WO2024131917A1 - 复合物及其用途 - Google Patents

复合物及其用途 Download PDF

Info

Publication number
WO2024131917A1
WO2024131917A1 PCT/CN2023/140808 CN2023140808W WO2024131917A1 WO 2024131917 A1 WO2024131917 A1 WO 2024131917A1 CN 2023140808 W CN2023140808 W CN 2023140808W WO 2024131917 A1 WO2024131917 A1 WO 2024131917A1
Authority
WO
WIPO (PCT)
Prior art keywords
domain
fusion
complex
nucleic acid
terminus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2023/140808
Other languages
English (en)
French (fr)
Inventor
毛少帅
臧赢
韦翔
罗浩
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Epigenic Therapeutics Inc
Original Assignee
Epigenic Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Epigenic Therapeutics Inc filed Critical Epigenic Therapeutics Inc
Priority to CN202380088637.8A priority Critical patent/CN120418279A/zh
Priority to JP2025537104A priority patent/JP2026500040A/ja
Priority to KR1020257024587A priority patent/KR20250124885A/ko
Priority to EP23906072.6A priority patent/EP4640697A1/en
Priority to AU2023412431A priority patent/AU2023412431A1/en
Publication of WO2024131917A1 publication Critical patent/WO2024131917A1/zh
Priority to US19/245,463 priority patent/US20250319203A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/117—Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C12N9/222—Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes
    • C12N9/226—Class 2 CAS enzyme complex, e.g. single CAS protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y201/00—Transferases transferring one-carbon groups (2.1)
    • C12Y201/01—Methyltransferases (2.1.1)
    • C12Y201/01037—DNA (cytosine-5-)-methyltransferase (2.1.1.37)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
    • C07K2319/81—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor containing a Zn-finger domain for DNA binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/90—Fusion polypeptide containing a motif for post-translational modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • the present application relates to the field of biomedicine, and specifically to a complex for gene editing and its use.
  • Abnormal epigenetic modification of the genome is closely related to the occurrence and development of many diseases, such as common metabolic disorders, cardiovascular diseases and cancers in life.
  • Gene epigenetic editing tools can achieve the purpose of corresponding gene transcription regulation without changing the gene sequence. This process will not cause permanent DNA damage, nor will it produce harmful mutations and off-target effects.
  • epigenetic therapy can provide better therapeutic effects by regulating the activity of multiple genes at the same time, making up for the shortcomings of gene therapy and providing new hope for the treatment of such diseases.
  • the development of gene site-directed modification technology makes it possible to carry out targeted epigenome editing and transcriptional regulation in the natural chromatin environment, especially the development of CRISPR/Cas9 technology.
  • the design of epigenetic modification and transcriptional regulation is mainly based on the combined use of nucleases, among which the combination of engineered defective nucleases (dCas9) is the most effective.
  • the design principle is to fuse various epigenetic regulatory effectors to dCas9, and achieve epigenome editing at specific genomic sites through the targeting and binding characteristics of the nuclease to the target DNA. These trans-regulatory domains and proteins work by hindering the binding of RNA polymerase to the dCas9 target site in the promoter region or recruiting endogenous transcription complexes.
  • a recent breakthrough in this field is the use of a dCas-SunTag system to fuse multiple copies of transcriptional activators or inhibitors to achieve target gene transcription regulation.
  • Hatada Izuho et al. (PCT/JP2021/006498) recruited fusions containing epigenetic modifiers (such as methyltransferases and histone acetyltransferases), transcriptional inhibitors (such as ZIM3) and antibodies through dCas9-GCN4 fusions to inhibit the expression of the target gene regulated by them.
  • epigenetic modifiers such as methyltransferases and histone acetyltransferases
  • transcriptional inhibitors such as ZIM3
  • antibodies through dCas9-GCN4 fusions to inhibit the expression of the target gene regulated by them.
  • the present application provides a compound for gene editing and its encoding nucleic acid, vector, composition, cell and other forms, which can achieve higher regulation efficiency and richer expression than the existing gene editing method based on SunTag recruitment strategy.
  • the modification and regulation range of target genes is rich.
  • the complex of the present application can be used to prepare products for inhibiting the expression of target genes and to prepare drugs.
  • the present application provides a complex comprising a first fusion and a second fusion, wherein the fusion of one of the first fusion and the second fusion comprises a DNA methylation domain and at least one recruitment domain A, and wherein the other fusion comprises a transcription repressor domain and at least one recruitment domain A'; wherein the recruitment domain A and the recruitment domain A' can interact with each other so that the fusion of one of the first fusion and the second fusion or a portion thereof can be recruited to the vicinity of the other fusion.
  • the first fusion or the second fusion comprises a nucleic acid binding domain.
  • the first fusion comprises a DNA methylation domain, a nucleic acid binding domain, and at least one recruitment domain A
  • the second fusion comprises a transcriptional repressor domain and at least one recruitment domain A'.
  • the first fusion comprises a DNA methylation domain, a nucleic acid binding domain, and a recruitment domain A in sequence from the N-terminus to the C-terminus.
  • the second fusion comprises a transcriptional repressor domain and a recruitment domain A' from the N-terminus to the C-terminus, or comprises a recruitment domain A' and a transcriptional repressor domain from the N-terminus to the C-terminus.
  • the first fusion comprises a transcriptional repressor domain, a nucleic acid binding domain, and at least one recruitment domain A
  • the second fusion comprises a DNA methylation domain and at least one recruitment domain A'.
  • the first fusion comprises a recruitment domain A, a nucleic acid binding domain, and a transcription repressor domain in sequence from the N-terminus to the C-terminus.
  • the second fusion comprises a DNA methylation domain and a recruitment domain A' in sequence from the N-terminus to the C-terminus, or comprises a recruitment domain A' and a DNA methylation domain in sequence from the N-terminus to the C-terminus.
  • the complex is characterized in that: 1) the first fusion comprises a DNA methylation domain, a nucleic acid binding domain and a recruitment domain A in sequence from the N-terminus to the C-terminus, and the second fusion comprises a transcription repressor domain and a recruitment domain A' in sequence from the N-terminus to the C-terminus; or 2) the first fusion comprises a DNA methylation domain, a nucleic acid binding domain and a recruitment domain A in sequence from the N-terminus to the C-terminus, and the second fusion comprises a recruitment domain A' and a transcription repressor domain in sequence from the N-terminus to the C-terminus; or 3) the first fusion comprises a recruitment domain A, a nucleic acid binding domain and a transcription repressor domain in sequence from the N-terminus to the C-terminus, and the second fusion comprises a DNA methylation domain and a recruitment domain A' in sequence from the N-terminus to the C-terminus; or
  • the nucleic acid binding domain is a DNA binding domain.
  • 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.
  • the DNA binding domain is capable of binding to a target sequence of a target locus.
  • the DNA binding domain is capable of binding to a guide RNA.
  • the guide RNA is capable of specifically recognizing and hybridizing to a target sequence of the target locus.
  • the DNA binding domain is a Cas protein
  • the Cas protein is a class II Cas nuclease
  • the Cas protein is selected from a class II, type II Cas nuclease and a class II, type V Cas nuclease.
  • the Cas protein is a Cas9 or Cas12 protein.
  • the Cas protein is an inactivated Cas9 (dCas9) protein or an inactivated Cas12 (dCas12) protein.
  • the DNA binding domain comprises an amino acid sequence shown in any one of SEQ ID NO:1-9, 343 and 344.
  • the recruitment domain A is selected from any one of the following two groups of domains
  • the recruitment domain A' is selected from any one of the other of the following two groups of domains: 1) general control non-derepressor protein 4 (GCN4), GFP11 fragment derived from split green fluorescent protein (GFP), and GVKESLV polypeptide; and 2) single-chain antibody (scFv), GFP1-10 fragment derived from split green fluorescent protein (GFP), and PDZ protein domain.
  • GCN4 general control non-derepressor protein 4
  • scFv single-chain antibody
  • the domain of one of the recruitment domain A and the recruitment domain A’ is GCN4, and the other domain is scFv; or 2) the domain of one of the recruitment domain A and the recruitment domain A’ is a GFP11 fragment, and the other domain is GFP1-10; or 3) the domain of one of the recruitment domain A and the recruitment domain A’ is GVKESLV, and the other domain is a PDZ protein domain.
  • the DNA methylation domain comprises at least one DNA methyltransferase or a functionally active fragment thereof.
  • the DNA methyltransferase is selected from DNMT3A, DNMT3B, DNMT3c, DNMT1, DNMT2, and DNMT3L.
  • the DNA methylation domain comprises at least one DNMT3A and at least one DNMT3L.
  • the DNA methyltransferase comprises the amino acid sequence shown in any one of SEQ ID NOs: 19-24.
  • the DNA methylation domain comprises a DNMT3A-DNMT3L domain or a DNMT3L-DNMT3A domain; wherein, - indicates that the domains at both ends are directly or indirectly connected in the order from N-terminus to C-terminus.
  • the transcriptional repressor is selected from one or more of the following domains: KRAB, ZIM3, ZNF680, ZNF554, ZNF264, ZNF582, ZNF324, ZNF669, ZNF354A, ZNF82, ZNF595, ZNF419, ZNF566, ZIM2, EHMT2, SUV39H1, ZFPM1, TRIM28, EZH2, MXD1, SID, LSD1, HP1a, HDAC3, HDAC1, PRMT1, SETDB1, hSIRT1, ZNF436, ZNF257, ZNF675, ZNF490, ZNF320, ZNF331, ZNF816, ZNF41, ZNF189, ZNF528, ZNF543, ZNF140, ZNF610, ZNF350 , ZNF8, ZNF30, ZNF98, ZNF677, ZNF596, ZNF214, ZNF37A, ZNF34, ZNF250, ZNF547, ZNF273, ZFP 82, ZNF224, ZNF33A
  • the transcription repressor domain comprises the amino acid sequence shown in any one of SEQ ID NOs: 25-50.
  • the fusion of one of the first fusion and the second fusion comprises a DNA methylation domain-dCas9 or dCas12 or TALE-n ⁇ GCN4, and the other fusion comprises a transcriptional repressor domain-scFv; or 2) the fusion of one of the first fusion and the second fusion comprises a DNA methylation domain-dCas9 or dCas12 or TALE-scFv, and the other fusion comprises a transcriptional repressor domain-GCN4; or 3) the fusion of one of the first fusion and the second fusion comprises a DNA methylation domain-dCas9 or dCas12 or TALE-n ⁇ GFP11, and the other fusion comprises a transcriptional repressor domain- domain-GFP1-10; or 4) the fusion of one of the first fusion and the second fusion comprises a DNA methylation domain-dCas9 or dCas12 or
  • the first fusion and/or the second fusion comprises an amino acid sequence as described in any of SEQ ID NOs: 51-76, 78-82, 85-93, 103-105, 110-115, 123, 124, 361 and 362.
  • the complex comprises the amino acid sequence shown in any one of SEQ ID NO:133-142, 153, 154, 158-163, 168, 345 and 346.
  • the fusion of one of the first fusion and the second fusion comprises n ⁇ GCN4-dCas9 or dCas12 or TALE-transcriptional repressor domain, and the other fusion comprises DNA methylation domain-scFv; or 2) the fusion of one of the first fusion and the second fusion comprises scFv-dCas9 or dCas12 or TALE-transcriptional repressor domain, and the other fusion comprises DNA methylation domain-GCN4; or 3) the fusion of one of the first fusion and the second fusion comprises n ⁇ GFP11-dCas9 or dCas12 or TALE-transcriptional repressor domain, and the other fusion comprises DNA methylation domain-GFP1-10; or 4) the first fusion and the second One of the fusions comprises GFP1-10-dCas9 or dCas12 or TALE-transcriptional repressor domain,
  • the first fusion and/or the second fusion comprises the amino acid sequence shown in any one of SEQ ID NO:83, 84, 94-102, 106-109, 116-122, 363 and 364.
  • the complex comprises the amino acid sequence shown in any one of SEQ ID NO:143-152, 155-157, 164-167, 347 and 348.
  • the complex further comprises a nuclear localization signal and/or a marker domain.
  • the present application provides a nucleic acid encoding the complex described in the present application.
  • the nucleic acid is a recombinant vector.
  • the recombinant vector further comprises a non-coding region.
  • the non-coding region is selected from the group consisting of introns, regulatory elements, promoters, enhancers, termination sequences, and 5' and 3' untranslated regions.
  • the nucleic acid comprises a first nucleic acid segment encoding the first fusion, and a second nucleic acid segment encoding the second fusion.
  • the first nucleic acid fragment is connected to the second nucleic acid fragment via a nucleic acid fragment encoding a cleavage peptide.
  • the cleavage peptide is a 2A peptide and/or an IRES.
  • the 2A peptide is selected from P2A, T2A, E2A, and F2A.
  • the nucleic acid comprises a nucleic acid sequence shown in any one of SEQ ID NO: 169-335 and 349-360.
  • the present application provides a delivery vector, comprising the complex described herein and/or the nucleic acid described herein, and optionally comprising a liposome and/or a lipid nanoparticle.
  • the present application provides a composition comprising the complex described herein, the nucleic acid described herein, and/or the delivery vector described herein.
  • the present application provides a cell, wherein the cell comprises the complex described herein, the nucleic acid described herein, the delivery vector described herein, and/or the composition described herein.
  • 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.
  • the present application provides a method for regulating target gene expression, the method comprising administering the complex described in the present application, the nucleic acid described in the present application, the delivery vector described in the present application, the composition described in the present application, the cell described in the present application and/or the kit described in the present application.
  • 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.
  • the method includes contacting the complex, the nucleic acid, the delivery vehicle, and/or the composition with a regulatory element near the target gene and/or the target gene.
  • 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.
  • the present application provides a method for treating or alleviating a disease or condition associated with abnormal target gene expression and/or abnormal target gene activity, the method comprising administering to a subject in need thereof an effective amount of the complex described herein, the nucleic acid described herein, the delivery vector described herein, the composition described herein, the cell described herein and/or the kit described herein.
  • the present application provides the use of the complex described herein, the nucleic acid described herein, the delivery vector described herein, the composition described herein, the cell described herein and/or the kit described herein for preparing a drug, wherein the drug is used to treat or alleviate a disease or its condition associated with abnormal target gene expression and/or abnormal target gene activity.
  • the present application provides the complex described herein, the nucleic acid described herein, the delivery vector described herein, the composition described herein, the cell described herein or the kit described herein, which is used to treat or alleviate diseases or symptoms associated with abnormal target gene expression and/or abnormal target gene activity.
  • FIG. 1 is a schematic diagram showing the structure of the composite of the present application.
  • FIG. 2 shows the inhibitory effect of the complex of the present application on the expression of the PTP1b gene.
  • FIG3 and FIG4 show the inhibitory effect of the complex of the present application on PCSK9 gene expression.
  • the term "recruitment” generally refers to the recruitment effect between protein molecules, which specifically refers to the recruitment of other molecules by proteins to perform specific biological functions.
  • This recruitment effect mainly depends on the affinity of intermolecular interactions, and its affinity is generally considered to be related to the spatial structure of protein molecules, which is relatively complex.
  • the interaction mechanism can illustratively include but is not limited to non-covalent bonds such as hydrogen bonds, ionic interactions, hydrophobic interactions, and van der Waals forces.
  • some proteins can recruit enzymes to catalyze chemical reactions, or recruit other proteins to form complexes. These recruitment effects are crucial for many cellular processes, such as signal transduction, DNA replication, and gene expression.
  • nucleic acid binding domain generally refers to a portion of a polypeptide or composition that is capable of binding to a specific nucleic acid, which may include a region that contacts a nucleic acid, a nucleic acid, and/or a protein substance.
  • 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 of CRISPR/CasX proteins, etc.
  • bZIP leucine zipper
  • DNA binding domain generally refers to an independently folded protein domain containing at least one motif that recognizes double-stranded or single-stranded DNA.
  • the DNA binding domain can recognize a specific DNA sequence (recognition or regulation sequence) or have a general affinity for DNA.
  • other domains of the DNA binding domain generally regulate the activity of the DNA binding domain; the DNA binding function can be structural or include transcriptional regulation, and sometimes these two effects overlap.
  • the DNA binding domain may include a (DNA) nuclease, such as a nuclease that can target DNA in a sequence-specific manner or can be guided or instructed to target DNA in a sequence-specific manner, such as a CRISPR-Cas system, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN) or a large range of nucleases.
  • a DNA binding domain is a DNA nuclease derived from the CRISPR-Cas system.
  • the DNA nuclease derived from the CRISPR-Cas system is a Cas protein.
  • TALE DNA binding domain or "TALE” is a polypeptide comprising one or more TALE repeat domains/units.
  • Naturally occurring TALE or wild-type TALE is a nucleic acid binding protein secreted by numerous species of proteobacteria.
  • the TALE polypeptide contains a nucleic acid binding domain composed of tandem repeats of highly conserved monomeric polypeptides, which are mainly 33, 34 or 35 amino acids in length and mainly differ from each other in amino acid positions 12 and 13.
  • the nucleic acid is DNA.
  • the polypeptide monomer of TALE is used to refer to a highly conserved repeat polypeptide sequence within the TALE nucleic acid binding domain, and the term "repeat variable diresidue” or “RVD” is used to refer to highly variable amino acids at positions 12 and 13 of the polypeptide monomer.
  • the general representation of the TALE monomer contained in the DNA binding domain is X 1-11 -(X 12 X 13 )-X 14-33 or 34 or 35 , wherein the subscript indicates the amino acid position, and X represents any amino acid.
  • X 12 X 13 indicates RVD.
  • the variable amino acid at position 13 is missing or absent, and in such monomers, the RVD consists of a single amino acid.
  • the RVD may alternatively be represented as X*, where X represents X12 , and (*) indicates that X13 is absent.
  • the DNA binding domain comprises several repeats of the TALE monomer, and this may be represented as ( X1-11- ( X12X13 )-X14-33 or 34 or 35 ) z , where in preferred embodiments, z is at least 5-40. In further preferred embodiments, z is at least 10-26.
  • TALE monomers have nucleotide binding affinity determined by the amino acid type within their RVD.
  • a polypeptide monomer with an RVD of NI preferentially binds to adenine (A)
  • a polypeptide monomer with an RVD of NG preferentially binds to thymine (T)
  • a polypeptide monomer with an RVD of HD preferentially binds to cytosine (C)
  • a monomer with an RVD of NN preferentially binds to adenine (A) and guanine (G).
  • a monomer with an RVD of IG preferentially binds to T.
  • the number and order of polypeptide monomer repeats in the nucleic acid binding domain of TALE determine its nucleic acid target specificity.
  • a monomer with an RVD of NS recognizes all four base pairs and can bind to A, T, G or C.
  • the structure and function of TALE are further described, for example, in Moscou et al., Science 326: 1501 (2009); Boch et al., Science 326: 1509-1512 (2009); and Zhang et al., Nature Biotechnology 29: 149-153 (2011), each of which is incorporated by reference in its entirety.
  • the repeat domain of TALE participates in the binding of TALE to its cognate target DNA sequence.
  • TALE Transcription activator-like effector
  • TALE Transcription activator-like effector
  • Non-limiting examples of methods for engineering DNA binding proteins are design and selection.
  • the designed DNA binding proteins are non-naturally occurring proteins whose design and/or composition are primarily derived from rational criteria.
  • Rational design criteria include the application of substitution rules and computerized algorithms for processing information in information databases storing existing TALE design and binding data. See, for example, U.S. Patents 6,140,081; 6,453,242; and 6,534,261; See also WO 98/53058; WO 98/53059; WO 98/53060; WO02/016536 and WO 03/016496 and US Publication No. 20110301073.
  • Cas enzyme can be used interchangeably with “Cas protein”, “CRISPR protein”, “CRISPR enzyme”, “CRISPR-Cas protein”, “CRISPR-Cas enzyme”, “Cas”, “CRISPR effector” or “Cas effector protein”, which generally refers to a class of enzymes that are complementary to the CRISPR sequence and can use the CRISPR sequence as a guide to recognize and cut a specific DNA strand.
  • Cas proteins include: Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and/or homologs thereof, or modified forms thereof.
  • These proteins are known, for example, the amino acid sequence of the Streptococcus pyogenes Cas9 protein can be found in
  • Class II Cas nuclease generally refers to a class of Cas proteins that perform recognition and/or cleavage functions in the form of a single protein, as defined by the updated classification scheme for CRISPR/Cas loci (Makarova et al., (2015) Nat Rev Microbiol, 13(11):722-36; Shmakov et al., (2015) Mol Cell, 60:385-397).
  • Class II Type II Cas nuclease and Class II Type V Cas nuclease generally refers to the single-protein, RNA-guided endonucleases in Class II Cas nucleases.
  • the V-B type Cas nucleases in Type II and Type V require the joint action of tracrRNA (trans-activating CRISPR RNA) and crRNA (CRISPR RNA) to function normally, and crRNA and tracrRNA can be artificially combined into a single guide RNA (sgRNA);
  • sgRNA single guide RNA
  • the V-A type Cas nuclease in Type V requires the use of crRNA alone to perform the guide function.
  • Class II type II Cas nucleases include Cas9 and its family-related nucleases
  • Class II type V Cas nucleases include Cas12a (also known as Cpf1), Cas12b (also known as C2c1), Cas12c (also known as C2c3), Cas12d (CasY), Cas12e (CasX), Cas12g, Cas12h, Cas12i, C2c1, C2c4, C2c5, C2c8, C2c9, C2c10, Cas14a, Cas14b, Cas14c nuclease and/or TnpB.
  • the term “dCas” may refer to a dCas protein or a fragment thereof.
  • “dCas9” may refer to a dCas9 protein or a fragment thereof.
  • the terms “iCas” and “dCas” are used interchangeably to refer to a CRISPR-associated protein without catalytic activity.
  • the dCas protein comprises one or more mutations in the DNA cleavage domain.
  • the dCas protein comprises one or more mutations in the RuvC or HNH domain.
  • the dCas molecule comprises one or more mutations in both the RuvC and HNH domains.
  • the dCas protein is a fragment of a wild-type Cas protein. In one embodiment, the dCas protein comprises a fragment from a wild-type Cas protein. , wherein the functional domain is selected from a Reel domain, a bridge helix domain or a PAM interaction domain. In one embodiment, the nuclease activity of 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.
  • the term “capable of binding” can be used interchangeably with “binding to”, “specifically identifying”, “targeting”, etc., and generally refers to that the binding molecule (for example, the gene expression regulatory molecule of the present application) is able to interact with the nucleotides on the target gene or target site, or the binding molecule (for example, the gene expression regulatory molecule of the present application) has sufficient affinity for the target gene or target site, and this interaction can be through conjugation, coupling, attachment, providing complementarity, providing covalent force or providing non-covalent force, increasing binding stability, etc.
  • guide RNA guide DNA
  • guide DNA guide DNA
  • gRNA a DNA molecule that can guide a nuclease (e.g., Argonaute, or Ago) to bind to and/or cleave a target gene.
  • the guide DNA may include: a single-stranded DNA molecule (ssDNA), a single-stranded DNA molecule that is phosphorylated at the 5' end, a single-stranded DNA molecule that is hydroxylated at the 5' end, a base fragment that can be complementary to the target gene and/or a length of 8-35nt.
  • ssDNA single-stranded DNA molecule
  • ssDNA single-stranded DNA molecule that is phosphorylated at the 5' end
  • a single-stranded DNA molecule that is hydroxylated at the 5' end a base fragment that can be complementary to the target gene and/or a length of 8-35nt.
  • guide RNA refers to an RNA comprising: (1) an "activation" nucleotide sequence that binds to a guide RNA-guided endonuclease (e.g., a class II Cas nuclease, such as a type II, type V or type VI Cas endonuclease) and activates the RNA-guided endonuclease; and (2) a "target" nucleotide sequence comprising a nucleotide sequence that hybridizes with a target nucleic acid.
  • a guide RNA-guided endonuclease e.g., a class II Cas nuclease, such as a type II, type V or type VI Cas endonuclease
  • the "activating" nucleotide sequence and the "target” nucleotide sequence can be on separate RNA molecules (e.g., “dual-guide RNAs”); or can be on the same RNA molecule ("single-guide RNA", also called sgRNA).
  • DNA methyltransferase generally refers to an enzyme that catalyzes the transfer of methyl groups to DNA.
  • Non-limiting examples of DNA methyltransferases include DNMT1, DNMT 3A, DNMT 3B, and DNMT 3L.
  • DNA methyltransferases can modify the activity of DNA fragments (e.g., regulate gene expression) without changing the DNA sequence.
  • a gene expression regulatory molecule may include one or more (e.g., two) DNA methyltransferases.
  • the DNA methyltransferase domain comprises a variant or homolog of an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to DNMT 3A.
  • the DNA methyltransferase domain comprises a variant or homolog of an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to DNMT 3L.
  • Variants or homologs of amino acid sequences having at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity.
  • the term "functionally active fragment” generally refers to a fragment having a partial region of a full-length protein or nucleic acid, but retaining or partially retaining the biological activity or function of the full-length protein or nucleic acid.
  • a functionally active fragment can retain or partially retain the ability of the full-length protein to bind to another molecule.
  • a functionally active fragment of a DNA methyltransferase can retain or partially retain the biological activity of the full-length DNA methyltransferase in catalyzing the transfer of methyl groups to DNA.
  • transcription repressor generally refers to a substance and/or agent that binds to a target nucleic acid sequence and causes a reduction in the expression level of a gene product associated with the target nucleic acid sequence, such as a protein (e.g., a transcription factor or a fragment thereof).
  • the gene product can be an RNA (e.g., mRNA) transcribed from a gene or a polypeptide translated from an mRNA transcribed from a gene.
  • mRNA RNA
  • an increase or decrease in mRNA levels results in an increase or decrease in the level of polypeptides translated therefrom. Standard techniques for measuring mRNA or protein can be used to determine expression levels.
  • Non-limiting examples of transcriptional repressors include: mSin3 interacting domain (SID) protein, methyl-CpG-binding domain 2 (MBD2), MBD3, DNA methyltransferase (DNMT) 1 (DNMT1), DNMT2A, DNMT3A, DNMT3B, DNMT3L, retinoblastoma protein (Rb), methyl CpG binding protein 2 (Mecp2), GATA-1 and its cofactor Fog1, MAT2 regulator (ROM2), Arabidopsis HD2A protein (AtHD2A), lysine-specific demethylase 1 (LSD1) and/or Krüppel-associated box (KRAB).
  • SID mSin3 interacting domain
  • MBD2 methyl-CpG-binding domain 2
  • DNMT1 DNA methyltransferase
  • DNMT1 DNA methyltransferase
  • DNMT2A DNA methyltransferase
  • KRAB is also referred to as "Kruper-associated box domain” or “Krüppel-associated box domain”, which generally refers to about 45 to about 75 amino acid residues of the transcriptional repression domain present in the transcription factor of the human zinc finger protein.
  • the KRAB domain can include a variant or homolog of an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the ZIM3KRAB domain or the KOX1KRAB domain.
  • split green fluorescent protein generally refers to a polypeptide that is able to split and immediately form active green fluorescent protein upon reassembly.
  • GCN4 refers to a transcription factor in Saccharomyces cerevisiae and a "master regulator” in the yeast genome, regulating nearly one-tenth of the yeast genome. It is a highly conserved protein and its homolog in mammals is Activating Transcription factor-4 (ATF4).
  • PDZ protein generally refers to a naturally occurring protein containing a PDZ domain.
  • exemplary PDZ proteins include CASK, MPP1, DLG1, DLG2, PSD95, NeDLG, TIP-33, SYN1a, TIP-43, LDP, LIM, LIMK1, LIMK2, MPP2, NOS1, AF6, PTN-4, prIL16, 41.8kD, KIAA0559, RGS12, KIAA0316, DVL1, TIP-40, TIAM1, MINT1, MAGI-1, MAGI-2, MAGI-3, KIAA0303, CBP, MINT3, TIP-2, KIAA0561 and/or TIP-I.
  • single-chain antibody or “scFv (Single Chain Antibody)” generally refers to a single-chain polypeptide containing one or more antigen binding sites.
  • H and L chains of the Fv fragment are encoded by different genes, they can be directly or through peptides.
  • Linkers can be used to connect the H and L chains into a single protein chain (called a single-chain antibody, sAb; Bird et al. 1988 Science 242: 423-426; and Huston et al. 1988 PNAS 85: 5879-5883) by recombinant methods.
  • the single-chain antibody is also included in the term "antibody” and can be used as a binding determinant in the design and manufacture of multispecific binding molecules, and the single-chain antibody can be prepared by recombinant technology or enzymatic or chemical cleavage of intact antibodies.
  • direct connection generally refers to direct connection.
  • the direct connection may be a situation where there is no spacer component (such as amino acid residues or derivatives thereof) between the connected substances (such as amino acid sequence segments) and they are directly connected; for example, amino acid sequence segment X is directly connected to another amino acid sequence segment Y through 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.
  • “Indirect connection” generally refers to a situation where there is a spacer component (such as amino acid residues or derivatives thereof) between the connected substances (such as amino acid sequence segments) and they are indirectly connected.
