WO2020057668A1 - 基于CRISPR/Cas系统对细胞进行基因编辑的方法 - Google Patents

基于CRISPR/Cas系统对细胞进行基因编辑的方法 Download PDF

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WO2020057668A1
WO2020057668A1 PCT/CN2019/107374 CN2019107374W WO2020057668A1 WO 2020057668 A1 WO2020057668 A1 WO 2020057668A1 CN 2019107374 W CN2019107374 W CN 2019107374W WO 2020057668 A1 WO2020057668 A1 WO 2020057668A1
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antigen
cells
gene
cell
grna
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PCT/CN2019/107374
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English (en)
French (fr)
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李宗海
廖朝晖
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科济生物医药（上海）有限公司
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Priority to CN201980058264.3A priority Critical patent/CN112805371A/zh
Priority to JP2021516699A priority patent/JP2022511345A/ja
Priority to AU2019341365A priority patent/AU2019341365A1/en
Priority to US17/277,930 priority patent/US20220017926A1/en
Priority to KR1020217011835A priority patent/KR20210065141A/ko
Priority to EP19863140.0A priority patent/EP3854871A4/en
Priority to CA3122131A priority patent/CA3122131A1/en
Publication of WO2020057668A1 publication Critical patent/WO2020057668A1/zh

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Definitions

  • the invention relates to a method of gene editing. More specifically, it relates to a method for gene editing of cells using the CRISPR / Cas system.
  • Gene editing includes altering the genome by deleting, inserting, mutating or replacing specific nucleic acid sequences.
  • the CRISPR-Cas system consists of regularly clustered spaced short palindromic repeats (CRISPR) and associated Cas proteins.
  • RNA-directed Cas endonucleases specifically target and cleave DNA in a sequence-dependent manner (Jinek, M.
  • the object of the present invention is to provide a method for quickly and efficiently knocking out genes in cells, in particular, a method capable of quickly and efficiently knocking out multiple genes at one time.
  • a method for gene editing of a cell based on a CRISPR / Cas system is provided.
  • a complex of Cas enzyme and gRNA is introduced into the cell for gene editing, wherein Cas in the complex is The ratio of enzyme to gRNA is 1: 3 to 1: 5.
  • the Cas enzyme is a Cas9 enzyme.
  • the Cas9 enzyme has an enzyme activity of 0.1 to 1 nmol, preferably 0.2 to 0.7 nmol, more preferably 0.3 to 0.5 nmol, and most preferably 0.37 nmol.
  • the Cas enzyme is a Cas9 enzyme.
  • the molar ratio of Cas9 enzyme to gRNA is 1: 1 to 1:10, preferably 1: 3 to 1: 5. Further, It is preferably 1: 4.
  • a Cas9 enzyme from NEB company can be used.
  • those skilled in the art can select other Cas9 enzymes having the same or similar functions.
  • the function that the Cas9 enzyme can achieve is that in a 30 ⁇ l reaction Cas9 enzyme reaction system (the reaction system includes: 20mM HEPES, 100mM NaCl, 5mM MgCl 2 , 0.1mM EDTA, and at 25 ° C. , PH 6.5), when containing 1nM PvuII linearized pBR322 DNA (one target site CGCTTGTTTCGGCGTGGGTA), 40nM sgRNA and 20nM Cas9 enzyme, incubate at 37 ° C for 1 hour, pass an agarose gel Electrophoresis confirmed that 90% of the pBR322 DNA was degraded.
  • the reaction system includes: 20mM HEPES, 100mM NaCl, 5mM MgCl 2 , 0.1mM EDTA, and at 25 ° C. , PH 6.5
  • 1nM PvuII linearized pBR322 DNA one target site CGCTTGTTTCGGCGTGGGTA
  • the amount of Cas9 enzyme that catalyzes the complete conversion of 1nmol substrate (PvuII linearized pBR322 DNA) into the product in one minute is 0.37nmol, and the number of grams of Cas9 enzyme is 59.57ng.
  • the Cas9 enzyme had an enzyme activity of 0.37 nmol (the amount of enzyme that catalyzes the conversion of 1 nmol substrate to product in 1 minute).
  • the enzyme activity of the enzyme is 0.37 nmol.
  • the molar ratio of Cas9 enzyme to gRNA to be introduced is calculated based on the Cas9 enzyme activity mentioned above, and the concentration of Cas9 enzyme in the introduction complex is confirmed.
  • the activity of Cas9 enzyme changes At this time, those skilled in the art can perform conversion based on the ratios determined herein based on the descriptions of the activities in different enzyme specifications to select the use concentration of Cas9 enzyme and its molar ratio to gRNA.
  • the Cas enzyme with an enzyme activity of 0.37 nmol is only an example.
