEQ ID NO:2861.

[0522] V19 Solution: NLS-3×GCN4-NLS-TALE HPT15-2×NLS-ZIM3-2a-NLS-DNMT3A-hDNMT3L-NLS-scFv, which can include the following components from the N-terminal to the C-terminal, and these components can be directly or indirectly connected to each other:

[0523] NLS (e.g., SEQ ID NO:2858), multiple GCN4s (e.g., SEQ ID NO:71), NLS (e.g., SEQ ID NO:2857), TALE HPT15 (e.g., SEQ ID NO:883), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857), Zim3 Krab (e.g., SEQ ID NO:2861), NLS (e.g., SEQ ID NO:2858), DNMT3A (e.g., SEQ ID NO:2860), hDNMT3L (e.g., SEQ ID NO:42), NLS (e.g., SEQ ID NO:2857), scFv (e.g., SEQ ID NO:2863), for example, scheme V19 may have 2, 3, or 4 GCN4s, each GCN4 being directly or indirectly connected to each other.

[0524] Furthermore, the V19 solution may further include an XTEN connector, for example, the N-terminal to the C-terminal may include the following components, which may be directly or indirectly connected to each other:

[0525] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0526] Multiple GCN4s (e.g., 3), such as the sequence shown in SEQ ID NO:71, are connected to each other via GS connectors (e.g., the sequence shown in SEQ ID NO:86);

[0527] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0528] TALE HPT15, for example, the sequence shown in SEQ ID NO:883;

[0529] One or more (e.g., two) sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0530] Linker10-XTEN 16 linker 2, for example, the sequence shown in SEQ ID NO:89;

[0531] Zim3 Krab, for example, the sequence shown in SEQ ID NO:2861;

[0532] P2A, for example, a sequence as shown in SEQ ID NO:2864;

[0533] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0534] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0535] Linker3-flexible peptide linker, for example, the sequence shown in SEQ ID NO:82;

[0536] hDNMT3L, for example, a sequence as shown in SEQ ID NO:42;

[0537] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0538] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0539] scFv, for example, the sequence shown in SEQ ID NO:2863.

[0540] The first fusion of the V19 scheme may include the following components from the N end to the C end, which may be directly or indirectly connected to each other: NLS (e.g., SEQ ID NO:2858), multiple GCN4 (e.g., SEQ ID NO:71), NLS (e.g., SEQ ID NO:2857), TALE HPT15 (e.g., SEQ ID NO:883), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857), and Zim3 Krab (e.g., SEQ ID NO:2861).

[0541] Furthermore, the first fusion component of the V19 solution, from the N-end to the C-end, may include the following components, which may be directly or indirectly connected to each other:

[0542] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0543] Multiple GCN4s (e.g., 3), such as the sequence shown in SEQ ID NO:71, are connected to each other via GS connectors (e.g., the sequence shown in SEQ ID NO:86);

[0544] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0545] TALE HPT15, for example, the sequence shown in SEQ ID NO:883;

[0546] One or more (e.g., two) sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0547] Linker10-XTEN 16 linker 2, for example, the sequence shown in SEQ ID NO:89;

[0548] Zim3 Krab, for example, the sequence shown in SEQ ID NO:2861.

[0549] The second fusion of the V19 scheme may include the following components from the N end to the C end, which may be directly or indirectly connected to each other: NLS (e.g., SEQ ID NO:2858), DNMT3A (e.g., SEQ ID NO:2860), hDNMT3L (e.g., SEQ ID NO:42), NLS (e.g., SEQ ID NO:2857), and scFv (e.g., SEQ ID NO:2863).

[0550] Furthermore, the second fusion component of the V19 scheme, from the N-end to the C-end, may include the following components, which may be directly or indirectly connected to each other:

[0551] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0552] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0553] Linker3-flexible peptide linker, for example, the sequence shown in SEQ ID NO:82;

[0554] hDNMT3L, for example, a sequence as shown in SEQ ID NO:42;

[0555] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0556] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0557] scFv, for example, the sequence shown in SEQ ID NO:2863.

[0558] V20 solution: NLS-scFV-hDNMT3L-DNMT3A-2a-NLS-3×GCN4-NLS-TALE HPT15-2×NLS-KRAB, which can include the following components from the N-terminal to the C-terminal, and these components can be directly or indirectly connected to each other:

[0559] NLS (e.g., SEQ ID NO:2858), scFv (e.g., SEQ ID NO:2863), hDNMT3L (e.g., SEQ ID NO:42), DNMT3A (e.g., SEQ ID NO:2860), NLS (e.g., SEQ ID NO:2858), multiple GCN4s (e.g., SEQ ID NO:71), NLS (e.g., SEQ ID NO:2857), TALE HPT15 (e.g., SEQ ID NO:883), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857), KRAB (e.g., SEQ ID NO:45), for example, scheme V20 may have 2, 3, or 4 GCN4s, each GCN4 being directly or indirectly connected to each other.

[0560] Furthermore, the V20 solution, from the N-end to the C-end, can include the following components, which can be directly or indirectly connected to each other:

[0561] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0562] scFv, for example, a sequence as shown in SEQ ID NO:2863;

[0563] hDNMT3L, for example, a sequence as shown in SEQ ID NO:42;

[0564] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0565] P2A, for example, a sequence as shown in SEQ ID NO:2864;

[0566] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0567] Multiple (e.g., 3) GCN4s, such as the sequence shown in SEQ ID NO:71, are connected to each other by a connector (e.g., SEQ ID NO:86);

[0568] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0569] TALE HPT15, for example, the sequence shown in SEQ ID NO:883;

[0570] Multiple sv40 NLS (e.g., 2), for example, the sequence shown in SEQ ID NO:2857;

[0571] Linker10-XTEN 16 linker 2, for example, the sequence shown in SEQ ID NO:89;

[0572] KRAB, for example, the sequence shown in SEQ ID NO:45.

[0573] The first fusion of the V20 scheme from the N end to the C end may include the following components, which may be directly or indirectly connected to each other: NLS (e.g., SEQ ID NO:2858), scFv (e.g., SEQ ID NO:2863), hDNMT3L (e.g., SEQ ID NO:42), and DNMT3A (e.g., SEQ ID NO:2860).

[0574] Furthermore, the first fusion component of the V20 solution, from the N-end to the C-end, may include the following components, which may be directly or indirectly connected to each other:

[0575] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0576] scFv, for example, a sequence as shown in SEQ ID NO:2863;

[0577] hDNMT3L, for example, a sequence as shown in SEQ ID NO:42;

[0578] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0579] The second fusion of the V20 scheme from the N end to the C end may include the following components, which may be directly or indirectly connected to each other: NLS, for example, the sequence shown in SEQ ID NO:2858; multiple (e.g., 3) GCN4, for example, the sequence shown in SEQ ID NO:71, each GCN4 connected to each other by a connector (e.g., SEQ ID NO:86); NLS, for example, the sequence shown in SEQ ID NO:2857; TALE HPT15, for example, the sequence shown in SEQ ID NO:883; multiple sv40 NLS (e.g., 2), for example, the sequence shown in SEQ ID NO:2857; KRAB, for example, the sequence shown in SEQ ID NO:45.

[0580] Furthermore, the second fusion component of the V20 solution, from the N-end to the C-end, may include the following components, which may be directly or indirectly connected to each other:

[0581] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0582] Multiple (e.g., 3) GCN4s, such as the sequence shown in SEQ ID NO:71, are connected to each other by a connector (e.g., SEQ ID NO:86);

[0583] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0584] TALE HPT15, for example, the sequence shown in SEQ ID NO:883;

[0585] Multiple sv40 NLS (e.g., 2), for example, the sequence shown in SEQ ID NO:2857;

[0586] Linker10-XTEN 16 linker 2, for example, the sequence shown in SEQ ID NO:89;

[0587] KRAB, for example, the sequence shown in SEQ ID NO:45.

