WO2022052877A1 - 对细胞中的靶位点进行基因编辑的方法 - Google Patents

对细胞中的靶位点进行基因编辑的方法 Download PDF

Info

Publication number
WO2022052877A1
WO2022052877A1 PCT/CN2021/116531 CN2021116531W WO2022052877A1 WO 2022052877 A1 WO2022052877 A1 WO 2022052877A1 CN 2021116531 W CN2021116531 W CN 2021116531W WO 2022052877 A1 WO2022052877 A1 WO 2022052877A1
Authority
WO
WIPO (PCT)
Prior art keywords
site
gene editing
cells
grna
cell
Prior art date
Application number
PCT/CN2021/116531
Other languages
English (en)
French (fr)
Inventor
张楫钦
杜冰
刘明耀
席在喜
Original Assignee
上海邦耀生物科技有限公司
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 上海邦耀生物科技有限公司 filed Critical 上海邦耀生物科技有限公司
Priority to US18/025,136 priority Critical patent/US20230323323A1/en
Priority to JP2023515399A priority patent/JP2023541028A/ja
Priority to EP21865931.6A priority patent/EP4212619A1/en
Publication of WO2022052877A1 publication Critical patent/WO2022052877A1/zh

Links

Images

Classifications

    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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 RNAses, DNAses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4636—Immune checkpoint inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464412—CD19 or B4
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • 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
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • 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
    • C12N15/1138—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 receptors or cell surface 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
    • 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 [CRISPRs]
    • 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
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to the field of biotechnology, in particular, to a method for gene editing of target sites in cells.
  • the CRISPR/Cas9 system is an adaptive immune mechanism derived from archaea and bacteria to resist the invasion of exogenous DNA fragments such as plasmids and bacteriophages.
  • the system mainly functions by the CRISPR sequence and the locus encoding the Cas protein.
  • the Cas9 nuclease of the type II family is the most commonly used. This system only needs a single effector protein to function.
  • the type II CRISPR system first integrates the invading DNA into the CRISPR repeats. Subsequently, the CRISPR RNA (crRNA) containing the invading DNA sequence is transcribed and processed.
  • crRNA CRISPR RNA
  • CRISPR RNA binds to crRNA and finally forms a complex with Cas9 protein.
  • the Cas9 protein acts as an endonuclease through its HNH and RuvC-like domains to initiate DNA double-strand breaks. Double-strand breaks in DNA trigger damage repair mechanisms.
  • cells will undergo precise homologous recombination repair, allowing insertion or replacement of exogenous sequences. So far, CRISPR/Cas9 technology has been successfully applied in many fields, showing broad prospects. However, the use of homologous recombination for precise gene therapy still faces many difficulties. One of the main limiting factors is that the efficiency of homologous recombination is very low.
  • Chimeric Antigen Receptor T-Cell Technology is a new type of adoptive immunotherapy emerging in recent years. It genetically modifies the patient's T cells in vitro, and then infuses them back into the patient after a certain expansion, so as to achieve targeted killing of tumors.
  • CAR-T products have been approved by the U.S. Food and Drug Administration for the clinical treatment of hematological tumors, marking the great success of CAR-T technology.
  • CAR-T technology still faces many challenges in various aspects, and there is huge room for improvement.
  • the most commonly used method is the introduction of foreign sequences using a viral system.
  • using the virus system has problems such as high preparation cost and potential safety hazards of random insertion.
  • One of the objects of the present invention is to provide a new method for improving the efficiency of homologous recombination of foreign sequences in cells, especially T cells.
  • the present invention provides a gene editing method for AAVS1, PD1, TRAC and B2M genes respectively.
  • a method for gene editing at a target site of a cell genome comprising:
  • the targeting sequence targeted by the gRNA comprises one or more of the sequences shown in SEQ ID No. 1-9.
  • the gene editing includes gene knockout and gene-targeted integration.
  • the gene editing is gene-targeted integration.
  • the gene editing is a gene knockout.
  • the gene editing enzyme is selected from the group consisting of CRISPR-associated protein (Cas) polypeptide, TALEN enzyme, ZFN enzyme, or a combination thereof.
  • Cas CRISPR-associated protein
  • the gene editing enzyme is derived from microorganisms; preferably from bacteria.
  • the source of the gene editing enzyme is selected from the group consisting of Streptococcus pyogenes, Staphylococcus aureus, Streptococcus canis, or a combination thereof.
  • the gene editing enzyme includes wild type or mutant gene editing enzyme.
  • the gene editing enzyme is selected from the group consisting of Cas9, Cas12, Cas13, Cms1, MAD7, Cas3, Cas8a, Cas8b, Cas10d, Cse1, Csy1, Csn2, Cas4, Cas10, Csm2, Cmr5, Fok1 , Cpf1, or a combination thereof.
  • the gRNA includes crRNA, tracrRNA, and sgRNA.
  • the gRNA targets and binds to the target site of the genome.
  • the gRNA includes unmodified and modified gRNA.
  • the modified gRNA includes chemical modification of bases.
  • the chemical modification includes methylation modification, methoxy modification, fluorination modification or thio modification.
  • the gene editing includes in vivo gene editing and in vitro gene editing.
  • the gene editing includes CRISPR-based gene editing.
  • the target site is selected from the group consisting of AAVS1, PD1, TRAC, B2M, or a combination thereof.
  • the targeting sequence of the gRNA targeting AAVS1 comprises the sequence shown in SEQ ID No.3.
  • the targeting sequence of the gRNA targeting PD1 comprises the sequence shown in SEQ ID NO.:1 and/or SEQ ID NO.:2.
  • the targeting sequence of the gRNA targeting TRAC comprises the sequence shown in SEQ ID NO.:4 and/or SEQ ID NO.:5.
  • the targeting sequence of the gRNA targeting B2M comprises the sequence shown in SEQ ID NO.:6.
  • the gene editing includes site-directed knock-in of donor DNA.
  • the donor DNA is double-stranded DNA.
  • the donor DNA includes a first homology arm and a second homology arm, wherein the first homology arm and the second homology arm can activate the donor DNA in the Cell-mediated homologous recombination at the target site of the cell genome, the sequence lengths of the first homology arm and the second homology arm are independently 200-2000bp, preferably 400-1000bp, more preferably ground, 700-900bp.
  • the first homology arm is homologous to the upstream (or left) sequence of the cleavage site of the target site in the genome
  • the second homology arm is homologous
  • the sequence on the downstream side (or right side) of the cleavage site of the target site in the genome is homologous.
  • the donor DNA also includes a target gene.
  • the target gene includes the coding sequence of chimeric antigen receptor or TCR.
  • sequence length of the donor DNA is 50bp-5000bp, preferably 80bp-4000bp.
  • the length of the target gene is 50bp-3000bp, preferably 1000bp-2000bp.
  • the length of the coding sequence of the chimeric antigen receptor or TCR is 50bp-3000bp, preferably, 1000bp-2000bp.
  • the ratio (D1/D2) of the sequence length D1 of the first homology arm and the sequence length D2 of the second homology arm is (0.8-1.2):(0.5-1.5), preferably Ground (0.9-1.1):(0.7-1.3), more preferably, 1:1.
  • the chimeric antigen receptor contains an antigen binding domain targeting tumor cell markers.
  • the chimeric antigen receptor includes an antigen binding domain targeting tumor cell markers, an optional hinge region, a transmembrane domain and an intracellular signaling binding domain.
  • the tumor cell marker is selected from the group consisting of ⁇ -folate receptor, 5T4, ⁇ v ⁇ 6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM , FAP, Fetal AchR, FR ⁇ , GD2, GD3, Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO -1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, IL-11R ⁇ , IL-13R
  • the hinge region is a hinge region of a protein selected from the group consisting of CD8, Ig (immunoglobulin) hinge, CD28, or a combination thereof.
  • the transmembrane domain is a transmembrane region of a protein selected from the group consisting of CD8 ⁇ , CD8 ⁇ , CD28, CD33, CD37, CD5, CD16, ICOS, CD9, CD22, CD134, CD137, CD154 , CD19, CD45, CD4, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , or a combination thereof.
  • the intracellular signal transduction binding domain includes a costimulatory signaling molecule and/or a primary signaling domain.
  • the costimulatory signaling molecule is selected from the group consisting of: OX40, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), LIGHT, DAP10, CDS, ICAM-1, CD278 (ICOS ), TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD54 (ICAM), CD83, LAT, NKD2C, SLP76, TRIM, ZAP70, or a combination thereof .
  • the primary signaling domain is selected from the group consisting of FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a, CD79b, CD66d, or a combination thereof.
  • sequence of the donor DNA is shown in any one of SEQ ID NO.: 12-38.
  • the cells include primary cells and passaged cells.
  • the cells include T cells,
  • the cells include CD3+ T cells, CD4+ helper T cells, CD4+ regulatory T cells, CD8+ T cells, and memory T cells.
  • the vector includes a viral vector.
  • the viral vector is selected from the group consisting of adeno-associated viral vector, lentiviral vector, or a combination thereof.
  • the introduction method includes electroporation, vector transformation, transfection, heat shock, electroporation, transduction, gene gun, microinjection, lipofection, and lentiviral infection.
  • the method is an in vitro method.
  • the method is non-diagnostic and non-therapeutic.
  • the second aspect of the present invention provides a gRNA that performs gene editing on a target site of the cell genome, and the targeting sequence targeted by the gRNA comprises one or more of the sequences shown in SEQ ID No. 1-9.
  • the targeting sequence targeted by the gRNA comprises one or more of the sequences shown in SEQ ID NO.: 1-6.
  • the targeting sequence targeted by the gRNA comprises one or more of the sequences shown in SEQ ID NO.: 7-9.
  • the gRNA includes unmodified and modified gRNA.
  • the modified gRNA includes chemical modification of bases.
  • the chemical modification includes methylation modification, methoxy modification, fluorination modification or thio modification.
  • a third aspect of the present invention provides a reaction system for performing gene editing on a target site of a cell genome, the reaction system comprising:
  • the targeting sequence targeted by the gRNA comprises one or more of the sequences shown in SEQ ID No. 1-9.
  • the gene editing includes gene knockout and gene-targeted integration.
  • the gene editing is gene-targeted integration.
  • the gene editing is a gene knockout.
  • the target site is selected from the group consisting of AAVS1, PD1, TRAC, B2M, or a combination thereof.
  • the targeting sequence of the gRNA targeting AAVS1 comprises the sequence shown in SEQ ID No.3.
  • the targeting sequence of the gRNA targeting PD1 comprises the sequence shown in SEQ ID NO.:1 and/or SEQ ID NO.:2.
  • the targeting sequence of the gRNA targeting TRAC comprises the sequence shown in SEQ ID NO.:4 and/or SEQ ID NO.:5.
  • the targeting sequence of the gRNA targeting B2M comprises the sequence shown in SEQ ID NO.:6.
  • the donor DNA includes a first homology arm and a second homology arm, wherein the first homology arm and the second homology arm can activate the donor DNA in the Homologous recombination mediated by immune cells at the target site of the immune cell genome, the sequence lengths of the first homology arm and the second homology arm are independently 200-2000bp, preferably 400-1000bp, More preferably, 700-900bp.
  • the donor DNA also includes a target gene.
  • the target gene includes the coding sequence of chimeric antigen receptor or TCR.
  • the chimeric antigen receptor contains an antigen binding domain targeting tumor cell markers.
  • the chimeric antigen receptor comprises an antigen binding domain targeting tumor cell markers, an optional hinge region, a transmembrane domain and an intracellular signaling binding domain.