  • the spacer component used in the present application may be a segment of amino acid residues, whose sequence is selected from the amino acid sequence shown in any one of SEQ ID NO:125-132 (SEQ ID NO:126 is GSG).
  • nuclear localization sequence generally refers to a peptide that directs a protein to the nucleus.
  • the NLS includes five basic positively charged amino acids.
  • the NLS can be located at any position on the peptide chain.
  • the NLS is an NLS derived from SV40.
  • the NLS includes the sequence shown in any one of SEQ ID NO:338-340.
  • the NLS has an amino acid sequence that is 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 NO:338-340.
  • the term "marker” refers to a peptide that can be introduced into an expression vector and can be used to allow deletion and/or purification of the expression product of one or more vector inserts.
  • markers are well known in the art and include radiolabeled amino acids or polypeptides attached to a biotin moiety that can be detected by a labeled avidin (e.g., containing a fluorescent marker or enzymatically active streptavidin that can be detected by optical or colorimetric methods).
  • Affinity markers such as FLAG, glutathione-S-transferase, maltose binding protein, cellulose binding domain, thioredoxin, NusA, mistin, chitin binding domain, cutinase, AGT, GFP and other markers that are widely used, such as those used in protein expression and purification systems.
  • polypeptides include, but are not limited to, the following: histidine tags, radioisotopes or radionuclides (e.g., 3H, 14C, 35S, 90Y, 99Tc, 111In, 125I, 177Lu, 166Ho or 153Sm); Fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphides), enzyme labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent labels; biotin groups; pendant polypeptide antigenic determinants recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, antigenic determinant labels); and magnetic agents, such as gadolinium chelates.
  • histidine tags e.g., radioisotopes or radionuclides
  • Fluorescent labels e.g., FITC, r
  • nucleic acid is used interchangeably with “polynucleotide”, “nucleotide”, “nucleotide sequence” and “oligonucleotide”, which generally refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and polymers thereof in single-stranded, double-stranded or multi-stranded form or their complements.
  • nucleotide can be a ribonucleotide, a deoxyribonucleotide or a modified version thereof.
  • the nucleotide can be a hybrid molecule of a mixture of single-stranded and double-stranded DNA, single-stranded and double-stranded RNA, and single-stranded and double-stranded DNA and RNA.
  • nucleotides can include, but are not limited to, any type of RNA, such as mRNA, siRNA, miRNA, sgRNA and guide RNA, as well as any type of DNA, genomic DNA, plasmid DNA and minicircle DNA and any fragments thereof.
  • the term also encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or bonds, which are synthetic, naturally occurring and non-naturally occurring.
  • sequence encoding or “nucleic acid encoding" generally refers to a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence encoding a protein.
  • the coding sequence may also include start and stop signals operably linked to regulatory elements, including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered.
  • the coding sequence may be codon optimized.
  • intron generally refers to a DNA fragment that is transcribed but removed from the RNA transcript by splicing together either end of the sequence (exon). Introns are considered to be intervening sequences within the protein coding region of a gene and generally do not contain the information represented by the protein produced by the gene.
  • the term "recombinant vector” generally refers to a nucleic acid molecule capable of transporting it and another nucleic acid connected thereto.
  • a vector which refers to a circular double-stranded DNA loop to which other DNA segments can be connected.
  • the vector can be linear.
  • a viral vector in which other DNA segments can be connected to the viral genome.
  • Specific vectors can be autonomously replicated in the host cell they are introduced into (for example, bacterial vectors and additional mammalian vectors with bacterial replication origins). Other vectors (for example, non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, and thus replicate together with the host genome.
  • regulatory element refers to a genetic element that can control the expression of a nucleic acid sequence.
  • splicing signals, promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, replication origins, internal ribosome entry sites ("IRES"), enhancers, etc. which together provide for replication, transcription, and translation of the coding sequence in the recipient cell. Not all of these control sequences need to be present.
  • promoter generally refers to a nucleotide sequence that controls or regulates an expression operably associated with the promoter.
  • a promoter is a nucleotide sequence that is transcribed by a promoter (e.g., a coding sequence).
  • a coding sequence controlled or regulated by a promoter can encode a polypeptide and/or a functional RNA.
  • a “promoter” refers to a nucleotide sequence that contains an RNA polymerase II binding site and directs the start of transcription.
  • a promoter is located 5' or upstream of the start of the coding region relative to the corresponding coding sequence.
  • a promoter may contain other elements that act as regulatory factors for gene expression; for example, a promoter region.
  • a promoter region may include at least one intron.
  • Promoters may include, for example, constitutive, inducible, time-regulated, developmentally regulated, chemically regulated, tissue-preferred and/or tissue-specific promoters for preparing recombinant nucleic acid molecules, such as "synthetic nucleic acid constructs" or "protein-RNA complexes". These different types of promoters are known in the art.
  • the term “enhancer” generally refers to a regulatory DNA sequence, such as 50-1500bp, that can be combined by protein (activator protein) to stimulate or enhance the transcription of a gene or several genes. These activators (also known as transcription factors) interact with the mediator complex and recruit polymerase II and general transcription factors, and then begin to transcribe the gene. Enhancers are usually cis-acting, but can be located upstream or downstream of the start site of the gene or the gene they regulate. In addition, enhancers can be forward or backward directions, and do not need to be located near the transcription start site to affect transcription, because some enhancers have been found to be located at hundreds of thousands of base pairs upstream or downstream of the start site. Enhancers can also be found in introns.
  • cleavage peptide refers to a class of polypeptides that can achieve the function of cleaving proteins.
  • the cleavage peptide can achieve protein cleavage via ribosome skipping rather than protease hydrolysis.
  • the cleavage peptide can be a cleavage 2A peptide, which can include T2A, F2A, P2A and/or E2A.
  • the term "delivery vector” generally refers to a transfer vehicle capable of delivering an agent (e.g., a nucleic acid molecule) to a target cell.
  • the delivery vector can deliver an agent to a specific cell subclass.
  • the delivery vector is targeted to certain types of cells by the inherent characteristics of the delivery vector or by the part coupled to the carrier, the part contained therein (or the part combined with the carrier, so that the part and the delivery vector are maintained together, and then the part is enough to target the delivery vector).
  • the delivery vector can also improve the in vivo half-life of the agent to be delivered and/or the bioavailability of the agent to be delivered.
  • the delivery vector can include a viral vector, a virus-like particle, a polycationic carrier, a peptide carrier, a liposome and/or a hybrid carrier.
  • the property e.g., size, charge and/or pH
  • the delivery vector can effectively deliver the delivery vector and/or the molecules encapsulated therein to the target cell, reduce immune clearance and/or promote to stay in the target cell.
  • the term "liposome” generally refers to a vesicle with an internal space that is separated from the external medium by one or more bilayer membranes.
  • the bilayer membrane can be formed by amphiphilic molecules, such as synthetic or naturally derived lipids containing spatially separated hydrophilic and hydrophobic domains; in other embodiments, the bilayer membrane can be formed by amphiphilic polymers and surfactants.
  • the liposome is a spherical vesicle structure composed of a single or multilayer lipid bilayer surrounding an internal aqueous compartment, and a relatively impermeable outer lipophilic phospholipid bilayer.
  • liposomes are biocompatible, non-toxic, and can deliver hydrophilic and lipophilic drugs.
  • Liposomes can be made of several different types of lipids, such as phospholipids. Liposomes can contain natural phospholipids and lipids (such as 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), sphingomyelin, egg phosphatidylcholine, monosialoganglioside or any combination thereof.
  • DSPC 1,2-distearoyl-sn-glycero-3-phosphatidylcholine
  • sphingomyelin such as 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), sphingomyelin, egg phosphatidylcholine, monosialoganglioside or any combination thereof.
  • DSPC 1,2-distearoyl-sn-glycero
  • liposomes can also contain cholesterol, sphingomyelin and/or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), for example, to increase stability and/or prevent leakage of liposome internal cargo.
  • DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • lipid nanoparticle generally refers to a particle comprising a plurality of (i.e., more than one) lipid molecules that are physically bound to each other (e.g., covalently or non-covalently) by intermolecular forces.
  • LNP can be, for example, a microsphere (including unilamellar and multilamellar vesicles, such as liposomes), a dispersed phase in an emulsion, a micelle, or an internal phase in a suspension.
  • LNP can encapsulate nucleic acids in cationic lipid particles (e.g., liposomes) and can be delivered to cells relatively easily.
  • lipid nanoparticles do not contain any viral components, which helps to minimize safety and immunogenicity issues.
  • the lipid particles can be used for in vitro, ex vivo, and in vivo delivery.
  • the lipid particles can also be used for cell populations of various sizes.
  • the LNP of the present application can be easily prepared by various methods known in the art, such as by mixing an organic phase with an aqueous phase. The mixing of the two phases can be achieved by a microfluidic device and an impinging flow reactor. The more fully the organic phase and the aqueous phase are mixed, the better the embedding rate and particle size distribution of the obtained LNP.
  • the particle size of the LNP can be adjusted by changing the mixing speed of the organic phase and the aqueous phase.
  • LNP can be used to deliver DNA molecules and/or RNA molecules (e.g., mRNA of Cas, sgRNA). In some cases, LNP can be used to deliver the RNP complex of Cas/gRNA. In some embodiments, LNP is used to deliver mRNA and gRNA.
  • the term "subject” generally refers to an animal, usually a mammal, such as a human, non-human primate (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), livestock (dogs and cats), farm animals (poultry such as chickens and ducks, horses, cattle, goats, sheep, pigs), and laboratory animals (mice, rats, rabbits, guinea pigs).
  • Human subjects include fetuses, newborns, infants, adolescents, and adult subjects.
  • Subjects include animal disease models, such as mice and other animal models of blood coagulation diseases (such as HemA), and other animal models known to those skilled in the art.
  • selected from generally refers to including selected objects and all combinations thereof.
  • selected from (:) A, B and C means including all combinations of A, B and C, for example, A, B, C, A+B, A+C, B+C or A+B+C.
  • the term "about” generally refers to a variation within a range of 0.5%-10% above or below a specified value, for example, 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.
  • the present application provides a complex comprising a first fusion and a second fusion, wherein the fusion of one of the first fusion and the second fusion comprises a DNA methylation domain and at least one recruitment domain A, and wherein the other fusion comprises a transcription repressor domain and at least one recruitment domain A'; wherein the recruitment domain A and the recruitment domain A' can interact with each other so that the fusion of one of the first fusion and the second fusion or a portion thereof can be recruited to the vicinity of the other fusion.
  • the present application provides a nucleic acid encoding the complex described in the present application.
  • the nucleic acid comprises DNA and/or mRNA.
  • the nucleic acid can be used to treat or alleviate a disease or condition associated with abnormal target gene expression and/or abnormal target gene activity.
  • the nucleic acid is mRNA; one or more modification techniques can be used to produce a more stable mRNA.
  • mRNA modification techniques can be roughly divided into three categories: synthesizing mRNA with artificially synthesized non-natural ribonucleic acids instead of natural ribonucleic acids; adding 5'caps, 3'poly (A) "tails” and UTR (untranslated region) sequences; using special new formulation technology to effectively protect mRNA.
  • the preferred mRNA modification technology can synthesize mRNA by replacing natural ribonucleic acids with artificially synthesized non-natural ribonucleic acids.
  • Chemical modifications on eukaryotic mRNA can be roughly divided into three categories: methylation, pseudouridine ( ⁇ ) and hypoxanthine.
  • the chemical modification can be selected from: pseudouridine, N1-methyl pseudouridine, N1-ethyl pseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thiol-1-methyl-1-deaza-pseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methyl-pseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine and 2'-O-methyluridine.
  • the nucleic acid is a recombinant vector
  • the recombinant vector comprises a nucleic acid encoding the complex described in the present application.
  • a recombinant vector can refer to a nucleic acid molecule capable of transporting another nucleic acid connected thereto.
  • Recombinant vectors can include single-stranded, double-stranded or partially double-stranded nucleic acid molecules; nucleic acid molecules containing one or more free ends, no free ends (e.g., circular); nucleic acid molecules containing DNA, RNA or both; and other types of polynucleotides known in the art.
  • viral vectors can be used.
  • Viral vectors can contain virally derived DNA or RNA sequences for packaging into viruses (e.g., retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses, and adeno-associated viruses AAV). Viruses and viral vectors can be used for in vitro, ex vivo and/or in vivo delivery.
  • viruses e.g., retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses, and adeno-associated viruses AAV.
  • viruses e.g., retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses, and adeno-associated viruses AAV.
  • viruses e.g., retroviruses, replication-defective retroviruses, adenoviruses, replication-defective aden
  • the application provides a kind of delivery vector
  • described delivery vector comprises complex described in the application and/or nucleic acid described in the application, and optionally comprises liposome and/or lipid nanoparticle.
  • delivery vector can be introduced into cell by physical delivery method.
  • the example of physical method comprises microinjection, electroporation and fluid dynamics delivery.
  • LNPs can be wrapped in cationic lipid particles (such as liposomes) by nucleic acid, and can be delivered to cell relatively easily.
  • lipid nanoparticle does not contain any viral component, and this helps to minimize safety and immunogenicity problems.
  • Lipid particles can be used for in vitro, ex vivo and in vivo delivery.
  • the components of LNP can include cationic lipids, ionizable lipids, PEGylated lipids and/or supporting lipids, and optionally a cholesterol component.
  • the present application provides a composition comprising the complex described in the present application, the nucleic acid described in the present application, and/or the delivery vector described in the present application.
  • the complex in the composition, the nucleic acid encoding the complex (or recombinant vector) and the delivery vector can be contained in one composition at the same time, or respectively contained in different compositions.
  • the nucleic acid encoding the complex (or recombinant vector) and/or the delivery vector they can be used simultaneously or separately.
  • the present application provides a cell, wherein the cell comprises the complex described herein, the nucleic acid described herein, the delivery vector described herein, and/or the composition described herein.
  • the present application provides a kit, the kit comprising the complex described in the present application, the nucleic acid described in the present application, the delivery vector described in the present application, the composition described in the present application, and/or the cell described in the present application.
  • the kit further comprises at least one container for placing the above components.
  • the kit comprises more than one of the above components, and it also comprises a second, third and/or other container other than the container, in which the more than one of the above components can be placed separately.
  • the kit can place various combinations of the above components in the container.
  • the kit further comprises a buffer reagent, a device for mixing, a device for measuring, a device for sorting and/or a device for marking.
  • the kit also comprises packaging for accommodating various containers.
  • the kit also comprises instructions for using the kit components.
  • the instructions include a paper-based physical form and/or a machine-readable electronic form.
  • the present application provides a method for regulating target gene expression, the method comprising administering the complex described in the present application, the nucleic acid described in the present application, the delivery vector described in the present application, the composition described in the present application, the cell described in the present application and/or the kit described in the present application.
  • the method for inhibiting target gene expression is to introduce the complex, the nucleic acid, the delivery vector, the composition, the cell and/or the kit into a cell containing the target gene.
  • the introduction of the cell can be introduced into the cell using a non-viral or virus-based transfection method.
  • the non-viral transfection method includes any suitable method for introducing the cell without using viral DNA or viral particles as a delivery system
  • non-limiting examples of non-viral transfection methods include nanoparticle encapsulation of nucleic acids encoding the complex (e.g., lipid nanoparticles, gold nanoparticles, etc.), calcium phosphate transfection, liposome transfection, nuclear transfection, sonoporation, transfection by heat shock, magnetic transfection and electroporation.
  • a viral-based transfection method includes any viral vector suitable for the method described in the present application, and its non-limiting examples include, but are not limited to, retrovirus, adenovirus, lentivirus and/or adeno-associated virus vectors.
  • the method for inhibiting the expression of a target gene further comprises introducing the complex, the nucleic acid, the delivery vector, the composition, the cell and/or the kit from an external environment into a cell.
  • the method for inhibiting the expression of a target gene comprises contacting the complex, the nucleic acid, the delivery vector, and/or the composition with a transcriptional regulatory element near the target gene and/or with the target gene.
  • the contact It refers to contacting the first fusion, the second fusion and the guide RNA described in the present application with the vicinity of the target gene and/or the transcriptional regulatory element of the target gene, and the guide RNA forms a complex with the fusion comprising the DNA binding domain, the complex specifically recognizes a specific region in the target gene and hybridizes with it, and the first fusion and the second fusion are recruited to the vicinity of the DNA binding domain through the direct or indirect interaction of their recruitment domain A and recruitment domain A', thereby regulating the expression of the target nucleic acid.
  • the method includes causing the first fusion, the second fusion and the guide RNA described in the present application to exist in the form of a complex (e.g., an assembled ribonucleoprotein complex), and contacting the complex with the vicinity of the target gene and/or the transcriptional regulatory element of the target gene.
  • a complex e.g., an assembled ribonucleoprotein complex
  • the present application provides a method for treating or alleviating a disease or a condition associated with abnormal target gene expression and/or abnormal target gene activity, the method comprising administering an effective amount of the complex described in the present application, the nucleic acid described in the present application, the delivery vector described in the present application, the composition described in the present application, the cell described in the present application, and/or the kit described in the present application to a subject in need.
  • the treatment method comprises mixing the complex, the nucleic acid, the delivery vector, the composition, the cell, and/or the kit with a therapeutic agent, and delivering the systemic drug to a subject in need, so that the subject is widely exposed to most of the body, and can be carried out by any means known in the art, including but not limited to intravenous, intra-arterial, subcutaneous, intracavitary, and intraperitoneal delivery.
  • the treatment method comprises mixing the complex, the nucleic acid, the delivery vector, the composition, the cell, and/or the kit with a therapeutic agent, and delivering the subject in need locally, so that the subject directly reaches the target site in the organism, and can be carried out by, for example, direct injection into the disease site (e.g., tumor or inflammation site) or target organ (e.g., liver, heart, pancreas, kidney, etc.).
  • the local delivery includes local application or local injection techniques, including but not limited to intramuscular, subcutaneous or intradermal injection.
  • the local delivery does not exclude systemic pharmacological effects.
  • the disease includes cardiovascular disease, nonalcoholic steatohepatitis, AMD, age-related macular degeneration, type 2 diabetes, obesity, liver failure, dyslipidemia, diabetic atherosclerosis and/or hypertension.
  • the present application provides the use of the complex described herein, the nucleic acid described herein, the delivery vector described herein, the composition described herein, the cell described herein and/or the kit described herein for preparing a drug, wherein the drug is used to treat or alleviate a disease or its condition associated with abnormal target gene expression and/or abnormal target gene activity.
  • the present application provides the complex described herein, the nucleic acid described herein, the delivery vector described herein, the composition described herein, the cell described herein or the kit described herein, which is used to treat or alleviate diseases or symptoms associated with abnormal target gene expression and/or abnormal target gene activity.
  • the first fusion and the second fusion of the complex of the present application can be generally divided into two situations: (1) one of the two fusions comprises a nucleic acid binding domain, a DNA methylation domain and a recruitment domain A, and the other One fusion contains the transcriptional repressor domain and the recruitment domain A', or (2) one of the two fusions contains the nucleic acid binding domain, the transcriptional repressor and the recruitment domain A, and the other fusion contains the DNA methylation domain and the recruitment domain A'.
  • one of the two fusions may include a DNA methylation domain, a nucleic acid binding domain, and a recruitment domain A in sequence from the N-terminus to the C-terminus.
  • one of the two fusions may include a recruitment domain A, a nucleic acid binding domain, and a transcription repressor domain in sequence from the N-terminus to the C-terminus.
  • the other of the two fusions may include a transcription repressor domain and a recruitment domain A' in sequence from the N-terminus to the C-terminus, or the recruitment domain A' and the transcription repressor domain, that is, the transcription repressor domain and the recruitment domain A' may be connected in sequence interchangeably.
  • the other of the two fusions may include a DNA methylation domain and a recruitment domain A' in sequence from the N-terminus to the C-terminus, or the recruitment domain A' and the DNA methylation domain, that is, the DNA methylation domain and the recruitment domain A' may be connected in sequence interchangeably.
  • the nucleic acid binding domain is a DNA binding domain.
  • the DNA binding domain may be selected from: a TALE domain, a zinc finger domain, a tetR domain, a large-range nuclease, a Cas protein, an Argonaute (Ago) protein, and homologs, modified forms or variants thereof.
  • the DNA binding domain may be a Cas protein
  • the Cas protein is a class II Cas nuclease.
  • the Cas protein may be selected from class II type II Cas nucleases and class II type V Cas nucleases; for example, the Cas protein may be a Cas9 or Cas12 protein.
  • the Cas protein may be an inactivated Cas9 (dCas9) protein or an inactivated Cas12 (dCas12) protein.
  • the DNA binding domain described in the present application may include, but is not limited to, the amino acid sequence shown in any one of SEQ ID NO: 1-9, 343 and 344.
  • the transcriptional repressor is selected from one or more of the following domains: KRAB, ZIM3, ZNF680, ZNF554, ZNF264, ZNF582, ZNF324, ZNF669, ZNF354A, ZNF82, ZNF595, ZNF419, ZNF566, ZIM2, EHMT2, SUV39H1, ZFPM1, TRIM28, EZH2, MXD1, SID, LSD1, HP1a, HDAC3, HDAC1, PRMT1, SETDB1, hSIRT1, ZNF436, ZNF257, ZNF675, ZNF490, ZNF320, ZNF331, ZNF816, ZNF41, ZNF189, ZNF528, ZNF543, ZNF140, ZNF610, ZNF3 50, ZNF8, ZNF30, ZNF98, ZNF677, ZNF596, ZNF214, ZNF37A, ZNF34, ZNF250, ZNF547, ZNF273, Z FP82, ZNF224, ZNF
  • the DNA methylation domain comprises at least one DNA methyltransferase or a functionally active fragment thereof.
  • the DNA methyltransferase is selected from DNMT3A, DNMT3B, DNMT3c, DNMT1, DNMT2 and DNMT3L.
  • the DNA methylation domain comprises at least one DNMT3A and at least one DNMT3L.
  • the at least one DNMT3A and the at least one DNMT3L are connected interchangeably and sequentially.
  • the DNA methylation domain comprises one DNMT3A and one DNMT3L, and they are connected interchangeably and sequentially.
  • the DNA methyltransferase comprises the amino acid sequence shown in any one of SEQ ID NO: 19-24.
  • the first fusion and the second fusion of the complex of the present application are complexes that form aggregates through the interaction between the recruitment domains contained in each. Therefore, the present application provides non-limiting examples of combinations of recruitment domain A and recruitment domain A': (1) the domain of one of the recruitment domain A and the recruitment domain A' is GCN4, and the other domain is scFv; or (2) the domain of one of the recruitment domain A and the recruitment domain A' is a GFP11 fragment, and the other domain is GFP1-10; or (3) the domain of one of the recruitment domain A and the recruitment domain A' is GVKESLV, and the other domain is a PDZ protein domain.
  • the situation that GFP11 and GFP1-10 are respectively derived from split GFP (SEQ ID NO: 15) to form the recruitment domain A and the recruitment domain A' can also be applied to other categories of fluorescent proteins, such as mCherry (SEQ ID NO: 16), eYFP (SEQ ID NO: 18), eCFP (SEQ ID NO: 17), etc., 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 complex provided in the present application.
  • one of the first fusion and the second fusion of the complex of the present application may include two or more recruitment domains, and they are connected by a linker sequence.
  • the amino acid sequence of an exemplary recruitment domain may include any one of SEQ ID NO: 10-14.
  • the present application may provide the following amino acid sequences of the first fusion or the second fusion:
  • the amino acid sequences of the epigenetic modification recruitment system and recruited elements (including Dnmt3a CD, Dnmt3l CD, dSpCas9 or TALE, KRAB) with HA epitope and P2A were optimized and synthesized by Genscript into nucleic acid sequences suitable for mammalian expression, and then cloned into the pLV-CAG vector with CAG promoter and WPRE, and the recruited elements and self-cleaved recruitment system fusion protein were expressed by the CAG promoter.
  • the different functional elements are optimized by Genscript into nucleic acid sequences suitable for mammalian expression and synthesized.
  • the vector other than the element to be replaced is amplified by PCR, and then the element to be replaced is amplified from the sequence synthesized by the company, and the homology arm sequence is introduced at the same time.
  • the different elements are recombined into the vector through NEBuilder reagent to construct the final expression plasmid.
  • the complex of the present application (the first fusion having the amino acid sequence shown in SEQ ID NO: 51 and the first fusion having the amino acid sequence shown in SEQ ID NO: NO:52) and different gRNAs targeting the mouse PTP1b gene (sg1, sg2, or an equal mixture of sg1 and sg2), the complementary nucleotide sequences of the gRNA targeting sequences are shown in SEQ ID NOs:336 and 337, and a control gRNA (NT gRNA, the complementary nucleotide sequence of its targeting sequence is shown in SEQ ID NO:365) were co-transfected into the mouse N2a cell line (700 ng editor + 300 ng gRNA, 24-well plate), and the transfected positive cells were sorted 72 hours after transfection, total RNA was extracted with Trizol, the relative expression of PTP1b was quantified by qPCR, and the relative inhibition efficiency of different tools and different gRNAs was calculated (Figure 2).
  • the prepared LNP was injected into mice through the tail vein at a dose of 4.5 mg per kg body weight. 4-10 days after the injection, blood was collected from the cheeks of the mice, and the content of PCSK9 protein in the blood was measured by Elisa.
  • the PBS group was a control group injected with an equal volume of PBS. The test results of each tool on the 4th day after injection are shown in FIG3 . Compared with the PBS control group, each group of tools in the present application showed a very prominent inhibitory effect on PCSK9 gene expression.
  • Example 3 According to the in vitro transcribed mRNA of Example 3 (see the table below for information on the tools used), the chemically synthesized sgRNA/mRNA mixture with a mass ratio of 1:1 (sgRNA sequence is the same as in Example 3), and the method for preparing LNP, the prepared LNP was added to Huh7 cells (1.25ug/mL dose), and 4 days after the addition of LNP, all cells were collected, total RNA was extracted with Trizol, and the relative expression of PCSK9 was quantified by qPCR to calculate the relative inhibition efficiency of different tools. The results are shown in Figure 4, where the NC group is a control without the addition of LNP. It can be seen that the fusion provided by the present application has a significant inhibitory effect on the expression of the PCSK9 gene.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Cell Biology (AREA)
  • Virology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicinal Preparation (AREA)

