Conjugates and their uses

Abstract

A complex comprising a first fusion and a second fusion, wherein one of the first fusion and the second fusion comprises a DNA methylation domain and at least one recruitment domain A, and the other fusion comprises a transcriptional repressor domain and at least one recruitment domain A', wherein the recruitment domain A and the recruitment domain A' are capable of interacting with each other such that one of the first fusion and the second fusion, or a portion thereof, can be recruited to the vicinity of the other fusion. Further provided is the use of the complex in the manufacture of a product for silencing expression of a target gene and in the manufacture of a medicament.

Description

[Technical Field]

[0001] The present application relates to the biomedical field, and in particular to a complex used in gene editing and its use. [Background technology]

[0002] Abnormalities in genome epigenetic modifications are closely related to the occurrence and progression of many diseases, including common metabolic disorders, cardiovascular diseases, and cancer. On the one hand, gene epigenetic editing tools can achieve the purpose of regulating the corresponding gene transcription without altering the gene sequence, without causing permanent DNA damage, harmful mutations, or off-target effects. Furthermore, epigenetic therapy can provide better therapeutic effects by simultaneously regulating the activity of multiple genes, thereby addressing the shortcomings of gene therapy and bringing new hope to the treatment of such diseases. On the other hand, the development of gene site-specific modification technologies, especially CRISPR / Cas9 technology, further enables targeted epigenetic editing and transcriptional regulation in the natural chromatin environment.

[0003] Currently, the design of epigenetic modifications and transcriptional regulation is primarily based on the combination of nucleases, with the most effective combination being the combination of engineered defective nucleases (dCas9). The design principle is to fuse various epigenetic regulatory effectors to dCas9, and use the nuclease's targeting and target DNA binding properties to achieve epigenome editing at specific genomic sites. These trans-regulatory domains and proteins function by interfering with RNA polymerase binding or recruiting endogenous transcription complexes to the dCas9 target site in the promoter region. Recent advances in this field include the use of the dCas-SunTag system to regulate the transcriptional repression of target genes by fusion of multiple copies of transcriptional activation or transcriptional repression proteins. For example, Hatada Izuho et al. (PCT / JP2021 / 006498) reported that dCas9-GCN4 fusion proteins were used to recruit fusion proteins containing epigenetic modifiers (e.g., methyltransferases and histone acetyltransferases), transcriptional repression regulators (e.g., ZIM3), and antibodies to suppress the expression of the target gene. Currently, there are only a few studies using transcriptional regulation and epigenetic site-specific modification techniques to treat diseases caused by epigenetic abnormalities in vivo, and existing editing tools have problems such as the suboptimal recruitment effect of the SunTag system, low transcriptional regulation efficiency, and limited scope of target gene modification (e.g., methylation modification). Summary of the Invention

[0004] The present application provides a gene editing complex and its encoding nucleic acid, vector, composition, cell, etc., which can achieve higher regulation efficiency and a broader target gene modification and regulation range compared to existing gene editing methods based on the SunTag mobilization policy. The complex of the present application can also be used in the manufacture of products that suppress the expression of target genes and in the manufacture of pharmaceuticals.

[0005] According to one aspect, the present application provides a complex comprising a first fusion and a second fusion, wherein one of the first and second fusions comprises a DNA methylation domain and at least one recruitment domain A, and the other fusion comprises a transcriptional repressor domain and at least one recruitment domain A', wherein the recruitment domain A and the recruitment domain A' are capable of interacting with each other such that one of the first and second fusions, or a part thereof, can be recruited to the vicinity of the other fusion.

[0006] In some embodiments, the first fusion or the second fusion comprises a nucleic acid binding domain. In some embodiments, the first fusion comprises a DNA methylation domain, a nucleic acid binding domain, and at least one recruitment domain A, and the second fusion comprises a transcriptional repressor domain and at least one recruitment domain A'. In some embodiments, the first fusion comprises, in N-terminus to C-terminus, a DNA methylation domain, a nucleic acid binding domain, and recruitment domain A. In some embodiments, the second fusion comprises, in N-terminal to C-terminal order, a transcriptional repressor domain and a recruitment domain A', or comprises, in N-terminal to C-terminal order, a recruitment domain A' and a transcriptional repressor domain.

[0007] In some embodiments, the first fusion comprises a transcriptional repressor domain, a nucleic acid binding domain, and at least one recruitment domain A, and the second fusion comprises a DNA methylation domain and at least one recruitment domain A'. In some embodiments, the first fusion comprises, in N- to C-terminal order, recruitment domain A, a nucleic acid binding domain, and a transcriptional repressor domain. In some embodiments, the second fusion comprises, in N-terminal to C-terminal order, a DNA methylation domain and a recruitment domain A', or comprises, in N-terminal to C-terminal order, a recruitment domain A' and a DNA methylation domain.

[0008] In some embodiments, the complex is characterized in that: 1) the first fusion comprises, in N-terminal to C-terminal order, a DNA methylation domain, a nucleic acid binding domain, and a recruitment domain A, and the second fusion comprises, in N-terminal to C-terminal order, a transcriptional repressor domain and a recruitment domain A'; or 2) the first fusion comprises, in N-terminal to C-terminal order, a DNA methylation domain, a nucleic acid binding domain, and a recruitment domain A, and the second fusion comprises, in N-terminal to C-terminal order, a recruitment domain A' and a transcriptional repressor domain; or 3) the first fusion comprises, in N-terminal to C-terminal order, a recruitment domain A, a nucleic acid binding domain, and a transcriptional repressor domain, and the second fusion comprises, in N-terminal to C-terminal order, a DNA methylation domain and a recruitment domain A'; or 4) the first fusion comprises, in N-terminal to C-terminal order, a recruitment domain A, a nucleic acid binding domain, and a transcriptional repressor domain, and the second fusion comprises, in N-terminal to C-terminal order, a recruitment domain A' and a DNA methylation domain.

[0009] In some embodiments, the nucleic acid binding domain is a DNA binding domain. In some embodiments, the DNA binding domain is selected from a TALE domain, a zinc finger domain, a tetR domain, a meganuclease, a Cas protein, an Argonaute (Ago) protein, and homologs, modified forms, or variants thereof. In some embodiments, the DNA binding domain is capable of binding to a target sequence in a target locus. In some embodiments, the DNA binding domain is capable of binding to a guide RNA.

[0010] In some embodiments, the guide RNA is capable of specifically recognizing and hybridizing to a target sequence in the target locus. In some embodiments, the DNA binding domain is a Cas protein, and the Cas protein is a class II Cas nuclease. In some embodiments, the Cas protein is selected from a class II type II Cas nuclease and a class II type V Cas nuclease. In some embodiments, the Cas protein is a Cas9 or Cas12 protein.

[0011] In some embodiments, the Cas protein is an inactivated Cas9 (dCas9) protein or an inactivated Cas12 (dCas12) protein. In some embodiments, the DNA-binding domain comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-9, 343, and 344. 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 groups of domains: 1) general control non-depressible protein 4 (GCN4), a GFP11 fragment derived from split green fluorescent protein (GFP), and a GVKESLV polypeptide; and 2) a single-chain antibody (scFv), a GFP1-10 fragment derived from split green fluorescent protein (GFP), and a PDZ protein domain.

[0012] In some embodiments, in the complex, 1) one of the recruitment domain A and the recruitment domain A' is GCN4 and the other domain is an 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) one of the recruitment domain A and the recruitment domain A' is GVKESLV and the other domain is a PDZ protein domain. In some embodiments, the DNA methylation domain comprises at least one DNA methyltransferase or a functionally active fragment thereof. In some embodiments, the DNA methyltransferase is selected from DNMT3A, DNMT3B, DNMT3c, DNMT1, DNMT2, and DNMT3L.

[0013] In some embodiments, the DNA methylation domain comprises at least one DNMT3A and at least one DNMT3L. In some embodiments, the DNA methyltransferase comprises the amino acid sequence set forth in any one of SEQ ID NOs: 19-24. In some embodiments, the DNA methylation domain comprises a DNMT3A-DNMT3L domain or a DNMT3L-DNMT3A domain, where - indicates that the domains are directly or indirectly linked at both ends in N-terminal to C-terminal order.

[0014] In some embodiments, the transcriptional repressor is selected from the group consisting of KRAB, ZIM3, ZNF680, ZNF554, ZNF264, ZNF582, ZNF324, ZNF669, ZNF354A, ZNF82, ZNF595, ZNF419, ZNF566, ZIM2, EHMT2, SUV39H1, ZFPM1, TRIM28, EZH2, MXD1, SID, LSD1, HP1a, HDAC3, HDAC1, PRMT1, SETDB1, hSIRT1, ZNF436, ZNF257, ZNF675, ZNF490, ZNF320, ZNF331, ZNF816, ZNF41, ZNF189, ZNF528, ZNF543, ZNF140, ZNF610, and ZNF350. , ZNF8, ZNF30, ZNF98, ZNF677, ZNF596, ZNF214, ZNF37A, ZNF34, ZNF250, ZNF547, ZNF273, ZFP 82, ZNF224, ZNF33A, ZNF45, ZNF175, ZNF184, ZFP28-1, ZFP28-2, ZNF18, ZNF213, ZNF394, ZFP 1, ZFP14, ZNF416, ZNF557, ZNF729, ZNF254, ZNF764, ZNF785, ZNF10, CBX5, RYBP, YAF2, MGA, C BX1, 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, Z NF7, ZN891, ZN337, Z705G, ZN529, ZN729, ZN419, Z705A, ZN302, ZN486, ZN621, ZN688, ZN33A, ZN554, ZN878, ZN772, Z N224, 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、Selected from one or more of MIXL1, SGT1, LMCD1, CEBPA, SOX14, WTIP, PRP19, NKX11, RBBP4, DMRT2, SMCA2, and functionally active fragments thereof.

[0015] In some embodiments, the transcriptional repressor domain comprises the amino acid sequence set forth in any one of SEQ ID NOs: 25-50.

