Fusions and their uses

Abstract

The present application further provides a fusion and its use, wherein the fusion comprises a nucleic acid binding domain and one or more effector domains, the effector domains comprising one or more epigenetic modification domains and / or transcriptional regulation domains, and the fusion comprises at least two types of epigenetic modification domains and / or transcriptional regulation domains. The present application also provides a use of the fusion in the manufacture of a product that inhibits expression of a target gene and in the manufacture of a related medicament.

Description

[Technical Field]

[0001] This application relates to the biomedicine field, and in particular to fusions used in gene editing and their uses. [Background technology]

[0002] Abnormalities in genome epigenetic modification are closely related to the occurrence and progression of many diseases, such as common metabolic disorders, cardiovascular diseases, and cancer. Gene epigenetic editing tools can achieve the purpose of regulating the transcription of corresponding genes without altering the gene sequence, and this process does not cause permanent DNA damage or harmful mutations or off-target effects. Furthermore, epigenetic therapy can provide better therapeutic effects by simultaneously regulating the activity of multiple genes, thereby overcoming the shortcomings of gene therapy and providing new prospects for the treatment of such diseases. Meanwhile, the development of gene site-specific modification technology has further enabled targeted epigenetic editing and transcriptional regulation in the natural chromatin environment, particularly the development of CRISPR / Cas9 technology.

[0003] Currently, the design of epigenetic modification and transcriptional regulation is primarily based on the binding of nucleases, with the binding of engineered defective nucleases (dCas9) being the most effective. The design principle is to fuse various epigenetic regulatory effectors to dCas9, and use the nuclease's target DNA binding and binding properties to achieve epigenetic editing at specific genomic sites. These trans-regulatory domains and proteins function by inhibiting RNA polymerase binding to the dCas9 target site in the promoter region or by recruiting endogenous transcriptional fusions. Currently, research into the use of transcriptional regulation and epigenetic site-specific modification technologies to treat diseases caused by epigenetic abnormalities in vivo is still limited, and traditional editing tools have problems such as low transcriptional regulation efficiency and limited scope of modification (e.g., methylation) of target genes. Summary of the Invention

[0004] The present application provides a fusion for gene editing, comprising a nucleic acid binding domain and an effector domain, and uses thereof. The effector domain comprises an epigenetic regulatory modifier and a transcriptional regulator, and the effector domain is linked to the N-terminus, C-terminus, or both the N-terminus and C-terminus of the nucleic acid binding domain. Compared with fusions of the same or similar domains and regulatory elements in the art, the fusion has a higher target gene regulation efficiency. By fusing different epigenetic modifiers and transcriptional regulators to the effector domain, the scope of epigenetic modification of the target gene can be enriched. The fusion of the present application can also be used in the manufacture of products that inhibit the expression of target genes and in the manufacture of pharmaceuticals.

[0005] In one aspect, the present application provides a fusion comprising a nucleic acid binding domain and one or more effector domains, wherein the effector domain comprises one or more of an epigenetic modification domain and / or a transcriptional regulation domain, and wherein the fusion comprises at least two types of epigenetic modification domains and / or transcriptional regulation domains. In some embodiments, the nucleic acid binding domain is a DNA binding domain.

[0006] In some embodiments, the DNA binding domain is selected from a TALE domain, a zinc finger domain, a tetR domain, a large-range nuclease, a Cas protein, an Argonaute (Ago) protein, and homologs or modified forms thereof.

[0007] In some embodiments, the DNA binding domain is capable of binding to a target sequence at a target locus. In some embodiments, the DNA binding domain is capable of binding to a guide RNA. In some embodiments, the guide RNA is capable of specifically recognizing and hybridizing to a target sequence of the target locus.

[0008] In some embodiments, the DNA binding domain is a Cas protein, and the Cas protein is a Class II Cas nuclease. In some embodiments, the Cas proteins are Class II Type II Cas nucleases and Class II Type V Cas nucleases. In some embodiments, the DNA binding domain is a Cas9 protein. In some embodiments, the Cas9 protein is an inactive (dead) Cas protein (dCas9).

[0009] In some embodiments, the nucleic acid binding domain comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-7, 353, and 354. 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. In some embodiments, the epigenetic modification domain comprises a DNA methyltransferase and / or a functionally active fragment thereof.

[0010] In some embodiments, the DNA methyltransferase is selected from DNMT3A, DNMT3B, Dnmt3c, DNMT1, DNMT2, and DNMT3L. In some embodiments, the epigenetic modification domain comprises a plurality of DNA methyltransferases and / or functionally active fragments thereof, and the plurality of DNA methyltransferases and / or functionally active fragments thereof are linked via a linker sequence. In some embodiments, the epigenetic modification domain comprises at least one DNMT3A and at least one DNMT3L.

[0011] In some embodiments, the DNA methyltransferase comprises the amino acid sequence set forth in any one of SEQ ID NOs: 8-21. In some embodiments, the transcriptional regulatory domain is a transcriptional activation domain or a transcriptional repressor domain.

[0012] In some embodiments, the transcriptional repressor domain is selected from the group consisting of KRAB, ZIM3, 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, ZNF 98, ZNF677, ZNF596, ZNF214, ZNF37A, ZNF34, ZNF250, ZNF547, ZNF273, ZFP82, ZNF224, Z NF33A, 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_2 N-terminal domain, HOXA13, HOXB13, HOXC13, HOXA11, HOXC11, HOXC10, HOXA10, HOXB9, HOXA9, ZFP28, ZN334, ZN568, ZN37A, ZN181, ZN510, ZN862, ZN140, ZN208, ZN248, ZN571, ZN699, ZN726, ZIK1, ZNF2, Z705F, ZNF14, ZN471, ZN624, ZNF84, ZNF7, ZN891, ZN337 , Z705G, ZN529, ZN729, ZN419, Z705A, ZN302, ZN486, ZN621, ZN688, ZN33A, ZN554, ZN878, ZN772, ZN224, ZN184, ZN544, ZNF57 ,ZN283,ZN549,ZN211,ZN615,ZN253,ZN226,ZN730,Z585A,ZN732,ZN681,ZN667,ZN649,ZN470,ZN484,ZN431,ZN382,ZN254,ZN124,ZN607,ZN317,ZN620,ZN141,ZN584,ZN540,ZN75D,ZN555,ZN658,ZN684,RBAK,ZN829,ZN582,ZN112,ZN716,HKR1,ZN350,ZN480,ZN416,ZNF92,ZN100,ZN736,ZNF74,ZN443,ZN195,ZN530,ZN782,ZN791,ZN331,Z354C,ZN157,ZN727,ZN550,ZN793,ZN235,ZN724,ZN573,ZN577,ZN789,ZN718,ZN300,ZN383,ZN429,ZN677,ZN850,ZN454,ZN257,ZN264,ZN485,ZN737,ZNF44,ZN596,ZN565,ZN543,ZFP69,SUMO1,ZNF12,ZN169,ZN433,ZN175,ZN347,ZNF25,ZN519,Z585B,ZN517,ZN846,ZN230,ZNF66,ZN713,ZN816,ZN426,ZN674,ZN627,ZNF20,Z587B,ZN316,ZN233,ZN611,ZN556,ZN234,ZN560,ZNF77,ZN682,ZN614,ZN785,ZN445,ZFP30,ZN225,ZN551,ZN610,ZN528,ZN284,ZN418,ZN490,ZN805,Z780B,ZN763,ZN285,ZNF85,ZN223,ZNF90,ZN557,ZN425,ZN229,ZN606,ZN155,ZN222,ZN442,ZNF91,ZN135,ZN778,ZN534,ZN586,ZN567,ZN440,ZN583,ZN441,ZNF43,ZN589,ZN563,ZN561,ZN136,ZN630,ZN527,ZN333,Z324B,ZN786,ZN709,ZN792,ZN599,ZN613,ZF69B,ZN799,ZN569,ZN564,ZN546,ZFP92,ZN723,ZN439,ZFP57,ZNF19,ZN404,ZN274,CBX3,ZN250,ZN570,ZN675,ZN695,ZN548,ZN132,ZN738,ZN420,ZN626,ZN559,ZN460,ZN268,ZN304,ZN605,ZN844,SUMO5,ZN101,ZN783,ZN417,ZN182,ZN823,ZN177,ZN197,ZN717,ZN669,ZN256,ZN251,CBX4,CDY2,CDYL2,ZN562,ZN461,Z324A,ZN766,ID2,ZN214,CBX7,ID1,CREM,SCX,ASCL1,ZN764,SCML2,TWST1,CREB1,TERF1,ID3,CBX8,GSX1,NKX22,ATF1,TWST2,ZNF17,TOX3,TOX4,ZMYM3,I2BP1,RHXF1,SSX2,I2BPL,ZN680,TRI68,HXA13,PHC3,TCF24,HXB13,HEY1,PHC2,ZNF81,FIGLA,SAM11,KMT2B,HEY2,JDP2,HXC13,ASCL4,HHEX,GSX2,ETV7,ASCL3,PHC1,OTP,I2BP2,VGLL2,HXA11,PDLI4,ASCL2,CDX4,ZN860,LMBL4,PDIP3,NKX25,CEBPB,ISL1,CDX2,PROP1,SIN3B,SMBT1,HXC11,HXC10,PRS6A,VSX1,NKX23,MTG16,HMX3,HMX1,KIF22,CSTF2,CEBPE,DLX2,PPARG,PRIC1,UNC4,BARX2,ALX3,TCF15,TERA,VSX2,HXD12,CDX1,TCF23,ALX1,HXA10,RX,CXXC5,SCML1,NFIL3,DLX6,MTG8,CEBPD,SEC13,FIP1,ALX4,LHX3,PRIC2,MAGI3,NELL1,PRRX1,MTG8R,RAX2,DLX3,DLX1,NKX26,NAB1,SAMD7,PITX3,WDR5,MEOX2,NAB2,DHX8,CBX6,EMX2,CPSF6,HXC12,KDM4B,LMBL3,PHX2A,EMX1,NC2B,DLX4,SRY,ZN777,ZN398,GATA3,BSH,SF3B4,TEAD1,TEAD3,RGAP1,PHF1,GATA2,FOXO3,ZN212,IRX4,ZBED6,LHX4,SIN3A,RBBP7,NKX61,R51A1,MB3L1,DLX5,NOTC1,TERF2,ZN282,RGS12,ZN840,SPI2B,PAX7,NKX62,ASXL2,FOXO1,GATA1,ZMYM5,LRP1,MIXL1,SGT1,LMCD1,CEBPA,SOX14,WTIP,Selected from PRP19, NKX11, RBBP4, DMRT2, SMCA2, and fragments thereof.

[0013] In some embodiments, the transcriptional repressor domain comprises the amino acid sequence set forth in any one of SEQ ID NOs: 22-46. In some embodiments, one or more of the nucleic acid binding domain, the epigenetic modification domain, and the transcriptional regulation domain are linked via a linker sequence.

[0014] In some embodiments, the linker sequence comprises at least 16 amino acids. In some embodiments, the linker sequence comprises an XTEN linker sequence. In some embodiments, the linker sequence comprises one or more fragment sequences excised from the amino acid sequence set forth in SEQ ID NO: 59, and the one or more fragment sequences comprise 16 or more consecutive amino acids.

[0015] In some embodiments, the linker sequence comprises a GS linking peptide, wherein the GS linking peptide comprises the sequence: (GS)a(GGS)b(GGGS)c(GGGGS)d, where 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. In some embodiments, the linker sequence comprises an amino acid sequence selected from one or more of SEQ ID NOs: 47-58.

[0016] In some embodiments, the one or more effector domains are N-terminal or C-terminal to the nucleic acid binding domain. In some embodiments, the effector domain comprises at least one type of the epigenetic modification domain and at least one type of the transcriptional regulatory domain.

[0017] In some embodiments, the epigenetic modification domain comprises DNMT3A and DNMT3L, and the C-terminus of the DNMT3A is linked to the N-terminus of the DNMT3L. In some embodiments, the epigenetic modification domain comprises DNMT3A and DNMT3L, and the C-terminus of the DNMT3L is linked to the N-terminus of the DNMT3A.

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

[0019] In some embodiments, the zinc finger protein-based transcription factor is selected from ZIM3 KRAB and KOX1 KRAB. In some embodiments, the transcriptional regulatory domain comprises two or more of the zinc finger protein-based transcription factors or functionally active fragments thereof. In some embodiments, the transcriptional regulatory domain comprises two or more of the KRAB domains described above, and the two or more of the KRAB domains described above are of the same type or different types.

[0020] In some embodiments, the two or more KRAB domains are linked by an XTEN linker sequence. In some embodiments, the one or more effector domains are N-terminal to the nucleic acid binding domain, and the epigenetic editing domain in the effector domain is N-terminal or C-terminal to the transcriptional regulatory domain.

[0021] In some embodiments, the fusion comprises the following entities linked in order from N-terminus to C-terminus: (1) at least one type of the epigenetic editing domain, at least one type of the transcriptional regulatory domain, and the nucleic acid binding domain; (2) at least one type of the transcriptional regulatory domain, at least one type of the epigenetic editing domain, and the nucleic acid binding domain; or (3) at least one type of the transcriptional regulatory domain, at least one type of the epigenetic editing domain, at least one type of the transcriptional regulatory domain, and the nucleic acid binding domain.

[0022] In some embodiments, the fusion comprises the following entities linked in order from N-terminus to C-terminus: (1) at least one type of DNA methyltransferase domain, at least one type of transcriptional repressor domain, and the nucleic acid binding domain; (2) at least one type of transcriptional repressor domain, at least one type of DNA methyltransferase domain, and the nucleic acid binding domain; or (3) at least one type of transcriptional repressor domain, at least one type of DNA methyltransferase domain, at least one type of transcriptional repressor domain, and the nucleic acid binding domain.