Abstract

一种复合物，其包含第一融合物和第二融合物，所述第一融合物和所述第二融合物其中之一的融合物包含DNA甲基化结构域和至少一个招募结构域A，并且其中另一个融合物包含转录阻遏物结构域和至少一个招募结构域A'；其中，所述招募结构域A和招募结构域A'能够相互作用，以使所述第一融合物和所述第二融合物其中之一的融合物或其部分能够被招募到另一个融合物附近。还提供了该复合物用于制备抑制靶基因表达的产品和制备药物的用途。

Description

复合物及其用途 技术领域
本申请涉及生物医药领域，具体的涉及一种用于基因编辑的复合物及其用途。
背景技术
基因组表观遗传修饰的异常与许多疾病的发生与发展息息相关，如生活中常见的代谢紊乱、心血管疾病和癌症等，而基因表观遗传编辑工具可在不改变基因序列的情况下达到相应基因转录调控目的，该过程不会造成永久性的DNA损伤，也不会产生有害突变和脱靶效应；并且表观遗传疗法可以通过同时调节多个基因活性来提供更好的治疗效果，弥补基因治疗存在的不足，以及为治疗这类疾病提供了新的希望。另一方面，基因定点修饰技术的发展更使得在天然染色质环境中有针对性地进行表观基因组编辑和转录调控成为可能，特别是CRISPR/Cas9技术的发展。
目前，表观遗传修饰和转录调控的设计主要基于核酸酶的结合运用，其中以工程化缺陷型核酸酶(dCas9)的结合最为有效，其设计原理在于把各种表观调控效应器融合到dCas9上，通过该核酸酶的靶向并结合目标DNA的特性实现特定基因组位点上表观基因组的编辑。这些反式调控结构域和蛋白是通过向启动子区域的dCas9靶向位点阻碍RNA聚合酶的结合或募集内源性转录复合物来发挥作用的。近期该领域一项突破性的工作是使用一种dCas-SunTag系统融合多个拷贝的转录激活或抑制蛋白实现目标基因转录的调控，例如Hatada Izuho等人(PCT/JP2021/006498)通过dCas9-GCN4融合物来招募包含有表观修饰因子(如甲基转移酶和组蛋白乙酰转移酶)、转录抑制调控因子(如ZIM3)以及抗体的融合物，从而对其调控的靶基因表达进行抑制。目前利用转录调控和表观遗传定点修饰技术在体内治疗表观遗传异常引起疾病的研究还屈指可数，且现有的编辑工具存在一定问题，例如SunTag系统招募效果不理想、转录调控效率低下和对靶基因的修饰(例如甲基化修饰)范围受限等。
发明内容
本申请提供了一种用于基因编辑的复合物以及其编码核酸、载体、组合物、细胞等形式，该复合物相比于现有基于SunTag招募策略的基因编辑方法能够取得更高的调控效率和更丰 富的靶基因的修饰和调控范围。并且本申请的复合物可以用于制备抑制靶基因表达的产品和制备药物的用途。
一方面，本申请提供一种复合物，其包含第一融合物和第二融合物，所述第一融合物和所述第二融合物其中之一的融合物包含DNA甲基化结构域和至少一个招募结构域A，并且其中另一个融合物包含转录阻遏物结构域和至少一个招募结构域A’；其中，所述招募结构域A和招募结构域A’能够相互作用，以使所述第一融合物和所述第二融合物其中之一的融合物或其部分能够被招募到另一个融合物附近。
在一些实施方案中，所述第一融合物或所述第二融合物包含核酸结合结构域。
在一些实施方案中，所述第一融合物包含DNA甲基化结构域、核酸结合结构域和至少一个招募结构域A，所述第二融合物包含转录阻遏物结构域和至少一个招募结构域A’。
在一些实施方案中，所述第一融合物中从N端到C端依次包含DNA甲基化结构域、核酸结合结构域和招募结构域A。
在一些实施方案中，所述第二融合物中从N端到C端依次包含转录阻遏物结构域和招募结构域A’，或者从N端到C端依次包含招募结构域A’和转录阻遏物结构域。
在一些实施方案中，所述第一融合物包含转录阻遏物结构域、核酸结合结构域和至少一个招募结构域A，所述第二融合物包含DNA甲基化结构域和至少一个招募结构域A’。
在一些实施方案中，所述第一融合物中从N端到C端依次包含招募结构域A、核酸结合结构域和转录阻遏物结构域。
在一些实施方案中，所述第二融合物中从N端到C端依次包含DNA甲基化结构域和招募结构域A’，或者从N端到C端依次包含招募结构域A’和DNA甲基化结构域。
在一些实施方案中，所述复合物的特征在于：1)所述第一融合物从N端到C端依次包含DNA甲基化结构域、核酸结合结构域和招募结构域A，所述第二融合物从N端到C端依次包含转录阻遏物结构域和招募结构域A’；或2)所述第一融合物从N端到C端依次包含DNA甲基化结构域、核酸结合结构域和招募结构域A，所述第二融合物从N端到C端依次包含招募结构域A’和转录阻遏物结构域；或3)第一融合物中从N端到C端依次包含招募结构域A、核酸结合结构域和转录阻遏物结构域，所述第二融合物中从N端到C端依次包含DNA甲基化结构域和招募结构域A’；或4)第一融合物中从N端到C端依次包含招募结构域A、核酸结合结构域和转录阻遏物结构域，所述第二融合物从N端到C端依次包含招募结构域A’和DNA甲基化结构域。
在一些实施方案中，所述核酸结合结构域为DNA结合结构域。
在一些实施方案中，所述DNA结合结构域选自：TALE结构域、锌指结构域、tetR结构域、大范围核酸酶、Cas蛋白、Argonaute(Ago)蛋白，以及其同系物、修饰形式或变体。
在一些实施方案中，所述DNA结合结构域能够结合靶基因座的目标序列。
在一些实施方案中，所述DNA结合结构域能够与引导RNA结合。
在一些实施方案中，所述引导RNA能够特异性识别所述靶基因座的目标序列并与其杂交。
在一些实施方案中，所述DNA结合结构域为Cas蛋白，且所述Cas蛋白为II类Cas核酸酶。
在一些实施方案中，所述Cas蛋白选自II类II型Cas核酸酶和II类V型Cas核酸酶。
在一些实施方案中，所述Cas蛋白为Cas9或Cas12蛋白。
在一些实施方案中，所述Cas蛋白为失活Cas9(dCas9)蛋白或失活Cas12(dCas12)蛋白。
在一些实施方案中，所述DNA结合结构域包含SEQ ID NO:1-9、343和344中任一项所示的氨基酸序列。
在一些实施方案中，所述招募结构域A选自下列两组结构域其中一组中的任一个，所述招募结构域A’选自下列两组结构域中另一组中的任一个：1)通用控制非去阻遏蛋白4(GCN4)、来源于分裂绿色荧光蛋白(GFP)的GFP11片段和GVKESLV多肽；和2)单链抗体(scFv)、来源于分裂绿色荧光蛋白(GFP)的GFP1-10片段和PDZ蛋白结构域。
在一些实施方案中，所述复合物中：1)所述招募结构域A和所述招募结构域A’其中之一的结构域为GCN4，并且其中另一个结构域为scFv；或2)所述招募结构域A和所述招募结构域A’其中之一的结构域为GFP11片段，并且其中另一个结构域为GFP1-10；或3)所述招募结构域A和所述招募结构域A’其中之一的结构域为GVKESLV，并且其中另一个结构域为PDZ蛋白结构域。
在一些实施方案中，所述DNA甲基化结构域包含至少一种DNA甲基转移酶或其功能活性片段。
在一些实施方案中，所述DNA甲基转移酶选自DNMT3A、DNMT3B、DNMT3c、DNMT1、DNMT2和DNMT3L。
在一些实施方案中，所述DNA甲基化结构域包含至少一个DNMT3A和至少一个DNMT3L。
在一些实施方案中，所述DNA甲基转移酶包含SEQ ID NO:19-24任一项所示的氨基酸序列。
在一些实施方案中，所述DNA甲基化结构域包含DNMT3A-DNMT3L结构域或DNMT3L-DNMT3A结构域；其中，-表示其两端的结构域按照从N端到C端的顺序直接或间接地连接。
在一些实施方案中，所述转录阻遏物选自下列所示结构域中的一种或多种：KRAB，ZIM3，ZNF680，ZNF554，ZNF264，ZNF582，ZNF324，ZNF669，ZNF354A，ZNF82，ZNF595，ZNF419，ZNF566，ZIM2，EHMT2，SUV39H1，ZFPM1，TRIM28，EZH2，MXD1，SID，LSD1，HP1a，HDAC3，HDAC1，PRMT1，SETDB1，hSIRT1，ZNF436，ZNF257，ZNF675，ZNF490，ZNF320，ZNF331，ZNF816，ZNF41，ZNF189，ZNF528，ZNF543，ZNF140，ZNF610，ZNF350，ZNF8，ZNF30，ZNF98，ZNF677，ZNF596，ZNF214，ZNF37A，ZNF34，ZNF250，ZNF547，ZNF273，ZFP82，ZNF224，ZNF33A，ZNF45，ZNF175，ZNF184，ZFP28-1，ZFP28-2，ZNF18，ZNF213，ZNF394，ZFP1，ZFP14，ZNF416，ZNF557，ZNF729，ZNF254，ZNF764，ZNF785，ZNF10，CBX5，RYBP，YAF2，MGA，CBX1，SCMH1，MPP8，SUMO3，HERC2，BIN1，PCGF2，TOX，FOXA1，FOXA2，IRF2BP1，IRF2BP2，IRF2BPL IRF-2BP1_2N-terminal domain，HOXA13，HOXB13，HOXC13，HOXA11，HOXC11，HOXC10，HOXA10，HOXB9，HOXA9，ZFP28，ZN334，ZN568，ZN37A，ZN181，ZN510，ZN862，ZN140，ZN208，ZN248，ZN571，ZN699，ZN726，ZIK1，ZNF2，Z705F，ZNF14，ZN471，ZN624，ZNF84，ZNF7，ZN891，ZN337，Z705G，ZN529，ZN729，ZN419，Z705A，ZN302，ZN486，ZN621，ZN688，ZN33A，ZN554，ZN878，ZN772，ZN224，ZN184，ZN544，ZNF57，ZN283，ZN549，ZN211，ZN615，ZN253，ZN226，ZN730，Z585A，ZN732，ZN681，ZN667，ZN649，ZN470，ZN484，ZN431，ZN382，ZN254，ZN124，ZN607，ZN317，ZN620，ZN141，ZN584，ZN540，ZN75D，ZN555，ZN658，ZN684，RBAK，ZN829，ZN582，ZN112，ZN716，HKR1，ZN350，ZN480，ZN416，ZNF92，ZN100，ZN736，ZNF74，ZN443，ZN195，ZN530，ZN782，ZN791，ZN331，Z354C，ZN157，ZN727，ZN550，ZN793，ZN235，ZN724，ZN573，ZN577，ZN789，ZN718，ZN300，ZN383，ZN429，ZN677，ZN850，ZN454，ZN257，ZN264，ZN485，ZN737，ZNF44，ZN596，ZN565，ZN543，ZFP69，SUMO1，ZNF12，ZN169，ZN433，ZN175，ZN347，ZNF25，ZN519，Z585B，ZN517，ZN846，ZN230，ZNF66，ZN713，ZN816，ZN426，ZN674，ZN627，ZNF20，Z587B，ZN316，ZN233，ZN611，ZN556，ZN234，ZN560，ZNF77，ZN682，ZN614，ZN785，ZN445，ZFP30，ZN225，ZN551，ZN610，ZN528，ZN284，ZN418，ZN490，ZN805，Z780B，ZN763，ZN285，ZNF85，ZN223，ZNF90，ZN557，ZN425，ZN229，ZN606，ZN155，ZN222，ZN442，ZNF91，ZN135，ZN778，ZN534，ZN586，ZN567，ZN440，ZN583，ZN441，ZNF43，ZN589， ZN563，ZN561，ZN136，ZN630，ZN527，ZN333，Z324B，ZN786，ZN709，ZN792，ZN599，ZN613，ZF69B，ZN799，ZN569，ZN564，ZN546，ZFP92，ZN723，ZN439，ZFP57，ZNF19，ZN404，ZN274，CBX3，ZN250，ZN570，ZN675，ZN695，ZN548，ZN132，ZN738，ZN420，ZN626，ZN559，ZN460，ZN268，ZN304，ZN605，ZN844，SUMO5，ZN101，ZN783，ZN417，ZN182，ZN823，ZN177，ZN197，ZN717，ZN669，ZN256，ZN251，CBX4，CDY2，CDYL2，ZN562，ZN461，Z324A，ZN766，ID2，ZN214，CBX7，ID1，CREM，SCX，ASCL1，ZN764，SCML2，TWST1，CREB1，TERF1，ID3，CBX8，GSX1，NKX22，ATF1，TWST2，ZNF17，TOX3，TOX4，ZMYM3，I2BP1，RHXF1，SSX2，I2BPL，ZN680，TRI68，HXA13，PHC3，TCF24，HXB13，HEY1，PHC2，ZNF81，FIGLA，SAM11，KMT2B，HEY2，JDP2，HXC13，ASCL4，HHEX，GSX2，ETV7，ASCL3，PHC1，OTP，I2BP2，VGLL2，HXA11，PDLI4，ASCL2，CDX4，ZN860，LMBL4，PDIP3，NKX25，CEBPB，ISL1，CDX2，PROP1，SIN3B，SMBT1，HXC11，HXC10，PRS6A，VSX1，NKX23，MTG16，HMX3，HMX1，KIF22，CSTF2，CEBPE，DLX2，PPARG，PRIC1，UNC4，BARX2，ALX3，TCF15，TERA，VSX2，HXD12，CDX1，TCF23，ALX1，HXA10，RX，CXXC5，SCML1，NFIL3，DLX6，MTG8，CEBPD，SEC13，FIP1，ALX4，LHX3，PRIC2，MAGI3，NELL1，PRRX1，MTG8R，RAX2，DLX3，DLX1，NKX26，NAB1，SAMD7，PITX3，WDR5，MEOX2，NAB2，DHX8，CBX6，EMX2，CPSF6，HXC12，KDM4B，LMBL3，PHX2A，EMX1，NC2B，DLX4，SRY，ZN777，ZN398，GATA3，BSH，SF3B4，TEAD1，TEAD3，RGAP1，PHF1，GATA2，FOXO3，ZN212，IRX4，ZBED6，LHX4，SIN3A，RBBP7，NKX61，R51A1，MB3L1，DLX5，NOTC1，TERF2，ZN282，RGS12，ZN840，SPI2B，PAX7，NKX62，ASXL2，FOXO1，GATA1，ZMYM5，LRP1，MIXL1，SGT1，LMCD1，CEBPA，SOX14，WTIP，PRP19，NKX11，RBBP4，DMRT2，SMCA2，以及其功能活性片段。
在一些实施方案中，所述转录阻遏物结构域包含SEQ ID NOs:25-50任一项所示的氨基酸序列。
在一些实施方案中，所述复合物中：1)所述第一融合物和所述第二融合物其中之一的融合物包含DNA甲基化结构域-dCas9或dCas12或TALE-n×GCN4，并且其中另一个融合物包含转录阻遏物结构域-scFv；或2)所述第一融合物和所述第二融合物其中之一的融合物包含DNA甲基化结构域-dCas9或dCas12或TALE-scFv，并且其中另一个融合物包含转录阻遏物结构域-GCN4；或3)所述第一融合物和所述第二融合物其中之一的融合物包含DNA甲基化结构域-dCas9或dCas12或TALE-n×GFP11，并且其中另一个融合物包含转录阻遏物结 构域-GFP1-10；或4)所述第一融合物和所述第二融合物其中之一的融合物包含DNA甲基化结构域-dCas9或dCas12或TALE-GFP1-10，并且其中另一个融合物包含转录阻遏物结构域-GFP11；或5)所述第一融合物和所述第二融合物其中之一的融合物包含DNA甲基化结构域-dCas9或dCas12或TALE-n×GCN4，并且其中另一个融合物包含scFv-转录阻遏物结构域；或6)所述第一融合物和所述第二融合物其中之一的融合物包含DNA甲基化结构域-dCas9或dCas12或TALE-scFv，并且其中另一个融合物包含GCN4-转录阻遏物结构域；或7)所述第一融合物和所述第二融合物其中之一的融合物包含DNA甲基化结构域-dCas9或dCas12或TALE-n×GFP11，并且其中另一个融合物包含GFP1-10-转录阻遏物结构域；或8)所述第一融合物和所述第二融合物其中之一的融合物包含DNA甲基化结构域-dCas9或dCas12或TALE-GFP1-10，并且其中另一个融合物包含GFP11-转录阻遏物结构域；其中，-表示其两端的结构域按照从N端到C端的顺序直接或间接地连接；n×GCN4或n×GFP11分别表示n个通过接头序列连接的GCN4拷贝或n个通过接头序列连接的GFP11拷贝，n选自1至20的任一整数。
在一些是实施方案中，所述第一融合物和/或第二融合物包含SEQ ID NO:51-76、78-82、85-93、103-105、110-115、123、124、361和362中任一项所述的氨基酸序列。
在一些实施方案中，所述复合物包含SEQ ID NO:133-142、153、154、158-163、168、345和346中任一项所示的氨基酸序列。
在一些实施方案中，所述复合物中：1)所述第一融合物和所述第二融合物其中之一的融合物包含n×GCN4-dCas9或dCas12或TALE-转录阻遏物结构域，并且其中另一个融合物包含DNA甲基化结构域-scFv；或2)所述第一融合物和所述第二融合物其中之一的融合物包含scFv-dCas9或dCas12或TALE-转录阻遏物结构域，并且其中另一个融合物包含DNA甲基化结构域-GCN4；或3)所述第一融合物和所述第二融合物其中之一的融合物包含n×GFP11-dCas9或dCas12或TALE-转录阻遏物结构域，并且其中另一个融合物包含DNA甲基化结构域-GFP1-10；或4)所述第一融合物和所述第二融合物其中之一的融合物包含GFP1-10-dCas9或dCas12或TALE-转录阻遏物结构域，并且其中另一个融合物包含DNA甲基化结构域-GFP11；或5)所述第一融合物和所述第二融合物其中之一的融合物包含n×GCN4-dCas9或dCas12或TALE-转录阻遏物结构域，并且其中另一个融合物包含scFv-DNA甲基化结构域；或6)所述第一融合物和所述第二融合物其中之一的融合物包含scFv-dCas9或dCas12或TALE-转录阻遏物结构域，并且其中另一个融合物包含GCN4-DNA甲基化结构域；或7)所述第一融合物和所述第二融合物其中之一的融合物包含n×GFP11-dCas9或dCas12 或TALE-转录阻遏物结构域，并且其中另一个融合物包含GFP1-10-DNA甲基化结构域；或8)所述第一融合物和所述第二融合物其中之一的融合物包含GFP1-10-dCas9或dCas12或TALE-转录阻遏物结构域，并且其中另一个融合物包含GFP11-DNA甲基化结构域；其中，-表示其两端的结构域按照从N端到C端的顺序直接或间接地连接；n×GCN4或n×GFP11分别表示n个通过接头序列连接的GCN4拷贝或n个通过接头序列连接的GFP11拷贝，n选自1至20的任一整数。
在一些实施方案中，所述第一融合物和/或第二融合物包含SEQ ID NO:83、84、94-102、106-109、116-122、363和364中任一项所示的氨基酸序列。
在一些实施方案中，所述复合物包含SEQ ID NO:143-152、155-157、164-167、347和348中任一项所示的氨基酸序列。
在一些实施方案中，所述复合物还包含核定位信号和/或标记物结构域。
另一方面，本申请提供编码本申请所述复合物的核酸。
在一些实施方案中，所述核酸为重组载体。
在一些实施方案中，所述重组载体还包括非编码区。
在一些实施方案中，所述非编码区选自内含子、调节元件、启动子、增强子、终止序列以及5’和3’非翻译区。
在一些实施方案中，所述核酸包含编码所述第一融合物的第一核酸片段，以及编码所述第二融合物的第二核酸片段。
在一些实施方案中，其中所述第一核酸片段与第二核酸片段通过编码剪切肽的核酸片段连接。
在一些实施方案中，所述剪切肽为2A肽和/或IRES。
在一些实施方案中，所述2A肽选自P2A、T2A、E2A和F2A。
在一些实施方案中，所述核酸包含SEQ ID NO:169-335和349-360中任一项所示的核酸序列。
另一方面，本申请提供一种递送载体，所述递送载体包含本申请所述的复合物和/或本申请所述的核酸，以及任选地包含脂质体和/或脂质纳米颗粒。
另一方面，本申请提供一种组合物，所述组合物包含本申请所述的复合物、本申请所述的核酸、和/或本申请所述的递送载体。
另一方面，本申请提供一种细胞，所述细胞包含本申请所述的复合物、本申请所述的核酸、本申请所述的递送载体、和/或本申请所述的组合物。
另一方面，本申请提供一种试剂盒，所述试剂盒包含本申请所述的复合物、本申请所述的核酸、本申请所述的递送载体、本申请所述的组合物、和/或本申请所述的细胞。
另一方面，本申请提供一种调控靶基因表达的方法，所述方法包括施用本申请所述的复合物、本申请所述的核酸、本申请所述的递送载体、本申请所述的组合物、本申请所述的细胞和/或本申请所述的试剂盒。
在一些实施方案中，所述方法包括将所述复合物、所述核酸、所述递送载体、所述组合物、所述细胞、和/或所述试剂盒引入含有所述靶基因的细胞。
在一些实施方案中，所述方法包括使所述复合物、所述核酸、所述递送载体、和/或所述组合物接触所述靶基因附近和/或所述靶基因的调控元件。
在一些实施方案中，所述调控元件包含核心启动子、近端启动子、远端增强子、沉默子、绝缘子元件、边界元件和/或基因座控制区。
另一方面，本申请提供一种治疗或缓解与靶基因表达异常和/或靶基因活性异常相关的疾病或其病症的方法，所述方法包括向有需要的受试者施用有效量的本申请所述的复合物、本申请所述的核酸、本申请所述的递送载体、本申请所述的组合物、本申请所述的细胞和/或本申请所述的试剂盒。
另一方面，本申请提供本申请所述的复合物、本申请所述的核酸、本申请所述的递送载体、本申请所述的组合物、本申请所述的细胞和/或本申请所述的试剂盒用于制备药物的用途，所述药物用于治疗或缓解与靶基因表达异常和/或靶基因活性异常相关的疾病或其病症。
另一方面，本申请提供本申请所述的复合物、本申请所述的核酸、本申请所述的递送载体、本申请所述的组合物、本申请所述的细胞或本申请所述的试剂盒，其用于治疗或缓解与靶基因表达异常和/或靶基因活性异常相关的疾病或其病症。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如 下：