[0016] In some embodiments, in the complex, 1) one of the first and second fusions comprises a DNA methylation domain-dCas9 or dCas12 or TALE-n×GCN4, and the other fusion comprises a transcriptional repressor domain-scFv, or 2) one of the first and second fusions comprises a DNA methylation domain-dCas9 or dCas12 or TALE-scFv, and the other fusion comprises a transcriptional repressor domain-GCN4, or 3) one of the first and second fusions comprises a DNA methylation domain-dCas9 or dCas12 or TALE-n×GFP11, and the other fusion comprises a transcriptional repressor domain-GFP1-10, or 4) one of the first and second fusions comprises a DNA methylation domain or 5) one of the first and second fusions comprises a DNA methylation domain-dCas9 or dCas12 or TALE-n×GCN4 and the other fusion comprises a scFv-transcriptional repressor domain, or 6) one of the first and second fusions comprises a DNA methylation domain-dCas9 or dCas12 or TALE-scFv and the other fusion comprises a GCN4-transcriptional repressor domain, or 7) one of the first and second fusions comprises a DNA methylation domain-dCas9 or dCas12 or TALE-n×GFP11 and the other fusion comprises a GFP1-10-transcriptional repressor domain, or 8)One of the first and second fusions contains a DNA methylation domain-dCas9 or dCas12 or TALE-GFP1-10, and the other fusion contains a GFP11-transcriptional repressor domain, where - indicates that the domains are linked at both ends, directly or indirectly, from the N-terminus to the C-terminus, and n×GCN4 or n×GFP11 represent n copies of GCN4 linked via a linker sequence or n copies of GFP11 linked via a linker sequence, respectively, and n is any integer selected from 1 to 20.

[0017] In some embodiments, the first fusion product and / or the second fusion product comprises the amino acid sequence set forth in any one of SEQ ID NOs: 51-76, 78-82, 85-93, 103-105, 110-115, 123, 124, 361, and 362. In some embodiments, the complex comprises an amino acid sequence set forth in any one of SEQ ID NOs: 133-142, 153, 154, 158-163, 168, 345, and 346.

[0018] In some embodiments, in the complex, 1) one of the first and second fusions comprises n×GCN4-dCas9 or dCas12 or TALE-transcriptional repressor domain, and the other fusion comprises DNA methylation domain-scFv, or 2) one of the first and second fusions comprises scFv-dCas9 or dCas12 or TALE-transcriptional repressor domain, and the other fusion comprises DNA methylation domain-GCN4, or 3) one of the first and second fusions comprises n×GFP11-dCas9 or dCas12 or TALE-transcriptional repressor domain, and the other fusion comprises DNA methylation domain-GFP1-10, or 4) one of the first and second fusions comprises GFP1-10-dCas or 5) one of the first and second fusions comprises n×GCN4-dCas9 or dCas12 or a TALE-transcriptional repressor domain and the other fusion comprises scFv-DNA methylation domain, or 6) one of the first and second fusions comprises scFv-dCas9 or dCas12 or a TALE-transcriptional repressor domain and the other fusion comprises GCN4-DNA methylation domain, or 7) one of the first and second fusions comprises n×GFP11-dCas9 or dCas12 or a TALE-transcriptional repressor domain and the other fusion comprises GFP1-10-DNA methylation domain, or 8)One of the first and second fusions comprises a GFP1-10-dCas9 or dCas12 or TALE-transcriptional repressor domain, and the other fusion comprises a GFP11-DNA methylation domain, where - indicates that the domains are linked at both ends, directly or indirectly, from the N-terminus to the C-terminus, and n×GCN4 or n×GFP11 represent n copies of GCN4 linked via a linker sequence or n copies of GFP11 linked via a linker sequence, respectively, and n is any integer selected from 1 to 20.

[0019] In some embodiments, the first fusion product and / or the second fusion product comprises the amino acid sequence set forth in any one of SEQ ID NOs: 83, 84, 94-102, 106-109, 116-122, 363, and 364. In some embodiments, the complex comprises an amino acid sequence set forth in any one of SEQ ID NOs: 143-152, 155-157, 164-167, 347, and 348. In some embodiments, the complex further comprises a nuclear localization signal and / or a label domain.

[0020] In another aspect, the present application provides a nucleic acid encoding the conjugate described herein. In some embodiments, the nucleic acid is a recombinant vector. In some embodiments, the recombinant vector further comprises a non-coding region. In some embodiments, the non-coding regions are selected from introns, regulatory elements, promoters, enhancers, termination sequences, and 5' and 3' untranslated regions.

[0021] In some embodiments, the nucleic acid comprises a first nucleic acid fragment encoding the first fusion and a second nucleic acid fragment encoding the second fusion. In some embodiments, wherein the first nucleic acid fragment and the second nucleic acid fragment are linked by a nucleic acid fragment encoding a cleavage peptide. In some embodiments, the truncation peptide is a 2A peptide and / or an IRES. In some embodiments, the 2A peptide is selected from P2A, T2A, E2A, and F2A. In some embodiments, the nucleic acid comprises a nucleic acid sequence set forth in any one of SEQ ID NOs: 169-335 and 349-360.

[0022] In another aspect, the present application provides a delivery vector comprising a complex described herein and / or a nucleic acid described herein, and optionally comprising a liposome and / or a lipid nanoparticle. In another aspect, the present application provides a composition comprising a complex described herein, a nucleic acid described herein, and / or a delivery vector described herein. In another aspect, the present application provides a cell comprising a complex described herein, a nucleic acid described herein, a delivery vector described herein, and / or a composition described herein. In another aspect, the present application provides a kit comprising a complex described herein, a nucleic acid described herein, a delivery vector described herein, a composition described herein, and / or a cell described herein.

[0023] In another aspect, the present application provides a method of modulating expression of a target gene, the method comprising administering a complex described herein, a nucleic acid described herein, a delivery vector described herein, a composition described herein, a cell described herein, and / or a kit described herein. In some embodiments, the method comprises introducing the complex, the nucleic acid, the delivery vector, the composition, the cell, and / or the kit into a cell containing the target gene.

[0024] In some embodiments, the method comprises contacting the complex, the nucleic acid, the delivery vector, and / or the composition with a regulatory element near and / or of the target gene. In some embodiments, the regulatory elements include a core promoter, a proximal promoter, a distal enhancer, a silencer, an insulator element, a boundary element, and / or a locus control region.

[0025] In another aspect, the present application provides a method for treating or alleviating a disease or disorder associated with aberrant target gene expression and / or aberrant target gene activity, the method comprising administering to a subject in need thereof an effective amount of a complex described herein, a nucleic acid described herein, a delivery vector described herein, a composition described herein, a cell described herein, and / or a kit described herein. In another aspect, the present application provides the use of a conjugate described herein, a nucleic acid described herein, a delivery vector described herein, a composition described herein, a cell described herein, and / or a kit described herein, wherein the medicament is in the manufacture of a medicament for treating or alleviating a disease or disorder associated with aberrant target gene expression and / or aberrant target gene activity.

[0026] In another aspect, the present application provides a conjugate described herein, a nucleic acid described herein, a delivery vector described herein, a composition described herein, a cell described herein, or a kit described herein for treating or alleviating a disease or disorder associated with aberrant target gene expression and / or aberrant target gene activity.

[0027] Those skilled in the art will readily appreciate other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the present application may enable those skilled in the art to modify the specific embodiments disclosed without departing from the spirit or scope of the present invention. Correspondingly, the drawings and description herein are illustrative only and not restrictive. [Brief explanation of the drawings]

[0028] Specific features of the present invention are set forth in the appended claims. A better understanding of the features and advantages of the present invention can be obtained by reference to the exemplary embodiments and drawings described in detail below, which are briefly described as follows: [Figure 1] 1 shows a structural diagram of the composite of the present invention. [Figure 2] 1 shows the inhibitory effect of the complex of the present invention on the expression of the PTP1b gene. [Figure 3-4] 1 shows the inhibitory effect of the complex of the present invention on the expression of the PCSK9 gene. DETAILED DESCRIPTION OF THE INVENTION

[0029] Hereinafter, embodiments of the present invention will be described with reference to specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0030] Definition of Terms In this application, the term "recruitment" generally refers to the recruitment between protein molecules, specifically to proteins recruiting other molecules to perform specific biological functions. This recruitment mainly depends on the affinity of intermolecular interactions, and the affinity is usually thought to be related to the spatial structure of protein molecules and is relatively complex. The interaction mechanism may illustratively include, but is not limited to, non-covalent interactions such as hydrogen bonds, ionic interactions, hydrophobic interactions, and van der Waals forces. For example, some proteins can recruit enzymes to catalyze chemical reactions or recruit other proteins to form complexes. These recruitment actions are crucial for many cellular processes, such as signal transduction, DNA replication, and gene expression.

[0031] As used herein, the term "nucleic acid binding domain" generally refers to a portion of a polypeptide or composition that can bind to a specific nucleic acid, and may include a region that contacts nucleic acids, nucleic acids, and / or proteinaceous materials. Examples of nucleic acid binding domains may include, but are not limited to, helix-turn-helix domains, zinc finger domains, leucine zipper (bZIP) domains, winged helix domains, winged helix-turn-helix domains, helix-loop-helix domains, HMG-box domains, Wor3 domains, immunoglobulin domains, B3 domains, TALE domains, and / or domains such as CRISPR / CasX proteins.

[0032] As used herein, the term "DNA-binding domain" generally refers to an independently folded protein domain containing 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 sequence or regulatory sequence) or have a general affinity for DNA. In certain cases, other domains of the DNA-binding domain typically regulate the activity of the DNA-binding domain, and the DNA-binding function may be structural or may include transcriptional regulation, or the two roles may overlap. In certain embodiments of the methods and gene expression regulatory molecules described herein, the DNA-binding domain may comprise a (DNA) nuclease, such as a nuclease that can sequence-specifically target DNA or that can be induced or directed to sequence-specifically target DNA, such as a CRISPR-Cas system, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), or meganuclease. In some embodiments, the DNA-binding domain is a DNA nuclease derived from a CRISPR-Cas system. For example, the DNA nuclease derived from the CRISPR-Cas system is a Cas protein.