  • the enzyme activity of the enzyme is different from the Cas enzyme, those skilled in the art can Activity was calculated to confirm the amount of Cas9 enzyme used and its molar ratio to gRNA.
  • the present invention relates to a method for editing two genes. Specifically, a complex of Cas9 enzyme and a first gRNA and a complex of Cas9 enzyme and a second gRNA are introduced into the cell. Perform gene editing.
  • a complex of Cas9 enzyme, a first gRNA and a second gRNA is simultaneously introduced into the cell for gene editing.
  • complex one and complex two are sequentially introduced into the cell for gene editing.
  • the molar ratio of Cas9 enzyme to gRNA is 1: 1 to 1:10, preferably 1: 3 to 1: 5, and more preferably 1: 4.
  • the molar ratio of Cas9 enzyme to gRNA in complex one is 1: 1 to 1:10, preferably 1: 3 to 1: 5, and more preferably 1: 4.
  • the mole of Cas9 enzyme and gRNA in complex two The ratio is 1: 1 to 1:10, preferably 1: 3 to 1: 5, and more preferably 1: 4.
  • the molar ratio of Cas9 enzyme to the sum of the first gRNA and the second gRNA is 1: 1 to 1:10, preferably 1: 3 to 1: 5, and more preferably 1: 4.
  • the molar ratio refers to the ratio between the amount of Cas9 enzyme and the amount of gRNA substance, wherein the amount of Cas9 enzyme or the enzyme activity is calculated based on the instructions of the Cas9 enzyme provided by the manufacturer, and the corresponding amount of gRNA is in accordance with The RNA base composition and the concentration of in vitro transcription were calculated.
  • the ratio of the Cas enzyme to the gRNA is 1: 4.
  • the cell is a eukaryotic cell; in a specific embodiment, the eukaryotic cell is an immune effector cell; in a specific embodiment, the immune effector cell is a T cell.
  • the concentration of the Cas enzyme is about 0.1 ⁇ M to 3 ⁇ M; preferably, about 0.125 ⁇ M to 3 ⁇ M; more preferably, about 0.2 ⁇ M to 3 ⁇ M; more preferably, about 0.25 ⁇ M to 3 ⁇ M; more preferably, about 0.5 ⁇ M to 3 ⁇ M.
  • the concentration of the Cas9 enzyme is about 0.1 ⁇ M to 3 ⁇ M; preferably, about 0.125 ⁇ M to 3 ⁇ M; more preferably, about 0.2 ⁇ M to 3 ⁇ M; more preferably, about 0.25 ⁇ M to 3 ⁇ M; more preferably, about 0.5 ⁇ M to 3 ⁇ M.
  • the concentration of the Cas enzyme is about 0.1 ⁇ M to 2 ⁇ M; preferably, about 0.125 ⁇ M to 2 ⁇ M; more preferably, about 0.5 ⁇ M to 2 ⁇ M; more preferably, about 0.5 ⁇ M to 2 ⁇ M; more preferably, about 0.5 ⁇ M to 2 ⁇ M.
  • the cell is a T cell
  • the CRISPR / Cas system edits the gene of the T cell.
  • the T and T chains of the TCR of the T cell are edited.
  • Gene editing of any one or two strands of genes; in specific embodiments, gene editing of TRAC; in specific embodiments, gene editing of constant regions of TRAC; in specific embodiments, TRAC includes There is a sequence shown in SEQ ID NO: 1 for gene editing.
  • the cell is a T cell, and the gene of the T cell is edited by using the CRISPR / Cas9 system; including:
  • Gene editing of genes in any one or both of the ⁇ and ⁇ chains of the TCR of the T cell using the CRISPR / Cas9 system preferably gene editing of TRAC; further preferably gene of the constant region of TRAC Editing; it is further preferred to perform gene editing on the sequence shown in SEQ ID NO: 45 in TRAC; it is further preferred to perform gene editing on the sequence shown in SEQ ID NO: 1 in TRAC, and / or
  • the CRISPR / Cas9 system is used to perform gene editing on the MHC genes of the T cells, preferably B2M genes are genetically edited, and it is further preferred to perform gene editing on the sequence shown in SEQ ID NO: 38 in the B2M genes, and it is further preferred to The B2M gene contains the sequence shown in SEQ ID NO: 10 for gene editing.
  • the gRNA is designed according to the PAM sequence in the sequence shown in SEQ ID NO: 1.
  • the gRNA is about 15-50bp, preferably, about 15-30bp, more preferably about 17-21bp; more preferably 20bp.
  • the gRNA used for editing the TRAC is a sequence including SEQ ID NO: 2, 3, 4, or 5; preferably, the gRNA used is a sequence including SEQ ID NO: 2 Shown sequence.