[0588] V21 solution: NLS-1×GCN4-ZIM3-2a-NLS-DNMT3A-hDNMT3L-NLS-TALE HPT15-2×NLS-scFv, which can include the following components from the N-terminal to the C-terminal, and the following components can be directly or indirectly connected to each other:

[0589] NLS (e.g., SEQ ID NO: 2858), GCN4 (e.g., SEQ ID NO: 71), Zim3 Krab (e.g., SEQ ID NO: 2861), NLS (e.g., SEQ ID NO: 2858), DNMT3A (e.g., SEQ ID NO: 2860), hDNMT3L (e.g., SEQ ID NO: 42), NLS (e.g., SEQ ID NO: 2857), TALE HPT15 (e.g., SEQ ID NO: 883), NLS (e.g., SEQ ID NO: 2857), NLS (e.g., SEQ ID NO: 2857), scFv (e.g., SEQ ID NO: 2863).

[0590] Furthermore, the V21 solution can include the following components from the N-end to the C-end, and these components can be directly or indirectly connected to each other:

[0591] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0592] GCN4, for example, the sequence shown in SEQ ID NO:71;

[0593] Zim3 Krab, for example, the sequence shown in SEQ ID NO:2861;

[0594] P2A, for example, a sequence as shown in SEQ ID NO:2864;

[0595] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0596] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0597] hDNMT3L, for example, a sequence as shown in SEQ ID NO:42;

[0598] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0599] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0600] TALE HPT15, for example, the sequence shown in SEQ ID NO:883;

[0601] Multiple (e.g., 2) sv40 NLS, for example, the sequence shown in SEQ ID NO:2857;

[0602] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0603] scFv, for example, the sequence shown in SEQ ID NO:2863.

[0604] The first fusion of the V21 scheme may include the following components from the N end to the C end, which may be directly or indirectly connected to each other: NLS (e.g., SEQ ID NO:2858), GCN4 (e.g., SEQ ID NO:71), and Zim3 Krab (e.g., SEQ ID NO:2861).

[0605] Furthermore, the first fusion component of the V21 scheme, from the N-end to the C-end, may include the following components, which may be directly or indirectly connected to each other:

[0606] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0607] GCN4, for example, the sequence shown in SEQ ID NO:71;

[0608] Zim3 Krab, for example, the sequence shown in SEQ ID NO:2861.

[0609] The second fusion of the V21 scheme may include the following components from the N end to the C end, which may be directly or indirectly connected to each other: NLS (e.g., SEQ ID NO:2858), DNMT3A (e.g., SEQ ID NO:2860), hDNMT3L (e.g., SEQ ID NO:42), NLS (e.g., SEQ ID NO:2857), TALE HPT15 (e.g., SEQ ID NO:883), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857), and scFv (e.g., SEQ ID NO:2863).

[0610] Furthermore, the second fusion component of the V21 scheme, from the N-end to the C-end, may include the following components, which may be directly or indirectly connected to each other:

[0611] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0612] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0613] hDNMT3L, for example, a sequence as shown in SEQ ID NO:42;

[0614] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0615] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0616] TALE HPT15, for example, the sequence shown in SEQ ID NO:883;

[0617] Multiple (e.g., 2) sv40 NLS, for example, the sequence shown in SEQ ID NO:2857;

[0618] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0619] scFv, for example, the sequence shown in SEQ ID NO:2863.

[0620] The V22 scheme: GFP1-10-KRAB-NLS-2a-NLS-DNMT3A-DNMT3L-NLS-TALE HPT15-2×NLS-7×GFP11-NLS, can include the following components from the N-terminus to the C-terminus, and these components can be directly or indirectly connected to each other:

[0621] GFP1-10 (e.g., SEQ ID NO:75), KRAB (e.g., SEQ ID NO:45), NLS (e.g., SEQ ID NO:2859), NLS (e.g., SEQ ID NO:2858), DNMT3A (e.g., SEQ ID NO:2860), DNMT3L-CD (e.g., SEQ ID NO:41), NLS (e.g., SEQ ID NO:2857), TALE HPT15 (e.g., SEQ ID NO:883), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857), multiple GFP11 (e.g., SEQ ID NO:72), for example, scheme V20 may have 2, 3, 4, 5, 6, 7 or 8 GFP11, each GFP11 being directly or indirectly connected to each other, NLS (e.g., SEQ ID NO:2859).

[0622] Furthermore, V22 from the N-terminal to the C-terminal may include the following components, which may be directly or indirectly connected to each other:

[0623] GFP1-10, for example, the sequence shown in SEQ ID NO:75;

[0624] KRAB, for example, a sequence as shown in SEQ ID NO:45;

[0625] BPSV40 NLS, for example, a sequence as shown in SEQ ID NO:2859;

[0626] P2A, for example, a sequence as shown in SEQ ID NO:2864;

[0627] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0628] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0629] DNMT3L-CD, for example, a sequence as shown in SEQ ID NO:41;

[0630] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0631] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0632] TALE HPT15, for example, the sequence shown in SEQ ID NO:883;

[0633] Multiple (e.g., 2) sv40 NLS, for example, the sequence shown in SEQ ID NO:2857;

[0634] Multiple (e.g., 7) GFP11, for example, the sequence shown in SEQ ID NO:72, each GFP11 is connected by a adapter (e.g., the sequence shown in SEQ ID NO:2856);

[0635] BPSV40 NLS, for example, the sequence shown in SEQ ID NO:2859.

[0636] The first fusion of the V22 scheme may include the following components from the N-terminus to the C-terminus, which may be directly or indirectly connected to each other: GFP1-10 (e.g., SEQ ID NO:75), KRAB (e.g., SEQ ID NO:45), and NLS (e.g., SEQ ID NO:2859).

[0637] Furthermore, the first fusion component of the V22 scheme, from the N-end to the C-end, may include the following components, which may be directly or indirectly connected to each other:

[0638] GFP1-10, for example, the sequence shown in SEQ ID NO:75;

[0639] KRAB, for example, a sequence as shown in SEQ ID NO:45;

[0640] BPSV40 NLS, for example, the sequence shown in SEQ ID NO:2859.

[0641] The second fusion of the V22 scheme may include the following components from the N end to the C end, which may be directly or indirectly connected to each other: NLS (e.g., SEQ ID NO:2858), DNMT3A (e.g., SEQ ID NO:2860), DNMT3L-CD (e.g., SEQ ID NO:41), NLS (e.g., SEQ ID NO:2857), TALE HPT15 (e.g., SEQ ID NO:883), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857), multiple GFP11 (e.g., SEQ ID NO:72), for example, there may be 2, 3, 4, 5, 6, 7 or 8 GFP11, each of which may be directly or indirectly connected to each other, and NLS (e.g., SEQ ID NO:2859).

[0642] Furthermore, the second fusion component of the V22 scheme, from the N-end to the C-end, may include the following components, which may be directly or indirectly connected to each other:

[0643] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0644] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0645] DNMT3L-CD, for example, a sequence as shown in SEQ ID NO:41;

[0646] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0647] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0648] TALE HPT15, for example, the sequence shown in SEQ ID NO:883;

[0649] Multiple (e.g., 2) sv40 NLS, for example, the sequence shown in SEQ ID NO:2857;

[0650] Multiple (e.g., 7) GFP11, for example, the sequence shown in SEQ ID NO:72, each GFP11 is connected by a adapter (e.g., the sequence shown in SEQ ID NO:2856);

[0651] BPSV40 NLS, for example, the sequence shown in SEQ ID NO:2859.

