Method for treating diseases by multi-target epigenetic editing

Abstract

A method for simultaneously regulating the expression and / or activity of a dual-target gene. The method comprises: providing a complex or a nucleic acid encoding the complex, wherein the complex comprises a DNA binding domain and a gene expression modulator. For example, the method simultaneously regulates the expression and / or activity of PCSK9 gene and ANGPTL3 gene. For example, the method simultaneously regulates the expression and / or activity of APOC3 gene and the ANGPTL3 gene. For example, the method simultaneously regulates the expression and / or activity of the PCSK9 gene and the APOC3 gene.

Description

Multi-target disease treatment via epigenetic editing Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a method for simultaneously regulating the expression and / or activity of dual-target genes. Background Art

[0002] Combined hyperlipidemia is the most common type of abnormal lipoprotein metabolism or operation in people with dyslipidemia. A series of clinical studies at home and abroad have confirmed that combined hyperlipidemia is a major cause of atherosclerotic cardiovascular disease (ASCVD), including coronary heart disease, stroke, and peripheral vascular disease. Combined hyperlipidemia is a state of dyslipidemia that includes both hypercholesterolemia and hypertriglyceridemia. The classification of this symptom is mainly based on the specific abnormal manifestations of blood lipid levels; among them, type II hyperlipidemia is the most common type, especially type IIb, which is characterized by elevated low-density lipoprotein cholesterol (LDL-C) and triglyceride levels, and is the most common form of mixed hyperlipidemia. Therefore, targeting groups with elevated LDL-C and triglyceride levels has become the main target for addressing mixed hyperlipidemia.

[0003] Currently, there are numerous therapeutic drugs targeting LDL-C. PCSK9, a popular target for lipid-lowering drug development, has garnered significant attention in recent years. Once produced in the body, PCSK9 degrades the low-density lipoprotein receptor (LDL-R) on the surface of liver cells, hindering the liver's effective clearance of cholesterol, particularly LDL-C, from the blood, ultimately leading to elevated blood lipid levels. Inhibiting PCSK9 expression offers a solution to this problem. By reducing PCSK9 expression, PCSK9 binding to LDL-R is reduced, thereby preventing LDL-R degradation on the surface of liver cells. This results in more LDL-R on the liver surface, enabling more effective clearance of LDL-C from the blood. Currently, drug strategies for reducing PCSK9 expression primarily fall into three categories: inhibitors, monoclonal antibodies, siRNA, and gene-editing drugs. These inhibitors, monoclonal antibodies, and siRNA all present challenges with long-term administration, requiring patients to administer them, which some patients cannot tolerate, and some patients with complications cannot receive treatment. Gene therapy drugs, such as Verve-101, developed by Verve Therapeutics in the United States, are base editors delivered to the liver via lipid nanoparticles. They replace one nucleotide in the PCSK9 gene with another, thereby inactivating the PCSK9 gene. However, the genetic risks associated with the genome-cleaving activity of these drugs remain to be evaluated. Therefore, there is an urgent need for a drug that can be administered once, is effective for life, and does not require genome-cleaving, thus avoiding genetic risks.

[0004] ANGPTL3 (vascular endothelial growth factor-like protein 3) is another protein that plays a key role in lipid metabolism and has also attracted widespread attention in the field of drug development. ANGPTL3 regulates lipid metabolism by inhibiting the activity of lipoprotein lipase (LPL) and endogenous lipoprotein lipase (EL), affecting triglyceride and cholesterol levels. ANGPTL3 can fill the gap left by PCSK9 in treating patients with homozygous hypercholesterolemia and can lower triglycerides while regulating cholesterol. ANGPTL3 can also fill the gap left by APOC3 in lowering LDL-C and can lower triglycerides while regulating cholesterol. Currently, drugs targeting the ANGPTL3 gene mainly focus on monoclonal antibodies, siRNA, and gene-editing drugs. Preliminary studies have shown that ANGPTL3 targeted therapy has a bright future, but its long-term efficacy and safety still need to be verified through more extensive clinical trials.

[0005] APOC3, or apolipoprotein C-III, is a protein that plays a key role in blood lipid regulation. It plays an important role in cardiovascular health, particularly in controlling triglyceride levels. It affects the hydrolysis and clearance of triglycerides by inhibiting the activity of lipoprotein lipase (LPL), an enzyme responsible for breaking down triglycerides in the blood. Due to its crucial role in triglyceride metabolism, APOC3 has become a potential drug target for the treatment of hypertriglyceridemia and related cardiovascular diseases. For example, volanesorsen is an antisense oligonucleotide drug targeting APOC3 that is used to lower triglyceride levels in patients with hypertriglyceridemia. This drug lowers triglyceride levels in the blood by reducing APOC3 production. Its development represents a major advancement in the treatment of hypertriglyceridemia. In recent years, gene editing technology has also been used to regulate APOC3, thereby lowering blood triglycerides. However, for patients with mixed hypercholesterolemia, lowering triglyceride levels alone cannot completely eliminate the risk of cardiovascular disease. Therefore, the therapeutic effect of APOC3 single-target drugs on this population is very limited. Summary of the Invention

[0006] On the one hand, in order to solve the technical difficulties of the existing technology for treating mixed hyperlipidemia, this article provides a method for introducing inhibitory epigenetic modifications in specific regulatory regions of PCSK9 and ANGPTL3 through an epigenetic editing tool (EPIREG), changing the transcriptional activity of PCSK9 and ANGPTL3, and achieving simultaneous inhibition of the expression of both PCSK9 and ANGPTL3 genes, thereby reducing the levels of lipoprotein cholesterol (LDL-C) and triglycerides in the blood, and achieving the purpose of treating mixed hyperlipidemia. The epigenetic editing tool provided in this application achieves genomic positioning through gRNA and recruits epigenetic modification proteins such as DNA methyltransferases (DNMTs), introduces epigenetic modifications at specific sites, changes the chromatin structure, and thereby adjusts the target gene to a transcriptional repression state, achieving silencing regulation of the target gene. In this process, DNA will not be cut, avoiding the possibility of generating double-strand breaks in the genome, and is relatively safe.

[0007] On the one hand, the present application provides a method for simultaneously regulating the expression and / or activity of PCSK9 gene and ANGPTL3 gene, the method comprising providing an epigenetic editing system; the epigenetic editing system comprises a DNA binding domain and a gene expression regulator.

[0008] In some embodiments, the epigenetic editing system comprises a complex, and the DNA binding domain and the gene expression regulator are contained in the complex; or the epigenetic editing system comprises a nucleic acid encoding the complex.

[0009] In some embodiments, the complex comprises a first fusion portion and a second fusion portion; wherein, one of the first fusion portion and the second fusion portion comprises the DNA binding domain, at least one of the gene expression regulators and the recruitment domain A, the other of the first fusion portion and the second fusion portion comprises at least one of the gene expression regulators and the recruitment domain A', and the recruitment domain A and the recruitment domain A' are capable of interacting with each other.

[0010] In some embodiments, the interaction between the recruitment domain A and the recruitment domain A′ enables the gene expression regulator to be recruited to the regulatory region of the PCSK9 gene and / or the ANGPTL3 gene or to the vicinity thereof.

[0011] In some embodiments, the gene expression regulator comprised by the first fusion moiety and the second fusion moiety is optionally a transcriptional repressor domain and an epigenetic modification domain, respectively.

[0012] In some embodiments, the transcriptional repressor domain is selected from: 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, ZNF98, ZNF677, ZNF596, ZNF214, ZNF37A, ZNF34, ZNF250, ZNF547, ZNF273, ZFP82, ZNF224, ZNF33A, ZNF45, ZNF175, ZNF184, ZFP28-1, ZFP28-2, ZNF18, ZNF213, ZNF394, ZFP1, ZFP14, ZNF416, ZNF557, ZNF729, ZNF254, ZNF764, ZNF785, ZNF10, CBX5, RYBP, YAF2, MGA, CBX1, SCMH1, MPP8, SUMO3, HERC2, BIN, 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, PRP19, NKX11, RBBP4, DMRT2, SMCA2, and their functionally active fragments.

[0013] In some embodiments, the epigenetic modification domain comprises one or more of: DNA methyltransferase activity, DNA demethylase activity, DNA deamination activity, DNA amination activity, DNA oxidation activity, DNA helicase activity, histone methyltransferase activity, histone demethylase activity, histone acetyltransferase activity, histone deacetylase activity, histone kinase activity, histone phosphatase activity, histone ubiquitin ligase activity, and histone deubiquitinating activity.

[0014] In some embodiments, the epigenetic modification domain comprises a DNA methyltransferase and / or a functionally active fragment thereof.

[0015] In some embodiments, the DNA methyltransferase is selected from DNMT3A, DNMT3B, Dnmt3c, DNMT1, DNMT2, and DNMT3L.

[0016] In some embodiments, the DNA methyltransferase comprises at least one DNMT3A and at least one DNMT3L.

[0017] In some embodiments, the DNA binding domain is selected from the group consisting of: a TALE domain, a zinc finger domain, a tetR domain, a meganuclease, a Cas nuclease, an Argonaute (Ago) protein, and homologs, modified forms, or variants thereof.

[0018] In some embodiments, the DNA binding domain is capable of specifically recognizing a target sequence on the PCSK9 gene and / or the ANGPTL3 gene.

[0019] In some embodiments, the DNA binding domain specifically recognizes the target sequence by binding to a guide RNA.

[0020] In some embodiments, the DNA binding domain is a class II Cas nuclease.

[0021] In some embodiments, the Cas nuclease is selected from a class II type II Cas nuclease and a class II type V Cas nuclease.

[0022] In some embodiments, the Cas nuclease is Cas9.

[0023] In some embodiments, the Cas nuclease is a deactivated Cas9 (dCas9).

[0024] In some embodiments, the epigenetic editing system further comprises a guide RNA, which is capable of specifically recognizing a target sequence on the PCSK9 gene and / or the ANGPTL3 gene.

[0025] In some embodiments, the recruitment domain A is selected from any one of the following two groups of domains, and the recruitment domain A' is selected from any one of the other of the following two groups of domains: 1) general control non-derepressor protein 4 (GCN4), GFP11 fragment derived from split green fluorescent protein (GFP), or GVKESLV polypeptide; and 2) single-chain antibody (scFv), GFP1-10 fragment derived from split green fluorescent protein (GFP), or PDZ protein domain.

[0026] In some embodiments, the method is characterized in that: 1) the domain of one of the recruitment domain A and the recruitment domain A' is GCN4, and the other domain is scFv; or 2) the domain of one of the recruitment domain A and the recruitment domain A' is a GFP11 fragment, and the other domain is GFP1-10; or 3) the domain of one of the recruitment domain A and the recruitment domain A' is GVKESLV, and the other domain is a PDZ protein domain.

[0027] In some embodiments, the method is characterized in that: 1) one of the first fusion moiety and the second fusion moiety comprises DNA methyltransferase-dCas9-n×GCN4, and the other comprises a transcriptional repressor domain-scFv or scFv-transcriptional repressor domain; or 2) one of the first fusion moiety and the second fusion moiety comprises DNA methyltransferase-dCas9-scFv, and the other comprises a transcriptional repressor domain-GCN4 or GCN4-transcriptional repressor domain; or 3) one of the first fusion moiety and the second fusion moiety comprises DNA methyltransferase-dCas9-n×GFP1 1, and the other comprises a transcription repressor domain-GFP1-10 or GFP1-10-transcription repressor domain; or 4) one of the first fusion portion and the second fusion portion comprises DNA methyltransferase-dCas9-GFP1-10, and the other comprises a transcription repressor domain-GFP11 or GFP11-transcription repressor domain; wherein, - indicates that the domains at both ends are directly or indirectly connected in the order from N-terminus to C-terminus; n×GCN4 or n×GFP11 respectively represents n copies of GCN4 connected by a linker sequence or n copies of GFP11 connected by a linker sequence, and n is selected from any integer from 1 to 20.

[0028] In some embodiments, the method is characterized in that: 1) one of the first fusion portion and the second fusion portion comprises an n×GCN4-dCas9-transcriptional repressor domain, and the other comprises a DNA methyltransferase-scFv or scFv-DNA methyltransferase; or 2) one of the first fusion portion and the second fusion portion comprises an scFv-dCas9-transcriptional repressor domain, and the other comprises a DNA methyltransferase-GCN4 or GCN4-DNA methyltransferase; or 3) one of the first fusion portion and the second fusion portion comprises an n×GFP11-dCas9-transcriptional repressor structure domain, and the other comprises DNA methyltransferase-GFP1-10 or GFP1-10-DNA methyltransferase; or 4) one of the first fusion part and the second fusion part comprises a GFP1-10-dCas9-transcriptional repressor domain, and the other comprises a DNA methyltransferase-GFP11 or GFP11-DNA methyltransferase; wherein, - indicates that the domains at both ends are directly or indirectly connected in the order from N-terminus to C-terminus; n×GCN4 or n×GFP11 respectively represent n copies of GCN4 connected by a linker sequence or n copies of GFP11 connected by a linker sequence, and n is selected from any integer from 1 to 20.

[0029] In some embodiments, the complex comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-40.

[0030] In some embodiments, the nucleic acid is a recombinant expression vector.

[0031] In some embodiments, the recombinant expression vector is a plasmid or a viral vector.

[0032] In some embodiments, the nucleic acid comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 41-160.

[0033] In some embodiments, the complex or the nucleic acid encoding the complex is disposed in the same or different delivery vehicles.

[0034] In some embodiments, the delivery vehicle comprises a liposome and / or a lipid nanoparticle.

[0035] On the other hand, the present application provides a complex, such as the complex provided in the method described in the present application, and the complex can simultaneously regulate the expression and / or activity of PCSK9 gene and ANGPTL3 gene without changing the function of their gene sequences.

[0036] In another aspect, the present application provides a nucleic acid encoding the complex described in the present application.

[0037] On the other hand, the present application provides a recombinant expression vector comprising the nucleic acid described in the present application.

[0038] On the other hand, the present application provides a delivery vector, which comprises the complex described in the present application, the nucleic acid described in the present application and / or the recombinant expression vector described in the present application.

[0039] On the other hand, the present application provides a pharmaceutical composition comprising the complex described herein, the nucleic acid described herein, the recombinant expression vector described herein and / or the delivery vector described herein, and at least one pharmaceutically acceptable carrier.

[0040] In some embodiments, the pharmaceutical composition further comprises a guide RNA, which is capable of specifically recognizing a target sequence on the PCSK9 gene and / or the ANGPTL3 gene.

[0041] On the other hand, the present application provides a cell comprising the complex described herein, the nucleic acid described herein, the recombinant expression vector described herein, the delivery vector described herein, and / or the pharmaceutical composition described herein.

[0042] On the other hand, the present application provides a kit comprising the complex described herein, the nucleic acid described herein, the recombinant expression vector described herein, the delivery vector described herein, the pharmaceutical composition described herein, and / or the cell described herein.

[0043] On the other hand, the present application provides a method for treating a disease, comprising providing an effective amount of the complex described herein, the nucleic acid described herein, the recombinant expression vector described herein, the delivery vector described herein, the pharmaceutical composition described herein, the cell described herein, and / or the kit described herein to a subject in need thereof; the disease is a disease associated with abnormal gene activity of PCSK9 and / or ANGPTL3.

[0044] In some embodiments, the disease comprises combined hyperlipidemia.

[0045] The method provided in this application has at least the following advantages: PCSK9, as a popular target for lipid-lowering drug development, mainly reduces the expression of LDL-R on the surface of liver cells, thereby preventing the degradation of LDL-R on the surface of liver cells. This allows more LDL-R to be present on the surface of the liver, thereby more effectively clearing LDL-C from the blood. The main function of PCSK9 is to lower cholesterol in the blood. For patients with mixed hypercholesterolemia, their triglyceride levels are also abnormally elevated. Lowering LDL-C alone cannot completely eliminate the risk of cardiovascular disease. ANGPTL3 regulates lipid metabolism by inhibiting the activity of lipoprotein lipase (LPL) and endogenous lipoprotein lipase (EL), which can not only reduce LDL-C levels, but also reduce blood triglyceride levels. Therefore, the method provided in this application can simultaneously inhibit PCSK9 and ANGPLT3, and can achieve the simultaneous reduction of LDL-C and triglycerides in the blood, thereby solving the problem that single-target drugs for patients with mixed hyperlipidemia cannot achieve ideal therapeutic effects.

[0046] On the other hand, in order to solve the technical difficulties of the existing technology for treating mixed hyperlipidemia, this article provides a method for introducing inhibitory epigenetic modifications in specific regulatory regions of APOC3 and ANGPTL3 through an epigenetic editing tool (EPIREG), changing the transcriptional activity of APOC3 and ANGPTL3, and achieving simultaneous inhibition of the expression of both APOC3 and ANGPTL3 genes, thereby reducing the levels of lipoprotein cholesterol (LDL-C) and triglycerides in the blood, and achieving the purpose of treating mixed hyperlipidemia. The epigenetic editing tool provided in this application achieves genomic positioning through gRNA and recruits epigenetic modification proteins such as DNA methyltransferases (DNMTs), introduces epigenetic modifications at specific sites, changes the chromatin structure, and thereby adjusts the target gene to a transcriptional repression state, achieving silencing regulation of the target gene. In this process, DNA will not be cut, avoiding the possibility of generating genomic double-strand breaks, and has higher safety.

[0047] In one aspect, the present application provides a method for simultaneously regulating the expression and / or activity of the APOC3 gene and the ANGPTL3 gene, the method comprising providing an epigenetic editing system; the epigenetic editing system comprises a DNA binding domain and a gene expression regulator.

[0048] In some embodiments, the epigenetic editing system comprises a complex, and the DNA binding domain and the gene expression regulator are contained in the complex; or the epigenetic editing system comprises a nucleic acid encoding the complex.

[0049] In some embodiments, the complex comprises a first fusion portion and a second fusion portion; wherein, one of the first fusion portion and the second fusion portion comprises the DNA binding domain, at least one of the gene expression regulators and the recruitment domain A, the other of the first fusion portion and the second fusion portion comprises at least one of the gene expression regulators and the recruitment domain A', and the recruitment domain A and the recruitment domain A' are capable of interacting with each other.

[0050] In some embodiments, the interaction between the recruitment domain A and the recruitment domain A′ enables the gene expression regulator to be recruited to the regulatory region of the APOC3 gene and / or the ANGPTL3 gene or to the vicinity thereof.

[0051] In some embodiments, the gene expression regulator comprised by the first fusion moiety and the second fusion moiety is optionally a transcriptional repressor domain and an epigenetic modification domain, respectively.