[0033] As used herein, the term "TALE DNA-binding domain" or "TALE" refers to a polypeptide comprising one or more TALE repeat domains / units. Naturally occurring TALEs or "wild-type TALEs" are nucleic acid-binding proteins secreted by numerous species of Proteobacteria. TALE polypeptides comprise a nucleic acid-binding domain composed of tandem repeats of highly conserved monomeric polypeptides, which are primarily 33, 34, or 35 amino acids in length and differ from each other primarily at amino acid positions 12 and 13. In a preferred embodiment, the nucleic acid is DNA. As used herein, a TALE polypeptide monomer is used to refer to a highly conserved repeated polypeptide sequence within a TALE nucleic acid-binding domain, and the term "repeated variable dinucleotide" or "RVD" is used to refer to the highly variable amino acids at positions 12 and 13 of the polypeptide monomer. A common representation of a TALE monomer contained within a DNA-binding domain is X. 1-11 -(X 12 X 13 )-X 14-33又は34又は35 where the subscripts indicate the amino acid positions and X represents any amino acid. 12 X 13 indicates the RVD. In some TALE polypeptide monomers, the variable amino acid at position 13 is deleted or absent, and in such monomers, the RVD is composed of a single amino acid. In such cases, the RVD can alternatively be represented as X*, where X is X. 12 and (*) represents X 13 The DNA binding domain contains several repeats of the TALE monomer, and this is shown to be the absence of (X 1-11 -(X 12 X 13 )-X 14-33又は34又は35 ) z where, in a preferred embodiment, z is at least 5 to 40. In a more preferred embodiment, z is at least 10 to 26.

[0034] TALE monomers have nucleotide binding affinities determined by the type of amino acids in their RVDs. For example, polypeptide monomers with RVDs having NI preferentially bind adenine (A), those with RVDs having NG preferentially bind thymine (T), those with RVDs having HD preferentially bind cytosine (C), and those with RVDs having NN preferentially bind adenine (A) and guanine (G). In other embodiments, monomers with RVDs having IG preferentially bind T. Thus, the number and order of polypeptide monomer repeats within the nucleic acid binding domain of a TALE determine its nucleic acid target specificity. In a further embodiment of the present application, monomers with RVDs having NS can recognize all four base pairs and bind to A, T, G, or C. The structure and function of TALEs are further described, for example, in Moscou et al., Science 326:1501 (2009); Boch et al., Science 326:1509-1512 (2009); and Zhang et al., Nature Biotechnology 29:149-153 (2011), which are incorporated by reference in their entireties. The repeat domain of a TALE is involved in binding of the TALE to its homologous target DNA sequence. These repeat units (also called "repeat sequences") exhibit at least some sequence homology with other TALE repeat sequences in naturally occurring TALE proteins. See, for example, US Patent Publication No. 20110301073. TALE binding domains in the context of the present application 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. Thus, engineered DNA-binding proteins (TALEs) are non-naturally occurring proteins. A non-limiting example of a method for engineering DNA-binding proteins is design and selection. Designed DNA-binding proteins are non-naturally occurring proteins whose design and / or composition are determined primarily by rational criteria.Rational design criteria include the application of substitution rules and computational algorithms to process information in information databases that store existing TALE design and binding data. See, e.g., U.S. Patents 6,140,081; 6,453,242; and 6,534,261, and further see WO98 / 53058; WO98 / 53059; WO98 / 53060; WO02 / 016536 and WO03 / 016496 and U.S. Publication No. 20110301073.

[0035] As used herein, "Cas enzyme" is used interchangeably with "Cas protein," "CRISPR protein," "CRISPR enzyme," "CRISPR-Cas protein," "CRISPR-Cas enzyme," "Cas," "CRISPR effector," or "Cas effector protein," and refers to a class of enzymes that are typically complementary to CRISPR sequences and can recognize and cleave specific DNA strands using the CRISPR sequence as a guide. Non-limiting examples of Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also called Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csf1, Csf2, Csf3, Csf4, and / or homologs 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.

[0036] In this application, the term "class II Cas nuclease" generally refers to a class of Cas proteins that perform the recognition and / or cleavage functions in a single protein form, as defined by the updated classification method of CRISPR / Cas loci (Makarova et al., (2015) Nat Rev Microbiol, 13(11):722-36; Shmakov et al., (2015) Mol Cell, 60:385-397).

[0037] In this application, the term "Class II type II Cas nuclease and Class II type V Cas nuclease" generally refers to Class II Cas nuclease, which is a single-protein, RNA-guided endonuclease. Here, Type II and Type V VB Cas nucleases require the cooperation of tracrRNA (trans-activating CRISPR RNA) and crRNA (CRISPR RNA) for normal function, and crRNA and tracrRNA can be artificially combined into a single guide RNA (sgRNA), while Type V VA Cas nucleases require crRNA alone to perform their guide 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 called Cpf1), Cas12b (also called C2c1), Cas12c (also called C2c3), Cas12d (CasY), Cas12e (CasX), Cas12g, Cas12h, Cas12i, C2c1, C2c4, C2c5, C2c8, C2c9, C2c10, Cas14a, Cas14b, Cas14c nuclease and / or TnpB.

[0038] As used herein, 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 and refer to catalytically inactive CRISPR-associated proteins. In one embodiment, the dCas protein comprises one or more mutations in the DNA cleavage domain. In one embodiment, the dCas protein comprises one or more mutations in the RuvC or domain. In one embodiment, the dCas molecule comprises one or more mutations in both the RuvC and HNH domains. In one embodiment, the dCas protein is a fragment of a wild-type Cas protein. In one embodiment, the dCas protein comprises a functional domain derived from a wild-type Cas protein selected from a Reel domain, a bridge helix domain, or a PAM-interacting domain. In one embodiment, the nuclease activity of dCas is reduced by at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% compared to the nuclease activity of the corresponding wild-type Cas protein.

[0039] In this application, the term "capable of binding" is used interchangeably with "bind to," "specifically recognize," "target," etc., and generally refers to the ability of a binding molecule (e.g., a gene expression regulating molecule of the present application) to interact with nucleotides in a target gene or target site, or the binding molecule (e.g., a gene expression regulating molecule of the present application) to have sufficient affinity for a target gene or target site, and such interaction can be achieved by conjugating, coupling, attaching, providing complementarity, providing covalent or non-covalent binding forces, improving binding stability, etc.

[0040] 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 comprise a single-stranded DNA molecule (ssDNA), a single-stranded DNA molecule phosphorylated at the 5' end, a single-stranded DNA molecule hydroxylated at the 5' end, a base fragment complementary to the target gene, and / or having a length of 8 to 35 nt. In some embodiments of this application, the term "guide RNA" refers to an RNA comprising (1) an "activating" nucleotide sequence that binds to a guide RNA-guided endonuclease (e.g., a class II Cas nuclease, e.g., type II, type V, or type VI Cas endonuclease) and activates the RNA-guided endonuclease, and (2) a "targeting" nucleotide sequence that comprises a nucleotide sequence that hybridizes with a target nucleic acid. The "activating" nucleotide sequence and the "targeting" nucleotide sequence can be in separate RNA molecules (e.g., a "dual guide RNA") or in the same RNA molecule (a "single guide RNA," also called sgRNA).

[0041] As used herein, the term "DNA methyltransferase" generally refers to an enzyme that catalyzes the transfer of a methyl group to DNA. Non-limiting examples of DNA methyltransferases include DNMT1, DNMT3A, DNMT3B, and DNMT3L. For example, by DNA methylation, DNA methyltransferases can modify the activity of DNA fragments (e.g., regulate gene expression) without altering the DNA sequence. As described herein, a gene expression regulatory molecule may 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 may be referred to as a "DNA methyltransferase domain." In various embodiments, the DNA methyltransferase domain comprises a variant or homologue of an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to DNMT3A. In various embodiments, the DNA methyltransferase domain comprises a variant or homologue of an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to DNMT3L.

[0042] As used herein, the term "functionally active fragment" generally refers to a fragment having a subregion of a full-length protein or nucleic acid, but which retains or partially retains the biological activity or function of the full-length protein or nucleic acid. For example, a functionally active fragment can retain or partially retain the ability of the full-length protein to bind to another molecule. For example, a functionally active fragment of a DNA methyltransferase can retain or partially retain the biological activity function of the full-length DNA methyltransferase, which catalyzes the transfer of a methyl group to DNA.

[0043] As used herein, the term "transcriptional repressor" generally refers to a substance and / or reagent, such as a protein (e.g., a transcription factor or fragment thereof), that binds to a target nucleic acid sequence and reduces the expression level of a gene product associated with the target nucleic acid sequence. For example, the gene product may be an RNA (e.g., mRNA) transcribed from a gene or a polypeptide translated from an mRNA transcribed from a gene. Typically, an increase or decrease in mRNA levels results in an increase or decrease in the level of the polypeptide translated therefrom. Expression levels can be measured using standard techniques for measuring mRNA or protein. Non-limiting examples of transcriptional repressors include mSin3-interacting domain (SID) protein, methyl-CpG-binding domain 2 (MBD2), MBD3, DNA methyltransferase (DNMT) 1 (DNMT1), DNMT2A, DNMT3A, DNMT3B, DNMT3L, retinoblastoma protein (Rb), methyl-CpG-binding protein 2 (Mecp2), GATA-1 and its cofactor Fog1, regulator of MAT2 (ROM2), Arabidopsis HD2A protein (AtHD2A), lysine-specific demethylase 1 (LSD1), and / or Kruppel-associated box (KRAB).

[0044] As used herein, the term "KRAB," also referred to as a "Kruper-associated box domain" or "Kruppel-associated box domain," typically refers to a transcriptional repressor domain of about 45 to about 75 amino acid residues present in human zinc finger protein transcription factors. In various embodiments, the KRAB domain may comprise 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 the ZIM3KRAB domain or the KOX1KRAB domain.

[0045] As used herein, the term "split green fluorescent protein" generally refers to a polypeptide that can be split and reconstituted to form active green fluorescent protein. As used herein, the term "GCN4" refers to a transcription factor in S. cerevisiae, a "master regulator" of the yeast genome that regulates approximately one-tenth of the yeast genome; it is a highly conserved protein whose mammalian homolog is Activating Transcription factor-4 (ATF4).