  • the tumor cell marker is selected from the group consisting of ⁇ -folate receptor, 5T4, ⁇ v ⁇ 6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM , FAP, Fetal AchR, FR ⁇ , GD2, GD3, Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO -1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, IL-11R ⁇ , IL-13R
  • the hinge region is a hinge region of a protein selected from the group consisting of CD8, Ig (immunoglobulin) hinge, CD28, or a combination thereof.
  • the transmembrane domain is a transmembrane region of a protein selected from the group consisting of CD8 ⁇ , CD8 ⁇ , CD28, CD33, CD37, CD5, CD16, ICOS, CD9, CD22, CD134, CD137, CD154 , CD19, CD45, CD4, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , or a combination thereof.
  • the intracellular signal transduction binding domain includes a costimulatory signaling molecule and/or a primary signaling domain.
  • the costimulatory signaling molecule is selected from the group consisting of: OX40, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), LIGHT, DAP10, CDS, ICAM-1, CD278 (ICOS ), TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD54 (ICAM), CD83, LAT, NKD2C, SLP76, TRIM, ZAP70, or a combination thereof .
  • the primary signaling domain is selected from the group consisting of FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a, CD79b, CD66d, or a combination thereof.
  • the cells include primary cells and passaged cells.
  • the cells include T cells,
  • the cells include CD3+ T cells, CD4+ helper T cells, CD4+ regulatory T cells, CD8+ T cells, and memory T cells.
  • the first homology arm is homologous to the upstream (or left) sequence of the cleavage site of the target site in the genome
  • the second homology arm is homologous
  • the sequence on the downstream side (or right side) of the cleavage site of the target site in the genome is homologous.
  • sequence length of the donor DNA is 50bp-5000bp, preferably, 80bp-4000bp.
  • the length of the target gene is 50bp-3000bp, preferably 1000bp-2000bp.
  • the length of the coding sequence of the chimeric antigen receptor or TCR is 50bp-3000bp, preferably, 1000bp-2000bp.
  • sequence of the donor DNA is shown in any one of SEQ ID NO.: 12-38.
  • the vector includes a viral vector.
  • the viral vector is selected from the group consisting of adeno-associated viral vector, lentiviral vector, or a combination thereof.
  • a fourth aspect of the present invention provides a kit for gene editing, the kit comprising:
  • first container, the second container and the third container are the same container or different containers.
  • the fifth aspect of the present invention provides a gene-edited cell prepared by the method described in the first aspect of the present invention.
  • the sixth aspect of the present invention provides an application of the cell according to the fifth aspect of the present invention in preparing tumor immunotherapy or cancer immunotherapy products.
  • Untreated T means untreated T cells
  • Control means T cells electroporated with homologous template and Cas9 only
  • LV-19bbz means CD19-CART cells prepared with lentivirus
  • AAVS1-19bbz indicates non-viral AAVS1 site-directed integration CD19-CART cells
  • PD1-19bbz indicates non-viral PD1 site-directed integration CD19-CART cells.
  • FIG. 1 is a schematic diagram of the principle of constructing non-viral site-specific integrated CAR-T cells.
  • TRAC-19bbz means non-viral TRAC site-directed integration CD19-CART cells
  • B2M-19bbz means non-viral B2M site-directed integration CD19-CART cells.
  • Figure 2 shows T cell survival after electroporation of plasmid or linear double-stranded DNA.
  • control group only plasmid or linear double-stranded DNA was electroporated, while in the sample group, template, Cas9 and sgRNA were electroporated at the same time.
  • Figure 3 shows the mTurquoise2 positive rate after electroporation using different templates.
  • the control group was electroporated with HDR template and Cas9 only, and the sample group was electroporated with linear double-stranded DNA template, Cas9 and sgRNA; pb added protective bases on both sides.
  • Figure 4 shows the mTurquoise2 positive rate and cell survival after electroporation of homology arm templates with different lengths.
  • the control group was electroporated with 800bp homologous template and Cas9 only, and the sample group was electroporated with different length homologous arm templates, Cas9 and sgRNA;
  • Picture A is the positive rate of mTurquoise2
  • Picture B is the relative number of live cells
  • Picture C is the relative number of mTurquoise2 positive live cells. quantity.
  • FIG. 5 shows AAVS1 site sgRNA screening.
  • Picture A shows the knockout rate of AAVS1 detected by T7E1
  • picture B shows the recombination rate of the fluorescent protein mTurquioise2 sequence at the AAVS1 site.
  • sgRNA-1 represents the AAVS1-sgRNA1 of the present application
  • sgRNA-2 represents the AAVS1-sgRNA2 of the present application
  • sgRNA-3 represents the AAVS1-sgRNA3 of the present application
  • sgRNA-4" represents the present application AAVS1-sgRNA4.
  • Figure 6 shows the PD1 site sgRNA screening, the recombination rate of the fluorescent protein mTurquioise2 sequence at the PD1 site.
  • sgRNA-1 indicates PD1-sgRNA1 of the present application
  • sgRNA-2 indicates PD1-sgRNA2 of the present application.
  • FIG. 7 shows TRAC site sgRNA screening.
  • Picture A shows the knockout rate of TRAC site detected by CD3 expression
  • picture B shows the recombination rate of fluorescent protein mTurquioise2 sequence at TRAC site.
  • sgRNA-1 indicates TRAC-sgRNA1 of the present application
  • sgRNA-2 indicates TRAC-sgRNA2 of the present application
  • sgRNA-3 indicates TRAC-sgRNA3 of the present application.
  • Figure 8 shows B2M site sgRNA screening.
  • Picture A shows the knockout rate of B2M site detected by B2M expression
  • picture B shows the recombination rate of fluorescent protein mTurquioise2 sequence at B2M site.
  • sgRNA-1 indicates B2M-sgRNA1 of the present application
  • sgRNA-2 indicates B2M-sgRNA2 of the present application.
  • Figure 9 shows the CAR positive rate and cell survival after electroporation of homology arm templates with different lengths.
  • the control group was electroporated with 800bp homologous template and Cas9 only, and the sample group was electroporated with homologous arm templates of different lengths, Cas9 and sgRNA;
  • A is the CAR positive rate
  • B is the relative number of live cells
  • C is the relative number of CAR-positive live cells. quantity.
  • Figure 10 shows the positive rate of AAVS1 site-specific integration of CD19-CART cells in CD3 + , CD4 + and CD8 + T cells
  • Figure 11 shows the positive rate and knockout rate of AAVS1 site-directed integration of CD19-CART cells.
  • Picture A shows the positive rate of AAVS1 site-directed integration of CD19-CART cells prepared from different human donor cells;
  • Picture B shows the positive rate and knockout rate of AAVS1 site-directed integration of CD19-CART cells prepared from five representative different human donor cells;
  • C The figure shows the positive rate of AAVS1 site-specific integration of CD19-CART cells by flow analysis.
  • the control group was electroporated homologous template and Cas9 only.
  • Figure 12 shows the results of DNA sequencing of AAVS1 site-directed integration CD19-CART cells.
  • HA stands for homology arm.
  • Figure 13 shows AAVS1 site-directed integration T cell survival.
  • Figure 14 shows in vitro expansion of AAVS1 site-directed integrating T cells.
  • Figure 15 shows the in vitro expansion of AAVS1 site-directed integration of CD19-CART cells co-cultured with target cells.
  • Figure 16 shows in vitro expansion of CD19-CART cells co-cultured with target cells for detection of AAVS1 site-directed integration by labeling.
  • the control group was electroporated homologous template and Cas9 only.
  • Figure 17 shows AAVS1 site-directed integration CD19-CART cell surface marker expression.
  • the control group was electroporated homologous template and Cas9 only.
  • Figure 18 shows AAVS1 site-directed integration of CD19-CART cell subtype ratios.
  • the control group was electroporated homologous template and Cas9 only.
  • Figure 19 shows the detection of AAVS1 site-directed integration of CD19-CART cells secreted factors.
  • the control group was electroporated homologous template and Cas9 only.
  • Figure 20 shows in vitro killing of CD19-CART cells by site-directed integration of AAVS1.
  • the control group was electroporated homologous template and Cas9 only.
  • Figure 21 shows in vivo killing of AAVS1 site-directed integration of CD19-CART cells.
  • the tumor target cells are Raji cells.
  • Figure 22 shows the positive rate and knockout rate of PD1 site-directed integration of CD19-CART cells.
  • Figure A shows the positive rate of PD1 site-directed integration of CD19-CART cells prepared from different human donor cells;
  • Picture B shows the positive rate and knockout rate of PD1 site-directed integration of CD19-CART cells prepared from five representative different human donor cells;
  • C The picture shows the positive rate of PD1 site-specific integration of CD19-CART cells by flow analysis;
  • D is the comparison of PD1 expression between PD1 site-specific integration of CD19-CART cells and lentivirus-produced CD19-CART cells.
  • the control group was electroporated homologous template and Cas9 only.
  • Figure 23 shows the results of DNA sequencing of PD1 site-directed integration CD19-CART cells.
  • HA stands for homology arm
  • Figure 24 shows the in vitro expansion of PD1 site-directed integration of CD19-CART cells co-cultured with target cells.
  • Figure 25 shows PD1 site-directed integration CD19-CART cell surface marker expression.
  • the control group was electroporated homologous template and Cas9 only.
  • Figure 26 shows PD1 site-directed integration detection of CD19-CART cells secreted factors.
  • the control group was electroporated homologous template and Cas9 only.
  • Figure 27 shows in vitro killing of CD19-CART cells by PD1 site-directed integration.
  • the control group was electroporated homologous template and Cas9 only.
  • Figure 28 shows PD1 site-directed integration killing of CD19-CART cells in vivo.
  • the tumor target cells were Raji cells overexpressing PDL1.
  • the inventors After extensive and in-depth research and extensive screening, the inventors have accidentally screened for the first time a gRNA that efficiently performs gene editing on target sites (eg: AAVS1, PD1, TRAC, B2M).
  • target sites eg: AAVS1, PD1, TRAC, B2M.
  • the inventors also unexpectedly determined the conditions for efficient integration of the exogenous nucleic acid cell genome in T cells (such as the first homology arm and the second homology arm of a specific length). Two homology arms allow for efficient integration of genomic target sites). The present invention has been completed on this basis.
  • CRISPR/Cas9 is an adaptive immune defense formed during the long-term evolution of bacteria and archaea, which can be used to fight against invading viruses and foreign DNA.
  • the CRISPR/Cas9 system provides immunity by integrating fragments of invading phage and plasmid DNA into CRISPR and utilizing the corresponding CRISPR RNAs, such as gRNAs, to direct the degradation of homologous sequences.
  • crRNA CRISPR-derived RNA
  • tracrRNA trans-activating RNA
  • tracrRNA trans-activating RNA
  • a single-guide RNA can be engineered to form a single-guide RNA, which is sufficient to guide the site-directed cleavage of DNA by Cas9.
  • the Cas9 effector nuclease was the first known unifying factor capable of co-localizing RNA, DNA and protein. Fusing the protein to Cas9 without nuclease (Cas9 nuclease-null) and expressing the appropriate gRNA can target any dsDNA sequence, and the end of the gRNA can be ligated to the target DNA without affecting the binding of Cas9. Thus, Cas9 can bring any fusion protein and RNA at any dsDNA sequence. This technology is called the CRISPR/Cas9 gene editing system.
  • a linearized donor ie, donor DNA or exogenous nucleic acid
  • the obtained transgenic donor contains homology arms of a certain length (eg, 700-900 bp); the donor DNA or exogenous nucleic acid is electroporated into cells together with Cas9 mRNA and guide RNA.
  • the targeted integration strategy provided by the present invention has higher integration efficiency.
  • a linearized dsDNA donor ie, donor DNA or exogenous nucleic acid
  • a transgene fragment ie, 700-900 bp homology arms on both sides in CRISPR-mediated genome editing
  • the targeted integration technology of the present invention has the highest recombination rate compared to MMEJ (microhomology arm mediated end joining) or HITI (non-homologous sequence end joining) mediated methods.