[0023] In some embodiments, the fusion comprises the following entities linked in order from N-terminus to C-terminus: (1) one or a combination of DNMT3A and DNMT3L, one or more of the above zinc finger protein-based transcription factors, and dCas9; (2) one or more of the above zinc finger protein-based transcription factors, one or a combination of DNMT3A and DNMT3L, and dCas9; or (3) one or more of the above zinc finger protein-based transcription factors, one or a combination of DNMT3A and DNMT3L, one or more of the above zinc finger protein-based transcription factors, and dCas9.

[0024] In some embodiments, the fusion comprises one or more of the following domains: DNMT3A-DNMT3L-KRAB-dCas9, KRAB-DNMT3A-DNMT3L-dCas9, DNMT3A-DNMT3L-ZNF582-dCas9, DNMT3A-DNMT3L-ZNF324-dCas9, DNMT3A-DNMT3L-ZNF680-dCas9, DNMT3A-DNMT3L-ZNF354a-d Cas9, DNMT3A-DNMT3L-ZNF419-dCas9, KRAB-DNMT3A-DNMT3L-KRAB-dCas9, KRAB-KRAB-DNMT3A-DNMT3L-dCas9, or DNMT3A-DNMT3L-KRAB-KRAB-dCas9, where - indicates that the domains of the fusion are directly and / or indirectly linked to each other and the domains are ordered from N-terminus to C-terminus.

[0025] In some embodiments, the one or more effector domains are C-terminal to the nucleic acid binding domain, and the epigenetic editing domain in the effector domain is N-terminal or C-terminal to the transcriptional regulatory domain. In some embodiments, the fusion comprises the following entities linked in order from N-terminus to C-terminus: (1) the nucleic acid binding domain, at least one type of the epigenetic editing domain, and at least one type of the transcriptional regulatory domain; (2) the nucleic acid binding domain, at least one type of the transcriptional regulatory domain, and at least one type of the epigenetic editing domain; or (3) the nucleic acid binding domain, at least one type of the transcriptional regulatory domain, at least one type of the epigenetic editing domain, and at least one type of the transcriptional regulatory domain.

[0026] In some embodiments, the fusion comprises the following entities linked in order from N-terminus to C-terminus: (1) the nucleic acid binding domain, at least one DNA methyltransferase domain, and at least one transcriptional repressor domain; (2) the nucleic acid binding domain, at least one type of transcriptional repressor domain, and at least one type of DNA methyltransferase domain; or (3) the nucleic acid binding domain, at least one type of transcriptional repressor domain, at least one type of DNA methyltransferase domain, and at least one type of transcriptional repressor domain.

[0027] In some embodiments, the fusion comprises the following entities linked in order from N-terminus to C-terminus: (1) dCas9, one or a combination of DNMT3A and DNMT3L, and one or more of the zinc finger protein-based transcription factors described above; (2) dCas9, one or more of the zinc finger protein-based transcription factors described above, and one or a combination of DNMT3A and DNMT3L; or (3) dCas9, one or more of the zinc finger protein-based transcription factors described above, and one or a combination of DNMT3A and DNMT3L, and one or more of the zinc finger protein-based transcription factors described above.

[0028] In some embodiments, the fusion comprises the following domains: dCas9-DNMT3A-DNMT3L-KRAB, dCas9-DNMT3L-DNMT3A-KRAB, dCas9-KRAB-DNMT3A-DNMT3L, dCas9-KRAB-DNMT3L-DNMT3A, dCas9-ZNF582-DNMT3L-DNMT3A, dCas9-ZNF324-DNMT3L-DNMT3A, dCas9-DNMT3L-DNMT3A-KRAB-KRAB, dCas9-DNMT3L-DNMT3A-KRAB-KRAB-KRAB, or dCas9-KRAB-DNMT3L-DNMT3A-KRAB, where - indicates that the domains of the fusion are directly and / or indirectly linked to each other and the domains are ordered from N-terminus to C-terminus.

[0029] In some embodiments, it comprises the amino acid sequence set forth in any one of SEQ ID NOs: 60-67, 70-107, and 337-344. In some embodiments, it comprises one or more effector domains, said effector domains being located N-terminal and C-terminal to said nucleic acid binding domain. In some embodiments, the one or more effector domains comprise at least one epigenetic editing domain. In some embodiments, the one or more effector domains comprise at least one epigenetic editing domain that provides a histone modification.

[0030] In some embodiments, the effector domain C-terminal to the nucleic acid binding domain comprises at least one type of epigenetic modification domain. In some embodiments, the effector domain C-terminal to the nucleic acid binding domain comprises at least one epigenetic modification domain that provides a histone modification.

[0031] In some embodiments, the effector domain N-terminal to the nucleic acid binding domain comprises one or more of an epigenetic modification domain and a transcriptional regulation domain. In some embodiments, the effector domain N-terminal to the nucleic acid binding domain comprises one or more of an epigenetic modification domain that provides a histone modification, an epigenetic modification domain that provides a DNA modification, and a transcriptional repressor domain.

[0032] In some embodiments, the effector domain N-terminal to the nucleic acid binding domain comprises (1) an epigenetic modification domain that provides a histone modification, (2) a transcriptional repressor domain, (3) an epigenetic modification domain that provides a DNA modification, or (4) a transcriptional repressor domain and an epigenetic modification domain that provides a DNA modification.

[0033] In some embodiments, the effector domain N-terminal to the nucleic acid binding domain comprises (1) one or more epigenetic modification domains selected from EZH2, HDAC3, HDAC1, EHMT2, PRMT1, PRMT5, SETDB1, hSIRT1, HP1a, LSD1, and functionally active fragments thereof; (2) KRAB or a functionally active fragment thereof; (3) DNMT3A, DNMT3L, or a combination of DNMT3A and DNMT3L; or (4) a combination of KRAB and DNMT3A, a combination of KRAB and DNMT3L, or a combination of KRAB, DNMT3A, and DNMT3L.

[0034] In some embodiments, the fusion comprises the following entities linked in order from N-terminus to C-terminus: (1) an epigenetic modification domain that provides a histone modification, the nucleic acid binding domain, and the epigenetic modification domain that provides a histone modification; (2) a transcriptional repressor domain, the nucleic acid binding domain, and the epigenetic modification domain that provides a histone modification; (3) an epigenetic modification domain that provides a DNA modification, the nucleic acid binding domain, and the epigenetic modification domain that provides a histone modification; (4) a transcriptional repressor domain, the epigenetic modification domain that provides a DNA modification, the nucleic acid binding domain, and the epigenetic modification domain that provides a histone modification; or (5) an epigenetic modification domain that provides a DNA modification, a transcriptional repressor domain, the nucleic acid binding domain, and the epigenetic modification domain that provides a histone modification.

[0035] In some embodiments, the epigenetic modification domain providing the histone modification is selected from EZH2, HDAC3, HDAC1, EHMT2, PRMT1, PRMT5, SETDB1, hSIRT1, HP1a, LSD1, and functionally active fragments thereof. In some embodiments, the epigenetic modification domain that provides the DNA modification is selected from DNMT3A, DNMT3L, a combination of DNMT3A and DNMT3L, or functionally active fragments thereof. In some embodiments, the transcriptional repressor domain is KRAB and / or a functionally active fragment thereof.

[0036] In some embodiments, the fusion comprises one of the following domains: DNMT3A-DNMT3L-dCas9-KRAB, HDAC3-dCas9-EZH2, KRAB-dCas9-EZH2, KRAB-DNMT3A-dCas9-EZH2, KRAB-DNMT3A-dCas9-HDAC3, DNMT3A-DNMT3L-dCas9-(EZH2) n=1-11 , DNMT3A-DNMT3L-dCas9-HDAC3, DNMT3A-DNMT3L-dCas9-EHMT2, DNMT3A-DNMT3L-dCas9-HDAC1, DNMT3A-DNMT3L-dCas9-PRMT1, DNMT3A-DNMT3L-dCas9-SETDB1, DNMT3A-DNMT3L-dCas9-hSIRT1, DNMT3A-DNMT3L-dCas9-PRMT5, DNMT3A-DNMT3L-dCas9-HP1a, DNMT3A-DNMT3L-dCas9-LSD1, or DNMT3A-DNMT3L-TALE-KRAB, where - indicates that the domains of the fusion are directly and / or indirectly linked to each other and the domains are ordered from N-terminus to C-terminus.

[0037] In some embodiments, it comprises the amino acid sequence set forth in any one of SEQ ID NOs: 68, 69, 108-126, 355, and 356. In some embodiments, the fusion further comprises a nuclear localization signal and / or a tag domain.

[0038] In another aspect, the present application provides a nucleic acid encoding a fusion described herein. In another aspect, the present application provides a recombinant vector comprising a nucleic acid described herein. In another aspect, the present application provides a delivery vector comprising a fusion described herein, a nucleic acid described herein, and / or a recombinant vector described herein, and optionally a liposome and / or a lipid nanoparticle. In another aspect, the present application provides a composition comprising a fusion described herein, a nucleic acid described herein, a recombinant vector described herein, and / or a delivery vector described herein.

[0039] In another aspect, the present application provides a cell comprising a fusion described herein, a nucleic acid described herein, a recombinant vector described herein, a delivery vector described herein, and / or a composition described herein. In another aspect, the present application provides a kit comprising a fusion described herein, a nucleic acid described herein, a recombinant vector described herein, a delivery vector described herein, a composition described herein, and / or a cell described herein.

[0040] In some embodiments, the kit further comprises at least one container for containing the fusion, the nucleic acid, the recombinant vector, the delivery vector, the composition, and / or the cells. In some embodiments, the kit further comprises instructions in physical form and / or machine-readable electronic form.

[0041] In another aspect, the present application provides a method of modulating expression of a target gene, comprising administering a fusion described herein, a nucleic acid described herein, a recombinant vector described herein, a delivery vector described herein, a composition described herein, a cell described herein, and / or a kit described herein.

[0042] In some embodiments, the method comprises introducing the fusion, the nucleic acid, the recombinant vector, the delivery vector, the composition, the cell, and / or the kit into a cell containing the target gene. In some embodiments, the method includes contacting the fusion, the nucleic acid, the recombinant vector, the delivery vector, and / or the composition with a regulatory element near and / or of the target gene. In some embodiments, the regulatory elements include a core promoter, a proximal promoter, a distal enhancer, a silencer, an insulator element, a boundary element, and / or a locus control region.

[0043] In another aspect, the present application provides a method for treating or alleviating a disease or symptom associated with aberrant target gene expression and / or aberrant target gene activity, the method comprising administering to a subject in need thereof an effective amount of a fusion described herein, a nucleic acid described herein, a recombinant vector described herein, a delivery vector described herein, a composition described herein, a cell described herein, and / or a kit described herein. In another aspect, the present application provides use of a fusion described herein, a nucleic acid described herein, a recombinant vector described herein, a delivery vector described herein, a composition described herein, a cell described herein, and / or a kit described herein in the manufacture of a medicament for treating or alleviating a disease or condition associated with aberrant target gene expression and / or aberrant target gene activity.

[0044] In another aspect, the present application provides a fusion described herein, a nucleic acid described herein, a recombinant vector described herein, a delivery vector described herein, a composition described herein, a cell described herein, or a kit described herein for use in treating or alleviating a disease or condition associated with aberrant target gene expression and / or aberrant target gene activity. Those skilled in the art will readily appreciate other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As will be apparent to those skilled in the art, the present application will enable them to make modifications to the specific embodiments disclosed without departing from the spirit and scope of the invention to which the present disclosure pertains. Correspondingly, the drawings and description in the present application are illustrative only and not restrictive. [Brief explanation of the drawings]

[0045] Specific features of the present invention are set forth in the appended claims. A better understanding of the features and advantages of the present invention can be obtained by reference to the exemplary embodiments and drawings described in detail below, the brief description of which follows.

[0046] [Figure 1] FIG. 1 is a schematic representation of the composition of an exemplary fusion of the present application. [Figure 2] FIG. 1 is a schematic representation of the composition of exemplary fusions comprising different transcriptional repressor domains of the present application. [Figure 3] 1 shows the inhibitory effect of exemplary fusions of the present application on the VEGFA gene. [Figure 4] 1 shows the inhibitory effect of exemplary fusions of the present application on the CLTA gene. [Figures 5A-5C] 1 shows the inhibitory effect of exemplary fusions of the present application on the CD151 gene. [Figures 6A-6F] 1 shows the inhibitory effect of exemplary fusions of the present application on the PCSK9 gene. [Figure 7] 1 shows the inhibitory effect of exemplary fusions of the present application on the PCSK9 gene. DETAILED DESCRIPTION OF THE INVENTION

[0047] Hereinafter, embodiments of the present invention will be described with reference to specific examples, and those skilled in the art will easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0048] Definition of Terms As used herein, the term "epigenetic modification domain" generally refers to a domain that can alter gene expression or cell phenotype of a cell population after administration. As will be understood, such changes refer to modifications related to one or more functions of the genome and do not involve changes in the nucleic acid sequence. Examples of such modifications are DNA methylation and histone modifications, both of which are important in regulating gene expression without altering the underlying DNA sequence.

[0049] As used herein, the term "DNA-binding domain" generally refers to an independently folding protein domain that recognizes at least one motif in double-stranded or single-stranded DNA. For example, the DNA-binding domain can recognize a specific DNA sequence (recognition or regulatory sequence) or have a general affinity for DNA. In some cases, other domains of the DNA-binding domain generally regulate the activity of the DNA-binding domain, and the DNA-binding function may be structural or include transcriptional regulation, sometimes with both roles overlapping. In certain embodiments of the methods and gene expression regulatory molecules provided herein, the DNA-binding domain may comprise a (DNA) nuclease, such as a nuclease that can target DNA in a sequence-specific manner or can be directed to target DNA in a sequence-specific manner, such as a CRISPR-Cas system, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), or large nuclease. In some embodiments, the DNA-binding domain is a DNA nuclease derived from a CRISPR-Cas system. For example, the DNA nuclease derived from the CRISPR-Cas system is a Cas protein.