  • the gRNA used for editing the TRAC includes a sequence represented by SEQ ID NO: 2, 3, 4, 5, 32, 33, 39, or 40; preferably, the adopted gRNA includes There is a sequence shown in SEQ ID NO: 2, 32 or 33.
  • the gRNA used for editing the TRAC is a sequence shown as SEQ ID NO: 2, 3, 4, 5, 32, 33, 39, or 40; preferably, the gRNA used is as shown in SEQ ID NO: 2, 32 or 33.
  • the first gRNA may include a sequence shown in SEQ ID NO: 2, 3, 4, 5, 32, 33, 39, or 40.
  • the concentration of the Cas enzyme is about 0.1 ⁇ M to 0.5 ⁇ M; preferably, about 0.125 ⁇ M to 0.5 ⁇ M, and more preferably, about 0.25 ⁇ M to 0.5 ⁇ M.
  • the cell is a T cell, and the CRISPR / Cas system performs gene editing on the B2M gene of the T cell.
  • the B2M gene includes SEQ ID NO: 10 Gene editing; in a specific embodiment, the gRNA is designed according to the PAM sequence in the sequence shown in SEQ ID NO: 10.
  • the gRNA used for editing the B2M gene comprises the sequence shown in SEQ ID NO: 11, 12, 13, or 14; preferably, the adopted gRNA contains the sequence shown in SEQ ID NO: 12 sequence.
  • the gRNA used for editing the B2M gene is a sequence shown as SEQ ID NO: 11, 12, 13, or 14; preferably, the gRNA used is shown as SEQ ID NO: 12 sequence.
  • the second gRNA may include a sequence shown in SEQ ID NO: 11, 12, 13, or 14.
  • the descriptions for complex one, complex two, or complex three are the same as above, and the descriptions for the first gRNA and the second gRNA are also the same as above, it should be understood that the complex, the complex one, and the complex two Or three complexes are intended to indicate different complexes, and there is no priority order for their numbering.
  • the same is true for the first gRNA and the second gRNA. It is intended to represent two different gRNAs.
  • One gRNA and another gRNA can also be used.
  • Means that one gRNA may include the sequence shown in SEQ ID NO: 2, 3, 4, 5, 32, 33, 39, or 40, and the other gRNA may include the sequence shown in SEQ ID NO: 11, 12, The sequence shown in 13, or 14.
  • the concentration of the Cas enzyme is about 0.25 ⁇ M to 3 ⁇ M, preferably about 0.5 ⁇ M to 3 ⁇ M, and more preferably about 1 ⁇ M to 3 ⁇ M.
  • the cell is a T cell
  • the CRISPR / Cas system performs gene editing on the TRAC and B2M genes of the T cells; in a specific embodiment, the first exon of the TRAC and B2M genes Perform gene editing.
  • the TRAC and / or B2M genes are subjected to gene editing, and the TRAC and / or B2M genes are silenced.
  • the gRNA used for editing the TRAC contains the sequence shown in SEQ ID NO: 2, 3, 4, or 5, and the gRNA used for editing the B2M gene contains SEQ ID NO: 11, The sequence shown in 12, 13, or 14; preferably, the gRNA used for editing the TRAC contains the sequence shown in SEQ ID NO: 2, and the gRNA used for editing the B2M gene contains the sequence shown in SEQ ID NO: 12 Shown sequence.
  • the gRNA is about 15-50bp, preferably, about 15-30bp, and more preferably about 20bp; in a specific embodiment, it is 20bp.
  • the ratio of the edited B2M gRNA and the edited TRAC gRNA is about 1.5: 1 to 0.5: 1; preferably, about 1: 1.
  • the concentration of the Cas enzyme is about 1 ⁇ M to 3 ⁇ M.
  • the T cells also express chimeric receptors, exogenous cytokines, inhibitory / activating receptors or ligands, and co-stimulatory factors; in specific embodiments, the T cells Cells also express chimeric antigen receptor receptors.
  • a method for gene editing of the TRAC gene of T cells based on the CRISPR / Cas system is provided.
  • a complex of Cas enzyme and gRNA is introduced into the cell for gene editing, wherein Cas enzyme The ratio to gRNA is 1: 3 to 1: 5.
  • the Cas enzyme is a Cas9 enzyme.
  • gene editing is performed on genes in any one or both of the ⁇ and ⁇ chains of the TCR of the T cell; in a specific embodiment, gene editing is performed on the TRAC of the T cell; in a specific embodiment, gene editing is performed on the constant region of the T cell TRAC; in a specific embodiment, gene editing is performed on the sequence of the T cell including the sequence shown in SEQ ID NO: 1; In a specific embodiment, the gRNA is designed according to the PAM sequence in the sequence shown in SEQ ID NO: 1.