  • the present invention provides a method for gene editing of a target site of a cell genome, comprising:
  • the targeting sequence targeted by the gRNA comprises one or more of the sequences shown in SEQ ID No. 1-9.
  • the method of the present invention can perform efficient gene knockout or gene-targeted integration of target sites (such as AAVS1, PD1, TRAC, B2M, etc.).
  • target sites such as AAVS1, PD1, TRAC, B2M, etc.
  • the present invention provides a reaction system for performing gene editing on a target site of a cell genome, the reaction system comprising:
  • the targeting sequence targeted by the gRNA comprises one or more of the sequences shown in SEQ ID No. 1-9.
  • the donor DNA includes a first homology arm and a second homology arm, wherein the first homology arm and the second homology arm can activate the donor DNA in the Homologous recombination mediated by immune cells at the target site of the immune cell genome, the sequence lengths of the first homology arm and the second homology arm are independently 200-2000bp, preferably 400-1000bp, More preferably, 700-900bp.
  • the sequence length of the donor DNA to be integrated can be 50bp-5000bp.
  • the length of the target gene (or called the target gene) in the donor DNA of the present invention can be 50bp-3000bp.
  • the target site is not particularly limited, and a preferred target site includes AAVS1, PD1, TRAC, and B2M.
  • the ratio (D1/D2) of the sequence length D1 of the first homology arm and the sequence length D2 of the second homology is (0.8-1.2):(0.5-1.5), Preferably (0.9-1.1):(0.7-1.3), more preferably, 1:1.
  • sequence lengths of the two homology arms are not required to be completely identical, and a certain length difference is allowed between the two.
  • the present invention can improve the site-specific integration efficiency of exogenous nucleic acid in T cells.
  • This method can successfully prepare non-viral site-specific integrated CAR-T cells with AAVS1, PD1, TRAC, B2M and other sites, which can avoid many disadvantages of virus preparation and improve the uniformity and stability of CAR-T products.
  • it also provides technical support for the preparation of enhanced, inducible and other diversified CAR-T products.
  • Electrotransduction of T cells is a technical method to achieve gene editing of T cells.
  • the steps refer to the P3 Primary Cell of Lonza Company. X kit
  • the kit is P3 Primary Cell 4D-Nucleofector TM X Kit, (Lonza, V4XP-3024)
  • 5Synthetic sgRNA (dissolve the synthesized sgRNA in TE buffer and dilute to a final concentration of 10ug/uL)
  • the CD19-CART comprises an extracellular domain targeting CD19, a transmembrane region selected from CD8 ⁇ , and an intracellular signaling domain selected from CD3 ⁇ and CD137;
  • the cells are added to the preheated cell culture medium and cultured in a cell culture incubator.
  • non-viral site-specific integrated CD19-CART cells including CAR-T cells integrated at AAVS1 safety site, CD19-CART cells integrated at immune checkpoint PD1 site can be constructed in one step.
  • CD19-CART cells integrated at the TRAC site, CD19-CART cells integrated at the B2M site can be constructed in one step.
  • AAVS1-sgRNA1 CCGGAGAGGACCCAGACACG (SEQ ID NO.:7)
  • AAVS1-sgRNA2 AGAGCTAGCACAGACTAGAG (SEQ ID NO. 3)
  • AAVS1-sgRNA3 AAAGCAAGAGGATGGAGAGG (SEQ ID NO.: 8)
  • AAVS1-sgRNA4 GAGAGGACCCAGACACGGGG (SEQ ID NO.: 9)
  • PD1-sgRNA1 CGACTGGCCAGGGCGCCTGT (SEQ ID NO.1)
  • PD1-sgRNA2 GGGCGGTGCTACAACTGGGC (SEQ ID NO.2)
  • TRAC-sgRNA1 ACAAAACTGTGCTAGACATG (SEQ ID NO.:4)
  • TRAC-sgRNA2 AGAGCAACAGTGCTGTGGCC (SEQ ID NO.:5)
  • TRAC-sgRNA3 AGAGTCTCTCAGCTGGTACA (SEQ ID NO.: 10)
  • B2M-sgRNA1 ACTCACGCTGGATAGCCTCC (SEQ ID NO. 11)
  • B2M-sgRNA2 GAGTAGCGCGAGCACAGCTA (SEQ ID NO.: 6)
  • the sgRNA was synthesized by the company, dissolved in TE buffer, and diluted to a final concentration of 10ug/uL
  • the kit is P3 Primary Cell 4D-Nucleofector TM X Kit, (Lonza, V4XP-3024)
  • AAVS1 site-directed integration CAR-T cells can be constructed without virus using the present invention, the CAR positive rate is about 10%-20% (Fig. 11), the knockout rate is about 65%-90% (Fig. 11), and This method can achieve site-directed integration of CAR elements in CD3+, CD4+, and CD8+ T cells ( Figure 10).
  • CD19-CART cells have the expression of cell surface markers, cell subtype changes, cytokine secretion, etc. exhibited similar properties ( Figures 17-19).
  • the AAVS1 site-specific integration CD19-CART cells constructed by the present invention can effectively kill tumor cells both inside and outside ( Figure 20, 21).
  • PD1 site-directed integration CAR-T cells can be constructed without virus.
  • the positive rate of CAR is about 10%-30% (Fig. 22), and the knockout rate is about 80%-95% (Fig. 22).
  • CD19-CART cells constructed by the present invention have stronger expansion ability than CD19-CART cells prepared with lentivirus ( FIG. 24 ).
  • CD19-CART cells can activate the expression of surface markers in response to the stimulation of tumor cells, and the expression level of CD137 increased more obviously (Figure 25).
  • CD19-CART cells can secrete cytokines in response to the stimulation of tumor cells, and the level of IFN- ⁇ secretion is more obvious (Figure 26).
  • CD19-CART cells prepared by lentivirus Compared with CD19-CART cells prepared by lentivirus, the PD1 site-directed integration CD19-CART cells constructed by the present invention showed better tumor killing ability in vitro and in vivo ( Figure 27, 28).
  • the vector is a circular structure, and the vector backbone is from the plasmid pmaxGFP TM , purchased from Lonza Company.
  • the "PD1 site 1 site-specific integration of the homology arm 800bp DNA donor sequence (HDR) of mTurquoise2" in the homologous double-stranded DNA sequence of the donor DNA sequence is as shown in SEQ ID NO.: 12.
  • the vector is a circular structure, and the vector backbone is from the plasmid pmaxGFP TM , purchased from Lonza Company.
  • PD1 site 2 integrates the homology arm 800bp DNA donor sequence (HDR) of mTurquoise2", that is, as shown in SEQ ID NO.: 13.
  • Linear double-stranded DNA (donor DNA) is shown in any one of SEQ ID NO.: 12-38.
  • the 800bp DNA donor sequence (HDR) of the homology arm of PD1 site-specific integration of mTurquoise2 is shown in SEQ ID NO.: 12, and the 800bp DNA donor sequence (HDR) of the homology arm of mTurquoise2 site-specific integration of PD1 site 2 is shown in SEQ ID NO.: 12 ID NO.: 13, PD1 site 1 site-specific integration of mTurquoise2 homology arm 200bp DNA donor sequence (HDR) as shown in SEQ ID NO.: 14, PD1 site 1 site-specific integration of mTurquoise2 homology arm 400bp DNA donor
  • the sequence (HDR) is shown in SEQ ID NO.: 15, the 1600bp DNA donor sequence (HDR) of the homology arm of mTurquoise2 is site-specifically integrated in PD1, and the 1600bp DNA donor sequence (HDR) is shown in SEQ ID NO.: 16, and PD1 site 2 is site-specifically integrated with mTurquo
  • the homology arm 200bp DNA donor sequence (HDR) is shown in SEQ ID NO.: 17, the PD1 site 2 site-specific integration of the homology arm 400bp DNA donor sequence (HDR) of mTurquoise2 is shown in SEQ ID NO.: 18, PD1 position
  • the 1600bp DNA donor sequence (HDR) of the homology arm of mTurquoise2 site-directed integration at point 2 is shown in SEQ ID NO.: 19
  • the 800bp DNA donor sequence (HDR) of the homology arm of mTurquoise2 site-directed integration at AAVS1 site 1/4 is shown in SEQ ID NO.
  • the 800bp DNA donor sequence (HDR) of the homology arm of AAVS1 site-specific integration of mTurquoise2 is as shown in SEQ ID NO.:21
  • the 800bp DNA donor sequence of the homology arm of AAVS1 site-specific integration of mTurquoise2 ( HDR) as shown in SEQ ID NO.: 22
  • the homology arm 800bp DNA donor sequence (HDR) of TRAC site 1 integrated mTurquoise2 is as shown in SEQ ID NO.: 23
  • the homology of mTurquoise2 is site-specifically integrated at TRAC site 2
  • the arm 800bp DNA donor sequence (HDR) is shown in SEQ ID NO.: 24, the homology arm 800 bp DNA donor sequence (HDR) of mTurquoise2 is site-specifically integrated at TRAC site 3, and the B2M site 2 is shown in SEQ ID NO.: 25
  • the sequence of the linear double-stranded DNA (donor DNA) as a comparative example is shown in any one of SEQ ID NO.: 39-44.
  • the 800bp DNA donor sequence (HITI-pb or writing "HITI(pb)") of the homology arm of mTurquoise2 site-specific integration of PD1 site 1 is shown in SEQ ID NO.: 39
  • 800bp DNA donor sequence (HITI) is shown in SEQ ID NO.: 40
  • PD1 site 1 site-specific integration of the homology arm 800bp DNA donor sequence (MMEJ) of mTurquoise2 is shown in SEQ ID NO.: 41
  • PD1 site 2 Site-directed integration of the homology arm 800bp DNA donor sequence of mTurquoise2 (HITI-pb or writing "HITI (pb)" as shown in SEQ ID NO.: 42
  • PD1 site 2 site-directed integration of the homology arm 800bp DNA donor sequence of mTurquoise2 ( HITI) as shown in SEQ ID NO.: 43,
  • Untreated T means untreated T cells
  • Control means T cells electroporated with homologous template and Cas9 only
  • LV-19bbz means CD19-CART cells prepared with lentivirus
  • AAVS1-19bbz indicates non-viral AAVS1 site-directed integration CD19-CART cells
  • PD1-19bbz indicates non-viral PD1 site-directed integration CD19-CART cells.
  • Example 1 The delivery method of linear double-stranded DNA as a homologous template is better than a plasmid; when the homology arm of the homologous template is set to be 800bp in length, it has the best recombination rate and can obtain the most positive cells, which is the best condition
  • the donor sequences of HITI, HITI(pb) and MMEJ at PD1 site 1 are shown in SEQ ID NO.:39-41 respectively.
  • PD1 The donor sequences of HITI, HITI(pb) and MMEJ at site 2 are shown in SEQ ID NO.: 42-44, respectively.
  • the results showed that the HDR template had the highest recombination rate (Fig. 3). Based on the above results, it was determined that the use of linear double-stranded DNA containing homology arms as a template to construct non-viral site-directed integration CAR-T cells was the best method ( Figure 1).
  • the effect of homology arm length was compared.
  • the fluorescent protein mTurquoise2 sequence was used as the exogenous sequence to carry out recombination experiments at two PD1 sites ( Figure 4).
  • the results showed that when the homology arm length was 200bp, 400bp, 800bp and 1600bp, all showed good integration effect, and with the increase of the homology arm length, the integration rate of mTurquoise2 increased, but the 1600bp and 800bp were not the same. No significant increase in recombination rate was shown.
  • the cell survival was significantly negatively correlated with the homology arm length. Combining the recombination rate and cell survival, we analyzed the number of mTurquoise2-positive viable cells.
  • CD19-CART cells prepared by lentivirus can activate the expression of surface markers and secrete cytokines in response to the stimulation of tumor cells, and the increase in the expression level of CD137 is more obvious ( Figure 25). , the increase in the level of IFN- ⁇ secretion was more significant ( Figure 26). Finally, in vitro and in vivo experiments proved that compared with CD19-CART cells prepared by lentivirus, the PD1 site-specific integrated CD19-CART cells constructed by the present invention have stronger tumor killing ability (Figure 27, 28).
  • the CRISPR/Cas9 gene editing tool can successfully construct AAVS1, PD1, TRAC, B2M site-directed integrated CAR-T cells.
  • the present invention proves that compared with the prior art, the site-directed integration CAR-T cells prepared by the method of the present invention have a higher positive rate and can function effectively.
  • this technical method can reduce the high cost of using virus in the preparation of CAR-T, reduce the potential safety hazards caused by random insertion of virus, and improve the uniformity of CAR-T products.
  • this method can also realize the diversification of CAR-T cells and enhance the anti-tumor ability of CAR-T cells.
  • This example proves the importance and value of the method for improving the site-directed integration of exogenous sequences in T cells protected by the present invention, but is not limited to the preparation of AAVS1, PD1, TRAC, B2M site-directed integration of CAR-T cells, which can be extended to other exogenous sites Site-directed integration of sequences and development of other T-cell immunotherapies.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Biochemistry (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Oncology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Hematology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