[0050] As used herein, the term "TALE DNA-binding domain" or "TALE" refers to a polypeptide comprising one or more TALE repeat domains / units. Naturally occurring TALEs or "wild-type TALEs" are nucleic acid-binding proteins secreted by numerous species of Proteobacteria. TALE polypeptides comprise 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 from each other primarily at amino acid positions 12 and 13. In a preferred embodiment, the nucleic acid is DNA. As used herein, a TALE polypeptide monomer is used to refer to a highly conserved repeat polypeptide sequence within a TALE nucleic acid-binding domain, and the term "repeated variable dinucleotide" or "RVD" is used to refer to the highly variable amino acids at positions 12 and 13 of the polypeptide monomer. A general representation of a TALE monomer contained within a DNA-binding domain is X 1-11 -(X 12 X 13 )-X 14-33又は34又は35 where the subscripts indicate the amino acid positions and X indicates any amino acid. 12 X 13 indicates the RVD. In some TALE polypeptide monomers, the variable amino acid at position 13 is deleted or absent, and in this type of monomer, the RVD consists of a single amino acid. In this case, the RVD can alternatively be represented as X*, where X is X. 12 and (*) represents X 13 The DNA binding domain contains several repeats of TALE monomers, and this is shown to be the absence of (X 1-11 -(X 12 X 13 )-X 14-33又は34又は35 ) z In a preferred embodiment, z is at least 5 to 40. In a more preferred embodiment, z is at least 10 to 26.

[0051] TALE monomers have nucleotide binding affinities determined by the types of amino acids in their RVDs. For example, polypeptide monomers of RVDs with NI preferentially bind adenine (A), polypeptide monomers of RVDs with NG preferentially bind thymine (T), polypeptide monomers of RVDs with HD preferentially bind cytosine (C), and monomers of RVDs with NN preferentially bind adenine (A) and guanine (G). In another embodiment, monomers of RVDs with IG preferentially bind T. Thus, the number and order of polypeptide monomer repeats in the nucleic acid binding domain of a TALE determine its nucleic acid target specificity. In a further embodiment of the present application, monomers of RVDs with NS can recognize all four base pairs and bind A, T, G, or C. The structure and function of TALEs are further described in, for example, Moscou et al., Science 326:1501 (2009), Boch et al., Science 326:1509-1512 (2009), and Zhang et al., Nature Biotechnology 29:149-153 (2011), each of which is incorporated by reference in its entirety. The repeat domain of a TALE is involved in binding of the TALE to its homologous target DNA sequence. These repeat units (or "repeat sequences") exhibit at least some sequence homology with other TALE repeat sequences in naturally occurring TALE proteins. See, for example, U.S. Patent Publication No. 20110301073. The TALE binding domains of the present application can be "engineered" to bind to a predetermined nucleotide sequence, for example, by manipulating the recognition helix region (modifying one or more amino acids) of a naturally occurring TALE protein. Thus, engineered DNA-binding proteins (TALEs) are non-naturally occurring proteins. A non-limiting example of a method for engineering designed DNA-binding proteins is design and selection. Designed DNA-binding proteins are proteins that do not occur in nature, and their design and / or composition are derived primarily from rational criteria. Rational design criteria include applying substitution rules and computational algorithms to process information from knowledge databases that store existing TALE designs and binding data.See, e.g., U.S. Patents 6,140,081; 6,453,242; and 6,534,261, and further see WO 98 / 53058; WO 98 / 53059; WO 98 / 53060; WO02 / 016536; and WO 03 / 016496, and U.S. Patent Publication No. 20110301073.

[0052] As used herein, "Cas enzyme" may be used interchangeably with "Cas protein," "CRISPR protein," "CRISPR enzyme," "CRISPR-Cas protein," "CRISPR-Cas enzyme," "Cas," "CRISPR effector," or "Cas effector protein," which generally refer to a class of enzymes that are complementary to CRISPR sequences and can use the CRISPR sequence as a guide to recognize and cleave specific DNA strands. Non-limiting examples of Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also referred to as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csf1, Csf2, Csf3, Csf4, and / or homologs or modified forms thereof. These proteins are known; for example, the amino acid sequence of the Streptococcus pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2.

[0053] In this application, the "dCas9 enzyme" is also referred to as an "inactive Cas9 protein" or "inactive Cas9 enzyme." Known methods for generating a Cas9 protein (or a fragment thereof) having an inactive DNA cleavage domain can be found, for example, in Jinek et al., Science. 337:816-821 (2012) and Qi et al., "Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression," Cell. 28, 152(5):1173-83 (2013), each of which is incorporated herein by reference in its entirety. For example, the DNA cleavage domain of Cas9 is known to contain two subdomains: the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, and the RuvC1 subdomain cleaves the non-complementary strand. Mutations in these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A and H840A completely inactivate the nuclease activity of Streptococcus pyogenes Cas9 (Jinek et al., Science. 337:816-821 (2012); Qi et al., Cell. 28;152(5):1173-83 (2013)). Suitable CRISPR-inactive or nicked DNA-binding domains include, but are not limited to, nuclease-inactive variant Cas9 domains, including D10A, D10A / D839A / H840A, and D10A / D839A / H840A / N863A mutant domains, as described in WO2015089406A1, which is incorporated herein by reference. In some cases, endonuclease-inactive dCas9 from Streptococcus pyogenes has been used to target genes in bacterial, yeast, and human cells with gRNAs, silencing gene expression by steric hindrance. As used herein, "dCas" can refer to a dCas protein or a fragment thereof. As used herein, "dCas9" can refer to a dCas9 protein or a fragment thereof.As used herein, "iCas" and "dCas" can be used interchangeably and refer to catalytically inactive CRISPR-associated proteins. In one embodiment, the dCas protein comprises one or more mutations in the DNA cleavage domain. In one embodiment, the dCas protein comprises one or more mutations in the RuvC or domain. In one embodiment, the dCas molecule comprises one or more mutations in both the RuvC and HNH domains. In one embodiment, the dCas protein is a fragment of a wild-type Cas protein. In one embodiment, the dCas protein comprises a functional domain derived from a wild-type Cas protein, the functional domain being selected from a Reel domain, a bridge helix domain, or a PAM-interacting domain. In one embodiment, the nuclease activity of dCas is reduced by at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% compared to the nuclease activity of the corresponding wild-type Cas protein.

[0054] A suitable dCas may be derived from a wild-type Cas protein. The Cas protein may be derived from a type I, type II, or type III CRISPR-Cas system. In one embodiment, a suitable dCas may be derived from Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, or Cas10. In one embodiment, the dCas is derived from a Cas9 protein. For example, dCas9 can be obtained by introducing point mutations (e.g., substitutions, deletions, or additions) into the DNA cleavage domain (e.g., the nuclease domain, e.g., the RuvC and / or HNH domain) of the Cas9 protein. See, e.g., Jinek et al., Science (2012) 337:816-21, the entire contents of which are incorporated herein by reference. For example, introduction of two point mutations in the RuvC and HNH domains reduces Cas9 nuclease activity while retaining Cas9 sgRNA and DNA binding activity. In one embodiment, the two point mutations in the RuvC and HNH active sites are the D10A and H840A mutations of S. pyogenes Cas9. Alternatively, D10 and H840 of S. pyogenes Cas9 can be deleted to eliminate Cas9 nuclease activity while retaining its sgRNA and DNA binding activities. In one embodiment, the two point mutations in the RuvC and HNH active sites are the D10A and N580A mutations of S. pyogenes Cas9.

[0055] In various embodiments, the present application relates to a dCas protein or any variant or mutant thereof. All variants and mutants of dCas9 can be used in the methods, compositions, fusion molecules, or kits disclosed herein, including, but not limited to, SpCas9 (Cas9 isolated from Streptococcus pyogenes), SaCas9 (Cas9 isolated from Staphylococcus aureus), StCas9 (Cas9 isolated from Streptomyces thermophilus), NmCas9 (Cas9 isolated from Neisseria meningitidis), FnCas9 (Cas9 isolated from Francisella novicida), CjCas9 (Cas9 isolated from Campylobacter jejuni), ScCas9 (Cas9 isolated from Streptococcus canis), and any variant or mutant form derived from the above Cas9s, including high-fidelity Cas9. (Kleinstiver et al., Nature. January 28, 2016) and enhanced SpCas9 (Slaymaker et al., Sciences. January 1, 2016). For example, the dCas9 sequences set forth in SEQ ID NOS: 1162-1179 herein provide only some exemplary options and are not intended to be exhaustive. In one embodiment, the dCas protein is a Streptococcus pyogenes dCas9 protein containing mutations at D10 and / or H840 (set forth in SEQ ID NO: 1162). In one embodiment, the dCas protein is a Streptococcus pyogenes dCas9 protein containing mutations at D10 and / or H840A (set forth in SEQ ID NO: 1162).In one embodiment, the dCas9 protein is a Staphylococcus aureus dCas9 protein and comprises the amino acid sequence set forth in SEQ ID NO: 1163 or 1164, a sequence substantially identical (e.g., 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 more sequence identity) to SEQ ID NO: 1163 or 1164, or a sequence having one, two, three, four, five or more alterations (e.g., amino acid substitutions, insertions, or deletions) relative to SEQ ID NO: 1163 or 1164, or any fragment thereof.

[0056] Similar mutations can be applied to any other naturally occurring Cas9 (e.g., Cas9 from other species) or engineered Cas9. In certain embodiments, the dCas9 is selected from the group consisting of Streptococcus pyogenes dCas9, Staphylococcus aureus dCas9, Campylobacter jejuni dCas9, Corynebacterium diphtheria dCas9, Eubacterium ventriosum dCas9, Streptococcus pasteurianus dCas9, Lactobacillus farciminis dCas9, Sphaerochaeta globus dCas9, and Azospirillum (e.g., strain B510). The present invention also includes dCas9, Gluconacetobacter diazotrophicus dCas9, Neisseria cinerea dCas9, Roseburia intestinalis dCas9, Parvibaculum lavamentivorans dCas9, Nitratifractor salsuginis (e.g., strain DSM 16511) dCas9, Campylobacter lari (e.g., strain CF89-12) dCas9, Streptococcus thermophilus (e.g., strain LMD-9) dCas9, or fragments thereof. In certain embodiments, the present application further provides a vector comprising nucleotides encoding the following protein molecules:Streptococcus pyogenes dCas9, Staphylococcus aureus dCas9, Campylobacter jejuni dCas9, Corynebacterium diphtheriae dCas9, Eubacterium ventriosum dCas9, Streptococcus pasteurianus dCas9, Lactobacillus farciminis dCas9, Sphaerochaeta globus dCas9, Azospirillum sp. (strain B510) dCas9, Gluconacetobacter diazotrophicus dCas9, Neisseria cinerea dCas9, Roseburia intestinalis dCas9, Parvibacrum lavamentivorans dCas9, Nitratifractor sarsuginis (strain DSM 16511) dCas9, Campylobacter lari (strain CF89-12) dCas9, Streptococcus thermophilus (strain LMD-9) dCas9, or a fragment thereof.

[0057] In the present application, the term "capable of binding" is used interchangeably with "binding," "specifically recognizing," "targeting," etc., and generally refers to a binding molecule (e.g., a gene expression regulating molecule of the present application) being able to interact with a nucleotide in a target gene or target site, or a binding molecule (e.g., a gene expression regulating molecule of the present application) having sufficient affinity for a target gene or target site, and this interaction may be in the form of conjugation, coupling, attachment, providing complementarity, providing covalent or non-covalent binding force, improving binding stability, etc.

[0058] As used herein, the terms "guide RNA," "guide DNA," and "gRNA" are used interchangeably and generally refer to a DNA molecule capable of directing a nuclease (e.g., Argonaute or Ago) to bind to and / or cleave a target gene. In some preferred embodiments, the guide DNA may be a single-stranded DNA molecule (ssDNA), a single-stranded DNA molecule phosphorylated at the 5' end, a single-stranded DNA molecule hydroxylated at the 5' end, a base fragment capable of being complementary to the target gene, and / or a length of 8 to 35 nt. In some embodiments, the term "guide RNA" includes the following RNAs: (1) an "activating" nucleotide sequence that binds to and activates a guide RNA-guided endonuclease (e.g., a Class II Cas nuclease, e.g., a Type II, Type V, or Type VI Cas endonuclease), and (2) a "targeting" nucleotide sequence that hybridizes to a target nucleic acid. The "activating" nucleotide sequence and the "targeting" nucleotide sequence can be on separate RNA molecules (e.g., a "dual guide RNA") or on the same RNA molecule (a "single guide RNA," also called sgRNA).

[0059] As used herein, the term "Class II Cas nuclease" generally refers to a class of Cas proteins that identify and / or perform cleavage function as a single protein form, as defined based on the updated classification scheme of CRISPR / Cas loci (Makarova et al., (2015) Nat Rev Microbiol, 13(11):722-36; Shmakov et al., (2015) Mol Cell, 60:385-397).

[0060] In this application, "Class II Type II Cas nuclease and Class II Type V Cas nuclease" generally refer to single-protein RNA-guided endonucleases among Class II Cas nucleases. Among them, Type II and Type V Cas nuclease VB, which are Type II and Type V, can function normally only when transactivating CRISPR RNA (tracrRNA) and crRNA (CRISPR RNA) act together, and crRNA and tracrRNA can be artificially combined into a single guide RNA (sgRNA). However, Type V-ACas nuclease, which is Type V, requires crRNA alone to function as a guide. Non-limiting examples of Class II Type II Cas nucleases include Cas9 and its family related nucleases, and non-limiting examples of Class II Type V Cas nucleases include Cas12a (also called Cpf1), Cas12b (also called C2c1), Cas12c (also called C2c3), Cas12d (CasY), Cas12e (CasX), Cas12g, Cas12h, Cas12i, C2c1, C2c4, C2c5, C2c8, C2c9, C2c10, Cas14a, Cas14b, Cas14c nuclease and / or TnpB.