  • the ratio of Cas enzyme to gRNA is 1: 4.
  • the concentration of the Cas enzyme is about 0.1 ⁇ M to 0.5 ⁇ M; preferably, about 0.125 ⁇ M to 0.5 ⁇ M, and more preferably, about 0.25 ⁇ M to 0.5 ⁇ M.
  • the gRNA used for editing the TRAC includes the sequence shown in SEQ ID NO: 2, 3, 4, or 5; preferably, the adopted gRNA includes the sequence shown in SEQ ID NO: 2 sequence.
  • the ratio of the Cas enzyme to gRNA is 1: 4; the concentration of the Cas enzyme is 0.25 ⁇ M to 0.5 ⁇ M; and the gRNA used is SEQ ID NO: 2 Shown sequence.
  • a method for gene editing of a B2M gene of a T cell based on a CRISPR / Cas system is provided.
  • a complex of Cas enzyme and gRNA is introduced into the cell for gene editing, wherein the Cas enzyme
  • the ratio to gRNA is 1: 3 to 1: 5.
  • the Cas enzyme is a Cas9 enzyme.
  • the B2M gene includes a sequence represented by SEQ ID NO: 10 for gene editing.
  • the gRNA is designed according to the PAM sequence in the sequence shown in SEQ ID NO: 10. In a specific embodiment, the ratio of Cas enzyme to gRNA is 1: 4.
  • the concentration of the Cas enzyme is about 0.25 ⁇ M to 3 ⁇ M, preferably, about 0.5 ⁇ M to 3 ⁇ M, and more preferably, about 1 ⁇ M to 3 ⁇ M.
  • the gRNA used for editing the B2M gene contains the sequence shown in SEQ ID NO: 11, 12, 13, or 14; preferably, the gRNA used contains the sequence shown in SEQ ID NO: 12 .
  • the ratio of the Cas enzyme to gRNA is 1: 4; the concentration of the Cas enzyme is 1 ⁇ M to 3 ⁇ M; the gRNA used is composed of SEQ ID NO: 12 Shown sequence.
  • a method for gene editing of a TRAC gene and a B2M gene of a T cell based on a CRISPR / Cas system is provided.
  • a complex of Cas enzyme and gRNA is introduced into the cell, wherein Cas enzyme The ratio to the total gRNA is 1: 3 to 1: 5.
  • the Cas enzyme is a Cas9 enzyme.
  • gene editing is performed on the B2M gene including the sequence shown in SEQ ID NO: 10; in a specific embodiment, in a specific embodiment, according to the PAM in the sequence shown in SEQ ID NO: 10 Sequence to design gRNA.
  • gene editing is performed on any one or both of the ⁇ and ⁇ chains of TCR; in a specific embodiment, gene editing is performed on TRAC;
  • gene editing is performed on the constant region of TRAC
  • gene editing is performed on the TRAC sequence including SEQ ID: NO: 1; in a specific embodiment, in a specific embodiment, according to the PAM in the sequence shown in SEQ ID: NO: 1 Sequence to design gRNA.
  • the ratio of the Cas enzyme to the total gRNA is 1: 4. In a specific embodiment, the concentration of the Cas enzyme is 1 ⁇ M to 3 ⁇ M.
  • the ratio of gRNA used for editing the B2M gene and editing TRAC is 0.5: 1 to 1.5: 1, preferably, 1: 1.
  • the gRNA used for editing the B2M gene contains the sequence shown in SEQ ID NO: 11, 12, 13, or 14; preferably, the gRNA used contains the sequence shown in SEQ ID NO: 12 .
  • the gRNA used for editing the TRAC includes the sequence shown in SEQ ID NO: 2, 3, 4, or 5; preferably, the adopted gRNA includes the sequence shown in SEQ ID NO: 2 sequence.
  • the ratio of the Cas enzyme to the total gRNA is 1: 4; the concentration of the Cas enzyme is 1 ⁇ M to 3 ⁇ M; the adopted gRNA is a sequence comprising SEQ ID NO: 12 and a sequence comprising SEQ ID NO: The sequence shown in 2.
  • the T cells described in the second aspect, the third aspect, and the fourth aspect further express a chimeric receptor that recognizes a tumor antigen or a pathogen antigen, and the chimeric receptor has an extracellular antigen binding domain, A transmembrane domain, and an intracellular domain that specifically recognizes a target antigen.