[0052] In some embodiments, the transcriptional repressor domain is selected from: 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, ZNF98, ZNF677, ZNF596, ZNF214, ZNF37A, ZNF34, ZNF250, ZNF547, ZNF273, ZFP82, ZNF224, ZNF33A, ZNF45, ZNF175, ZNF184, ZFP28-1, ZFP28-2, ZNF18, ZNF213, ZNF394, ZFP1, ZFP14, ZNF416, ZNF557, ZNF729, ZNF254, ZNF764, ZNF785, ZNF10, CBX5, RYBP, YAF2, MGA, CBX1, SCMH1, MPP8, SUMO3, HERC2, BIN1, PCGF2, TOX, FOXA1, FOXA2, IRF2BP1, IRF2BP2, IRF2BPL IRF-2BP1_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, ZNZN254,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,CREB 1,TERF1,ID3,CBX8,GSX1,NKX22,ATF1,TWST2,ZNF17,TOX3,TOX4,ZMYM3,I2BP1,RHXF1,SSX2,I2BPL,ZN680,TRI68,HXA13,PHC3,TCF24,HXB 13,HEY1,PHC2,ZNF81,FIGLA,SAM11,KMT2B,HEY2,JDP2,HXC13,ASCL4,HHEX,GSX2,ETV7,ASCL3,PHC1,OTP,I2BP2,VGLL2,HXA11,PDLI4,ASCL2,CDX4,ZN860,LMBL4,PDIP3,NKX25,CEBPB,ISL1,CDX2,PROP1,SIN3B,SMBT1,HXC11,HXC10,PRS6A,VSX1,NKX23,MTG16,HMX3,HMX1,KIF22,CSTF2,CEBPE,DLX2,PPARG,PRIC1,UNC4,BARX2,ALX3,TCF15,TERA,VSX2,HXD12,CDX1,TCF23,ALX1,HXA10,RX,CXXC5,SCML1,NFIL3,DLX6,MTG8,CEBPD,SEC13,FIP1,ALX4,LHX3,PRIC2,MAGI3,NELL1,PRRX1,MTG8R,RAX2,DLX3,DLX1,NKX26,NAB1,SAMD7,PITX3,WDR5,MEOX2,NAB2,DHX8,CBX6,EMX2,CPSF6,HXC12,KDM4B,LMBL3,PHX2A,EMX1,NC2B,DLX4,SRY,ZN777,ZN398,GATA3,BSH,SF3B4,TEAD1,TEAD3,RGAP1,PHF1,GATA2,FOXO3,ZN212,IRX4,ZBED6,LHX4,SIN3A,RBBP7,NKX61,R51A1,MB3L1,DLX5,NOTC1,TERF2,ZN282,RGS12,ZN840,SPI2B,PAX7,NKX62,ASXL2,FOXO1,GATA1,ZMYM5,LRP1,MIXL1,SGT1,LMCD1,CEBPA,SOX14, WTIP, PRP19, NKX11, RBBP4, DMRT2, SMCA2, and their functionally active fragments.

[0053] In some embodiments, the epigenetic modification domain comprises one or more of: DNA methyltransferase activity, DNA demethylase activity, DNA deamination activity, DNA amination activity, DNA oxidation activity, DNA helicase activity, histone methyltransferase activity, histone demethylase activity, histone acetyltransferase activity, histone deacetylase activity, histone kinase activity, histone phosphatase activity, histone ubiquitin ligase activity, and histone deubiquitinating activity.

[0054] In some embodiments, the epigenetic modification domain comprises a DNA methyltransferase and / or a functionally active fragment thereof.

[0055] In some embodiments, the DNA methyltransferase is selected from DNMT3A, DNMT3B, Dnmt3c, DNMT1, DNMT2, and DNMT3L.

[0056] In some embodiments, the DNA methyltransferase comprises at least one DNMT3A and at least one DNMT3L.

[0057] In some embodiments, the DNA binding domain is selected from the group consisting of: a TALE domain, a zinc finger domain, a tetR domain, a meganuclease, a Cas nuclease, an Argonaute (Ago) protein, and homologs, modified forms, or variants thereof.

[0058] In some embodiments, the DNA binding domain is capable of specifically recognizing a target sequence on the APOC3 gene and / or the ANGPTL3 gene.

[0059] In some embodiments, the DNA binding domain specifically recognizes the target sequence by binding to a guide RNA.

[0060] In some embodiments, the DNA binding domain is a class II Cas nuclease.

[0061] In some embodiments, the Cas nuclease is selected from a class II type II Cas nuclease and a class II type V Cas nuclease.

[0062] In some embodiments, the Cas nuclease is Cas9.

[0063] In some embodiments, the Cas nuclease is a deactivated Cas9 (dCas9).

[0064] In some embodiments, the epigenetic editing system further comprises a guide RNA, which is capable of specifically recognizing a target sequence on the APOC3 gene and / or the ANGPTL3 gene.

[0065] In some embodiments, the recruitment domain A is selected from any one of the following two groups of domains, and the recruitment domain A' is selected from any one of the other of the following two groups of domains: 1) general control non-derepressor protein 4 (GCN4), GFP11 fragment derived from split green fluorescent protein (GFP), or GVKESLV polypeptide; and 2) single-chain antibody (scFv), GFP1-10 fragment derived from split green fluorescent protein (GFP), or PDZ protein domain.

[0066] In some embodiments, the method is characterized in that: 1) the domain of one of the recruitment domain A and the recruitment domain A' is GCN4, and the other domain is scFv; or 2) the domain of one of the recruitment domain A and the recruitment domain A' is a GFP11 fragment, and the other domain is GFP1-10; or 3) the domain of one of the recruitment domain A and the recruitment domain A' is GVKESLV, and the other domain is a PDZ protein domain.

[0067] In some embodiments, the method is characterized in that: 1) one of the first fusion moiety and the second fusion moiety comprises DNA methyltransferase-dCas9-n×GCN4, and the other comprises a transcriptional repressor domain-scFv or scFv-transcriptional repressor domain; or 2) one of the first fusion moiety and the second fusion moiety comprises DNA methyltransferase-dCas9-scFv, and the other comprises a transcriptional repressor domain-GCN4 or GCN4-transcriptional repressor domain; or 3) one of the first fusion moiety and the second fusion moiety comprises DNA methyltransferase-dCas9-n×GFP1 1, and the other comprises a transcription repressor domain-GFP1-10 or GFP1-10-transcription repressor domain; or 4) one of the first fusion portion and the second fusion portion comprises DNA methyltransferase-dCas9-GFP1-10, and the other comprises a transcription repressor domain-GFP11 or GFP11-transcription repressor domain; wherein, - indicates that the domains at both ends are directly or indirectly connected in the order from N-terminus to C-terminus; n×GCN4 or n×GFP11 respectively represents n copies of GCN4 connected by a linker sequence or n copies of GFP11 connected by a linker sequence, and n is selected from any integer from 1 to 20.

[0068] In some embodiments, the method is characterized in that: 1) one of the first fusion portion and the second fusion portion comprises an n×GCN4-dCas9-transcriptional repressor domain, and the other comprises a DNA methyltransferase-scFv or scFv-DNA methyltransferase; or 2) one of the first fusion portion and the second fusion portion comprises an scFv-dCas9-transcriptional repressor domain, and the other comprises a DNA methyltransferase-GCN4 or GCN4-DNA methyltransferase; or 3) one of the first fusion portion and the second fusion portion comprises an n×GFP11-dCas9-transcriptional repressor structure domain, and the other comprises DNA methyltransferase-GFP1-10 or GFP1-10-DNA methyltransferase; or 4) one of the first fusion part and the second fusion part comprises a GFP1-10-dCas9-transcriptional repressor domain, and the other comprises a DNA methyltransferase-GFP11 or GFP11-DNA methyltransferase; wherein, - indicates that the domains at both ends are directly or indirectly connected in the order from N-terminus to C-terminus; n×GCN4 or n×GFP11 respectively represent n copies of GCN4 connected by a linker sequence or n copies of GFP11 connected by a linker sequence, and n is selected from any integer from 1 to 20.

[0069] In some embodiments, the complex comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-40.

[0070] In some embodiments, the nucleic acid is a recombinant expression vector.

[0071] In some embodiments, the recombinant expression vector is a plasmid or a viral vector.

[0072] In some embodiments, the nucleic acid comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 41-160.

[0073] In some embodiments, the complex or the nucleic acid encoding the complex is disposed in the same or different delivery vehicles.

[0074] In some embodiments, the delivery vehicle comprises a liposome and / or a lipid nanoparticle.

[0075] On the other hand, the present application provides a complex, such as the complex provided in the method described in the present application, and the complex can simultaneously regulate the expression and / or activity of the APOC3 gene and the ANGPTL3 gene without changing the function of their gene sequences.

[0076] In another aspect, the present application provides a nucleic acid encoding the complex described in the present application.

[0077] On the other hand, the present application provides a recombinant expression vector comprising the nucleic acid described in the present application.

[0078] On the other hand, the present application provides a delivery vector, which comprises the complex described in the present application, the nucleic acid described in the present application and / or the recombinant expression vector described in the present application.

[0079] On the other hand, the present application provides a pharmaceutical composition comprising the complex described herein, the nucleic acid described herein, the recombinant expression vector described herein and / or the delivery vector described herein, and at least one pharmaceutically acceptable carrier.

[0080] In some embodiments, the pharmaceutical composition further comprises a guide RNA, which is capable of specifically recognizing a target sequence on the APOC3 gene and / or the ANGPTL3 gene.

[0081] On the other hand, the present application provides a cell comprising the complex described herein, the nucleic acid described herein, the recombinant expression vector described herein, the delivery vector described herein, and / or the pharmaceutical composition described herein.

[0082] On the other hand, the present application provides a kit comprising the complex described herein, the nucleic acid described herein, the recombinant expression vector described herein, the delivery vector described herein, the pharmaceutical composition described herein, and / or the cell described herein.

[0083] On the other hand, the present application provides a method for treating a disease, comprising providing an effective amount of the complex described herein, the nucleic acid described herein, the recombinant expression vector described herein, the delivery vector described herein, the pharmaceutical composition described herein, the cell described herein, and / or the kit described herein to a subject in need thereof; the disease is a disease associated with abnormal gene activity of APOC3 and / or ANGPTL3.

[0084] In some embodiments, the disease comprises combined hyperlipidemia.

[0085] The method provided in the present application has at least the following advantages: APOC3, as a popular target for the development of lipid-lowering drugs, affects the hydrolysis and clearance of triglycerides by inhibiting the activity of lipoprotein lipase (LPL). Inhibiting APOC3 can reduce triglycerides in the blood. For patients with mixed hypercholesterolemia, their LDL-C levels are also abnormally elevated, and lowering triglycerides alone cannot completely eliminate the risk of cardiovascular disease. ANGPTL3 regulates lipid metabolism by inhibiting the activity of lipoprotein lipase (LPL) and endogenous lipoprotein lipase (EL), which can not only reduce the level of LDL-C, but also reduce the level of blood triglycerides. Therefore, the method provided in the present application can simultaneously inhibit APOC3 and ANGPLT3 dual targets, and can achieve the simultaneous reduction of LDL-C and triglycerides in the blood, thereby solving the problem that single-target drugs for patients with mixed hyperlipidemia cannot achieve ideal therapeutic effects.

[0086] On the other hand, in order to solve the technical difficulties of the existing technology for treating mixed hyperlipidemia, this article provides a method for introducing inhibitory epigenetic modifications in specific regulatory regions of PCSK9 and APOC3 through an epigenetic editing tool (EPIREG), changing the transcriptional activity of PCSK9 and APOC3, and achieving simultaneous inhibition of the expression of both PCSK9 and APOC3 genes, thereby reducing the levels of lipoprotein cholesterol (LDL-C) and triglycerides in the blood, and achieving the purpose of treating mixed hyperlipidemia. The epigenetic editing tool provided in this application achieves genomic positioning through gRNA and recruits epigenetic modification proteins such as DNA methyltransferases (DNMTs), introduces epigenetic modifications at specific sites, changes the chromatin structure, and thereby adjusts the target gene to a transcriptional repression state, achieving silencing regulation of the target gene. In this process, DNA will not be cut, avoiding the possibility of generating double-strand breaks in the genome, and is relatively safe.

[0087] On the one hand, the present application provides a method for simultaneously regulating the expression and / or activity of PCSK9 gene and APOC3 gene, the method comprising providing an epigenetic editing system; the epigenetic editing system comprises a DNA binding domain and a gene expression regulator.

[0088] In some embodiments, the epigenetic editing system comprises a complex, and the DNA binding domain and the gene expression regulator are contained in the complex; or the epigenetic editing system comprises a nucleic acid encoding the complex.

[0089] In some embodiments, the complex comprises a first fusion portion and a second fusion portion; wherein, one of the first fusion portion and the second fusion portion comprises the DNA binding domain, at least one of the gene expression regulators and the recruitment domain A, the other of the first fusion portion and the second fusion portion comprises at least one of the gene expression regulators and the recruitment domain A', and the recruitment domain A and the recruitment domain A' are capable of interacting with each other.

[0090] In some embodiments, the interaction between the recruitment domain A and the recruitment domain A' enables the gene expression regulator to be recruited to the regulatory region of the PCSK9 gene and / or the APOC3 gene or its vicinity.

[0091] In some embodiments, the gene expression regulator comprised by the first fusion moiety and the second fusion moiety is optionally a transcriptional repressor domain and an epigenetic modification domain, respectively.

[0092] In some embodiments, the transcriptional repressor domain is selected from: 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, ZNF98, ZNF677, ZNF596, ZNF214, ZNF37A, ZNF34, ZNF250, ZNF547, ZNF273, ZFP82, ZNF224, ZNF33A, ZNF45, ZNF175, ZNF184, ZFP28-1, ZFP28-2, ZNF18, ZNF213, ZNF394, ZFP1, ZFP14, ZNF416, ZNF557, ZNF729, ZNF254, ZNF764, ZNF785, ZNF10, CBX-5, 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, ZN-554, ZN878, ZN772, ZN224, ZN184, ZN544, ZNF57, ZN283, ZN549, ZN211, ZN6-15, ZN253, ZN226, ZN730, Z585A, ZN732, ZN681, ZN667, ZN649, ZN470, ZN484, ZN431, ZN382,ZN254,ZN124,ZN607,ZN317,ZN620,ZN141,ZN584,ZN540,ZN75D,ZN555,ZN658,ZN684,RBAK,ZN829,ZN582,ZN112,ZN716,HKR1,ZN350,ZN480,ZN416,ZNF92,ZN100,ZN736,ZNF74,ZN443,ZN195,ZN530,ZN782,ZN791,ZN331,Z354C,ZN157,ZN727,ZN550,ZN793,ZN235,ZN724,ZN573,ZN577,ZN789,ZN718,ZN300,ZN383,ZN429,ZN677,ZN850,ZN454,ZN257,ZN264,ZN485,ZN737,ZNF44,ZN596,ZN565,ZN543,ZFP69,SUMO1,ZNF12,ZN169,ZN433,ZN175,ZN347,ZNF25,ZN519,Z585B,ZN517,ZN846,ZN230,ZNF66,ZN713,ZN816,ZN426,ZN674,ZN627,ZNF20,Z587B,ZN316,ZN233,ZN611,ZN556,ZN234,ZN560,ZNF77,ZN682,ZN614,ZN785,ZN445,ZFP30,ZN225,ZN551,ZN610,ZN528,ZN284,ZN418,ZN490,ZN805,Z780B,ZN763,ZN285,ZNF85,ZN223,ZNF90,ZN557,ZN425,ZN229,ZN606,ZN155,ZN222,ZN442,ZNF91,ZN135,ZN778,ZN534,ZN586,ZN567,ZN440,ZN583,ZN441,ZNF43,ZN589,ZN563,ZN561,ZN136,ZN630,ZN527,ZN333,Z324B,ZN786,ZN709,ZN792,ZN599,ZN613,ZF69B,ZN799,ZN569,ZN564,ZN546,ZFP92,ZN723,ZN439,ZFP57,ZNF19,ZN404,ZN274,CBX3,ZN250,ZN570,ZN675,ZN695,ZN548,ZN132,ZN738,ZN420,ZN626,ZN559,ZN460,ZN268,ZN304,ZN605,ZN844,SUMO5,ZN101,ZN783,ZN417,ZN182,ZN823,ZN177,ZN197,ZN717,ZN669,ZN256,ZN251,CBX4,CDY2,CDYL2,ZN562,ZN461,Z324A,ZN766,ID2,ZN214,CBX7,ID1,CREM,SCX,ASCL1,ZN764,SCML2,TWST1,CREB1,TERF1,ID3,CBX8,GSX1,NKX22,ATF1,TWST2,ZNF17,TOX3,TOX4,ZMYM3,I2BP1,RHXF1,SSX2,I2BPL,ZN680,TRI68,HXA13,PHC3,TCF24,HXB13,HEY1,PHC2,ZNF81,FIGLA,SAM11,KMT2B,HEY2,JDP2,HXC13,ASCL4,HHEX,GSX2,ETV7,ASCL3,PHC1,OTP,I2BP2,VGLL2,HXA11,PDLI4,ASCL2,CDX4,ZN860,LMBL4,PDIP3,NKX25,CEBPB,ISL1,CDX2,PROP1,SIN3B,SMBT1,HXC11,HXC10,PRS6A,VSX1,NKX23,MTG16,HMX3,HMX1,KIF22,CSTF2,CEBPE,DLX2,PPARG,PRIC1,UNC4,BARX2,ALX3,TCF15,TERA,VSX2,HXD12,CDX1,TCF23,ALX1,HXA10,RX,CXXC5,SCML1,NFIL3,DLX6,MTG8,CEBPD,SEC13,FIP1,ALX4,LHX3,PRIC2,MAGI3,NELL1,PRRX1,MTG8R,RAX2,DLX3,DLX1,NKX26,NAB1,SAMD7,PITX3,WDR5,MEOX2,NAB2,DHX8,CBX6,EMX2,CPSF6,HXC12,KDM4B,LMBL3,PHX2A,EMX1,NC2B,DLX4,SRY,ZN777,ZN398,GATA3,BSH,SF3B4,TEAD1,TEAD3,RGAP1,PHF1,GATA2,FOXO3,ZN212,IRX4,ZBED6,LHX4,SIN3A,RBBP7,NKX61,R51A1,MB3L1,DLX5,NOTC1,TERF2,ZN282,RGS12,ZN840,SPI2B,PAX7,NKX62,ASXL2,FOXO1,GATA1,ZMYM5,LRP1,MIXL1,SGT1,LMCD1,CEBPA,SOX14,WTIP, PRP19, NKX11, RBBP4, DMRT2, SMCA2, and their functionally active fragments.

[0093] In some embodiments, the epigenetic modification domain comprises one or more of: DNA methyltransferase activity, DNA demethylase activity, DNA deamination activity, DNA amination activity, DNA oxidation activity, DNA helicase activity, histone methyltransferase activity, histone demethylase activity, histone acetyltransferase activity, histone deacetylase activity, histone kinase activity, histone phosphatase activity, histone ubiquitin ligase activity, and histone deubiquitinating activity.

[0094] In some embodiments, the epigenetic modification domain comprises a DNA methyltransferase and / or a functionally active fragment thereof.

[0095] In some embodiments, the DNA methyltransferase is selected from DNMT3A, DNMT3B, Dnmt3c, DNMT1, DNMT2, and DNMT3L.

[0096] In some embodiments, the DNA methyltransferase comprises at least one DNMT3A and at least one DNMT3L.

[0097] In some embodiments, the DNA binding domain is selected from the group consisting of: a TALE domain, a zinc finger domain, a tetR domain, a meganuclease, a Cas nuclease, an Argonaute (Ago) protein, and homologs, modified forms, or variants thereof.

[0098] In some embodiments, the DNA binding domain is capable of specifically recognizing target sequences on the PCSK9 gene and / or the APOC3 gene.

[0099] In some embodiments, the DNA binding domain specifically recognizes the target sequence by binding to a guide RNA.

[0100] In some embodiments, the DNA binding domain is a class II Cas nuclease.

[0101] In some embodiments, the Cas nuclease is selected from a class II type II Cas nuclease and a class II type V Cas nuclease.

[0102] In some embodiments, the Cas nuclease is Cas9.

[0103] In some embodiments, the Cas nuclease is a deactivated Cas9 (dCas9).

[0104] In some embodiments, the epigenetic editing system further comprises a guide RNA, which can specifically recognize a target sequence on the PCSK9 gene and / or the APOC3 gene.

[0105] In some embodiments, the recruitment domain A is selected from any one of the following two groups of domains, and the recruitment domain A' is selected from any one of the other of the following two groups of domains: 1) general control non-derepressor protein 4 (GCN4), GFP11 fragment derived from split green fluorescent protein (GFP), or GVKESLV polypeptide; and 2) single-chain antibody (scFv), GFP1-10 fragment derived from split green fluorescent protein (GFP), or PDZ protein domain.

[0106] In some embodiments, the method is characterized in that: 1) the domain of one of the recruitment domain A and the recruitment domain A' is GCN4, and the other domain is scFv; or 2) the domain of one of the recruitment domain A and the recruitment domain A' is a GFP11 fragment, and the other domain is GFP1-10; or 3) the domain of one of the recruitment domain A and the recruitment domain A' is GVKESLV, and the other domain is a PDZ protein domain.