图1显示的是本申请复合物的结构示意图。
图2显示的是本申请复合物对PTP1b基因表达的抑制效果。
图3和图4显示的是本申请复合物对PCSK9基因表达的抑制效果。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“招募”通常是针对蛋白质分子间的招募作用，其具体是指蛋白质招募其他分子来执行特定的生物学功能。这种招募作用主要依赖分子间相互作用的亲和性，且通常其亲和性被认为与蛋白分子的空间结构相关，较为复杂。相互作用机制示例性地可包含但不限于氢键、离子相互作用、疏水相互作用、范德华力等非共价键作用，。例如，一些蛋白质可以招募酶来催化化学反应，或者招募其他蛋白质来形成复合物。这些招募作用对于许多细胞过程至关重要，如信号转导、DNA复制和基因表达等。
在本申请中，术语“核酸结合结构域”通常是指能够与特定核酸结合的多肽或组合物的部分，其可以包含接触核酸的区域、核酸和/或蛋白质物质。核酸结合结构域的示例可包括但不限于：螺旋-转-螺旋结构域、锌指结构域、亮氨酸拉链(bZIP)结构域、有翼的螺旋结构域、有翼的螺旋转螺旋结构域、螺旋-环-螺旋结构域、HMG-box结构域、Wor3结构域、免疫球蛋白结构域、B3结构域、TALE结构域和/或CRISPR/CasX蛋白等的结构域。
在本申请中，术语“DNA结合结构域”通常是指独立折叠的蛋白质结构域，其含有识别双链或单链DNA的至少一个基序。例如，所述DNA结合域可识别特异性DNA序列(识别或调节序列)或具有对DNA的一般亲和性。在某些情形下，DNA结合域的其他结构域通常调节DNA结合域的活性；DNA结合功能可以是结构性的或者包括转录调节，有时这两种作用是重叠的。在根据本申请所提供的方法和基因表达调节分子的某些实施方案中，DNA结合域可包含(DNA)核酸酶，诸如能够以序列特异性方式靶向DNA或者能够被指导或指示以序列特异性方式靶向DNA的核酸酶，诸如CRISPR-Cas系统、锌指核酸酶(ZFN)、转录激活子样效应因子核酸酶(TALEN)或大范围核酸酶。在一些实施方案中，DNA结合域是源自CRISPR-Cas系统的DNA核酸酶。例如，该源自CRISPR-Cas系统的DNA核酸酶是Cas蛋白。
在本申请中，术语“TALE DNA结合结构域”或“TALE”是包含一个或多个TALE重复结构域/单元的多肽。天然存在的TALE或“野生型TALE”是由变形菌的众多物种分泌的核酸结合蛋白。TALE多肽含有由高度保守的单体多肽的串联重复构成的核酸结合结构域，所述单体多肽长度主要为33、34或35个氨基酸，并且主要在氨基酸位置12和13中彼此不同。在优选的实施方案中，所述核酸是DNA。如本文使用的，TALE的多肽单体用于指在TALE核酸结合结构域内高度保守的重复多肽序列，并且术语“重复可变双残基”或“RVD”用于指在多肽单体的位置12和13处高度可变的氨基酸。包含在DNA结合结构域内的TALE单体的一般表示是X1-11-(X12X13)-X14-33或34或35，其中下标指示氨基酸位置，并且X表示任何氨基酸。X12X13指示RVD。在一些TALE多肽单体中，在位置13处的可变氨基酸缺失或不存在，并且在此类单体中，RVD由单个氨基酸组成。在此类情况下，RVD可以可替代地表示为X*，其中X表示X12，并且(*)指示X13不存在。DNA结合结构域包含TALE单体的几个重复，并且这可以表示为(X1-11-(X12X13)-X14-33或34或35)z，其中在优选的实施方案中，z是至少5-40。在进一步优选的实施方案中，z是至少10-26。
TALE单体具有由在其RVD内的氨基酸类型决定的核苷酸结合亲和力。例如，具有NI的RVD的多肽单体优先与腺嘌呤(A)结合，具有NG的RVD的多肽单体优先与胸腺嘧啶(T)结合，具有HD的RVD的多肽单体优先与胞嘧啶(C)结合，并且具有NN的RVD的单体优先与腺嘌呤(A)和鸟嘌呤(G)结合。在另外一些实施方案中，具有IG的RVD的单体优先与T结合。因此，在TALE的核酸结合结构域中的多肽单体重复的数目和次序决定其核酸靶特异性。在本申请进一步的实施方案中，具有NS的RVD的单体识别所有四个碱基对，并且可以与A、T、G或C结合。TALE的结构和功能例如在Moscou等人，Science326:1501(2009)；Boch等人，Science326:1509-1512(2009)；和Zhang等人，NatureBiotechnology29:149-153(2011)中进一步描述，所述参考文献各自整体通过引用并入。TALE的重复结构域参与TALE与其同源靶DNA序列的结合。这些重复单元(或称“重复序列”)展现与天然存在的TALE蛋白内的其它TALE重复序列的至少一些序列同源性。参见例如美国专利公布号20110301073。本申请涉及的TALE结合结构域可以“工程改造”以结合于预定核苷酸序列，例如经由天然存在的TALE蛋白的识别螺旋区域的工程改造(改变一个或多个氨基酸)。因此，工程改造的DNA结合蛋白(TALE)是非天然存在的蛋白。用于工程改造DNA结合蛋白的方法的非限制性实例是设计和选择。所设计的DNA结合蛋白是非天然存在的蛋白，其设计和/或组成主要源于合理的标准。合理的设计标准包括应用替换规则和用于处理储存现有的TALE设计和结合数据的信息数据库中的信息的计算化算法。参见例如美国专利6,140,081；6,453,242；和6,534,261； 还参见WO 98/53058；WO 98/53059；WO 98/53060；WO02/016536和WO 03/016496以及美国公布号20110301073。
在本申请中，“Cas酶”可与“Cas蛋白”、“CRISPR蛋白”、“CRISPR酶”、“CRISPR-Cas蛋白”、“CRISPR-Cas酶”、“Cas”、“CRISPR效应子”或“Cas效应子蛋白”互换地使用，其通常是指与CRISPR序列互补的一类酶，能够使用CRISPR序列作为指导(guide)，从而识别和切割特定的DNA链。Cas蛋白的非限制性实例包括：Casl、CaslB、Cas2、Cas3、Cas4、Cas5、Cas6、Cas7、Cas8、Cas9(也称为Csnl和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，和/或他们的同系物、或其修饰形式。这些蛋白是已知的，例如，化脓链球菌Cas9蛋白的氨基酸序列可见于SwissProt数据库登录号Q99ZW2下。
在本申请中，术语“II类Cas核酸酶”通常是指根据CRISPR/Cas基因座的更新分类方案(Makarova等人，(2015)Nat Rev Microbiol[自然微生物学综述]，13(11):722-36；Shmakov等人，(2015)Mol Cell[分子细胞]，60:385-397)所定义的，以单一蛋白形式发挥识别和/或切割功能的一类Cas蛋白。
在本申请中，术语“II类II型Cas核酸酶和II类V型Cas核酸酶”通常是指II类Cas核酸酶中，是单蛋白的、RNA指导的内切核酸酶。在这其中，II型和V型中的V-B型Cas核酸酶需要tracrRNA(反式激活CRISPR RNA)和crRNA(CRISPR RNA)共同作用才能正常发挥功能，且crRNA和tracrRNA可以人工组合成一个单一的向导RNA(sgRNA)；V型中的V-A型Cas核酸酶则需要单独使用crRNA行使向导功能。II类II型Cas核酸酶的非限制性示例包括Cas9及其家族相关核酸酶，II类V型Cas核酸酶的非限制性示例包括Cas12a(也称为Cpf1)、Cas12b(也称为C2c1)、Cas12c(也称为C2c3)、Cas12d(CasY)、Cas12e(CasX)、Cas12g、Cas12h、Cas12i、C2c1、C2c4、C2c5、C2c8、C2c9、C2c10、Cas14a、Cas14b、Cas14c核酸酶和/或TnpB。
在本申请中，术语“dCas”可指dCas蛋白或其片段。例如，如本文中所用，“dCas9”可指dCas9蛋白或其片段。如本文中所用，术语“iCas”和“dCas”可互换使用，指无催化活性的CRISPR相关蛋白。在一个实施方案中，dCas蛋白在DNA切割结构域中包含一个或多个突变。在一个实施方案中，dCas蛋白在RuvC或结构域中包含一个或多个突变。在一个实施方案中，dCas分子在RuvC和HNH结构域中都包含一个或多个突变。在一个实施方案中，dCas蛋白是野生型Cas蛋白的片段。在一个实施方案中，dCas蛋白包含来自野生型Cas蛋白 的功能结构域，其中该功能结构域选自Reel结构域、桥螺旋结构域或PAM相互作用结构域。在一个实施方案中，与相应的野生型Cas蛋白的核酸酶活性相比，dCas的核酸酶活性降低了至少40％、至少45％、至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％或至少99％。
在本申请中，术语“能够结合”可与“结合于”、“特异性地识别”、“靶向”等互换地使用，通常是指结合分子(例如，本申请的基因表达调节分子)能够与靶基因或靶位点上的核苷酸相互作用，或者该结合分子(例如，本申请的基因表达调节分子)对靶基因或靶位点具有足够的亲和力，这种相互作用可以是通过缀合、偶联、附着、提供互补性、提供共价作用力或提供非共价作用力、提高结合稳定性等方式。
在本申请中，术语“引导RNA”、“向导DNA”和“gRNA”可互换地使用，其通常是指能够指导核酸酶(例如Argonaute，或Ago)结合和/或剪切靶标基因的DNA分子。在一些优选的实施方案中，向导DNA可以包括：为单链DNA分子(ssDNA)、为5’端磷酸化的单链DNA分子、为5’端羟基化的单链DNA分子、具有能够和靶标基因互补的碱基片段和/或具有8-35nt的长度。在本申请的一些实施方案中，术语“引导RNA”是指包含以下的RNA：(1)结合于向导RNA指导的核酸内切酶(例如II类Cas核酸酶，例如II型、V型或VI型Cas核酸内切酶)且活化RNA指导的核酸内切酶的“活化”核苷酸序列；和(2)包含与靶核酸杂交的核苷酸序列的“靶”核苷酸序列。“活化”核苷酸序列和“靶”核苷酸序列可以在分开的RNA分子(例如“双向导RNA”)上；或可以在相同的RNA分子(“单向导RNA”，也称为sgRNA)上。
在本申请中，术语“DNA甲基转移酶”通常是指催化甲基转移至DNA的酶。DNA甲基转移酶的非限制性实例包括DNMT1、DNMT 3A、DNMT 3B和DNMT 3L。例如，通过DNA甲基化，DNA甲基转移酶可以在不更改DNA序列的情况下修饰DNA片段的活性(例如调控基因表达)。如本文所述，基因表达调节分子可以包括一个或多个(例如两个)DNA甲基转移酶。当DNA甲基转移酶作为基因表达调节分子的一部分包括在内时，DNA甲基转移酶可以被称为“DNA甲基转移酶结构域”。在各方面中，DNA甲基转移酶结构域包含与DNMT 3A具有至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列一致性的氨基酸序列的变异体或同源物。在各方面中，DNA甲基转移酶结构域包含与DNMT 3L具有至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少 94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列一致性的氨基酸序列的变异体或同源物。
在本申请中，术语“功能活性片段”通常是指具有全长蛋白质或核酸的部分区域，但保留或部分保留全长蛋白质或核酸的生物活性或功能的片段。例如，功能活性片段可以保留或部分保留全长蛋白质结合另一种分子的能力。例如，DNA甲基转移酶的功能活性片段，可以保留或部分保留全长DNA甲基转移酶的催化甲基基团转移到DNA的生物活性功能。
在本申请中，术语“转录阻遏物”通常是指结合靶核酸序列且导致与靶核酸序列有关的基因产物的表达水平降低的物质和/或试剂，如蛋白质(例如转录因子或其片段)。例如，所述基因产物可以是从基因转录的RNA(例如mRNA)或从自基因转录的mRNA翻译的多肽。通常mRNA水平中的增加或降低导致从其翻译的多肽水平的增加或降低。可以使用测量mRNA或蛋白的标准技术来测定表达水平。非限制性的转录阻遏物实例包括：mSin3相互作用结构域(SID)蛋白、甲基-CpG-结合结构域2(MBD2)、MBD3、DNA甲基转移酶(DNMT)1(DNMT1)、DNMT2A、DNMT3A、DNMT3B、DNMT3L、视网膜母细胞瘤蛋白(Rb)、甲基CpG结合蛋白2(Mecp2)、GATA-1及其辅助因子Fog1、MAT2调节剂(ROM2)、拟南芥HD2A蛋白(AtHD2A)、赖氨酸特异性的脱甲基酶1(LSD1)和/或Krüppel-相关盒(KRAB)。
在本申请中，术语“KRAB”也称为“克鲁珀相关盒结构域”或“Krüppel相关盒结构域”，其通常是指存在于人锌指蛋白的转录因子中的转录抑制结构域的约45至约75个氨基酸残基。在各方面中，KRAB结构域可以包括与ZIM3KRAB结构域或KOX1KRAB结构域具有至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列一致性的氨基酸序列的变异体或同源物。
在本申请中，术语“分裂绿色荧光蛋白”通常是指能够分裂并在重新组合时立即形成活性绿色荧光蛋白的多肽。
在本申请中，术语“GCN4”是酿酒酵母(S.cerevisiae)中的一种转录因子，是酵母基因组中的“主调节因子”("master regulator")，调节接近十分之一的酵母基因组，它是一种高度保守蛋白，其在哺乳动物中的同源物是转录激活因子(Activating Transcription factor)-4(ATF4)。
在本申请中，术语“PDZ蛋白”通常是指天然存在的含有PDZ结构域的蛋白。示例性的PDZ蛋白包括CASK、MPPl、DLGl、DLG2、PSD95、NeDLG、TIP-33、SYNla、TIP-43、LDP、LIM、LIMK1、LIMK2、MPP2、N0S l、AF6、PTN_4、prIL16、41.8kD、KIAA0559、RGS 12、KIAA0316、DVL1、TIP-40、TIAMl、MINTl、MAGI-I、MAGI-2、MAGI-3、KIAA0303、 CBP、MINT3、TIP-2、KIAA0561和/或TIP-I。
在本申请中，术语“单链抗体”或“scFv(Single Chain Antibody)”通常是指含有一个或多个抗原结合部位的单链多肽。另外，尽管Fv片段的H和L链是由不同的基因编码的，它们可直接或通过肽而连接在一起，例如，通过重组的方法，可用合成的衔接物(linker)将H和L链连接成单一蛋白链(称为单链抗体，sAb；Bird et al.1988Science242：423-426；and Huston et al.1988PNAS 85：5879-5883)。该单链抗体也被包括在术语“抗体”之中，可在设计和制造多特异性结合分子中用作结合决定簇，并且通过重组技术或完整抗体的酶促或化学切割可制备所述单链抗体。
在本申请中，术语“直接或间接结合”通常是指相对的“直接相连”或“间接相连”。“直接相连”通常是指直接连接。例如，所述直接相连可以为相连的物质(例如氨基酸序列区段)之间没有间隔成分(例如氨基酸残基或其衍生物)而直接相连接的情况；例如氨基酸序列区段X与另一氨基酸序列区段Y通过氨基酸序列区段X的C端氨基酸与氨基酸序列区段Y的N端氨基酸形成的酰胺键直接连接。“间接相连”通常是指相连的物质(例如氨基酸序列区段)之间有间隔成分(例如氨基酸残基或其衍生物)而间接相连接的情况。例如，本申请所用的间隔成分可以是一段氨基酸残基，其序列选自SEQ ID NO:125-132(SEQ ID NO:126为GSG)中任一项所示的氨基酸序列。
在本申请中，“核定位序列”或“NLS”通常是指将蛋白导向至细胞核的肽。在某些实施方案中，NLS包括五个碱性带正电的氨基酸。NLS可以位于肽链上的任何位置。在某些实施方案中，NLS为衍生自SV40的NLS。在某些实施方案中，NLS包括SEQ ID NO:338-340中任一项所示的序列。在一些实施方案中，NLS具有与SEQ ID NO:338-340中任一项至少50％、55％、60％、65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列一致性的氨基酸序列。
在本申请中，术语“标记物”指的是肽，可以将其引入表达载体中，其可以用来允许一个或多个载体插入片段的表达产物的删除和/或纯化。这样的标记物是本领域公知的并包括放射性标记的氨基酸或连接可以通过标记的抗生物素蛋白(例如，含有可以通过光学或比色方法检测的荧光标记或酶活性的抗生蛋白链菌素)检测的生物素部分的多肽。亲和性标记物如FLAG，谷胱甘肽-S-转移酶，麦芽糖结合蛋白，纤维素结合结构域，硫氧还蛋白，NusA，mistin，几丁质结合结构域，角质酶，AGT，GFP和广泛使用的其它标记物，如蛋白表达和纯化系统中所用的。用于多肽的更多非限制性实例包括，但不限于以下的：组氨酸标记物，放射性同位素或放射性核素(利如，3H，14C，35S，90Y，99Tc，111In，125I，177Lu，166Ho或153Sm)； 荧光标记物(例如，FITC，若丹明，镧系磷)，酶标记物(例如，辣根过氧化物酶，莹光素酶，碱性磷酸酶)；化学发光标记物；生物素基团；由第二报告子识别的悬垂多肽抗原决定部位(例如，亮氨酸拉链对序列，用于二抗的结合位点，金属结合结构域，抗原决定部位标记物)；和磁性试剂，如钆螯合物。
在本申请中，术语“核酸”与“多核苷酸”、“核苷酸”、“核苷酸序列”和“寡核苷酸”可互换地使用，其通常是指核苷酸(例如，脱氧核糖核苷酸或核糖核苷酸)和其呈单链、双链或多链形式的聚合物或其互补物。例如，核苷酸可以为核糖核苷酸、脱氧核糖核苷酸或其修饰版本。例如，核苷酸可以为单链和双链DNA、单链和双链RNA以及具有单链和双链DNA和RNA的混合物的杂交分子。例如，核苷酸可以包括但不限于任何类型的RNA，例如mRNA、siRNA、miRNA、sgRNA和引导RNA，以及任何类型的DNA、基因组DNA、质粒DNA和微环DNA以及其任何片段。所述术语还涵盖含有已知核苷酸类似物或经修饰的主链残基或键的核酸，所述核酸为合成的、天然存在的和非天然存在的。
在本申请中，术语“编码……的序列”或“编码……的核酸”通常是指包含编码蛋白质的核苷酸序列的核酸(RNA或DNA分子)。编码序列还可包括与调控元件可操作地连接的起始和终止信号，所述调控元件包含能够在对其施用了核酸的个体或哺乳动物的细胞中指导表达的启动子和多腺苷酸化信号。可对编码序列进行密码子优化。在本申请中，术语“内含子”通常是指包括经过转录的，但却从RNA转录本中通过将序列(外显子)两端的任一端拼接在一起而被去除的DNA片段。内含子被认为是基因的蛋白编码区内的干扰序列，且通常不含有由该基因产生的蛋白所代表的信息。
在本申请中，术语“重组载体”通常是指能够转运它以及与之连接的另一种核酸的核酸分子。一种类型的载体是“质粒”，这指另外的DNA区段可以连接到其内的环状双链DNA环。可替代地，载体可以是线性的。另一种类型的载体是病毒载体，其中另外的DNA区段可以连接到病毒基因组内。特定载体能够在它们引入其内的宿主细胞内自主复制(例如，具有细菌复制起点的细菌载体和附加型哺乳动物载体)。其他载体(例如，非附加型哺乳动物载体)可以在引入宿主细胞内后整合到宿主细胞的基因组内，并且从而连同宿主基因组一起复制。
在本申请中，术语“调节元件”是指能够控制核酸序列表达的遗传元件。例如，剪接信号、启动子序列、多腺苷酸化信号、转录终止序列、上游调节结构域、复制起点、内部核糖体进入位点(“IRES”)、增强子等，它们共同提供了编码序列在受体细胞中的复制、转录和翻译。并非所有这些控制序列都需要存在。
在本申请中，术语“启动子”通常是指控制或调节与启动子可操作地关联的核苷酸序列 (例如，编码序列)的转录的核苷酸序列。由启动子控制或调节的编码序列可以编码多肽和/或功能性RNA。通常，“启动子”是指包含RNA聚合酶II结合位点并指导转录起始的核苷酸序列。通常，启动子位于相对于相应编码序列的编码区起点的5’或上游。启动子可以包含其他作为基因表达调节因子的元件；例如，启动子区域。在一些实施方案中，启动子区域可包括至少一个内含子。启动子可以包括例如组成型、诱导型、时间调节的、发育调节的、化学调节的、组织优选的和/或组织特异性的启动子，用于制备重组核酸分子，例如“合成核酸构建体”或“蛋白质-RNA复合物”。这些不同类型的启动子是本领域已知的。
在本申请中，术语“增强子”通常是指可以被蛋白质(激活蛋白)结合以刺激或增强一种基因或若干种基因的转录的调控DNA序列，例如50-1500bp。这些激活蛋白(又称转录因子)与介体复合物相互作用并募集聚合酶II和一般转录因子，然后开始转录基因。增强子通常是顺式作用的，但可以位于基因或它们调控的基因的起始位点的上游或下游。此外，增强子可以是向前或向后方向，并且不需要位于转录起始位点附近以影响转录，因为已发现一些增强子位于起始位点上游或下游的数十万个碱基对。内含子中也可以发现增强子。
在本申请中，术语“剪切肽”是指能够实现剪切蛋白的功能的一类多肽。例如，所述剪切肽可以经核糖体跳跃而非蛋白酶水解来实现蛋白质剪切。例如，所述剪切肽可为剪切2A肽，其可包括T2A，F2A、P2A和/或E2A。
在本申请中，术语“递送载体”通常是指能够将试剂(例如，核酸分子)递送至靶细胞的转移媒介物。递送载体可以将试剂递送到特定的细胞亚类。例如，借助递送载体的固有特征或者通过与载体相偶联的部分、包含在其内的部分(或者与载体结合的部分，从而使得该部分和该递送载体维持在一起，进而使得该部分足以靶向递送载体)使递送载体靶向某些类型的细胞。递送载体还可提高要递送的试剂的体内半衰期和/或要递送的试剂的生物利用度。递送载体可包括病毒载体、病毒样颗粒、聚阳离子载体、肽载体、脂质体和/或杂交载体。例如，如果靶细胞是肝细胞，所述递送载体的性质(例如，尺寸、电荷和/或pH)可以有效地将所述递送载体和/或其中包载的分子递送至靶细胞、降低免疫清除和/或促进在该靶细胞中停留。