[0046] As used herein, the term "PDZ protein" generally refers to a naturally occurring protein that contains a PDZ domain. Exemplary PDZ proteins include CASK, MPPl, DLGl, DLG2, PSD95, NeDLG, TIP-33, SYNla, TIP-43, LDP, LIM, LIMKl, LIMK2, MPP2, NOSl, AF6, PTN_4, prIL16, 41.8kD, KIAA0559, RGS12, KIAA0316, DVL1, TIP-40, TIAMl, MINTl, MAGI-I, MAGI-2, MAGI-3, KIAA0303, CBP, MINT3, TIP-2, KIAA0561, and / or TIP-I.

[0047] As used herein, the term "single-chain antibody" or "scFv (Single Chain Antibody)" generally refers to a single-chain polypeptide comprising one or more antigen-binding sites. Furthermore, although the heavy and light chains of an Fv fragment are encoded by different genes, they can be linked directly or via a peptide. For example, the heavy and light chains can be linked by recombinant methods using a synthetic linker to form a single protein chain (also called a single-chain antibody, or 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 within the term "antibody" and can be used as binding determinants in the design and production of multispecific binding molecules. They can be produced by recombinant techniques or by enzymatic or chemical cleavage of intact antibodies.

[0048] In the present application, the term "directly or indirectly linked" generally refers to the relative terms "directly linked" or "indirectly linked." "Directly linked" generally means directly connected. For example, the direct link may be a case where linked substances (e.g., amino acid sequence segments) are directly connected without a spacer component (e.g., amino acid residues or derivatives thereof) between them. For example, amino acid sequence segment X and another amino acid sequence segment Y are directly linked via an amide bond formed by the C-terminal amino acid of amino acid sequence segment X and the N-terminal amino acid of amino acid sequence segment Y. "Indirectly linked" generally refers to a case where linked substances (e.g., amino acid sequence segments) are indirectly linked with a spacer component (e.g., amino acid residues or derivatives thereof) between them. For example, the spacer component used in the present application may be an amino acid residue having a sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 125 to 132 (SEQ ID NO: 126 is GSG).

[0049] As used herein, the term "nuclear localization sequence" or "NLS" generally refers to a peptide that targets a protein to the cell nucleus. In certain embodiments, the NLS comprises five positively charged basic amino acids. The NLS may be located at any position in the peptide chain. In certain embodiments, the NLS is an SV40-derived NLS. In certain embodiments, the NLS comprises a sequence set forth in any one of SEQ ID NOs: 338-340. In some embodiments, the NLS comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 338-340.

[0050] As used herein, the term "marker" refers to a peptide that can be introduced into an expression vector and used to enable removal and / or purification of the expression product of one or more vector inserts. Such markers are well known in the art and include radiolabeled amino acids or polypeptides linked to a biotin moiety that can be detected by labeled avidin (e.g., streptavidin containing a fluorescent label or enzymatic activity that can be detected by optical or colorimetric methods). Affinity markers are, for example, FLAG, glutathione-S-transferase, maltose-binding protein, cellulose-binding domain, thioredoxin, NusA, mistin, chitin-binding domain, cutinase, AGT, GFP, and other widely used markers used in protein expression and purification systems. Further non-limiting examples of markers for use in polypeptides include, but are not limited to, histidine markers, radioisotopes or radionuclides (e.g., H, C, S, Y, Tc, In, I, Lu, Ho, or Sm), fluorescent markers (e.g., FITC, rhodamine, lanthanoid phosphorus), enzymatic markers (e.g., horseradish peroxidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotin groups, antigenic sites on pendant polypeptides recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, antigenic site markers), and magnetic reagents such as gadolinium chelates.

[0051] As used herein, 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 or their complements in single-, double-, or multi-stranded form. For example, nucleotides may be ribonucleotides, deoxyribonucleotides, or modified versions thereof. For example, nucleotides may be single- and double-stranded DNA, single- and double-stranded RNA, and hybrid molecules comprising mixtures of single- and double-stranded DNA and RNA. For example, nucleotides may include, but are not limited to, any type of RNA, such as mRNA, siRNA, miRNA, sgRNA, and guide RNA, and any type of DNA, genomic DNA, plasmid DNA, minicircle DNA, and any fragment thereof. The term also includes nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, including synthetic, natural, and non-natural nucleic acids.

[0052] As used herein, the terms "sequence encoding ..." or "nucleic acid encoding ..." generally refer to a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence encoding a protein. The coding sequence may further comprise initiation and termination signals operably linked to regulatory elements, including promoters and polyadenylation signals capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered. Codon optimization of the coding sequence may be performed. As used herein, the term "intron" generally refers to a DNA segment that is transcribed but removed from the RNA transcript by splicing one of the sequences (exons) at either end. Introns are considered to be intervening sequences within the protein-coding region of a gene and generally do not contain information about the protein produced by that gene.

[0053] As used herein, the term "recombinant vector" generally refers to a nucleic acid molecule capable of transporting and to which another nucleic acid has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Alternatively, the vector may be linear. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell after introduction into the host cell, and thereby are replicated along with the host genome.

[0054] As used herein, the term "regulatory element" refers to a genetic element capable of controlling the expression of a nucleic acid sequence, such as splicing signals, promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which together provide for the replication, transcription, and translation of a coding sequence in a recipient cell. Not all of these control sequences need be present.

[0055] As used herein, the term "promoter" generally refers to a nucleotide sequence that controls or regulates the transcription of a nucleotide sequence (e.g., a coding sequence) with which it is operably associated. The coding sequence controlled or regulated by a promoter can encode a polypeptide and / or functional RNA. A "promoter" generally refers to a nucleotide sequence that contains an RNA polymerase II binding site and directs the initiation of transcription. A promoter is typically located 5' or upstream of the origin of a coding region relative to the corresponding coding sequence. A promoter may also contain other elements as gene expression regulators, such as a promoter region. In some embodiments, a promoter region may contain at least one intron. Promoters may include constitutive, inducible, temporally-regulated, developmentally-regulated, chemically-regulated, tissue-preferential, and / or tissue-specific promoters, for example, to produce recombinant nucleic acid molecules such as "synthetic nucleic acid constructs" or "protein-RNA complexes." These different types of promoters are known in the art.

[0056] As used herein, the term "enhancer" generally refers to a regulatory DNA sequence, typically 50 to 1500 bp, that can be bound by a protein (activator protein) to stimulate or enhance transcription of a gene or genes. These activator proteins (also called transcription factors) interact with the Mediator complex, recruiting polymerase II and general transcription factors, which then initiate gene transcription. Enhancers are typically cis-acting but can be located upstream or downstream of the start site of a gene or the genes they regulate. Furthermore, enhancers can be oriented in either a forward or reverse direction and do not need to be located near the transcription start site to affect transcription, as some enhancers have been found located hundreds of thousands of base pairs upstream or downstream of the start site. Enhancers can also be found within introns.

[0057] As used herein, the term "cleavage peptide" refers to a class of polypeptides that can achieve the function of a cleaved protein. For example, the cleavage peptide can achieve protein cleavage by ribosomal skipping rather than protease hydrolysis. For example, the cleavage peptide can be a cleavage 2A peptide, including T2A, F2A, P2A, and / or E2A.

[0058] As used herein, the term "delivery vector" generally refers to a transport vehicle capable of delivering a drug (e.g., a nucleic acid molecule) to a target cell. A delivery vector can deliver a drug to a specific cell subset. For example, a delivery vector can be targeted to a specific cell type by its inherent characteristics or by a moiety attached to or contained within the vector (or attached to the vector so that the moiety and the delivery vector are maintained together, and the moiety is sufficient to target the delivery vector). A delivery vector can also improve the in vivo half-life and / or bioavailability of a delivered reagent. Delivery vectors may include viral vectors, virus-like particles, polycationic vectors, peptide vectors, liposomes, and / or hybrid vectors. For example, when the target cell is a hepatocyte, the properties of the delivery vector (e.g., size, charge, and / or pH) can effectively deliver the delivery vector and / or molecules encapsulated therein to the target cell, reduce immune clearance, and / or promote retention within the target cell.

[0059] As used herein, the term "liposome" generally refers to a vesicle having an internal space separated from the external medium by one or more bilayer membranes. In some embodiments, the bilayer membrane can be formed by amphiphilic molecules, such as synthetic or naturally occurring lipids containing spatially separated hydrophilic and hydrophobic domains. In other embodiments, the bilayer membrane can be formed by an amphiphilic polymer and a surfactant. In some embodiments, the liposome is a spherical vesicular structure consisting of a monolayer or multilayer lipid bilayer surrounding an internal aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. In some embodiments, liposomes are biocompatible and nontoxic, and can deliver hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their cargo across biological membranes and the blood-brain barrier (BBB). Liposomes can be made from several different types of lipids, such as phospholipids. Liposomes may contain natural phospholipids and lipids (e.g., 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC)), sphingomyelin, egg phosphatidylcholine, monosialoganglioside, or any combination thereof. Several other additives can be added to the liposomes to modify the structure and properties of the liposomes. For example, the liposomes may further contain, for example, cholesterol, sphingomyelin, and / or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) to improve stability and / or prevent leakage of the cargo inside the liposomes.

[0060] The term "lipid nanoparticle (LNP)" generally refers to a particle comprising multiple (i.e., more than one) lipid molecules physically bound to one another (e.g., covalently or non-covalently) by intermolecular forces. LNPs may be, for example, microspheres (including unilamellar and multilamellar vesicles such as liposomes), the dispersed phase in an emulsion, micelles, or the internal phase in a suspension. LNPs can encapsulate nucleic acids within cationic lipid particles (e.g., liposomes) and deliver them relatively easily to cells. In some instances, lipid nanoparticles do not contain any viral components, thereby minimizing safety and immunogenicity concerns. The lipid particles can be used for in vivo, ex vivo, and in vivo delivery. The lipid particles can also be used for cell populations of various sizes. The LNPs of the present application can be easily produced by various methods known in the art, for example, by mixing an organic phase with an aqueous phase. Mixing of the two phases can be achieved by microfluidic devices and impinging flow reactors. The more thoroughly the organic phase and aqueous phase are mixed, the higher the encapsulation efficiency and particle size distribution of the resulting LNPs. Preferably, the particle size of the LNPs can be adjusted by varying the mixing speed of the organic and aqueous phases. The faster the mixing speed, the smaller the particle size of the LNPs produced. The encapsulation efficiency can be optimized by adjusting the N / P (ionizable lipid / nucleic acid) ratio of the LNP system. In some instances, LNPs can be used to deliver DNA molecules and / or RNA molecules (e.g., mRNA of Cas, sgRNA). In certain cases, LNPs can be used to deliver RNP complexes of Cas / gRNA. In some embodiments, LNPs are used to deliver mRNA and gRNA.