[0107] In some embodiments, the method is characterized in that: 1) one of the first fusion moiety and the second fusion moiety comprises DNA methyltransferase-dCas9-n×GCN4, and the other comprises a transcriptional repressor domain-scFv or scFv-transcriptional repressor domain; or 2) one of the first fusion moiety and the second fusion moiety comprises DNA methyltransferase-dCas9-scFv, and the other comprises a transcriptional repressor domain-GCN4 or GCN4-transcriptional repressor domain; or 3) one of the first fusion moiety and the second fusion moiety comprises DNA methyltransferase-dCas9-n×GFP1 1, and the other comprises a transcription repressor domain-GFP1-10 or GFP1-10-transcription repressor domain; or 4) one of the first fusion portion and the second fusion portion comprises DNA methyltransferase-dCas9-GFP1-10, and the other comprises a transcription repressor domain-GFP11 or GFP11-transcription repressor domain; wherein, - indicates that the domains at both ends are directly or indirectly connected in the order from N-terminus to C-terminus; n×GCN4 or n×GFP11 respectively represents n copies of GCN4 connected by a linker sequence or n copies of GFP11 connected by a linker sequence, and n is selected from any integer from 1 to 20.

[0108] In some embodiments, the method is characterized in that: 1) one of the first fusion portion and the second fusion portion comprises an n×GCN4-dCas9-transcriptional repressor domain, and the other comprises a DNA methyltransferase-scFv or scFv-DNA methyltransferase; or 2) one of the first fusion portion and the second fusion portion comprises an scFv-dCas9-transcriptional repressor domain, and the other comprises a DNA methyltransferase-GCN4 or GCN4-DNA methyltransferase; or 3) one of the first fusion portion and the second fusion portion comprises an n×GFP11-dCas9-transcriptional repressor structure domain, and the other comprises DNA methyltransferase-GFP1-10 or GFP1-10-DNA methyltransferase; or 4) one of the first fusion part and the second fusion part comprises a GFP1-10-dCas9-transcriptional repressor domain, and the other comprises a DNA methyltransferase-GFP11 or GFP11-DNA methyltransferase; wherein, - indicates that the domains at both ends are directly or indirectly connected in the order from N-terminus to C-terminus; n×GCN4 or n×GFP11 respectively represent n copies of GCN4 connected by a linker sequence or n copies of GFP11 connected by a linker sequence, and n is selected from any integer from 1 to 20.

[0109] In some embodiments, the complex comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-40.

[0110] In some embodiments, the nucleic acid is a recombinant expression vector.

[0111] In some embodiments, the recombinant expression vector is a plasmid or a viral vector.

[0112] In some embodiments, the nucleic acid comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 41-160.

[0113] In some embodiments, the complex or the nucleic acid encoding the complex is disposed in the same or different delivery vehicles.

[0114] In some embodiments, the delivery vehicle comprises a liposome and / or a lipid nanoparticle.

[0115] On the other hand, the present application provides a complex, such as the complex provided in the method described in the present application, and the complex can simultaneously regulate the expression and / or activity of PCSK9 gene and APOC3 gene without changing the function of their gene sequences.

[0116] In another aspect, the present application provides a nucleic acid encoding the complex described in the present application.

[0117] On the other hand, the present application provides a recombinant expression vector comprising the nucleic acid described in the present application.

[0118] On the other hand, the present application provides a delivery vector, which comprises the complex described in the present application, the nucleic acid described in the present application and / or the recombinant expression vector described in the present application.

[0119] On the other hand, the present application provides a pharmaceutical composition comprising the complex described herein, the nucleic acid described herein, the recombinant expression vector described herein and / or the delivery vector described herein, and at least one pharmaceutically acceptable carrier.

[0120] In some embodiments, the pharmaceutical composition further comprises a guide RNA, which is capable of specifically recognizing a target sequence on the PCSK9 gene and / or the APOC3 gene.

[0121] On the other hand, the present application provides a cell comprising the complex described herein, the nucleic acid described herein, the recombinant expression vector described herein, the delivery vector described herein, and / or the pharmaceutical composition described herein.

[0122] On the other hand, the present application provides a kit comprising the complex described herein, the nucleic acid described herein, the recombinant expression vector described herein, the delivery vector described herein, the pharmaceutical composition described herein, and / or the cell described herein.

[0123] On the other hand, the present application provides a method for treating a disease, comprising providing an effective amount of the complex described herein, the nucleic acid described herein, the recombinant expression vector described herein, the delivery vector described herein, the pharmaceutical composition described herein, the cell described herein, and / or the kit described herein to a subject in need thereof; the disease is a disease associated with abnormal gene activity of PCSK9 and / or APOC3.

[0124] In some embodiments, the disease comprises combined hyperlipidemia.

[0125] The method provided in the present application has at least the following advantages: PCSK9, as a popular target for the development of lipid-lowering drugs, affects the hydrolysis and clearance of triglycerides by inhibiting the activity of lipoprotein lipase (LPL). Inhibiting PCSK9 can reduce triglycerides in the blood. For patients with mixed hypercholesterolemia, their LDL-C levels are also abnormally elevated, and lowering triglycerides alone cannot completely eliminate the risk of cardiovascular disease. APOC3 can affect the hydrolysis and clearance of triglycerides by inhibiting the activity of lipoprotein lipase (LPL). Therefore, the method provided in the present application can inhibit both PCSK9 and APOC3 targets at the same time, and can achieve the simultaneous reduction of LDL-C and triglycerides in the blood, thereby solving the problem that single-target drugs for patients with mixed hyperlipidemia cannot achieve ideal therapeutic effects.

[0126] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0127] The specific features of the inventions of this application are set forth in the appended claims. The features and advantages of the inventions of this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0128] FIG1 shows that the method described in the present application reduces the mRNA expression level of the target genes PCSK9 and / or ANGPTL3.

[0129] FIG2 shows that the method described in the present application reduces the mRNA expression level of the target gene APOC3 and / or ANGPTL3.

[0130] FIG3 shows that the method described in the present application reduces the mRNA expression level of the target genes PCSK9 and / or APOC3. DETAILED DESCRIPTION

[0131] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0132] Definition of terms

[0133] In this application, the term "nucleic acid" is used interchangeably with "polynucleotide", "nucleotide", "nucleotide sequence" and "oligonucleotide" and generally refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and polymers thereof in single-stranded, double-stranded or multi-stranded form or their complements. For example, a nucleotide can be a ribonucleotide, a deoxyribonucleotide or a modified version thereof. For example, a nucleotide can be a single-stranded and double-stranded DNA, a single-stranded and double-stranded RNA, and a hybrid molecule having a mixture of single-stranded and double-stranded DNA and RNA. For example, a nucleotide can include, but is not limited to, any type of RNA, such as mRNA, siRNA, miRNA, sgRNA and guide RNA, and any type of DNA, genomic DNA, plasmid DNA and minicircle DNA, and any fragments thereof. The term also encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or bonds, which are synthetic, naturally occurring, and non-naturally occurring.

[0134] In this application, the term "sequence encoding..." or "nucleic acid encoding..." generally refers to a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence that encodes a protein. The coding sequence may also include start and stop signals operably linked to regulatory elements, including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered. The coding sequence may be codon optimized. In this application, the term "intron" generally refers to a segment of DNA that is transcribed but removed from the RNA transcript by splicing together either end of the sequence (exons). Introns are considered to be intervening sequences within the protein coding region of a gene and generally do not contain the information represented by the protein produced by that gene.

[0135] In this application, the term "recruitment" generally refers to the recruitment effect between protein molecules, which specifically refers to the recruitment of other molecules by proteins to perform specific biological functions. This recruitment effect mainly depends on the affinity of intermolecular interactions, and its affinity is generally considered to be related to the spatial structure of protein molecules and is relatively complex. The interaction mechanism can illustratively include but is not limited to non-covalent bonds such as hydrogen bonds, ionic interactions, hydrophobic interactions, and van der Waals forces. For example, some proteins can recruit enzymes to catalyze chemical reactions, or recruit other proteins to form complexes. These recruitment effects are crucial for many cellular processes, such as signal transduction, DNA replication, and gene expression.

[0136] In the present application, the term "DNA binding domain" generally refers to an independently folded protein domain that contains at least one motif that recognizes double-stranded or single-stranded DNA. For example, the DNA binding domain can recognize a specific DNA sequence (recognition or regulatory sequence) or has a general affinity for DNA. In some cases, other domains of the DNA binding domain typically regulate the activity of the DNA binding domain; the DNA binding function can be structural or include transcriptional regulation, and sometimes these two effects are overlapping. In certain embodiments of the methods and gene expression regulatory molecules provided herein, the DNA binding domain may include a (DNA) nuclease, such as a nuclease that can target DNA in a sequence-specific manner or can be guided or instructed 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 a large range of nucleases. In some embodiments, the DNA binding domain is a DNA nuclease derived from the CRISPR-Cas system. For example, the DNA nuclease derived from the CRISPR-Cas system is a Cas protein.

[0137] In this application, the term "TALE domain" is a polypeptide comprising one or more TALE repeat domains / units. Naturally occurring TALEs or "wild-type TALEs" are nucleic acid binding proteins secreted by numerous species of Proteobacteria. TALE polypeptides contain a nucleic acid binding domain composed of tandem repeats of highly conserved monomeric polypeptides that are primarily 33, 34, or 35 amino acids in length and differ from each other primarily in amino acid positions 12 and 13. In a preferred embodiment, the nucleic acid is DNA. As used herein, the polypeptide monomer of a TALE is used to refer to a highly conserved repeat polypeptide sequence within a TALE nucleic acid binding domain, and the term "repeat variable diresidue" or "RVD" is used to refer to highly variable amino acids at positions 12 and 13 of a polypeptide monomer. A general representation of a TALE monomer contained within a DNA binding domain is X 1-11 -(X 12 X 13 )-X 14-33或34或35 , where the subscripts indicate amino acid positions and X represents any amino acid. 12 X 13 Indicates RVD. In some TALE polypeptide monomers, the variable amino acid at position 13 is missing or absent, and in such monomers, the RVD consists of a single amino acid. In such cases, the RVD may alternatively be represented as X*, where X represents X 12 , and (*) indicates X 13 Not present. The DNA binding domain contains several repeats of the TALE monomer, and this can be represented as (X 1-11 -(X 12X 13 )-X 14-33或34或35 ) z , wherein in a preferred embodiment, z is at least 5-40. In a further preferred embodiment, z is at least 10-26.

[0138] TALE monomers have nucleotide binding affinity determined by the type of amino acids within their RVDs. For example, a polypeptide monomer with an RVD of NI preferentially binds to adenine (A), a polypeptide monomer with an RVD of NG preferentially binds to thymine (T), a polypeptide monomer with an RVD of HD preferentially binds to cytosine (C), and a monomer with an RVD of NN preferentially binds to adenine (A) and guanine (G). In other embodiments, a monomer with an RVD of IG preferentially binds to T. Therefore, the number and order of polypeptide monomer repeats in the TALEN nucleic acid binding domain determine its nucleic acid target specificity. In a further embodiment of the present application, a monomer with an RVD of NS recognizes all four base pairs and can bind to A, T, G or C. The structure and function of TALE are further described, for example, in Moscou et al., Science 326: 1501 (2009); Boch et al., Science 326: 1509-1512 (2009); and Zhang et al., Nature Biotechnology 29: 149-153 (2011), each of which is incorporated by reference in its entirety. The repeat domain of TALE participates in the binding of TALE to its cognate target DNA sequence. These repeat units (or "repeat sequences") exhibit at least some sequence homology to other TALE repeat sequences within naturally occurring TALE proteins. See, for example, U.S. Patent Publication No. 20110301073. The TALE binding domains to which this application relates can be "engineered" to bind to a predetermined nucleotide sequence, for example, by engineering (changing one or more amino acids) the recognition helix region of a naturally occurring TALE protein. Therefore, engineered DNA binding proteins (TALEs) are non-naturally occurring proteins. Non-limiting examples of methods for engineering DNA binding proteins are design and selection. The designed DNA binding proteins are non-naturally occurring proteins whose design and / or composition are primarily derived from rational criteria. Rational design criteria include applying substitution rules and computational algorithms for processing information in information databases storing existing TALE design and binding data. See, for example, U.S. Patents 6,140,081; 6,453,242; and 6,534,261; also see WO 98 / 53058; WO 98 / 53059; WO 98 / 53060; WO 02 / 016536 and WO 03 / 016496 and U.S. Publication No. 20110301073.

[0139] In this application, "Cas (nucleic acid) enzyme" can 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 refers to a class of enzymes that are complementary to the CRISPR sequence and can use the CRISPR sequence as a guide to recognize and cut specific DNA strands. Non-limiting examples of Cas proteins include: Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and / or homologs thereof, 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.

[0140] In this application, the term "class II Cas nuclease" generally refers to a class of Cas proteins that perform recognition and / or cleavage functions in the form of a single protein, as defined by the updated classification scheme for CRISPR / Cas loci (Makarova et al., (2015) Nat Rev Microbiol, 13(11):722-36; Shmakov et al., (2015) Mol Cell, 60:385-397).

[0141] In this application, the term "Class II Type II Cas nuclease and Class II Type V Cas nuclease" generally refers to the single-protein, RNA-guided endonucleases in Class II Cas nucleases. Among them, the VB-type Cas nucleases in Type II and Type V require the combined action of tracrRNA (trans-activating CRISPR RNA) and crRNA (CRISPR RNA) to function properly, and crRNA and tracrRNA can be artificially combined into a single guide RNA (sgRNA); the VA-type Cas nuclease in Type V requires the use of crRNA alone to perform its guiding function. 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 known as Cpf1), Cas12b (also known as C2c1), Cas12c (also known as C2c3), Cas12d (CasY), Cas12e (CasX), Cas12g, Cas12h, Cas12i, C2c1, C2c4, C2c5, C2c8, C2c9, C2c10, Cas14a, Cas14b, Cas14c nuclease and / or TnpB.

[0142] In this application, the term "dCas" may refer to a dCas protein or a fragment thereof. For example, as used herein, "dCas9" may refer to a dCas9 protein or a fragment thereof. As used herein, the terms "iCas" and "dCas" are used interchangeably to refer to a CRISPR-associated protein without catalytic activity. 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 HNH 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 from a wild-type Cas protein, wherein the functional domain is selected from a Reel domain, a bridge helix domain, or a PAM interaction domain. In one embodiment, the nuclease activity of the 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.

[0143] In this application, the term "transcription repressor" generally refers to a substance and / or agent that binds to a target nucleic acid sequence and causes a decrease in the expression level of a gene product related to the target nucleic acid sequence, such as a protein (e.g., a transcription factor or a fragment thereof). For example, the gene product can be an RNA (e.g., mRNA) transcribed from a gene or a polypeptide translated from an mRNA transcribed from a gene. Typically, an increase or decrease in mRNA levels results in an increase or decrease in the level of polypeptides translated therefrom. Standard techniques for measuring mRNA or protein can be used to determine expression levels. 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 Krüppel-associated box (KRAB).

[0144] In this application, the term "DNA methyltransferase" generally refers to an enzyme that catalyzes the transfer of methyl groups to DNA. Non-limiting examples of DNA methyltransferases include DNMT1, DNMT2, DNMT 3A, DNMT 3B, Dnmt3c, and DNMT 3L. For example, through DNA methylation, DNA methyltransferases can modify the activity of DNA fragments (e.g., regulate gene expression) without changing 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 aspects, the DNA methyltransferase domain comprises a variant or homolog of an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to DNMT 3A. In various aspects, the DNA methyltransferase domain comprises a variant or homolog of an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to DNMT 3L.

[0145] As used herein, the term "functionally active fragment" generally refers to a fragment that has a partial region of a full-length protein or nucleic acid but retains or partially retains the biological activity or function of the full-length protein or nucleic acid. For example, a functionally active fragment may retain or partially retain the ability of the full-length protein to bind to another molecule. For example, a functionally active fragment of a DNA methyltransferase may retain or partially retain the biological activity of the full-length DNA methyltransferase in catalyzing the transfer of methyl groups to DNA.

[0146] In this application, the terms "specifically recognize", "capable of binding", "binding to", "targeting", etc. are used interchangeably, and generally mean that the binding molecule (for example, the gene expression regulatory molecule of the present application) can interact with the nucleotides on the target gene or target site, or the binding molecule (for example, the gene expression regulatory molecule of the present application) has sufficient affinity for the target gene or target site. This interaction can be through conjugation, coupling, attachment, providing complementarity, providing covalent force or providing non-covalent force, improving binding stability, etc.

[0147] In this application, the terms "guide RNA", "guide DNA" and "gRNA" are used interchangeably, and generally refer to a DNA molecule that can guide a nuclease (e.g., Argonaute, or Ago) to bind to and / or cleave a target gene. In some preferred embodiments, the guide DNA may include: a single-stranded DNA molecule (ssDNA), a single-stranded DNA molecule that is phosphorylated at the 5' end, a single-stranded DNA molecule that is hydroxylated at the 5' end, a base segment that is complementary to the target gene and / or has a length of 8-35nt. In some embodiments of the present application, the term "guide RNA" refers to an RNA comprising the following: (1) an "activation" nucleotide sequence that binds to a guide RNA-guided nuclease (e.g., a class II Cas nuclease, such as a type II, type V or type VI Cas nuclease) and activates the RNA-guided nuclease; and (2) a "target" nucleotide sequence comprising a nucleotide sequence that hybridizes with a target nucleic acid. The "activating" nucleotide sequence and the "target" nucleotide sequence can be on separate RNA molecules (e.g., "dual-guide RNAs"); or can be on the same RNA molecule ("single-guide RNA," also called sgRNA).

[0148] In the present application, the terms "target sequence" and "target DNA" or "pre-spacer sequence" are used interchangeably, and generally refer to a nucleotide sequence present in a target nucleic acid, which comprises a core base sequence complementary to the oligonucleotides (e.g., guide RNA) of the present application. In some cases, the target sequence is composed of a region complementary to the continuous nucleotide sequence of the oligonucleotides of the present application on the target nucleic acid. In some cases, the target sequence is longer than the complementary sequence of a single oligonucleotide and can, for example, represent an optional region of the target nucleic acid that can be targeted by several oligonucleotides of the present application. In some cases, "target sequence" can mean a part of a target gene, such as one or more exon sequences of a target gene, an intron sequence, or a regulatory sequence of a target gene, or a combination of exons and intron sequences, introns and regulatory sequences, exons and regulatory sequences, or exons, introns and regulatory sequences of a target gene. In the context of the formation of the CRISPR complex of the present application or the system, "target DNA" refers to a sequence that a guide RNA sequence is designed to have complementarity thereto, wherein the hybridization between the target DNA and the guide RNA sequence promotes the formation of a CRISPR complex or system. In some embodiments, the target DNA is located in the nucleus or cytoplasm of the cell. A CRISPR / Cas9-based system can include at least one gRNA, wherein the gRNAs target different DNA sequences. The target DNA sequences can be overlapping. The target sequence or pre-spacer sequence is followed by a PAM sequence located at the 3' end of the pre-spacer sequence. Different type II systems have different PAM requirements. For example, the type II Streptococcus pyogenes system uses an "NGG" sequence, where "N" can be any nucleotide.

[0149] In this application, the term "GCN4" refers to a transcription factor in Saccharomyces cerevisiae (S. cerevisiae), a "master regulator" in the yeast genome, regulating nearly one-tenth of the yeast genome. It is a highly conserved protein, and its homolog in mammals is Activating Transcription factor-4 (ATF4).

[0150] In this application, the term "split green fluorescent protein" generally refers to a polypeptide that is capable of splitting and immediately forming active green fluorescent protein upon reassembly.

[0151] In this application, the term "PDZ protein" generally refers to naturally occurring proteins that contain a PDZ domain. Exemplary PDZ proteins include CASK, MPP1, DLG1, DLG2, PSD95, NeDLG, TIP-33, SYN1a, TIP-43, LDP, LIM, LIMK1, LIMK2, MPP2, NOS1, AF6, PTN-4, prIL16, 41.8kD, KIAA0559, RGS12, KIAA0316, DVL1, TIP-40, TIAM1, MINT1, MAGI-1, MAGI-2, MAGI-3, KIAA0303, CBP, MINT3, TIP-2, KIAA0561 and / or TIP-1.