在本申请中，术语“脂质体”通常是指通过一个或多个双层的膜与外部介质隔离的具有内部空间的囊泡。在一些实施方案中，所述双层的膜可以通过两性分子形成，如包含空间隔离的亲水性和疏水性结构域的合成或天然来源的脂质；在另一些实施方案中，所述双层的膜可以通过两亲性聚合物和表面活性剂形成。在一些实施方案中，所述脂质体是球形囊泡结构，其由围绕内部水性区室的单层或多层脂质双分子层、和相对不可渗透的外部亲脂性磷脂双分子层组成。在一些实施方案中，脂质体是生物相容的、无毒的，可以递送亲水性和亲脂性药 物分子，保护它们的运载物不被血浆酶降解，并且将它们的负载运输穿过生物膜和血脑屏障(BBB)。脂质体可由几种不同类型的脂质例如磷脂制成。脂质体可包含天然磷脂和脂质(诸如1,2-二硬脂酰基-sn-甘油-3-磷脂酰胆碱(DSPC)、鞘磷脂、卵磷脂酰胆碱、单唾液酸神经节苷脂或其任意组合。为了改变脂质体的结构和性质，可向脂质体中加入几种其它添加剂。例如，脂质体还可包含胆固醇、鞘磷脂和/或1,2-二油酰基-sn-甘油-3-磷酸乙醇胺(DOPE)，例如，以增加稳定性和/或防止脂质体内部运载物的泄漏。
术语“脂质纳米颗粒(LNP)”通常是指包含通过分子间力彼此物理结合(例如，共价或非共价)的多个(即多于一个)脂质分子的颗粒。LNP可以是例如微球(包括单层和多层囊泡，例如脂质体)、乳液中的分散相、胶团或悬浮液中的内相。LNP可将核酸包封在阳离子脂质颗粒(例如，脂质体)内，并且可被相对容易地递送至细胞。在一些实例中，脂质纳米颗粒不含任何病毒组分，这有助于最小化安全性和免疫原性问题。所述脂质颗粒可用于体外、离体和体内递送。所述脂质颗粒还可用于各种规模的细胞群。本申请的LNP可通过本领域已知的各种方法，例如通过混合有机相与水相来容易地制备。两相的混合可通过微流体装置和撞击流反应器来实现。有机相和水相混合越充分，获得的LNP的包埋率和粒径分布就越好。优选地，LNP的粒径可通过改变有机相与水相的混合速度来调节。混合速度越快，制备的LNP的粒径将越小。包埋效率可通过调节LNP系统的N/P(可电离脂质/核酸)比值来优化。在一些实例中，LNP可用于递送DNA分子和/或RNA分子(例如，Cas、sgRNA的mRNA)。在某些情况下，LNP可用于递送Cas/gRNA的RNP复合物。在一些实施方案中，LNP用于递送mRNA和gRNA。
在本申请中，术语“受试者”通常是指动物，通常是哺乳动物，诸如人、非人灵长类动物(猿、长臂猿、大猩猩、黑猩猩、猩猩、猕猴)、家畜(狗和猫)、农场动物(家禽如鸡和鸭、马、牛、山羊、绵羊、猪)和实验动物(小鼠、大鼠、兔、豚鼠)。人受试者包括胎儿、新生儿、婴儿、青少年和成人受试者。受试者包括动物疾病模型，例如小鼠和血液凝固疾病(诸如HemA)的其它动物模型，和本领域技术人员已知的其它动物模型。
在本申请中，术语“包含”通常是指包括明确指定的特征，但不排除其他要素。
在本申请中，术语“选自”通常是指包括选择的对象以及其所有组合。例如“选自(：)A、B和C”意指包括A、B和C的所有组合，例如，A、B、C、A+B、A+C、B+C或A+B+C。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下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％、或10％的范围内变动。
发明详述
一方面，本申请提供一种复合物，其包含第一融合物和第二融合物，所述第一融合物和所述第二融合物其中之一的融合物包含DNA甲基化结构域和至少一个招募结构域A，并且其中另一个融合物包含转录阻遏物结构域和至少一个招募结构域A’；其中，所述招募结构域A和招募结构域A’能够相互作用，以使所述第一融合物和所述第二融合物其中之一的融合物或其部分能够被招募到另一个融合物附近。
另一方面，本申请提供编码本申请所述复合物的核酸。例如，所述核酸包含DNA和/或mRNA。例如，所述核酸可用于治疗或缓解与靶基因表达异常和/或靶基因活性异常相关的疾病或其病症。在一些实施方案中，所述核酸为mRNA；可使用一种或多种修饰技术用于产生更稳定的mRNA。已知的mRNA修饰技术大致可分为三类：用人工合成的非天然核糖核酸代替天然核糖核酸合成mRNA；添加5’caps、3’poly(A)“尾”和UTR(未翻译区)序列；采用特殊的新型配方技术，有效保护mRNA。其中，优选的mRNA修饰技术可以通过人工合成非天然核糖核酸取代天然核糖核酸合成mRNA。真核mRNA上的化学修饰大致可以分为三类：甲基化、伪尿苷(Ψ)和次黄嘌呤。例如，所述化学修饰可选自：假尿苷、N1-甲基假尿苷、N1-乙基假尿苷、2-硫代尿苷、4’-硫代尿苷、5-甲基胞嘧啶、2-硫代-1-甲基-1-脱氮-假尿苷、2-硫代-1-甲基-假尿苷、2-硫代-5-氮杂-尿苷、2-硫代-二氢假尿苷、2-硫代-二氢尿苷、2-硫代-假尿苷、4-甲氧基-2-硫代-假尿苷、4-甲氧基-假尿苷、4-硫代-1-甲基-假尿苷、4-硫代-假尿苷、5-氮杂-尿苷、二氢假尿苷、5-甲基尿苷、5-甲氧基尿苷和2’-O-甲基尿苷。例如，所述核酸为重组载体，所述重组载体包含编码本申请所述复合物的核酸。例如，重组载体可以是指能够转运与其连接的另一种核酸的核酸分子。重组载体可以包括单链、双链或部分双链的核酸分子；包含一个或多个游离端，没有游离端(例如，环状)的核酸分子；包含DNA、RNA或两者的核酸分子；和本领域已知的其他种类的多核苷酸。例如，可以使用病毒载体。病毒载体可包含病毒衍生的DNA或RNA序列，用于包装成病毒(例如逆转录病毒、复制缺陷型逆转录病毒、腺病毒、复制缺陷型腺病毒、和腺相关病毒AAV)。病毒和病毒载体可用于体外、离体和/或体内递送。
另一方面，本申请提供一种递送载体，所述递送载体包含本申请所述的复合物和/或本申请所述的核酸，以及任选地包含脂质体和/或脂质纳米颗粒。例如，可以通过物理递送方法将递送载体引入细胞。物理方法的例子包括显微注射、电穿孔和流体动力学递送。例如，LNPs可以将核酸包裹在阳离子脂质颗粒(例如脂质体)中，并且可以相对容易地递送至细胞。在一些例子中，脂质纳米颗粒不含任何病毒成分，这有助于最大限度地减少安全性和免疫原性问题。 脂质颗粒可用于体外、离体和体内递送。LNP的成分可包括阳离子脂质，可电离的脂质，聚乙二醇化脂质和/或支持脂质，以及任选的胆固醇组分。
另一方面，本申请提供一种组合物，所述组合物包含本申请所述的复合物、本申请所述的核酸、和/或本申请所述的递送载体。例如，组合物中的复合物、编码所述复合物的核酸(或重组载体)和递送载体可以同时包含在一个组合物中，或者分别包含在不同组合物中。例如，当使用组合物中的复合物、编码所述复合物的核酸(或重组载体)和/或递送载体时，可以同时使用，或者分开使用。
另一方面，本申请提供一种细胞，所述细胞包含本申请所述的复合物、本申请所述的核酸、本申请所述的递送载体、和/或本申请所述的组合物。
另一方面，本申请提供一种试剂盒，所述试剂盒包含本申请所述的复合物、本申请所述的核酸、本申请所述的递送载体、本申请所述的组合物、和/或本申请所述的细胞。例如，所述试剂盒还包含至少一个放置上述组分的容器。例如，所述试剂盒包含一种以上的上述组分，其还包含所述容器以外的第二个、第三个和/或其他容器，其内可以分开放置所述一种以上的上述组分。例如，所述试剂盒可以在容器中放置上述组分的各种组合形式。例如，所述试剂盒还进一步包括缓冲试剂、用于混合的装置、用于测量的装置、用于分选的装置和/或用于标记的装置。例如，所述试剂盒还包括用于容纳各种容器的包装。例如，所述试剂盒还包括关于使用试剂盒组分的说明书。例如，所述说明书包括纸质的实体形式和/或可机读的电子形式。
另一方面，本申请提供一种调控靶基因表达的方法，所述方法包括施用本申请所述的复合物、本申请所述的核酸、本申请所述的递送载体、本申请所述的组合物、本申请所述的细胞和/或本申请所述的试剂盒。例如，所述抑制靶基因表达的方法是将所述复合物、所述核酸、所述递送载体、所述组合物、所述细胞和/或所述试剂盒引入含有靶基因的细胞。例如，所述引入细胞可以是使用非病毒或基于病毒的转染方式引入细胞。例如，所述非病毒转染方法包括不使用病毒DNA或病毒颗粒作为递送系统引入细胞的任何适当方法，非限制性的非病毒转染方法示例包括编码复合物的核酸的纳米颗粒封装(例如脂质纳米颗粒、金纳米颗粒等)、磷酸钙转染、脂质体转染、核转染、声穿孔、通过热休克转染、磁转染和电穿孔。例如，基于病毒的转染方法包括任何适用于本申请所述方法的病毒载体，其非限制性示例包括但不限于反转录病毒、腺病毒、慢病毒和/或腺相关病毒载体。例如，所述抑制靶基因表达的方法还包括将所述复合物、所述核酸、所述递送载体、所述组合物、所述细胞和/或所述试剂盒从外部环境引入细胞中。再例如，所述抑制靶基因表达的方法包括使所述复合物、所述核酸、所述递送载体、和/或所述组合物接触靶基因附近和/或所述靶基因的转录调控元件。例如，所述接触 是指使本申请所述的第一融合物、第二融合物以及向导RNA与靶基因附近和/或所述靶基因的转录调控元件接触，并且向导RNA与包含DNA结合结构域的融合物形成复合物，该复合物特异识别靶基因中的特定区域并与之杂交，同时第一融合物和第二融合物通过其招募结构域A和招募结构域A’直接或间接的相互作用而被募集到DNA结合结构域附近，从而对靶核酸的表达进行调控。例如，所述方法包括使本申请所述的第一融合物、第二融合物与向导RNA以复合物(例如，组装的核糖核蛋白复合物)的形式存在，并且使该复合物接触靶基因附近和/或所述靶基因的转录调控元件。
另一方面，本申请提供一种治疗或缓解与靶基因表达异常和/或靶基因活性异常相关的疾病或其病症的方法，所述方法包括向有需要的受试者施用有效量的本申请所述的复合物、本申请所述的核酸、本申请所述的递送载体、本申请所述的组合物、本申请所述的细胞和/或本申请所述的试剂盒。例如，所述治疗方法包含将所述复合物、所述核酸、所述递送载体、所述组合物、所述细胞和/或所述试剂盒与治疗剂混合，并对有需要的受试者进行全身递送，使其广泛暴露于身体的大部分，可以通过本领域已知的任何手段进行，包括但不限于向静脉内、动脉内、皮下、腔内和腹膜内递送。例如，所述治疗方法包含将所述复合物、所述核酸、所述递送载体、所述组合物、所述细胞和/或所述试剂盒与治疗剂混合，并对有需要的受试者进行局部递送，使其直接到达有机体内的靶部位，可以通过例如直接注射到疾病部位(如，肿瘤或炎症部位)或靶器官(如，肝脏、心脏、胰腺、肾脏等)中来进行。例如，所述局部递送包括局部施用或局部注射技术，包括但不限于肌内、皮下或皮内注射。例如，所述局部递送不排除全身性的药理学作用。例如，所述疾病包括心血管疾病、非酒精性脂肪肝炎、AMD、老年黄斑病变、2型糖尿病、肥胖症、肝衰竭、血脂异常(dislipidemia)、糖尿病性动脉粥样硬化和/或高血压。
另一方面，本申请提供本申请所述的复合物、本申请所述的核酸、本申请所述的递送载体、本申请所述的组合物、本申请所述的细胞和/或本申请所述的试剂盒用于制备药物的用途，所述药物用于治疗或缓解与靶基因表达异常和/或靶基因活性异常相关的疾病或其病症。
另一方面，本申请提供本申请所述的复合物、本申请所述的核酸、本申请所述的递送载体、本申请所述的组合物、本申请所述的细胞或本申请所述的试剂盒，其用于治疗或缓解与靶基因表达异常和/或靶基因活性异常相关的疾病或其病症。
第一融合物或第二融合物
在一些实施方案中，本申请复合物的第一融合物和第二融合物总体上可分为两种情形：(1)两个融合物其中之一包含核酸结合结构域、DNA甲基化结构域和招募结构域A，且另 一个融合物包含转录阻遏物结构域和招募结构域A’，或者(2)两个融合物其中之一包含核酸结合结构域、转录阻遏物和招募结构域A，且另一个融合物包含DNA甲基化结构域和招募结构域A’。
具体地，在上述(1)情形下的一些实施方案中，所述两个融合物其中之一从N端到C端依次可包含DNA甲基化结构域、核酸结合结构域和招募结构域A。例如，在上述(2)情形下的一些实施方案中，所述两个融合物其中之一从N端到C端依次可包含招募结构域A、核酸结合结构域和转录阻遏物结构域。例如，在上述(1)情形下的一些实施方案中，所述两个融合物中的另一个融合物从N端到C端依次可包含转录阻遏物结构域和招募结构域A’，或者招募结构域A’和转录阻遏物结构域，即转录阻遏物结构域和招募结构域A’可互换顺序地连接。例如，在上述(2)情形下的一些实施方案中，所述两个融合物中的另一个融合物从N端到C端依次可包含DNA甲基化结构域和招募结构域A’，或者招募结构域A’和DNA甲基化结构域，即DNA甲基化结构域和招募结构域A’可互换顺序地连接。
在一些更具体的实施方案中，所述核酸结合结构域为DNA结合结构域。例如，所述DNA结合结构域可选自：TALE结构域、锌指结构域、tetR结构域、大范围核酸酶、Cas蛋白、Argonaute(Ago)蛋白，以及其同系物、修饰形式或变体。例如，所述DNA结合结构域可以是Cas蛋白，且所述Cas蛋白为II类Cas核酸酶。进一步地，所述Cas蛋白可选自II类II型Cas核酸酶和II类V型Cas核酸酶；例如，所述Cas蛋白可以是Cas9或Cas12蛋白。在某些实施方案中，所述Cas蛋白可为失活Cas9(dCas9)蛋白或失活Cas12(dCas12)蛋白。例如，本申请所述DNA结合结构域可包含但不限于SEQ ID NO:1-9、343和344中任一项所示的氨基酸序列。
在一些更具体的实施方案中，所述转录阻遏物选自下列所示结构域中的一种或多种：KRAB，ZIM3，ZNF680，ZNF554，ZNF264，ZNF582，ZNF324，ZNF669，ZNF354A，ZNF82，ZNF595，ZNF419，ZNF566，ZIM2，EHMT2，SUV39H1，ZFPM1，TRIM28，EZH2，MXD1，SID，LSD1，HP1a，HDAC3，HDAC1，PRMT1，SETDB1，hSIRT1，ZNF436，ZNF257，ZNF675，ZNF490，ZNF320，ZNF331，ZNF816，ZNF41，ZNF189，ZNF528，ZNF543，ZNF140，ZNF610，ZNF350，ZNF8，ZNF30，ZNF98，ZNF677，ZNF596，ZNF214，ZNF37A，ZNF34，ZNF250，ZNF547，ZNF273，ZFP82，ZNF224，ZNF33A，ZNF45，ZNF175，ZNF184，ZFP28-1，ZFP28-2，ZNF18，ZNF213，ZNF394，ZFP1，ZFP14，ZNF416，ZNF557，ZNF729，ZNF254，ZNF764，ZNF785，ZNF10，CBX5，RYBP，YAF2，MGA，CBX1，SCMH1，MPP8，SUMO3，HERC2，BIN1，PCGF2，TOX，FOXA1，FOXA2，IRF2BP1，IRF2BP2，IRF2BPL IRF-2BP1_2N-terminal  domain，HOXA13，HOXB13，HOXC13，HOXA11，HOXC11，HOXC10，HOXA10，HOXB9，HOXA9，ZFP28，ZN334，ZN568，ZN37A，ZN181，ZN510，ZN862，ZN140，ZN208，ZN248，ZN571，ZN699，ZN726，ZIK1，ZNF2，Z705F，ZNF14，ZN471，ZN624，ZNF84，ZNF7，ZN891，ZN337，Z705G，ZN529，ZN729，ZN419，Z705A，ZN302，ZN486，ZN621，ZN688，ZN33A，ZN554，ZN878，ZN772，ZN224，ZN184，ZN544，ZNF57，ZN283，ZN549，ZN211，ZN615，ZN253，ZN226，ZN730，Z585A，ZN732，ZN681，ZN667，ZN649，ZN470，ZN484，ZN431，ZN382，ZN254，ZN124，ZN607，ZN317，ZN620，ZN141，ZN584，ZN540，ZN75D，ZN555，ZN658，ZN684，RBAK，ZN829，ZN582，ZN112，ZN716，HKR1，ZN350，ZN480，ZN416，ZNF92，ZN100，ZN736，ZNF74，ZN443，ZN195，ZN530，ZN782，ZN791，ZN331，Z354C，ZN157，ZN727，ZN550，ZN793，ZN235，ZN724，ZN573，ZN577，ZN789，ZN718，ZN300，ZN383，ZN429，ZN677，ZN850，ZN454，ZN257，ZN264，ZN485，ZN737，ZNF44，ZN596，ZN565，ZN543，ZFP69，SUMO1，ZNF12，ZN169，ZN433，ZN175，ZN347，ZNF25，ZN519，Z585B，ZN517，ZN846，ZN230，ZNF66，ZN713，ZN816，ZN426，ZN674，ZN627，ZNF20，Z587B，ZN316，ZN233，ZN611，ZN556，ZN234，ZN560，ZNF77，ZN682，ZN614，ZN785，ZN445，ZFP30，ZN225，ZN551，ZN610，ZN528，ZN284，ZN418，ZN490，ZN805，Z780B，ZN763，ZN285，ZNF85，ZN223，ZNF90，ZN557，ZN425，ZN229，ZN606，ZN155，ZN222，ZN442，ZNF91，ZN135，ZN778，ZN534，ZN586，ZN567，ZN440，ZN583，ZN441，ZNF43，ZN589，ZN563，ZN561，ZN136，ZN630，ZN527，ZN333，Z324B，ZN786，ZN709，ZN792，ZN599，ZN613，ZF69B，ZN799，ZN569，ZN564，ZN546，ZFP92，ZN723，ZN439，ZFP57，ZNF19，ZN404，ZN274，CBX3，ZN250，ZN570，ZN675，ZN695，ZN548，ZN132，ZN738，ZN420，ZN626，ZN559，ZN460，ZN268，ZN304，ZN605，ZN844，SUMO5，ZN101，ZN783，ZN417，ZN182，ZN823，ZN177，ZN197，ZN717，ZN669，ZN256，ZN251，CBX4，CDY2，CDYL2，ZN562，ZN461，Z324A，ZN766，ID2，ZN214，CBX7，ID1，CREM，SCX，ASCL1，ZN764，SCML2，TWST1，CREB1，TERF1，ID3，CBX8，GSX1，NKX22，ATF1，TWST2，ZNF17，TOX3，TOX4，ZMYM3，I2BP1，RHXF1，SSX2，I2BPL，ZN680，TRI68，HXA13，PHC3，TCF24，HXB13，HEY1，PHC2，ZNF81，FIGLA，SAM11，KMT2B，HEY2，JDP2，HXC13，ASCL4，HHEX，GSX2，ETV7，ASCL3，PHC1，OTP，I2BP2，VGLL2，HXA11，PDLI4，ASCL2，CDX4，ZN860，LMBL4，PDIP3，NKX25，CEBPB，ISL1，CDX2，PROP1，SIN3B，SMBT1，HXC11，HXC10，PRS6A，VSX1，NKX23，MTG16，HMX3，HMX1，KIF22，CSTF2，CEBPE，DLX2，PPARG，PRIC1，UNC4，BARX2，ALX3，TCF15，TERA， VSX2，HXD12，CDX1，TCF23，ALX1，HXA10，RX，CXXC5，SCML1，NFIL3，DLX6，MTG8，CEBPD，SEC13，FIP1，ALX4，LHX3，PRIC2，MAGI3，NELL1，PRRX1，MTG8R，RAX2，DLX3，DLX1，NKX26，NAB1，SAMD7，PITX3，WDR5，MEOX2，NAB2，DHX8，CBX6，EMX2，CPSF6，HXC12，KDM4B，LMBL3，PHX2A，EMX1，NC2B，DLX4，SRY，ZN777，ZN398，GATA3，BSH，SF3B4，TEAD1，TEAD3，RGAP1，PHF1，GATA2，FOXO3，ZN212，IRX4，ZBED6，LHX4，SIN3A，RBBP7，NKX61，R51A1，MB3L1，DLX5，NOTC1，TERF2，ZN282，RGS12，ZN840，SPI2B，PAX7，NKX62，ASXL2，FOXO1，GATA1，ZMYM5，LRP1，MIXL1，SGT1，LMCD1，CEBPA，SOX14，WTIP，PRP19，NKX11，RBBP4，DMRT2，SMCA2，以及其功能活性片段。
在一些更具体的实施方案中，所述DNA甲基化结构域包含至少一种DNA甲基转移酶或其功能活性片段。例如，所述DNA甲基转移酶选自DNMT3A、DNMT3B、DNMT3c、DNMT1、DNMT2和DNMT3L。例如，所述DNA甲基化结构域包含至少一个DNMT3A和至少一个DNMT3L。例如，所述至少一个DNMT3A和所述至少一个DNMT3L可互换顺序地连接。例如，所述DNA甲基化结构域包含一个DNMT3A和一个DNMT3L，且它们可互换顺序地连接。例如，所述DNA甲基转移酶包含SEQ ID NO:19-24任一项所示的氨基酸序列。
本申请复合物的第一融合物和第二融合物是通过各自包含的招募结构域间的相互作用进而形成聚集的复合物。因此，本申请提供了非限制性的招募结构域A和招募结构域A’的组合示例：(1)所述招募结构域A和所述招募结构域A’其中之一的结构域为GCN4，并且其中另一个结构域为scFv；或(2)所述招募结构域A和所述招募结构域A’其中之一的结构域为GFP11片段，并且其中另一个结构域为GFP1-10；或(3)所述招募结构域A和所述招募结构域A’其中之一的结构域为GVKESLV，并且其中另一个结构域为PDZ蛋白结构域。同理于GFP11和GFP1-10分别源自分裂GFP(SEQ ID NO:15)形成所述招募结构域A和所述招募结构域A’的情形同样可适用于其他类别的荧光蛋白，例如mCherry(SEQ ID NO:16)、eYFP(SEQ ID NO:18)、eCFP(SEQ ID NO:17)等，即可通过分裂mCherry、分裂eYFP、或分裂eCFP分别获取不同组的招募结构域A和招募结构域A’用于本申请提供的复合物中。在一些实施方案中，本申请复合物的所述第一融合物和所述第二融合物其中之一可包含两个或两个以上的招募结构域，且它们是通过接头序列连接的。示例性的招募结构域的氨基酸序列可包含如SEQ ID NO:10-14中任一项所示。
综合以上情形，本申请可提供下列所示的第一融合物或第二融合物的氨基酸序列：


















