一种对细胞中的靶位点进行基因编辑的方法，具体地，一种对细胞基因组的靶位点进行基因编辑的方法，包括：（a）提供一待基因编辑的细胞；（b）将（i）基因编辑酶或其编码核酸或表达所述基因编辑酶的第一表达载体；和（ii）gRNA或表达所述gRNA的第二表达载体导入所述细胞中，对所述细胞基因组的靶位点进行基因编辑，所述gRNA引导基因编辑酶对靶位点进行定点切割；其中，所述gRNA靶向的靶向序列包含SEQ ID No.1-9所示的序列中的一个或多个。该方法可对靶位点进行高效基因编辑。

Description

对细胞中的靶位点进行基因编辑的方法 技术领域
本发明涉及生物技术领域，具体地，涉及对细胞中的靶位点进行基因编辑的方法。
背景技术
CRISPR/Cas9系统是来源于古细菌和细菌的一种抵御质粒、噬菌体等外源DNA片段入侵的获得性免疫机制。该系统主要由CRISPR序列和编码Cas蛋白的基因座发挥功能，目前最常用的是Ⅱ型家族的Cas9核酸酶，这一系统只需要单一的效应蛋白就可发挥作用。当外源DNA入侵细菌时,Ⅱ型CRISPR系统首先将入侵DNA整合到CRISPR重复序列之间。随后,包含入侵DNA序列的CRISPR RNA(crRNA)被转录和加工出来。此后，反式激活crRNA(transactivating CRISPR RNA,tracrRNA)与crRNA结合,并最终与Cas9蛋白形成复合物。最后，Cas9蛋白通过其HNH和RuvC样结构域发挥核酸内切酶作用引发DNA双链断裂。DNA的双链断裂会引发损伤修复机制，当有同源模板存在的情况下，细胞会发生精确的同源重组修复，从而可以实现外源序列的插入或替换。迄今，CRISPR/Cas9技术已经在很多领域得以成功应用，显示出广阔的前景。但是，利用同源重组方式进行精确的基因治疗依然面临很多困难，其中一个主要限制因素就是同源重组的效率非常低。虽然有不少研究报道了多种提高同源重组率的方法，但越来越多的证据表明这些方法并不适用于所有类型的细胞且效果也并不一致。因此，在特定细胞中找到有效提高同源重组的个性化方法对基于细胞类型的细胞治疗尤为重要。
嵌合抗原受体T细胞技术(Chimeric Antigen Receptor T-Cell,CAR-T)是近年来兴起的一种新型过继免疫疗法。它将患者T细胞在体外进行基因改造，经过一定扩增后回输至患者体内，从而实现肿瘤的靶向杀伤。目前，已有三款CAR-T产品被美国食品药品监督管理局批准用于血液肿瘤的临床治疗，标志着CAR-T技术的巨大成功。但是，作为新兴的技术，CAR-T技术在各方面依然面临着很多挑战，有巨大的改进空间。首先，现在最常用的方法是利用病毒系统导入外源序列。但是，使用病毒系统存在制备成本高、有随机插入的安全隐患等问题。其次，包括病毒制备在内的传统技术无法实现外源序列在基因组中的 精确插入，这样会造成细胞均一性差而影响疗效，也无法对T细胞进行更多的改造。另外，目前CAR-T疗法都是个性化治疗，存在生产成本高、周期长、效果有限等问题。因此，利用CRISPR/Cas9等技术实现CAR等人工改造元件在T细胞中的定点整合，对推动现有T细胞疗法的发展具有重要意义。一方面它可以避免外源序列随机插入带来的不确定性，提高细胞产品的均一性和稳定性。另一方面它可以实现T细胞的多样化改造，既可以一步构建通用型CAR-T细胞，通过调控内源基因构建增强型细胞产品，也可以实现人工元件的动态化表达，对拓展现有技术的应用有很大的帮助。但是，由于T细胞本身细胞类型的特殊性，通过同源重组实现外源序列定点整合的效率一直很低。因此，本领域迫切需要开发一种提高T细胞中同源重组效率的方法。
发明内容
本发明的目的之一就是提供一种提高细胞(尤其是T细胞)中外源序列同源重组的效率的新方法。
本发明提供了分别针对AAVS1、PD1、TRAC、B2M基因进行基因编辑的方法。
在本发明的第一方面，提供了一种对细胞基因组的靶位点进行基因基因编辑的方法，包括：
(a)提供一待基因编辑的细胞；
(b)将(i)基因编辑酶或其编码核酸或表达所述基因编辑酶的第一表达载体；和(ii)gRNA或表达所述gRNA的第二表达载体导入所述细胞中，对所述细胞基因组的靶位点进行基因编辑，所述gRNA引导基因编辑酶对靶位点进行定点切割；
其中，所述gRNA靶向的靶向序列包含SEQ ID No.1-9所示的序列中的一个或多个。
在另一优选例中，所述基因编辑包括基因敲除和基因靶向整合。
在另一优选例中，所述gRNA靶向的靶向序列包含SEQ ID NO.:1－6所示的序列中的一个或多个时，所述基因编辑为基因靶向整合。
在另一优选例中，所述gRNA靶向的靶向序列包含SEQ ID NO.:7－9所示序列中的一个或多个时，所述基因编辑为基因敲除。
在另一优选例中，所述基因编辑酶选自下组：CRISPR相关蛋白(Cas)多肽、TALEN酶、ZFN酶、或其组合。
在另一优选例中，所述基因编辑酶来源于微生物；优选地来源于细菌。
在另一优选例中，所述基因编辑酶的来源选自下组：酿脓链球菌(Streptococcus pyogenes)、葡萄球菌(Staphylococcus aureus)、犬链球菌(Streptococcus canis)、或其组合。
在另一优选例中，所述基因编辑酶包括野生型或突变型的基因编辑酶。
在另一优选例中，所述基因编辑酶选自下组：Cas9、Cas12、Cas13、Cms1、MAD7、Cas3、Cas8a、Cas8b、Cas10d、Cse1、Csy1、Csn2、Cas4、Cas10、Csm2、Cmr5、Fok1、Cpf1、或其组合。
在另一优选例中，所述gRNA包括crRNA、tracrRNA、sgRNA。
在另一优选例中，所述的gRNA靶向结合于所述基因组的靶位点。
在另一优选例中，所述gRNA包括未修饰和经修饰的gRNA。
在另一优选例中，所述经修饰的gRNA包括碱基的化学修饰。
在另一优选例中，所述化学修饰包括甲基化修饰、甲氧基修饰、氟化修饰或硫代修饰。
在另一优选例中，所述的基因编辑包括体内基因编辑、体外基因编辑。
在另一优选例中，所述的基因编辑包括基于CRISPR的基因编辑。
在另一优选例中，所述靶位点选自下组：AAVS1、PD1、TRAC、B2M、或其组合。
在另一优选例中，所述gRNA靶向AAVS1的靶向序列包含SEQ ID No.3所示的序列。
在另一优选例中，所述gRNA靶向PD1的靶向序列包含SEQ ID NO.:1和/或SEQ ID NO.:2所示的序列。
在另一优选例中，所述gRNA靶向TRAC的靶向序列包含SEQ ID NO.:4和/或SEQ ID NO.:5所示的序列。
在另一优选例中，所述gRNA靶向B2M的靶向序列包含SEQ ID NO.:6所示的序列。
在另一优选例中，所述基因编辑包括定点敲入供体DNA。
在另一优选例中，所述供体DNA为双链DNA。
在另一优选例中，所述的供体DNA包括第一同源臂和第二同源臂，其中所述第一同源臂和第二同源臂能够启动所述供体DNA在所述细胞基因组的靶位点处由细胞介导的同源重组，所述第一同源臂和第二同源臂的序列长度各自独立的为 200-2000bp，较佳地，400-1000bp，更佳地，700-900bp。
在另一优选例中，所述的第一同源臂与所述基因组的靶位点的切割位点的上游侧(或左侧)序列是同源的，而所述的第二同源臂与所述基因组的靶位点的切割位点的下游侧(或右侧)序列是同源的。
在另一优选例中，所述供体DNA还包括目的基因。
在另一优选例中，所述目的基因包括嵌合抗原受体或TCR的编码序列。
在另一优选例中，所述供体DNA的序列长度为50bp－5000bp，较佳地，80bp－4000bp。
在另一优选例中，所述目的基因的长度为50bp-3000bp，较佳地，1000bp-2000bp。
在另一优选例中，所述嵌合抗原受体或TCR的编码序列的长度为50bp-3000bp，较佳地，1000bp-2000bp。
在另一优选例中，所述第一同源臂的序列长度D1和第二同源臂的序列长度D2之比(D1/D2)为(0.8-1.2):(0.5-1.5)，较佳地(0.9-1.1):(0.7-1.3)，更佳地，1:1。
在另一优选例中，所述嵌合抗原受体含有靶向肿瘤细胞标志物的抗原结合结构域。
在另一优选例中，所述嵌合抗原受体包括靶向肿瘤细胞标志物的抗原结合结构域、任选的绞链区、跨膜结构域和细胞内信号传导结合域。
在另一优选例中，所述肿瘤细胞标志物选自下组：α叶酸受体、5T4、αvβ6整联蛋白、BCMA、B7-H3、B7-H6、CAIX、CD16、CD19、CD20、CD22、CD30、CD33、CD44、CD44v6、CD44v7/8、CD70、CD79a、CD79b、CD123、CD138、CD171、CEA、CSPG4、EGFR、包含ErbB2(HER2)的EGFR家族、EGFRvIII、EGP2、EGP40、EPCAM、EphA2、EpCAM、FAP、胎儿AchR、FRα、GD2、GD3、磷脂酰肌醇蛋白聚糖-3(GPC3)、HLA-A1+MAGE1、HLA-A2+MAGE1、HLA-A3+MAGE1、HLA-A1+NY-ESO-1、HLA-A2+NY-ESO-1、HLA-A3+NY-ESO-1、IL-11Rα、IL-13Rα2、Lambda、Lewis-Y、Kappa、间皮素、Muc1、Muc16、NCAM、NKG2D配体、NY-ESO-1、PRAME、PSCA、PSMA、ROR1、SSX、存活蛋白、TAG72、TEM、VEGFR2、WT-1、或其组合。
在另一优选例中，所述绞链区为选自下组的蛋白的铰链区：CD8、Ig(免疫球蛋白)铰链、CD28、或其组合。
在另一优选例中，所述跨膜结构域为选自下组的蛋白的跨膜区：CD8α、CD8β、CD28、CD33、CD37、CD5、CD16、ICOS、CD9、CD22、CD134、CD137、CD154、 CD19、CD45、CD4、CD3ε、CD3γ、CD3ζ、或其组合。
在另一优选例中，所述细胞内信号传导结合域包括共刺激信号分子和/或初级信号传导结构域。
在另一优选例中，所述共刺激信号分子选自下组：OX40、CD28、CD30、CD40、CD70、CD134、4-1BB(CD137)、LIGHT、DAP10、CDS、ICAM-1、CD278(ICOS)、TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、CARD11、CD2、CD7、CD27、CD54(ICAM)、CD83、LAT、NKD2C、SLP76、TRIM、ZAP70、或其组合。
在另一优选例中，所述初级信号传导结构域选自下组：FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b、CD66d、或其组合。
在另一优选例中，所述供体DNA的序列如SEQ ID NO.:12－38中任一项所示。
在另一优选例中，所述的细胞包括原代细胞和传代的细胞。
在另一优选例中，所述细胞包括T细胞，
在另一优选例中，所述细胞包括CD3+T细胞、CD4+辅助T细胞、CD4+调节T细胞、CD8+T细胞、记忆性T细胞。
在另一优选例中，所述载体包括病毒载体。
在另一优选例中，所述病毒载体选自下组：腺相关病毒载体、慢病毒载体、或其组合。
在另一优选例中，所述导入方法包括电转、载体转化、转染、热休克、电穿孔、转导、基因枪、显微注射、脂质体转染、慢病毒感染。
在另一优选例中，所述方法是体外方法。
在另一优选例中，所述方法是非诊断性和非治疗性的。
本发明第二方面提供了一种对细胞基因组的靶位点进行基因编辑的gRNA，所述gRNA靶向的靶向序列包含SEQ ID No.1-9所示的序列中的一个或多个。
在另一优选例中，所述gRNA靶向的靶向序列包含SEQ ID NO.:1－6所示的序列中的一个或多个。
在另一优选例中，所述gRNA靶向的靶向序列包含SEQ ID NO.:7－9所示的序列中的一个或多个。
在另一优选例中，所述gRNA包括未修饰和经修饰的gRNA。
在另一优选例中，所述经修饰的gRNA包括碱基的化学修饰。
在另一优选例中，所述化学修饰包括甲基化修饰、甲氧基修饰、氟化修饰或硫代修饰。
本发明第三方面提供了一种用于对细胞基因组的靶位点进行基因编辑的反应体系，所述反应体系包括：
(a)供体DNA，所述供体DNA为双链DNA；
(b)基因编辑酶或其编码核酸或表达所述基因编辑酶的第一表达载体；
(c)gRNA或表达所述gRNA的第二表达载体；
其中，所述gRNA靶向的靶向序列包含SEQ ID No.1-9所示的序列中的一个或多个。
在另一优选例中，所述基因编辑包括基因敲除和基因靶向整合。
在另一优选例中，所述gRNA靶向的靶向序列包含SEQ ID NO.:1－6所示的序列的一个或多个时，所述基因编辑为基因靶向整合。
在另一优选例中，所述gRNA靶向的靶向序列包含SEQ ID NO.:7－9所示序列的一个或多个时，所述基因编辑为基因敲除。
在另一优选例中，所述靶位点选自下组：AAVS1、PD1、TRAC、B2M、或其组合。
在另一优选例中，所述gRNA靶向AAVS1的靶向序列包含SEQ ID No.3所示的序列。
在另一优选例中，所述gRNA靶向PD1的靶向序列包含SEQ ID NO.:1和/或SEQ ID NO.:2所示的序列。
在另一优选例中，所述gRNA靶向TRAC的靶向序列包含SEQ ID NO.:4和/或SEQ ID NO.:5所示的序列。
