WO2022152266A1 - 用于基因编辑的组合物及其应用 - Google Patents

用于基因编辑的组合物及其应用 Download PDF

Info

Publication number
WO2022152266A1
WO2022152266A1 PCT/CN2022/072130 CN2022072130W WO2022152266A1 WO 2022152266 A1 WO2022152266 A1 WO 2022152266A1 CN 2022072130 W CN2022072130 W CN 2022072130W WO 2022152266 A1 WO2022152266 A1 WO 2022152266A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
gene editing
another preferred
sequence
seq
Prior art date
Application number
PCT/CN2022/072130
Other languages
English (en)
French (fr)
Inventor
张楫钦
田悦
张林杰
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
Publication of WO2022152266A1 publication Critical patent/WO2022152266A1/zh

Links

Images

Classifications

    • 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
    • C07K14/70521—CD28, CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • 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
    • 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
    • A61K39/464429—Molecules with a "CD" designation not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • 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
    • C12N15/11—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
    • 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
    • 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
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • 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
    • 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
    • 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
    • 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
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • 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 composition for gene editing and its application.
  • 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.
  • the purpose of the present invention is to provide a method for improving the efficiency of homologous recombination in T cells, and to provide a target sequence derived from the PD1 gene that can specifically bind to gRNA, and a gRNA site for efficient integration and cleavage of this target sequence.
  • a target sequence selected from the group consisting of:
  • nucleotide sequence of the 40th-130th position (preferably the 50th-122nd position) shown in SEQ ID NO.:1;
  • the target sequence is selected from the following group:
  • the target sequence comprises the sequence shown in any one of SEQ ID NO.:3-15.
  • the target sequence is derived from PD1 gene.
  • the second aspect of the present invention provides a gRNA sequence, the gRNA sequence is complementary to the target sequence described in the first aspect of the present invention.
  • the gRNA includes crRNA, tracrRNA, and sgRNA.
  • 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 targeting sequence of the gRNA comprises the sequence shown in any one of SEQ ID NO.:16-28.
  • a third aspect of the present invention provides a gene editing system, the system comprising a gene editing enzyme and a gRNA, the gRNA is complementary to the target sequence described in the first aspect of the present invention.
  • 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 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 or site-directed knock-in of donor DNA.
  • the target sequence is selected from the following group of target sites: PD1.
  • the targeting sequence of the gRNA comprises the sequence shown in any one of SEQ ID NO.:16-28.
  • composition further includes 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 genome Cell-mediated homologous recombination at the target site.
  • sequence lengths of the first homology arm and the second homology arm are independently 200-2000 bp, preferably 400-1000 bp, more preferably 700-900 bp.
  • 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 to the genome The sequence downstream (or right) of the cleavage site of the target site is homologous.
  • the target site includes PD1.
  • the donor DNA also includes a target gene.
  • the target gene comprises 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-2200bp.
  • 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.: 29-31.
  • the fourth aspect of the present invention provides a cell obtained by editing the gene editing system according to the third aspect of the present invention.
  • the cells are cells cultured in vitro.
  • 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.
  • a fifth aspect of the present invention provides a composition, comprising:
  • a pharmaceutically acceptable carrier is selected from:
  • the composition further includes an expression complex, and the expression complex includes a nucleic acid molecule encoding an exogenous protein.
  • the exogenous protein is a protein that can be expressed in cells.
  • the exogenous protein is a recombinant receptor that can be expressed on the cell surface.
  • the recombinant receptor includes chimeric antigen receptor (CAR), TCR, and chimeric surface receptor.
  • CAR chimeric antigen receptor
  • TCR TCR
  • chimeric surface receptor chimeric antigen receptor
  • the composition includes a pharmaceutical composition.
  • the pharmaceutical composition is liquid.
  • the dosage form of the pharmaceutical composition includes injection or injection.
  • the dosage form of the pharmaceutical composition is an intravenous injection dosage form or an intratumoral injection dosage form.
  • the system according to the third aspect of the present invention or the cells according to the fourth aspect of the present invention account for 1-99 wt % of the total weight of the composition, preferably 10 wt %. -90wt%, more preferably 30-70wt%.
  • a sixth aspect of the present invention provides a medicine box, comprising:
  • said active ingredient comprising the system of the third aspect of the invention, or the invention
  • the active ingredient or the drug is a single preparation.
  • the dosage form of the active ingredient or the drug is an injection dosage form or an injection.
  • the dosage form of the active ingredient or the drug is an intravenous injection dosage form or an intratumoral injection dosage form.
  • the kit further contains instructions, and the instructions describe the instructions for administering the active ingredient or the drug to the administration object, thereby preventing and/or treating a disease.
  • the administration object is a cell.
  • the administration target is T cells.
  • the administration object is a human or a non-human mammal.
  • the administration is contacting the active ingredient or the drug with T cells, or injecting the active ingredient or the drug into the human body by means of injection.
  • the seventh aspect of the present invention provides a method for gene editing of cells, comprising contacting the cells with the gene editing system described in the third aspect of the present invention or the composition described in the fifth aspect of the present invention or the sixth aspect of the present invention.
  • the kit of the aspect is described in the third aspect of the present invention or the composition described in the fifth aspect of the present invention or the sixth aspect of the present invention.
  • the contacting is performed in vitro.
  • the cells are cells cultured in vitro.
  • 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 eighth aspect of the present invention provides a kit for gene editing, the kit comprising the gene editing system of the third aspect of the present invention or the composition of the fifth aspect of the present invention or the sixth aspect of the present invention the said kit.
  • the kit further includes labels or instructions.
  • the label or the instructions describe the instructions for administering the system or the composition or the kit to the editing subject to perform gene editing.
  • the editing object includes 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 ninth aspect of the present invention provides the gene editing system of the third aspect of the present invention, the cell of the fourth aspect of the present invention, the composition of the fifth aspect of the present invention, or the medicine of the sixth aspect of the present invention Use of the cassette for the preparation of a medicament for preventing and/or treating a disease.
  • the disease includes cancer.
  • the cancer includes solid tumors and hematological tumors.
  • the cancer includes tumors with high PD-L1 expression.
  • the cancer is selected from the group consisting of leukemia, lymphoma, liver cancer, lung cancer, kidney cancer, breast cancer, colorectal cancer, gastric cancer, brain cancer, melanoma, ovarian cancer, prostate cancer, pancreatic cancer , mesothelioma, or a combination thereof.