不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的复合物、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例1
设计和构建包含本申请的复合物的质粒
带有HA表位、P2A的表观修饰招募系统和被招募元件(包括Dnmt3a CD，Dnmt3l CD，dSpCas9或TALE，KRAB)的氨基酸序列是由Genscript公司优化成适于哺乳动物表达的核酸序列并合成，然后克隆到带有CAG启动子和WPRE的pLV-CAG载体，由CAG启动子表达被招募元件和自剪切的招募系统融合蛋白。
在优化不同功能元件时，不同功能元件是由Genscript公司优化成适于哺乳动物表达的核酸序列并合成。先通过PCR将需要替换的元件以外的载体扩增，再将需要替换的元件从公司合成的序列上扩增，同时引入同源臂序列，最后通过NEBuilder试剂将不同元件重组到载体中，构建成最终的表达质粒。
实施例2
本申请的复合物对PTP1b基因表达的抑制作用
将包含本申请复合物(具有SEQ ID NO:51所示氨基酸序列的第一融合物与具有SEQ ID  NO:52所示氨基酸序列的第二融合物结合而成的复合物)的编辑工具和不同靶向到小鼠PTP1b基因的gRNA(sg1、sg2，或者sg1和sg2等量混合)，gRNA靶向序列的互补核苷酸序列如SEQ ID NO:336和337所示，以及对照gRNA(NT gRNA，其靶向序列的互补核苷酸序列如SEQ IF NO:365所示)共同转染到小鼠N2a细胞系(700ng编辑器+300ng gRNA，24孔板)，转染72小时后分选转染阳性的细胞，用Trizol提取总RNA，通过qPCR定量PTP1b的相对表达量，计算不同工具和不同gRNA的相对抑制效率(图2)。结果表明，相比较于核酸结合结构域dCas9、甲基化因子DNMT3A和DNMT3L、以及转录阻遏物KRAB直接融合在一起的对照组(图2右)实施方案(DNMT3A-DNMT3L-dCas9-KRAB)，本申请的复合物对靶向PTP1b基因的gRNA，即sg1和/或sg2，均获得了更高或相当的抑制效率。
实施例3
本申请的复合物对PCSK9基因表达的抑制作用
将不同版本的工具体外转录成mRNA(待测工具信息参见下表总结)，然后和化学合成的sgRNA 1:1质量比混合(第一融合物和第二融合物的mRNA与sgRNA的质量比为0.5:0.5:1；sgRNA靶向序列的互补核苷酸序列如SEQ ID NO:341和/或342所示)，制备成LNP(LNP引用文献：Musunuru,K.,Chadwick,A.C.,Mizoguchi,T.et al.In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates.Nature 593,429–434(2021).)。将制备好的LNP通过尾静脉注射到小鼠体内，注射剂量为4.5mg每kg体重，注射4-10天后，从小鼠脸颊取血，通过Elisa测定血液中PCSK9蛋白的含量。PBS组为注射等体积PBS的对照组。各工具在注射后第4天的检测结果如图3所示，与PBS对照组相比，本申请的各组工具均对PCSK9基因表达显现出极为突出的抑制效果。