[0061] As used herein, the term "DNA methyltransferase" generally refers to an enzyme that catalyzes the transfer of a methyl group to DNA. Non-limiting examples of DNA methyltransferases include DNMT1, DNMT 3A, DNMT 3B, and DNMT 3L. For example, by DNA methylation, DNA methyltransferases can modify the activity of DNA fragments (e.g., regulate gene expression) without altering the DNA sequence. As described herein, a gene expression regulatory molecule can include one or more (e.g., two) DNA methyltransferases. When a DNA methyltransferase is included as part of a gene expression regulatory molecule, the DNA methyltransferase can be referred to as a "DNA methyltransferase domain." In various embodiments, the DNA methyltransferase domain comprises a variant or homologue of an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to DNMT 3A. In various embodiments, the DNA methyltransferase domain comprises a variant or homologue of an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to DNMT 3L.

[0062] As used herein, the term "functionally active fragment" generally refers to a fragment having a subregion of a full-length protein or nucleic acid, but which retains or partially retains the biological activity or function of the full-length protein or nucleic acid. For example, a functionally active fragment can retain or partially retain the ability of the full-length protein to bind to another molecule. For example, a functionally active fragment of a DNA methyltransferase can retain or partially retain the biological activity function of the full-length DNA methyltransferase, which catalyzes the transfer of a methyl group to DNA.

[0063] As used herein, the term "linker" generally refers to a linker connecting two or more moieties. In various embodiments, the linker is linked at the N-terminus and C-terminus to the amino acid sequence of the remainder of the compound (e.g., the fusion proteins provided herein). For example, as used herein, the terms "XTEN," "XTEN linker," or "XTEN polypeptide" refer to a recombinant polypeptide lacking hydrophobic amino acid residues (e.g., an unstructured recombinant peptide). 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.

[0064] As used herein, the terms "inhibition," "suppression," "silencing," and the like generally refer to a reduction in gene expression and / or activity. For example, administering a substance of the present application can negatively affect (e.g., decrease) the activity of a nucleic acid sequence compared to the activity of the nucleic acid sequence in the absence (control) of the substance (e.g., a fusion protein, a conjugate, a nucleic acid, a vector). For example, inhibition can refer to a reduction in a disease or disease symptom. For example, inhibition can include at least partially, partially, or completely blocking activation (e.g., transcription) of a nucleic acid sequence, or reducing, preventing, or delaying activation of a nucleic acid sequence. For example, inhibitory activity can be 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or less of the control.

[0065] As used herein, the term "transcriptional repressor" generally refers to a substance and / or reagent, such as a protein (e.g., a transcription factor or fragment thereof), that binds to a target nucleic acid sequence and results in a decrease in the expression level of a gene product associated with the target nucleic acid sequence. For example, the gene product may be an RNA (e.g., mRNA) transcribed from a gene, or a polypeptide translated from an mRNA transcribed from a gene. Typically, an increase or decrease in mRNA levels results in an increase or decrease in the level of the polypeptide translated therefrom. Expression levels can be measured using standard techniques for measuring mRNA or protein. Non-limiting examples of transcriptional repressors include mSin3-interacting domain (SID) protein, methyl-CpG-binding domain 2 (MBD2), MBD3, DNA methyltransferase (DNMT) 1 (DNMT1), DNMT2A, DNMT3A, DNMT3B, DNMT3L, retinoblastoma protein (Rb), methyl-CpG-binding protein 2 (Mecp2), GATA-1 and its cofactor Fog1, MAT2 regulator (ROM2), Arabidopsis HD2A protein (AtHD2A), lysine-specific demethylase 1 (LSD1), and / or Kruppel-associated box (KRAB).

[0066] As used herein, the term "KRAB," also referred to as "Kruppel-associated box domain" or "Kruppel-associated box domain," generally refers to a transcription inhibitory domain of about 45 to about 75 amino acid residues present in human zinc finger protein transcription factors. In various embodiments, the KRAB domain may include variants or homologs of amino acid sequences that share at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the ZIM3 KRAB domain or the KOX1 KRAB domain.

[0067] As used herein, "nuclear localization sequence" or "nuclear localization signal" or "NLS" generally refers to a peptide that guides a protein to the cell nucleus. For example, an NLS contains five basic, positively charged amino acids. For example, an NLS can be located anywhere along the peptide chain.

[0068] As used herein, the term "tag" refers to a peptide that can be introduced into an expression vector and used to enable deletion and / or purification of the expression product of one or more vector inserts. Such tags are known in the art and include radiolabeled amino acids or polypeptides linked to a biotin moiety that can be detected by labeled avidin (e.g., streptavidin containing a fluorescent label or enzymatic activity that can be detected optically or colorimetrically). Affinity tags include FLAG, glutathione-S-transferase, maltose-binding protein, cellulose-binding domain, thioredoxin, NusA, mistin, chitin-binding domain, cutinase, AGT, GFP, and other commonly used tags used in protein expression and purification systems. Further non-limiting examples of polypeptides include, but are not limited to, histidine tags, radioisotopes or radionuclides (e.g., H, C, S, Y, Tc, In, I, Lu, Ho, or Sm), fluorescent tags (e.g., FITC, rhodamine, lanthanide phosphorus), enzymatic tags (e.g., horseradish peroxidase, luciferase, alkaline phosphatase), chemiluminescent tags, biotin groups, pendant polypeptide epitopes recognized by a second reporter (e.g., leucine zipper motifs, binding sites for secondary antibodies, metal binding domains, epitope tags), and magnetic reagents such as gadolinium chelates.

[0069] As used herein, the specific proteins (e.g., KRAB, dCas9, Dnmt3A, Dnmt3L) may include any naturally occurring form of the protein or may include variants or homologs that maintain the protein activity (e.g., having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% activity compared to the naturally occurring protein). In each embodiment, the variant or homolog has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 consecutive amino acids) compared to the naturally occurring form.

[0070] As used herein, the term "nucleic acid" is used interchangeably with "polynucleotide," "nucleotide," "nucleotide sequence," and "oligonucleotide," and generally refers to a polymer of nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and their complements in single-, double-, or multi-stranded form. For example, nucleotides may be ribonucleotides, deoxyribonucleotides, or modified versions thereof. For example, nucleotides may be single- and double-stranded DNA, single- and double-stranded RNA, and hybridized molecules comprising mixtures of single- and double-stranded DNA and RNA. For example, nucleotides may include, but are not limited to, any type of RNA, such as mRNA, siRNA, miRNA, sgRNA, and guide RNA, as well as any type of DNA, including genomic DNA, plasmid DNA, and minicircle DNA, and any fragment thereof. The term also encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, including synthetic, naturally occurring, and non-naturally occurring nucleic acids.

[0071] As used herein, the terms "sequence encoding ..." or "nucleic acid encoding ..." generally refer to a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence that encodes a protein. The coding sequence may further comprise initiation and termination signals operably linked to regulatory elements, including promoters and polyadenylation signals, that direct expression in cells of an individual or mammal to which the nucleic acid is administered. The coding sequence may be codon-optimized.

[0072] As used herein, the term "delivery vector" generally refers to a transport vehicle capable of delivering a reagent (e.g., a nucleic acid molecule) to a target cell. A delivery vector can deliver a reagent to a specific cell subset. For example, a delivery vector targets a specific cell type by its inherent characteristics or by a moiety bound to or contained within the vehicle (or by a moiety that binds to the vehicle and is sufficient to maintain the delivery vector and thereby target the delivery vector). A delivery vector can also improve the in vivo half-life and / or bioavailability of the delivered reagent. Delivery vectors include viral vectors, virus-like particles, polycationic vectors, peptide vectors, liposomes, and / or hybridized vectors. For example, when the target cell is a hepatocyte, the characteristics of the delivery vector (e.g., size, charge, and / or pH) are effective in delivering the delivery vector and / or molecules entrapped therein to the target cell, reducing immune clearance, and / or promoting retention in the target cell.

[0073] As used herein, the term "liposome" generally refers to a vesicle having an internal space separated from the external medium by one or more bilayer membranes. In some embodiments, the bilayer membrane may be formed by amphiphilic molecules, such as synthetic or naturally occurring lipids, containing spatially separated hydrophilic and hydrophobic domains; in other embodiments, the bilayer membrane may be formed by amphiphilic polymers and surfactants. In some embodiments, the liposome is a spherical vesicular structure consisting of a monolayer or multilayer lipid bilayer surrounding an internal aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. In some embodiments, liposomes are biocompatible, nontoxic, and capable of delivering hydrophilic and lipophilic drug molecules, protecting their cargo from degradation by plasma enzymes, and transporting their carriers 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 (e.g., 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)), sphingomyelin, egg yolk phosphatidylcholine, monosialoganglioside, or any combination thereof. Several other additives may be added to the liposomes to modify the structure and properties of the liposomes. For example, the liposomes may further contain cholesterol, sphingomyelin, and / or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) to increase stability and / or prevent leakage of the cargo inside the liposomes.

[0074] The term "lipid nanoparticle (LNP)" generally refers to a particle comprising multiple (i.e., more than one) lipid molecules physically bound to each other by intermolecular forces (e.g., covalent or non-covalent bonds). LNPs may be, for example, microspheres (including unilamellar and multilamellar vesicles, e.g., liposomes), the dispersed phase in an emulsion, micelles, or the internal phase in a suspension. LNPs can encapsulate nucleic acids within cationic lipid particles (e.g., liposomes) and can be relatively easily delivered to cells. In some instances, lipid nanoparticles do not contain any viral components, which helps minimize safety and immunogenicity issues. The lipid particles are useful for in vitro, ex vivo, and in vivo delivery. The lipid particles can also be used to target cell populations of various sizes. The LNPs of the present application can be easily prepared by various methods known in the art, for example, by mixing an organic phase with an aqueous phase. Mixing of the two phases may be achieved using microfluidic devices and impinging flow reactors. The more thoroughly the organic and aqueous phases are mixed, the better the capture rate and particle size distribution of the resulting LNPs. Preferably, the particle size of the LNPs can be adjusted by varying the mixing speed of the organic and aqueous phases. A faster mixing speed results in smaller particle sizes of the prepared LNPs. The capture 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 (e.g., molecules containing coding sequences for DNA-binding proteins and / or sgRNAs) and / or RNA molecules (e.g., mRNA for Cas and sgRNAs). In certain cases, LNPs can be used to deliver RNP complexes of Cas / gRNAs. In some embodiments, LNPs can be used to deliver mRNAs and gRNAs (e.g., mRNA fusion molecules containing DNMT3A-DNMT3L (3A-3L)-dCas9-KRAB and at least one type of sgRNA targeting a target gene).

[0075] As used herein, the term "recombinant vector" generally refers to a nucleic acid molecule capable of transporting itself and another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Alternatively, the vector may be linear. Another type of vector is a viral vector, into which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell after introduction into the host cell, and are replicated along with the host genome.

[0076] As used herein, the term "regulatory element" refers to a genetic element capable of controlling the expression of a nucleic acid sequence. For example, splicing signals, promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, replication origins, internal ribosome entry sites ("IRES"), enhancers, etc., cooperate to provide for the replication, transcription, and translation of a coding sequence in a recipient cell. Not all of these regulatory sequences need to be present. Transcriptional control signals in eukaryotes typically include "promoter" and "enhancer" elements. Promoters and enhancers are composed of short arrays of DNA sequences, where a promoter is a regulatory element that promotes the initiation of transcription of an operably linked coding region, and an enhancer is a regulatory element that increases the rate of genetic transcription by increasing the activity of the nearest promoter located 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 variety of eukaryotic sources, including genes in yeast, insect, and mammalian cells, as well as viruses (similar regulatory sequences, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer depends on the type of recipient cell. Some eukaryotic promoters and enhancers have a broad host range, while others function in a limited subset of cell types (for reviews, see, e.g., Voss et al., Trends Biochem. Sci., 11:287 (1986); and Maniatis et al., ibid., incorporated herein by reference in their entireties). For example, the SV40 early gene enhancer is highly active in a variety of cell types derived 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 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)), the long terminal repeat 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 above references are incorporated herein by reference in their entireties. Promoters and enhancers can occur alone or together in nature. For example, retroviral long terminal repeats contain promoter and enhancer elements. Generally, the role of promoters and enhancers is independent of the gene being transcribed or translated. Thus, the enhancers and promoters used may be "endogenous," "exogenous," or "heterologous" with respect to the gene to which they are operably linked. An "endogenous" enhancer / promoter is one that is naturally linked to a given gene in the genome. An "exogenous" or "heterologous" enhancer or promoter is one that has been juxtaposed with a gene by genetic engineering (i.e., molecular biology techniques), such that transcription of the gene is directed by the juxtaposed enhancer / promoter. The presence of "splicing signals" on an expression vector usually results in high-level expression of the recombinant transcript. In certain embodiments, a "splicing signal" mediates the removal of an intron from a primary RNA transcript and is composed of a splice donor and acceptor 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).Common splice donor and acceptor sites are the splice sites of SV40 16S RNA. In certain embodiments, a "transcription termination signal" is typically located downstream of a polyadenylation signal and is several hundred nucleotides in length. For example, the term "poly A signal" or "poly A sequence" refers to a DNA sequence that directs the termination and polyadenylation of a nascent RNA transcript. Efficient polyadenylation of recombinant transcripts is often required because transcripts lacking a poly A signal are unstable and rapidly degraded. The poly A signal used in an expression vector may be "heterologous" or "endogenous." An endogenous poly A signal is one that is naturally present at the 3' end of the coding region of a given gene in the genome. A heterologous poly A signal is one that is isolated from one gene and operably linked to the 3' end of another gene. A common heterologous poly A signal is the SV40 poly A signal. The SV40 poly A signal is contained on a 237 bp BamHI / BclI restriction fragment and directs termination and polyadenylation (Sambrook et al., supra, 16.6-16.7, incorporated by reference in its entirety).