[0061] As used herein, the term "subject" generally refers to an animal, typically a mammal such as a human, non-human primate (monkey, gibbon, gorilla, chimpanzee, orangutan, macaque), livestock (dogs and cats), farm animals (chickens and domestic animals such as ducks, horses, cows, goats, sheep, and pigs), and laboratory animals (mice, rats, rabbits, and guinea pigs). Human subjects include fetal, neonatal, infant, adolescent, and adult subjects. Subjects also include animal disease models, such as mice and other animal models of blood clotting disorders (such as HemA), and other animal models known to those skilled in the art.

[0062] In this application, the term "comprising" generally means including the explicitly specified features but not excluding other elements. As used herein, the term "chosen from" generally means inclusive of the selected object and all combinations thereof. For example, "(chosen from) A, B, and C" means inclusive of all combinations of A, B, and C, such as A, B, C, A+B, A+C, B+C, or A+B+C.

[0063] In this application, the term "about" generally refers to a variation within 0.5% to 10% above or below a specified value, for example, a variation within 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0064] Detailed Description of the Invention According to one aspect, the present application provides a complex comprising a first fusion and a second fusion, wherein one of the first and second fusions comprises a DNA methylation domain and at least one recruitment domain A, and the other fusion comprises a transcriptional repressor domain and at least one recruitment domain A', wherein the recruitment domain A and the recruitment domain A' are capable of interacting with each other such that one of the first and second fusions, or a part thereof, can be recruited to the vicinity of the other fusion.

[0065] In another aspect, the present application provides a nucleic acid encoding the conjugate described herein. For example, the nucleic acid may include DNA and / or mRNA. For example, the nucleic acid may be used to treat or alleviate a disease or disorder associated with abnormal target gene expression and / or abnormal target gene activity. In some embodiments, the nucleic acid is mRNA, and one or more modification techniques may be used to produce a more stable mRNA. Known mRNA modification techniques can be broadly divided into three categories: synthesizing mRNA using artificially synthesized non-natural ribonucleic acids instead of natural ribonucleic acids; adding 5' caps, 3' poly(A) "tails," and UTR (untranslated region) sequences; and effectively protecting mRNA using special novel formulation techniques. Here, a preferred mRNA modification technique is synthesizing mRNA using artificially synthesized non-natural ribonucleic acids instead of natural ribonucleic acids. Chemical modifications on eukaryotic mRNA can be broadly divided into three categories: methylation, pseudouridine (Ψ), and hypoxanthine. For example, the chemical modification may be selected from pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine. For example, the nucleic acid may be a recombinant vector containing a nucleic acid encoding the conjugate described herein. For example, the recombinant vector may be a nucleic acid molecule capable of transporting another nucleic acid linked to it. Recombinant vectors may include nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded, nucleic acid molecules that contain one or more free ends, nucleic acid molecules that have no free ends (e.g., circular), nucleic acid molecules that contain DNA, RNA, or both, and other types of polynucleotides known in the art.For example, viral vectors may be used. Viral vectors may include virally derived DNA or RNA sequences for packaging into viruses (e.g., retroviruses, replication-deficient retroviruses, adenoviruses, replication-deficient adenoviruses, and adeno-associated viruses (AAV)). Viruses and viral vectors can be used for in vitro, ex vivo, and / or in vivo delivery.

[0066] In another aspect, the present application provides a delivery vector comprising the complexes and / or nucleic acids described herein, and optionally comprising liposomes and / or lipid nanoparticles. For example, the delivery vector can be introduced into cells using physical delivery methods. Examples of physical methods include microinjection, electroporation, and hydrodynamic delivery. For example, LNPs can encapsulate nucleic acids within cationic lipid particles (e.g., liposomes) and deliver them relatively easily to cells. In some instances, lipid nanoparticles do not contain any viral components, thereby minimizing safety and immunogenicity issues. Lipid particles can be used for in vivo, ex vivo, and in vivo delivery. Components of LNPs may include cationic lipids, ionized lipids, PEG-2 lipids and / or supporting lipids, and optionally, a cholesterol component.

[0067] In another aspect, the present application provides a composition comprising the complex described herein, the nucleic acid described herein, and / or the delivery vector described herein. For example, the complex, the nucleic acid (or recombinant vector) encoding the complex, and the delivery vector in the composition may be contained simultaneously in one composition, or may be contained in different compositions. For example, when the complex, the nucleic acid (or recombinant vector) encoding the complex, and / or the delivery vector in the composition are used, they may be used simultaneously or separately. In another aspect, the present application provides a cell comprising a complex described herein, a nucleic acid described herein, a delivery vector described herein, and / or a composition described herein.

[0068] In another aspect, the present application provides a kit comprising the complex described herein, the nucleic acid described herein, the delivery vector described herein, the composition described herein, and / or the cell described herein. For example, the kit further comprises at least one container for disposing the components. For example, the kit may further comprise second, third, and / or other containers other than the container that contain one or more of the components and in which the one or more components can be separately disposed. For example, the kit may also dispose various combinations of the components in the containers. For example, the kit may further comprise buffer reagents, mixing devices, measuring devices, sorting devices, and / or labeling devices. For example, the kit may further comprise packaging for housing the various containers. For example, the kit may further comprise instructions for using the components of the kit. For example, the instructions may comprise paper form and / or machine-readable electronic form.

[0069] In another aspect, the present application provides a method for modulating expression of a target gene, the method comprising administering a complex described herein, a nucleic acid described herein, a delivery vector described herein, a composition described herein, a cell described herein, and / or a kit described herein. For example, the method for suppressing expression of the target gene comprises introducing the complex, the nucleic acid, the delivery vector, the composition, the cell, and / or the kit into a cell containing the target gene. For example, the introduction into the cell may be by non-viral or viral-based transfection. For example, the non-viral transfection method includes any suitable method for transfecting cells without using viral DNA or viral particles as a delivery system. Non-limiting examples of non-viral transfection methods include nanoparticle encapsulation of nucleic acid encoding the complex (e.g., lipid nanoparticles, gold nanoparticles, etc.), calcium phosphate transfection, liposome transfection, nucleofection, sonoporation, heat shock transfection, magnetofection, and electroporation. For example, viral-based transfection methods include any viral vector suitable for use in the methods described herein, non-limiting examples of which include retroviral, adenoviral, lentiviral, and / or adeno-associated viral vectors. For example, the method of suppressing expression of a target gene further includes introducing the complex, the nucleic acid, the delivery vector, the composition, the cell, and / or the kit into a cell from an external environment. Also, for example, the method of suppressing expression of a target gene includes contacting the complex, the nucleic acid, the delivery vector, and / or the composition with a transcriptional regulatory element near and / or of the target gene.For example, the contacting refers to contacting the first fusion, second fusion, and guide RNA described herein with a transcriptional regulatory element near a target gene and / or the target gene, and the guide RNA and the fusion comprising a DNA-binding domain form a complex, which specifically recognizes and hybridizes to a specific region of the target gene, and simultaneously the first fusion and the second fusion interact directly or indirectly via their recruitment domains A and A' to be recruited to the vicinity of the DNA-binding domain, thereby regulating the expression of the target nucleic acid. For example, the method includes causing the first fusion, second fusion, and guide RNA described herein to exist in the form of a complex (e.g., an assembled ribonucleoprotein complex) and contacting the complex with a transcriptional regulatory element near a target gene and / or the target gene.

[0070] In another aspect, the present application provides a method for treating or alleviating a disease or disorder associated with aberrant target gene expression and / or aberrant target gene activity, the method comprising administering to a subject in need thereof an effective amount of a complex described herein, a nucleic acid described herein, a delivery vector described herein, a composition described herein, a cell described herein, and / or a kit described herein. For example, the treatment method may comprise mixing the complex, nucleic acid, delivery vector, composition, cell, and / or kit with a therapeutic agent and delivering the mixture to a subject in need thereof systemically to expose the mixture to most of the body, which may be performed by any means known in the art, including, but not limited to, intravenous, intraarterial, subcutaneous, intracavitary, and intraperitoneal delivery. For example, the treatment method may comprise mixing the complex, nucleic acid, delivery vector, composition, cell, and / or kit with a therapeutic agent and delivering the mixture to a subject in need thereof locally to directly reach a target site in the body, which may be performed by, for example, direct injection into a disease site (e.g., a tumor or an inflammatory site) or a target organ (e.g., the liver, heart, pancreas, kidney, etc.). For example, the local delivery includes local administration or local injection techniques, including, but not limited to, intramuscular, subcutaneous, or intradermal injection. For example, the local delivery does not exclude systemic pharmacological effects. For example, the disease includes cardiovascular disease, nonalcoholic steatohepatitis, AMD, age-related macular degeneration, type 2 diabetes, obesity, liver failure, dyslipidemia, diabetic atherosclerosis, and / or hypertension.

[0071] In another aspect, the present application provides use of a conjugate described herein, a nucleic acid described herein, a delivery vector described herein, a composition described herein, a cell described herein, and / or a kit described herein in the manufacture of a medicament for treating or alleviating a disease or disorder associated with aberrant target gene expression and / or aberrant target gene activity. In another aspect, the present application provides a conjugate described herein, a nucleic acid described herein, a delivery vector described herein, a composition described herein, a cell described herein, or a kit described herein for treating or alleviating a disease or disorder associated with aberrant target gene expression and / or aberrant target gene activity.