  • the target antigen is a tumor antigen selected from the group consisting of: thyroid stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD22; CD30; CD70; CD123; CD138; CD33; CD44; CD44v7 / 8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); Interleukin 13 receptor Subunit ⁇ (IL-13R ⁇ ); Interleukin 11 receptor ⁇ (IL-11R ⁇ ); Prostate Stem Cell Antigen (PSCA); Prostate Specific Membrane Antigen (PSMA); Carcinoembryonic Antigen (CEA); NY-ESO-1; HIV -1Gag; MART-1; gp100; tyrosinase; mesothelin; EpCAM; protease serine 21 (PRSS21); vascular endothelial growth
  • TSHR
  • the target antigen is a pathogen antigen
  • the pathogen antigen is selected from: an antigen of a virus, a bacterium, a fungus, a protozoan, or a parasite
  • the viral antigen is selected from: a giant cell Viral antigen, Epstein-Barr virus antigen, human immunodeficiency virus antigen or influenza virus antigen.
  • the chimeric receptor is selected from a chimeric antigen receptor (CAR) or a T cell antigen coupler (TAC).
  • CAR chimeric antigen receptor
  • TAC T cell antigen coupler
  • the chimeric receptor is a chimeric antigen receptor.
  • the chimeric antigen receptor includes:
  • the chimeric receptor is TAC and includes:
  • the extracellular domain includes an antibody domain having an antigen-binding domain, and a single-chain antibody that binds to CD3;
  • the antibody that specifically binds to a tumor antigen of the chimeric antigen receptor is a full-length antibody, scFv, Fab, (Fab '), or single domain antibody.
  • the use of the T cells according to the second aspect, the third aspect, and the fourth aspect is provided for preparing a T cell expressing a chimeric receptor, the chimeric receptor has Extracellular antigen-binding domains, transmembrane domains, and intracellular domains that specifically recognize target antigens.
  • the target antigen is a tumor antigen or a pathogen antigen.
  • the target antigen is a tumor antigen selected from the group consisting of: thyroid stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD22; CD30; CD70; CD123; CD138; CD33; CD44; CD44v7 / 8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); Interleukin 13 receptor Subunit ⁇ (IL-13R ⁇ ); Interleukin 11 receptor ⁇ (IL-11R ⁇ ); Prostate Stem Cell Antigen (PSCA); Prostate Specific Membrane Antigen (PSMA); Carcinoembryonic Antigen (CEA); NY-ESO-1; HIV -1Gag; MART-1; gp100; tyrosinase; mesothelin; EpCAM; protease serine 21 (PRSS21); vascular endothelial growth
  • TSHR
  • the target antigen is a pathogen antigen
  • the pathogen antigen is selected from: an antigen of a virus, a bacterium, a fungus, a protozoan, or a parasite
  • the viral antigen is selected from: giant cells Viral antigen, Epstein-Barr virus antigen, human immunodeficiency virus antigen or influenza virus antigen.
  • the chimeric receptor is selected from a chimeric antigen receptor (CAR) or a T cell antigen coupler (TAC).
  • CAR chimeric antigen receptor
  • TAC T cell antigen coupler
  • the chimeric receptor is a chimeric antigen receptor (CAR);
  • the chimeric antigen receptor includes:
  • the chimeric receptor is TAC and includes:
  • the extracellular domain includes an antibody domain having an antigen-binding domain, and a single-chain antibody that binds to CD3;
  • the antibody that specifically binds to a tumor antigen of the chimeric antigen receptor is a full-length antibody, scFv, Fab, (Fab '), or single domain antibody.
  • the electric conversion conditions may be, for example, 150-600V, 0.5ms-20ms, and may be preferably 150V-300V, 2ms-15ms, for example.
  • the molar ratio of the gene editing gRNA for the TCR gene and the gRNA for the gene editing of the MHC gene is about 1: 5 to 5: 1, preferably 1: 2 to 2: 1; more preferably It's about 1: 1.
  • the T cells are as described above.
  • the chimeric receptor is a chimeric antigen receptor (CAR), and the chimeric antigen receptor is as described in the above aspect.
  • CAR chimeric antigen receptor
  • a seventh aspect of the present invention it relates to a universal T cell constructed by the method of the present invention described above.
  • the present invention relates to a universal T cell whose TRAC and / or B2M genes are silenced.
  • the TRAC gene is silenced by performing gene editing on the sequence containing SEQ ID NO: 1, and it is further preferred that the TRAC gene is silenced by including the sequence shown by SEQ ID NO: 1.
  • the sequence shown in SEQ ID NO: 45 is implemented by gene editing;
  • the B2M gene is silenced by performing gene editing on the sequence containing SEQ ID ID: 10, it is further preferred that the B2M gene is silenced on the sequence containing SEQ ID ID NO: 10 as SEQ ID ID NO:
  • the sequence shown in 38 was implemented by gene editing.