  • the tenth aspect of the present invention provides a method for treating a disease, comprising administering to a subject an effective amount of the cells of the fourth aspect of the present invention, the composition of the fifth aspect of the present invention, or the sixth aspect of the present invention. described medicine box.
  • the disease selection includes cancer.
  • the cancer includes solid tumors and hematological tumors.
  • the cancer includes tumors with high PD-L1 expression.
  • the cancer is selected from the group consisting of leukemia, lymphoma, liver cancer, lung cancer, kidney cancer, breast cancer, colorectal cancer, gastric cancer, brain cancer, melanoma, ovarian cancer, prostate cancer, pancreatic cancer , mesothelioma, or a combination thereof.
  • the administration includes injection administration.
  • the subject is a human or a non-human mammal.
  • the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.
  • Untreated T indicates untreated T cells
  • Control indicates T cells electroporated with homologous template and Cas9 only
  • LV-19bbz indicates CD19-CART cells prepared with lentivirus
  • PD1-19bbz means non-viral PD1 site-specific integration CD19-CART cells.
  • Figure 1 shows the efficient integration of sgRNA site screening of PD1 gene, which is the recombination rate of CD19-CAR sequence at PD1 site.
  • Figure 2 shows the screening of PD1 gene CRISPR/Cas9 efficient cleavage sgRNA sites to detect PD1 site knockout rate by ICE method.
  • Figure 3 shows the efficient integration of PD1 gene sgRNA site screening, the recombination rate of mTurquoise2 fluorescent protein sequence at PD1 site.
  • Figure 4 shows the efficient integration of sgRNA site screening of PD1 gene, which is the recombination rate of CD19-CAR sequence at PD1 site.
  • Figure 5 shows the screening of PD1 gene CRISPR/Cas9 efficient cleavage sgRNA sites to detect PD1 site knockout rate by ICE method.
  • Figure 6 shows the results of DNA sequencing of PD1 site-directed integration CD19-CART cells.
  • HA stands for homology arm.
  • Figure 7 shows the positive rate and knockout rate of PD1 site-directed integration of CD19-CART cells.
  • Picture A shows the positive rate of PD1 site-directed integration CD19-CART cells prepared from T cells from different healthy donors;
  • Picture B shows the positive rate and knockout rate of PD1 site-directed integration CD19-CART cells prepared from five representative T cells from different healthy donors ;
  • Panel C shows the positive rate of PD1 site-specific integration of CD19-CART cells by flow analysis, and the control group is only electroporated with homologous template and Cas9;
  • Panel D shows PD1 expression of PD1 site-specific integration of CD19-CART cells and lentivirus-prepared CD19-CART cells Compare.
  • Figure 8 shows the in vitro expansion of PD1 site-directed integration of CD19-CART cells co-cultured with target cells.
  • Figure 9 shows PD1 site-directed integration CD19-CART cell surface marker expression.
  • the control group was electroporated with homologous template and Cas9 only.
  • Figure 10 shows the detection of PD1 site-directed integration of CD19-CART cells secreted factors.
  • the control group was electroporated homologous template and Cas9 only.
  • Figure 11 shows in vitro killing of CD19-CART cells by PD1 site-directed integration.
  • the control group was electroporated homologous template and Cas9 only.
  • Figure 12 shows PD1 site-directed integration killing of CD19-CART cells in vivo.
  • the tumor target cells were PD-L1-overexpressing Raji cells.
  • the inventors After extensive and in-depth research and extensive screening, the inventors have accidentally screened for the first time a target sequence derived from the PD1 gene that can specifically bind to the gRNA, and creatively designed a gRNA for efficient gene editing of PD1 for the target sequence.
  • the present invention also finds for the first time that the gene editing composition containing the gRNA, gene editing enzyme (and donor DNA in some cases) has high knockout rate and recombination rate. 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. Fusion of the protein to nuclease-free Cas9 (Cas9nuclease-null) and expression of the appropriate gRNA can target any dsDNA sequence, and the gRNA end can be ligated to the target DNA without affecting Cas9 binding. Thus, Cas9 can bring any fusion protein and RNA at any dsDNA sequence. This technique 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 (for example, 700-900 bp); the donor DNA or exogenous nucleic acid is electroporated into cells together with Cas9 protein and guide RNA.
  • the targeted integration strategy provided by the present invention has higher integration efficiency.
  • the present invention provides a gene editing system comprising a gene editing enzyme and a gRNA complementary to a target sequence derived from the PD1 gene.
  • the target sequence is selected from the group consisting of:
  • the target sequence is selected from the group consisting of:
  • the target sequence is shown in SEQ ID NO.: 3-15.
  • the gene editing system of the present invention further contains donor DNA.
  • the gene editing system of the present invention can significantly improve the knockout rate and recombination rate.
  • compositions and methods of administration are provided.
  • the present invention also provides a pharmaceutical composition, which contains (a) a safe and effective amount of the gene editing system of the present invention or the cells described in the fourth aspect of the present invention; and (b) pharmaceutically acceptable carrier or excipient.
  • the dosage of the gene editing system of the present invention is usually 10 micrograms-100 mg/dose, preferably 100-1000 micrograms/dose.
  • the concentration of the cells in the pharmaceutical composition is 1 ⁇ 10 3 -1 ⁇ 10 8 cells/Kg body weight, more preferably 1 ⁇ 10 5 -1 ⁇ 10 7 cells/Kg weight.
  • an effective dose is about 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg body weight of the gene editing system of the present invention administered to an individual.
  • the gene editing system of the present invention can be used alone or in combination with other therapeutic agents (eg, formulated in the same pharmaceutical composition).
  • the pharmaceutical composition of the present invention can be administered in the form of a cell therapy agent.
  • the gene editing system of the present invention can be used to modify cells, and then the modified cells are administered to those in need by intravenous or intratumoral injection. subject.
  • the gene editing systems of the present invention can be used to engineer cells in vivo or in isolation.
  • the cells can be T cells and the modification can include gene editing.
  • the pharmaceutical composition may also contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier for administration of a therapeutic agent.
  • the term refers to pharmaceutical carriers that do not themselves induce the production of antibodies detrimental to the individual receiving the composition, and are not undue toxicity upon administration. These vectors are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).
  • Such carriers include, but are not limited to: saline, buffers, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.
  • Pharmaceutically acceptable carriers in therapeutic compositions can contain liquids such as water, saline, glycerol and ethanol.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.
  • therapeutic compositions can be prepared as injectables, eg, liquid solutions or suspensions; solid forms suitable for solution or suspension, liquid carriers, prior to injection can also be prepared.
  • compositions of the present invention can be administered by conventional routes including, but not limited to, intratumoral, intramuscular, intravenous, subcutaneous, intradermal, or topical administration.
  • the subject to be prevented or treated can be an animal; especially a human.
  • the pharmaceutical composition of the present invention When used for actual treatment, various pharmaceutical compositions in different dosage forms can be adopted according to the usage. Preferably, it is an intravenous drug preparation or an intratumoral drug injection.
  • compositions can be formulated according to conventional methods by mixing, diluting or dissolving, with occasional addition of suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicity isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizers, and the formulation process can be carried out in a conventional manner according to the dosage form.
  • suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicity isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizers, and the formulation process can be carried out in a conventional manner according to the dosage form.
  • compositions of the present invention can also be administered in the form of sustained release formulations.
  • the gene editing system of the present invention can be incorporated into a pellet or microcapsule with a slow-release polymer as a carrier, and the pellet or microcapsule is then surgically implanted into the tissue to be treated.
  • sustained-release polymers ethylene-vinyl acetate copolymer, polyhydrometaacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymer, Lactic acid-glycolic acid copolymers and the like are preferably exemplified by biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers.
  • the pharmaceutical composition may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg, aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, sulfate buffered saline, etc.
  • carbohydrates such as glucose, mannose, sucrose or dextran, mannitol
  • proteins polypeptides or amino acids
  • antioxidants such as glycine
  • chelating agents such as EDTA or glutathione
  • adjuvants eg, aluminum hydroxide
  • preservatives e.g, aluminum hydroxide
  • the dose of the gene editing system of the present invention as an active ingredient can be reasonably determined according to the weight, age, sex, and symptom level of each patient to be treated.
  • the gene editing system containing a specific gRNA of the present invention can significantly improve the knockout rate and the site-specific integration efficiency of exogenous nucleic acid.
  • the present invention can improve the site-specific integration efficiency of exogenous sequences in T cells by screening the obtained gRNA sites for efficient integration of exogenous sequences
  • the present invention can improve the efficiency of T cell gene knockout by screening the gRNA sites that are efficiently cleaved by CRISPR/Cas9
  • Electrotransduction of T cells is a technical method to achieve T cell gene editing.
  • the steps refer to Lonza's P3Primary Cell 4D- X kit
  • the kit is P3Primary 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.
  • the target sequence of the present invention can be as follows, i.e. SEQ ID NO.: 1:
  • the target sequence of the present invention can be as follows, i.e. SEQ ID NO.: 2:
  • the target sequence of the present invention may comprise the sequence shown in any one of SEQ ID NO.: 3-15:
  • DNA-sg1 (ie SEQ ID NO.: 3): ACAGGCGCCCTGGCCAGTCG.
  • DNA-sg2 (ie SEQ ID NO.: 4): CAGGCGCCCTGGCCAGTCGT.
  • DNA-sg3 (ie SEQ ID NO.: 5): GGGCGCCTGTGGGATCTGCA.
  • DNA-sg4 (ie SEQ ID NO.: 6): TGGCCAGTCGTCTGGGCGGT.
  • DNA-sg5 (ie SEQ ID NO.: 7): GGCCAGTCGTCTGGGCGGTG.
  • DNA-sg6 (ie SEQ ID NO.: 8): GTGGGATCTGCATGCCTGGA.
  • DNA-sg7 (ie SEQ ID NO.: 9): GGCATGCAGATCCCCACAGGC.
  • DNA-sg8 (ie SEQ ID NO.: 10): GCCCAGTTGTAGCACCGCCC.
  • DNA-sg9 (ie SEQ ID NO.: 11): CCAGCCCAGTTGTAGCACCG.
  • DNA-sg10 ie SEQ ID NO.: 12
  • GGCCGCCAGCCCAGTTGTAG GGCCGCCAGCCCAGTTGTAG
  • DNA-sg11 (ie SEQ ID NO.: 13): TCCTGGCCGCCAGCCCAGTT.
  • DNA-sg12 (ie SEQ ID NO.: 14): AGTTGTAGCACCGCCCAGAC.
  • DNA-sg13 (ie SEQ ID NO.: 15): GTTGTAGCACCGCCCAGACG.
  • the targeting sequence of the gRNA of the present invention may comprise the sequence shown in any one of SEQ ID NO.: 16-28:
  • PD1-sg1 ie SEQ ID NO.: 16: CGACTGGCCAGGGCGCCTGT.
  • PD1-sg2 (ie SEQ ID NO.: 17): ACGACTGGCCAGGGCGCCTG.
  • PD1-sg3 (ie SEQ ID NO.: 18): TGCAGATCCCACAGGCGCCC.
  • PD1-sg4 (ie SEQ ID NO.: 19): ACCGCCCAGACGACTGGCCA.
  • PD1-sg5 (ie SEQ ID NO.: 20): CACCGCCCAGACGACTGGCC.
  • PD1-sg6 (ie SEQ ID NO.: 21): TCCAGGCATGCAGATCCCCAC.
  • PD1-sg7 ie SEQ ID NO.: 22: GCCTGTGGGATCTGCATGCC.
  • PD1-sg8 (ie SEQ ID NO.: 23): GGGCGGTGCTACAACTGGGC.
  • PD1-sg9 (ie SEQ ID NO.: 24): CGGTGCTACAACTGGGCTGG.
  • PD1-sg10 (ie SEQ ID NO.: 25): CTACAACTGGGCTGGCGGCC.
  • PD1-sg11 (ie SEQ ID NO.: 26): AACTGGGCTGGCGGCCAGGA.
  • PD1-sg12 (ie SEQ ID NO.: 27): GTCTGGGCGGTGCTACAACT.
  • PD1-sg13 (ie SEQ ID NO.: 28): CGTCTGGGCGGTGCTACAAC.
  • a group of PD1 sites were selected, and spCas9 (TrueCut TM Cas9 Protein v2, Thermofisher), sgRNA and CAR element template were introduced into the stimulated T cells, and the sequence of the CAR element template was shown in SEQ ID NO.:29.
  • the positive rate of CAR was detected by flow analysis to determine the site of efficient integration of foreign sequences.
  • the results showed that the CAR elements were integrated to different degrees in multiple PD1 sites ( Figure 1).
  • the ICE method was used to detect the PD1 knockout rate of this group of loci. The results showed that some PD1 sites had a higher knockout rate ( Figure 2).
  • spCas9 TrueCut TM Cas9 Protein v2, Thermofisher
  • sgRNA a template containing the sequence of fluorescent protein mTurquoise2
  • the sequence is shown in SEQ ID NO.:30.
  • the recombination rate of fluorescent proteins was detected by flow cytometry to identify sites for efficient integration of foreign sequences. The results showed that the fluorescent protein sequences were integrated to different degrees at multiple sites of PD1 ( Figure 3).
  • SEQ ID NO.: 29 that is, the donor DNA sequence containing the CAR sequence used for the detection of the first group of PD1 sites (PD1-sg1 to PD1-sg7), in this sequence, the nucleotides at positions 1-800 are The first homology arm sequence, the nucleotides at positions 2954-3753 are the second homology arm sequence:
  • SEQ ID NO.: 30 that is, the donor DNA sequence containing the mTurquoise2 sequence used for the detection of PD1 second group sites (PD1-sg8 to PD1-sg13), in this sequence, the nucleotides at positions 1-800 are The first homology arm sequence, the nucleotides at positions 2213-3012 are the second homology arm sequence:
  • SEQ ID NO.: 31 that is, the donor DNA sequence containing the CAR sequence used for the detection of the second group of PD1 sites (PD1-sg8 to PD1-sg13), in this sequence, the nucleotides at positions 1-800 are The first homology arm sequence, the nucleotides at positions 2954-3753 are the second homology arm sequence:
  • the PD1 site-directed integration type CD19-CART cells were successfully constructed using T cells of different healthy donors (the donor DNA sequence used is shown in SEQ ID NO.: 29), and the Sequencing verification (Fig. 6), the total positive rate was about 10%-30% (Fig. 7A, C), the knockout rate was about 80%-95% (Fig. 7B), and the expression level of PD1 was significantly decreased (Fig. 7D), reflecting Good preparation stability was obtained. On this basis, the biological function of PD1 site-specific integrated CD19-CART was tested.
  • the experimental results show that, after contacting with PD-L1 high-expressing tumor target cells (in this example, lentivirus-infected PD-L1 stable over-expressing Raji tumor target cells are used as an example), PD1 site-directed integration CD19-CART cells have more potency than lentiviruses.
  • the prepared CAR-T cells had stronger expansion ability ( Figure 8). Similar to CD19-CART cells prepared by lentivirus, PD1 site-specific integration CD19-CART cells can respond to PD-L1 high-expressing tumor target cells (this example uses lentivirus-infected PD-L1 stable over-expressing Raji tumor target cells as an example.
  • PD1 site-integrated CD19-CART cells can secrete a large amount of cytokines after contacting with PD-L1 high-expressing tumor target cells (this example uses lentivirus-infected PD-L1 stable over-expressing Raji tumor target cells as an example).
  • IL-2, TNF- ⁇ , IFN- ⁇ Fig. 10A-C, among which the increase in the secretion level of IFN- ⁇ was more significant.
  • PD1 site-directed integration CAR-T cells can be successfully constructed.
  • the present invention proves that, compared with the prior art, the site-directed integration CAR-T cells prepared by using the sequences 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 method for improving site-directed integration of exogenous sequences in T cells provided by the present invention, as well as the importance and value of efficient integration and cleavage of gRNA sites and DNA target sequences in protected T cells, but not limited to PD1 site-directed integration of CAR-
  • the preparation of T cells can be extended to the 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)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • General Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Oncology (AREA)
  • Biophysics (AREA)
  • Hematology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Virology (AREA)
  • Hospice & Palliative Care (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