[0072] First fusion or second fusion In some embodiments, the first and second fusions of the complexes described herein can be generally divided into two cases: (1) one of the two fusions comprises a nucleic acid binding domain, a DNA methylation domain, and a recruitment domain A, while the other fusion comprises a transcriptional repressor domain and a recruitment domain A'; or (2) one of the two fusions comprises a nucleic acid binding domain, a transcriptional repressor domain, and a recruitment domain A, while the other fusion comprises a DNA methylation domain and a recruitment domain A'.

[0073] Specifically, in some embodiments of (1) above, one of the two fusions may comprise, in N- to C-terminal order, a DNA methylation domain, a nucleic acid binding domain, and a recruitment domain A. For example, in some embodiments of (2) above, one of the two fusions may comprise, in N- to C-terminal order, a recruitment domain A, a nucleic acid binding domain, and a transcriptional repressor domain. For example, in some embodiments of (1) above, the other of the two fusions may comprise, in N- to C-terminal order, a transcriptional repressor domain and a recruitment domain A', or a recruitment domain A' and a transcriptional repressor domain, i.e., the binding order of the transcriptional repressor domain and the recruitment domain A' is interchangeable. For example, in some embodiments of (2) above, the other of the two fusions may comprise, in N- to C-terminal order, a DNA methylation domain and a recruitment domain A', or a recruitment domain A' and a DNA methylation domain, i.e., the binding order of the DNA methylation domain and the recruitment domain A' is interchangeable.

[0074] In some more specific embodiments, the nucleic acid-binding domain is a DNA-binding domain. For example, the DNA-binding domain may be selected from a TALE domain, a zinc finger domain, a tetR domain, a meganuclease, a Cas protein, an Argonaute (Ago) protein, and homologs, modified forms, or variants thereof. For example, the DNA-binding domain may be a Cas protein that is a class II Cas nuclease. Furthermore, the Cas protein may be selected from a class II type II Cas nuclease and a class II type V Cas nuclease. For example, the Cas protein may be a Cas9 or Cas12 protein. In certain embodiments, the Cas protein may be an inactivated Cas9 (dCas9) protein or an inactivated Cas12 (dCas12) protein. For example, the DNA-binding domain described herein may comprise, but is not limited to, any one of the amino acid sequences set forth in SEQ ID NOs: 1-9, 343, and 344.

[0075] In some more specific embodiments, the transcriptional repressor is selected from the group consisting of KRAB, ZIM3, ZNF680, ZNF554, ZNF264, ZNF582, ZNF324, ZNF669, ZNF354A, ZNF82, ZNF595, ZNF419, ZNF566, ZIM2, EHMT2, SUV39H1, ZFPM1, TRIM28, EZH2, MXD1, SID, LSD1, HP1a, HDAC3, HDAC1, PRMT1, SETDB1, hSIRT1, ZNF436, ZNF257, ZNF675, ZNF490, ZNF320, ZNF331, ZNF816, ZNF41, ZNF189, ZNF528, ZNF543, ZNF140, ZNF610, ZNF 350, ZNF8, ZNF30, ZNF98, ZNF677, ZNF596, ZNF214, ZNF37A, ZNF34, ZNF250, ZNF547, ZNF273, Z FP82, ZNF224, ZNF33A, ZNF45, ZNF175, ZNF184, ZFP28-1, ZFP28-2, ZNF18, ZNF213, ZNF394, ZF P1, ZFP14, ZNF416, ZNF557, ZNF729, ZNF254, ZNF764, ZNF785, ZNF10, CBX5, RYBP, YAF2, MGA, C BX1, 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, ZNF8 4, ZNF7, ZN891, ZN337, Z705G, ZN529, ZN729, ZN419, Z705A, ZN302, ZN486, ZN621, ZN688, ZN33A, ZN554, ZN878, ZN 772, 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、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、Selected from one or more of LRP1, MIXL1, SGT1, LMCD1, CEBPA, SOX14, WTIP, PRP19, NKX11, RBBP4, DMRT2, SMCA2, and functionally active fragments thereof.

[0076] In some more specific embodiments, the DNA methylation domain comprises at least one DNA methyltransferase or a functionally active fragment thereof. For example, the DNA methyltransferase is selected from DNMT3A, DNMT3B, DNMT3c, DNMT1, DNMT2, and DNMT3L. For example, the DNA methylation domain comprises at least one DNMT3A and at least one DNMT3L. For example, the binding order of the at least one DNMT3A and the at least one DNMT3L is interchangeable. For example, the DNA methylation domain comprises one DNMT3A and one DNMT3L, and the binding order thereof is interchangeable. For example, the DNA methyltransferase comprises the amino acid sequence set forth in any one of SEQ ID NOs: 19 to 24.

[0077] The first fusion complex and the second fusion complex of the present application further form an aggregated complex through interaction between the recruitment domains contained therein. Thus, the present application provides non-limiting examples of combinations of recruitment domain A and recruitment domain A', such as (1) one of the recruitment domain A and the recruitment domain A' is GCN4 and the other domain is an scFv, (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) one of the recruitment domain A and the recruitment domain A' is GVKESLV and the other domain is a PDZ protein domain. Similarly, the case in which GFP11 and GFP1-10 are derived from split GFP (SEQ ID NO: 15) to form the recruitment domain A and the recruitment domain A', respectively, can also be applied to other types of fluorescent proteins, such as mCherry (SEQ ID NO: 16), eYFP (SEQ ID NO: 18), and eCFP (SEQ ID NO: 17). That is, different groups of recruitment domain A and recruitment domain A' can be obtained by splitting mCherry, splitting eYFP, or splitting eCFP for use in the conjugates provided herein. In some embodiments, one of the first fusion and the second fusion of the conjugates of the present application may contain two or more recruitment domains linked via a linker sequence. The amino acid sequence of an exemplary recruitment domain may include any one of SEQ ID NOs: 10-14.

[0078] In summary, the present application can provide the amino acid sequences of the first fusion or second fusion shown below. [Table 1-1] [Table 1-2] [Table 1-3] Table 1-4 Table 1-5 Table 1-6 Table 1-7 Table 1-8 Table 1-9 Table 1-10 Table 1-11 Table 1-12 Table 1-13 Table 1-14 Table 1-15 Table 1-16 Table 1-17 Table 1-18 Table 1-19 Table 1-20 Table 1-21 Table 1-22 Table 1-23 Table 1-24 Table 1-25 Table 1-26 Table 1-27 Table 1-28 Table 1-29 Table 1-30 Table 1-31 Table 1-32 Table 1-33 Table 1-34 Table 1-35 Table 1-36 Table 1-37 Table 1-38 Table 1-39 Table 1-40 Table 1-41 Table 1-42 Table 1-43 Table 1-44 Table 1-45 Table 1-46 Table 1-47 Table 1-48 Table 1-49 Table 1-50 Table 1-51 Table 1-52 Table 1-53 Table 1-54 Table 1-55 Table 1-56 Table 1-57 Table 1-58 Table 1-59 [Table 1-60] [Table 1-61] [Table 1-62] [Table 1-63] [Table 1-64] [Table 1-65] [Table 1-66] [Table 1-67] [Table 1-68]

[0079] Without being bound by any theory, the following examples are intended to merely illustrate the conjugates, methods of preparation, and uses of the present invention, and are not intended to limit the scope of the present invention. [Example]

[0080] Example 1 Design and construction of plasmids containing the complexes of the present application The amino acid sequences containing the HA epitope, the P2A epigenetic modification mobilization system, and the mobilized elements (including Dnmt3a CD, Dnmt3l CD, dSpCas9 or TALE, and KRAB) were optimized and synthesized by Genscript Inc. into nucleic acid sequences suitable for mammalian expression, and then cloned into the pLV-CAG vector containing the CAG promoter and WPRE. The fusion proteins of the mobilized elements and the self-cleaving mobilization system were expressed by the CAG promoter. To optimize the different functional elements, Genscript synthesized them into nucleic acid sequences suitable for mammalian expression. First, the vector was amplified by PCR, excluding the elements requiring substitution. Then, the elements requiring substitution were amplified from a sequence synthesized in-house, and homologous arm sequences were simultaneously introduced. Finally, the different elements were recombined into the vector using the NEBuilder reagent to construct the final expression plasmid.

[0081] Example 2 Inhibitory effect of the complex of the present invention on the expression of the PTP1b gene An editing tool containing the present complex (a complex combining a first fusion having the amino acid sequence shown in SEQ ID NO: 51 and a second fusion having the amino acid sequence shown in SEQ ID NO: 52) was co-transfected with different gRNAs (sg1, sg2, or an equal mixture of sg1 and sg2) targeting the mouse PTP1b gene, gRNA target sequences whose complementary nucleotide sequences are shown in SEQ ID NOs: 336 and 337, and a control gRNA (NTgRNA, the complementary nucleotide sequence of whose target sequence is shown in SEQ ID NO: 365) into the mouse N2a cell line (700 ng of editor + 300 ng of gRNA, 24-well plate). 72 hours after transfection, transfected-positive cells were selected, total RNA was extracted using Trizol, and the relative expression levels of PTP1b were quantified by qPCR. The relative silencing efficiencies of the different tools and different gRNAs were calculated (Figure 2). The results showed that the present complex achieved higher or equivalent repression efficiency for gRNAs targeting the PTP1b gene, i.e., sg1 and / or sg2, compared to a control embodiment (Figure 2, right) in which the nucleic acid binding domain dCas9, the methylation factors DNMT3A and DNMT3L, and the transcriptional repressor KRAB were directly fused (DNMT3A-DNMT3L-dCas9-KRAB).

[0082] Example 3 Inhibitory effect of the complex of the present invention on PCSK9 gene expression Different versions of the tool were transcribed into mRNA in vitro (see the table below for a summary of the tool information measured), then mixed with a chemically synthesized sgRNA at a 1:1 mass ratio (the mass ratio of mRNA to sgRNA for the first and second fusions was 0.5:0.5:1, and the complementary nucleotide sequences of the sgRNA target sequences are shown in SEQ ID NOs: 341 and / or 342). LNPs were then produced (LNP reference: Musunuru, K., Chadwick, A.C., Mizoguchi, T. et al. In vivo CRISPR-based editing of PCSK9 durably lowers cholesterol in primates. Nature 593, 429-434 (2021)). The LNPs were injected into mice via the tail vein at a dose of 4.5 mg / kg body weight. Blood was collected from the cheek of the mice 4 to 10 days after injection, and the PCSK9 protein content in the blood was measured by ELISA. The PBS group was a control group injected with an equal volume of PBS. The detection results of each tool on day 4 after injection are shown in Figure 3. Compared with the PBS control group, all of the tools in each group of the present application showed a significantly significant inhibitory effect on PCSK9 gene expression.