[0077] As used herein, the term "subject" generally refers to animals, typically mammals such as humans, non-human primates (monkeys, gibbons, gorillas, chimpanzees, orangutans, rhesus monkeys), livestock (dogs and cats), farm animals (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs), and laboratory animals (mice, rats, rabbits, guinea pigs). Human subjects include fetal, neonatal, infant, adolescent, and adult subjects. Subjects also include animal disease models, e.g., mouse and other animal models of blood clotting disorders (such as HemA), as well as other animal models known to those skilled in the art.

[0078] In this application, the term "comprising" generally means including the features explicitly specified, but not excluding other elements. As used herein, the term "selected from" generally means to include the objects of selection and all combinations thereof. For example, "(:) selected from A, B, and C" means to include all combinations of A, B, and C, e.g., A, B, C, A+B, A+C, B+C, or A+B+C.

[0079] As used herein, the term "about" generally refers to a variation within 0.5% to 10% above or below the specified numerical value, for example, a variation within 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified numerical value.

[0080] In one aspect, the present application provides a fusion comprising a nucleic acid binding domain and one or more effector domains, wherein the effector domain comprises one or more of an epigenetic modification domain and / or a transcriptional regulation domain, and wherein the fusion comprises at least two types of epigenetic modification domains and / or transcriptional regulation domains.

[0081] In another aspect, the present application provides a nucleic acid encoding the fusion product described herein. For example, the nucleic acid may include DNA and / or mRNA. For example, the nucleic acid may 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, and one or more modification techniques can be used to generate more stable mRNA. Known mRNA modification techniques can be broadly divided into three types: synthesizing mRNA using artificially synthesized non-natural ribonucleic acid instead of natural ribonucleic acid; adding 5' caps, 3' poly(A) "tails," and UTR (untranslated region) sequences; and using special novel formulation techniques to effectively protect mRNA. A preferred mRNA modification technique is synthesizing mRNA using artificially synthesized non-natural ribonucleic acid instead of natural ribonucleic acid. Chemical modifications on eukaryotic mRNA can be broadly classified into three types: methylation, pseudouridine (Ψ), and hypoxanthine. For example, the chemical modification may be selected from pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine and 2'-O-methyluridine.

[0082] In another aspect, the present application provides a recombinant vector comprising a nucleic acid described herein. For example, a recombinant vector can refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. Recombinant vectors may include single-stranded, double-stranded, or partially double-stranded nucleic acid molecules, nucleic acid molecules containing one or more free ends, nucleic acid molecules without free ends (e.g., circular), nucleic acid molecules comprising DNA, RNA, or both, and other types of polynucleotides known in the art. For example, viral vectors can be used. Viral vectors may include DNA or RNA sequences derived from viruses used for packaging into viruses (e.g., retroviruses, replication-deficient retroviruses, adenoviruses, replication-deficient adenoviruses, and adeno-associated viruses (AAV)). Viruses and viral vectors are useful for in vitro, ex vivo, and / or in vivo delivery.

[0083] In another aspect, the present application provides a delivery vector comprising a fusion construct described herein, a nucleic acid described herein, and / or a recombinant vector described herein, and optionally a liposome and / or a lipid nanoparticle. For example, the delivery vector can comprise one or more Cas proteins and one or more guide RNAs in the form of a ribonucleoprotein complex (RNP). For example, ribonucleoproteins can be delivered by polypeptide-based shuttle factors. For example, ribonucleoproteins can be delivered using synthetic peptides. 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 within cationic lipid particles (e.g., liposomes) and can be delivered relatively easily to cells. In some instances, lipid nanoparticles do not contain any viral components, which helps minimize safety and immunogenicity issues. Lipid particles are useful for in vitro, ex vivo, and in vivo delivery. Components of LNPs may include cationic lipids, ionizable lipids, PEGylated lipids, and / or supporting lipids, and optionally, cholesterol components. In some embodiments, LNPs may include ionizable lipids (20%-70%, molar ratio), PEGylated lipids (0%-30%, molar ratio), supporting lipids (30%-50%, molar ratio), and cholesterol (10%-50%, molar ratio).

[0084] In another aspect, the present application provides a composition comprising a fusion described herein, a nucleic acid described herein, a recombinant vector described herein, and / or a delivery vector described herein. For example, the fusion, the nucleic acid encoding the fusion, the recombinant vector, and the delivery vector in the composition may be contained together in one composition, or may be contained in different compositions. For example, when the fusion, the nucleic acid encoding the fusion, the recombinant vector, and the delivery vector in the composition are used, they may be used together or separately.

[0085] In another aspect, the present application provides a cell comprising a fusion described herein, a nucleic acid described herein, a recombinant vector described herein, a delivery vector described herein, and / or a composition described herein. In another aspect, the present application provides a kit comprising a fusion described herein, a nucleic acid described herein, a recombinant vector described herein, a delivery vector described herein, a composition described herein, and / or a cell described herein.

[0086] In another aspect, the present application provides a method for modulating expression of a target gene, the method comprising administering a fusion described herein, a nucleic acid described herein, a recombinant vector described herein, a delivery vector described herein, a composition described herein, a cell described herein, and / or a kit described herein. For example, the method may be a non-therapeutic method. For example, the method may not directly involve the human body. For example, the method may be an in vitro or ex vivo method. For example, the method may be a therapeutic method. For example, the method may be an in vivo method. For example, the method may reduce the expression and / or activity of the target gene. For example, administering a substance described herein can negatively affect (e.g., decrease) the activity of a nucleic acid sequence compared to the expression and / or activity of the target gene in the absence of the substance, and may include, for example, at least partially, partially, or completely blocking activation (e.g., transcription) of the nucleic acid sequence, or reducing, preventing, or delaying activation of the nucleic acid sequence. For example, the inhibitory activity may be about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10% or less of that in a control.

[0087] In another aspect, the present application provides methods for treating or alleviating a disease or symptom associated with aberrant target gene expression and / or aberrant target gene activity, the method comprising administering to a subject in need thereof an effective amount of a fusion described herein, a nucleic acid described herein, a recombinant vector described herein, a delivery vector described herein, a composition described herein, a cell described herein, and / or a kit described herein. In some embodiments, the treatment is for an organ disease / disorder, illustratively including liver disease, eye disease, muscle disease, heart disease, blood disease, brain disease, kidney disease, or may include treatment of autoimmune diseases, central nervous system diseases, cancer and other proliferative diseases, neurodegenerative diseases, inflammatory diseases, metabolic diseases, musculoskeletal diseases, etc.

[0088] fusion In some embodiments, the one or more effector domains are N-terminal or C-terminal to the nucleic acid binding domain. For example, the one or more effector domains are all located at the N-terminus of the nucleic acid binding domain. In some embodiments, the effector domain may include at least one type of epigenetic modification domain and at least one type of transcriptional regulation domain, and the epigenetic editing domain in the effector domain may be located at the N-terminus of the transcriptional regulation domain or at the C-terminus of the transcriptional regulation domain. In these cases, the epigenetic modification domain may include DNMT3A and DNMT3L, with the C-terminus of DNMT3A linked to the N-terminus of DNMT3L, or the C-terminus of DNMT3L linked to the N-terminus of DNMT3A. In a similar situation, the transcriptional regulation domain may include a transcriptional repression domain. For example, the transcriptional regulation domain may include a zinc finger protein-based transcription factor or a functionally active fragment thereof. Specifically, the zinc finger protein-based transcription factor may include KRAB, and for example, in some embodiments of the present application, the zinc finger protein-based transcription factor may be selected from ZIM3 KRAB and KOX1 KRAB.

[0089] In some embodiments of the above situation, the transcriptional regulatory domain may comprise two or more KRAB domains, and the two or more KRAB domains are the same type or different types.For example, the transcriptional regulatory domain comprises two or more ZIM3 KRABs, two or more KOX1 KRABs, or ZIM3 KRABs and KOX1 KRABs at the same time, and the total number thereof is two or more.In these cases, the two or more (same type or different type) KRAB domains may be linked via an XTEN linker sequence.

[0090] The following are exemplary fusions of the present application, with the amino acid sequences of the components as follows: bold - DNMT3A, underlined bold - DNMT3L, italics - linker sequence, bold italics - transcriptional regulatory domain, underlined bold italics - dCas9, * indicates any amino acid.

[0091] The amino acid sequence of DNMT3A-DNMT3L-XTEN16-ZIM3-XTEN80-dCas9 (V7 or V7' form, SEQ ID NO: 60 or 337) is as follows:

[0092] [ka] [ka]

[0093] or V7' form: [ka] [ka]

[0094] The amino acid sequence of ZIM3-XTEN16-DNMT3A-DNMT3L-XTEN80-dCas9 (V8 or V8' form, SEQ ID NO: 61 or 338) is as follows:

[0095] [ka] [ka]

[0096] or V8' form: [ka] [ka]

[0097] The amino acid sequence of DNMT3A-DNMT3L-XTEN16-KRAB-XTEN80-dCas9 (V19 form, SEQ ID NO: 70) is as follows:

[0098] [ka] [ka]

[0099] The amino acid sequence of DNMT3A-(human)DNMT3L-XTEN16-ZIM3-XTEN80-dCas9 (V20 form, SEQ ID NO: 71) is as follows:

[0100] [ka] [ka]

[0101] The amino acid sequence of ZIM3-XTEN16-DNMT3A-(human)DNMT3L-XTEN80-dCas9 (V21 form, SEQ ID NO: 72) is as follows:

[0102] [ka] [ka]

[0103] The amino acid sequence of KRAB-XTEN16-DNMT3A-DNMT3L-XTEN80-dCas9 (V22 form, SEQ ID NO: 73) is as follows:

[0104] [ka] [ka]

[0105] The amino acid sequence of ZIM3-BFP-DNMT3A-(human)DNMT3L-XTEN80-dCas9 (V38 form, SEQ ID NO: 89) is as follows (underlined, dashed, bold italics - BFP, SEQ ID NO: 55):

[0106] [ka] [ka]

[0107] The amino acid sequence of DNMT3A-(human)DNMT3L-XTEN80-ZIM3-XTEN80-dCas9 (V39 form, SEQ ID NO: 90) is as follows:

[0108] [ka] [ka]

[0109] The amino acid sequence of DNMT3A-(human)DNMT3L-XTEN80-ZNF582-XTEN80-dCas9 (V40 form, SEQ ID NO: 91) is as follows:

[0110] [ka] [ka]

[0111] The amino acid sequence of DNMT3A-(human)DNMT3L-XTEN80-ZNF324-XTEN80-dCas9 (V41 form, SEQ ID NO: 92) is as follows:

[0112] [ka] [ka]

[0113] The amino acid sequence of DNMT3A-(human)DNMT3L-BFP-XTEN80-ZIM3-XTEN80-dCas9 (V42 form, SEQ ID NO: 93) is as follows (underlined, dashed, bold italics - BFP, SEQ ID NO: 55):

[0114] [ka] [ka]

[0115] The amino acid sequence of DNMT3A-(human)DNMT3L-Beta2-XTEN80-ZIM3-XTEN80-dCas9 (V43 form, SEQ ID NO: 94) is as follows:

[0116] [ka] [ka]

[0117] The amino acid sequence of DNMT3A-(human)DNMT3L-beta1-XTEN80-ZIM3-XTEN80-dCas9 (V44 form, SEQ ID NO: 95) is as follows:

[0118] [ka] [ka]

[0119] The amino acid sequence of DNMT3A-(human)DNMT3L-XTEN80-ZIM3-GS-XTEN80-dCas9 (V45 form, SEQ ID NO: 96) is as follows:

[0120] [ka] [ka]

[0121] The amino acid sequence of DNMT3A-(human)DNMT3L-XTEN80-xten-ZIM3-GS-xten80-dCas9 (V46 form, SEQ ID NO: 97) is as follows:

[0122] [ka] [ka]

[0123] The amino acid sequence of DNMT3A-(human)DNMT3L-XTEN80-ZNF680-GS-XTEN80-dCas9 (V47 form, SEQ ID NO: 98) is as follows:

[0124] [ka] [ka]

[0125] The amino acid sequence of DNMT3A-(human)DNMT3L-XTEN80-ZNF354a-GS-XTEN80-dCas9 (V48 form, SEQ ID NO: 99) is as follows:

[0126] [ka] [ka]

[0127] The amino acid sequence of DNMT3A-(human)DNMT3L-XTEN80-ZNF419-GS-XTEN80-dCas9 (V49 form, SEQ ID NO: 100) is as follows:

[0128] [ka] [ka]

[0129] The amino acid sequence of KRAB-XTEN80-DNMT3A-(human)DNMT3L-XTEN16-ZIM3-dCas9 (V54 form, SEQ ID NO: 105) is as follows:

[0130] [ka] [ka]

[0131] The amino acid sequence of KRAB-XTEN80-ZIM3-XTEN16-DNMT3A-(human)DNMT3L-dCas9 (V55 form, SEQ ID NO: 106) is as follows:

[0132] [ka] [ka]

[0133] The amino acid sequence of DNMT3A-(human)DNMT3L-XTEN80-KRAB-XTEN80-ZIM3-XTEN80-dCas9 (V56 form, SEQ ID NO: 107) is as follows:

[0134] [ka] [ka]

[0135] Furthermore, for example, the one or more effector domains are all located at the N-terminus of the nucleic acid binding domain. In some embodiments, the effector domain may include at least one type of epigenetic modification domain and at least one type of transcriptional regulation domain, and the epigenetic editing domain in the effector domain may be located at the N-terminus of the transcriptional regulation domain or at the C-terminus of the transcriptional regulation domain. In these cases, the epigenetic modification domain may include DNMT3A and DNMT3L, and the C-terminus of DNMT3A may be linked to the N-terminus of DNMT3L, or the C-terminus of DNMT3L may be linked to the N-terminus of DNMT3A. In a similar situation, the transcriptional regulation domain may include a transcriptional repression domain. For example, the transcriptional regulation domain may include a zinc finger protein-based transcription factor or a functionally active fragment thereof. Specifically, the zinc finger protein-based transcription factor may include KRAB, and for example, in some embodiments of the present application, the zinc finger protein-based transcription factor may be selected from ZIM3 KRAB and KOX1 KRAB.