提供了一种源自PD1基因的靶序列，与该靶序列特异性结合的gRNA以及含有该gRNA的基因编辑系统。该基因编辑系统可显著提高敲除率和重组率，可用于制备CAR-T以及预防和/或治疗疾病的组合物和药盒。

Description

用于基因编辑的组合物及其应用 技术领域
本发明涉及生物技术领域，具体地，涉及用于基因编辑的组合物及其应用。
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方法并筛选高效整合和切割的位点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，同时也需要筛选高效整合和切割的位点。
发明内容
本发明的目的在于提供提高T细胞中同源重组效率的方法，并提供可特异性结合gRNA的源自PD1基因的靶序列，以及针对这段靶序列可实现高效整合和切割的gRNA位点。
在本发明的第一方面，提供了一种靶序列，所述靶序列选自下组：
(1)SEQ ID NO.:1所示的第40-130位(较佳地第50-122位)的核苷酸序列；
(2)将SEQ ID NO.:1所示的第40-130位(较佳地第50-122位)的核苷酸序列经过一个或多个(≤10个，如2-8个，优选为3-5个)核苷酸的取代、缺失或添加而形成的衍生核苷酸，并且所述靶序列特异性与gRNA结合。
在另一优选例中，所述靶序列选自下组：
(1)具有SEQ ID NO.:2所示核苷酸序列的多核苷酸；
(2)与SEQ ID NO.:2所示核苷酸序列的同源性≥95％，较佳地≥98％，更佳地≥99％的多核苷酸；
(3)与上述(1)-(2)互补的多核苷酸。
在另一优选例中，所述靶序列包含如SEQ ID NO.:3-15中任一项所示的序列。
在另一优选例中，所述靶序列来源于PD1基因。
本发明第二方面提供了一种gRNA序列，所述gRNA序列与本发明第一方面 所述的靶序列互补。
在另一优选例中，所述gRNA包括crRNA、tracrRNA、sgRNA。
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gRNA的靶向序列包含SEQ ID NO.:16-28中任一所示的序列。
本发明第三方面提供了一种基因编辑系统，所述系统包括基因编辑酶和gRNA，所述gRNA与本发明第一方面所述的靶序列互补。
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的基因编辑或定点敲入供体DNA。
在另一优选例中，所述靶序列选自下组的靶位点：PD1。
在另一优选例中，所述gRNA的靶向序列包含SEQ ID NO.:16-28中任一项所示的序列。
在另一优选例中，所述组合物还包括供体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在基因组的靶位点处由细胞介导的同源重组。
在另一优选例中，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，所述靶位点包括PD1。
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，所述目的基因包含嵌合抗原受体或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-2200bp。
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.:29-31中任一项所示。
本发明第四方面提供了一种细胞，所述细胞经本发明第三方面所述的基因编辑系统进行编辑后获得。
在另一优选例中，所述细胞为体外培养的细胞。
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。
本发明第五方面提供了一种组合物，包括：
本发明第三方面所述的系统或本发明第四方面所述的细胞；和
药学上可接受的载体。
在另一优选例中，所述组合物还包括表达复合物，所述表达复合物包括编码外源蛋白质的核酸分子。
在另一优选例中，所述外源蛋白质是能够在细胞中表达的蛋白。
在另一优选例中，所述外源蛋白质是能够在细胞表面上表达的重组受体。
在另一优选例中，所述重组受体包括嵌合抗原受体(CAR)、TCR、嵌合表面受体。
在另一优选例中，所述组合物包括药物组合物。
在另一优选例中，所述药物组合物为液态。
在另一优选例中，所述药物组合物的剂型包括注射剂或针剂。
在另一优选例中，所述药物组合物的剂型为静脉注射剂型或瘤内注射剂型。
在另一优选例中，所述的组合物中，本发明第三方面所述的系统或本发明第四方面所述的细胞占所述组合物总重量的1-99wt％，较佳地10-90wt％，更佳地30-70wt％。
本发明第六方面提供了一种药盒，包括：
第一容器，以及活性成分或含有所述活性成分的药物，所述活性成分或所述药物位于所述第一容器中，所述活性成分包含本发明第三方面所述的系统、或本发明第四方面所述的细胞、或本发明第五方面所述的组合物。
在另一优选例中，所述活性成分或所述药物为单方制剂。
在另一优选例中，所述活性成分或所述药物的剂型为注射剂型或针剂。
在另一优选例中，所述活性成分或所述药物的剂型为静脉注射剂型或瘤内注射剂型。在另一优选例中，所述药盒还含有说明书，所述说明书中记载了向施用对象给予所述活性成分或所述药物，从而预防和/或治疗疾病的说明。
在另一优选例中，所述施用对象为细胞。
在另一优选例中，所述施用对象为T细胞。
在另一优选例中，所述施用对象为人或非人哺乳动物。
在另一优选例中，所述施用为将所述活性成分或所述药物与T细胞接触，或者是将所述活性成分或所述药物通过注射方式注入人体内。
本发明第七方面提供了一种对细胞进行基因编辑的方法，包括将所述细胞接触本发明第三方面所述的基因编辑系统或本发明第五方面所述的组合物或本发明第六方面所述的药盒。
在另一优选例中，所述接触在体外进行。
在另一优选例中，所述细胞为体外培养的细胞。
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。
本发明第八方面提供了一种用于基因编辑的试剂盒，所述试剂盒包括本发明第三方面所述的基因编辑系统或本发明第五方面所述的组合物或本发明第六方面所述的药盒。
在另一优选例中，所述试剂盒还包括标签或说明书。
在另一优选例中，所述标签或所述说明书中记载了向编辑对象给予所述系统或所述组合物或所述药盒，从而进行基因编辑的说明。
在另一优选例中，所述编辑对象包括细胞。
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。
本发明第九方面提供了一种本发明第三方面所述的基因编辑系统、本发明第四方面所述的细胞、本发明第五方面所述的组合物或本发明第六方面所述药盒的用途，用于制备用于预防和/或治疗疾病的药物。
在另一优选例中，所述疾病包括癌症。
在另一优选例中，所述癌症包括实体肿瘤和血液肿瘤。
在另一优选例中，所述癌症包括PD-L1高表达的肿瘤。
在另一优选例中，所述癌症选自下组：白血病、淋巴瘤、肝癌、肺癌、肾癌、乳腺癌、结直肠癌、胃癌、脑癌、黑色素瘤、卵巢癌、前列腺癌、胰腺癌、间皮素瘤、或其组合。
本发明第十方面提供了一种治疗疾病的方法，包括向受试者施用有效量的本发明第四方面所述的细胞、本发明第五方面所述的组合物或本发明第六方面所述的药盒。