[0652] The V23 scheme: GFP1-10-KRAB-NLS-2a-NLS-DNMT3A-DNMT3L-NLS-TALE HPT15-2×NLS-1×GFP11-NLS, can include the following components from the N-terminus to the C-terminus, and these components can be directly or indirectly connected to each other:

[0653] GFP1-10 (e.g., SEQ ID NO:75), KRAB (e.g., SEQ ID NO:45), NLS (e.g., SEQ ID NO:2859), NLS (e.g., SEQ ID NO:2858), DNMT3A (e.g., SEQ ID NO:2860), DNMT3L-CD (e.g., SEQ ID NO:41), NLS (e.g., SEQ ID NO:2857), TALE HPT15 (e.g., SEQ ID NO:883), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857), GFP11 (e.g., SEQ ID NO:72), NLS (e.g., SEQ ID NO:2859).

[0654] Furthermore, the V23 solution can include the following components from the N-end to the C-end, and these components can be directly or indirectly connected to each other:

[0655] GFP1-10, for example, the sequence shown in SEQ ID NO:75;

[0656] KRAB, for example, a sequence as shown in SEQ ID NO:45;

[0657] BPSV40 NLS, for example, a sequence as shown in SEQ ID NO:2859;

[0658] P2A, for example, a sequence as shown in SEQ ID NO:2864;

[0659] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0660] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0661] DNMT3L-CD, for example, a sequence as shown in SEQ ID NO:41;

[0662] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0663] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0664] TALE HPT15, for example, the sequence shown in SEQ ID NO:883;

[0665] Multiple (e.g., 2) sv40 NLS, such as the sequence shown in SEQ ID NO:2857;

[0666] GFP11, for example, the sequence shown in SEQ ID NO:72;

[0667] BPSV40 NLS, for example, the sequence shown in SEQ ID NO:2859.

[0668] The first fusion of the V23 scheme may include the following components from the N-terminus to the C-terminus, which may be directly or indirectly connected to each other: GFP1-10 (e.g., SEQ ID NO:75), KRAB (e.g., SEQ ID NO:45), and NLS (e.g., SEQ ID NO:2859).

[0669] Furthermore, the first fusion component of the V23 scheme, from the N-end to the C-end, may include the following components, which may be directly or indirectly connected to each other:

[0670] GFP1-10, for example, the sequence shown in SEQ ID NO:75;

[0671] KRAB, for example, a sequence as shown in SEQ ID NO:45;

[0672] BPSV40 NLS, for example, the sequence shown in SEQ ID NO:2859.

[0673] The second fusion of the V23 scheme may include the following components from the N end to the C end, which may be directly or indirectly connected to each other: NLS (e.g., SEQ ID NO:2858), DNMT3A (e.g., SEQ ID NO:2860), DNMT3L-CD (e.g., SEQ ID NO:41), NLS (e.g., SEQ ID NO:2857), TALE HPT15 (e.g., SEQ ID NO:883), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857), GFP11 (e.g., SEQ ID NO:72), and NLS (e.g., SEQ ID NO:2859).

[0674] Furthermore, the second fusion component of the V23 scheme, from the N-end to the C-end, may include the following components, which may be directly or indirectly connected to each other:

[0675] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0676] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0677] DNMT3L-CD, for example, a sequence as shown in SEQ ID NO:41;

[0678] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0679] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0680] TALE HPT15, for example, the sequence shown in SEQ ID NO:883;

[0681] Multiple (e.g., 2) sv40 NLS, such as the sequence shown in SEQ ID NO:2857;

[0682] GFP11, for example, the sequence shown in SEQ ID NO:72;

[0683] BPSV40 NLS, for example, the sequence shown in SEQ ID NO:2859.

[0684] The V24 scheme: NLS-7×GFP11-NLS-NLS-TALE HPT15-2×NLS-ZIM3-2a-NLS-DNMT3A-hDNMT3L-NLS-GFP1-10, which can include the following components from the N-terminus to the C-terminus, and these components can be directly or indirectly connected to each other:

[0685] NLS (e.g., SEQ ID NO: 2858), multiple GFP11 (e.g., SEQ ID NO: 72) (e.g., 2, 3, 4, 5, 6, 7 or 8 GFP11, each GFP11 directly or indirectly connected to each other), NLS (e.g., SEQ ID NO: 2859), NLS (e.g., SEQ ID NO: 2857), TALE HPT15 (e.g., SEQ ID NO: 883), NLS (e.g., SEQ ID NO: 2857), NLS (e.g., SEQ ID NO: 2857), Zim3 Krab (e.g., SEQ ID NO: 2861), NLS (e.g., SEQ ID NO: 2858), DNMT3A (e.g., SEQ ID NO: 2860), hDNMT3L (e.g., SEQ ID NO: 42), NLS (e.g., SEQ ID NO: 2857), GFP1-10 (e.g., SEQ ID NO: 75).

[0686] Furthermore, the V24 solution can include the following components from the N-end to the C-end, and these components can be directly or indirectly connected to each other:

[0687] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0688] Multiple (e.g., 7) GFP11s, such as the sequence shown in SEQ ID NO:72, are linked together by adapters (e.g., the sequence shown in SEQ ID NO:2856);

[0689] BPSV40 NLS, for example, a sequence as shown in SEQ ID NO:2859;

[0690] Linker 26-GS linker, for example, the sequence shown in SEQ ID NO:2871;

[0691] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0692] TALE HPT15, for example, the sequence shown in SEQ ID NO:883;

[0693] Multiple (e.g., two) sv40 NLS, for example, the sequence shown in SEQ ID NO:2857;

[0694] Linker10-XTEN 16 linker 2, for example, the sequence shown in SEQ ID NO:89;

[0695] Zim3 Krab, for example, the sequence shown in SEQ ID NO:2861;

[0696] P2A, for example, a sequence as shown in SEQ ID NO:2864;

[0697] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0698] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0699] hDNMT3L, for example, a sequence as shown in SEQ ID NO:42;

[0700] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0701] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0702] GFP1-10, for example, the sequence shown in SEQ ID NO:75.

[0703] The first fusion of the V24 scheme may include the following components from the N-terminus to the C-terminus, which may be directly or indirectly connected to each other: NLS (e.g., SEQ ID NO: 2858), multiple GFP11 (e.g., SEQ ID NO: 72) (e.g., 2, 3, 4, 5, 6, 7 or 8 GFP11, each of which is directly or indirectly connected to each other), NLS (e.g., SEQ ID NO: 2859), NLS (e.g., SEQ ID NO: 2857), TALE HPT15 (e.g., SEQ ID NO: 883), NLS (e.g., SEQ ID NO: 2857), NLS (e.g., SEQ ID NO: 2857), and Zim3 Krab (e.g., SEQ ID NO: 2861).