[0152] In this application, the term "single-chain antibody" or "scFv (Single Chain Antibody)" generally refers to a single-chain polypeptide containing one or more antigen-binding sites. In addition, although the H and L chains of the Fv fragment are encoded by different genes, they can be linked together directly or through a peptide, for example, by recombinant methods, the H and L chains can be linked into a single protein chain using a synthetic linker (called a single-chain antibody, sAb; Bird et al. 1988 Science 242: 423-426; and Huston et al. 1988 PNAS 85: 5879-5883). Such single-chain antibodies are also included in the term "antibody" and can be used as binding determinants in the design and manufacture of multispecific binding molecules. They can be prepared by recombinant technology or enzymatic or chemical cleavage of intact antibodies.

[0153] In this application, the term "recombinant expression vector" generally refers to a genetically modified oligonucleotide or polynucleotide construct that, when the construct comprises a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide, and when the vector is contacted with a host cell under conditions sufficient to express the mRNA, protein, polypeptide, or peptide in the host cell, allows the host cell to express the mRNA, protein, polypeptide, or peptide. For example, a recombinant expression vector comprising a coding sequence for a protein can be produced and used to produce large quantities of the protein. Recombinant expression vectors comprising a nucleic acid sequence encoding a protein of the present invention or a complex thereof can be routinely produced. Those skilled in the art can isolate or synthesize nucleic acids encoding the protein of the present invention and insert them into an expression vector using standard techniques and readily available starting materials. The coding sequence is operatively linked to the necessary regulatory sequences. Expression vectors are well known and readily available. Examples of expression vectors include plasmids, phages, viral vectors, and other nucleic acid molecules or nucleic acid molecule vehicles used to transform host cells and promote expression of the coding sequence. "Plasmid" refers to a circular double-stranded DNA loop into which additional DNA segments can be attached. Alternatively, the vector can be linear. The carrier of another type is a viral vector, in which other DNA segments can be connected to the viral genome. Specific vectors can be autonomously replicated in the host cell they introduce therein (for example, bacterial vectors and additional mammalian vectors with bacterial replication origins). Other vectors (for example, non-additional mammalian vectors) can be integrated into the genome of the host cell after introducing the host cell, and thereby replicate together with the host genome.

[0154] In the present application, term " delivery vector " generally refers to the transfer vehicle that reagent (for example, nucleic acid molecule) can be delivered to target cell.Delivery vector can deliver reagent to specific cell subclass.For example, by the intrinsic characteristics of delivery vector or by the part coupled with carrier, the part contained therein (or the part combined with carrier, so that this part and this delivery vector are maintained together, and then make this part enough to target delivery vector) make delivery vector target certain type of cell.Delivery vector also can improve the half-life in vivo of the reagent to be sent and / or the bioavailability of the reagent to be sent.Delivery vector can comprise viral vector, virus-like particle, polycationic carrier, peptide carrier, liposome and / or hybrid carrier.For example, if target cell is hepatocyte, the character (for example, size, charge and / or pH) of described delivery vector can effectively be delivered to target cell, reduce immune clearance and / or promote to stay in this target cell by the molecule of described delivery vector and / or wherein encapsulated.

[0155] In this application, the term "liposome" generally refers to a vesicle with an internal space that is isolated from an external medium by one or more bilayer membranes. In some embodiments, the bilayer membrane can be formed by amphiphilic molecules, such as synthetic or naturally derived lipids comprising spatially isolated hydrophilic and hydrophobic domains; in other embodiments, the bilayer membrane can be formed by amphiphilic polymers and surfactants. In some embodiments, the liposome is a spherical vesicle structure consisting of a monolayer or multilayer lipid bilayer surrounding an internal aqueous compartment and a relatively impermeable external lipophilic phospholipid bilayer. In some embodiments, liposomes are biocompatible, non-toxic, can deliver hydrophilic and lipophilic drug molecules, protect their cargo from being degraded by plasma enzymes, and transport their loads across biological membranes and the blood-brain barrier (BBB). Liposomes can be made of several different types of lipids, such as phospholipids. Liposomes can comprise natural phospholipids and lipids such as 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), sphingomyelin, egg phosphatidylcholine, monosialoganglioside, or any combination thereof. In order to modify the structure and properties of the liposomes, several other additives can be added to the liposomes. For example, the liposomes can also comprise cholesterol, sphingomyelin, and / or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), for example, to increase stability and / or prevent leakage of cargo inside the liposomes.

[0156] The term "lipid nanoparticle (LNP)" generally refers to a particle comprising a plurality (i.e., more than one) lipid molecules that are physically bound to each other (e.g., covalently or non-covalently) by intermolecular forces. LNP can be, for example, a microsphere (including unilamellar and multilamellar vesicles, such as liposomes), a dispersed phase in an emulsion, a micelle, or an internal phase in a suspension. LNP can encapsulate nucleic acids in cationic lipid particles (e.g., liposomes) and can be delivered to cells relatively easily. In some instances, lipid nanoparticles do not contain any viral components, which helps to minimize safety and immunogenicity issues. The lipid particles can be used for in vitro, ex vivo, and in vivo delivery. The lipid particles can also be used for cell populations of various sizes. The LNP of the present application can be easily prepared by various methods known in the art, such as by mixing an organic phase with an aqueous phase. The mixing of the two phases can be achieved by a microfluidic device and an impinging stream reactor. The more fully the organic phase and the aqueous phase are mixed, the better the embedding efficiency and particle size distribution of the LNP obtained. Preferably, the particle size of the LNP can be adjusted by changing the mixing speed of the organic phase and the aqueous phase. The faster the mixing speed, the smaller the particle size of the LNP prepared. The embedding efficiency can be optimized by adjusting the N / P (ionizable lipid / nucleic acid) ratio of the LNP system. In some instances, LNP can be used to deliver DNA molecules and / or RNA molecules (e.g., mRNA of Cas, sgRNA). In some cases, LNP can be used to deliver the RNP complex of Cas / gRNA. In some embodiments, LNP is used to deliver mRNA and gRNA.

[0157] In this application, the term "pharmaceutically acceptable carrier" generally refers to a carrier for administering therapeutic agents, such as antibodies or polypeptides, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition and that can be administered without excessive toxicity. Suitable carriers can be large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polyamino acids, amino acid copolymers, lipid aggregates, and inactivated viral particles. These carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in therapeutic compositions can include liquids such as water, saline, glycerol, and ethanol. Auxiliary substances, such as wetting agents or emulsifiers, pH buffering substances, etc., may also be present in these carriers.

[0158] In this application, the term "subject" generally refers to an animal, typically a mammal, such as a human, non-human primate (ape, gibbon, gorilla, chimpanzee, orangutan, macaque), livestock (dogs and cats), farm animals (poultry such as chickens and ducks, horses, cattle, goats, sheep, pigs), and laboratory animals (mice, rats, rabbits, guinea pigs). Human subjects include fetuses, newborns, infants, adolescents, and adult subjects. Subjects include animal disease models, for example, mice and other animal models of blood coagulation diseases (such as HemA), and other animal models known to those skilled in the art.

[0159] In this application, the term "comprising" generally means including the features specifically stated, but not excluding other elements.

[0160] In this application, the term "selected from" generally refers to the selected objects and all combinations thereof. For example, "selected from A, B and C" means all combinations of A, B and C, for example, A, B, C, A+B, A+C, B+C or A+B+C.

[0161] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0162] Detailed Description of the Invention

[0163] Epigenetic editing systems regulating PCSK9 and ANGPTL3 genes

[0164] In one aspect, the present application provides a method for simultaneously regulating the expression and / or activity of PCSK9 and ANGPTL3 genes, the method comprising providing an epigenetic editing system; the epigenetic editing system comprises a DNA binding domain and a gene expression regulator. In some embodiments, the epigenetic editing system comprises a complex, wherein the DNA binding domain and the gene expression regulator are contained in the complex; or the epigenetic editing system comprises a nucleic acid encoding the complex.

[0165] On the other hand, the present application provides a complex, such as the complex provided in the method described in the present application, and the complex can simultaneously regulate the expression and / or activity of PCSK9 gene and ANGPTL3 gene without changing the function of their gene sequences.

[0166] The composite of the present application

[0167] In some embodiments, the complex described herein comprises a first fusion moiety and a second fusion moiety; wherein one of the first fusion moiety and the second fusion moiety comprises the DNA binding domain, at least one gene expression regulator, and a recruitment domain A, and the other of the first fusion moiety and the second fusion moiety comprises at least one gene expression regulator and a recruitment domain A', and the recruitment domain A and the recruitment domain A' are capable of interacting. For example, the gene expression regulators contained in the first fusion moiety and the second fusion moiety are optionally a transcriptional repressor domain and an epigenetic modification domain, respectively. That is, in some specific embodiments, the first fusion moiety and the second fusion moiety of the complex of the present application can be generally divided into two situations: 1) one of the two fusion moieties comprises a DNA binding domain, an epigenetic modification domain, and a recruitment domain A, and the other fusion moiety comprises a transcriptional repressor domain and a recruitment domain A', or 2) one of the two fusion moieties comprises a DNA binding domain, a transcriptional repressor, and a recruitment domain A, and the other fusion moiety comprises an epigenetic modification domain and a recruitment domain A'.

[0168] Specifically, in some embodiments of the above-mentioned situation 1), one of the two fusion parts may comprise, from N-terminus to C-terminus, an epigenetic modification domain, a DNA binding domain, and a recruitment domain A. For example, in some embodiments of the above-mentioned situation 2), one of the two fusion parts may comprise, from N-terminus to C-terminus, a recruitment domain A, a DNA binding domain, and a transcriptional repressor domain. For example, in some embodiments of the above-mentioned situation 1), the other fusion part of the two fusion parts may comprise, from N-terminus to C-terminus, a transcriptional repressor domain and a recruitment domain A', or the recruitment domain A' and the transcriptional repressor domain, i.e., the transcriptional repressor domain and the recruitment domain A', may be connected in sequence interchangeably. For example, in some embodiments of the above-mentioned situation 2), the other fusion part of the two fusion parts may comprise, from N-terminus to C-terminus, an epigenetic modification domain and a recruitment domain A', or the recruitment domain A' and the epigenetic modification domain, i.e., the epigenetic modification domain and the recruitment domain A', may be connected in sequence interchangeably.

[0169] For example, the DNA binding domain is selected from the group consisting of: a TALE domain, a zinc finger domain, a tetR domain, a large-range nuclease, a Cas nuclease, an Argonaute (Ago) protein, and homologs, modified forms, or variants thereof. In some embodiments, the DNA binding domain is capable of specifically recognizing a target sequence on the PCSK9 gene and / or the ANGPTL3 gene. For example, the DNA binding domain specifically recognizes the target sequence by binding to a guide RNA. For example, the DNA binding domain is a class II Cas nuclease. Further, the Cas nuclease is selected from class II type II Cas nucleases and class II type V Cas nucleases; for example, the Cas nuclease is Cas9 or Cas12. In certain embodiments, the Cas nuclease is an inactivated Cas9 (dCas9) or an inactivated Cas12 (dCas12).

[0170] For example, the transcriptional repressor is selected from one or more of the following domains: KRAB, ZIM3, ZNF680, ZNF554, ZNF264, ZNF582, ZNF324, ZNF669, ZNF354A, ZNF82, ZNF595, ZNF419, ZNF566, ZIM2, EHMT2, SUV39H1, ZFPM1, TRIM28, EZH2, MXD1, SID, LSD1, HP1a, HDAC3, HDAC1, PRMT1, SETDB1, hSIRT1, ZNF436, ZNF257, ZNF675, ZNF490, ZNF320, ZNF331, ZNF816, ZNF41, ZNF189, ZNF528, ZNF543, ZNF140, ZNF610, ZNF350, ZNF8, ZNF30, ZNF98, ZNF677, ZNF596, ZNF214, ZNF37A, ZNF34, ZNF250, ZNF547, ZNF273, ZFP82, ZNF224, ZNF33A, ZNF45, ZNF175, ZNF184, ZFP28-1, ZFP28-2, ZNF18, ZNF213, ZNF394, ZFP1, ZFP14, ZNF416, ZNF557, ZNF729, ZNF254, ZNF764, ZNF785, ZNF10, CBX5, RYBP, YAF2, MGA, CBX1, SCMH1, MPP8, SUMO3, HERC2, BIN1, PCGF2, TOX, FOXA1, FOXA2, IRF2BP1, IRF2BP2, 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,<h2 style=";text-align:left;direction:ltr">ZN101,ZN783,ZN417,ZN182,ZN823,ZN177,ZN197,ZN717,ZN669,ZN256,ZN 251,CBX4,CDY2,CDYL2,ZN562,ZN461,Z324A,ZN766,ID2,ZN214,CBX7,ID1 ,CREM,SCX,ASCL1,ZN764,SCML2,TWST1,CREB1,TERF1,ID3,CBX8,GSX1,NK X22 , Atf1 , Twst2 , znf17 , tox3 , tox4 , zmym3 , i2BP1 , rhxf1 , ssx2 , i2bpl , zn6 80,TRI68,HXA13,PHC3,TCF24,HXB13,HEY1,PHC2,ZNF81,FIGLA,SAM11,KM T2B,HEY2,JDP2,HXC13,ASCL4,HHEX,GSX2,ETV7,ASCL3,PHC1,OTP,I2BP2, VGLL2,HXA11,PDLI4,ASCL2,CDX4,ZN860,LMBL4,PDIP3,NKX25,CEBPB,ISL 1,CDX2,PROP1,SIN3B,SMBT1,HXC11,HXC10,PRS6A,VSX1,NKX23,MTG16,HMX 3,HMX1,KIF22,CSTF2,CEBPE,DLX2,PPARG,PRIC1,UNC4,BARX2,ALX3,TCF1 5,TERA,VSX2,HXD12,CDX1,TCF23,ALX1,HXA10,RX,CXXC5,SCML1,NFIL3,D LX6,MTG8,CEBPD,SEC13,FIP1,ALX4,LHX3,PRIC2,MAGI3,NELL1,PRRX1,MT G8R,RAX2,DLX3,DLX1,NKX26,NAB1,SAMD7,PITX3,WDR5,MEOX2,NAB2,DHX8, CBX6,EMX2,CPSF6,HXC12,KDM4B,LMBL3,PHX2A,EMX1,NC2B,DLX4,SRY,ZN7 77,ZN398,GATA3,BSH,SF3B4,TEAD1,TEAD3,RGAP1,PHF1,GATA2,FOXO3,ZN2 12,IRX4,ZBED6,LHX4,SIN3A,RBBP7,NKX61,R51A1,MB3L1,DLX5,NOTC1,TE RF2,ZN282,RGS12,ZN840,SPI2B,PAX7,NKX62,ASXL2,FOXO1,GATA1,ZMYM5,LRP1, MIXL1, SGT1, LMCD1, CEBPA, SOX14, WTIP, PRP19, NKX11, RBBP4, DMRT2, SMCA2, and their functionally active fragments.

[0171] For example, the epigenetic modification domain comprises: DNA methyltransferase activity, DNA demethylase activity, DNA deamination activity, DNA amination activity, DNA oxidation activity, DNA helicase activity, histone methyltransferase activity, histone demethylase activity, histone acetyltransferase activity, histone deacetylase activity, histone kinase activity, histone phosphatase activity, histone ubiquitin ligase activity, and one or more of histone deubiquitinating activity. In some embodiments, the epigenetic modification domain comprises a DNA methyltransferase and / or a functionally active fragment thereof. In some embodiments, the DNA methyltransferase is selected from DNMT3A, DNMT3B, Dnmt3c, DNMT1, DNMT2, and DNMT3L. For example, the DNA methyltransferase comprises at least one DNMT3A and at least one DNMT3L.

[0172] The first fusion portion and the second fusion portion of the complex of the present application are formed by the interaction between the recruitment domains contained in each of them to form an aggregated complex. In some embodiments, the interaction between the recruitment domain A and the recruitment domain A' can enable the gene expression regulator to be recruited to the regulatory region of the PCSK9 gene and / or the ANGPTL3 gene or its vicinity. Thus, the present application provides non-limiting examples of combinations of recruitment domain A and recruitment domain A': 1) the domain of one of the recruitment domain A and the recruitment domain A' is GCN4, and the other domain is scFv; or 2) the domain of one of the recruitment domain A and the recruitment domain A' is a GFP11 fragment, and the other domain is GFP1-10; or 3) the domain of one of the recruitment domain A and the recruitment domain A' is GVKESLV, and the other domain is a PDZ protein domain. Similarly, the situation in which GFP11 and GFP1-10 are respectively derived from splitting GFP to form the recruitment domain A and the recruitment domain A' can also be applied to other categories of fluorescent proteins, such as mCherry, eYFP, eCFP, etc., that is, different groups of recruitment domains A and recruitment domains A' can be obtained by splitting mCherry, splitting eYFP, or splitting eCFP for use in the complex provided by the present application. In some embodiments, one of the first fusion part and the second fusion part of the complex of the present application may include two or more recruitment domains, and they are connected by a linker sequence. Exemplary two or more recruitment domains can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 GCN4s connected by a linker sequence.

[0173] In some embodiments, the method is characterized in that: 1) one of the first fusion moiety and the second fusion moiety comprises DNA methyltransferase-dCas9-n×GCN4, and the other comprises a transcriptional repressor domain-scFv or scFv-transcriptional repressor domain; or 2) one of the first fusion moiety and the second fusion moiety comprises DNA methyltransferase-dCas9-scFv, and the other comprises a transcriptional repressor domain-GCN4 or GCN4-transcriptional repressor domain; or 3) one of the first fusion moiety and the second fusion moiety comprises DNA methyltransferase-dCas9-n×GFP1 1, and the other comprises a transcription repressor domain-GFP1-10 or GFP1-10-transcription repressor domain; or 4) one of the first fusion portion and the second fusion portion comprises DNA methyltransferase-dCas9-GFP1-10, and the other comprises a transcription repressor domain-GFP11 or GFP11-transcription repressor domain; wherein, - indicates that the domains at both ends are directly or indirectly connected in the order from N-terminus to C-terminus; n×GCN4 or n×GFP11 respectively represents n copies of GCN4 connected by a linker sequence or n copies of GFP11 connected by a linker sequence, and n is selected from any integer from 1 to 20.

[0174] In other embodiments, the method is characterized in that: 1) one of the first fusion portion and the second fusion portion comprises an n×GCN4-dCas9-transcriptional repressor domain, and the other comprises a DNA methyltransferase-scFv or scFv-DNA methyltransferase; or 2) one of the first fusion portion and the second fusion portion comprises an scFv-dCas9-transcriptional repressor domain, and the other comprises a DNA methyltransferase-GCN4 or GCN4-DNA methyltransferase; or 3) one of the first fusion portion and the second fusion portion comprises an n×GFP11-dCas9-transcriptional repressor domain. domain, and the other comprises DNA methyltransferase-GFP1-10 or GFP1-10-DNA methyltransferase; or 4) one of the first fusion part and the second fusion part comprises a GFP1-10-dCas9-transcriptional repressor domain, and the other comprises a DNA methyltransferase-GFP11 or GFP11-DNA methyltransferase; wherein, - indicates that the domains at both ends are directly or indirectly connected in the order from N-terminus to C-terminus; n×GCN4 or n×GFP11 respectively represent n copies of GCN4 connected by a linker sequence or n copies of GFP11 connected by a linker sequence, and n is selected from any integer from 1 to 20.

[0175] Epigenetic editing systems regulating APOC3 and ANGPTL3 genes

[0176] In one aspect, the present application provides a method for simultaneously regulating the expression and / or activity of the APOC3 gene and the ANGPTL3 gene, the method comprising providing an epigenetic editing system; the epigenetic editing system comprises a DNA binding domain and a gene expression regulator. In some embodiments, the epigenetic editing system comprises a complex, wherein the DNA binding domain and the gene expression regulator are contained in the complex; or the epigenetic editing system comprises a nucleic acid encoding the complex.