[0083] [Table 2-1] [Table 2-2] [Table 2-3]

[0084] Example 4 Inhibitory effect of the complex of the present invention on PCSK9 gene expression The LNPs prepared using the inhydrotranscribed mRNA (see the table below for information on the tools used) from Example 3, a chemically synthesized sgRNA / mRNA mixture with a mass ratio of 1:1 (sgRNA sequence the same as in Example 3), and the LNP manufacturing method were added to Huh7 cells (1.25 μg / mL). Four days after LNP addition, all cells were harvested, total RNA was extracted using Trizol, and the relative expression level of PCSK9 was quantified by qPCR to calculate the relative inhibitory efficiency of the different tools. The results are shown in Figure 4, where the NC group is a control without LNP addition, demonstrating that the fusions of the present invention have a significant inhibitory effect on PCSK9 gene expression. [Table 3]

Claims

1. A complex comprising a first fusion and a second fusion, a complex in which one of the first and second fusions comprises a DNA methylation domain and at least one recruitment domain A, and the other fusion comprises a transcriptional repressor domain and at least one recruitment domain A', wherein the recruitment domain A and the recruitment domain A' are capable of interacting with each other such that one of the first and second fusions, or a portion thereof, can be recruited to the vicinity of the other fusion.

2. The complex of claim 1 , wherein the first fusion or the second fusion comprises a nucleic acid binding domain.

3. 3. The complex of claim 1 or 2, wherein the first fusion comprises a DNA methylation domain, a nucleic acid binding domain, and at least one recruitment domain A, and the second fusion comprises a transcriptional repressor domain and at least one recruitment domain A'.

4. The complex of any one of claims 1 to 3, wherein the first fusion comprises, in order from N-terminus to C-terminus, a DNA methylation domain, a nucleic acid binding domain, and a recruitment domain A.

5. 5. The complex of claim 1, wherein the second fusion comprises, in N-terminal to C-terminal order, a transcriptional repressor domain and a recruitment domain A', or comprises, in N-terminal to C-terminal order, a recruitment domain A' and a transcriptional repressor domain.

6. 3. The complex of claim 1 or 2, wherein the first fusion comprises a transcriptional repressor domain, a nucleic acid binding domain, and at least one recruitment domain A, and the second fusion comprises a DNA methylation domain and at least one recruitment domain A'.

7. 7. The complex of any one of claims 1, 2 and 6, wherein the first fusion comprises, in N- to C-terminal order, a recruitment domain A, a nucleic acid binding domain and a transcription repressor domain.

8. 8. The complex of any one of claims 1, 2, 6 and 7, wherein the second fusion comprises, in N-terminal to C-terminal order, a DNA methylation domain and a recruitment domain A', or comprises, in N-terminal to C-terminal order, a recruitment domain A' and a DNA methylation domain.

9. 1) the first fusion comprises, in N- to C-terminal order, a DNA methylation domain, a nucleic acid binding domain, and a recruitment domain A, and the second fusion comprises, in N- to C-terminal order, a transcriptional repressor domain and a recruitment domain A'; or 2) the first fusion comprises, in N- to C-terminal order, a DNA methylation domain, a nucleic acid binding domain, and a recruitment domain A, and the second fusion comprises, in N- to C-terminal order, a recruitment domain A' and a transcriptional repressor domain; or 3) the first fusion comprises, in N- to C-terminal order, a recruitment domain A, a nucleic acid binding domain, and a transcriptional repressor domain, and the second fusion comprises, in N- to C-terminal order, a DNA methylation domain and a recruitment domain A'; or 4) The complex according to any one of claims 1 to 8, wherein the first fusion comprises, in order from the N-terminus to the C-terminus, a recruitment domain A, a nucleic acid binding domain, and a transcriptional repressor domain, and the second fusion comprises, in order from the N-terminus to the C-terminus, a recruitment domain A' and a DNA methylation domain.

10. The complex according to any one of claims 2 to 4, 6, 7 and 9, wherein the nucleic acid binding domain is a DNA binding domain.

11. The complex of claim 10, wherein the DNA binding domain is selected from a TALE domain, a zinc finger domain, a tetR domain, a meganuclease, a Cas protein, an Argonaute (Ago) protein, and homologs, modified forms, or variants thereof.

12. 12. The complex of claim 10 or 11, wherein the DNA binding domain is capable of binding to a target sequence in a target locus.

13. The complex of any one of claims 10 to 12, wherein the DNA-binding domain is capable of binding to a guide RNA.

14. 14. The complex of claim 13, wherein the guide RNA is capable of specifically recognizing and hybridizing with a target sequence of the target locus.

15. The complex of any one of claims 10 to 14, wherein the DNA binding domain is a Cas protein, and the Cas protein is a class II Cas nuclease.

16. 16. The complex of claim 15, wherein the Cas protein is selected from a Class II type II Cas nuclease and a Class II type V Cas nuclease.

17. The complex of claim 15 or 16, wherein the Cas protein is a Cas9 or Cas12 protein.

18. The complex according to any one of claims 15 to 17, wherein the Cas protein is an inactivated (dead) Cas9 (dCas9) protein or an inactivated (dead) Cas12 (dCas12) protein.

19. The complex according to any one of claims 10 to 14, wherein the DNA-binding domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 9, 343 and 344.

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

21. 1) one of the recruitment domain A and the recruitment domain A' is GCN4 and the other domain is an scFv; or 2) one of the recruitment domain A and the recruitment domain A' is a GFP11 fragment and the other domain is GFP 1-10; or 3) The complex of claim 20, wherein one of the recruitment domain A and the recruitment domain A' is GVKESLV and the other domain is a PDZ protein domain.

22. The complex of any one of claims 1 to 21, wherein the DNA methylation domain comprises at least one DNA methyltransferase or a functionally active fragment thereof.

23. 23. The complex of claim 22, wherein the DNA methyltransferase is selected from DNMT3A, DNMT3B, DNMT3c, DNMT1, DNMT2 and DNMT3L.

24. The complex of any one of claims 1 to 23, wherein the DNA methylation domain comprises at least one DNMT3A and at least one DNMT3L.

25. The complex according to claim 22 or 23, wherein the DNA methyltransferase comprises an amino acid sequence set forth in any one of SEQ ID NOs: 19 to 24.

26. The complex according to any one of claims 1 to 25, wherein the DNA methylation domain comprises a DNMT3A-DNMT3L domain or a DNMT3L-DNMT3A domain, where - indicates that the domains are linked at both ends, directly or indirectly, in N-terminal to C-terminal order.

27. The transcriptional repressors contain 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, Z NF30, ZNF98, ZNF677, ZNF596, ZNF214, ZNF37A, ZNF34, ZNF250, ZNF547, ZNF273, ZFP82, ZN F224, ZNF33A, ZNF45, ZNF175, ZNF184, ZFP28-1, ZFP28-2, ZNF18, ZNF213, ZNF394, ZFP1, ZF P14, ZNF416, ZNF557, ZNF729, ZNF254, ZNF764, ZNF785, ZNF10, CBX5, RYBP, YAF2, MGA, CBX 1, 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, ZN 891, ZN337, Z705G, ZN529, ZN729, ZN419, Z705A, ZN302, ZN4 86, ZN621, ZN688, ZN33A, ZN554, ZN878, ZN772, ZN224, ZN18 4, ZN544, ZNF57, ZN283, ZN549, ZN211, ZN615, ZN253, ZN226 , ZN730, Z585A, ZN732, ZN681, ZN667, ZN649, ZN470, ZN484,ZN431、ZN382、ZN254、ZN124、ZN607、ZN317、ZN620、ZN141、ZN584、ZN540、ZN75D、ZN555、ZN658、ZN684、RBAK、ZN829、ZN582、ZN112、ZN716、HKR1、ZN350、ZN480、ZN416、ZNF92、ZN100、ZN736、ZNF74、ZN443、ZN195、ZN530、ZN782、ZN791、ZN331、Z354C、ZN157、ZN727、ZN550、ZN793、ZN235、ZN724、ZN573、ZN577、ZN789、ZN718、ZN300、ZN383、ZN429、ZN677、ZN850、ZN454、ZN257、ZN264、ZN485、ZN737、ZNF44、ZN596、ZN565、ZN543、ZFP69、SUMO1、ZNF12、ZN169、ZN433、ZN175、ZN347、ZNF25、ZN519、Z585B、ZN517、ZN846、ZN230、ZNF66、ZN713、ZN816、ZN426、ZN674、ZN627、ZNF20、Z587B、ZN316、ZN233、ZN611、ZN556、ZN234、ZN560、ZNF77、ZN682、ZN614、ZN785、ZN445、ZFP30、ZN225、ZN551、ZN610、ZN528、ZN284、ZN418、ZN490、ZN805、Z780B、ZN763、ZN285、ZNF85、ZN223、ZNF90、ZN557、ZN425、ZN229、ZN606、ZN155、ZN222、ZN442、ZNF91、ZN135、ZN778、ZN534、ZN586、ZN567、ZN440、ZN583、ZN441、ZNF43、ZN589、ZN563、ZN561、ZN136、ZN630、ZN527、ZN333、Z324B、ZN786、ZN709、ZN792、ZN599、ZN613、ZF69B、ZN799、ZN569、ZN564、ZN546、ZFP92、ZN723、ZN439、ZFP57、ZNF19、ZN404、ZN274、CBX3、ZN250、ZN570、ZN675、ZN695、ZN548、ZN132、ZN738、ZN420、ZN626、ZN559、ZN460、ZN268、ZN304、ZN605、ZN844、SUMO5、ZN101、ZN783、ZN417、ZN182、ZN823、ZN177、ZN197、ZN717、ZN669、ZN256、ZN251、CBX4、CDY2、CDYL2、ZN562、ZN461、Z324A、ZN766、ID2、ZN214、CBX7、ID1、CREM、SCX、ASCL1、ZN764、SCML2、TWST1、CREB1、TERF1、ID3、CBX8、GSX1、NKX22、ATF1、TWST2、ZNF17、TOX3、TOX4、ZMYM3、I2BP1、RHXF1、SSX2、I2BPL、ZN680、TRI68、HXA13、PHC3、TCF24、HXB13、HEY1、PHC2、ZNF81、FIGLA、SAM11、KMT2B、HEY2、JDP2、HXC13、ASCL4、HHEX、GSX2、ETV7、ASCL3、PHC1、OTP、I2BP2、VGLL2、HXA11、PDLI4、ASCL2、CDX4、ZN860、LMBL4、PDIP3、NKX25、CEBPB、ISL1、CDX2、PROP1、SIN3B、SMBT1、HXC11、HXC10、PRS6A、VSX1、NKX23、MTG16、HMX3、HMX1、KIF22、CSTF2、CEBPE、DLX2、PPARG、PRIC1、UNC4、BARX2、ALX3、TCF15、TERA、VSX2、HXD12、CDX1、TCF23、ALX1、HXA10、RX、CXXC5、SCML1、NFIL3、DLX6、MTG8、CEBPD、SEC13、FIP1、ALX4、LHX3、PRIC2、MAGI3、NELL1、PRRX1、MTG8R、RAX2、DLX3、DLX1、NKX26、NAB1、SAMD7、PITX3、WDR5、MEOX2、NAB2、DHX8、CBX6、EMX2、CPSF6、HXC12、KDM4B、LMBL3、PHX2A、EMX1、NC2B、DLX4、SRY、ZN777、ZN398、GATA3、BSH、SF3B4、TEAD1、TEAD3、RGAP1、PHF1、GATA2、FOXO3、ZN212、IRX4、ZBED6、LHX4、SIN3A、RBBP7、NKX61、R51A1、MB3L1、DLX5、NOTC1、TERF2、ZN282、RGS12、ZN840、SPI2B、PAX7、NKX62、ASXL2、FOXO1、GATA1、ZMYM5、LRP1、MIXL1、SGT1、LMCD1、The complex according to any one of claims 1 to 26, which is selected from one or more of CEBPA, SOX14, WTIP, PRP19, NKX11, RBBP4, DMRT2, SMCA2, and functionally active fragments thereof.