[0136] In some embodiments of the above situation, the transcriptional regulatory domain may comprise two or more KRAB domains, and the two or more KRAB domains are the same type or different types.For example, the transcriptional regulatory domain comprises two or more ZIM3 KRABs, two or more KOX1 KRABs, or ZIM3 KRABs and KOX1 KRABs at the same time, and the total number thereof is two or more.In these cases, the two or more (same type or different type) KRAB domains may be linked via an XTEN linker sequence.

[0137] The following is another exemplary fusion of the present application, with the amino acid sequences of the components as follows: bold - DNMT3A, underlined bold - DNMT3L, italics - linker sequence, bold italics - transcriptional regulatory domain, underlined bold italics - dCas9, * indicates any amino acid.

[0138] The amino acid sequence of dCas9-XTEN80-DNMT3A-DNMT3L-XTEN16-ZIM3 (V9 or V9' form, SEQ ID NO: 62 or 339) is as follows:

[0139] [ka] [ka]

[0140] or V9' form: [ka] [ka]

[0141] The amino acid sequence of dCas9-XTEN80-ZIM3-XTEN16-DNMT3A-DNMT3L (V10 or V10' form, SEQ ID NO: 63 or 340) is as follows:

[0142] [ka] [ka]

[0143] or V10' form: [ka] [ka]

[0144] The amino acid sequence of dCas9-XTEN80-ZIM3-XTEN16-DNMT3L-DNMT3A (V11 or V11' form, SEQ ID NO: 64 or 341) is as follows:

[0145] [ka] [ka]

[0146] or V11' form: [ka] [ka]

[0147] The amino acid sequence of dCas9-XTEN80-ZIM3-XTEN80-DNMT3L-DNMT3A (V14 or V14' form, SEQ ID NO: 65 or 342) is as follows:

[0148] [ka] [ka]

[0149] or V14' form: [ka] [ka]

[0150] The amino acid sequence of dCas9-XTEN80-ZIM3-NEW XTEN-DNMT3L-DNMT3A (V15 or V15' form, SEQ ID NO: 66 or 343) is as follows:

[0151] [ka] [ka]

[0152] or V15' form: [ka] [ka]

[0153] The amino acid sequence of dCas9-XTEN80-ZIM3-XTEN80-(human)DNMT3L-DNMT3A (V16 or V16' form, SEQ ID NO: 67 or 344) is as follows:

[0154] [ka] [ka]

[0155] or V16' form: [ka] [ka]

[0156] The amino acid sequence of dCas9-XTEN80-DNMT3L-DNMT3A-XTEN16-ZIM3 (V23 form, SEQ ID NO: 74) is as follows:

[0157] [ka] [ka]

[0158] The amino acid sequence of dCas9-XTEN80-DNMT3A-DNMT3L-XTEN16-KRAB (V24 form, SEQ ID NO: 75) is as follows:

[0159] [ka] [ka]

[0160] The amino acid sequence of dCas9-XTEN80-DNMT3L-DNMT3A-XTEN16-KRAB (V25 form, SEQ ID NO: 76) is as follows:

[0161] [ka] [ka]

[0162] The amino acid sequence of dCas9-XTEN80-KRAB-NEW XTEN-DNMT3L-DNMT3A (V26 form, SEQ ID NO: 77) is as follows:

[0163] [ka] [ka]

[0164] The amino acid sequence of dCas9-XTEN80-KRAB-NEW XTEN-DNMT3A-DNMT3L (V27 form, SEQ ID NO: 78) is as follows:

[0165] [ka] [ka]

[0166] The amino acid sequence of dCas9-XTEN80-KRAB-XTEN80-(human)DNMT3L-DNMT3A (V28 form, SEQ ID NO: 79) is as follows:

[0167] [ka] [ka]

[0168] The amino acid sequence of dCas9-XTEN80-ZNF582-XTEN80-(human)DNMT3L-DNMT3A (V29 form, SEQ ID NO: 80) is as follows:

[0169] [ka] [ka]

[0170] The amino acid sequence of dCas9-XTEN80-ZNF324-XTEN80-(human)DNMT3L-DNMT3A (V30 form, SEQ ID NO: 81) is as follows:

[0171] [ka] [ka]

[0172] The amino acid sequence of dCas9-NEW XTEN-KRAB-NEW XTEN-(human)DNMT3L-DNMT3A (V31 form, SEQ ID NO: 82) is as follows:

[0173] [ka] [ka]

[0174] The amino acid sequence of dCas9-NEW XTEN-(human)DNMT3L-DNMT3A-NEW XTEN-KRAB (V32 form, SEQ ID NO: 83) is as follows:

[0175] [ka] [ka]

[0176] The amino acid sequence of dCas9-XTEN80-ZIM3-XTEN80-DNMT3A-(human)DNMT3L (V33 form, SEQ ID NO: 84) is as follows:

[0177] [ka] [ka]

[0178] The amino acid sequence of dCas9-BFP-ZIM3-XTEN80-DNMT3A-(human)DNMT3L (V34 form, SEQ ID NO: 85) is as follows (underlined, dashed, bold italics - BFP, SEQ ID NO: 55):

[0179] [ka] [ka]

[0180] The amino acid sequence of dCas9-BFP-ZIM3-XTEN80-(human)DNMT3L-DNMT3A (V35 form, SEQ ID NO: 86) is as follows (underlined, dashed, bold italics - BFP, SEQ ID NO: 55):

[0181] [ka] [ka]

[0182] The amino acid sequence of dCas9-BFP-ZNF582-XTEN80-(human)DNMT3L-DNMT3A (V36 form, SEQ ID NO: 87) is as follows (underlined, dashed, bold italics - BFP, SEQ ID NO: 55):

[0183] [ka] [ka]

[0184] The amino acid sequence of dCas9-BFP-ZNF324-XTEN80-(human)DNMT3L-DNMT3A (V37 form, SEQ ID NO: 88) is as follows (underlined, dashed, bold italics - BFP, SEQ ID NO: 55):

[0185] [ka] [ka]

[0186] The amino acid sequence of dCas9-XTEN80-DNMT3L-DNMT3A-XTEN16-KRAB-XTEN16-KRAB (V50 form, SEQ ID NO: 101) is as follows:

[0187] [ka] [ka]

[0188] The amino acid sequence of dCas9-XTEN80-DNMT3L-DNMT3A-XTEN16-KRAB-XTEN16-KRAB-XTEN16-KRAB (V51 form, SEQ ID NO: 102) is as follows:

[0189] [ka] [ka]

[0190] The amino acid sequence of dCas9-XTEN80-KRAB-NEW XTEN-DNMT3L-DNMT3A-XTEN16-KRAB (V52 form, SEQ ID NO: 103) is as follows:

[0191] [ka] [ka]

[0192] The amino acid sequence of dCas9-XTEN80-ZIM3-NEW XTEN-DNMT3L-DNMT3A-XTEN16-KRAB (V53 form, SEQ ID NO: 104) is as follows:

[0193] [ka] [ka]

[0194] In another embodiment, the fusion herein may include one or more effector domains, the effector domains being located N-terminal and C-terminal to the nucleic acid binding domain. For example, the effector domain C-terminal to the nucleic acid-binding domain may include at least one type of epigenetic modification domain, specifically, the effector domain C-terminal to the nucleic acid-binding domain may include an epigenetic modification domain that provides at least one type of histone modification.

[0195] For example, the effector domain located at the N-terminus of the nucleic acid binding domain may include one or more of an epigenetic modification domain and a transcriptional regulation domain. Specifically, the effector domain located at the N-terminus of the nucleic acid binding domain may include at least one type of epigenetic modification domain, at least one type of transcriptional regulation domain, or at least one type of epigenetic modification domain and at least one type of transcriptional regulation domain. More specifically, the effector domain N-terminal to the nucleic acid binding domain may include one or more of an epigenetic modification domain that provides a histone modification, an epigenetic modification domain that provides a DNA modification, and a transcriptional repressor domain, for example, an epigenetic modification domain that provides at least one type of histone modification, an epigenetic modification domain that provides at least one type of DNA modification, at least one type of transcriptional repressor domain, an epigenetic modification domain that provides at least one type of histone modification and an epigenetic modification domain that provides at least one type of DNA modification, an epigenetic modification domain that provides at least one type of histone modification and at least one type of transcriptional regulatory domain, and an epigenetic modification domain that provides at least one type of DNA modification and at least one type of transcriptional regulatory domain.

[0196] In some embodiments in the above situation, the effector domain N-terminal to the nucleic acid binding domain comprises (1) an epigenetic modification domain that provides a histone modification, (2) a transcriptional repressor domain, (3) an epigenetic modification domain that provides a DNA modification, or (4) a transcriptional repressor domain and an epigenetic modification domain that provides a DNA modification. Specifically, in some embodiments within these contexts, the effector domain N-terminal to the nucleic acid binding domain comprises (1) one or more epigenetic modification domains selected from EZH2, HDAC3, HDAC1, EHMT2, PRMT1, PRMT5, SETDB1, hSIRT1, HP1a, LSD1, and functionally active fragments thereof; (2) KRAB or a functionally active fragment thereof; (3) DNMT3A, DNMT3L, or a combination of DNMT3A and DNMT3L; or (4) a combination of KRAB and DNMT3A, a combination of KRAB and DNMT3L, or a combination of KRAB, DNMT3A, and DNMT3L.

[0197] The following are some exemplary fusions of the present application, with each component part in the amino acid sequence as follows: bold - epigenetic modification domain (e.g., DNMT) that provides DNA modification; underlined bold - epigenetic modification domain (e.g., HDAC) that provides histone modification; italic - linker sequence; bold italic - transcriptional regulatory domain; underlined bold italic - dCas9 or TALE; * denotes any amino acid.

[0198] The amino acid sequence of DNMT3A-DNMT3L-XTEN80-dCas9-XTEN16-ZIM3 (V17 form, SEQ ID NO: 68) is as follows:

[0199] [ka] [ka]

[0200] The amino acid sequence of DNMT3A-(human)DNMT3L-XTEN80-dCas9-XTEN16-KRAB (V18 form, SEQ ID NO: 69) is as follows:

[0201] [ka] [ka]

[0202] The amino acid sequence of HDAC3-dCas9-EZH2 (V57 form, SEQ ID NO: 108) is as follows:

[0203] [ka] [ka]

[0204] The amino acid sequence of KRAB-dCas9-EZH2 (V58 form, SEQ ID NO: 109) is as follows:

[0205] [ka] [ka]

[0206] The amino acid sequence of KRAB-DNMT3A-dCas9-EZH2 (V59 form, SEQ ID NO: 110) is as follows:

[0207] [ka] [ka]

[0208] The amino acid sequence of KRAB-DNMT3A-dCas9-HDAC3 (V60 form, SEQ ID NO: 111) is as follows:

[0209] [ka] [ka]

[0210] The amino acid sequence of DNMT3A-DNMT3L-XTEN80-dCas9-XTEN16-EZH2x11 (V61 form, SEQ ID NO: 112) is as follows:

[0211] [ka] [ka]

[0212] The amino acid sequence of DNMT3A-DNMT3L-XTEN80-dCas9-XTEN16-EZH2 (V62 form, SEQ ID NO: 113) is as follows:

[0213] [ka] [ka]

[0214] The amino acid sequence of DNMT3A-DNMT3L-XTEN80-dCas9-XTEN16-EZH2 (core) (V63 form, SEQ ID NO: 114) is as follows:

[0215] [ka] [ka]

[0216] The amino acid sequence of DNMT3A-DNMT3L-XTEN80-dCas9-XTEN16-HDAC3 (V64 form, SEQ ID NO: 115) is as follows:

[0217] [ka] [ka]

[0218] The amino acid sequence of DNMT3A-DNMT3L-XTEN80-dCas9-XTEN16-HDAC3(core 1) (V65 form, SEQ ID NO: 116) is as follows:

[0219] [ka] [ka]

[0220] The amino acid sequence of DNMT3A-DNMT3L-XTEN80-dCas9-XTEN16-HDAC3(core2) (V66 form, SEQ ID NO: 117) is as follows:

[0221] [ka] [ka]

[0222] The amino acid sequence of DNMT3A-DNMT3L-XTEN80-dCas9-XTEN16-EHMT2 (V67 form, SEQ ID NO: 118) is as follows:

[0223] [ka] [ka]

[0224] The amino acid sequence of DNMT3A-DNMT3L-XTEN80-dCas9-XTEN16-HDAC1 (V68 form, SEQ ID NO: 119) is as follows:

[0225] [ka] [ka]

[0226] The amino acid sequence of DNMT3A-DNMT3L-XTEN80-dCas9-XTEN16-PRMT1 (V69 form, SEQ ID NO: 120) is as follows:

[0227] [ka] [ka]

[0228] The amino acid sequence of DNMT3A-DNMT3L-XTEN80-dCas9-XTEN16-SETDB1 (V70 form, SEQ ID NO: 121) is as follows:

[0229] [ka] [ka]

[0230] The amino acid sequence of DNMT3A-DNMT3L-XTEN80-dCas9-XTEN16-hSIRT1 (V71 form, SEQ ID NO: 122) is as follows:

[0231] [ka] [ka]

[0232] The amino acid sequence of DNMT3A-DNMT3L-XTEN80-dCas9-XTEN16-PRMT5 (V72 form, SEQ ID NO: 123) is as follows:

[0233] [ka] [ka]

[0234] The amino acid sequence of DNMT3A-DNMT3L-XTEN80-dCas9-XTEN16-HP1a (V73 form, SEQ ID NO: 124) is as follows:

[0235] [ka] [ka]

[0236] The amino acid sequence of DNMT3A-DNMT3L-XTEN80-dCas9-XTEN16-PRMT5(short) (V74 form, SEQ ID NO: 125) is as follows:

[0237] [ka] [ka]

[0238] The amino acid sequence of DNMT3A-DNMT3L-XTEN80-dCas9-XTEN16-LSD1 (V75 form, SEQ ID NO: 126) is as follows:

[0239] [ka] [ka]

[0240] The amino acid sequence of DNMT3A-DNMT3L-XTEN80-TALE8-XTEN16-KRAB (V76 form, SEQ ID NO: 355) is as follows:

[0241] [ka]

[0242] The amino acid sequence of DNMT3A-DNMT3L-XTEN80-TALE6-XTEN16-KRAB (V77 form, SEQ ID NO: 356) is as follows:

[0243] [ka]

[0244] Without wishing to be bound by any theory, the following examples are merely intended to illustrate the fusion proteins, production methods, uses, etc. of the present application, and are not intended to limit the scope of the present invention. [Example]

[0245] Example 1 Design and construction of plasmids containing the present fusions The amino acid sequences of epigenetic modification editors (including Dnmt3a CD, Dnmt3l CD, dSpCas9 or TALE, and KRAB) containing HA epidope, P2A, and BFP were optimized and synthesized by Genscript Inc. into nucleic acid sequences suitable for mammalian expression, and then cloned into the pLV-CAG vector containing the CAG promoter and WPRE. The complete epigenetic modification editor and self-cleaving BFP were expressed under the CAG promoter. When optimizing the order of different elements, the different fragments are amplified using PCR primers with homology arms, and then the NEBuilder reagents are used to reassemble the different fragments into a specific order depending on the order. When optimizing different functional elements, the elements are synthesized by Genscript, Inc., and optimized to a nucleic acid sequence suitable for mammalian expression. The vector is first amplified by PCR, excluding the elements requiring substitution. Next, the elements requiring substitution are amplified from the sequences synthesized by Genscript, simultaneously introducing homologous arm sequences. Finally, the different elements are recombined into the vector using the NEBuilder reagent to construct the final expression plasmid.