[0177] On the other hand, the present application provides a complex, such as the complex provided in the method described in the present application, and the complex can simultaneously regulate the expression and / or activity of the APOC3 gene and the ANGPTL3 gene without changing the function of their gene sequences.

[0178] The composite of the present application

[0179] In some embodiments, the complex described herein comprises a first fusion moiety and a second fusion moiety; wherein one of the first fusion moiety and the second fusion moiety comprises the DNA binding domain, at least one gene expression regulator, and a recruitment domain A, and the other of the first fusion moiety and the second fusion moiety comprises at least one gene expression regulator and a recruitment domain A', and the recruitment domain A and the recruitment domain A' are capable of interacting. For example, the gene expression regulators contained in the first fusion moiety and the second fusion moiety are optionally a transcriptional repressor domain and an epigenetic modification domain, respectively. That is, in some specific embodiments, the first fusion moiety and the second fusion moiety of the complex of the present application can be generally divided into two situations: 1) one of the two fusion moieties comprises a DNA binding domain, an epigenetic modification domain, and a recruitment domain A, and the other fusion moiety comprises a transcriptional repressor domain and a recruitment domain A', or 2) one of the two fusion moieties comprises a DNA binding domain, a transcriptional repressor, and a recruitment domain A, and the other fusion moiety comprises an epigenetic modification domain and a recruitment domain A'.

[0180] Specifically, in some embodiments of the above-mentioned situation 1), one of the two fusion parts may comprise, from N-terminus to C-terminus, an epigenetic modification domain, a DNA binding domain, and a recruitment domain A. For example, in some embodiments of the above-mentioned situation 2), one of the two fusion parts may comprise, from N-terminus to C-terminus, a recruitment domain A, a DNA binding domain, and a transcriptional repressor domain. For example, in some embodiments of the above-mentioned situation 1), the other fusion part of the two fusion parts may comprise, from N-terminus to C-terminus, a transcriptional repressor domain and a recruitment domain A', or the recruitment domain A' and the transcriptional repressor domain, i.e., the transcriptional repressor domain and the recruitment domain A', may be connected in sequence interchangeably. For example, in some embodiments of the above-mentioned situation 2), the other fusion part of the two fusion parts may comprise, from N-terminus to C-terminus, an epigenetic modification domain and a recruitment domain A', or the recruitment domain A' and the epigenetic modification domain, i.e., the epigenetic modification domain and the recruitment domain A', may be connected in sequence interchangeably.

[0181] For example, the DNA binding domain is selected from the group consisting of: a TALE domain, a zinc finger domain, a tetR domain, a meganuclease, a Cas nuclease, an Argonaute (Ago) protein, and homologs, modified forms, or variants thereof. In some embodiments, the DNA binding domain is capable of specifically recognizing a target sequence on the APOC3 gene and / or the ANGPTL3 gene. For example, the DNA binding domain specifically recognizes the target sequence by binding to a guide RNA. For example, the DNA binding domain is a class II Cas nuclease. Further, the Cas nuclease is selected from class II type II Cas nucleases and class II type V Cas nucleases; for example, the Cas nuclease is Cas9 or Cas12. In certain embodiments, the Cas nuclease is an inactivated Cas9 (dCas9) or an inactivated Cas12 (dCas12).

[0182] For example, the transcriptional repressor is selected from one or more of the following domains: KRAB, ZIM3, ZNF680, ZNF554, ZNF264, ZNF582, ZNF324, ZNF669, ZNF354A, ZNF82, ZNF595, ZNF419, ZNF566, ZIM2, EHMT2, SUV39H1, ZFPM1, TRIM28, EZH2, MXD1, SID, LSD1, HP1a, HDAC3, HDAC1, PRMT1, SETDB1, hSIRT1, ZNF436, ZNF257, ZNF675, ZNF490, ZNF320, ZNF331, ZNF816, ZNF41, ZNF189, ZNF528, ZNF543, ZNF140, ZNF610, ZNF350, ZNF8, ZNF30, ZNF98, ZNF677, ZNF596, ZNF214, ZNF37A, ZNF34, ZNF250, ZNF547, ZNF273, ZFP82, ZNF224, ZNF33A, ZNF45, ZNF175, ZNF184, ZFP28-1, ZFP28-2, ZNF18, ZNF213, ZNF394, ZFP1, ZFP14, ZNF416, ZNF557, ZNF729, ZNF254, ZNF764, ZNF785, ZNF10, CBX5, RYBP, YAF2, MGA, CBX1, SCMH1, MPP8, SUMO3, HERC2, BIN1, PCGF2, TOX, FOXA1, FOXA2, IRF2BP1, IRF2BP2, 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,<h2 style=";text-align:left;direction:ltr">ZN101,ZN783,ZN417,ZN182,ZN823,ZN177,ZN197,ZN717,ZN669,ZN256,ZN 251,CBX4,CDY2,CDYL2,ZN562,ZN461,Z324A,ZN766,ID2,ZN214,CBX7,ID1 ,CREM,SCX,ASCL1,ZN764,SCML2,TWST1,CREB1,TERF1,ID3,CBX8,GSX1,NK X22 , Atf1 , Twst2 , znf17 , tox3 , tox4 , zmym3 , i2BP1 , rhxf1 , ssx2 , i2bpl , zn6 80,TRI68,HXA13,PHC3,TCF24,HXB13,HEY1,PHC2,ZNF81,FIGLA,SAM11,KM T2B,HEY2,JDP2,HXC13,ASCL4,HHEX,GSX2,ETV7,ASCL3,PHC1,OTP,I2BP2, VGLL2,HXA11,PDLI4,ASCL2,CDX4,ZN860,LMBL4,PDIP3,NKX25,CEBPB,ISL 1,CDX2,PROP1,SIN3B,SMBT1,HXC11,HXC10,PRS6A,VSX1,NKX23,MTG16,HMX 3,HMX1,KIF22,CSTF2,CEBPE,DLX2,PPARG,PRIC1,UNC4,BARX2,ALX3,TCF1 5,TERA,VSX2,HXD12,CDX1,TCF23,ALX1,HXA10,RX,CXXC5,SCML1,NFIL3,D LX6,MTG8,CEBPD,SEC13,FIP1,ALX4,LHX3,PRIC2,MAGI3,NELL1,PRRX1,MT G8R,RAX2,DLX3,DLX1,NKX26,NAB1,SAMD7,PITX3,WDR5,MEOX2,NAB2,DHX8, CBX6,EMX2,CPSF6,HXC12,KDM4B,LMBL3,PHX2A,EMX1,NC2B,DLX4,SRY,ZN7 77,ZN398,GATA3,BSH,SF3B4,TEAD1,TEAD3,RGAP1,PHF1,GATA2,FOXO3,ZN2 12,IRX4,ZBED6,LHX4,SIN3A,RBBP7,NKX61,R51A1,MB3L1,DLX5,NOTC1,TE RF2,ZN282,RGS12,ZN840,SPI2B,PAX7,NKX62,ASXL2,FOXO1,GATA1,ZMYM5,LRP1, MIXL1, SGT1, LMCD1, CEBPA, SOX14, WTIP, PRP19, NKX11, RBBP4, DMRT2, SMCA2, and their functionally active fragments.

[0183] For example, the epigenetic modification domain comprises: DNA methyltransferase activity, DNA demethylase activity, DNA deamination activity, DNA amination activity, DNA oxidation activity, DNA helicase activity, histone methyltransferase activity, histone demethylase activity, histone acetyltransferase activity, histone deacetylase activity, histone kinase activity, histone phosphatase activity, histone ubiquitin ligase activity, and one or more of histone deubiquitinating activity. In some embodiments, the epigenetic modification domain comprises a DNA methyltransferase and / or a functionally active fragment thereof. In some embodiments, the DNA methyltransferase is selected from DNMT3A, DNMT3B, Dnmt3c, DNMT1, DNMT2, and DNMT3L. For example, the DNA methyltransferase comprises at least one DNMT3A and at least one DNMT3L.

[0184] The first fusion portion and the second fusion portion of the complex of the present application are formed by the interaction between the recruitment domains contained in each of them to form an aggregated complex. In some embodiments, the interaction between the recruitment domain A and the recruitment domain A' can enable the gene expression regulator to be recruited to the regulatory region of the APOC3 gene and / or the ANGPTL3 gene or its vicinity. Thus, the present application provides non-limiting examples of combinations of recruitment domain A and recruitment domain A': 1) the domain of one of the recruitment domain A and the recruitment domain A' is GCN4, and the other domain is scFv; or 2) the domain of one of the recruitment domain A and the recruitment domain A' is a GFP11 fragment, and the other domain is GFP1-10; or 3) the domain of one of the recruitment domain A and the recruitment domain A' is GVKESLV, and the other domain is a PDZ protein domain. Similarly, the situation in which GFP11 and GFP1-10 are respectively derived from splitting GFP to form the recruitment domain A and the recruitment domain A' can also be applied to other categories of fluorescent proteins, such as mCherry, eYFP, eCFP, etc., that is, different groups of recruitment domains A and recruitment domains A' can be obtained by splitting mCherry, splitting eYFP, or splitting eCFP for use in the complex provided by the present application. In some embodiments, one of the first fusion part and the second fusion part of the complex of the present application may include two or more recruitment domains, and they are connected by a linker sequence. Exemplary two or more recruitment domains can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 GCN4s connected by a linker sequence.

[0185] In some embodiments, the method is characterized in that: 1) one of the first fusion moiety and the second fusion moiety comprises DNA methyltransferase-dCas9-n×GCN4, and the other comprises a transcriptional repressor domain-scFv or scFv-transcriptional repressor domain; or 2) one of the first fusion moiety and the second fusion moiety comprises DNA methyltransferase-dCas9-scFv, and the other comprises a transcriptional repressor domain-GCN4 or GCN4-transcriptional repressor domain; or 3) one of the first fusion moiety and the second fusion moiety comprises DNA methyltransferase-dCas9-n×GFP1 1, and the other comprises a transcription repressor domain-GFP1-10 or GFP1-10-transcription repressor domain; or 4) one of the first fusion portion and the second fusion portion comprises DNA methyltransferase-dCas9-GFP1-10, and the other comprises a transcription repressor domain-GFP11 or GFP11-transcription repressor domain; wherein, - indicates that the domains at both ends are directly or indirectly connected in the order from N-terminus to C-terminus; n×GCN4 or n×GFP11 respectively represents n copies of GCN4 connected by a linker sequence or n copies of GFP11 connected by a linker sequence, and n is selected from any integer from 1 to 20.

[0186] In other embodiments, the method is characterized in that: 1) one of the first fusion portion and the second fusion portion comprises an n×GCN4-dCas9-transcriptional repressor domain, and the other comprises a DNA methyltransferase-scFv or scFv-DNA methyltransferase; or 2) one of the first fusion portion and the second fusion portion comprises an scFv-dCas9-transcriptional repressor domain, and the other comprises a DNA methyltransferase-GCN4 or GCN4-DNA methyltransferase; or 3) one of the first fusion portion and the second fusion portion comprises an n×GFP11-dCas9-transcriptional repressor domain. domain, and the other comprises DNA methyltransferase-GFP1-10 or GFP1-10-DNA methyltransferase; or 4) one of the first fusion part and the second fusion part comprises a GFP1-10-dCas9-transcriptional repressor domain, and the other comprises a DNA methyltransferase-GFP11 or GFP11-DNA methyltransferase; wherein, - indicates that the domains at both ends are directly or indirectly connected in the order from N-terminus to C-terminus; n×GCN4 or n×GFP11 respectively represent n copies of GCN4 connected by a linker sequence or n copies of GFP11 connected by a linker sequence, and n is selected from any integer from 1 to 20.

[0187] Epigenetic editing systems regulating PCSK9 and APOC3 genes

[0188] On the other hand, the present application provides a method for simultaneously regulating the expression and / or activity of the PCSK9 gene and the APOC3 gene, the method comprising providing an epigenetic editing system; the epigenetic editing system comprises a DNA binding domain and a gene expression regulator. In some embodiments, the epigenetic editing system comprises a complex, wherein the DNA binding domain and the gene expression regulator are contained in the complex; or the epigenetic editing system comprises a nucleic acid encoding the complex.

[0189] On the other hand, the present application provides a complex, such as the complex provided in the method described in the present application, and the complex can simultaneously regulate the expression and / or activity of PCSK9 gene and APOC3 gene without changing the function of their gene sequences.

[0190] The composite of the present application

[0191] In some embodiments, the complex described herein comprises a first fusion moiety and a second fusion moiety; wherein one of the first fusion moiety and the second fusion moiety comprises the DNA binding domain, at least one gene expression regulator, and a recruitment domain A, and the other of the first fusion moiety and the second fusion moiety comprises at least one gene expression regulator and a recruitment domain A', and the recruitment domain A and the recruitment domain A' are capable of interacting. For example, the gene expression regulators contained in the first fusion moiety and the second fusion moiety are optionally a transcriptional repressor domain and an epigenetic modification domain, respectively. That is, in some specific embodiments, the first fusion moiety and the second fusion moiety of the complex of the present application can be generally divided into two situations: 1) one of the two fusion moieties comprises a DNA binding domain, an epigenetic modification domain, and a recruitment domain A, and the other fusion moiety comprises a transcriptional repressor domain and a recruitment domain A', or 2) one of the two fusion moieties comprises a DNA binding domain, a transcriptional repressor, and a recruitment domain A, and the other fusion moiety comprises an epigenetic modification domain and a recruitment domain A'.

[0192] Specifically, in some embodiments of the above-mentioned situation 1), one of the two fusion parts may comprise, from N-terminus to C-terminus, an epigenetic modification domain, a DNA binding domain, and a recruitment domain A. For example, in some embodiments of the above-mentioned situation 2), one of the two fusion parts may comprise, from N-terminus to C-terminus, a recruitment domain A, a DNA binding domain, and a transcriptional repressor domain. For example, in some embodiments of the above-mentioned situation 1), the other fusion part of the two fusion parts may comprise, from N-terminus to C-terminus, a transcriptional repressor domain and a recruitment domain A', or the recruitment domain A' and the transcriptional repressor domain, i.e., the transcriptional repressor domain and the recruitment domain A', may be connected in sequence interchangeably. For example, in some embodiments of the above-mentioned situation 2), the other fusion part of the two fusion parts may comprise, from N-terminus to C-terminus, an epigenetic modification domain and a recruitment domain A', or the recruitment domain A' and the epigenetic modification domain, i.e., the epigenetic modification domain and the recruitment domain A', may be connected in sequence interchangeably.

[0193] For example, the DNA binding domain is selected from the group consisting of: a TALE domain, a zinc finger domain, a tetR domain, a large-range nuclease, a Cas nuclease, an Argonaute (Ago) protein, and homologs, modified forms or variants thereof. In some embodiments, the DNA binding domain is capable of specifically recognizing a target sequence on the PCSK9 gene and / or the APOC3 gene. For example, the DNA binding domain specifically recognizes the target sequence by binding to a guide RNA. For example, the DNA binding domain is a Class II Cas nuclease. Further, the Cas nuclease is selected from Class II Type II Cas nuclease and Class II Type V Cas nuclease; for example, the Cas nuclease is Cas9 or Cas12. In certain embodiments, the Cas nuclease is an inactivated Cas9 (dCas9) or an inactivated Cas12 (dCas12).

[0194] For example, the transcriptional repressor is selected from one or more of the following domains: KRAB, ZIM3, ZNF680, ZNF554, ZNF264, ZNF582, ZNF324, ZNF669, ZNF354A, ZNF82, ZNF595, ZNF419, ZNF566, ZIM2, EHMT2, SUV39H1, ZFPM1, TRIM28, EZH2, MXD1, SID, LSD1, HP1a, HDAC3, HDAC1, PRMT1, SETDB1, hSIRT1, ZNF436, ZNF257, ZNF675, ZNF490, ZNF320, ZNF331, ZNF816, ZNF41, ZNF189, ZNF528, ZNF543, ZNF140, ZNF610, ZNF350, ZNF8, ZNF30, ZNF98, ZNF677, ZNF596, ZNF214, ZNF37A, ZNF34, ZNF250, ZNF547, ZNF273, ZFP82, ZNF224, ZNF33A, ZNF45, ZNF175, ZNF184, ZFP28-1, ZFP28-2, ZNF18, ZNF213, ZNF394, ZFP1, ZFP14, ZNF416, ZNF557, ZNF729, ZNF254, ZNF764, ZNF785, ZNF10, CBX5, RYBP, YAF2, MGA, CBX1, SCMH1, MPP8, SUMO3, HERC2, BIN1, PCGF2, TOX, FOXA1, FOXA2, IRF2BP1, IRF2BP2, 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,<h2 style=";text-align:left;direction:ltr">ZN101,ZN783,ZN417,ZN182,ZN823,ZN177,ZN197,ZN717,ZN669,ZN256,ZN 251,CBX4,CDY2,CDYL2,ZN562,ZN461,Z324A,ZN766,ID2,ZN214,CBX7,ID1 ,CREM,SCX,ASCL1,ZN764,SCML2,TWST1,CREB1,TERF1,ID3,CBX8,GSX1,NK X22 , Atf1 , Twst2 , znf17 , tox3 , tox4 , zmym3 , i2BP1 , rhxf1 , ssx2 , i2bpl , zn6 80,TRI68,HXA13,PHC3,TCF24,HXB13,HEY1,PHC2,ZNF81,FIGLA,SAM11,KM T2B,HEY2,JDP2,HXC13,ASCL4,HHEX,GSX2,ETV7,ASCL3,PHC1,OTP,I2BP2, VGLL2,HXA11,PDLI4,ASCL2,CDX4,ZN860,LMBL4,PDIP3,NKX25,CEBPB,ISL 1,CDX2,PROP1,SIN3B,SMBT1,HXC11,HXC10,PRS6A,VSX1,NKX23,MTG16,HMX 3,HMX1,KIF22,CSTF2,CEBPE,DLX2,PPARG,PRIC1,UNC4,BARX2,ALX3,TCF1 5,TERA,VSX2,HXD12,CDX1,TCF23,ALX1,HXA10,RX,CXXC5,SCML1,NFIL3,D LX6,MTG8,CEBPD,SEC13,FIP1,ALX4,LHX3,PRIC2,MAGI3,NELL1,PRRX1,MT G8R,RAX2,DLX3,DLX1,NKX26,NAB1,SAMD7,PITX3,WDR5,MEOX2,NAB2,DHX8, CBX6,EMX2,CPSF6,HXC12,KDM4B,LMBL3,PHX2A,EMX1,NC2B,DLX4,SRY,ZN7 77,ZN398,GATA3,BSH,SF3B4,TEAD1,TEAD3,RGAP1,PHF1,GATA2,FOXO3,ZN2 12,IRX4,ZBED6,LHX4,SIN3A,RBBP7,NKX61,R51A1,MB3L1,DLX5,NOTC1,TE RF2,ZN282,RGS12,ZN840,SPI2B,PAX7,NKX62,ASXL2,FOXO1,GATA1,ZMYM5,LRP1, MIXL1, SGT1, LMCD1, CEBPA, SOX14, WTIP, PRP19, NKX11, RBBP4, DMRT2, SMCA2, and their functionally active fragments.

[0195] For example, the epigenetic modification domain comprises: DNA methyltransferase activity, DNA demethylase activity, DNA deamination activity, DNA amination activity, DNA oxidation activity, DNA helicase activity, histone methyltransferase activity, histone demethylase activity, histone acetyltransferase activity, histone deacetylase activity, histone kinase activity, histone phosphatase activity, histone ubiquitin ligase activity, and one or more of histone deubiquitinating activity. In some embodiments, the epigenetic modification domain comprises a DNA methyltransferase and / or a functionally active fragment thereof. In some embodiments, the DNA methyltransferase is selected from DNMT3A, DNMT3B, Dnmt3c, DNMT1, DNMT2, and DNMT3L. For example, the DNA methyltransferase comprises at least one DNMT3A and at least one DNMT3L.