  • the TRAC gene is silenced by using the gRNA of the sequence shown in SEQ ID NO: 2, 32, or 33 to edit the TRAC gene
  • the B2M gene is silenced by using the SEQ ID NO:
  • the gRNA of the sequence shown in Fig. 12 was implemented by gene editing of the B2M gene.
  • the T cells further express a chimeric antigen receptor, preferably the T cells also express a chimeric receptor that recognizes a tumor antigen or a pathogen antigen, and the chimeric receptor has extracellular antigen binding Domain, transmembrane domain, and intracellular domain, the extracellular antigen binding domain specifically recognizes a target antigen.
  • the T cells are as described above.
  • the chimeric antigen receptor is as described above.
  • a gRNA construct comprising a core selected from one of SEQ ID NO: 2, 3, 4, 5, 32, 33, 39, 40, 11, 12, 13, or 14 Nucleotide sequence.
  • the gRNA construct of the present invention comprises: a nucleotide sequence selected from one of SEQ ID NO: 2, 3, 4, 5, 32, 33, 39, or 40, and selected from SEQ ID NO: a nucleotide sequence of one of 11, 12, 13, or 14.
  • the gRNA construct of the present invention comprises: a sequence selected from the group consisting of SEQ ID NO: 2, 32, or 33, and / or a sequence represented by SEQ ID NO: 12.
  • the invention relates to the modification of T cells by using gene editing technology, and can effectively inhibit the functions of T cell antigen receptor (TCR) and major histocompatibility complex (MHC) in T cells through the knockout of multiple genes; encoding TCR
  • TCR T cell antigen receptor
  • MHC major histocompatibility complex
  • the gene is TRAC and the gene encoding MHC I is B2M.
  • RNP RNA nucleic acid and protein complex
  • Figure 1 is a schematic diagram of the binding sites of sgRNA and TRAC gene
  • Figure 2 shows the effect of RNP with different composition ratios on the TRAC knockout effect
  • Figure 3 shows the effect of different gRNA sequences on the effect of TRAC knockout
  • FIG. 4 shows the effect of different concentrations of Cas9 enzyme on the effect of TRAC knockout
  • Figure 5 shows a schematic diagram of gRNA and B2M gene binding sites
  • Figure 6 shows the effect of different gRNAs on B2M gene knockout effect
  • FIG. 7 shows the effect of different concentrations of Cas9 enzyme on B2M gene knockout effect
  • Figure 8 shows the effects of different gRNA components on TRAC and B2M double knockout when TRAC and B2M are knocked out simultaneously;
  • Figure 9 shows the effect of the concentration of the RNP complex formed between the gRNA mixture consisting of the TRAC and B2M genes and the Cas9 enzyme on the knockout efficiency
  • FIG. 10 (a)-(d) show the prediction efficiency of TRAC and B2M gene mutations by Tide online software
  • Figure 11 shows the results of clone sequencing to verify TRAC and B2M gene mutation results.
  • FIG. 12 shows the TRAC and B2M gene knockout efficiency in CAR T cells targeted to BCMA
  • the inventor's research found that when using the CRISPR / Cas9 system for gene editing, the selection of gRNA, the ratio of Cas9 enzymes and gRNA, etc. have a great effect on the editing efficiency, and the present invention was completed on the basis of this.
  • the term about refers to the usual error range of each value that is readily known to those skilled in the art.
  • References herein to "about” a value or parameter include (and describe) embodiments that point to the value or parameter itself.
  • the description about “about X” includes a description of "X”.
  • “about” or “including” may mean that it is within 1 or more than 1 according to the actual standard deviation in the field.
  • “about” or “including” may mean a range of up to 10% (ie, ⁇ 10%).
  • about 5 ⁇ M may include any number between 4.5 ⁇ M and 5.5 ⁇ M.
  • gene editing refers to the ability to allow humans to "edit” a target gene to achieve the deletion, addition, etc. of a specific DNA fragment.
  • molencing refers to a phenomenon in which a gene is not expressed or underexpressed without damaging the original DNA due to various reasons. Gene silencing occurs at two levels, one is gene silencing at the transcription level due to DNA methylation, heterochromatinization, and position effects, and the other is post-transcriptional gene silencing, which is the level after gene transcription Gene inactivation is performed by specifically inhibiting target RNA, including antisense RNA, co-suppression, gene suppression, RNA interference and microRNA-mediated translation suppression.
  • CRISPR Clustered Regularly Interspaced Short PalindromicRepeats
  • Cas9 CRISPRassociated Nuclease
  • CCRISPR / Cas9 CRISPR-related nuclease
  • Cas9 Nuclease CRISPR-related nuclease
  • CCRISPR / Cas9 CRISPR-guided, using Cas9 Nuclease to target genes Editing techniques.