在另一优选例中，所述gRNA靶向B2M的靶向序列包含SEQ ID NO.:6所示的序列。
在另一优选例中，所述的供体DNA包括第一同源臂和第二同源臂，其中所述第一同源臂和第二同源臂能够启动所述供体DNA在所述免疫细胞基因组的靶位点处由免疫细胞介导的同源重组，所述第一同源臂和第二同源臂的序列长度各自独立的为200-2000bp，较佳地，400-1000bp，更佳地，700-900bp。
在另一优选例中，所述供体DNA还包括目的基因。
在另一优选例中，所述目的基因包括嵌合抗原受体或TCR的编码序列。
在另一优选例中，所述嵌合抗原受体含有靶向肿瘤细胞标志物的抗原结合结构域。
在另一优选例中，所述嵌合抗原受体包括靶向肿瘤细胞标志物的抗原结合结构 域、任选的绞链区、跨膜结构域和细胞内信号传导结合域。
在另一优选例中，所述肿瘤细胞标志物选自下组：α叶酸受体、5T4、αvβ6整联蛋白、BCMA、B7-H3、B7-H6、CAIX、CD16、CD19、CD20、CD22、CD30、CD33、CD44、CD44v6、CD44v7/8、CD70、CD79a、CD79b、CD123、CD138、CD171、CEA、CSPG4、EGFR、包含ErbB2(HER2)的EGFR家族、EGFRvIII、EGP2、EGP40、EPCAM、EphA2、EpCAM、FAP、胎儿AchR、FRα、GD2、GD3、磷脂酰肌醇蛋白聚糖-3(GPC3)、HLA-A1+MAGE1、HLA-A2+MAGE1、HLA-A3+MAGE1、HLA-A1+NY-ESO-1、HLA-A2+NY-ESO-1、HLA-A3+NY-ESO-1、IL-11Rα、IL-13Rα2、Lambda、Lewis-Y、Kappa、间皮素、Muc1、Muc16、NCAM、NKG2D配体、NY-ESO-1、PRAME、PSCA、PSMA、ROR1、SSX、存活蛋白、TAG72、TEM、VEGFR2、WT-1、或其组合。
在另一优选例中，所述绞链区为选自下组的蛋白的铰链区：CD8、Ig(免疫球蛋白)铰链、CD28、或其组合。
在另一优选例中，所述跨膜结构域为选自下组的蛋白的跨膜区：CD8α、CD8β、CD28、CD33、CD37、CD5、CD16、ICOS、CD9、CD22、CD134、CD137、CD154、CD19、CD45、CD4、CD3ε、CD3γ、CD3ζ、或其组合。
在另一优选例中，所述细胞内信号传导结合域包括共刺激信号分子和/或初级信号传导结构域。
在另一优选例中，所述共刺激信号分子选自下组：OX40、CD28、CD30、CD40、CD70、CD134、4-1BB(CD137)、LIGHT、DAP10、CDS、ICAM-1、CD278(ICOS)、TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、CARD11、CD2、CD7、CD27、CD54(ICAM)、CD83、LAT、NKD2C、SLP76、TRIM、ZAP70、或其组合。
在另一优选例中，所述初级信号传导结构域选自下组：FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b、CD66d、或其组合。
在另一优选例中，所述的细胞包括原代细胞和传代的细胞。
在另一优选例中，所述细胞包括T细胞，
在另一优选例中，所述细胞包括CD3+T细胞、CD4+辅助T细胞、CD4+调节T细胞、CD8+T细胞、记忆性T细胞。
在另一优选例中，所述的第一同源臂与所述基因组的靶位点的切割位点的上游侧(或左侧)序列是同源的，而所述的第二同源臂与所述基因组的靶位点的切割位点的下游侧(或右侧)序列是同源的。
在另一优选例中，所述供体DNA的序列长度为50bp－5000bp，较佳地，80bp －4000bp。
在另一优选例中，所述目的基因的长度为50bp-3000bp，较佳地，1000bp-2000bp。
在另一优选例中，所述嵌合抗原受体或TCR的编码序列的长度为50bp-3000bp，较佳地，1000bp-2000bp。
在另一优选例中，所述供体DNA的序列如SEQ ID NO.:12－38中任一项所示。
在另一优选例中，所述载体包括病毒载体。
在另一优选例中，所述病毒载体选自下组：腺相关病毒载体、慢病毒载体、或其组合。
本发明第四方面提供了一种用于基因编辑的试剂盒，所述试剂盒包括：
i)第一容器以及位于第一容器内的供体DNA，所述供体DNA为双链DNA；
ii)第二容器以及位于第二容器内的基因编辑酶或其编码核酸或表达所述基因编辑酶的第一表达载体；和
iii)第三容器以及位于第三容器内的gRNA或表达所述gRNA的第二表达载体，并且所述gRNA靶向的靶向序列包含SEQ ID No.1-9所示的序列的一个或多个。
在另一优选例中，所述第一容器、第二容器、第三容器为同一容器或不同的容器。
本发明第五方面提供了一种本发明第一方面所述的方法制备得到的经基因编辑的细胞。
本发明第六方面提供了一种本发明第五方面所述的细胞在制备肿瘤免疫治疗或癌症免疫治疗产品中的应用。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
本申请的附图中，“Untreated T”表示未处理的T细胞，“Control”表示只电转了同源模板和Cas9的T细胞，“LV-19bbz”表示用慢病毒制备的CD19-CART细胞，“AAVS1-19bbz”表示非病毒AAVS1定点整合型CD19-CART细胞，“PD1-19bbz”表示非病毒PD1定点整合型CD19-CART细胞。
图1为构建非病毒定点整合型CAR-T细胞原理示意图。“TRAC-19bbz”表示非病毒TRAC定点整合型CD19-CART细胞，“B2M-19bbz”表示非病毒B2M定点整合型CD19-CART细胞。
图2显示了质粒或线性双链DNA电转后T细胞存活。对照组只电转质粒或线性双链DNA，样品组同时电转了模板、Cas9和sgRNA。
图3显示了使用不同模板电转后mTurquoise2阳性率。对照组为只电转HDR模板和Cas9，样品组为电转线性双链DNA模板、Cas9和sgRNA；pb为在两侧添加了保护碱基。
图4显示了不同长度同源臂模板电转后mTurquoise2阳性率和细胞存活。对照组为只电转800bp同源模板和Cas9，样品组为电转不同长度同源臂模板、Cas9和sgRNA；A图为mTurquoise2阳性率、B图为活细胞相对数量、C图为mTurquoise2阳性活细胞相对数量。
图5显示了AAVS1位点sgRNA筛选。A图为T7E1检测AAVS1位点敲除率、B图为荧光蛋白mTurquioise2序列在AAVS1位点重组率。图中，“sgRNA-1”表示本申请的AAVS1-sgRNA1，“sgRNA-2”表示本申请的AAVS1-sgRNA2，“sgRNA-3”表示本申请的AAVS1-sgRNA3，“sgRNA-4”表示本申请的AAVS1-sgRNA4。
图6显示了PD1位点sgRNA筛选，为荧光蛋白mTurquioise2序列在PD1位点重组率。图中，“sgRNA-1”表示本申请的PD1-sgRNA1，“sgRNA-2”表示本申请的PD1-sgRNA2。
图7显示了TRAC位点sgRNA筛选。A图为通过CD3表达检测TRAC位点敲除率、B图为荧光蛋白mTurquioise2序列在TRAC位点重组率。图中，“sgRNA-1”表示本申请的TRAC-sgRNA1，“sgRNA-2”表示本申请的TRAC-sgRNA2，“sgRNA-3”表示本申请的TRAC-sgRNA3。
图8显示了B2M位点sgRNA筛选。A图为通过B2M表达检测B2M位点敲除率、B图为荧光蛋白mTurquioise2序列在B2M位点重组率。图中，“sgRNA-1”表示本申请的B2M-sgRNA1，“sgRNA-2”表示本申请的B2M-sgRNA2。
图9显示了不同长度同源臂模板电转后CAR阳性率和细胞存活。对照组为只电转800bp同源模板和Cas9，样品组为电转不同长度同源臂模板、Cas9和sgRNA；A图为CAR阳性率、B图为活细胞相对数量、C图为CAR阳性活细胞相对数量。
图10显示了CD3 +、CD4 +和CD8 +T细胞中AAVS1定点整合CD19-CART细胞阳 性率
图11显示了AAVS1定点整合CD19-CART细胞阳性率和敲除率。A图为不同人供体细胞制备的AAVS1定点整合CD19-CART细胞阳性率；B图为五个代表性的不同人供体细胞制备的AAVS1定点整合CD19-CART细胞阳性率和敲除率；C图为流式分析显示AAVS1定点整合CD19-CART细胞阳性率。对照组为只电转同源模板和Cas9。
图12显示了AAVS1定点整合CD19-CART细胞DNA测序结果。HA代表同源臂。
图13显示了AAVS1定点整合T细胞存活率。
图14显示了AAVS1定点整合T细胞体外扩增。
图15显示了AAVS1定点整合CD19-CART细胞与靶细胞共培养体外扩增。
图16显示了用标记法检测AAVS1定点整合CD19-CART细胞与靶细胞共培养体外扩增。对照组为只电转同源模板和Cas9。
图17显示了AAVS1定点整合CD19-CART细胞表面标志物表达。对照组为只电转同源模板和Cas9。
图18显示了AAVS1定点整合CD19-CART细胞亚型比例。对照组为只电转同源模板和Cas9。
图19显示了AAVS1定点整合CD19-CART细胞分泌因子检测。对照组为只电转同源模板和Cas9。
图20显示了AAVS1定点整合CD19-CART细胞体外杀伤。对照组为只电转同源模板和Cas9。
图21显示了AAVS1定点整合CD19-CART细胞体内杀伤。肿瘤靶细胞为Raji细胞。
图22显示了PD1定点整合CD19-CART细胞阳性率和敲除率。A图为不同人供体细胞制备的PD1定点整合CD19-CART细胞阳性率；B图为五个代表性的不同人供体细胞制备的PD1定点整合CD19-CART细胞阳性率和敲除率；C图为流式分析显示PD1定点整合CD19-CART细胞阳性率；D图为PD1定点整合CD19-CART细胞与慢病毒制备CD19-CART细胞的PD1表达比较。对照组为只电转同源模板和Cas9。
图23显示了PD1定点整合CD19-CART细胞DNA测序结果。HA代表同源臂
图24显示了PD1定点整合CD19-CART细胞与靶细胞共培养体外扩增。
图25显示了PD1定点整合CD19-CART细胞表面标志物表达。对照组为只电转同源模板和Cas9。
图26显示了PD1定点整合CD19-CART细胞分泌因子检测。对照组为只电转同源模板和Cas9。
图27显示了PD1定点整合CD19-CART细胞体外杀伤。对照组为只电转同源模板和Cas9。
图28显示了PD1定点整合CD19-CART细胞体内杀伤。肿瘤靶细胞为PDL1过表达的Raji细胞。
具体实施方式
本发明人经过广泛而深入的研究，通过大量筛选，首次意外的筛选到了对靶位点(如：AAVS1、PD1、TRAC、B2M)高效进行基因编辑的gRNA。此外，本发明人通过比较不同长度的第一同源臂和第二同源臂，还意外的确定了T细胞中外源核酸细胞基因组高效整合的条件(比如特定长度的第一同源臂和第二同源臂可进行基因组靶位点的高效整合)。在此基础上完成了本发明。
术语
CRISPR/Cas9介导的基因编辑方法
CRISPR/Cas9是细菌和古细菌在长期演化过程中形成的一种适应性免疫防御，可用来对抗入侵的病毒及外源DNA。CRISPR/Cas9系统通过将入侵噬菌体和质粒DNA的片段整合到CRISPR中，并利用相应的CRISPR RNAs(比如gRNAs)来指导同源序列的降解，从而提供免疫性。
此系统的工作原理是crRNA(CRISPR-derived RNA)通过碱基配对与tracrRNA(trans-activating RNA)结合形成tracrRNA/crRNA复合物，此复合物引导核酸酶Cas9蛋白在与crRNA配对的序列靶位点剪切双链DNA。而通过人工设计这两种RNA，可以改造形成具有引导作用的gRNA(single-guide RNA)，足以引导Cas9对DNA的定点切割。
作为一种RNA导向的dsDNA结合蛋白，Cas9效应物核酸酶是已知的第一个统一因子(unifying factor)，能够共定位RNA、DNA和蛋白。将蛋白与无核酸酶的Cas9(Cas9 nuclease-null)融合，并表达适当的gRNA，可靶定任何dsDNA序列，而gRNA的末端可连接到目标DNA，不影响Cas9的结合。因此， Cas9能在任何dsDNA序列处带来任何融合蛋白及RNA。这种技术被称为CRISPR/Cas9基因编辑系统。
靶向整合机制
在本发明的靶向整合策略中，提供了一种含有特定长度的同源臂的线性化供体(即供体DNA或外源核酸)，这种供体是通过PCR扩增或者精确酶切获得的含有一定长度(比如，700-900bp)同源臂的转基因供体；将供体DNA或外源核酸与Cas9mRNA以及guide RNA一起电转到细胞中。本发明提供的靶向整合策略相比于已有的基因靶向策略具有更高的整合效率。
在本发明中，在CRISPR介导的基因组编辑中由转基因片段以及两边的700-900bp同源臂组成的线性化dsDNA供体(即供体DNA或外源核酸)是实现高效靶向整合的关键因素。与MMEJ(微同源臂介导的末端连接)或HITI(非同源序列的末端连接)介导的方法相比，本发明的靶向整合技术具有最高的重组率。