在另一优选例中，所述疾病选包括癌症。
在另一优选例中，所述癌症包括实体肿瘤和血液肿瘤。
在另一优选例中，所述癌症包括PD-L1高表达的肿瘤。
在另一优选例中，所述癌症选自下组：白血病、淋巴瘤、肝癌、肺癌、肾癌、乳腺癌、结直肠癌、胃癌、脑癌、黑色素瘤、卵巢癌、前列腺癌、胰腺癌、间皮素瘤、或其组合。
在另一优选例中，所述施用包括注射施用。
在另一优选例中，所述受试者为人或非人哺乳动物。
在另一优选例中，所述所述非人哺乳动物包括啮齿动物和灵长目动物，优 选小鼠、大鼠、兔、猴。
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，“PD1-19bbz”表示非病毒PD1定点整合型CD19-CART细胞。
图1显示了PD1基因高效整合sgRNA位点筛选，为CD19-CAR序列在PD1位点重组率。
图2显示了PD1基因CRISPR/Cas9高效切割sgRNA位点筛选，为通过ICE方法检测PD1位点敲除率。
图3显示了PD1基因高效整合sgRNA位点筛选，为mTurquoise2荧光蛋白序列在PD1位点重组率。
图4显示了PD1基因高效整合sgRNA位点筛选，为CD19-CAR序列在PD1位点重组率。
图5显示了PD1基因CRISPR/Cas9高效切割sgRNA位点筛选，为通过ICE方法检测PD1位点敲除率。
图6显示了PD1定点整合CD19-CART细胞DNA测序结果。HA代表同源臂。
图7显示了PD1定点整合CD19-CART细胞阳性率和敲除率。A图为不同健康供体T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健康供体T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，对照组为只电转同源模板和Cas9；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。
图8显示了PD1定点整合CD19-CART细胞与靶细胞共培养体外扩增。
图9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10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11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12显示了PD1定点整合CD19-CART细胞体内杀伤。肿瘤靶细胞为PD-L1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可特异性结合gRNA的源自PD1基因的靶序列，并且针对该靶序列创造性地设计对PD1进行高效基因编辑的gRNA，本发明还首次发现含有该gRNA、基因编辑酶(在某些情况下还含有供体DNA)的基因编辑组合物具有很高的敲除率和重组率。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(Cas9nuclease-nul l)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Cas9蛋白以及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。
基因编辑系统
本发明提供了一种基因编辑系统，所述系统包括基因编辑酶和gRNA，所述gRNA与来源于PD1基因的靶序列互补。
在一优选实施方式中，所述靶序列选自下组：
(1)SEQ ID NO.1所示的第40-130位(较佳地第50-122位)的核苷酸序列；
(2)将SEQ ID NO.1所示的第40-130位(较佳地第50-122位)的核苷酸序列经过一个或多个(≤10个，如2-8个，优选为3-5个)核苷酸的取代、缺失或添加而形成的衍生核苷酸，并且所述靶序列特异性与gRNA结合。
在一优选实施方式中，所述靶序列选自下组：
(1)具有SEQ ID NO:2所示核苷酸序列的多核苷酸；
(2)与SEQ ID NO:2所示核苷酸序列的同源性≥95％，较佳地≥98％，更佳地≥99％的多核苷酸；
(3)与上述(1)-(2)互补的多核苷酸。
在一优选实施方式中，所述靶序列如SEQ ID NO.:3-15所示。
在一优选实施方式中，本发明的基因编辑系统还含有供体DNA。
本发明的基因编辑系统可显著提高敲除率和重组率。
药物组合物和施用方法
另一方面，本发明还提供了一种药物组合物，它含有(a)安全有效量的本发明的基因编辑系统或本发明第四方面所述的细胞；以及(b)药学上可接受的载体或赋形剂。本发明的基因编辑系统的剂量通常为10微克-100毫克/剂，较佳地为100-1000微克/剂。
在一优选实施方式中，所述药物组合物中所述细胞的浓度为1×10 3-1×10 8个细胞/Kg体重，更优地1×10 5-1×10 7个细胞/Kg体重。为了本发明的目的，有效的剂量为给予个体约0.01毫克/千克至50毫克/千克，较佳地0.05毫克/千克至10毫克/千克体重的本发明的基因编辑系统。此外，本发明的基因编辑系统可以单用，也可与其他治疗剂一起使用(如配制在同一药物组合物中)。
在本发明中，本发明的药物组合物可以细胞治疗剂的形式施用，例如本发明的基因编辑系统可用于改造细胞，然后将改造好的细胞通过静脉或瘤内注射的方式施用于有需要的受试者。在某些实施方式中，本发明的基因编辑系统可用于改造体内的或分离的细胞。在某些实施方式中，所述细胞可以为T细胞，所述改造可以包括基因编辑。
药物组合物还可含有药学上可接受的载体。术语“药学上可接受的载体”指用于治疗剂给药的载体。该术语指这样一些药剂载体：它们本身不诱导产生对接受该组合物的个体有害的抗体，且给药后没有过分的毒性。这些载体是本领域普通技术人员所熟知的。在Remington’s Pharmaceutical Sciences(Mack Pub.Co.，N.J.1991)中可找到关于药学上可接受的赋形剂的充分讨论。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、佐剂及其组合。
治疗性组合物中药学上可接受的载体可含有液体，如水、盐水、甘油和乙醇。另外，这些载体中还可能存在辅助性的物质，如润湿剂或乳化剂、pH缓冲物质等。
通常，可将治疗性组合物制成可注射剂，例如液体溶液或悬液；还可制成在注射前适合配入溶液或悬液中、液体载体的固体形式。
一旦配成本发明的组合物，可将其通过常规途径进行给药，其中包括(但并不限于)：瘤内、肌内、静脉内、皮下、皮内、或局部给药。待预防或治疗的对象可以是动物；尤其是人。
当本发明的药物组合物被用于实际治疗时，可根据使用情况而采用各种不同剂型的药物组合物。较佳地为静脉用药制剂或瘤内用药注射剂。
这些药物组合物可根据常规方法通过混合、稀释或溶解而进行配制，并且偶尔添加合适的药物添加剂，如赋形剂、崩解剂、粘合剂、润滑剂、稀释剂、缓冲剂、等渗剂(isotonicities)、防腐剂、润湿剂、乳化剂、分散剂、稳定剂和助溶剂，而且该配制过程可根据剂型用惯常方式进行。