[0196] The first fusion part and the second fusion part of the complex of the present application are formed by the interaction between the recruitment domains respectively contained in the complex. In some embodiments, the interaction between the recruitment domain A and the recruitment domain A' can enable the gene expression regulator to be recruited to the regulatory region of the PCSK9 gene and / or the APOC3 gene or its vicinity. Thus, the present application provides non-limiting examples of combinations of recruitment domain A and recruitment domain A': 1) the domain of one of the recruitment domain A and the recruitment domain A' is GCN4, and the other domain is scFv; or 2) the domain of one of the recruitment domain A and the recruitment domain A' is a GFP11 fragment, and the other domain is GFP1-10; or 3) the domain of one of the recruitment domain A and the recruitment domain A' is GVKESLV, and the other domain is a PDZ protein domain. Similarly, the situation in which GFP11 and GFP1-10 are respectively derived from splitting GFP to form the recruitment domain A and the recruitment domain A' can also be applied to other categories of fluorescent proteins, such as mCherry, eYFP, eCFP, etc., that is, different groups of recruitment domains A and recruitment domains A' can be obtained by splitting mCherry, splitting eYFP, or splitting eCFP for use in the complex provided by the present application. In some embodiments, one of the first fusion part and the second fusion part of the complex of the present application may include two or more recruitment domains, and they are connected by a linker sequence. Exemplary two or more recruitment domains can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 GCN4s connected by a linker sequence.

[0197] In some embodiments, the method is characterized in that: 1) one of the first fusion moiety and the second fusion moiety comprises DNA methyltransferase-dCas9-n×GCN4, and the other comprises a transcriptional repressor domain-scFv or scFv-transcriptional repressor domain; or 2) one of the first fusion moiety and the second fusion moiety comprises DNA methyltransferase-dCas9-scFv, and the other comprises a transcriptional repressor domain-GCN4 or GCN4-transcriptional repressor domain; or 3) one of the first fusion moiety and the second fusion moiety comprises DNA methyltransferase-dCas9-n×GFP1 1, and the other comprises a transcription repressor domain-GFP1-10 or GFP1-10-transcription repressor domain; or 4) one of the first fusion portion and the second fusion portion comprises DNA methyltransferase-dCas9-GFP1-10, and the other comprises a transcription repressor domain-GFP11 or GFP11-transcription repressor domain; wherein, - indicates that the domains at both ends are directly or indirectly connected in the order from N-terminus to C-terminus; n×GCN4 or n×GFP11 respectively represents n copies of GCN4 connected by a linker sequence or n copies of GFP11 connected by a linker sequence, and n is selected from any integer from 1 to 20.

[0198] In other embodiments, the method is characterized in that: 1) one of the first fusion portion and the second fusion portion comprises an n×GCN4-dCas9-transcriptional repressor domain, and the other comprises a DNA methyltransferase-scFv or scFv-DNA methyltransferase; or 2) one of the first fusion portion and the second fusion portion comprises an scFv-dCas9-transcriptional repressor domain, and the other comprises a DNA methyltransferase-GCN4 or GCN4-DNA methyltransferase; or 3) one of the first fusion portion and the second fusion portion comprises an n×GFP11-dCas9-transcriptional repressor domain. domain, and the other comprises DNA methyltransferase-GFP1-10 or GFP1-10-DNA methyltransferase; or 4) one of the first fusion part and the second fusion part comprises a GFP1-10-dCas9-transcriptional repressor domain, and the other comprises a DNA methyltransferase-GFP11 or GFP11-DNA methyltransferase; wherein, - indicates that the domains at both ends are directly or indirectly connected in the order from N-terminus to C-terminus; n×GCN4 or n×GFP11 respectively represent n copies of GCN4 connected by a linker sequence or n copies of GFP11 connected by a linker sequence, and n is selected from any integer from 1 to 20.

[0199] Based on the above, the present application may provide exemplary amino acid sequences of the first fusion moiety or the second fusion moiety:

[0200] In another aspect, the present application provides a nucleic acid encoding the complex described herein. For example, the nucleic acid comprises DNA and / or mRNA. For example, the nucleic acid can be used to treat or alleviate a disease or condition associated with abnormal target gene expression and / or abnormal target gene activity, wherein the target gene is PCSK9 and / or ANGPTL3. For example, the nucleic acid can be used to treat or alleviate a disease or condition associated with abnormal target gene expression and / or abnormal target gene activity, wherein the target gene is APOC3 and / or ANGPTL3. For example, the nucleic acid can be used to treat or alleviate a disease or condition associated with abnormal target gene expression and / or abnormal target gene activity, wherein the target gene is PCSK9 and / or APOC3. In some embodiments, the nucleic acid is mRNA; one or more modification techniques can be used to produce a more stable mRNA. In some embodiments, the nucleic acid is mRNA; one or more modification techniques can be used to produce a more stable mRNA. Known mRNA modification technologies can be broadly categorized into three types: synthesizing mRNA using synthetic non-natural RNAs instead of natural RNAs; adding 5' caps, 3' poly(A) tails, and UTR (untranslated region) sequences; and employing specialized novel formulation technologies to effectively protect mRNA. Among these, the preferred mRNA modification technology involves synthesizing mRNA using synthetic non-natural RNAs instead of natural RNAs. Chemical modifications on eukaryotic mRNA can be broadly categorized into three types: methylation, pseudouridine (Ψ), and hypoxanthine. For example, the chemical modification can be selected from the group consisting of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thiol-1-methyl-1-deaza-pseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydro-pseudouridine, 2-thiol-dihydro-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methyl-pseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydro-pseudouridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine. For example, the nucleic acid is a recombinant expression vector comprising a nucleic acid encoding the complex described herein. For example, a recombinant expression vector can refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. Recombinant expression vectors can include single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules comprising one or more free ends, no 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 can contain virally derived DNA or RNA sequences for packaging into viruses (e.g., retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses, and adeno-associated viruses AAV).Viruses and viral vectors can be used for delivery in vitro, ex vivo, and / or in vivo.

[0201] On the other hand, the present application provides a recombinant expression vector comprising the nucleic acid described in the present application.

[0202] On the other hand, the application provides a kind of delivery vector, described delivery vector comprises complex as described in the application, nucleic acid as described in the application and / or recombinant expression vector as described in the application.For example, described delivery vector optionally comprises liposome and / or lipid nanoparticle (LNP).For example, delivery vector can be introduced into cell by physical delivery method.The example of physical method comprises microinjection, electroporation and hydrodynamic delivery.For example, LNPs can be wrapped in nucleic acid in cationic lipid granule (for example liposome), and can be delivered to cell relatively easily.In some examples, lipid nanoparticle does not contain any viral component, and this helps to reduce safety and immunogenicity problem to greatest extent.Lipid granule can be used for external, ex vivo and in vivo delivery.The composition of LNP can comprise cationic lipid, ionizable lipid, pegylated lipid and / or support lipid, and optional cholesterol component.

[0203] On the other hand, the present application provides a pharmaceutical composition comprising the complex described herein, the nucleic acid described herein, the recombinant expression vector described herein and / or the delivery vector described herein, and at least one pharmaceutically acceptable carrier.

[0204] On the other hand, the present application provides a cell comprising the complex described herein, the nucleic acid described herein, the recombinant expression vector described herein, the delivery vector described herein, and / or the pharmaceutical composition described herein.

[0205] On the other hand, the present application provides a kit comprising the complex described herein, the nucleic acid described herein, the recombinant expression vector described herein, the delivery vector described herein, the pharmaceutical composition described herein, and / or the cell described herein. For example, the kit further comprises at least one container for placing the above-mentioned components. For example, the kit comprises more than one of the above-mentioned components, and further comprises a second, third and / or other container other than the container, in which the more than one above-mentioned components can be placed separately. For example, the kit can place various combinations of the above-mentioned components in the container. For example, the kit further comprises a buffer reagent, a device for mixing, a device for measuring, a device for sorting and / or a device for labeling. For example, the kit further comprises packaging for accommodating various containers. For example, the kit further comprises instructions for using the kit components. For example, the instructions comprise a physical paper form and / or a machine-readable electronic form.

[0206] In another aspect, the present application provides a method for treating a disease, comprising providing an effective amount of the complex described herein, the nucleic acid described herein, the recombinant expression vector described herein, the delivery vector described herein, the pharmaceutical composition described herein, the cell described herein, and / or the kit described herein to a subject in need thereof; the disease is a disease associated with abnormal gene activity of PCSK9 and / or ANGPTL3. For example, the method comprises introducing the complex, the nucleic acid, the recombinant expression vector, the delivery vector, the pharmaceutical composition, the cell described herein, and / or the kit described herein into a cell containing a target gene; the target gene is PCSK9 and / or ANGPTL3. In another aspect, the present application provides a method for treating a disease, comprising providing an effective amount of the complex described herein, the nucleic acid described herein, the recombinant expression vector described herein, the delivery vector described herein, the pharmaceutical composition, the cell described herein, and / or the kit described herein to a subject in need thereof; the disease is a disease associated with abnormal gene activity of APOC3 and / or ANGPTL3. For example, the method comprises introducing the complex, the nucleic acid, the recombinant expression vector, the delivery vector, the pharmaceutical composition, the cell, and / or the kit into a cell containing a target gene; the target gene is APOC3 and / or ANGPTL3. In another aspect, the present application provides a method for treating a disease, comprising providing an effective amount of the complex, the nucleic acid, the recombinant expression vector, the delivery vector, the pharmaceutical composition, the cell, and / or the kit described herein to a subject in need thereof; the disease is a disease associated with abnormal gene activity of PCSK9 and / or APOC3. For example, the method comprises introducing the complex, the nucleic acid, the recombinant expression vector, the delivery vector, the pharmaceutical composition, the cell, and / or the kit into a cell containing a target gene; the target gene is PCSK9 and / or APOC3. For example, the introduction into the cell can be performed using a non-viral or viral-based transfection method. For example, the non-viral transfection method includes any suitable method for introducing the cell without using viral DNA or viral particles as a delivery system, and non-limiting examples of non-viral transfection methods include nanoparticle encapsulation of nucleic acids encoding the complex (e.g., lipid nanoparticles, gold nanoparticles, etc.), calcium phosphate transfection, liposome transfection, nucleofection, sonoporation, transfection by heat shock, magnetofection, and electroporation. For example, viral-based transfection methods include any viral vector suitable for the methods described herein, non-limiting examples of which include, but are not limited to, retrovirus, adenovirus, lentivirus, and / or adeno-associated virus vectors.For example, the method for treating a disease further comprises introducing the complex, the nucleic acid, the recombinant expression vector, the delivery vector, the pharmaceutical composition, the cell, and / or the kit from an external environment into a cell. For another example, the method for treating a disease comprises contacting the complex, the nucleic acid, the recombinant expression vector, the delivery vector, and / or the pharmaceutical composition with a target gene near the target gene and / or with a transcriptional regulatory element of the target gene; the target gene is PCSK9 and / or ANGPTL3. For another example, the method for treating a disease comprises contacting the complex, the nucleic acid, the recombinant expression vector, the delivery vector, and / or the pharmaceutical composition with a target gene near the target gene and / or with a transcriptional regulatory element of the target gene; the target gene is APOC3 and / or ANGPTL3. For another example, the method for treating a disease comprises contacting the complex, the nucleic acid, the recombinant expression vector, the delivery vector, and / or the pharmaceutical composition with a target gene near the target gene and / or with a transcriptional regulatory element of the target gene; the target gene is PCSK9 and / or APOC3. For example, the contact refers to contacting the first fusion portion, the second fusion portion and the guide RNA of the complex described herein with the vicinity of the target gene and / or the transcriptional regulatory element of the target gene, and the guide RNA forms a complex with the fusion comprising the DNA binding domain, the complex specifically recognizes a specific region in the target gene and hybridizes therewith, and the first fusion portion and the second fusion portion are recruited to the vicinity of the DNA binding domain through direct or indirect interaction of their recruitment domain A and recruitment domain A', thereby regulating the expression of the target nucleic acid. For example, the method includes causing the first fusion portion, the second fusion portion and the guide RNA described herein to exist in the form of a complex (e.g., an assembled ribonucleoprotein complex), and causing the complex to contact the vicinity of the target gene and / or the transcriptional regulatory element of the target gene.

[0207] Without intending to be bound by any theory, the following examples are merely intended to illustrate the methods and uses of regulating gene expression of the present application, and are not intended to limit the scope of the present invention.

[0208] Example

[0209] Example 1

[0210] The present invention regulates the gene expression levels of PCSK9 and ANGPTL3 in cells

[0211] The epigenetic editing tool mRNA (SEQ ID NO: 116) and the corresponding sgRNA (NT control: SEQ ID NO: 161, sequences targeting PCSK9 and ANGPTL3: SEQ ID NOs: 162 and 164, respectively) were prepared by LNP embedding at a mass ratio of 1:1 to obtain LNP test samples containing the editing tool combined with different sgRNAs. Among them, the dual-target sample was prepared by LNP embedding with the epigenetic editing tool mRNA: Pcsk9-sgRNA: Angptl3-sgRNA = 1:0.5:0.5 (LNP preparation reference: https: / / doi.org / 10.1038 / s41586-021-03534-y). The test cells used were the human hepatoma cell line Huh7. 50,000 cells were seeded per well in a 24-well plate. After 12 hours, LNP samples were added to the plate at a dose of 2.5 μg / ml. After 4-6 hours, fresh DMEM + 10% FBS medium was replaced and the cells were cultured. After 7 days, the cells were harvested for mRNA extraction and reverse transcribed into cDNA. qPCR (primer sequences are shown in SEQ ID NOs: 165-168, 172, and 173) was used to measure the mRNA expression levels of the target genes PCSK9 and ANGPTL3. The gene silencing efficiency of the target genes was determined by comparison with a blank control group (without mRNA and sgRNA). The results in Figure 1 demonstrate that the dual-target sample group significantly inhibited the mRNA expression of the target genes. Furthermore, compared to the single-target sample group, the dual-target sample group used half the amount of sgRNA for each target, yet the inhibitory effect was essentially equivalent to that of the single-target sample group.

[0212] Example 2

[0213] The present method regulates the gene expression levels of PCSK9 and ANGPTL3 in cynomolgus monkey blood

[0214] This example uses cynomolgus macaques as a research model. The epigenetic editing tool mRNA (SEQ ID NO: 116) and the corresponding sgRNA (SEQ ID NOs: 163 and 164 targeting PCSK9 and ANGPTL3, respectively) were prepared by LNP encapsulation at a mass ratio of 1:1 to obtain LNP test samples containing the editing tool combined with different sgRNAs. The dual-target sample was prepared by LNP encapsulation with a ratio of 1:0.5:0.5 of epigenetic editing tool mRNA:Pcsk9-sgRNA:Angptl3-sgRNA (LNP preparation reference: https: / / doi.org / 10.1038 / s41586-021-03534-y).

[0215] One week before dosing, serum samples from cynomolgus monkeys were collected for baseline PCSK9 and ANGPTL3 levels. The animals were given two injections of an antihistamine one day and one hour before dosing. Dosing was performed intravenously according to body weight. Seven and 14 days after dosing, blood PCSK9 and ANGPTL3 protein expression levels were measured (qPCR primer sequences are shown in SEQ ID NOs: 169, 166, and 170-173). The expression levels were compared with baseline values ​​to determine the gene silencing efficacy of different sample combinations. Blood cholesterol and triglyceride levels were further measured to confirm the synergistic effect of the dual targets.

[0216] Example 3

[0217] The present invention regulates the gene expression levels of APOC3 and ANGPTL3 in cells

[0218] The epigenetic editing tool mRNA (SEQ ID NO: 116) and the corresponding sgRNA (NT control: SEQ ID NO: 161, sequences targeting APOC3 and ANGPTL3: SEQ ID NOs: 252 and 164, respectively) were prepared by LNP embedding at a mass ratio of 1:1 to obtain LNP test samples containing the editing tool combined with different sgRNAs. Among them, the dual-target sample was prepared by LNP embedding with the epigenetic editing tool mRNA:Apoc3-sgRNA:Angptl3-sgRNA = 1:0.5:0.5 (LNP preparation reference: https: / / doi.org / 10.1038 / s41586-021-03534-y). The test cells used were the human hepatoma cell line Huh7. 50,000 cells were seeded per well in a 24-well plate. After 12 hours, LNP samples were added to the plate at a dose of 2.5 μg / ml. After 4-6 hours, fresh DMEM + 10% FBS medium was replaced and the cells were cultured. After 7 days, the cells were harvested for mRNA extraction and reverse transcribed into cDNA. qPCR (primer sequences are shown in SEQ ID NOs: 167-168, 253, 254, 172, and 173) was used to measure the mRNA expression levels of the target genes APOC3 and ANGPTL3. The gene silencing efficiencies of the target genes were compared with those of the empty control group (without mRNA and sgRNA). The results in Figure 2 demonstrate that the dual-target sample group significantly inhibited the mRNA expression of the target genes. Furthermore, although the amount of sgRNA for each target in the dual-target sample group was halved compared to the single-target sample group, the inhibitory efficacy was essentially equivalent to that of the single-target sample group.

[0219] Example 4

[0220] The present method regulates the gene expression levels of APOC3 and ANGPTL3 in cynomolgus monkey blood

[0221] This example uses cynomolgus macaques as a research model. The epigenetic editing tool mRNA (SEQ ID NO: 116) and the corresponding sgRNA (SEQ ID NOs: 252 and 164 targeting APOC3 and ANGPTL3, respectively) were prepared by LNP encapsulation at a mass ratio of 1:1 to obtain LNP test samples containing the editing tool combined with different sgRNAs. The dual-target sample was prepared by LNP encapsulation with a ratio of 1:0.5:0.5 of epigenetic editing tool mRNA:Apoc3-sgRNA:Angptl3-sgRNA (LNP preparation reference: https: / / doi.org / 10.1038 / s41586-021-03534-y).

[0222] One week before dosing, serum samples from cynomolgus macaques were collected for baseline APOC3 and ANGPTL3 levels. The animals were given two injections of an antihistamine one day and one hour before dosing. Dosing was performed intravenously according to body weight. Seven and 14 days after dosing, blood APOC3 and ANGPTL3 protein expression levels were measured (qPCR primer sequences are shown in SEQ ID NOs: 170, 171, 255, 256, 172, and 173). These levels were compared with baseline values ​​to determine gene silencing efficiencies for different sample combinations. Blood cholesterol and triglyceride levels were further measured to confirm the synergistic effect of the dual targets.

[0223] Example 5

[0224] The present invention regulates the gene expression levels of PCSK9 and APOC3 in cells

[0225] The epigenetic editing tool mRNA (SEQ ID NO: 116) and the corresponding sgRNA (NT control: SEQ ID NO: 161, sequences targeting PCSK9 and APOC3: SEQ ID NOs: 162 and 252, respectively) were prepared by LNP embedding at a mass ratio of 1:1 to obtain LNP test samples containing the editing tool combined with different sgRNAs. Among them, the dual-target sample was prepared by LNP embedding with the epigenetic editing tool mRNA: PCSK9-sgRNA: APOC3-sgRNA = 1:0.5:0.5 (LNP preparation reference: https: / / doi.org / 10.1038 / s41586-021-03534-y). The test cells used were the human hepatoma cell line Huh7. 50,000 cells were seeded per well in a 24-well plate. After 12 hours, LNP samples were added to the plate at a dose of 2.5 μg / ml. After 4-6 hours, fresh DMEM + 10% FBS medium was replaced and the cells were cultured. After 7 days, the cells were harvested for mRNA extraction and reverse transcribed into cDNA. qPCR (primer sequences are shown in SEQ ID NOs: 165, 166, 253, 254, 172, and 173) was used to measure the mRNA expression levels of the target genes PCSK9 and APOC3. The gene silencing efficiency of the different target samples was determined by comparison with a control group containing no mRNA and sgRNA. The results in Figure 3 show that the dual-target sample group produced a highly significant inhibitory effect on the mRNA expression of the target genes. Furthermore, compared to the single-target sample group, the amount of sgRNA used for each target in the dual-target sample group was halved, but the inhibitory effect was essentially equivalent to that of the single-target sample group.