对细胞基因组的靶位点进行基因编辑的方法
本发明提供了一种对细胞基因组的靶位点进行基因基因编辑的方法，包括：
(a)提供一待基因编辑的细胞；
(b)将(i)基因编辑酶或其编码核酸或表达所述基因编辑酶的第一表达载体；和(ii)gRNA或表达所述gRNA的第二表达载体导入所述细胞中，对所述细胞基因组的靶位点进行基因编辑，所述gRNA引导基因编辑酶对靶位点进行定点切割；
其中，所述gRNA靶向的靶向序列包含SEQ ID No.1-9所示的序列中的一个或多个。
本发明的方法可对靶位点(比如AAVS1、PD1、TRAC、B2M等)进行高效的基因敲除或基因靶向整合。
反应体系
本发明提供了一种用于对细胞基因组的靶位点进行基因编辑的反应体系，所述的反应体系包括：
(a)供体DNA，所述供体DNA为双链DNA；
(b)基因编辑酶或其编码核酸或表达所述基因编辑酶的第一表达载体；
(c)gRNA或表达所述gRNA的第二表达载体；
其中，所述gRNA靶向的靶向序列包含SEQ ID No.1-9所示的序列中的一个或多个。
在一优选实施方式中，所述的供体DNA包括第一同源臂和第二同源臂，其中所述第一同源臂和第二同源臂能够启动所述供体DNA在所述免疫细胞基因组的靶位点处由免疫细胞介导的同源重组，所述第一同源臂和第二同源臂的序列长度各自独立的为200-2000bp，较佳地，400-1000bp，更佳地，700-900bp。
在本发明中，待整合的供体DNA的序列长度可以为50bp-5000bp。本发明的供体DNA中靶基因(或者称为目的基因)的长度可以是50bp-3000bp。
在本发明中，对靶位点没有特别限制，一种优选的靶位点包括AAVS1、PD1、TRAC、B2M。
在本发明的一优选实施方式中，所述第一同源臂的序列长度D1和第二同源的序列长度D2之比(D1/D2)为(0.8-1.2):(0.5-1.5)，较佳地(0.9-1.1):(0.7-1.3)，更佳地，1：1。
在本反应体系中，对两个同源臂的序列长度并不要求完全一致，其两者之间允许存在一定的长度差。
本发明的主要优点包括：
1)本发明方法的基因编辑(如定点整合)效率显著高于其他现有的基因编辑技术。
2)导入方式简便，可通过电转等方式导入。
3)本发明能提高外源核酸在T细胞中的定点整合效率。
4)使用本发明的方法，使定点整合型T细胞产品的制备成为可能。
5)本方法可以成功制备AAVS1、PD1、TRAC、B2M等位点的非病毒定点整合型CAR-T细胞，能避免病毒制备存在的诸多弊端，能提高CAR-T产品的均一性和稳定性。另外，也为制备增强型、可诱导型等多样化CAR-T产品提供技术支持。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York： Cold Spring Harbor Laboratory Press，1989)中所述的条件，或按照《微生物：实验手册》(James Cappuccino和Natalie Sherman编，Pearson Education出版社)中所述的条件，或按照制造厂商所建议的条件。本专利涉及实验中使用的人供体细胞都获自上海赛笠生物科技有限公司。
如无特别说明，实施例所用的材料和试剂均为市售产品。
通用方法
I.电转T细胞
电转T细胞是实现T细胞基因编辑的技术方法，步骤参照Lonza公司P3 Primary Cell
Figure PCTCN2021116531-appb-000001
X试剂盒
仪器与材料：
①Lonza 4D-Nucleofector TM System细胞核转仪
②试剂盒为P3 Primary Cell 4D-Nucleofector TM X Kit，(Lonza,V4XP-3024)
③CD3/CD28磁珠刺激后2-4天的T细胞
④商品化spCas9蛋白(5ug/ul)(TrueCut TM Cas9 Protein v2，Thermofisher)
⑤合成的sgRNA(将合成的sgRNA溶解于TE缓冲液中，稀释成终浓度10ug/uL)
⑥含同源模板的线性化双链DNA
具体操作步骤：
适用于100μl规格的电转杯：
(1)按照82μl Solution+18μl supplement/每个电转杯，按电转总数，配置一个电转液混合液，混匀，放室温。
(2)将Cas9蛋白和sgRNA9进行共孵育，室温放置10min，形成RNP。
(3)RNP中加入线性化的同源模板双链DNA(包括含PD1两个位点同源臂的荧光蛋白mTurquoise2序列、含AAVS1、PD1、TRAC和B2M同源臂的CD19-CART序列)，室温孵育2min。
所述CD19-CART包括靶向CD19的胞外结构域，选自CD8α的跨膜区以及选自CD3ζ和CD137的胞内信号传导结构域；
(4)收集激活状态的T细胞，计数为5×10 6进行一个电转反应。
(5)将细胞与“RNP+同源模板”充分混合重悬，加入电转杯。
(6)打开电转仪，将电转杯放入槽孔，选择相应程序EO115进行电转。
(7)将细胞加入已预热的细胞培养基中，于细胞培养箱中培养。
II.外源序列重组率检测
1)取1×10 6细胞于无菌的1.5ml离心管中，离心后弃掉上清；
2)向细胞沉淀中加入流式缓冲液(含2％血清的PBS)清洗细胞；
3)置于室温离心机中，离心后尽量吸尽上清；
4)与检测抗体或蛋白进行共孵育，冰上孵育30分钟；
5)用流式缓冲液清洗细胞两次，离心后尽量吸尽上清；
6)用合适体积流式缓冲液重悬细胞，进行流式上机分析；
III.活细胞数量检测
使用Promega公司Celltiter One Solution Cell Proliferation Assay试剂盒检测活细胞数量，操作步骤参照说明书。
1)将MTS试剂平衡至室温；
2)设置三副孔重复，取100μl细胞悬液至96孔板中，加入20μl MTS试剂；
3)置于37℃，含有5％CO2的培养箱中孵育4h；
4)酶标仪490nm检测，统计分析数据。
举例使用本发明的方法，结合CRISPR/Cas9技术，可以一步构建非病毒定点整合型CD19-CART细胞(包括AAVS1安全位点整合的CAR-T细胞、免疫检查点PD1位点整合的CD19-CART细胞、TRAC位点整合的CD19-CART细胞、B2M位点整合的CD19-CART细胞)
1、靶点序列信息：
AAVS1-sgRNA1：CCGGAGAGGACCCAGACACG(SEQ ID NO.:7)
AAVS1-sgRNA2：AGAGCTAGCACAGACTAGAG(SEQ ID NO.3)
AAVS1-sgRNA3：AAAGCAAGAGGATGGAGAGG(SEQ ID NO.:8)
AAVS1-sgRNA4：GAGAGGACCCAGACACGGGG(SEQ ID NO.:9)
PD1-sgRNA1：CGACTGGCCAGGGCGCCTGT(SEQ ID NO.1)
PD1-sgRNA2：GGGCGGTGCTACAACTGGGC(SEQ ID NO.2)
TRAC-sgRNA1：ACAAAACTGTGCTAGACATG(SEQ ID NO.:4)
TRAC-sgRNA2：AGAGCAACAGTGCTGTGGCC(SEQ ID NO.:5)
TRAC-sgRNA3：AGAGTCTCTCAGCTGGTACA(SEQ ID NO.:10)
B2M-sgRNA1：ACTCACGCTGGATAGCCTCC(SEQ ID NO.11)
B2M-sgRNA2：GAGTAGCGCGAGCACAGCTA(SEQ ID NO.:6)
2、由公司合成sgRNA，溶解于TE缓冲液中，稀释成终浓度10ug/uL
3、电转制备非病毒定点整合CD19-CART
仪器与材料：①Lonza 4D-Nucleofector TM System细胞核转仪
②试剂盒为P3 Primary Cell 4D-Nucleofector TM X Kit，(Lonza,V4XP-3024)
③CD3/CD28磁珠刺激后2-3天的T细胞
④商品化spCas9蛋白(5ug/ul)(TrueCut TM Cas9 Protein v2，Thermofisher)⑤公司合成的sgRNA
⑥含同源模板的线性化双链DNA
具体操作步骤：
适用于100μl规格的电转杯：
(1)按照82μl Solution+18μl supplement/每个电转杯，按电转总数，配置一个电转液混合液，混匀，放室温。
(2)将将Cas9蛋白分别与AAVS1-sgRNA、PD1-sgRNA1、PD1-sgRNA2、TRAC-sgRNA和B2M-sgRNA进行共孵育，室温放置10min，形成RNP
(3)RNP中加入包含各位点同源臂的线性化双链DNA，室温孵育2min
(4)收集激活状态的T细胞，计数为5×10 6进行一个电转反应
(5)将细胞与”RNP+同源模板”充分混合重悬，加入电转杯
(6)打开电转仪，将电转杯放入槽孔，选择相应程序EO115进行电转
(7)将细胞加入已预热的细胞培养基中，于细胞培养箱中培养
4、对AAVS1定点整合CD19-CART细胞进行评价
(1)使用本发明可不使用病毒构建AAVS1定点整合型CAR-T细胞，CAR阳性 率大约在10％-20％(图11)，敲除率大约在65％-90％(图11)，且该方法在CD3+、CD4+、CD8+T细胞中均可实现CAR元件的定点整合(图10)。
(2)使用本发明构建的AAVS1定点整合型CD19-CART细胞具有较高的存活率(图13)。虽然电转步骤本身造成了一定数量的细胞死亡，但并不影响AAVS1定点整合型CD19-CART细胞的有效扩增(图14-16)。
(3)虽然存在部分不同，但总体来看，AAVS1定点整合型CD19-CART细胞与慢病毒制备的CD19-CART细胞相比，在细胞表面标志物表达、细胞亚型改变、细胞因子分泌等方面呈现出相似的特性(图17-19)。
(4)与慢病毒制备的CD19-CART细胞相似，使用本发明构建的AAVS1定点整合型CD19-CART细胞在内外均能有效杀伤肿瘤细胞(图20、21)。
5、对PD1定点整合CD19-CART细胞进行评价
(1)使用本发明可不使用病毒构建PD1定点整合型CAR-T细胞，CAR阳性率大约在10％-30％(图22)，敲除率大约在80％-95％(图22)。
(2)使用本发明构建的PD1定点整合型CD19-CART细胞具有比慢病毒制备的CD19-CART细胞更强的扩增能力(图24)。
(3)与慢病毒制备的CD19-CART细胞相似，PD1定点整合型CD19-CART细胞能响应肿瘤细胞的刺激而激活表面标志物的表达，其中CD137表达水平的上升更为明显(图25)。
(4)与慢病毒制备的CD19-CART细胞相似，PD1定点整合型CD19-CART细胞能响应肿瘤细胞的刺激而分泌细胞因子，其中IFN-γ分泌水平的上升更为明显(图26)。
(5)与慢病毒制备的CD19-CART细胞相比，使用本发明构建的PD1定点整合型CD19-CART细胞在体内外均体现出更好的肿瘤杀伤能力(图27、28)。
此外，需要说明的是，本申请以如下所示的线性双链DNA(供体DNA)为例，进行实验。
其中，对质粒序列作如下说明：
(1)以PD1位点1定点整合mTurquoise2的同源臂800bpDNA质粒供体序列为例说明载体和供体DNA的情况：
载体是环状结构，载体骨架来自质粒pmaxGFP TM，购于Lonza公司。
供体DNA序列同线性双链DNA序列中的“PD1位点1定点整合mTurquoise2的同源臂800bpDNA供体序列(HDR)”，即如SEQ ID NO.:12所示。
(2)以PD1位点2定点整合mTurquoise2的同源臂800bpDNA质粒供体序列为例说明载体和供体DNA的情况：
载体是环状结构，载体骨架来自质粒pmaxGFP TM，购于Lonza公司。
供体DNA序列同线性双链DNA序列中“PD1位点2定点整合mTurquoise2的同源臂800bpDNA供体序列(HDR)”，即如SEQ ID NO.:13所示。
线性双链DNA(供体DNA)如SEQ ID NO.:12－38中任一项所示。