  • CRISPER / Cas9 system are collectively referred to as transcripts and other elements involved in the expression or directing of the Cas9 enzyme gene, including sequences encoding the Cas9 gene, tracr (trans-activated CRISPR) sequences (such as tracrRNA or active part tracrRNA), and tracr pair Sequences (covering "codirectional repeats” and parts of tracrRNA processing in the context of endogenous CRISPR systems), guide sequences (also known as “spacers” (gRNAs) in the context of endogenous CRISPR systems) , Or other sequences and transcripts from CRISPR loci.
  • CRISPR systems are characterized by the promotion of CRISPR complexes (also called anterior compartments in the context of endogenous CRISPR systems) at the site of the target sequence. Formed elements.
  • target sequence refers to a sequence that is complementary to a guide sequence, and the complementary pairing between the target sequence and the guide sequence promotes the formation of a CRISPR complex. Full complementarity is not required, provided that there is sufficient complementarity to cause hybridization and promote the formation of a CRISPR complex.
  • a target sequence may comprise any polynucleotide, such as a DNA or RNA polynucleotide. In some embodiments, the target sequence is located in the nucleus or cytoplasm of a cell.
  • a guide sequence is any polynucleotide sequence that is sufficiently complementary to a target polynucleotide sequence to hybridize to the target sequence and direct the CRISPR complex to specifically bind to the sequence of the target sequence.
  • the degree of complementarity between the guide sequence and its corresponding target sequence is about or more than about 50%, 60%, 75%, 80% , 85%, 90%, 95%, 97.5%, 99%, or more.
  • Any suitable algorithm for aligning sequences can be used to determine the optimal alignment, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, Algorithms based on Burrows-Wheeler Transform (e.g. Burrows-Wheeler Aligner), ClustalW, ClustalX, BLAT, Novoalign (Novocraft Technology Corporation), ELAND (Emilion) Corporation (Illumina), San Diego, California), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).
  • Burrows-Wheeler Aligner e.g. Burrows-Wheeler Aligner
  • ClustalW ClustalX
  • BLAT Novoalign
  • ELAND Emilion Corporation
  • SOAP available at soap.genomics.org.cn
  • Maq available at maq.sourceforge.net.
  • the CRISPR enzyme is one or more heterologous protein domains (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more domains).
  • a CRISPR enzyme fusion protein may comprise any other protein, and optionally a linker sequence between any two domains.
  • protein domains that can be fused to CRISPR enzymes include, but are not limited to, epitope tags, reporter gene sequences, and one or more protein domains having the following activities: methylase activity, demethylase activity, Transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity.
  • Non-limiting examples of epitope tags include histidine (His) tag, V5 tag, FLAG tag, influenza virus hemagglutinin (HA) tag, Myc tag, VSV-G tag, and thioredoxin (Trx) tag .
  • reporter genes include, but are not limited to, glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), ⁇ -galactosidase, ⁇ -glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, green fluorescent protein (CFP), yellow fluorescent protein (YFP), spontaneous including blue fluorescent protein (BFP) Fluorescent protein.
  • GST glutathione-S-transferase
  • HRP horseradish peroxidase
  • CAT chloramphenicol acetyltransferase
  • CAT chloramphenicol acetyltransferase
  • CAT chloramphenicol acetyltransferase
  • ⁇ -galactosidase ⁇ -galactosidase
  • ⁇ -glucuronidase luciferase
  • CRISPR enzymes can be fused to a gene sequence encoding a protein or protein fragment that binds to a DNA molecule or to other cellular molecules, including, but not limited to, maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusion, a GAL4 DNA binding domain fusion, and a herpes simplex virus (HSV) BP16 protein fusion. Additional domains that can form part of a fusion protein comprising a CRISPR enzyme are described in US 20110059502, which is incorporated herein by reference.
  • Cas9 enzyme can be wild-type Cas9 or any modified version of Cas9, including any naturally occurring bacterial Cas9 as well as any chimera, mutant, homolog, or ortholog.
  • the Cas9 enzyme can contain one or more mutations and can be used as a universal DNA binding protein with or without fusion to a functional domain. These mutations can be artificially introduced mutations or acquired and lost functional mutations. These mutations may include, but are not limited to, mutations in one of the catalytic domains (D10 and H840) in the RuvC and HNH catalytic domains, respectively.
  • a Cas9 enzyme from NEB company can be used.
  • the function that Cas9 enzyme can achieve is that in a 30 ⁇ l reaction Cas9 enzyme reaction system (the reaction system includes: 20mM HEPES, 100mM NaCl, 5mM MgCl 2 , 0.1mM EDTA, and the pH is 6.5 at 25 ° C)
  • the reaction system includes: 20mM HEPES, 100mM NaCl, 5mM MgCl 2 , 0.1mM EDTA, and the pH is 6.5 at 25 ° C
  • 1nM PvuII linearized pBR322 DNA one target site CGCTTGTTTCGGCGTGGGTA
  • 40nM sgRNA and 20nM Cas9 enzyme 90% was confirmed by agarose gel electrophoresis under incubation at 37 ° C for 1 hour.