本发明的药物组合物还可以缓释剂形式给药。例如，本发明的基因编辑系统可被掺入以缓释聚合物为载体的药丸或微囊中，然后将该药丸或微囊通过手术植入待治疗的组织。作为缓释聚合物的例子，可例举的有乙烯-乙烯基乙酸酯共聚物、聚羟基甲基丙烯酸酯(polyhydrometaacrylate)、聚丙烯酰胺、聚乙烯吡咯烷酮、甲基纤维素、乳酸聚合物、乳酸-乙醇酸共聚物等，较佳地可例举的是可生物降解的聚合物如乳酸聚合物和乳酸-乙醇酸共聚物。
在一个实施方式中，所述药物组合物可包括缓冲液诸如中性缓冲盐水、硫酸盐缓冲盐水等等；碳水化合物诸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇；蛋白质；多肽或氨基酸诸如甘氨酸；抗氧化剂；螯合剂诸如EDTA或谷胱甘肽；佐剂(例如，氢氧化铝)；和防腐剂。本发明的药物组合物优选配制用于静脉内施用。
当本发明的药物组合物被用于实际治疗时，作为活性成分的本发明的基因编辑系统的剂量，可根据待治疗的每个病人的体重、年龄、性别、症状程度而合理地加以确定。
本发明的主要优点包括：
(1)本发明的含有特定gRNA的基因编辑系统可显著提高敲除率和外源核酸的定点整合效率。
(2)通过筛选得到的外源序列高效整合的gRNA位点，本发明能提高外源序列在T细胞中定点整合效率
(3)通过筛选得到的CRISPR/Cas9高效切割的gRNA位点，本发明能提高T细胞基因敲除的效率
(4)使用本发明方法，使定点整合型T细胞产品的制备成为可能，也能更有效地制备基因敲除的T细胞产品
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P3Primary Cell 4D-
Figure PCTCN2022072130-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P3Primary 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，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5×106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1×106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.敲除率检测
(1)使用“血液/细胞/组织基因组DNA提取试剂盒”(TIANGEN，DP304)提取未处理和电转后T细胞基因组DNA
(2)通过PCR扩增切割位点区域DNA
(3)使用“普通琼脂糖凝胶DNA回收试剂盒”(TIANGEN，DP209)纯化PCR产物
(4)将纯化的PCR产物进行一代测序
(5)通过ICE工具(Synthego)分析敲除率
在某些实施方式中，本发明的靶序列可以如下所示，即SEQ ID NO.:1：
Figure PCTCN2022072130-appb-000002
Figure PCTCN2022072130-appb-000003
在其他实施方式中，本发明的靶序列可以如下所示，即SEQ ID NO.:2：
Figure PCTCN2022072130-appb-000004
在另一些实施方式中，本发明的靶序列可以包含如SEQ ID NO.:3-15中任一项所示的序列：
DNA-sg1(即SEQ ID NO.:3)：ACAGGCGCCCTGGCCAGTCG。
DNA-sg2(即SEQ ID NO.:4)：CAGGCGCCCTGGCCAGTCGT。
DNA-sg3(即SEQ ID NO.:5)：GGGCGCCTGTGGGATCTGCA。
DNA-sg4(即SEQ ID NO.:6)：TGGCCAGTCGTCTGGGCGGT。
DNA-sg5(即SEQ ID NO.:7)：GGCCAGTCGTCTGGGCGGTG。
DNA-sg6(即SEQ ID NO.:8)：GTGGGATCTGCATGCCTGGA。
DNA-sg7(即SEQ ID NO.:9)：GGCATGCAGATCCCACAGGC。
DNA-sg8(即SEQ ID NO.:10)：GCCCAGTTGTAGCACCGCCC。
DNA-sg9(即SEQ ID NO.:11)：CCAGCCCAGTTGTAGCACCG。
DNA-sg10(即SEQ ID NO.:12)：GGCCGCCAGCCCAGTTGTAG。
DNA-sg11(即SEQ ID NO.:13)：TCCTGGCCGCCAGCCCAGTT。
DNA-sg12(即SEQ ID NO.:14)：AGTTGTAGCACCGCCCAGAC。
DNA-sg13(即SEQ ID NO.:15)：GTTGTAGCACCGCCCAGACG。
在某些实施方式中，本发明的gRNA的靶向序列可以包含如SEQ ID NO.:16-28中任一项所示的序列：
PD1-sg1(即SEQ ID NO.:16)：CGACTGGCCAGGGCGCCTGT。
PD1-sg2(即SEQ ID NO.:17)：ACGACTGGCCAGGGCGCCTG。
PD1-sg3(即SEQ ID NO.:18)：TGCAGATCCCACAGGCGCCC。
PD1-sg4(即SEQ ID NO.:19)：ACCGCCCAGACGACTGGCCA。
PD1-sg5(即SEQ ID NO.:20)：CACCGCCCAGACGACTGGCC。
PD1-sg6(即SEQ ID NO.:21)：TCCAGGCATGCAGATCCCAC。
PD1-sg7(即SEQ ID NO.:22)：GCCTGTGGGATCTGCATGCC。
PD1-sg8(即SEQ ID NO.:23)：GGGCGGTGCTACAACTGGGC。
PD1-sg9(即SEQ ID NO.:24)：CGGTGCTACAACTGGGCTGG。
PD1-sg10(即SEQ ID NO.:25)：CTACAACTGGGCTGGCGGCC。
PD1-sg11(即SEQ ID NO.:26)：AACTGGGCTGGCGGCCAGGA。
PD1-sg12(即SEQ ID NO.:27)：GTCTGGGCGGTGCTACAACT。
PD1-sg13(即SEQ ID NO.:28)：CGTCTGGGCGGTGCTACAAC。
实施例1 sgRNA的筛选实验
首先选择了一组PD1位点，在刺激后的T细胞中导入spCas9(TrueCut TM Cas9 Protein v2，Thermofisher)、sgRNA和CAR元件模板，其中CAR元件模板的序列如SEQ ID NO.:29所示。通过流式分析检测CAR阳性率，以确定外源序列高效整合的位点。结果显示，CAR元件在PD1多个位点均有不同程度的整合(图1)。同时，利用ICE方法检测这组位点的PD1敲除率。结果显示PD1部分位点有较高的敲除率(图2)。