[0704] Furthermore, the first fusion component of the V24 scheme, from the N-end to the C-end, may include the following components, which may be directly or indirectly connected to each other:

[0705] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0706] Multiple (e.g., 7) GFP11s, such as the sequence shown in SEQ ID NO:72, are linked together by adapters (e.g., the sequence shown in SEQ ID NO:2856);

[0707] BPSV40 NLS, for example, a sequence as shown in SEQ ID NO:2859;

[0708] Linker 26-GS linker, for example, the sequence shown in SEQ ID NO:2871;

[0709] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0710] TALE HPT15, for example, the sequence shown in SEQ ID NO:883;

[0711] Multiple (e.g., two) sv40 NLS, for example, the sequence shown in SEQ ID NO:2857;

[0712] Linker10-XTEN 16 linker 2, for example, the sequence shown in SEQ ID NO:89;

[0713] Zim3 Krab, for example, the sequence shown in SEQ ID NO:2861.

[0714] The second fusion of the V24 scheme may include the following components from the N-terminus to the C-terminus, which may be directly or indirectly connected to each other: DNMT3A (e.g., SEQ ID NO:2860), hDNMT3L (e.g., SEQ ID NO:42), and GFP1-10 (e.g., SEQ ID NO:75).

[0715] Furthermore, the second fusion component of the V24 scheme, from the N-end to the C-end, may include the following components, which may be directly or indirectly connected to each other:

[0716] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0717] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0718] hDNMT3L, for example, a sequence as shown in SEQ ID NO:42;

[0719] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0720] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0721] GFP1-10, for example, the sequence shown in SEQ ID NO:75.

[0722] The V28 solution: NLS-DNMT3A-DNMT3L-scFv-2a-NLS-1×GCN4-NLS-TALE HPT21-NLS-NLS-KRAB, can include the following components from the N-terminal to the C-terminal, and these components can be directly or indirectly connected to each other:

[0723] NLS (e.g., SEQ ID NO:2858), DNMT3A (e.g., SEQ ID NO:2860), DNMT3L-CD (e.g., SEQ ID NO:41), scFv (e.g., SEQ ID NO:2863), NLS (e.g., SEQ ID NO:2858), GCN4 (e.g., SEQ ID NO:71), NLS (e.g., SEQ ID NO:2857), TALE HPT21 (e.g., SEQ ID NO:819), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857), KRAB (e.g., SEQ ID NO:45).

[0724] Furthermore, the V28 solution can include the following components from the N-end to the C-end, and these components can be directly or indirectly connected to each other:

[0725] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0726] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0727] DNMT3L-CD, for example, a sequence as shown in SEQ ID NO:41;

[0728] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0729] scFv, for example, a sequence as shown in SEQ ID NO:2863;

[0730] P2A, for example, a sequence as shown in SEQ ID NO:2864;

[0731] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0732] GCN4, for example, the sequence shown in SEQ ID NO:71;

[0733] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0734] TALE HPT21, for example, the sequence shown in SEQ ID NO:819;

[0735] Multiple (e.g., 2) sv40 NLS, for example, the sequence shown in SEQ ID NO:2857;

[0736] Linker10-XTEN 16 linker 2, for example, the sequence shown in SEQ ID NO:89;

[0737] KRAB, for example, the sequence shown in SEQ ID NO:45.

[0738] The first fusion of the V28 scheme may include the following components from the N end to the C end, which may be directly or indirectly connected to each other: NLS (e.g., SEQ ID NO:2858), DNMT3A (e.g., SEQ ID NO:2860), hDNMT3L-CD (e.g., SEQ ID NO:41), and scFv (e.g., SEQ ID NO:2863).

[0739] Furthermore, the first fusion component of the V28 solution, from the N-end to the C-end, may include the following components, which may be directly or indirectly connected to each other:

[0740] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0741] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0742] DNMT3L-CD, for example, a sequence as shown in SEQ ID NO:41;

[0743] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0744] scFv, for example, the sequence shown in SEQ ID NO:2863.

[0745] The second fusion of the V28 scheme may include the following components from the N end to the C end, which may be directly or indirectly connected to each other: NLS (e.g., SEQ ID NO:2858), GCN4 (e.g., SEQ ID NO:71), NLS (e.g., SEQ ID NO:2857), TALE HPT21 (e.g., SEQ ID NO:819), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857), and KRAB (e.g., SEQ ID NO:45).

[0746] Furthermore, the second fusion component of the V28 scheme, from the N-terminal to the C-terminal, may include the following components, which may be directly or indirectly connected to each other:

[0747] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0748] GCN4, for example, the sequence shown in SEQ ID NO:71;

[0749] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0750] TALE HPT21, for example, the sequence shown in SEQ ID NO:819;

[0751] Multiple (e.g., 2) sv40 NLS, for example, the sequence shown in SEQ ID NO:2857;

[0752] Linker10-XTEN 16 linker 2, for example, the sequence shown in SEQ ID NO:89;

[0753] KRAB, for example, the sequence shown in SEQ ID NO:45.

[0754] The V31 solution: NLS-DNMT3A-DNMT3L-scFv-2a-NLS-1×GCN4-NLS-TALE HPT25-NLS-NLS-KRAB, can include the following components from the N-terminal to the C-terminal, and these components can be directly or indirectly connected to each other:

[0755] NLS (e.g., SEQ ID NO:2858), DNMT3A (e.g., SEQ ID NO:2860), DNMT3L-CD (e.g., SEQ ID NO:41), scFv (e.g., SEQ ID NO:2863), NLS (e.g., SEQ ID NO:2858), GCN4 (e.g., SEQ ID NO:71), NLS (e.g., SEQ ID NO:2857), TALE HPT25 (e.g., SEQ ID NO:958), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857), KRAB (e.g., SEQ ID NO:45).

[0756] Furthermore, the V31 solution can include the following components from the N-end to the C-end, and these components can be directly or indirectly connected to each other:

[0757] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0758] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0759] DNMT3L-CD, for example, a sequence as shown in SEQ ID NO:41;

[0760] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0761] scFv, for example, a sequence as shown in SEQ ID NO:2863;

[0762] P2A, for example, a sequence as shown in SEQ ID NO:2864;

[0763] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0764] GCN4, for example, the sequence shown in SEQ ID NO:71;

[0765] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0766] TALE HPT25, for example, the sequence shown in SEQ ID NO:958;

[0767] HA tag, for example, a sequence as shown in SEQ ID NO:2862;

[0768] Linker5-GS linker, for example, the sequence shown in SEQ ID NO:84;

[0769] Multiple (e.g., 2) sv40 NLS, for example, the sequence shown in SEQ ID NO:2857;

[0770] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0771] Linker10-XTEN 16 linker 2, for example, the sequence shown in SEQ ID NO:89;

[0772] KRAB, for example, the sequence shown in SEQ ID NO:45.

[0773] The first fusion of the V31 scheme may include the following components from the N end to the C end, which may be directly or indirectly connected to each other: NLS (e.g., SEQ ID NO:2858), DNMT3A (e.g., SEQ ID NO:2860), hDNMT3L-CD (e.g., SEQ ID NO:41), and scFv (e.g., SEQ ID NO:2863).

[0774] Furthermore, the first fusion component of the V31 scheme, from the N-end to the C-end, may include the following components, which may be directly or indirectly connected to each other:

[0775] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0776] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0777] DNMT3L-CD, for example, a sequence as shown in SEQ ID NO:41;

[0778] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0779] scFv, for example, the sequence shown in SEQ ID NO:2863.

[0780] The second fusion of the V31 scheme may include the following components from the N end to the C end, which may be directly or indirectly connected to each other: NLS (e.g., SEQ ID NO:2858), GCN4 (e.g., SEQ ID NO:71), NLS (e.g., SEQ ID NO:2857), TALE HPT25 (e.g., SEQ ID NO:958), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857), and KRAB (e.g., SEQ ID NO:45).