实施例4
本申请的复合物对PCSK9基因表达的抑制作用
根据实施例3的体外转录mRNA(所用工具信息参见下表)、化学合成质量比为1:1的sgRNA/mRNA混合物(sgRNA序列同实施例3)、以及制备LNP的方法，将制备好的LNP加入到Huh7细胞中(1.25ug/mL剂量)，LNP加入4天后，收取所有细胞，用Trizol提取总RNA，通过qPCR定量PCSK9的相对表达量，计算不同工具的相对抑制效率。结果如图4所示，其中NC组为未加入LNP的对照，可以看出本申请提供的融合物对PCSK9基因的表达有着显著的抑制作用。

Claims (53)

  1. 一种复合物，其包含第一融合物和第二融合物，所述第一融合物和所述第二融合物其中之一的融合物包含DNA甲基化结构域和至少一个招募结构域A，并且其中另一个融合物包含转录阻遏物结构域和至少一个招募结构域A’；其中，所述招募结构域A和招募结构域A’能够相互作用，以使所述第一融合物和所述第二融合物其中之一的融合物或其部分能够被招募到另一个融合物附近。
  2. 根据权利要求1所述的复合物，所述第一融合物或所述第二融合物包含核酸结合结构域。
  3. 根据权利要求1或2所述的复合物，所述第一融合物包含DNA甲基化结构域、核酸结合结构域和至少一个招募结构域A，所述第二融合物包含转录阻遏物结构域和至少一个招募结构域A’。
  4. 根据权利要求1-3中任一项所述的复合物，所述第一融合物中从N端到C端依次包含DNA甲基化结构域、核酸结合结构域和招募结构域A。
  5. 根据权利要求1-4中任一项所述的复合物，所述第二融合物中从N端到C端依次包含转录阻遏物结构域和招募结构域A’，或者从N端到C端依次包含招募结构域A’和转录阻遏物结构域。
  6. 根据权利要求1或2所述的复合物，所述第一融合物包含转录阻遏物结构域、核酸结合结构域和至少一个招募结构域A，所述第二融合物包含DNA甲基化结构域和至少一个招募结构域A’。
  7. 根据权利要求1、2和6中任一项所述的复合物，所述第一融合物中从N端到C端依次包含招募结构域A、核酸结合结构域和转录阻遏物结构域。
  8. 根据权利要求1、2、6和7中任一项所述的复合物，所述第二融合物中从N端到C端依次包含DNA甲基化结构域和招募结构域A’，或者从N端到C端依次包含招募结构域A’和DNA甲基化结构域。
  9. 根据权利要求1-8中任一项所述的复合物，其特征在于：
    1)所述第一融合物从N端到C端依次包含DNA甲基化结构域、核酸结合结构域和招募结构域A，所述第二融合物从N端到C端依次包含转录阻遏物结构域和招募结构域A’；或
    2)所述第一融合物从N端到C端依次包含DNA甲基化结构域、核酸结合结构域和招募结构域A，所述第二融合物从N端到C端依次包含招募结构域A’和转录阻遏物结构域；或
    3)第一融合物中从N端到C端依次包含招募结构域A、核酸结合结构域和转录阻遏物结构域，所述第二融合物中从N端到C端依次包含DNA甲基化结构域和招募结构域A’； 或
    4)第一融合物中从N端到C端依次包含招募结构域A、核酸结合结构域和转录阻遏物结构域，所述第二融合物从N端到C端依次包含招募结构域A’和DNA甲基化结构域。
  10. 根据权利要求2-4、6、7和9中任一项所述的复合物，所述核酸结合结构域为DNA结合结构域。
  11. 根据权利要求10所述的复合物，所述DNA结合结构域选自：TALE结构域、锌指结构域、tetR结构域、大范围核酸酶、Cas蛋白、Argonaute(Ago)蛋白，以及其同系物、修饰形式或变体。
  12. 根据权利要求10或11所述的复合物，所述DNA结合结构域能够结合靶基因座的目标序列。
  13. 根据权利要求10-12中任一项所述的复合物，所述DNA结合结构域能够与引导RNA结合。
  14. 根据权利要求13所述的复合物，所述引导RNA能够特异性识别所述靶基因座的目标序列并与其杂交。
  15. 根据权利要求10-14中任一项所述的复合物，所述DNA结合结构域为Cas蛋白，且所述Cas蛋白为II类Cas核酸酶。
  16. 根据权利要求15所述的复合物，所述Cas蛋白选自II类II型Cas核酸酶和II类V型Cas核酸酶。
  17. 根据权利要求15或16所述的复合物，所述Cas蛋白为Cas9或Cas12蛋白。
  18. 根据权利要求15-17中任一项所述的复合物，所述Cas蛋白为失活Cas9(dCas9)蛋白或失活Cas12(dCas12)蛋白。
  19. 根据权利要求10-14中任一项所述的复合物，所述DNA结合结构域包含SEQ ID NO:1-9、343和344中任一项所示的氨基酸序列。
  20. 根据权利要求1-19中任一项所述的复合物，所述招募结构域A选自下列两组结构域其中一组中的任一个，所述招募结构域A’选自下列两组结构域中另一组中的任一个：
    1)通用控制非去阻遏蛋白4(GCN4)、来源于分裂绿色荧光蛋白(GFP)的GFP11片段或GVKESLV多肽；和
    2)单链抗体(scFv)、来源于分裂绿色荧光蛋白(GFP)的GFP1-10片段或PDZ蛋白结构域。
  21. 根据权利要求20所述的复合物，其中：
    1)所述招募结构域A和所述招募结构域A’其中之一的结构域为GCN4，并且其中另一个结构域为scFv；或
    2)所述招募结构域A和所述招募结构域A’其中之一的结构域为GFP11片段，并且其中另一个结构域为GFP1-10；或
    3)所述招募结构域A和所述招募结构域A’其中之一的结构域为GVKESLV，并且其中另一个结构域为PDZ蛋白结构域。
  22. 根据权利要求1-21中任一项所述的复合物，所述DNA甲基化结构域包含至少一种DNA甲基转移酶或其功能活性片段。
  23. 根据权利要求22所述的复合物，所述DNA甲基转移酶选自DNMT3A、DNMT3B、DNMT3c、DNMT1、DNMT2和DNMT3L。
  24. 根据权利要求1-23中任一项所述的复合物，所述DNA甲基化结构域包含至少一个DNMT3A和至少一个DNMT3L。
  25. 根据权利要求22或23所述的复合物，所述DNA甲基转移酶包含SEQ ID NO:19-24中任一项所示的氨基酸序列。
  26. 根据权利要求1-25中任一项所述的复合物，所述DNA甲基化结构域包含DNMT3A-DNMT3L结构域或DNMT3L-DNMT3A结构域；其中，-表示其两端的结构域按照从N端到C端的顺序直接或间接地连接。
  27. 根据权利要求1-26中任一项所述的复合物，所述转录阻遏物选自下列所示结构域中的一种或多种：KRAB，ZIM3，ZNF680，ZNF554，ZNF264，ZNF582，ZNF324，ZNF669，ZNF354A，ZNF82，ZNF595，ZNF419，ZNF566，ZIM2，EHMT2，SUV39H1，ZFPM1，TRIM28，EZH2，MXD1，SID，LSD1，HP1a，HDAC3，HDAC1，PRMT1，SETDB1，hSIRT1，ZNF436，ZNF257，ZNF675，ZNF490，ZNF320，ZNF331，ZNF816，ZNF41，ZNF189，ZNF528，ZNF543，ZNF140，ZNF610，ZNF350，ZNF8，ZNF30，ZNF98，ZNF677，ZNF596，ZNF214，ZNF37A，ZNF34，ZNF250，ZNF547，ZNF273，ZFP82，ZNF224，ZNF33A，ZNF45，ZNF175，ZNF184，ZFP28-1，ZFP28-2，ZNF18，ZNF213，ZNF394，ZFP1，ZFP14，ZNF416，ZNF557，ZNF729，ZNF254，ZNF764，ZNF785，ZNF10，CBX5，RYBP，YAF2，MGA，CBX1，SCMH1，MPP8，SUMO3，HERC2，BIN1，PCGF2，TOX，FOXA1，FOXA2，IRF2BP1，IRF2BP2，IRF2BPL IRF-2BP1_2 N-terminal domain，HOXA13，HOXB13，HOXC13，HOXA11，HOXC11，HOXC10，HOXA10，HOXB9，HOXA9，ZFP28，ZN334，ZN568，ZN37A，ZN181，ZN510，ZN862，ZN140，ZN208，ZN248，ZN571，ZN699，ZN726，ZIK1，ZNF2，Z705F，ZNF14，ZN471，ZN624，ZNF84，ZNF7，ZN891，ZN337，Z705G，ZN529，ZN729，ZN419，Z705A，ZN302，ZN486，ZN621，ZN688，ZN33A，ZN554，ZN878，ZN772，ZN224，ZN184，ZN544，ZNF57，ZN283，ZN549， ZN211，ZN615，ZN253，ZN226，ZN730，Z585A，ZN732，ZN681，ZN667，ZN649，ZN470，ZN484，ZN431，ZN382，ZN254，ZN124，ZN607，ZN317，ZN620，ZN141，ZN584，ZN540，ZN75D，ZN555，ZN658，ZN684，RBAK，ZN829，ZN582，ZN112，ZN716，HKR1，ZN350，ZN480，ZN416，ZNF92，ZN100，ZN736，ZNF74，ZN443，ZN195，ZN530，ZN782，ZN791，ZN331，Z354C，ZN157，ZN727，ZN550，ZN793，ZN235，ZN724，ZN573，ZN577，ZN789，ZN718，ZN300，ZN383，ZN429，ZN677，ZN850，ZN454，ZN257，ZN264，ZN485，ZN737，ZNF44，ZN596，ZN565，ZN543，ZFP69，SUMO1，ZNF12，ZN169，ZN433，ZN175，ZN347，ZNF25，ZN519，Z585B，ZN517，ZN846，ZN230，ZNF66，ZN713，ZN816，ZN426，ZN674，ZN627，ZNF20，Z587B，ZN316，ZN233，ZN611，ZN556，ZN234，ZN560，ZNF77，ZN682，ZN614，ZN785，ZN445，ZFP30，ZN225，ZN551，ZN610，ZN528，ZN284，ZN418，ZN490，ZN805，Z780B，ZN763，ZN285，ZNF85，ZN223，ZNF90，ZN557，ZN425，ZN229，ZN606，ZN155，ZN222，ZN442，ZNF91，ZN135，ZN778，ZN534，ZN586，ZN567，ZN440，ZN583，ZN441，ZNF43，ZN589，ZN563，ZN561，ZN136，ZN630，ZN527，ZN333，Z324B，ZN786，ZN709，ZN792，ZN599，ZN613，ZF69B，ZN799，ZN569，ZN564，ZN546，ZFP92，ZN723，ZN439，ZFP57，ZNF19，ZN404，ZN274，CBX3，ZN250，ZN570，ZN675，ZN695，ZN548，ZN132，ZN738，ZN420，ZN626，ZN559，ZN460，ZN268，ZN304，ZN605，ZN844，SUMO5，ZN101，ZN783，ZN417，ZN182，ZN823，ZN177，ZN197，ZN717，ZN669，ZN256，ZN251，CBX4，CDY2，CDYL2，ZN562，ZN461，Z324A，ZN766，ID2，ZN214，CBX7，ID1，CREM，SCX，ASCL1，ZN764，SCML2，TWST1，CREB1，TERF1，ID3，CBX8，GSX1，NKX22，ATF1，TWST2，ZNF17，TOX3，TOX4，ZMYM3，I2BP1，RHXF1，SSX2，I2BPL，ZN680，TRI68，HXA13，PHC3，TCF24，HXB13，HEY1，PHC2，ZNF81，FIGLA，SAM11，KMT2B，HEY2，JDP2，HXC13，ASCL4，HHEX，GSX2，ETV7，ASCL3，PHC1，OTP，I2BP2，VGLL2，HXA11，PDLI4，ASCL2，CDX4，ZN860，LMBL4，PDIP3，NKX25，CEBPB，ISL1，CDX2，PROP1，SIN3B，SMBT1，HXC11，HXC10，PRS6A，VSX1，NKX23，MTG16，HMX3，HMX1，KIF22，CSTF2，CEBPE，DLX2，PPARG，PRIC1，UNC4，BARX2，ALX3，TCF15，TERA，VSX2，HXD12，CDX1，TCF23，ALX1，HXA10，RX，CXXC5，SCML1，NFIL3，DLX6，MTG8，CEBPD，SEC13，FIP1，ALX4，LHX3，PRIC2，MAGI3，NELL1，PRRX1，MTG8R，RAX2，DLX3，DLX1，NKX26，NAB1，SAMD7，PITX3，WDR5，MEOX2，NAB2，DHX8，CBX6，EMX2，CPSF6，HXC12，KDM4B，LMBL3，PHX2A，EMX1， NC2B，DLX4，SRY，ZN777，ZN398，GATA3，BSH，SF3B4，TEAD1，TEAD3，RGAP1，PHF1，GATA2，FOXO3，ZN212，IRX4，ZBED6，LHX4，SIN3A，RBBP7，NKX61，R51A1，MB3L1，DLX5，NOTC1，TERF2，ZN282，RGS12，ZN840，SPI2B，PAX7，NKX62，ASXL2，FOXO1，GATA1，ZMYM5，LRP1，MIXL1，SGT1，LMCD1，CEBPA，SOX14，WTIP，PRP19，NKX11，RBBP4，DMRT2，SMCA2，以及其功能活性片段。
  28. 根据权利要求1-27中任一项所述的复合物，所述转录阻遏物结构域包含SEQ ID NOs:25-50中任一项所示的氨基酸序列。
  29. 根据权利要求1-28中任一项所述的复合物，其中：
    1)所述第一融合物和所述第二融合物其中之一的融合物包含DNA甲基化结构域-dCas9或dCas12或TALE-n×GCN4，并且其中另一个融合物包含转录阻遏物结构域-scFv；或
    2)所述第一融合物和所述第二融合物其中之一的融合物包含DNA甲基化结构域-dCas9或dCas12或TALE-scFv，并且其中另一个融合物包含转录阻遏物结构域-GCN4；或
    3)所述第一融合物和所述第二融合物其中之一的融合物包含DNA甲基化结构域-dCas9或dCas12或TALE-n×GFP11，并且其中另一个融合物包含转录阻遏物结构域-GFP1-10；或
    4)所述第一融合物和所述第二融合物其中之一的融合物包含DNA甲基化结构域-dCas9或dCas12或TALE-GFP1-10，并且其中另一个融合物包含转录阻遏物结构域-GFP11；或
    5)所述第一融合物和所述第二融合物其中之一的融合物包含DNA甲基化结构域-dCas9或dCas12或TALE-n×GCN4，并且其中另一个融合物包含scFv-转录阻遏物结构域；或
    6)所述第一融合物和所述第二融合物其中之一的融合物包含DNA甲基化结构域-dCas9或dCas12或TALE-scFv，并且其中另一个融合物包含GCN4-转录阻遏物结构域；或
    7)所述第一融合物和所述第二融合物其中之一的融合物包含DNA甲基化结构域-dCas9或dCas12或TALE-n×GFP11，并且其中另一个融合物包含GFP1-10-转录阻遏物结构域；或
    8)所述第一融合物和所述第二融合物其中之一的融合物包含DNA甲基化结构域-dCas9或dCas12或TALE-GFP1-10，并且其中另一个融合物包含GFP11-转录阻遏物结构域；
    其中，-表示其两端的结构域按照从N端到C端的顺序直接或间接地连接；n×GCN4或n×GFP11分别表示n个通过接头序列连接的GCN4拷贝或n个通过接头序列连接的GFP11拷贝，n选自1至20的任一整数。
  30. 根据权利要求1-29中任一项所述的复合物，所述第一融合物和/或第二融合物包含SEQ ID NO:51-76、78-82、85-93、103-105、110-115、123、124、361和362中任一项所述的氨基酸序列。
  31. 根据权利要求1-30中任一项所述的复合物，其包含SEQ ID NO:133-142、153、154、158-163、168、345和346中任一项所示的氨基酸序列。
  32. 根据权利要求1-28中任一项所述的复合物，其中：
    1)所述第一融合物和所述第二融合物其中之一的融合物包含n×GCN4-dCas9或dCas12或TALE-转录阻遏物结构域，并且其中另一个融合物包含DNA甲基化结构域-scFv；或
    2)所述第一融合物和所述第二融合物其中之一的融合物包含scFv-dCas9或dCas12或TALE-转录阻遏物结构域，并且其中另一个融合物包含DNA甲基化结构域-GCN4；或
    3)所述第一融合物和所述第二融合物其中之一的融合物包含n×GFP11-dCas9或dCas12或TALE-转录阻遏物结构域，并且其中另一个融合物包含DNA甲基化结构域-GFP1-10；或
    4)所述第一融合物和所述第二融合物其中之一的融合物包含GFP1-10-dCas9或dCas12或TALE-转录阻遏物结构域，并且其中另一个融合物包含DNA甲基化结构域-GFP11；或
    5)所述第一融合物和所述第二融合物其中之一的融合物包含n×GCN4-dCas9或dCas12或TALE-转录阻遏物结构域，并且其中另一个融合物包含scFv-DNA甲基化结构域；或
    6)所述第一融合物和所述第二融合物其中之一的融合物包含scFv-dCas9或dCas12或TALE-转录阻遏物结构域，并且其中另一个融合物包含GCN4-DNA甲基化结构域；或
    7)所述第一融合物和所述第二融合物其中之一的融合物包含n×GFP11-dCas9或dCas12或TALE-转录阻遏物结构域，并且其中另一个融合物包含GFP1-10-DNA甲基化结构域；或
    8)所述第一融合物和所述第二融合物其中之一的融合物包含GFP1-10-dCas9或dCas12或TALE-转录阻遏物结构域，并且其中另一个融合物包含GFP11-DNA甲基化结构域；
    其中，-表示其两端的结构域按照从N端到C端的顺序直接或间接地连接；n×GCN4或n×GFP11分别表示n个通过接头序列连接的GCN4拷贝或n个通过接头序列连接的GFP11拷贝，n选自1至20的任一整数。
  33. 根据权利要求1-28和32中任一项所述的复合物，所述第一融合物和/或第二融合物包含SEQ ID NO:83、84、94-102、106-109、116-122、363和364中任一项所示的氨基酸序列。
  34. 根据权利要求1-28、32和33中任一项所述的复合物，其包含SEQ ID NO:143-152、155- 157、164-167、347和348中任一项所示的氨基酸序列。
  35. 根据权利要求1-34中任一项所述的复合物，所述复合物还包含核定位信号和/或标记物结构域。
  36. 编码权利要求1-35中任一项所述复合物的核酸。
  37. 根据权利要求36所述的核酸，其为重组载体。
  38. 根据权利要求37所述的重组载体，其还包括非编码区。
  39. 根据权利要求38所述的非编码区，其选自内含子、调节元件、启动子、增强子、终止序列以及5’和3’非翻译区。
  40. 根据权利要求36-39中任一项所述的核酸，其包含编码所述第一融合物的第一核酸片段，以及编码所述第二融合物的第二核酸片段。
  41. 根据权利要求40所述的核酸，其中所述第一核酸片段与第二核酸片段通过编码剪切肽的核酸片段连接。
  42. 根据权利要求41所述的核酸，所述剪切肽为2A肽和/或IRES。
  43. 根据权利要求42所述的核酸，所述2A肽选自P2A、T2A、E2A和F2A。
  44. 根据权利要求36-43中任一项所述的核酸，其包含SEQ ID NO:169-335和349-360中任一项所示的核酸序列。
  45. 一种递送载体，所述递送载体包含权利要求1-35中任一项所述的复合物和/或权利要求36-44中任一项所述的核酸，以及任选地包含脂质体和/或脂质纳米颗粒。
  46. 一种组合物，所述组合物包含权利要求1-35中任一项所述的复合物、权利要求36-44中任一项所述的核酸、和/或权利要求45所述的递送载体。
  47. 一种细胞，所述细胞包含权利要求1-35中任一项所述的复合物、权利要求36-44中任一项所述的核酸、权利要求45所述的递送载体、和/或权利要求46所述的组合物。
  48. 一种试剂盒，所述试剂盒包含权利要求1-35中任一项所述的复合物、权利要求36-44中任一项所述的核酸、权利要求45所述的递送载体、权利要求46所述的组合物、和/或权利要求47所述的细胞。
  49. 一种调控靶基因表达的方法，所述方法包括施用权利要求1-35中任一项所述的复合物、权利要求36-44中任一项所述的核酸、权利要求45所述的递送载体、权利要求46所述的组合物、权利要求47所述的细胞和/或权利要求48所述的试剂盒。
  50. 根据权利要求49所述的方法，所述方法包括将所述复合物、所述核酸、所述递送载体、所述组合物、所述细胞、和/或所述试剂盒引入含有所述靶基因的细胞。
  51. 根据权利要求49所述的方法，所述方法包括使所述复合物、所述核酸、所述递送载体、 和/或所述组合物接触所述靶基因附近和/或所述靶基因的调控元件。
  52. 根据权利要求51所述的方法，所述调控元件包含核心启动子、近端启动子、远端增强子、沉默子、绝缘子元件、边界元件和/或基因座控制区。
  53. 权利要求1-35中任一项所述的复合物、权利要求36-44中任一项所述的核酸、权利要求45所述的递送载体、权利要求46所述的组合物、权利要求47所述的细胞和/或权利要求48所述的试剂盒用于制备药物的用途，所述药物用于治疗或缓解与靶基因表达异常和/或靶基因活性异常相关的疾病或其病症。
PCT/CN2023/140808 2022-12-23 2023-12-22 复合物及其用途 Ceased WO2024131917A1 (zh)