其中，PD1位点1定点整合mTurquoise2的同源臂800bpDNA供体序列(HDR)如SEQ ID NO.:12所示，PD1位点2定点整合mTurquoise2的同源臂800bpDNA供体序列(HDR)如SEQ ID NO.:13所示，PD1位点1定点整合mTurquoise2的同源臂200bpDNA供体序列(HDR)如SEQ ID NO.:14所示，PD1位点1定点整合mTurquoise2的同源臂400bpDNA供体序列(HDR)如SEQ ID NO.:15所示，PD1位点1定点整合mTurquoise2的同源臂1600bpDNA供体序列(HDR)如SEQ ID NO.:16所示，PD1位点2定点整合mTurquoise2的同源臂200bpDNA供体序列(HDR)如SEQ ID NO.:17所示，PD1位点2定点整合mTurquoise2的同源臂400bpDNA供体序列(HDR)如SEQ ID NO.:18所示，PD1位点2定点整合mTurquoise2的同源臂1600bpDNA供体序列(HDR)如SEQ ID NO.:19所示，AAVS1位点1/4定点整合mTurquoise2的同源臂800bpDNA供体序列(HDR)如SEQ ID NO.:20所示，AAVS1位点2定点整合mTurquoise2的同源臂800bpDNA供体序列(HDR)如SEQ ID NO.:21所示，AAVS1位点3定点整合mTurquoise2的同源臂800bpDNA供体序列(HDR)如SEQ ID NO.:22所示，TRAC位点1定点整合mTurquoise2的同源臂800bpDNA供体序列(HDR)如SEQ ID NO.:23所示，TRAC位点2定点整合mTurquoise2的同源臂800bpDNA供体序列(HDR)如SEQ ID NO.:24所示，TRAC位点3定点整合mTurquoise2的同源臂800bpDNA供体序列(HDR)如SEQ ID NO.:25所示，B2M位点2定点整合mTurquoise2的同源臂800bpDNA供体序列(HDR)如SEQ ID NO.:26所示，AAVS1位点2定点整合CD19-CAR的同源臂200bpDNA供体序列(HDR)如SEQ ID NO.:27所示，AAVS1位点2 定点整合CD19-CAR的同源臂400bpDNA供体序列(HDR)如SEQ ID NO.:28所示，AAVS1位点2定点整合CD19-CAR的同源臂800bpDNA供体序列(HDR)如SEQ ID NO.:29所示，PD1位点1定点整合CD19-CAR的同源臂200bpDNA供体序列(HDR)如SEQ ID NO.:30所示，PD1位点1定点整合CD19-CAR的同源臂400bpDNA供体序列(HDR)如SEQ ID NO.:31所示，PD1位点1定点整合CD19-CAR的同源臂800bpDNA供体序列(HDR)如SEQ ID NO.:32所示，TRAC位点2定点整合CD19-CAR的同源臂200bpDNA供体序列(HDR)如SEQ ID NO.:33所示，TRAC位点2定点整合CD19-CAR的同源臂400bpDNA供体序列(HDR)如SEQ ID NO.:34所示，TRAC位点2定点整合CD19-CAR的同源臂800bpDNA供体序列(HDR)如SEQ ID NO.:35所示，B2M位点2定点整合CD19-CAR的同源臂200bpDNA供体序列(HDR)如SEQ ID NO.:36所示，B2M位点2定点整合CD19-CAR的同源臂400bpDNA供体序列(HDR)如SEQ ID NO.:37所示，B2M位点2定点整合CD19-CAR的同源臂800bpDNA供体序列(HDR)如SEQ ID NO.:38所示，
作为对比例的线性双链DNA(供体DNA)的序列如SEQ ID NO.:39－44中任一项所示。
其中，PD1位点1定点整合mTurquoise2的同源臂800bpDNA供体序列(HITI-pb或者写作“HITI(pb)”)如SEQ ID NO.:39所示，PD1位点1定点整合mTurquoise2的同源臂800bpDNA供体序列(HITI)如SEQ ID NO.:40所示，PD1位点1定点整合mTurquoise2的同源臂800bpDNA供体序列(MMEJ)如SEQ ID NO.:41所示，PD1位点2定点整合mTurquoise2的同源臂800bpDNA供体序列(HITI-pb或者写作“HITI(pb)”)如SEQ ID NO.:42所示，PD1位点2定点整合mTurquoise2的同源臂800bpDNA供体序列(HITI)如SEQ ID NO.:43所示，PD1位点2定点整合mTurquoise2的同源臂800bpDNA供体序列(MMEJ)如SEQ ID NO.:44所示。
本申请的附图中，“Untreated T”表示未处理的T细胞，“Control”表示只电转了同源模板和Cas9的T细胞，“LV-19bbz”表示用慢病毒制备的CD19-CART细胞，“AAVS1-19bbz”表示非病毒AAVS1定点整合型CD19-CART细胞，“PD1-19bbz”表示非病毒PD1定点整合型CD19-CART细胞。
实施例1线性双链DNA作为同源模板递送方式优于质粒；设定同源模板的同源臂在800bp长度时，具有最佳的重组率，能得到最多的阳性细胞，为最佳条件
首先比较了质粒和线性双链DNA形式递送模板对T细胞的影响。结果显示当以线性双链DNA的形式递送模板时，细胞的存活率明显高于质粒(图2)。其次，对不同的模板设计对重组率的影响进行了比较，其中HDR为含800bp同源臂模板，MMEJ为含20bp微同源臂模板，HITI为两侧含sgRNA靶点但无同源臂模板，HITI(pb)在HITI基础上两端加上了50bp保护碱基，PD1位点1的HITI、HITI(pb)、MMEJ的供体序列分别如SEQ ID NO.:39－41所示，PD1位点2的HITI、HITI(pb)、MMEJ的供体序列分别如SEQ ID NO.:42－44所示。结果显示，HDR模板具有最高的重组率(图3)。综合以上结果，确定以包含同源臂的线性双链DNA作为模板构建非病毒定点整合型CAR-T细胞为最佳方法(图1)。
之后，对同源臂长度的影响进行了比较。先以荧光蛋白mTurquoise2序列作为外源序列，在两个PD1位点开展了重组实验(图4)。结果显示，同源臂长度为200bp、400bp、800bp、1600bp时，都呈现出良好的整合效果，并且随着同源臂长度的增加，mTurquoise2的整合率随之上升，但1600bp与800bp相比并未显示出重组率的明显提高。而细胞的存活与同源臂长度呈显著负相关。综合重组率和细胞存活的情况，我们对mTurquoise2阳性的活细胞数量进行了分析。结果显示当同源臂长度为800bp时，可以获得最多的阳性细胞。在此基础上，对PD1高效整合位点进行了再次验证并对AAVS1、TRAC、B2M的高效整合位点进行了筛选。通过敲除率和mTurquoise2重组率的比较，最终确定PD1-sgRNA1、AAVS1-sgRNA2、TRAC-sgRNA2、B2M-sgRNA2为优选序列(图5-8)。最后，以CAR元件作为外源序列，分别构建AAVS1、PD1、TRAC、B2M定点整合型CD19-CART细胞并比较不同长度同源臂的影响。与mTurquoise2重组结果相一致，同源臂为800bp时能制备出最多数量的阳性细胞(图9)。因此，我们确定含800bp同源臂的HDR模板为构建定点整合型T细胞的最佳条件。
之前实验我们主要对CD3 +T细胞的定点整合条件进行了摸索和优化。在此基础上，我们尝试使用同样方法对CD4 +和CD8 +T细胞进行编辑。结果显示我们的方法在CD3 +、CD4 +和CD8 +T细胞中具有相近的定点整合率(图10)。
实施例2使用本发明方法构建非病毒AAVS1定点整合型CD19-CART细胞， 并对其功能进行检测
使用本发明方法，我们在不同的供体细胞中成功构建了AAVS1定点整合型CD19-CART细胞并进行了测序验证，总阳性率大约在10％-20％，敲除率大约在65％-90％，体现出了较好的制备稳定性(图11、12)。在此基础上，我们对AAVS1定点整合型CD19-CART的生物学功能进行了检测。实验结果显示，AAVS1定点整合型CD19-CART细胞具有较高的存活率(图13)。虽然电转步骤本身造成了一定数量的细胞死亡，但T细胞的体外扩增能力并无受到明显影响(图14)。与肿瘤靶细胞接触后，AAVS1定点整合型CD19-CART细胞也能很好地扩增(图15、16)。之后，对细胞表面标志物表达、细胞亚型改变、细胞因子分泌等方面进行了检测。总体来看，与慢病毒制备的CD19-CART细胞相似，AAVS1定点整合型CD19-CART细胞能对肿瘤细胞产生明显的应答(图17-19)。但在某些方面，两者之间还是存在一定的差异。最后，对AAVS1定点整合型CD19-CART细胞的肿瘤杀伤能力进行了评估。体内外结果证明与慢病毒制备的CD19-CART细胞一样，它对肿瘤细胞有很好的杀伤作用(图20、21)。
实施例3使用本发明方法构建非病毒PD1定点整合型CD19-CART细胞，并对其功能进行检测
使用本发明方法，我们在不同的供体细胞中成功构建了PD1定点整合型CD19-CART细胞并进行了测序验证，总阳性率大约在10％-30％，敲除率大约在80％-95％，体现出了较好的制备稳定性(图22、23)。在此基础上，我们对PD1定点整合型CD19-CART的生物学功能进行了检测。实验结果显示与肿瘤靶细胞接触后，PD1定点整合型CD19-CART细胞具有比慢病毒制备的CAR-T细胞更强的扩增能力(图24)。与慢病毒制备的CD19-CART细胞相似，PD1定点整合型CD19-CART细胞能响应肿瘤细胞的刺激而激活表面标志物的表达并分泌细胞因子，其中CD137表达水平的上升更为明显(图25)，IFN-γ分泌水平的上升更为显著(图26)。最后，通过体内外实验证明了与慢病毒制备的CD19-CART细胞相比，使用本发明构建的PD1定点整合型CD19-CART细胞具有更强的肿瘤杀伤能力(图27、28)。
综上，使用本发明方法并结合位点筛选，利用CRISPR/Cas9基因编辑工具可成功构建AAVS1、PD1、TRAC、B2M定点整合型CAR-T细胞。本发明证明了与 已有技术相比，使用本发明方法制备的定点整合型CAR-T细胞具有较高的阳性率，并可以有效地发挥功能。该技术方法与传统慢病毒制备方法相比，可减少CAR-T制备过程中使用病毒带来的高昂成本，并减少病毒随机插入带来的安全隐患，也提高了CAR-T产品的均一性。另外，该方法还可实现CAR-T细胞的多样化改造，增强CAR-T细胞的抗肿瘤能力。该举例证明了本发明保护的提高T细胞中外源序列定点整合方法的重要性和价值，但不限于AAVS1、PD1、TRAC、B2M定点整合CAR-T细胞的制备，可拓展到其他位点外源序列的定点整合及其他T细胞免疫疗法的开发。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种对细胞基因组的靶位点进行基因编辑的方法，其特征在于，包括：
    (a)提供一待基因编辑的细胞；
    (b)将(i)基因编辑酶或其编码核酸或表达所述基因编辑酶的第一表达载体；和(ii)gRNA或表达所述gRNA的第二表达载体导入所述细胞中，对所述细胞基因组的靶位点进行基因编辑，所述gRNA引导基因编辑酶对靶位点进行定点切割；
    其中，所述gRNA靶向的靶向序列包含SEQ ID No.1-9所示的序列中的一个或多个。
  2. 如权利要求1所述的方法，其特征在于，所述基因编辑包括基因敲除和基因靶向整合。
  3. 如权利要求1所述的方法，其特征在于，所述基因编辑酶选自下组：CRISPR相关蛋白(Cas)多肽、TALEN酶、ZFN酶、或其组合。
  4. 如权利要求1所述的方法，其特征在于，所述靶位点选自下组：AAVS1、PD1、TRAC、B2M、或其组合。
  5. 如权利要求1所述的方法，其特征在于，所述基因编辑包括定点敲入供体DNA。
  6. 一种对细胞基因组的靶位点进行基因编辑的gRNA，其特征在于，所述gRNA靶向的靶向序列包含SEQ ID No.1-9所示的序列中的一个或多个。
  7. 一种用于对细胞基因组的靶位点进行基因编辑的反应体系，其特征在于，所述反应体系包括：
    (a)供体DNA，所述供体DNA为双链DNA；
    (b)基因编辑酶或其编码核酸或表达所述基因编辑酶的第一表达载体；
    (c)gRNA或表达所述gRNA的第二表达载体；
    其中，所述gRNA靶向的靶向序列包含SEQ ID No.1-9所示的序列中的一个或多个。
  8. 一种用于基因编辑的试剂盒，其特征在于，所述试剂盒包括：
    i)第一容器以及位于第一容器内的供体DNA，所述供体DNA为双链DNA；
    ii)第二容器以及位于第二容器内的基因编辑酶或其编码核酸或表达所述基因编辑酶的第一表达载体；和
    iii)第三容器以及位于第三容器内的gRNA或表达所述gRNA的第二表达载体， 并且所述gRNA靶向的靶向序列包含SEQ ID No.1-9所示的序列的一个或多个。
  9. 一种权利要求1所述的方法制备得到的经基因编辑的细胞。
  10. 一种权利要求9所述的细胞在制备肿瘤免疫治疗或癌症免疫治疗产品中的应用。
PCT/CN2021/116531 2020-09-10 2021-09-03 对细胞中的靶位点进行基因编辑的方法 WO2022052877A1 (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US18/025,136 US20230323323A1 (en) 2020-09-10 2021-09-03 Method for performing gene editing on target site in cell
JP2023515399A JP2023541028A (ja) 2020-09-10 2021-09-03 細胞内の標的部位を遺伝子編集する方法
EP21865931.6A EP4212619A1 (en) 2020-09-10 2021-09-03 Method for performing gene editing on target site in cell