[0781] Furthermore, the second fusion component of the V31 scheme, from the N-end to the C-end, may include the following components, which may be directly or indirectly connected to each other:

[0782] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0783] GCN4, for example, the sequence shown in SEQ ID NO:71;

[0784] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0785] TALE HPT25, for example, the sequence shown in SEQ ID NO:958;

[0786] HA tag, for example, a sequence as shown in SEQ ID NO:2862;

[0787] Linker5-GS linker, for example, the sequence shown in SEQ ID NO:84;

[0788] Multiple (e.g., 2) sv40 NLS, for example, the sequence shown in SEQ ID NO:2857;

[0789] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0790] Linker10-XTEN 16 linker 2, for example, the sequence shown in SEQ ID NO:89;

[0791] KRAB, for example, the sequence shown in SEQ ID NO:45.

[0792] V35 solution

[0793] V35 solution: NLS-scFv-ZIM3-2a-NLS-DNMT3A-hDNMT3L-NLS-TALE HPT15-2×NLS-1×GCN4-NLS, which can include the following components from the N end to the C end, and the following components can be directly or indirectly connected to each other:

[0794] NLS (e.g., SEQ ID NO: 2858), scFv (e.g., SEQ ID NO: 2863), Zim3 Krab (e.g., SEQ ID NO: 2861), NLS (e.g., SEQ ID NO: 2858), DNMT3A (e.g., SEQ ID NO: 2860), hDNMT3L (e.g., SEQ ID NO: 42), NLS (e.g., SEQ ID NO: 2857), TALE HPT15 (e.g., SEQ ID NO: 883), NLS (e.g., SEQ ID NO: 2857), NLS (e.g., SEQ ID NO: 2857), GCN4 (e.g., SEQ ID NO: 71), NLS (e.g., SEQ ID NO: 2859).

[0795] Furthermore, the V35 solution from the N-end to the C-end can include the following components, which can be directly or indirectly connected to each other:

[0796] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0797] scFv, for example, a sequence as shown in SEQ ID NO:2863;

[0798] Zim3 Krab, for example, the sequence shown in SEQ ID NO:2861;

[0799] P2A, for example, a sequence as shown in SEQ ID NO:2864;

[0800] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0801] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0802] hDNMT3L, for example, a sequence as shown in SEQ ID NO:42;

[0803] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0804] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0805] TALE HPT15, for example, the sequence shown in SEQ ID NO:883;

[0806] Multiple (e.g., two) sv40 NLS, for example, the sequence shown in SEQ ID NO:2857;

[0807] GCN4, for example, SEQ ID NO:71;

[0808] BPSV40 NLS, for example, the sequence shown in SEQ ID NO:2859.

[0809] The first fusion of the V35 scheme may include the following components from the N end to the C end, which may be directly or indirectly connected to each other: NLS (e.g., SEQ ID NO:2858), scFv (e.g., SEQ ID NO:2863), and Zim3 Krab (e.g., SEQ ID NO:2861).

[0810] Furthermore, the first fusion component of the V35 solution, from the N-terminal to the C-terminal, may include the following components, which may be directly or indirectly connected to each other:

[0811] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0812] scFv, for example, a sequence as shown in SEQ ID NO:2863;

[0813] Zim3 Krab, for example, the sequence shown in SEQ ID NO:2861.

[0814] The second fusion of the V35 scheme may include the following components from the N end to the C end, which may be directly or indirectly connected to each other: NLS (e.g., SEQ ID NO:2858), DNMT3A (e.g., SEQ ID NO:2860), hDNMT3L (e.g., SEQ ID NO:42), NLS (e.g., SEQ ID NO:2857), TALE HPT15 (e.g., SEQ ID NO:883), NLS (e.g., SEQ ID NO:2857), NLS (e.g., SEQ ID NO:2857), GCN4 (e.g., SEQ ID NO:71), BPSV40 NLS (e.g., the sequence shown in SEQ ID NO:2859).

[0815] Furthermore, the second fusion of the V35 scheme (SEQ ID NO:2845) may include the following components from the N-terminus to the C-terminus, and these components may be directly or indirectly connected to each other:

[0816] c-myc NLS, for example, the sequence shown in SEQ ID NO:2858;

[0817] DNMT3A, for example, a sequence as shown in SEQ ID NO:2860;

[0818] hDNMT3L, for example, a sequence as shown in SEQ ID NO:42;

[0819] linker4-XTEN 80linker 1, for example, the sequence shown in SEQ ID NO:83;

[0820] sv40 NLS, for example, a sequence as shown in SEQ ID NO:2857;

[0821] TALE HPT15, for example, the sequence shown in SEQ ID NO:883;

[0822] Multiple (e.g., two) sv40 NLS, for example, the sequence shown in SEQ ID NO:2857;

[0823] GCN4, for example, SEQ ID NO:71;

[0824] BPSV40 NLS, for example, the sequence shown in SEQ ID NO:2859.

[0825] In summary, this application may provide the following amino acid sequences of the first or second fusion compound (bold—DNMT3A, underlined bold—DNMT3L, italic—recruitment domain, <underlined bold>—<transcriptional regulatory domain>, underlined bold italic—TALE):

[0826] 1) The complex peptide scFv-ZIM3KRAB-2a-DNMT3A-(human)DNMT3L-TALE HPT15-10×GCN4 (V15 protocol, SEQ ID NO:15; TALE domain sequence is SEQ ID NO:883), which contains the fusion product: a) scFv-ZIM3KRAB (SEQ ID NO:99):

[0827] and b) DNMT3A-(human)DNMT3L-TALE HPT15-10×GCN4 (SEQ ID NO:100):

[0828] 2) The complex peptide KRAB-scFv-2a-DNMT3A-DNMT3L-TALE HPT15-2×GCN4 (V16 protocol, SEQ ID NO:16; TALE domain sequence is SEQ ID NO:883), which includes the fusion product: a) KRAB-scFv (SEQ ID NO:101):

[0829] and b) DNMT3A-DNMT3L-TALE HPT15-2×GCN4 (SEQ ID NO:102):

[0830] 3) The complex peptide DNMT3A-DNMT3L-scFv-2a-1×GCN4-TALE HPT15-KRAB (V17 protocol, SEQ ID NO:17; TALE domain sequence is SEQ ID NO:883), comprising the fusion product: a) DNMT3A-DNMT3L-scFv (V13 protocol, SEQ ID NO:13):

[0831] and b) 1×GCN4-TALE HPT15-KRAB (SEQ ID NO:103):

[0832] 4) The complex peptide DNMT3A-(human)DNMT3L-scFv-2a-3×GCN4-TALE HPT15-ZIM3 KRAB (V18 protocol, SEQ ID NO:18; TALE domain sequence is SEQ ID NO:883), which includes the fusion product: a) DNMT3A-(human)DNMT3L-scFv (SEQ ID NO:104):

[0833] and b) 3×GCN4-TALE HPT15-ZIM3 KRAB (SEQ ID NO:105):

[0834] 5) The complex peptide 3×GCN4-TALE HPT15-ZIM3 KRAB-2a-DNMT3A-(human)DNMT3L-scFv (V19 protocol, SEQ ID NO:19; TALE domain sequence is SEQ ID NO:883), which contains the fusion product: a) 3×GCN4-TALE HPT15-ZIM3 KRAB (SEQ ID NO:2846):

[0835] and b) DNMT3A-(human)DNMT3L-scFv (SEQ ID NO:2847):

[0836] 6) The complex peptide scFv-(human)DNMT3L-DNMT3A-2a-3×GCN4-TALE HPT15-KRAB (V20 protocol, SEQ ID NO:20; TALE domain sequence is SEQ ID NO:883), comprising the fusion product: a) scFv-(human)DNMT3L-DNMT3A (SEQ ID NO:106):