28. The complex of any one of claims 1 to 27, wherein the transcriptional repressor domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 25 to 50.

29. 1) one of the first and second fusions comprises a DNA methylation domain-dCas9 or dCasl2 or a TALE-nxGCN4, and the other fusion comprises a transcriptional repressor domain-scFv; or 2) one of the first and second fusions comprises a DNA methylation domain-dCas9 or dCasl2 or a TALE-scFv, and the other fusion comprises a transcriptional repressor domain-GCN4; or 3) one of the first and second fusions comprises a DNA methylation domain-dCas9 or dCas12 or a TALE-nxGFP11, and the other fusion comprises a transcriptional repressor domain-GFP1-10; or 4) one of the first and second fusions comprises a DNA methylation domain-dCas9 or dCas12 or a TALE-GFP1-10, and the other fusion comprises a transcriptional repressor domain-GFP11; or 5) one of the first and second fusions comprises a DNA methylation domain-dCas9 or dCasl2 or TALE-nxGCN4, and the other fusion comprises an scFv-transcriptional repressor domain; or 6) one of the first and second fusions comprises a DNA methylation domain-dCas9 or dCasl2 or TALE-scFv, and the other fusion comprises a GCN4-transcriptional repressor domain; or 7) one of the first and second fusions comprises a DNA methylation domain-dCas9 or dCas12 or TALE-nxGFP11, and the other fusion comprises a GFP1-10-transcriptional repressor domain; or 8) one of the first and second fusions comprises a DNA methylation domain-dCas9 or dCas12 or TALE-GFP1-10, and the other fusion comprises a GFP11-transcriptional repressor domain; Here, - indicates that the domains are linked directly or indirectly at both ends in the order from the N-terminus to the C-terminus, and n×GCN4 or n×GFP11 represents n copies of GCN4 linked via a linker sequence or n copies of GFP11 linked via a linker sequence, respectively, and n is any integer selected from 1 to 20. The complex according to any one of claims 1 to 28.

30. The first fusion and / or the second fusion comprises an amino acid sequence set forth in any one of SEQ ID NOs: 51-76, 78-82, 85-93, 103-105, 110-115, 123, 124, 361 and 362. The conjugate of any one of claims 1 to 29.

31. The complex according to any one of claims 1 to 30, comprising an amino acid sequence set forth in any one of SEQ ID NOs: 133 to 142, 153, 154, 158 to 163, 168, 345 and 346.

32. 1) one of the first and second fusions comprises nxGCN4-dCas9 or dCasl2 or a TALE-transcriptional repressor domain, and the other fusion comprises a DNA methylation domain-scFv; or 2) one of the first and second fusions comprises scFv-dCas9 or dCasl2 or TALE-transcriptional repressor domain, and the other fusion comprises DNA methylation domain-GCN4; or 3) one of the first and second fusions comprises nxGFP11-dCas9 or dCas12 or a TALE-transcriptional repressor domain, and the other fusion comprises a DNA methylation domain-GFP1-10; or 4) one of the first and second fusions comprises GFP1-10-dCas9 or dCas12 or a TALE-transcriptional repressor domain, and the other fusion comprises a DNA methylation domain-GFP11; or 5) one of the first and second fusions comprises nxGCN4-dCas9 or dCasl2 or a TALE-transcriptional repressor domain, and the other fusion comprises an scFv-DNA methylation domain; or 6) one of the first and second fusions comprises a scFv-dCas9 or dCasl2 or TALE-transcriptional repressor domain, and the other fusion comprises a GCN4-DNA methylation domain; or 7) one of the first and second fusions comprises nxGFP11-dCas9 or dCas12 or a TALE-transcriptional repressor domain, and the other fusion comprises a GFP1-10-DNA methylation domain; or 8) one of the first and second fusions comprises a GFP1-10-dCas9 or dCas12 or TALE-transcriptional repressor domain, and the other fusion comprises a GFP11-DNA methylation domain; Here, - indicates that the domains are linked directly or indirectly at both ends in the order from the N-terminus to the C-terminus, and n×GCN4 or n×GFP11 represents n copies of GCN4 linked via a linker sequence or n copies of GFP11 linked via a linker sequence, respectively, and n is any integer selected from 1 to 20. The complex according to any one of claims 1 to 28.

33. The conjugate of any one of claims 1 to 28 and 32, wherein the first fusion and / or the second fusion comprises an amino acid sequence set forth in any one of SEQ ID NOs: 83, 84, 94-102, 106-109, 116-122, 363 and 364.

34. The complex according to any one of claims 1 to 28, 32 and 33, comprising an amino acid sequence set forth in any one of SEQ ID NOs: 143 to 152, 155 to 157, 164 to 167, 347 and 348.

35. The complex according to any one of claims 1 to 34, further comprising a nuclear localization signal and / or a label domain.

36. A nucleic acid encoding the complex of any one of claims 1 to 35.

37. 37. The nucleic acid of claim 36, which is a recombinant vector.

38. 38. The recombinant vector of claim 37, further comprising a non-coding region.

39. 39. The non-coding region of claim 38, selected from introns, regulatory elements, promoters, enhancers, termination sequences and 5' and 3' untranslated regions.

40. 40. The nucleic acid of any one of claims 36 to 39, comprising a first nucleic acid fragment encoding the first fusion product and a second nucleic acid fragment encoding the second fusion product.

41. 41. The nucleic acid of claim 40, wherein the first and second nucleic acid fragments are linked by a nucleic acid fragment encoding a cleavage peptide.

42. 42. The nucleic acid of claim 41, wherein the truncation peptide is a 2A peptide and / or an IRES.

43. 43. The nucleic acid of claim 42, wherein the 2A peptide is selected from P2A, T2A, E2A, and F2A.

44. The nucleic acid according to any one of claims 36 to 43, comprising a nucleic acid sequence set forth in any one of SEQ ID NOs: 169 to 335 and 349 to 360.

45. 45. A delivery vector comprising a complex according to any one of claims 1 to 35 and / or a nucleic acid according to any one of claims 36 to 44, and optionally comprising a liposome and / or a lipid nanoparticle.

46. A composition comprising a complex according to any one of claims 1 to 35, a nucleic acid according to any one of claims 36 to 44, and / or a delivery vector according to claim 45.

47. A cell comprising a complex according to any one of claims 1 to 35, a nucleic acid according to any one of claims 36 to 44, a delivery vector according to claim 45, and / or a composition according to claim 46.

48. 48. A kit comprising a complex according to any one of claims 1 to 35, a nucleic acid according to any one of claims 36 to 44, a delivery vector according to claim 45, a composition according to claim 46, and / or a cell according to claim 47.

49. 1. A method for modulating expression of a target gene, comprising:

47. A method comprising administering a complex according to any one of claims 1 to 35, a nucleic acid according to any one of claims 36 to 44, a delivery vector according to claim 45, a composition according to claim 46, a cell according to claim 47 and / or a kit according to claim 48.

50. 50. The method of claim 49, comprising introducing the complex, the nucleic acid, the delivery vector, the composition, the cell, and / or the kit into a cell containing the target gene.

51. 50. The method of claim 49, comprising contacting the complex, the nucleic acid, the delivery vector, and / or the composition with a regulatory element near / or of the target gene.

52. 52. The method of claim 51 , wherein the regulatory elements comprise a core promoter, a proximal promoter, a distal enhancer, a silencer, an insulator element, a boundary element, and / or a locus control region.

53. Use of a complex according to any one of claims 1 to 35, a nucleic acid according to any one of claims 36 to 44, a delivery vector according to claim 45, a composition according to claim 46, a cell according to claim 47 and / or a kit according to claim 48, Use in the manufacture of a medicament for treating or alleviating a disease or disorder associated with abnormal expression and / or abnormal activity of a target gene.

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