Priority Applications (6)

Application Number Priority Date Filing Date Title
CN202380088637.8A CN120418279A (zh) 2022-12-23 2023-12-22 复合物及其用途
JP2025537104A JP2026500040A (ja) 2022-12-23 2023-12-22 複合体及びその使用
KR1020257024587A KR20250124885A (ko) 2022-12-23 2023-12-22 복합체 및 이의 용도
EP23906072.6A EP4640697A1 (en) 2022-12-23 2023-12-22 Complex and use thereof
AU2023412431A AU2023412431A1 (en) 2022-12-23 2023-12-22 Complex and use thereof
US19/245,463 US20250319203A1 (en) 2022-12-23 2025-06-23 Complex and use thereof

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN202211663018.0 2022-12-23
CN202211663018 2022-12-23
CN202311453875.2 2023-11-02
CN202311453875 2023-11-02

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US19/245,463 Continuation US20250319203A1 (en) 2022-12-23 2025-06-23 Complex and use thereof

Publications (1)

Publication Number Publication Date
WO2024131917A1 true WO2024131917A1 (zh) 2024-06-27

Family

ID=91587703

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/140808 Ceased WO2024131917A1 (zh) 2022-12-23 2023-12-22 复合物及其用途

Country Status (8)

Country Link
US (1) US20250319203A1 (zh)
EP (1) EP4640697A1 (zh)
JP (1) JP2026500040A (zh)
KR (1) KR20250124885A (zh)
CN (1) CN120418279A (zh)
AU (1) AU2023412431A1 (zh)
TW (1) TW202440930A (zh)
WO (1) WO2024131917A1 (zh)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025261474A1 (zh) * 2024-06-21 2025-12-26 益杰立科(上海)生物科技有限公司 靶向抑制素亚基βE(INHBE)的工程化基因转录阻抑工具及其用途
WO2025261475A1 (zh) * 2024-06-21 2025-12-26 益杰立科(上海)生物科技有限公司 靶向血管内皮生长因子的工程化基因转录阻抑工具及用途
WO2025261493A1 (zh) * 2024-06-21 2025-12-26 益杰立科(上海)生物科技有限公司 通过表观编辑靶向调节pcsk9的方法及其用途
WO2025261473A1 (zh) * 2024-06-21 2025-12-26 益杰立科(上海)生物科技有限公司 工程化基因转录阻抑工具及其用途

Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998053060A1 (en) 1997-05-23 1998-11-26 Gendaq Limited Nucleic acid binding proteins
WO1998053059A1 (en) 1997-05-23 1998-11-26 Medical Research Council Nucleic acid binding proteins
US6140081A (en) 1998-10-16 2000-10-31 The Scripps Research Institute Zinc finger binding domains for GNN
WO2002016536A1 (fr) 2000-08-23 2002-02-28 Kao Corporation Detergent bactericide antisalissures, destine aux surfaces dures
US6453242B1 (en) 1999-01-12 2002-09-17 Sangamo Biosciences, Inc. Selection of sites for targeting by zinc finger proteins and methods of designing zinc finger proteins to bind to preselected sites
WO2003016496A2 (en) 2001-08-20 2003-02-27 The Scripps Research Institute Zinc finger binding domains for cnn
US6534261B1 (en) 1999-01-12 2003-03-18 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US20110301073A1 (en) 2010-05-17 2011-12-08 Sangamo Biosciences, Inc. Novel DNA-binding proteins and uses thereof
CN109929865A (zh) * 2019-03-11 2019-06-25 东南大学 基于gal4-uas系统的crispr辅助反式增强子激活基因表达的方法及其应用
US20200199552A1 (en) * 2015-09-17 2020-06-25 The Regents Of The University Of California Variant cas9 polypeptides comprising internal insertions
WO2021167101A1 (ja) * 2020-02-21 2021-08-26 国立大学法人群馬大学 脱メチル化酵素と転写関連因子またはクロマチン関連因子を用いた特定遺伝子の相乗的発現誘導法
CN113950526A (zh) * 2019-04-09 2022-01-18 加利福尼亚大学董事会 通过靶向体内表观遗传阻遏实现的持久镇痛

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20240011120A (ko) * 2020-12-22 2024-01-25 크로마 메디슨, 인크. 후성유전학적 편집을 위한 조성물 및 방법

Patent Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998053058A1 (en) 1997-05-23 1998-11-26 Gendaq Limited Nucleic acid binding proteins
WO1998053059A1 (en) 1997-05-23 1998-11-26 Medical Research Council Nucleic acid binding proteins
WO1998053060A1 (en) 1997-05-23 1998-11-26 Gendaq Limited Nucleic acid binding proteins
US6140081A (en) 1998-10-16 2000-10-31 The Scripps Research Institute Zinc finger binding domains for GNN
US6534261B1 (en) 1999-01-12 2003-03-18 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US6453242B1 (en) 1999-01-12 2002-09-17 Sangamo Biosciences, Inc. Selection of sites for targeting by zinc finger proteins and methods of designing zinc finger proteins to bind to preselected sites
WO2002016536A1 (fr) 2000-08-23 2002-02-28 Kao Corporation Detergent bactericide antisalissures, destine aux surfaces dures
WO2003016496A2 (en) 2001-08-20 2003-02-27 The Scripps Research Institute Zinc finger binding domains for cnn
US20110301073A1 (en) 2010-05-17 2011-12-08 Sangamo Biosciences, Inc. Novel DNA-binding proteins and uses thereof
US20200199552A1 (en) * 2015-09-17 2020-06-25 The Regents Of The University Of California Variant cas9 polypeptides comprising internal insertions
CN109929865A (zh) * 2019-03-11 2019-06-25 东南大学 基于gal4-uas系统的crispr辅助反式增强子激活基因表达的方法及其应用
CN113950526A (zh) * 2019-04-09 2022-01-18 加利福尼亚大学董事会 通过靶向体内表观遗传阻遏实现的持久镇痛
WO2021167101A1 (ja) * 2020-02-21 2021-08-26 国立大学法人群馬大学 脱メチル化酵素と転写関連因子またはクロマチン関連因子を用いた特定遺伝子の相乗的発現誘導法

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
BIRD ET AL., SCIENCE, vol. 242, 1988, pages 423 - 426
HUSTON ET AL., PNAS, vol. 85, 1988, pages 5879 - 5883
JIANG, WEN ET AL.: "BE-PLUS: a New Base Editing Tool with Broadened Editing Window and Enhanced Fidelity", CELL RESEARCH, vol. 28, 6 June 2018 (2018-06-06), pages 855 - 861, XP036675688, DOI: 10.1038/s41422-018-0052-4 *
MAKAROVA ET AL., NAT REV MICROBIOL, vol. 13, no. 11, 2015, pages 722 - 36
MOSCOU ET AL., SCIENCE, vol. 326, 2009, pages 1509 - 1512
MUSUNURU, K.CHADWICK, A.C.MIZOGUCHI, T ET AL.: "In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates", NATURE, vol. 593, 2021, pages 429 - 434, XP037513148, DOI: 10.1038/s41586-021-03534-y
See also references of EP4640697A1
SHMAKOV ET AL., MOL CELL, vol. 60, 2015, pages 385 - 397
ZHANG ET AL., NATURE BIOTECHNOLOGY, vol. 29, 2011, pages 149 - 153
方凯伦等 (FANG, KAILUN ET AL.): "CRISPR/Cas工具的开发和应用 (Advances and Applications of CRISPR/Cas Toolbox)", 科学通报 (CHINESE SCIENCE BULLETIN), vol. 65, no. 11, 11 February 2020 (2020-02-11), pages 973 - 990, XP055937416, DOI: 10.1360/TB-2019-0806 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025261474A1 (zh) * 2024-06-21 2025-12-26 益杰立科(上海)生物科技有限公司 靶向抑制素亚基βE(INHBE)的工程化基因转录阻抑工具及其用途
WO2025261475A1 (zh) * 2024-06-21 2025-12-26 益杰立科(上海)生物科技有限公司 靶向血管内皮生长因子的工程化基因转录阻抑工具及用途
WO2025261493A1 (zh) * 2024-06-21 2025-12-26 益杰立科(上海)生物科技有限公司 通过表观编辑靶向调节pcsk9的方法及其用途
WO2025261473A1 (zh) * 2024-06-21 2025-12-26 益杰立科(上海)生物科技有限公司 工程化基因转录阻抑工具及其用途

Also Published As

Publication number Publication date
AU2023412431A1 (en) 2025-08-07
CN120418279A (zh) 2025-08-01
EP4640697A1 (en) 2025-10-29
KR20250124885A (ko) 2025-08-20
JP2026500040A (ja) 2026-01-05
TW202440930A (zh) 2024-10-16
US20250319203A1 (en) 2025-10-16

Similar Documents

Publication Publication Date Title
US12410435B2 (en) Compositions and methods of use of CRISPR-Cas systems in nucleotide repeat disorders
WO2024131917A1 (zh) 复合物及其用途
JP2022127638A (ja) 最適化機能ＣＲＩＳＰＲ－Ｃａｓ系による配列操作のための系、方法および組成物
US20170349914A1 (en) DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF CRISPR SYSTEMS AND COMPOSITIONS FOR GENOME EDITING AS TO HEMATOPOIETIC STEM CELLS (HSCs)
EP4640835A1 (en) Fusion and use thereof
CN105899657A (zh) 用于改变基因产物表达的crispr-cas系统和方法、结构信息以及诱导型模块化cas酶
WO2025167914A1 (zh) 通过表观编辑多靶点治疗疾病的方法
TW202606727A (zh) 靶向抑制素亞基βＥ（ＩＮＨＢＥ）的工程化基因轉錄阻抑工具及其用途
WO2025261474A1 (zh) 靶向抑制素亚基βE(INHBE)的工程化基因转录阻抑工具及其用途
WO2025261475A1 (zh) 靶向血管内皮生长因子的工程化基因转录阻抑工具及用途
WO2025261473A1 (zh) 工程化基因转录阻抑工具及其用途
CN121568968A (zh) 靶向乙型肝炎病毒基因的工程化基因转录阻抑工具及其用途
CN121358854A (zh) 靶向pcsk9的工程化基因转录阻抑工具及其用途
HK40130129A (zh) 靶向pcsk9的工程化基因转录阻抑工具及其用途
WO2024168253A1 (en) Delivery of an rna guided recombination system
WO2024168265A1 (en) Aav delivery of rna guided recombination system
WO2025261493A1 (zh) 通过表观编辑靶向调节pcsk9的方法及其用途
TW202610664A (zh) 通過表觀編輯靶向調節pcsk9的方法及其用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23906072

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2025537104

Country of ref document: JP

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2025537104

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 202380088637.8

Country of ref document: CN

ENP Entry into the national phase

Ref document number: 1020257024587

Country of ref document: KR

Free format text: ST27 STATUS EVENT CODE: A-0-1-A10-A15-NAP-PA0105 (AS PROVIDED BY THE NATIONAL OFFICE)

WWE Wipo information: entry into national phase

Ref document number: 1020257024587

Country of ref document: KR

Ref document number: AU2023412431

Country of ref document: AU

Ref document number: 823604

Country of ref document: NZ

WWE Wipo information: entry into national phase

Ref document number: 2023906072

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

WWP Wipo information: published in national office

Ref document number: 823604

Country of ref document: NZ

WWE Wipo information: entry into national phase

Ref document number: 11202504192V

Country of ref document: SG

WWP Wipo information: published in national office

Ref document number: 11202504192V

Country of ref document: SG

WWP Wipo information: published in national office

Ref document number: 202380088637.8

Country of ref document: CN

ENP Entry into the national phase

Ref document number: 2023906072

Country of ref document: EP

Effective date: 20250723

ENP Entry into the national phase

Ref document number: 2023412431

Country of ref document: AU

Date of ref document: 20231222

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2023906072

Country of ref document: EP

Effective date: 20250723

WWP Wipo information: published in national office

Ref document number: 1020257024587

Country of ref document: KR

WWP Wipo information: published in national office

Ref document number: 2023906072

Country of ref document: EP