[0837] and b) 3×GCN4-TALE HPT15-KRAB (V14 scheme, SEQ ID NO:14):

[0838] 7) A complex peptide 1×GCN4-ZIM3 KRAB-2a-DNMT3A-(human)DNMT3L-TALE HPT15-scFv (V21 protocol, SEQ ID NO:21; TALE domain sequence is SEQ ID NO:883), comprising the fusion product: a) 1×GCN4-ZIM3

[0839] and b) DNMT3A-(human)DNMT3L-TALE HPT15-scFv (SEQ ID NO: 108):

[0840] 8) The complex peptide GFP1-10-KRAB-2a-DNMT3A-DNMT3L-TALE HPT15-7×GFP11 (V22 protocol, SEQ ID NO:22; TALE domain sequence is SEQ ID NO:883), which contains the fusion product: a) GFP1-10-KRAB (SEQ ID NO:109):

[0841] and b) DNMT3A-DNMT3L-TALE HPT15-7×GFP11 (SEQ ID NO: 110)

[0842] 9) The complex peptide GFP1-10-KRAB-2a-DNMT3A-DNMT3L-TALE HPT15-1×GFP11 (V23 protocol, SEQ ID NO:23; TALE domain sequence is SEQ ID NO:883), comprising the following fusions: a) fusion a) shown in GFP1-10-KRAB (see above 8), i.e. SEQ ID NO:109) and b) DNMT3A-DNMT3L-TALE HPT15-1×GFP11 (SEQ ID NO:111):

[0843] 10) The complex peptide 7×GFP11-TALE HPT15-ZIM3 KRAB-2a-DNMT3A-(human)DNMT3L-GFP1-10 (V24 protocol, SEQ ID NO:24; TALE domain sequence is SEQ ID NO:883), comprising the fusion product: a) 7×GFP11-TALE HPT15-ZIM3 KRAB (SEQ ID NO:112):

[0844] and b) DNMT3A-(human)DNMT3L-GFP1-10 (SEQ ID NO: 113):

[0845] 11) The complex peptide DNMT3A-DNMT3L-scFv-2a-1×GCN4-TALE HPT21-KRAB (V28 scheme, SEQ ID NO:28; TALE domain sequence is SEQ ID NO:819), comprising the following fusions: a) the V13 scheme shown in DNMT3A-DNMT3L-scFv (see above 3), i.e. SEQ ID NO:13) and b) 1×GCN4-TALE HPT21-KRAB (SEQ ID NO:114):

[0846] 12) The complex peptide DNMT3A-DNMT3L-scFv-2a-1×GCN4-TALE HPT25-KRAB (V31 scheme, SEQ ID NO:31; TALE domain sequence is SEQ ID NO:958), comprising the following fusions: a) the V13 scheme shown in DNMT3A-DNMT3L-scFv (see above 3), i.e. SEQ ID NO:13) and b) 1×GCN4-TALE HPT25-KRAB (SEQ ID NO:115):

[0847] 13) The complex peptide scFv-ZIM3 KRAB-2a-DNMT3A-(human)DNMT3L-TALE-1×GCN4 (V35 scheme, SEQ ID NO:2842; TALE domain sequence is SEQ ID NO:883), which includes fusion products: a) fusion product a) shown in scFv-ZIM3 KRAB (see above 1), i.e. SEQ ID NO:99 or 2872, where the difference between sequence 99 and 2872 is that the starting amino acid is M or MV, and the other parts are structurally identical) and b) DNMT3A-(human)DNMT3L-TALE-1×GCN4 (SEQ ID NO:2845):

[0848] 14) Fusion scFv-KRAB (V11 scheme, SEQ ID NO:11):

[0849] 15) Fusion DNMT3A-DNMT3L-TALE HPT15-4×GCN4 (V12 scheme, SEQ ID NO:12; TALE field sequence is SEQ ID NO:883):

[0850] In some specific embodiments, the TALE sequence (underlined bold italic portion) in the fusion sequence of the above-described example complex peptide may also be replaced with an amino acid sequence selected from any one of SEQ ID NOs:35-38 and 661-2840, 2865, 2867-2868.

[0851] The structure of the epigenetic editing agent provided in this application may also be selected from (but is 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, terms such as "nucleic acid binding domain", "DNA binding domain", "CasN", "dCas9", etc., can be replaced with the TALE domain of the amino acid sequence contained in any one of SEQ ID NO:35-38 and 661-2840 provided in this application), the disclosures of the above-mentioned patent applications are hereby incorporated by reference.

[0852] 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.

[0853] Example

[0854] Example 1

[0855] The epigenetic editing agent of this application exhibits high inhibition efficiency of PCSK9 gene expression in mice.

[0856] Different versions of the tool were transcribed into mRNA in vitro and prepared as LNPs according to the reference (LNP cited in: Musunuru, K., Chadwick, AC, Mizoguchi, T. et al. In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates. Nature 593, 429–434 (2021)). The prepared LNPs were injected into PCSK9 humanized mice via tail vein at doses of 1 mg / kg body weight or 3 mg / kg body weight. Blood was collected from the cheeks of mice 4 or 7 days after injection, and the PCSK9 protein content in the blood was measured by ELISA. The PBS group served as a control group injected with an equal volume of PBS. Most tools showed increased PCSK9 gene expression inhibition efficiency compared to the PBS control group, with the highest reaching ~99%.

[0857] Table 2 Information on appearance editing tools

[0858] Table 3 shows the appearance editing tool information in Figure 1 (injection dose: 3 mg, injection days: 4 days).

[0859] Table 4 shows the appearance editing tool information in Figure 2 (injection dose: 3 mg, injection days: 4 days).

[0860] Table 5 shows the appearance editing tool information in Figure 3 (injection dose: 1 mg, injection days: 4 days). Note: The amino acid sequences and encoding mRNA sequences of the tools in groups V26-223 are shown in SEQ ID NO:26 and 2844, respectively; the amino acid sequences and encoding mRNA sequences of the tools in group V5-223 are shown in SEQ ID NO:5 and 2841, respectively; and the amino acid sequences and encoding mRNA sequences of the tools in group V35-223 are shown in SEQ ID NO:2842 and 2843, respectively.

[0861] Table 6 shows the appearance editing tool information in Figure 4 (injection dose: 1 mg, injection days: 4 days).

[0862] Table 7 shows the appearance editing tool information in Figure 5 (injection dose: 1 mg, injection days: 7 days).

[0863] Table 8 shows the appearance editing tool information in Figure 6 (injection dose: 1 mg, injection days: 7 days).

[0864] Example 2: Drugability Study of V35-223

[0865] To further elucidate the drug potential of the V35-223 molecule, the levels of PCSK9 in the blood of primates (cynomolgus monkeys) and the levels of LDL-c in the blood (venous blood collected 6 hours after fasting) were persistently measured after intravenous administration of 1.5 mg / kg of V35-223. The ELISA kit was purchased from R&D Systems, catalog number: DPC900. The assay method was performed according to the kit instructions. LDL-c was measured using a biochemical analyzer. Using the average PCSK9 and LDL-c levels on day-2 and day-1 before administration as a baseline, during the approximately 300-day study, PCSK9 maintained an inhibitory efficiency of approximately 78% (see Table 8 and Figure 7), and LDL-c levels decreased by approximately 60% (see Table 9 and Figure 8). This demonstrates that the V35-223 molecule exhibits good in vivo inhibitory efficiency and persistence, making it a potential drug for heterozygous hypercholesterolemia.