之后，又选择了另一组PD1位点，在刺激后的T细胞中导入spCas9(TrueCut TM Cas9 Protein v2，Thermofisher)、sgRNA和含荧光蛋白mTurquoise2序列的模板,其中含荧光蛋白mTurquoise2序列的模板的序列如SEQ ID NO.:30所示。通过流式分析检测荧光蛋白的重组率，以确定外源序列高效整合的位点。结果显示，荧光蛋白序列在PD1多个位点均有不同程度的整合(图3)。在另一个实验中，在刺激后的T细胞中导入spCas9(TrueCut TM Cas9 Protein v2，Thermofisher)、sgRNA和CAR元件模板，其中CAR元件模板的序列如SEQ ID NO.:31所示。通过流式分析检测CAR阳性率，以确定外源序列高效整合的位点。与荧光蛋白重组检测结果相似，观察到CAR元件在PD1多个位点均有不同程度的整合(图4)。同时，利用ICE方法检测这组PD1位点的敲除率。结果显示PD1部分位点有较高的敲除率(图5)。
本实施例中提到的序列SEQ ID NO.:29-31中的任一项具体如下：
SEQ ID NO.:29，即PD1第一组位点(PD1-sg1到PD1-sg7)检测使用的含CAR序列的供体DNA序列，在该序列中，第1-800位的核苷酸为第一同源臂序列，第2954-3753位的核苷酸为第二同源臂序列：
Figure PCTCN2022072130-appb-000005
Figure PCTCN2022072130-appb-000006
Figure PCTCN2022072130-appb-000007
Figure PCTCN2022072130-appb-000008
SEQ ID NO.:30，即PD1第二组位点(PD1-sg8到PD1-sg13)检测使用的含mTurquoise2序列的供体DNA序列，在该序列中，第1-800位的核苷酸为第一同源臂序列，第2213-3012位的核苷酸为第二同源臂序列：
Figure PCTCN2022072130-appb-000009
Figure PCTCN2022072130-appb-000010
Figure PCTCN2022072130-appb-000011
SEQ ID NO.:31，即PD1第二组位点(PD1-sg8到PD1-sg13)检测使用的含CAR序列的供体DNA序列，在该序列中，第1-800位的核苷酸为第一同源臂序列，第2954-3753位的核苷酸为第二同源臂序列：
Figure PCTCN2022072130-appb-000012
Figure PCTCN2022072130-appb-000013
Figure PCTCN2022072130-appb-000014
实施例2 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PD1-sg1序列，使用不同健康供者的T细胞成功构建了PD1定点整合型CD19-CART细胞(其中用到的供体DNA序列如SEQ ID NO.:29所示)，并进行了测序验证(图6)，总阳性率大约在10％-30％(图7A、C)，敲除率大约在80％-95％(图7B)，PD1表达水平明显下降(图7D)，体现出了较好的制备稳定性。在此基础上，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PD-L1高表达肿瘤靶细胞(本实施例以慢病毒感染的PD-L1稳定过表达Raji肿瘤靶细胞为例)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8)。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PD-L1高表达肿瘤靶细胞(本实施例以慢病毒感染的PD-L1稳定过表达Raji肿瘤靶细胞为例)的刺激而激活表面标志物CD69、CD137、CD25的表达(图9A-C)，其中CD137表达水平的上升更为明显。相一致地，PD1定点整合型CD19-CART细胞与PD-L1高表达肿瘤靶细胞(本实施例以慢病毒感染的PD-L1稳定过表达Raji肿瘤靶细胞为例)接触后能大量分泌细胞因子IL-2、TNF-α、IFN-γ(图10A-C)，其中IFN-γ分泌水平的上升更为显著。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细胞具有对PD-L1高表达肿瘤靶细胞(本实施例以慢病毒感染的PD-L1稳定过表达Raji肿瘤靶细胞为例)更强的肿瘤杀伤能力(图11、12)。
综上，使用本发明序列并利用CRISPR/Cas9基因编辑工具可成功构建PD1定点整合型CAR-T细胞。本发明证明了与现有技术相比，使用本发明序列制备的定点整合型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提供的提高T细胞中外源序列定点整合的方法，以及保护的T细胞中高效整合、切割gRNA位点和DNA靶序列的重要性和价值，但不限于PD1定点整合CAR-T细胞的制备，可拓展到其他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. 一种靶序列，其特征在于，所述靶序列选自下组：
    (1)SEQ ID NO.:1所示的第40-130位的核苷酸序列；
    (2)将SEQ ID NO.:1所示的第40-130位的核苷酸序列经过一个或多个(≤10个，如2-8个，优选为3-5个)核苷酸的取代、缺失或添加而形成的衍生核苷酸，并且所述靶序列特异性与gRNA结合。
  2. 如权利要求1所述的靶序列，其特征在于，所述靶序列选自下组：
    (1)具有SEQ ID NO.:2所示核苷酸序列的多核苷酸；
    (2)与SEQ ID NO.:2所示核苷酸序列的同源性≥95％，较佳地≥98％，更佳地≥99％的多核苷酸；
    (3)与上述(1)-(2)互补的多核苷酸。
  3. 一种gRNA序列，其特征在于，所述gRNA序列与权利要求1所述的靶序列互补。
  4. 一种基因编辑系统，其特征在于，所述系统包括基因编辑酶和gRNA，所述gRNA与权利要求1所述的靶序列互补。
  5. 一种细胞，其特征在于，所述细胞经权利要求4所述的基因编辑系统进行编辑后获得。
  6. 一种组合物，其特征在于，包括：
    权利要求4所述的系统或权利要求5所述的细胞；和
    药学上可接受的载体。
  7. 一种药盒，其特征在于，包括：
    第一容器，以及活性成分或含有所述活性成分的药物，所述活性成分或所述药物位于所述第一容器中，所述活性成分包含权利要求4所述的系统、或权利要求5所述的细胞、或权利要求6所述的组合物。
  8. 一种对细胞进行基因编辑的方法，其特征在于，包括将所述细胞接触权利要求4所述的基因编辑系统或权利要求6所述的组合物或权利要求7所述的药盒。
  9. 一种用于基因编辑的试剂盒，其特征在于，所述试剂盒包括权利要求4所述的基因编辑系统或权利要求6所述的组合物或权利要求7所述的药盒。
  10. 一种权利要求4所述的基因编辑系统、权利要求5所述的细胞、权利要求6所述的组合物或权利要求7所述药盒的用途，其特征在于，用于制备用 于预防和/或治疗疾病的药物。
PCT/CN2022/072130 2021-01-18 2022-01-14 用于基因编辑的组合物及其应用 WO2022152266A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110064928.6A CN114807155A (zh) 2021-01-18 2021-01-18 用于基因编辑的组合物及其应用
CN202110064928.6 2021-01-18