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010949701.5A CN114164231B (zh) 2020-09-10 2020-09-10 对细胞中的靶位点进行基因编辑的方法
CN202010949701.5 2020-09-10

Publications (1)

Publication Number Publication Date
WO2022052877A1 true WO2022052877A1 (zh) 2022-03-17

Family

ID=80475876

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/116531 WO2022052877A1 (zh) 2020-09-10 2021-09-03 对细胞中的靶位点进行基因编辑的方法

Country Status (5)

Country Link
US (1) US20230323323A1 (zh)
EP (1) EP4212619A1 (zh)
JP (1) JP2023541028A (zh)
CN (1) CN114164231B (zh)
WO (1) WO2022052877A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024105162A1 (en) * 2022-11-16 2024-05-23 Alia Therapeutics Srl Type ii cas proteins and applications thereof

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110863015B (zh) * 2019-11-25 2023-06-16 重庆精准生物产业技术研究院有限公司 对t细胞低敏感性的靶细胞及其制备方法和应用
CN114807155A (zh) * 2021-01-18 2022-07-29 华东师范大学 用于基因编辑的组合物及其应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107586777A (zh) * 2016-07-08 2018-01-16 上海吉倍生物技术有限公司 人PDCD1基因sgRNA的用途及其相关药物
CN108998419A (zh) * 2018-08-13 2018-12-14 北京呈诺医学科技有限公司 一种低免疫原性、可诱导凋亡的ips细胞制备方法
CN109628493A (zh) * 2017-10-09 2019-04-16 广东赤萌医疗科技有限公司 一种用于制备可异体移植t细胞的基因编辑系统
CN109652380A (zh) * 2019-01-25 2019-04-19 苏州茂行生物科技有限公司 基于碱基编辑靶向LewisY的CAR-T细胞及其制备方法和应用
CN109777782A (zh) * 2019-02-15 2019-05-21 北京门罗生物科技有限公司 一种通用型car-t细胞及其制备方法和用途
WO2019210280A1 (en) * 2018-04-27 2019-10-31 Casebia Therapeutics Limited Liability Partnership Anti-bcma car-t-cells for plasma cell depletion
CN110616187A (zh) * 2018-06-20 2019-12-27 西安桑尼赛尔生物医药有限公司 CRISPR-Cas9高效敲入嵌合抗原受体基因到T细胞特定基因组位点的方法及应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107586777A (zh) * 2016-07-08 2018-01-16 上海吉倍生物技术有限公司 人PDCD1基因sgRNA的用途及其相关药物
CN109628493A (zh) * 2017-10-09 2019-04-16 广东赤萌医疗科技有限公司 一种用于制备可异体移植t细胞的基因编辑系统
WO2019210280A1 (en) * 2018-04-27 2019-10-31 Casebia Therapeutics Limited Liability Partnership Anti-bcma car-t-cells for plasma cell depletion
CN110616187A (zh) * 2018-06-20 2019-12-27 西安桑尼赛尔生物医药有限公司 CRISPR-Cas9高效敲入嵌合抗原受体基因到T细胞特定基因组位点的方法及应用
CN108998419A (zh) * 2018-08-13 2018-12-14 北京呈诺医学科技有限公司 一种低免疫原性、可诱导凋亡的ips细胞制备方法
CN109652380A (zh) * 2019-01-25 2019-04-19 苏州茂行生物科技有限公司 基于碱基编辑靶向LewisY的CAR-T细胞及其制备方法和应用
CN109777782A (zh) * 2019-02-15 2019-05-21 北京门罗生物科技有限公司 一种通用型car-t细胞及其制备方法和用途

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JAMES CAPPUCCINONATALIE SHERMAN: "Microbiology: Laboratory Manual", PEARSON EDUCATION PRESS
SAMBROOK ET AL.: "Molecular Cloning: Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024105162A1 (en) * 2022-11-16 2024-05-23 Alia Therapeutics Srl Type ii cas proteins and applications thereof

Also Published As

Publication number Publication date
US20230323323A1 (en) 2023-10-12
CN114164231B (zh) 2024-05-17
EP4212619A1 (en) 2023-07-19
JP2023541028A (ja) 2023-09-27
CN114164231A (zh) 2022-03-11

Similar Documents

Publication Publication Date Title
WO2022052877A1 (zh) 对细胞中的靶位点进行基因编辑的方法
JP7142571B2 (ja) リンパ球における高レベル且つ安定な遺伝子移入のための方法
ES2768984T3 (es) Meganucleasas diseñadas con secuencias de reconocimiento encontradas en el gen de la región constante alfa del receptor de células T humanas
US20230310502A1 (en) Targeted replacement of endogenous t cell receptors
CN114729368A (zh) 用于免疫疗法的组合物和方法
WO2019005957A1 (en) GENETICALLY MODIFIED T CELLS COMPRISING A MODIFIED INTRON IN THE ALPHA T CELL RECEPTOR GENE
WO2021082784A1 (zh) 一种基于腺病毒的基因编辑方法
WO2021012960A1 (zh) 一种引导PD1基因切割实现外源序列高效整合的sgRNA
Narayanan et al. DNA modification and functional delivery into human cells using Escherichia coli DH10B
JP2019503653A (ja) トランスポゾン系、それを含むキット及びそれらの使用
WO2019173942A1 (en) Engineered chimeric guide rna and uses thereof
WO2021119006A1 (en) Crispr abasic restricted nucleotides and crispr accuracy via analogs
WO2022152266A1 (zh) 用于基因编辑的组合物及其应用
CN115552015A (zh) 经由同时敲入和基因破坏来改造免疫细胞
US11998567B2 (en) Engineered chimeric guide RNA and uses thereof
CN116732099B (zh) 一种干细胞多重CRISPR/Cas基因组编辑方法
WO2021095847A1 (ja) 抗原特異的受容体遺伝子を環状ｄｎａを用いてｔ細胞ゲノムに導入する方法
WO2023066243A1 (zh) 一种将目的基因定点整合至免疫细胞特定位点的方法及其应用
CN114728021A (zh) 同种异体t细胞和其产生方法
WO2023081811A1 (en) Immune cells with chimeric antigen receptors or chimeric autoantibody receptors
CN117355607A (zh) 非病毒同源性介导的末端连接
WO2023023635A1 (en) Nonviral generation of genome edited chimeric antigen receptor t cells

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: 21865931

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2023515399

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2021865931

Country of ref document: EP

Effective date: 20230411