[0866] Table 9. PCSK9 levels in primate blood after V35-223 administration.

[0867] Table 10 LDL-c levels in primate blood after administration of V35-223

Claims

1. An epigenetic editing agent comprising a transcription activator-like effector (TALE) domain, at least one epigenetic modification domain, and at least one transcriptional regulatory domain, wherein: 1) The TALE domain, the at least one epigenetic modification domain, and the at least one transcriptional regulatory domain are directly or indirectly connected; or 2) The TALE domain, the at least one epigenetic modification domain, and at least one recruitment domain A are directly or indirectly connected to form a first fusion, and the at least one transcriptional regulatory domain is directly or indirectly connected to at least one recruitment domain A' to form a second fusion; or 3) The TALE domain, the at least one transcriptional regulatory domain, and the at least one recruitment domain A are directly or indirectly connected to form a first fusion, and the at least one epigenetic modification domain is directly or indirectly connected to the at least one recruitment domain A' to form a second fusion; and: The recruitment domains A and A' described in 2) and 3) can interact to enable a fusion of the first fusion and the second fusion, or a portion thereof, to be recruited to the vicinity of the other fusion; The TALE domain can specifically bind to target nucleotide sequences on the PCSK9 gene and / or the regulatory elements of the PCSK9 gene.

2. The epigenetic editing agent according to claim 1, wherein the target nucleotide sequence is selected from any one of SEQ ID NO:116-660.

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

4. The epigenetic editing agent according to any one of claims 1-3, wherein the TALE domain comprises the amino acid sequence of any one of SEQ ID NO:35-38 and 661-2840, 2865, and 2867-2868.

5. The epigenetic editing agent according to any one of claims 1-4, wherein the epigenetic modification domain is selected from: DNA deamination activity, DNA methyltransferase activity, DNA demethylase activity, DNA amination activity, DNA oxidation 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 deubiquitination activity.

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

7. The epigenetic editing agent according to claim 6, wherein the DNA methyltransferase is selected from DNMT3A, DNMT3B, DNMT3C, DNMT1, DNMT2 and DNMT3L.

8. The epigenetic editing agent according to any one of claims 1-7, wherein the epigenetic modification domain comprises a plurality of DNA methyltransferases and / or their functionally active fragments, and the plurality of DNA methyltransferases and / or their functionally active fragments are linked by adapter sequences.

9. The epigenetic editing agent according to 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 according to any one of claims 6-8, wherein the DNA methyltransferase comprises the amino acid sequence shown in any one of SEQ ID NOs:39-44, 2860.

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

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

13. The epigenetic editing agent according to claim 12, wherein the transcriptional repressor domain is selected from: KRAB, ZIM3 KRAB, ZNF680, ZNF554, ZNF264, ZNF582, ZNF324, ZNF669, ZNF354A, ZNF82, ZNF595, ZN F419, ZNF566, ZIM2, EHMT2, SUV39H1, ZFPM1, TRIM28, EZH2, MXD1, SID, LSD1, HP1a, HDA C3, ZNF436, ZNF257, ZNF675, ZNF490, ZNF320, ZNF331, ZNF816, ZNF41, ZNF189, ZNF52 8. ZNF543, ZNF140, ZNF610, ZNF350, ZNF8, ZNF30, ZNF98, ZNF677, ZNF596, ZNF214, ZNF 37A, ZNF34, ZNF250, ZNF547, ZNF273, ZFP82, ZNF224, ZNF33A, ZNF45, ZNF175, ZNF184 , ZFP28-1, ZFP28-2, ZNF18, ZNF213, ZNF394, ZFP1, ZFP14, ZNF416, ZNF557, ZNF729, ZN F254, ZNF764, ZNF785, ZNF10, CBX5, RYBP, YAF2, MGA, CBX1, SCMH1, MPP8, SUMO3, HERC2, BIN1, PCGF2, TOX, FOXA1, FOXA2, IRF2BP1, IRF2BP2, IRF2BPLIRF-2BP1_2N-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, ZN8 91, ZN337, Z705G, ZN529, ZN729, ZN419, Z705A, ZN302, ZN486, ZN621, ZN688, ZN33A, ZN554, ZN878, ZN772, ZN224, ZN18 4. 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 their functionally active fragments.

14. The epigenetic editing agent according to claim 12 or 13, wherein the transcriptional repressor domain comprises the amino acid sequence shown in any one of SEQ ID NOs:45-70, 2861.

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

16. The epigenetic editing agent according to claim 15, wherein the zinc finger protein-based transcription factor is Related inhibitors (KRAB) or KRAB domains derived from ZIM3 (ZIM3 KRAB).

17. The epigenetic editing agent according to any one of claims 1-16, wherein the transcriptional regulatory domain comprises two or more of the zinc finger protein-based transcription factors or their functionally active fragments, wherein the two or more zinc finger protein-based transcription factors are of the same type or different types.

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

19. The epigenetic editing agent according to claim 18, wherein the adapter sequence is an XTEN adapter sequence.

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

21. The epigenetic editing agent according to claim 20, wherein the histone modification domain is selected from: EZH2, HDAC3, HDAC1, EHMT2, PRMT1, PRMT5, SETDB1, hSIRT1, HP1a, LSD1, and their functionally active fragments.

22. The epigenetic editing agent according to any one of claims 1-21, wherein it is a fusion peptide.

23. The epigenetic editing agent according to claim 22, wherein the epigenetic modification domain and the transcriptional regulatory domain are both located at the N-terminus or C-terminus of the TALE domain.

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

25. The epigenetic editing agent according to any one of claims 22-24, wherein the fusion peptide is sequentially linked from the N-terminus to the C-terminus to: 1) The epigenetic modification domain, the transcriptional regulatory domain, and the TALE domain; or 2) The transcriptional regulatory domain, the epigenetic modification domain, and the TALE domain; or 3) The TALE domain, the epigenetic modification domain, and the transcriptional regulatory domain; or 4) The TALE domain, the transcriptional regulatory domain, and the epigenetic modification domain; or 5) The epigenetic modification domain, the TALE domain, and the transcriptional regulatory domain; or 6) The transcriptional regulatory domain, the TALE domain, and the epigenetic modification domain.

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

27. The epigenetic editing agent according to any one of claims 22-26, wherein 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, DNMT3 A-DNMT3L-TALE-HDAC3, DNMT3A-DNMT3L-TALE-HP1a, DNMT3A-DNMT3L-TALE-KRAB, DNMT3A-DNMT3L-TALE-ZIM3 KRAB, among which, - indicates that the structural domains of the fusion are directly and / or indirectly connected, and that the structural domains are arranged in order from the N end to the C end.

28. The epigenetic editing agent according to any one of claims 1-21, wherein it is a complex peptide comprising the first fusion compound and the second fusion compound.

29. The epigenetic editing agent according to claim 28, wherein the first fusion comprises, from the N-terminus to the C-terminus, an epigenetic modification domain, a TALE domain, and a recruitment domain A.

30. The epigenetic editing agent according to claim 28 or 29, wherein the second fusion comprises, from the N-terminus to the C-terminus, a transcriptional repressor domain and a recruitment domain A', or from the N-terminus to the C-terminus, a recruitment domain A' and a transcriptional repressor domain.

31. The epigenetic editing agent according to claim 28, wherein the first fusion comprises, from the N-terminus to the C-terminus, a recruitment domain A, a TALE domain, and a transcriptional repressor domain.