Publications (1)

Publication Number Publication Date
WO2022152266A1 true WO2022152266A1 (zh) 2022-07-21

Family

ID=82446949

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/072130 WO2022152266A1 (zh) 2021-01-18 2022-01-14 用于基因编辑的组合物及其应用

Country Status (2)

Country Link
CN (1) CN114807155A (zh)
WO (1) WO2022152266A1 (zh)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107164377A (zh) * 2017-06-12 2017-09-15 王小平 基于碱基编辑的基因敲除方法及其应用
CN109722437A (zh) * 2018-12-29 2019-05-07 广州百暨基因科技有限公司 一种通用型car-t细胞及其制备方法和用途
CN109797171A (zh) * 2017-05-08 2019-05-24 北京东方略细胞技术有限公司 经修饰的t细胞、其制备方法及用途
CN109844125A (zh) * 2016-08-31 2019-06-04 南京凯地生物科技有限公司 人pd-1基因敲除的cldn18．2特异性嵌合抗原受体t细胞的制备方法以及应用
CN110785488A (zh) * 2017-04-21 2020-02-11 圣拉斐尔医院有限责任公司 基因疗法

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106480097A (zh) * 2016-10-13 2017-03-08 南京凯地生物科技有限公司 利用CRISPR/Cas9技术敲除人PD‑1基因构建可靶向MSLN新型CAR‑T细胞的方法及其应用
CN109517841B (zh) * 2018-12-05 2020-10-30 华东师范大学 一种用于核苷酸序列修饰的组合物、方法与应用
CN109652380B (zh) * 2019-01-25 2023-04-28 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
CN112239769B (zh) * 2019-07-19 2023-11-07 上海邦耀生物科技有限公司 一种引导PD1基因切割实现外源序列高效整合的sgRNA
CN114164231B (zh) * 2020-09-10 2024-05-17 上海邦耀生物科技有限公司 对细胞中的靶位点进行基因编辑的方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109844125A (zh) * 2016-08-31 2019-06-04 南京凯地生物科技有限公司 人pd-1基因敲除的cldn18．2特异性嵌合抗原受体t细胞的制备方法以及应用
CN110785488A (zh) * 2017-04-21 2020-02-11 圣拉斐尔医院有限责任公司 基因疗法
US20200078408A1 (en) * 2017-04-21 2020-03-12 Ospedale San Raffaele S.R.L. Gene therapy
CN109797171A (zh) * 2017-05-08 2019-05-24 北京东方略细胞技术有限公司 经修饰的t细胞、其制备方法及用途
CN107164377A (zh) * 2017-06-12 2017-09-15 王小平 基于碱基编辑的基因敲除方法及其应用
CN109722437A (zh) * 2018-12-29 2019-05-07 广州百暨基因科技有限公司 一种通用型car-t细胞及其制备方法和用途

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE Nucleotide NCBI; 28 December 1994 (1994-12-28), ANONYMOUS : "Human PD-1 gene, complete cds", XP055951482, Database accession no. L27440 *
SI JINGWEN; SHI XIANGJUN; SUN SHUHAO; ZOU BIN; LI YAOPENG; AN DONGJIE; LIN XINGYU; GAO YAN; LONG FEI; PANG BO; LIU XING; LIU TIAN;: "Hematopoietic Progenitor Kinase1 (HPK1) Mediates T Cell Dysfunction and Is a Druggable Target for T Cell-Based Immunotherapies", CANCER CELL, CELL PRESS, US, vol. 38, no. 4, 28 August 2020 (2020-08-28), US , pages 551, XP086291390, ISSN: 1535-6108, DOI: 10.1016/j.ccell.2020.08.001 *
YU QING, WANG YUFEI; JIA ANNA; WANG YUEXIN; YANG QIULI; LIU GUANGWEI: "New progress in tumor immunotherapy", BULLETIN OF BIOLOGY, vol. 55, no. 9, 20 September 2020 (2020-09-20), pages 1 - 6, XP055951485, ISSN: 0006-3193 *

Also Published As

Publication number Publication date
CN114807155A (zh) 2022-07-29

Similar Documents

Publication Publication Date Title
US20230174621A1 (en) Modified epidermal growth factor receptor peptides for use in genetically-modified cells
CN110616187B (zh) 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
US20210277373A1 (en) Genetically-modified t cells comprising a modified intron in the t cell receptor alpha gene
CN111479917A (zh) 经基因修饰以消除T细胞受体和β2-微球蛋白表达的永生化CAR-T细胞
AU2019252527B2 (en) Optimized engineered nucleases having specificity for the human T cell receptor alpha constant region gene
EP3448995A1 (en) Allele editing and applications thereof
KR102338993B1 (ko) 인위적으로 조작된 조작면역세포
JP7471289B2 (ja) 抗ｌｉｖ１免疫細胞癌療法
CN111263808B (zh) 一种靶向HPK1的gRNA和一种编辑HPK1基因的方法
BR112020020073A2 (pt) Células t que expressam um receptor recombinante, polinucleotídeos relacionados e métodos
WO2022052877A1 (zh) 对细胞中的靶位点进行基因编辑的方法
WO2021082784A1 (zh) 一种基于腺病毒的基因编辑方法
JP2023547887A (ja) セーフハーバー遺伝子座
WO2022152266A1 (zh) 用于基因编辑的组合物及其应用
WO2023081811A1 (en) Immune cells with chimeric antigen receptors or chimeric autoantibody receptors
WO2021170089A1 (en) Engineering immune cells via simultaneous knock-in and gene disruption
WO2022266538A2 (en) Compositions and methods for targeting, editing or modifying human genes
WO2024148167A1 (en) Optimized engineered meganucleases having specificity for the human t cell receptor alpha constant region gene

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: 22739141

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22739141

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 14.02.2024)