[0226] Example 6

[0227] The present invention regulates the gene expression levels of PCSK9 and APOC3 in the blood of cynomolgus monkeys

[0228] This example uses cynomolgus monkeys as a research model. The epigenetic editing tool mRNA (SEQ ID NO: 116) and the corresponding sgRNA (SEQ ID NOs: 163 and 252 targeting PCSK9 and APOC3, respectively) were prepared by LNP embedding at a mass ratio of 1:1 to obtain LNP test samples containing the editing tool combined with different sgRNAs. The dual-target sample was prepared by LNP embedding with the epigenetic editing tool mRNA: PCSK9-sgRNA: APOC3-sgRNA ratio of 1:0.5:0.5 (LNP preparation reference: https: / / doi.org / 10.1038 / s41586-021-03534-y).

[0229] One week before dosing, serum samples of cynomolgus macaques were collected for baseline blood PCSK9 and APOC3 levels. Two injections of antihistamines were administered to the animals one day and one hour before dosing. The drug was administered intravenously according to body weight. Seven and 14 days after dosing, the expression levels of PCSK9 and APOC3 proteins in the blood were measured (qPCR primer sequences such as SEQ ID NOs: 169, 166 and 255, 256, 172, and 173), and compared with the baseline values ​​to obtain the gene silencing efficiency of different sample combinations. The cholesterol and triglyceride levels in the blood were further tested to determine the synergistic effect of the dual targets.

Claims

1. A method for simultaneously regulating the expression and / or activity of PCSK9 and ANGPTL3 genes, the method comprising providing an epigenetic editing system; the epigenetic editing system comprises a DNA binding domain and a gene expression regulator.

2. The method according to claim 1, wherein the epigenetic editing system comprises a complex, wherein the DNA binding domain and the gene expression regulator are contained in the complex; or the epigenetic editing system comprises a nucleic acid encoding the complex.

3. The method of claim 2, wherein the complex comprises a first fusogenic moiety and a second fusogenic moiety; One of the first fusion part and the second fusion part comprises the DNA binding domain, at least one gene expression regulator and the recruitment domain A, and the other of the first fusion part and the second fusion part comprises at least one gene expression regulator and the recruitment domain A', and the recruitment domain A and the recruitment domain A' are capable of interacting with each other.

4. The method according to claim 3, wherein the interaction between the recruitment domain A and the recruitment domain A' enables the gene expression regulator to be recruited to the regulatory region of the PCSK9 gene and / or the ANGPTL3 gene or its vicinity.

5. The method according to claim 3 or 4, wherein the gene expression regulators comprised by the first fusion moiety and the second fusion moiety are optionally transcriptional repressor domains and epigenetic modification domains, respectively.

6. The method according to claim 5, wherein the transcriptional repressor domain is selected from: 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, ZNF98, ZNF677, ZNF596, ZNF214, ZNF37A, ZNF34, ZNF250, ZNF547, ZNF273, ZFP82, ZNF224, ZNF33A, ZNF45, ZNF175, ZNF184, ZFP28-1, ZFP28-2, ZNF18, ZNF213, ZNF394, ZFP1, ZFP14, ZNF416, ZNF557, ZNF729, ZNF254, ZNF764, ZNF785, ZNF10, CBX5, RYBP, YAF2, MGA, CBX1, SCMH1, MPP8, SUMO3, HERC2, BIN1, PCGF2, TOX, FOXA1, FOXA2, IRF2BP1, IRF2BP2, IRF2BPLIRF-2BP1_2N-terminal domain, HOXA13, HOXB13, HOXC13, HOXA11, HOXC11, HOXC10, HOXA10, HOXB9, HOXA9, ZFP28, ZN334, ZN568, ZN37A, ZN181, ZN510, ZN862, ZN140, ZN208, ZN248, ZN571, ZN699, ZN726, ZIK1, ZNF2, Z705F, ZNF14, ZN471, ZN624, ZNF84, ZNF7, ZN891, ZN337, Z705G, ZN529, ZN729, ZN419, Z705A, ZN302, ZN486, ZN621, ZN688, ZN33A, ZN554, ZN878, ZN772, ZN224, ZN184, ZN544, ZNF57, ZN283, ZN549, ZN211, ZN615, ZN253, ZN226, ZN730, Z585A, ZN732, ZN681, ZN667, ZN649, ZN470, ZN484, ZN431, ZN382,ZN254,ZN124,ZN607,ZN317,ZN620,ZN141,ZN584,ZN540,ZN75D,ZN555,ZN658,ZN684,RBAK,ZN829,ZN582,ZN112,ZN716,HKR1,ZN350,ZN480,ZN416,ZNF92,ZN100,ZN736,ZNF74,ZN443,ZN195,ZN530,ZN782,ZN791,ZN331,Z354C,ZN157,ZN727,ZN550,ZN793,ZN235,ZN724,ZN573,ZN577,ZN789,ZN718,ZN300,ZN383,ZN429,ZN677,ZN850,ZN454,ZN257,ZN264,ZN485,ZN737,ZNF44,ZN596,ZN565,ZN543,ZFP69,SUMO1,ZNF12,ZN169,ZN433,ZN175,ZN347,ZNF25,ZN519,Z585B,ZN517,ZN846,ZN230,ZNF66,ZN713,ZN816,ZN426,ZN674,ZN627,ZNF20,Z587B,ZN316,ZN233,ZN611,ZN556,ZN234,ZN560,ZNF77,ZN682,ZN614,ZN785,ZN445,ZFP30,ZN225,ZN551,ZN610,ZN528,ZN284,ZN418,ZN490,ZN805,Z780B,ZN763,ZN285,ZNF85,ZN223,ZNF90,ZN557,ZN425,ZN229,ZN606,ZN155,ZN222,ZN442,ZNF91,ZN135,ZN778,ZN534,ZN586,ZN567,ZN440,ZN583,ZN441,ZNF43,ZN589,ZN563,ZN561,ZN136,ZN630,ZN527,ZN333,Z324B,ZN786,ZN709,ZN792,ZN599,ZN613,ZF69B,ZN799,ZN569,ZN564,ZN546,ZFP92,ZN723,ZN439,ZFP57,ZNF19,ZN404,ZN274,CBX3,ZN250,ZN570,ZN675,ZN695,ZN548,ZN132,ZN738,ZN420,ZN626,ZN559,ZN460,ZN268,ZN304,ZN605,ZN844,SUMO5,ZN101,ZN783,ZN417,ZN182,ZN823,ZN177,ZN197,ZN717,ZN669,ZN256,ZN251,CBX4,CDY2,CDYL2,ZN562,ZN461,Z324A,ZN766,ID2,ZN214,CBX7,ID1,CREM,SCX,ASCL1,ZN764,SCML2,TWST1,CREB1,TERF1,ID3,CBX8,GSX1,NKX22,ATF1,TWST2,ZNF17,TOX3,TOX4,ZMYM3,I2BP1,RHXF1,SSX2,I2BPL,ZN680,TRI68,HXA13,PHC3,TCF24,HXB13,HEY1,PHC2,ZNF81,FIGLA,SAM11,KMT2B,HEY2,JDP2,HXC13,ASCL4,HHEX,GSX2,ETV7,ASCL3,PHC1,OTP,I2BP2,VGLL2,HXA11,PDLI4,ASCL2,CDX4,ZN860,LMBL4,PDIP3,NKX25,CEBPB,ISL1,CDX2,PROP1,SIN3B,SMBT1,HXC11,HXC10,PRS6A,VSX1,NKX23,MTG16,HMX3,HMX1,KIF22,CSTF2,CEBPE,DLX2,PPARG,PRIC1,UNC4,BARX2,ALX3,TCF15,TERA,VSX2,HXD12,CDX1,TCF23,ALX1,HXA10,RX,CXXC5,SCML1,NFIL3,DLX6,MTG8,CEBPD,SEC13,FIP1,ALX4,LHX3,PRIC2,MAGI3,NELL1,PRRX1,MTG8R,RAX2,DLX3,DLX1,NKX26,NAB1,SAMD7,PITX3,WDR5,MEOX2,NAB2,DHX8,CBX6,EMX2,CPSF6,HXC12,KDM4B,LMBL3,PHX2A,EMX1,NC2B,DLX4,SRY,ZN777,ZN398,GATA3,BSH,SF3B4,TEAD1,TEAD3,RGAP1,PHF1,GATA2,FOXO3,ZN212,IRX4,ZBED6,LHX4,SIN3A,RBBP7,NKX61,R51A1,MB3L1,DLX5,NOTC1,TERF2,ZN282,RGS12,ZN840,SPI2B,PAX7,NKX62,ASXL2,FOXO1,GATA1,ZMYM5,LRP1,MIXL1,SGT1,LMCD1,CEBPA,SOX14,WTIP, PRP19, NKX11, RBBP4, DMRT2, SMCA2, and their functionally active fragments.

7. The method according to claim 5, wherein the epigenetic modification domain comprises one or more of DNA methyltransferase activity, DNA demethylase activity, DNA deamination activity, DNA amination activity, DNA oxidation activity, DNA helicase activity, histone methyltransferase activity, histone demethylase activity, histone acetyltransferase activity, histone deacetylase activity, histone kinase activity, histone phosphatase activity, histone ubiquitin ligase activity, and histone deubiquitinating activity.

8. The method according to claim 5 or 7, wherein the epigenetic modification domain comprises a DNA methyltransferase and / or a functionally active fragment thereof.

9. The method according to claim 8, wherein the DNA methyltransferase is selected from the group consisting of DNMT3A, DNMT3B, Dnmt3c, DNMT1, DNMT2 and DNMT3L.

10. The method according to claim 8 or 9, wherein the DNA methyltransferase comprises at least one DNMT3A and at least one DNMT3L.

11. The method according to any one of claims 2-10, wherein the DNA binding domain is selected from the group consisting of: a TALE domain, a zinc finger domain, a tetR domain, a large range of nucleases, a Cas nuclease, an Argonaute (Ago) protein, and homologs, modified forms or variants thereof.

12. The method according to any one of claims 2 to 11, wherein the DNA binding domain is capable of specifically recognizing a target sequence on the PCSK9 gene and / or the ANGPTL3 gene.

13. The method according to any one of claims 2 to 12, wherein the DNA binding domain specifically recognizes the target sequence by binding to a guide RNA.

14. The method of any one of claims 2-13, wherein the DNA binding domain is a class II Cas nuclease.

15. The method of claim 11 or 14, wherein the Cas nuclease is selected from class II type II Cas nuclease and class II type V Cas nuclease.

16. The method according to any one of claims 11, 14 and 15, wherein the Cas nuclease is Cas9.

17. The method according to any one of claims 11 and 14-16, wherein the Cas nuclease is a deactivated Cas9 (dCas9).

18. The method according to any one of claims 2 to 17, wherein the epigenetic editing system further comprises a guide RNA, wherein the guide RNA is capable of specifically recognizing a target sequence on the PCSK9 gene and / or the ANGPTL3 gene.

19. The method according to any one of claims 3 to 10, wherein the recruitment domain A is selected from any one of the following two groups of domains, and the recruitment domain A' is selected from any one of the other of the following two groups of domains: 1) general control non-derepressor protein 4 (GCN4), GFP11 fragment derived from split green fluorescent protein (GFP), or GVKESLV polypeptide; and 2) Single-chain Fv (scFv), GFP1-10 fragments derived from split green fluorescent protein (GFP), or PDZ protein domains.

20. The method of claim 19, wherein: 1) one of the recruitment domain A and the recruitment domain A' is GCN4, and the other domain is scFv; or 2) one of the recruitment domain A and the recruitment domain A' is a GFP11 fragment, and the other domain is GFP1-10; or 3) The structure of one of the recruitment domain A and the recruitment domain A' is GVKESLV, and the other one is a PDZ protein domain.

21. The method according to claim 20, wherein: 1) one of the first fusion moiety and the second fusion moiety comprises DNA methyltransferase-dCas9-n×GCN4, and the other comprises a transcriptional repressor domain-scFv or scFv-transcriptional repressor domain; or 2) one of the first fusion moiety and the second fusion moiety comprises DNA methyltransferase-dCas9-scFv, and the other comprises a transcriptional repressor domain-GCN4 or a GCN4-transcriptional repressor domain; or 3) one of the first fusion moiety and the second fusion moiety comprises DNA methyltransferase-dCas9-n×GFP11, and the other comprises a transcriptional repressor domain-GFP1-10 or GFP1-10-transcriptional repressor domain; or 4) one of the first fusion moiety and the second fusion moiety comprises DNA methyltransferase-dCas9-GFP1-10, and the other comprises a transcriptional repressor domain-GFP11 or GFP11-transcriptional repressor domain; Wherein, - indicates that the domains at both ends are directly or indirectly connected in the order from N-terminus to C-terminus; n×GCN4 or n×GFP11 respectively represents n copies of GCN4 connected by a linker sequence or n copies of GFP11 connected by a linker sequence, and n is any integer selected from 1 to 20.

22. The method of claim 20, wherein: 1) one of the first fusion moiety and the second fusion moiety comprises an n×GCN4-dCas9-transcriptional repressor domain, and the other comprises a DNA methyltransferase-scFv or scFv-DNA methyltransferase; or 2) one of the first fusion moiety and the second fusion moiety comprises a scFv-dCas9-transcriptional repressor domain, and the other comprises a DNA methyltransferase-GCN4 or a GCN4-DNA methyltransferase; or 3) one of the first fusion moiety and the second fusion moiety comprises n×GFP11-dCas9-transcriptional repressor domain, and the other comprises DNA methyltransferase-GFP1-10 or GFP1-10-DNA methyltransferase; or 4) one of the first fusion moiety and the second fusion moiety comprises a GFP1-10-dCas9-transcriptional repressor domain, and the other comprises a DNA methyltransferase-GFP11 or a GFP11-DNA methyltransferase; Wherein, - indicates that the domains at both ends are directly or indirectly connected in the order from N-terminus to C-terminus; n×GCN4 or n×GFP11 respectively represents n copies of GCN4 connected by a linker sequence or n copies of GFP11 connected by a linker sequence, and n is any integer selected from 1 to 20.

23. The method according to claim 21 or 22, wherein the complex comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-40.

24. The method according to any one of claims 2 to 23, wherein the nucleic acid is a recombinant expression vector. The method according to claim 24 , wherein the recombinant expression vector is a plasmid or a viral vector.

26. The method according to any one of claims 2 to 25, wherein the nucleic acid comprises the nucleotide sequence shown in any one of SEQ ID NOs: 41 to 160.

27. The method according to any one of claims 2 to 26, wherein the complex or the nucleic acid encoding the complex is disposed in the same or different delivery vehicles.

28. The method of claim 27, wherein the delivery vehicle comprises a liposome and / or a lipid nanoparticle.

29. A complex, wherein the complex is the complex provided by the method according to any one of claims 2 to 28, and the complex is capable of simultaneously regulating the expression and / or activity of PCSK9 gene and ANGPTL3 gene without changing the function of their gene sequences.

30. A nucleic acid encoding the complex of claim 29. A recombinant expression vector comprising the nucleic acid of claim 30 .

32. A delivery vector comprising the complex of claim 29, the nucleic acid of claim 30 and / or the recombinant expression vector of claim 31.

33. A pharmaceutical composition comprising the complex of claim 29, the nucleic acid of claim 30, the recombinant expression vector of claim 31 and / or the delivery vector of claim 32, and at least one pharmaceutically acceptable carrier.

34. The pharmaceutical composition according to claim 33, further comprising a guide RNA, wherein the guide RNA is capable of specifically recognizing a target sequence on the PCSK9 gene and / or the ANGPTL3 gene.

35. A cell comprising the complex of claim 29, the nucleic acid of claim 30, the recombinant expression vector of claim 31, the delivery vector of claim 32, and / or the pharmaceutical composition of claim 33 or 34.

36. A kit comprising the complex of claim 29, the nucleic acid of claim 30, the recombinant expression vector of claim 31, the delivery vector of claim 32, the pharmaceutical composition of claim 33 or 34, and / or the cell of claim 35.

37. A method for simultaneously regulating the expression and / or activity of APOC3 and ANGPTL3 genes, the method comprising providing an epigenetic editing system; the epigenetic editing system comprises a DNA binding domain and a gene expression regulator.

38. The method of claim 37, wherein the epigenetic editing system comprises a complex, wherein the DNA binding domain and the gene expression regulator are contained in the complex; or the epigenetic editing system comprises a nucleic acid encoding the complex.

39. The method of claim 38, wherein the complex comprises a first fusogenic moiety and a second fusogenic moiety; wherein One of the first fusion part and the second fusion part comprises the DNA binding domain, at least one gene expression regulator and the recruitment domain A, and the other of the first fusion part and the second fusion part comprises at least one gene expression regulator and the recruitment domain A', and the recruitment domain A and the recruitment domain A' are capable of interacting with each other.

40. The method according to claim 39, wherein the interaction between the recruitment domain A and the recruitment domain A' enables the gene expression regulator to be recruited to the regulatory region of the APOC3 gene and / or the ANGPTL3 gene or to the vicinity thereof.

41. The method of claim 39 or 40, wherein the gene expression regulator comprised by the first fusion moiety and the second fusion moiety is optionally a transcriptional repressor domain and an epigenetic modification domain, respectively.