32. The epigenetic editing agent according to claim 28 or 31, wherein the second fusion comprises, from the N-terminus to the C-terminus, an epigenetic modification domain and a recruitment domain A', or from the N-terminus to the C-terminus, a recruitment domain A' and an epigenetic modification domain.

33. The epigenetic editing agent according to any one of claims 28-32, wherein the complex peptide comprises the following characteristics: 1) The first fusion compound contains, from N-terminus to C-terminus, an epigenetic modification domain, a TALE domain, and a recruitment domain A; the second fusion compound contains, from N-terminus to C-terminus, a transcriptional repressor domain and a recruitment domain A'; or 2) The first fusion compound contains, from N-terminus to C-terminus, an epigenetic modification domain, a TALE domain, and a recruitment domain A; the second fusion compound contains, from N-terminus to C-terminus, a recruitment domain A' and a transcriptional repressor domain; or 3) The first fusion compound contains, from N-terminus to C-terminus, a recruitment domain A, a TALE domain, and a transcriptional repressor domain; the second fusion compound contains, from N-terminus to C-terminus, an epigenetic modification domain and a recruitment domain A'; or 4) The first fusion contains, from N-terminus to C-terminus, a recruitment domain A, a TALE domain, and a transcriptional repressor domain, while the second fusion contains, from N-terminus to C-terminus, a recruitment domain A' and an epigenetic modification domain.

34. The epigenetic editing agent according to any one of claims 1-33, wherein the recruitment domain A is selected from one of two groups of domains, and the recruitment domain A' is selected from the other of two groups of domains: 1) Universally controlled non-derepressor protein 4 (GCN4), a GFP11 fragment derived from splitting green fluorescent protein (GFP), or a GVKESLV polypeptide; and 2) Single-chain antibody (scFv), GFP1-10 fragments derived from split green fluorescent protein (GFP), or PDZ protein domain.

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

36. The epigenetic editing agent according to any one of claims 28-35, wherein the complex peptide comprises the following characteristics: 1) One of the first fusion and the second fusion contains DNMT(3A-3L)-TALE-n×GCN4, and the other fusion contains a transcriptional repressor domain -scFv; or 2) One of the first fusion and the second fusion contains DNMT(3A-3L)-TALE-scFv, and the other fusion contains a transcriptional repressor domain -GCN4; or 3) One of the first fusion and the second fusion contains DNMT(3A-3L)-TALE-n×GFP11, and the other fusion contains a transcriptional repressor domain -GFP1-10; or 4) One of the first fusion and the second fusion contains DNMT(3A-3L)-TALE-GFP1-10, and the other fusion contains a transcriptional repressor domain-GFP11; or 5) One of the first fusion and the second fusion contains DNMT(3A-3L)-TALE-n×GCN4, and the other fusion contains an scFv-transcriptional repressor domain; or 6) One of the first fusion and the second fusion contains DNMT(3A-3L)-TALE-scFv, and the other fusion contains a GCN4-transcriptional repressor domain; or 7) One of the first fusion and the second fusion contains DNMT(3A-3L)-TALE-n×GFP11, and the other fusion contains a GFP1-10-transcriptional repressor domain; or 8) One of the first fusion and the second fusion contains DNMT(3A-3L)-TALE-GFP1-10, and the other fusion contains a GFP11-transcriptional repressor domain; or 9) One of the first fusion and the second fusion contains an n×GCN4-TALE-transcriptional repressor domain, and the other fusion contains DNMT(3A-3L)-scFv; or 10) One of the first fusion and the second fusion contains the scFv-TALE-transcriptional repressor domain, and the other fusion contains DNMT(3A-3L)-GCN4; or 11) One of the first fusion and the second fusion contains an n×GFP11-TALE-transcriptional repressor domain, and the other fusion contains DNMT(3A-3L)-GFP1-10; or 12) One of the first fusion and the second fusion contains a GFP1-10-TALE-transcriptional repressor domain, and the other fusion contains DNMT(3A-3L)-GFP11; or 13) One of the first fusion and the second fusion contains an n×GCN4-TALE-transcriptional repressor domain, and the other fusion contains scFv-DNMT(3A-3L); or 14) One of the first fusion and the second fusion contains the scFv-TALE-transcriptional repressor domain, and the other fusion contains GCN4-DNMT(3A-3L); or 15) One of the first fusion and the second fusion contains an n×GFP11-TALE-transcriptional repressor domain, and the other fusion contains GFP1-10-DNMT(3A-3L); or 16) One of the first fusion and the second fusion contains a GFP1-10-TALE-transcriptional repressor domain, and the other fusion contains a GFP11-DNMT(3A-3L); in, DNMT(3A-3L) indicates that DNMT3A and DNMT3L are directly or indirectly connected in any order, and - indicates that the domains at both ends are directly or indirectly connected in order from the N end to the C end; n×GCN4 or n×GFP11 represent n copies of GCN4 connected by the adapter sequence or n copies of GFP11 connected by the adapter sequence, respectively, where n is selected from any integer from 1 to 20.

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

38. The epigenetic editing agent according to any one of claims 1-37, which is capable of providing modification of at least one nucleotide in the vicinity of the PCSK9 gene and / or within the regulatory element of the PCSK9 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 fragment encoding the first fusion compound and a second nucleic acid fragment encoding the second fusion compound, wherein the first nucleic acid fragment and the second nucleic acid fragment are linked by a nucleic acid fragment encoding a cleavage peptide.

41. The nucleic acid according to claim 40, wherein the cleavage peptide is a 2A peptide and / or IRES.

42. The nucleic acid according to claim 41, wherein the 2A peptide is selected from P2A, T2A, E2A and F2A.

43. The nucleic acid according to any one of claims 39-42, wherein it is a recombinant vector.

44. The nucleic acid according to claim 43, wherein the recombinant vector further comprises a non-coding region.

45. The nucleic acid according to claim 44, wherein the non-coding region is selected from introns, regulatory elements, promoters, enhancers, termination sequences, and 5' and 3' untranslated regions.

46. ​​A delivery vector comprising an epigenetic editing agent according to any one of claims 1-38 and / or a nucleic acid according to any one of claims 39-45, and optionally comprising liposomes and / or lipid nanoparticles.

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 vector 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 vector 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 vector of claim 46, the composition of claim 47, and / or the cells of claim 48.

50. A method for regulating the expression of the PCSK9 gene product, the method comprising administering an epigenetic editing agent according to any one of claims 1-38, a nucleic acid according to any one of claims 39-45, a delivery vector according to claim 46, a composition according to claim 47, a cell according to claim 48, and / or a kit according to claim 49.

51. The method of claim 50, wherein the method comprises introducing the epigenetic modifier, the nucleic acid, the delivery vector, the composition, the cell, and / or the kit into cells containing the PCSK9 gene.

52. The method of claim 51, wherein the method comprises contacting the epigenetic modifier, the nucleic acid, the delivery vector, and / or the composition with the PCSK9 gene and / or the regulatory elements of the PCSK9 gene.

53. The method according to claim 52, wherein 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.

54. A method for treating or alleviating a disease or symptom thereof associated with abnormal PCSK9 gene expression and / or abnormal PCSK9 gene activity, the method comprising administering to a subject in need an effective amount of any one of claims 1-38, any one of claims 39-45, any one of claims 46, any one of claims 47, any one of claims 48, and / or any one of claims 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 vector of claim 46, the composition of claim 47, the cell of claim 48, and / or the kit of claim 49 for the preparation of a medicament for the treatment or relief of a disease or condition associated with abnormal PCSK9 gene expression and / or abnormal PCSK9 gene activity.

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