[0246] Example 2 The fusions of the present application can inhibit the expression of the VEGFA gene. To evaluate the effect of the fusion constructs provided herein (comprising the amino acid sequences set forth in SEQ ID NOs: 337-342) on inhibiting target gene expression, two sets of experiments were designed. Seven sets of editor plasmids and one control group were constructed according to the method described in Example 1. Two sets of experiments were conducted. Two different gRNAs (comprising the nucleotide sequences set forth in SEQ ID NOs: 332 and 333) targeting the mouse VEGFA gene and seven different editor plasmids were co-transfected into HEK293T cell lines (tools: 350 ng editor, 150 ng gRNA, 48-well plate) using PEI. A mouse VEGFA reporter gene was inserted into the 293T cell line, and GFP expression was regulated by the VEGFA regulatory region. Eleven days after transfection, the average green fluorescence intensity of different samples was analyzed and divided by the fluorescence intensity of the control group (transfection control NT gRNA, sequence shown in SEQ ID NO: 364) to obtain the relative inhibition efficiency (Figure 3). The results show that the present fusions all achieved higher or equal inhibition efficiency on GFP expression compared to the control group.

[0247] Example 3 The fusions of the present application can inhibit the expression of the CTLA-1 gene According to the method of Example 1, different editing tools (containing the amino acid sequences set forth in SEQ ID NOS: 337-344) were constructed, and a control group V1 (in which a methylation factor and a transcriptional repressor were fused to the N- and C-termini of the nucleic acid binding domain, respectively, and containing the amino acid sequence set forth in SEQ ID NOS: 328) was designated. These editing tools and a gRNA targeting the human CLTA gene (containing the nucleotide sequence set forth in SEQ ID NOS: 334) or a control gRNA (NT gRNA, sequence set forth in SEQ ID NOS: 364) were co-transfected into HEK293T cells (tool: 700 ng editor, 300 ng gRNA, 24-well plate). 72 hours after transfection, positively transfected cells were selected, total RNA was extracted with Trizol, and the relative expression levels of CLTA were quantified by qPCR to obtain the relative inhibitory efficiencies of the different tools (Figure 4). The results showed that the fusions of this application all exhibited comparable inhibitory efficiencies against mRNA transcribed from the CLTA gene compared to group V1.

[0248] Example 4 The fusion of the present application can inhibit the expression of the CD151 gene. According to the method of Example 1, different versions of epigenetic editing tools in the V15' form (original amino acid sequence shown in SEQ ID NO: 343) were constructed, in which (1) the effector domain contains DNMT3L factors from different sources (amino acid sequences shown in SEQ ID NOs: 13-21), such as the 3L-1 to 3L-9 versions shown in Figure 5A; (2) the effector domain contains transcriptional repressors based on different zinc finger proteins (amino acid sequences shown in SEQ ID NOs: 22-35), such as the versions of ZIM3, ZNF680, etc. shown in Figure 5B; and (3) the effector domain contains different types of transcriptional repressors (amino acid sequences shown in SEQ ID NOs: 36-46), such as the versions of EHMT2, SUV39H1, etc. shown in Figure 5C. The different editing tools and a gRNA plasmid targeting CD151 or a control gRNA (NT gRNA, sequence shown in SEQ ID NO: 364) were co-transfected into HEK293T cells (tool: 700 ng, gRNA: 300 ng, 24-well plate). 48 hours after transfection, positively transfected cells were selected using a flow cytometer and cultured. Nine days after transfection, cells were harvested and CD151 expression was detected using a flow cytometer. Cells with lower CD151 expression levels than untransfected cells were defined as inhibited cells. The effects of different versions of the epigenetic editing tools were compared based on the percentage of inhibited cells (Figures 5A-5C). The results show that the inhibitory effects of the present fusions containing different methylation factors or different transcriptional repressors were higher or equivalent to those of the control group (NT gRNA and unfused DNMT3L factor or unfused transcriptional repressor).

[0249] Example 5 The fusions of the present application can inhibit expression of the PCSK9 gene. The different versions of the tool were transcribed in vitro into mRNA (see SEQ ID NOS: 261-327 for mRNA sequences) and then mixed with chemically synthesized sgRNAs (sgRNA sequences are SEQ ID NOS: 335 and 336) at a 1:1 mass ratio to prepare LNPs (LNP reference: Musunuru, K., Chadwick, A.C., Mizoguchi, T. et al. In vivo CRISPR-based editing of PCSK9 durably lowers cholesterol in primates. Nature 593, 429-434 (2021)). The prepared LNPs were injected into mice via the tail vein at a dose of 4.5 mg / kg body weight. Blood was collected from the cheeks of the mice 4 to 10 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. The results are shown in Figures 6A to 6F, and demonstrate that the fusion product provided by the present application has a significant inhibitory effect on the expression of the PCSK9 gene.

[0250] Example 6 The fusions of the present application can inhibit expression of the PCSK9 gene. Based on the in vitro transcription of mRNA (mRNA sequences are shown in SEQ ID NOs: 261-327), a chemically synthesized 1:1 mass ratio sgRNA / mRNA mixture (sgRNA sequence is shown in SEQ ID NO: 363), and the LNP preparation method described in Example 5, the prepared LNP was added to Huh7 cells (1.25 μg / mL dose). Seven days after LNP addition, all cells were harvested, total RNA was extracted with Trizol, and the relative expression level of PCSK9 was quantified by qPCR. The relative inhibitory efficiency of different tools was calculated. The results are shown in Figure 7, where the NC group is a control without LNP addition, and the NT group is a control with NT gRNA (sequence is shown in SEQ ID NO: 364). It was found that the fusion provided herein has a significant inhibitory effect on PCSK9 gene expression.

Claims

1. A fusion comprising a nucleic acid binding domain and one or more effector domains, wherein the effector domain comprises one or more of an epigenetic modification domain and / or a transcriptional regulatory domain, and wherein the fusion comprises at least two types of epigenetic modification domains and / or transcriptional regulatory domains.

2. The fusion of claim 1 , wherein the nucleic acid binding domain is a DNA binding domain.

3. 3. The fusion of claim 2, wherein the DNA binding domain is selected from a TALE domain, a zinc finger domain, a tetR domain, a large-range nuclease, a Cas protein, an Argonaute (Ago) protein, and homologs or modified forms thereof.

4. The fusion of any one of claims 2 to 3, wherein the DNA binding domain is capable of binding to a target sequence in a target locus.

5. The fusion of claim 4, wherein the DNA binding domain is capable of binding to a guide RNA.

6. The fusion of claim 5, wherein the guide RNA specifically recognizes and hybridizes with a target sequence of the target locus.

7. The fusion of any one of claims 2 to 6, wherein the DNA binding domain is a Cas protein, and the Cas protein is a class II Cas nuclease.

8. The fusion of claim 7, wherein the Cas protein is selected from a class II type II Cas nuclease and a class II type V Cas nuclease.

9. The fusion of any one of claims 2 to 8, wherein the DNA binding domain is a Cas9 protein.

10. The fusion of claim 9, wherein the Cas9 protein is an inactive (dead) Cas9 protein (dCas9).

11. The fusion product according to any one of claims 1 to 6, wherein the nucleic acid binding domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 7, 353 and 354.

12. 12. The fusion of any one of claims 1 to 11, 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.

13. The fusion of any one of claims 1 to 12, wherein the epigenetic modification domain comprises a DNA methyltransferase and / or a functionally active fragment thereof.

14. The fusion of claim 13, wherein the DNA methyltransferase is selected from DNMT3A, DNMT3B, Dnmt3c, DNMT1, DNMT2 and DNMT3L.

15. 15. The fusion of any one of claims 1 to 14, wherein the epigenetic modification domain comprises a plurality of DNA methyltransferases and / or functionally active fragments thereof, and the plurality of DNA methyltransferases and / or functionally active fragments thereof are linked via a linker sequence.

16. The fusion of any one of claims 1 to 15, wherein the epigenetic modification domain comprises at least one DNMT3A and at least one DNMT3L.

17. The fusion product according to any one of claims 13 to 15, wherein the DNA methyltransferase comprises an amino acid sequence shown in any one of SEQ ID NOs: 8 to 21.

18. The fusion of any one of claims 1 to 17, wherein the transcriptional regulatory domain is a transcriptional activation domain or a transcriptional repressor domain.

19. The transcriptional repressor domain may be any of KRAB, ZIM3, ZNF680, ZNF554, ZNF264, ZNF582, ZNF324, ZNF669, ZNF354A, ZNF82, ZNF595, ZNF419, ZNF566, ZIM2, EHMT2, SUV39H1, ZFPM1, TRIM28, EZH2, M XD1, SID, LSD1, HP1a, HDAC3, ZNF436, ZNF257, ZNF675, ZNF490, ZNF320, ZNF331, ZNF81 6, ZNF41, ZNF189, ZNF528, ZNF543, ZNF140, ZNF610, ZNF350, ZNF8, ZNF30, ZNF98, ZNF6 77, ZNF596, ZNF214, ZNF37A, ZNF34, ZNF250, ZNF547, ZNF273, ZFP82, ZNF224, ZNF33A , ZNF45, ZNF175, ZNF184, ZFP28-1, ZFP28-2, ZNF18, ZNF213, ZNF394, ZFP1, ZFP14, ZNF 416, ZNF557, ZNF729, ZNF254, ZNF764, ZNF785, ZNF10, CBX5, RYBP, YAF2, MGA, CBX1, SC MH1, MPP8, SUMO3, HERC2, BIN1, PCGF2, TOX, FOXA1, FOXA2, IRF2BP1, IRF2BP2, IRF2BPL IRF-2BP1_2 N-terminal domain, HOXA13, HOXB13, HOXC13, HOXA11, HOXC11, HOXC10, HOXA10, HOXB9, HOXA9, ZFP28, ZN334, ZN568, ZN37A, ZN181, ZN510, ZN862, ZN140, ZN208, ZN248, ZN571, ZN699, ZN726, ZIK1, ZNF2, Z705F, ZNF14, ZN471, ZN624, ZNF84, ZNF7, ZN891, ZN337, Z705G , ZN529, ZN729, ZN419, Z705A, ZN302, ZN486, ZN621, ZN688, ZN3 3A, ZN554, ZN878, ZN772, ZN224, ZN184, ZN544, ZNF57, ZN283, ZN 549, ZN211, ZN615, ZN253, ZN226, ZN730, Z585A, ZN732, ZN681, Z N667, 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、The fusion of claim 18, wherein the fusion is selected from RBBP4, DMRT2, SMCA2, and functionally active fragments thereof.

20. The fusion of claim 18 or 19, wherein the transcriptional repressor domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 22 to 46.

21. The fusion product of any one of claims 1 to 20, wherein one or more of the nucleic acid binding domain, the epigenetic modification domain and the transcriptional regulatory domain are linked via a linker sequence.

22. The fusion of claim 21 , wherein the linker sequence comprises at least 16 amino acids.

23. 23. The fusion of claim 21 or 22, wherein the linker sequence comprises an XTEN linker sequence.

24. The fusion product according to any one of claims 21 to 23, wherein the linker sequence comprises one or more fragment sequences excised from the amino acid sequence set forth in SEQ ID NO: 59, and the one or more fragment sequences comprise 16 or more consecutive amino acids.

25. The linker sequence comprises a GS linking peptide, the GS linking peptide having the sequence: (GS) ａ (GGS) ｂ (GGGS) ｃ (GGGGS) ｄ wherein G represents a glycine residue (Gly), S represents a serine residue (Ser), and a, b, c, and d represent integers of 0 or greater.

26. The fusion of any one of claims 21 to 25, wherein the linker sequence comprises an amino acid sequence selected from one or more of SEQ ID NOs: 47 to 58.

27. The fusion of any one of claims 1 to 26, wherein the one or more effector domains are N-terminal or C-terminal to the nucleic acid binding domain.

28. The fusion of claim 27 , wherein the effector domain comprises at least one type of epigenetic modification domain and at least one type of transcriptional regulatory domain.