42. The method according to claim 41, wherein the transcriptional repressor domain is selected from: 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, ZNF98, ZNF677, ZNF596, ZNF214, ZNF37A, ZNF34, ZNF250, ZNF547, ZNF273, ZFP82, ZNF224, ZNF33A, ZNF45, ZNF175, ZNF184, ZFP28-1, ZFP28-2, ZNF18, ZNF213, ZNF394, ZFP1, ZFP14, ZNF416, ZNF557, ZNF729, ZNF254, ZNF764, ZNF785, ZNF10, CBX5, RYBP, YAF2, MGA, CBX1, SCMH1, MPP8, SUMO3, HERC2, BIN1, PCGF2, TOX, FOXA1, FOXA2, IRF2BP1, IRF2BP2, IRF2BPLIRF-2BP1_2N-terminal domain, HOXA13, HOXB13, HOXC13, HOXA11, HOXC11, HOXC10, HOXA10, HOXB9, HOXA9, ZFP28, ZN334, ZN568, ZN37A, ZN181, ZN510, ZN862, ZN140, ZN208, ZN248, ZN571, ZN699, ZN726, ZIK1, ZNF2, Z705F, ZNF14, ZN471, ZN624, ZNF84, ZNF7, ZN891, ZN337, Z705G, ZN529, ZN729, ZN419, Z705A, ZN302, ZN486, ZN621, ZN688, ZN33A, ZN554, ZN878, ZN772, ZN224, ZN184, ZN544, ZNF57, ZN283, ZN549, ZN211, ZN615, ZN253, ZN226, ZN730, Z585A, ZN732, ZN681, ZN667, ZN649, ZN470, ZN484, ZN431,ZN382,ZN254,ZN124,ZN607,ZN317,ZN620,ZN141,ZN584,ZN540,ZN75D,ZN555,ZN658,ZN684,RBAK,ZN829,ZN582,ZN112,ZN716,HKR1,ZN350,ZN480,ZN416,ZNF92,ZN100,ZN736,ZNF74,ZN443,ZN195,ZN530,ZN782,ZN791,ZN331,Z354C,ZN157,ZN727,ZN550,ZN793,ZN235,ZN724,ZN573,ZN577,ZN789,ZN718,ZN300,ZN383,ZN429,ZN677,ZN850,ZN454,ZN257,ZN264,ZN485,ZN737,ZNF44,ZN596,ZN565,ZN543,ZFP69,SUMO1,ZNF12,ZN169,ZN433,ZN175,ZN347,ZNF25,ZN519,Z585B,ZN517,ZN846,ZN230,ZNF66,ZN713,ZN816,ZN426,ZN674,ZN627,ZNF20,Z587B,ZN316,ZN233,ZN611,ZN556,ZN234,ZN560,ZNF77,ZN682,ZN614,ZN785,ZN445,ZFP30,ZN225,ZN551,ZN610,ZN528,ZN284,ZN418,ZN490,ZN805,Z780B,ZN763,ZN285,ZNF85,ZN223,ZNF90,ZN557,ZN425,ZN229,ZN606,ZN155,ZN222,ZN442,ZNF91,ZN135,ZN778,ZN534,ZN586,ZN567,ZN440,ZN583,ZN441,ZNF43,ZN589,ZN563,ZN561,ZN136,ZN630,ZN527,ZN333,Z324B,ZN786,ZN709,ZN792,ZN599,ZN613,ZF69B,ZN799,ZN569,ZN564,ZN546,ZFP92,ZN723,ZN439,ZFP57,ZNF19,ZN404,ZN274,CBX3,ZN250,ZN570,ZN675,ZN695,ZN548,ZN132,ZN738,ZN420,ZN626,ZN559,ZN460,ZN268,ZN304,ZN605,ZN844,SUMO5,ZN101,ZN783,ZN417,ZN182,ZN823,ZN177,ZN197,ZN717,ZN669,ZN256,ZN251,CBX4,CDY2,CDYL2,ZN562,ZN461,Z324A,ZN766,ID2,ZN214,CBX7,ID1,CREM,SCX,ASCL1,ZN764,SCML2,TWST1,CREB1,TERF1,ID3,CBX8,GSX1,NKX22,ATF1,TWST2,ZNF17,TOX3,TOX4,ZMYM3,I2BP1,RHXF1,SSX2,I2BPL,ZN680,TRI68,HXA13,PHC3,TCF24,HXB13,HEY1,PHC2,ZNF81,FIGLA,SAM11,KMT2B,HEY2,JDP2,HXC13,ASCL4,HHEX,GSX2,ETV7,ASCL3,PHC1,OTP,I2BP2,VGLL2,HXA11,PDLI4,ASCL2,CDX4,ZN860,LMBL4,PDIP3,NKX25,CEBPB,ISL1,CDX2,PROP1,SIN3B,SMBT1,HXC11,HXC10,PRS6A,VSX1,NKX23,MTG16,HMX3,HMX1,KIF22,CSTF2,CEBPE,DLX2,PPARG,PRIC1,UNC4,BARX2,ALX3,TCF15,TERA,VSX2,HXD12,CDX1,TCF23,ALX1,HXA10,RX,CXXC5,SCML1,NFIL3,DLX6,MTG8,CEBPD,SEC13,FIP1,ALX4,LHX3,PRIC2,MAGI3,NELL1,PRRX1,MTG8R,RAX2,DLX3,DLX1,NKX26,NAB1,SAMD7,PITX3,WDR5,MEOX2,NAB2,DHX8,CBX6,EMX2,CPSF6,HXC12,KDM4B,LMBL3,PHX2A,EMX1,NC2B,DLX4,SRY,ZN777,ZN398,GATA3,BSH,SF3B4,TEAD1,TEAD3,RGAP1,PHF1,GATA2,FOXO3,ZN212,IRX4,ZBED6,LHX4,SIN3A,RBBP7,NKX61,R51A1,MB3L1,DLX5,NOTC1,TERF2,ZN282,RGS12,ZN840,SPI2B,PAX7,NKX62,ASXL2,FOXO1,GATA1,ZMYM5,LRP1,MIXL1,SGT1,LMCD1,CEBPA,SOX14, WTIP, PRP19, NKX11, RBBP4, DMRT2, SMCA2, and their functionally active fragments.

43. The method of claim 41, wherein the epigenetic modification domain comprises one or more of DNA methyltransferase activity, DNA demethylase activity, DNA deamination activity, DNA amination activity, DNA oxidation activity, DNA helicase activity, histone methyltransferase activity, histone demethylase activity, histone acetyltransferase activity, histone deacetylase activity, histone kinase activity, histone phosphatase activity, histone ubiquitin ligase activity, and histone deubiquitinating activity.

44. The method according to claim 41 or 43, wherein the epigenetic modification domain comprises a DNA methyltransferase and / or a functionally active fragment thereof.

45. The method of claim 44, wherein the DNA methyltransferase is selected from the group consisting of DNMT3A, DNMT3B, Dnmt3c, DNMT1, DNMT2, and DNMT3L.

46. The method of claim 44 or 45, wherein the DNA methyltransferase comprises at least one DNMT3A and at least one DNMT3L.

47. The method of any one of claims 38-46, wherein the DNA binding domain is selected from the group consisting of a TALE domain, a zinc finger domain, a tetR domain, a meganuclease, a Cas nuclease, an Argonaute (Ago) protein, and homologs, modified forms, or variants thereof.

48. The method according to any one of claims 38 to 47, wherein the DNA binding domain is capable of specifically recognizing a target sequence on the APOC3 gene and / or the ANGPTL3 gene.

49. The method according to any one of claims 38 to 48, wherein the DNA binding domain specifically recognizes the target sequence by binding to a guide RNA.

50. The method of any one of claims 38-49, wherein the DNA binding domain is a Class II Cas nuclease.

51. The method of claim 47 or 50, wherein the Cas nuclease is selected from a class II type II Cas nuclease and a class II type V Cas nuclease.

52. The method of any one of claims 47, 50, and 51, wherein the Cas nuclease is Cas9.

53. The method of any one of claims 47 and 50-52, wherein the Cas nuclease is a deactivated Cas9 (dCas9).

54. The method according to any one of claims 38 to 53, wherein the epigenetic editing system further comprises a guide RNA, wherein the guide RNA is capable of specifically recognizing a target sequence on the APOC3 gene and / or the ANGPTL3 gene.

55. The method of any one of claims 39-46, wherein the recruitment domain A is selected from either of the following two groups of domains, and the recruitment domain A' is selected from either of the other of the following two groups of domains: 1) general control non-derepressor protein 4 (GCN4), GFP11 fragment derived from split green fluorescent protein (GFP), or GVKESLV polypeptide; and 2) Single-chain Fv (scFv), GFP1-10 fragments derived from split green fluorescent protein (GFP), or PDZ protein domains.

56. The method of claim 55, wherein: 1) one of the recruitment domain A and the recruitment domain A' is GCN4, and the other domain is scFv; or 2) one of the recruitment domain A and the recruitment domain A' is a GFP11 fragment, and the other domain is GFP1-10; or 3) The structure of one of the recruitment domain A and the recruitment domain A' is GVKESLV, and the other one is a PDZ protein domain.

57. The method of claim 56, wherein: 1) one of the first fusion moiety and the second fusion moiety comprises DNA methyltransferase-dCas9-n×GCN4, and the other comprises a transcriptional repressor domain-scFv or scFv-transcriptional repressor domain; or 2) one of the first fusion moiety and the second fusion moiety comprises DNA methyltransferase-dCas9-scFv, and the other comprises a transcriptional repressor domain-GCN4 or a GCN4-transcriptional repressor domain; or 3) one of the first fusion moiety and the second fusion moiety comprises DNA methyltransferase-dCas9-n×GFP11, and the other comprises a transcriptional repressor domain-GFP1-10 or GFP1-10-transcriptional repressor domain; or 4) one of the first fusion moiety and the second fusion moiety comprises DNA methyltransferase-dCas9-GFP1-10, and the other comprises a transcriptional repressor domain-GFP11 or GFP11-transcriptional repressor domain; Wherein, - indicates that the domains at both ends are directly or indirectly connected in the order from N-terminus to C-terminus; n×GCN4 or n×GFP11 respectively represents n copies of GCN4 connected by a linker sequence or n copies of GFP11 connected by a linker sequence, and n is any integer selected from 1 to 20.

58. The method of claim 56, wherein: 1) one of the first fusion moiety and the second fusion moiety comprises an n×GCN4-dCas9-transcriptional repressor domain, and the other comprises a DNA methyltransferase-scFv or scFv-DNA methyltransferase; or 2) one of the first fusion moiety and the second fusion moiety comprises a scFv-dCas9-transcriptional repressor domain, and the other comprises a DNA methyltransferase-GCN4 or a GCN4-DNA methyltransferase; or 3) one of the first fusion moiety and the second fusion moiety comprises n×GFP11-dCas9-transcriptional repressor domain, and the other comprises DNA methyltransferase-GFP1-10 or GFP1-10-DNA methyltransferase; or 4) one of the first fusion moiety and the second fusion moiety comprises a GFP1-10-dCas9-transcriptional repressor domain, and the other comprises a DNA methyltransferase-GFP11 or a GFP11-DNA methyltransferase; Wherein, - indicates that the domains at both ends are directly or indirectly connected in the order from N-terminus to C-terminus; n×GCN4 or n×GFP11 respectively represents n copies of GCN4 connected by a linker sequence or n copies of GFP11 connected by a linker sequence, and n is any integer selected from 1 to 20.

59. The method according to claim 57 or 58, wherein the complex comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-40.

60. The method of any one of claims 38-59, wherein the nucleic acid is a recombinant expression vector. The method according to claim 60 , wherein the recombinant expression vector is a plasmid or a viral vector.

62. The method according to any one of claims 38-61, wherein the nucleic acid comprises the nucleotide sequence shown in any one of SEQ ID NOs:41-160.

63. The method of any one of claims 38-62, wherein the complex or the nucleic acid encoding the complex is disposed in the same or different delivery vehicles.

64. The method of claim 63, wherein the delivery vehicle comprises a liposome and / or a lipid nanoparticle.

65. A complex, wherein the complex is the complex provided by the method according to any one of claims 38 to 64, and the complex is capable of simultaneously regulating the expression and / or activity of the APOC3 gene and the ANGPTL3 gene without changing the functions of their gene sequences.

66. A nucleic acid encoding the complex of claim 65.

67. A recombinant expression vector comprising the nucleic acid of claim 66.

68. A delivery vector comprising the complex of claim 65, the nucleic acid of claim 66 and / or the recombinant expression vector of claim 67.

69. A pharmaceutical composition comprising the complex of claim 65, the nucleic acid of claim 66, the recombinant expression vector of claim 67 and / or the delivery vector of claim 68, and at least one pharmaceutically acceptable carrier.

70. The pharmaceutical composition according to claim 69, further comprising a guide RNA, wherein the guide RNA is capable of specifically recognizing a target sequence on the APOC3 gene and / or the ANGPTL3 gene.

71. A cell comprising the complex of claim 65, the nucleic acid of claim 66, the recombinant expression vector of claim 67, the delivery vector of claim 68, and / or the pharmaceutical composition of claim 69 or 70.

72. A kit comprising the complex of claim 65, the nucleic acid of claim 66, the recombinant expression vector of claim 67, the delivery vector of claim 68, the pharmaceutical composition of claim 69 or 70, and / or the cell of claim 71.

73. A method for simultaneously regulating the expression and / or activity of PCSK9 gene and APOC3 gene, the method comprising providing an epigenetic editing system; the epigenetic editing system comprises a DNA binding domain and a gene expression regulator.

74. The method of claim 73, wherein the epigenetic editing system comprises a complex, wherein the DNA binding domain and the gene expression regulator are contained in the complex; or the epigenetic editing system comprises a nucleic acid encoding the complex.

75. The method of claim 74, wherein the complex comprises a first fusogenic moiety and a second fusogenic moiety; wherein One of the first fusion part and the second fusion part comprises the DNA binding domain, at least one gene expression regulator and the recruitment domain A, and the other of the first fusion part and the second fusion part comprises at least one gene expression regulator and the recruitment domain A', and the recruitment domain A and the recruitment domain A' are capable of interacting with each other.

76. The method according to claim 75, wherein the interaction between the recruitment domain A and the recruitment domain A' enables the gene expression regulator to be recruited to the regulatory region of the PCSK9 gene and / or the APOC3 gene or its vicinity.

77. The method of claim 75 or 76, wherein the gene expression regulators comprised by the first fusion moiety and the second fusion moiety are optionally transcriptional repressor domains and epigenetic modification domains, respectively.

78. The method according to claim 77, wherein the transcriptional repressor domain is selected from: 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, ZNF98, ZNF677, ZNF596, ZNF214, ZNF37A, ZNF34, ZNF250, ZNF547, ZNF273, ZFP82, ZNF224, ZNF33A, ZNF45, ZNF175, ZNF184, ZFP28-1, ZFP28-2, ZNF18, ZNF213, ZNF394, ZFP1, ZFP14, ZNF416, ZNF557, ZNF729, ZNF254, ZNF764, ZNF785, ZNF10, CBX5, RYBP, YAF2, MGA, CBX1, SCMH1, MPP8, SUMO3, HERC2, BIN1, PCGF2, TOX, FOXA1, FOXA2, IRF2BP1, IRF2BP2, IRF2BPLIRF-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, PRP19, NKX11, RBBP4, DMRT2, SMCA2, and their functionally active fragments.

79. The method of claim 77, wherein the epigenetic modification domain comprises one or more of DNA methyltransferase activity, DNA demethylase activity, DNA deamination activity, DNA amination activity, DNA oxidation activity, DNA helicase activity, histone methyltransferase activity, histone demethylase activity, histone acetyltransferase activity, histone deacetylase activity, histone kinase activity, histone phosphatase activity, histone ubiquitin ligase activity, and histone deubiquitinating activity.

80. The method of claim 77 or 79, wherein the epigenetic modification domain comprises a DNA methyltransferase and / or a functionally active fragment thereof.

81. The method of claim 80, wherein the DNA methyltransferase is selected from the group consisting of DNMT3A, DNMT3B, Dnmt3c, DNMT1, DNMT2, and DNMT3L.

82. The method of claim 80 or 81, wherein the DNA methyltransferase comprises at least one DNMT3A and at least one DNMT3L.

83. The method of any one of claims 74-82, wherein the DNA binding domain is selected from the group consisting of a TALE domain, a zinc finger domain, a tetR domain, a meganuclease, a Cas nuclease, an Argonaute (Ago) protein, and homologs, modified forms, or variants thereof.

84. The method according to any one of claims 74-83, wherein the DNA binding domain is capable of specifically recognizing a target sequence on the PCSK9 gene and / or the APOC3 gene.

85. The method of any one of claims 74-84, wherein the DNA binding domain specifically recognizes the target sequence by binding to a guide RNA.

86. The method of any one of claims 74-85, wherein the DNA binding domain is a Class II Cas nuclease.

87. The method of claim 83 or 86, wherein the Cas nuclease is selected from a Class II, Type II Cas nuclease and a Class II, Type V Cas nuclease.

88. The method of claims 83, 86 and 87, wherein the Cas nuclease is Cas9.

89. The method of any one of claims 83 and 86-88, wherein the Cas nuclease is a deactivated Cas9 (dCas9).

90. According to the method according to any one of claims 74-89, the epigenetic editing system further comprises a guide RNA, and the guide RNA can specifically recognize the target sequence on the PCSK9 gene and / or the APOC3 gene.

91. The method of any one of claims 75-82, wherein the recruitment domain A is selected from either of the following two groups of domains, and the recruitment domain A' is selected from either of the other of the following two groups of domains: 1) general control non-derepressor protein 4 (GCN4), GFP11 fragment derived from split green fluorescent protein (GFP), or GVKESLV polypeptide; and 2) Single-chain Fv (scFv), GFP1-10 fragments derived from split green fluorescent protein (GFP), or PDZ protein domains.

92. The method of claim 91, wherein: 1) one of the recruitment domain A and the recruitment domain A' is GCN4, and the other domain is scFv; or 2) one of the recruitment domain A and the recruitment domain A' is a GFP11 fragment, and the other domain is GFP1-10; or 3) The structure of one of the recruitment domain A and the recruitment domain A' is GVKESLV, and the other one is a PDZ protein domain.

93. The method of claim 92, wherein: 1) one of the first fusion moiety and the second fusion moiety comprises DNA methyltransferase-dCas9-n×GCN4, and the other comprises a transcriptional repressor domain-scFv or scFv-transcriptional repressor domain; or 2) one of the first fusion moiety and the second fusion moiety comprises DNA methyltransferase-dCas9-scFv, and the other comprises a transcriptional repressor domain-GCN4 or a GCN4-transcriptional repressor domain; or 3) one of the first fusion moiety and the second fusion moiety comprises DNA methyltransferase-dCas9-n×GFP11, and the other comprises a transcriptional repressor domain-GFP1-10 or GFP1-10-transcriptional repressor domain; or 4) one of the first fusion moiety and the second fusion moiety comprises DNA methyltransferase-dCas9-GFP1-10, and the other comprises a transcriptional repressor domain-GFP11 or GFP11-transcriptional repressor domain; Wherein, - indicates that the domains at both ends are directly or indirectly connected in the order from N-terminus to C-terminus; n×GCN4 or n×GFP11 respectively represents n copies of GCN4 connected by a linker sequence or n copies of GFP11 connected by a linker sequence, and n is any integer selected from 1 to 20.

94. The method of claim 92, wherein: 1) one of the first fusion moiety and the second fusion moiety comprises an n×GCN4-dCas9-transcriptional repressor domain, and the other comprises a DNA methyltransferase-scFv or scFv-DNA methyltransferase; or 2) one of the first fusion moiety and the second fusion moiety comprises a scFv-dCas9-transcriptional repressor domain, and the other comprises a DNA methyltransferase-GCN4 or a GCN4-DNA methyltransferase; or 3) one of the first fusion moiety and the second fusion moiety comprises n×GFP11-dCas9-transcriptional repressor domain, and the other comprises DNA methyltransferase-GFP1-10 or GFP1-10-DNA methyltransferase; or 4) one of the first fusion moiety and the second fusion moiety comprises a GFP1-10-dCas9-transcriptional repressor domain, and the other comprises a DNA methyltransferase-GFP11 or a GFP11-DNA methyltransferase; Wherein, - indicates that the domains at both ends are directly or indirectly connected in the order from N-terminus to C-terminus; n×GCN4 or n×GFP11 respectively represents n copies of GCN4 connected by a linker sequence or n copies of GFP11 connected by a linker sequence, and n is any integer selected from 1 to 20.

95. The method of claim 93 or 94, wherein the complex comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-40.

96. The method of any one of claims 74-95, wherein the nucleic acid is a recombinant expression vector.

97. The method according to claim 96, wherein the recombinant expression vector is a plasmid or a viral vector.

98. The method of any one of claims 74-97, wherein the nucleic acid comprises the nucleotide sequence shown in any one of SEQ ID NOs: 41-160.

99. The method of any one of claims 74-98, wherein the complex or the nucleic acid encoding the complex is disposed in the same or different delivery vehicles.

100. The method of claim 99, wherein the delivery vehicle comprises a liposome and / or a lipid nanoparticle.

101. A complex, wherein the complex is the complex provided by the method according to any one of claims 74-100, and the complex is capable of simultaneously regulating the expression and / or activity of PCSK9 gene and APOC3 gene without changing the function of their gene sequences.

102. A nucleic acid encoding the complex of claim 101.

103. A recombinant expression vector comprising the nucleic acid of claim 102.

104. A delivery vector comprising the complex of claim 101, the nucleic acid of claim 102 and / or the recombinant expression vector of claim 103.

105. A pharmaceutical composition comprising the complex of claim 101, the nucleic acid of claim 102, the recombinant expression vector of claim 103 and / or the delivery vector of claim 104, and at least one pharmaceutically acceptable carrier.

106. The pharmaceutical composition of claim 105, further comprising a guide RNA capable of specifically recognizing a target sequence on the PCSK9 gene and / or the APOC3 gene.

107. A cell comprising the complex of claim 101, the nucleic acid of claim 102, the recombinant expression vector of claim 103, the delivery vector of claim 104, and / or the pharmaceutical composition of claim 105 or 106.

108. A kit comprising the complex of claim 101, the nucleic acid of claim 102, the recombinant expression vector of claim 103, the delivery vector of claim 104, the pharmaceutical composition of claim 105 or 106, and / or the cell of claim 107.

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