29. The fusion of claim 27 or 28, wherein the epigenetic modification domain comprises DNMT3A and DNMT3L, and the C-terminus of DNMT3A is linked to the N-terminus of DNMT3L.

30. The fusion of claim 27 or 28, wherein the epigenetic modification domain comprises DNMT3A and DNMT3L, and the C-terminus of DNMT3L is linked to the N-terminus of DNMT3A.

31. The fusion of any one of claims 27 to 30, wherein the transcriptional regulatory domain comprises a transcriptional repressor domain.

32. The fusion of any one of claims 27 to 31, wherein the transcriptional regulatory domain comprises a zinc finger protein-based transcription factor or a functionally active fragment thereof.

33. 33. The fusion of claim 32, wherein the zinc finger protein-based transcription factor comprises KRAB.

34. 34. The fusion of claim 32 or 33, wherein the zinc finger protein-based transcription factor is selected from ZIM3 KRAB and KOX1 KRAB.

35. The fusion of any one of claims 27 to 34, wherein the transcriptional regulatory domain comprises two or more of the zinc finger protein-based transcription factors or functionally active fragments thereof.

36. The fusion of any one of claims 27 to 35, wherein the transcriptional regulatory domain comprises two or more of the above KRAB domains, and the two or more of the above KRAB domains are of the same type or different types.

37. The fusion of claim 36, wherein the two or more KRAB domains are linked by an XTEN linker sequence.

38. 38. The fusion of any one of claims 27 to 37, wherein the one or more effector domains are N-terminal to the nucleic acid binding domain, and the epigenetic editing domain in the effector domain is N-terminal or C-terminal to the transcriptional regulatory domain.

39. The following substances linked in order from N-terminus to C-terminus: (1) at least one type of said epigenetic editing domain, at least one type of said transcriptional regulatory domain, and said nucleic acid binding domain; (2) at least one type of transcriptional regulatory domain, at least one type of epigenetic editing domain, and the nucleic acid binding domain; or (3) at least one type of transcriptional regulatory domain, at least one type of epigenetic editing domain, at least one type of transcriptional regulatory domain and the nucleic acid binding domain; The fusion product according to any one of claims 27 to 38, comprising:

40. The following substances linked in order from N-terminus to C-terminus: (1) at least one DNA methyltransferase domain, at least one transcriptional repressor domain, and the nucleic acid binding domain; (2) at least one transcriptional repressor domain, at least one DNA methyltransferase domain, and the nucleic acid binding domain; or (3) at least one transcriptional repressor domain, at least one DNA methyltransferase domain, at least one transcriptional repressor domain and the nucleic acid binding domain; The fusion product of any one of claims 27 to 39, comprising:

41. The following substances linked in order from N-terminus to C-terminus: (1) one or a combination of DNMT3A and DNMT3L, one or more of the above zinc finger protein-based transcription factors, and dCas9; (2) one or a combination of one or more of the above zinc finger protein-based transcription factors, DNMT3A and DNMT3L, and dCas9; or (3) one or more of the above zinc finger protein-based transcription factors, one or a combination of DNMT3A and DNMT3L, one or more of the above zinc finger protein-based transcription factors, and dCas9; The fusion product according to any one of claims 27 to 40, comprising:

42. The fusion comprises the following domains: DNMT3A-DNMT3L-KRAB-dCas9, KRAB-DNMT3A-DNMT3L-dCas9, DNMT3A-DNMT3L-ZNF582-dCas9, DNMT3A-DNMT3L- ZNF324-dCas9, DNMT3A-DNMT3L-ZNF680-dCas9, DNMT3A-DNMT3L-ZNF354a-dCas9, DNMT3A-DNMT3L-ZNF419-dCa 42. The fusion of any one of claims 27 to 41, comprising KRAB-DNMT3A-DNMT3L-KRAB-dCas9, KRAB-KRAB-DNMT3A-DNMT3L-dCas9, or DNMT3A-DNMT3L-KRAB-KRAB-dCas9, wherein - indicates that the domains of the fusion are linked directly and / or indirectly to each other and that the domains are in N-terminal to C-terminal order.

43. 38. The fusion of any one of claims 27 to 37, wherein the one or more effector domains are C-terminal to the nucleic acid binding domain, and the epigenetic editing domain in the effector domain is N-terminal or C-terminal to the transcriptional regulatory domain.

44. The following substances linked in order from N-terminus to C-terminus: (1) the nucleic acid binding domain, at least one of the epigenetic editing domains, and at least one type of the transcriptional regulatory domain; (2) the nucleic acid binding domain, at least one type of the transcriptional regulatory domain, and at least one type of the epigenetic editing domain; or (3) the nucleic acid binding domain, at least one type of the transcriptional regulatory domain, at least one type of the epigenetic editing domain, and at least one type of the transcriptional regulatory domain; The fusion product of any one of claims 27 to 37 and 43, comprising:

45. The following substances linked in order from N-terminus to C-terminus: (1) the nucleic acid binding domain, at least one DNA methyltransferase domain, and at least one transcriptional repressor domain; (2) the nucleic acid binding domain, at least one transcriptional repressor domain, and at least one DNA methyltransferase domain; or (3) the nucleic acid binding domain, at least one transcriptional repressor domain, at least one DNA methyltransferase domain, and at least one transcriptional repressor domain; The fusion product according to any one of claims 27 to 37 and 43 to 44, comprising:

46. The following substances linked in order from N-terminus to C-terminus: (1) one or a combination of dCas9, DNMT3A, and DNMT3L, and one or more of the above zinc finger protein-based transcription factors; (2) dCas9, one or more of the above zinc finger protein-based transcription factors, and one or a combination of DNMT3A and DNMT3L; or (3) dCas9, one or more of the above zinc finger protein-based transcription factors, one or a combination of DNMT3A and DNMT3L, and one or more of the above zinc finger protein-based transcription factors; The fusion product according to any one of claims 27 to 37 and 43 to 45, comprising:

47. The fusion comprises the following domains: dCas9-DNMT3A-DNMT3L-KRAB, dCas9-DNMT3L-DNMT3A-KRAB, dCas9-KRAB-DNMT3A-DNMT3L, dCas9-KRAB- DNMT3L-DNMT3A, dCas9-ZNF582-DNMT3L-DNMT3A, dCas9-ZNF324-DNMT3L-DNMT3A, dCas9-DNMT3L-DNMT3A 47. The fusion of any one of claims 27-37 and 43-46, comprising -KRAB-KRAB, dCas9-DNMT3L-DNMT3A-KRAB-KRAB-KRAB, or dCas9-KRAB-DNMT3L-DNMT3A-KRAB, where - indicates that the domains of the fusion are linked directly and / or indirectly to each other and that the domains are in N-terminal to C-terminal order.

48. The fusion product according to any one of claims 27 to 47, comprising an amino acid sequence shown in any one of SEQ ID NOs: 60 to 67, 70 to 107 and 337 to 344.

49. 27. The fusion of any one of claims 1 to 26, wherein the fusion comprises one or more effector domains, the effector domains being located N-terminal and C-terminal to the nucleic acid binding domain.

50. 50. The fusion of claim 49, wherein the one or more effector domains comprise at least one epigenetic editing domain.

51. 51. The fusion of claim 50, wherein the one or more effector domains comprise at least one epigenetic editing domain that provides a histone modification.

52. The fusion of any one of claims 49 to 51, wherein the effector domain C-terminal to the nucleic acid binding domain comprises at least one type of epigenetic modification domain.

53. The fusion of any one of claims 49 to 52, wherein the effector domain C-terminal to the nucleic acid binding domain comprises at least one epigenetic modification domain that provides a histone modification.

54. The fusion of any one of claims 49 to 53, wherein the effector domain N-terminal to the nucleic acid binding domain comprises one or more of an epigenetic modification domain and a transcriptional regulatory domain.

55. 55. The fusion of any one of claims 49 to 54, wherein the effector domain N-terminal to the nucleic acid binding domain comprises one or more of an epigenetic modification domain that provides a histone modification, an epigenetic modification domain that provides a DNA modification, and a transcriptional repressor domain.

56. The effector domain, which is N-terminal to the nucleic acid binding domain, (1) an epigenetic modification domain that provides histone modifications; (2) a transcriptional repressor domain, (3) an epigenetic modification domain that provides a DNA modification; or (4) a transcriptional repressor domain and an epigenetic modification domain that provides DNA modification; 56. The fusion of any one of claims 49 to 55, comprising:

57. The effector domain, which is N-terminal to the nucleic acid binding domain, (1) one or more epigenetic modification domains selected from EZH2, HDAC3, HDAC1, EHMT2, PRMT1, PRMT5, SETDB1, hSIRT1, HP1a, LSD1, and functionally active fragments thereof; (2) KRAB or a functionally active fragment thereof; (3) DNMT3A, DNMT3L, or a combination of DNMT3A and DNMT3L; or (4) The fusion product according to any one of claims 49 to 56, comprising a combination of KRAB and DNMT3A, a combination of KRAB and DNMT3L, or a combination of KRAB, DNMT3A, and DNMT3L.

58. The following substances linked in order from N-terminus to C-terminus: (1) an epigenetic modification domain that provides a histone modification; the nucleic acid binding domain and the epigenetic modification domain that provides a histone modification; (2) a transcriptional repressor domain, the nucleic acid binding domain, and an epigenetic modification domain that provides a histone modification; (3) an epigenetic modification domain that provides DNA modification, the nucleic acid binding domain, and an epigenetic modification domain that provides histone modification; (4) a transcriptional repressor domain, an epigenetic modification domain that provides DNA modification, the nucleic acid binding domain, and an epigenetic modification domain that provides histone modification; or (5) an epigenetic modification domain that provides DNA modification, a transcriptional repressor domain, the nucleic acid binding domain, and an epigenetic modification domain that provides histone modification; 58. The fusion of any one of claims 49 to 57, comprising:

59. The fusion of claim 58, wherein the epigenetic modification domain that provides the histone modification is selected from EZH2, HDAC3, HDAC1, EHMT2, PRMT1, PRMT5, SETDB1, hSIRT1, HP1a, LSD1, and functionally active fragments thereof.

60. 59. The fusion of claim 58, wherein the epigenetic modification domain that provides the DNA modification is selected from DNMT3A, DNMT3L, a combination of DNMT3A and DNMT3L, or functionally active fragments thereof.

61. 59. The fusion of claim 58, wherein the transcriptional repressor domain is KRAB and / or a functionally active fragment thereof.

62. The fusion comprises the following domains: DNMT3A-DNMT3L-dCas9-KRAB, HDAC3-dCas9-EZH2, KRAB-dCas9-EZH2, KRAB-DN MT3A-dCas9-EZH2, KRAB-DNMT3A-dCas9-HDAC3, DNMT3A-DNMT3L-dCas9-(EZH2) ｎ＝１－１１ , DNMT3A-DNMT3L-dCas9-HDAC3, DNMT3A-DNMT3L-dCas9-EHMT2, DNMT3A-DNMT3L-dCas9-HDAC1, DNMT3A- DNMT3L-dCas9-PRMT1, DNMT3A-DNMT3L-dCas9-SETDB1, DNMT3A-DNMT3L-dCas9-hSIRT1, DNMT3A-DNMT3L 62. The fusion of any one of claims 49-61, comprising -dCas9-PRMT5, DNMT3A-DNMT3L-dCas9-HP1a, DNMT3A-DNMT3L-dCas9-LSD1, or DNMT3A-DNMT3L-TALE-KRAB, wherein - indicates that the domains of the fusion are linked directly and / or indirectly to each other, and the domains are in N-terminal to C-terminal order.

63. The fusion of any one of claims 49 to 62, comprising an amino acid sequence set forth in any one of SEQ ID NOs: 68, 69, 108-126, 355 and 356.

64. The fusion of any one of claims 49 to 63, further comprising a nuclear localization signal and / or a tag domain.

65. A nucleic acid encoding the fusion of any one of claims 1 to 64.

66. 66. A recombinant vector comprising the nucleic acid of claim 65.

67. 67. A delivery vector comprising a fusion according to any one of claims 1 to 64, a nucleic acid according to claim 65, and / or a recombinant vector according to claim 66, and optionally comprising a liposome and / or a lipid nanoparticle.

68. A composition comprising a fusion according to any one of claims 1 to 64, a nucleic acid according to claim 65, a recombinant vector according to claim 66, and / or a delivery vector according to claim 67.

69. A cell comprising a fusion according to any one of claims 1 to 64, a nucleic acid according to claim 65, a recombinant vector according to claim 66, a delivery vector according to claim 67, and / or a composition according to claim 68.

70. 69. A kit comprising a fusion according to any one of claims 1 to 64, a nucleic acid according to claim 65, a recombinant vector according to claim 66, a delivery vector according to claim 67, a composition according to claim 68, and / or a cell according to claim 69.

71. 71. A method for modulating expression of a target gene comprising administering a fusion according to any one of claims 1 to 64, a nucleic acid according to claim 65, a recombinant vector according to claim 66, a delivery vector according to claim 67, a composition according to claim 68, a cell according to claim 69 and / or a kit according to claim 70.

72. 72. The method of claim 71, comprising introducing the fusion, the nucleic acid, the recombinant vector, the delivery vector, the composition, the cell, and / or the kit into a cell containing the target gene.

73. 72. The method of claim 71, comprising contacting the fusion, the nucleic acid, the recombinant vector, the delivery vector, and / or the composition with a regulatory element near and / or of the target gene.

74. 74. The method of claim 73, wherein the regulatory elements comprise a core promoter, a proximal promoter, a distal enhancer, a silencer, an insulator element, a boundary element, and / or a locus control region.

75. A method for treating or alleviating a disease or symptom associated with abnormal target gene expression and / or abnormal target gene activity, comprising administering to a subject in need thereof an effective amount of the fusion of any one of claims 1 to 64, the nucleic acid of claim 65, the recombinant vector of claim 66, the delivery vector of claim 67, the composition of claim 68, the cell of claim 69 and / or the kit of claim 70.

Images