  • the pBR322 DNA was degraded.
  • the amount of Cas9 enzyme that catalyzes the complete conversion of 1nmol substrate (PvuII linearized pBR322 DNA) into the product in one minute is 0.37nmol, and the number of grams of Cas9 enzyme is 59.57ng.
  • the Cas9 enzyme had an enzyme activity of 0.37 nmol (the amount of enzyme that catalyzes the conversion of 1 nmol substrate to product in 1 minute).
  • the molar ratio of Cas9 enzyme to gRNA to be introduced is calculated based on the Cas9 enzyme activity mentioned above, and the concentration of Cas9 enzyme in the introduction complex is confirmed.
  • the activity of Cas9 enzyme changes At this time, those skilled in the art can perform conversion based on the ratios determined herein based on the descriptions of the activities in different enzyme specifications to select the use concentration of Cas9 enzyme and its molar ratio to gRNA.
  • the Cas enzyme is a nicking enzyme.
  • the Cas9 is delivered to the cell as an mRNA. This allows transient expression of the enzyme, thereby reducing toxicity.
  • Cas9 can also be delivered to cells in a nucleotide construct encoding and expressing the Cas9 enzyme. Alternatively, Cas9 can be expressed under the control of an inducible promoter.
  • CRISPR and Cas enzymes are generally used interchangeably herein unless otherwise stated.
  • many residue numbers used herein refer to the Cas9 enzyme from the type II CRISPR locus in Streptococcus pyogenes.
  • the present invention includes more Cas9 from other microbial species, such as SpCas9, SaCa9, St1Cas9, and the like.
  • sgRNA refers to short gRNA.
  • the administered gRNA, tracr pairing sequence, and tracr sequence may be administered separately, or a complete RNA sequence may be given.
  • Cas9 protein can bind to gRNA to cut DNA at specific sites.
  • the CRISPR / Cas system derived from Streptococcus pyogenes recognizes 23bp and can target 20bp.
  • the last 3 NGG sequences of its recognition site are called PAM ( protospacer (adjacent motif) sequence.
  • Cas enzyme CRISPR enzyme
  • CRISPR protein CRISPR protein
  • Cas protein CRISPRCas
  • Cas transgenes can be delivered by vectors (eg, AAV, adenovirus, lentivirus), and / or particles and / or nanoparticles, and / or electrotransformation.
  • vectors eg, AAV, adenovirus, lentivirus
  • exons of corresponding coding genes in the constant regions of one or both of the ⁇ and ⁇ chains of the TCR are knocked out using CRISPER / Cas technology, so that the endogenous TCR is inactive,
  • the first exon of the constant region of the endogenous TCR ⁇ chain is site-directed.
  • “Inhibiting” or “suppressing” the expression of B2M or TCR means that the expression of B2M or TCR in a cell is reduced by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, At least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100%.
  • inhibiting B2M expression refers to a reduction in B2M content in a cell by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50% , At least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%.
  • the expression or content of a protein in a cell can be determined by any suitable method known in the art, such as ELISA, immunohistochemistry, Western Blotting, or flow cytometry using B2M or TCR specific antibodies.
  • T cell receptor is a cell surface receptor involved in T cell activation in response to the presentation of an antigen.
  • TCR usually consists of two chains, ⁇ and ⁇ , which can be assembled to form a heterodimer and associated with the CD3 transduction subunit to form a T cell receptor complex present on the cell surface.
  • the alpha and beta chains of TCR are composed of immunoglobulin-like N-terminal variable (V) and constant (C) regions, hydrophobic transmembrane domains, and short cytoplasmic regions.
  • variable regions of the alpha and beta chains are produced recombinantly by V (D) J, resulting in a large number of diverse antigen specificities within a population of T cells.
  • T cells are activated by processed peptide fragments associated with MHC molecules, and T cells introduce additional dimensions into antigen recognition, called MHC restriction. Recognition of MHC differences between donors and recipients through T cell receptors leads to cell proliferation and potential development of GVHD. It has been shown that the normal surface expression of TCR relies on the synergistic synthesis and assembly of all seven components of the complex (Ashwell and Klusner 1990). Inactivation of TCR ⁇ or TCR ⁇ can lead to the elimination of TCR from the surface of T cells, preventing the recognition of alloantigens and the resulting GVHD.
  • MHC is a histocompatibility complex. It is a collective name for all gene groups encoding biocompatible complex antigens. MHC antigens are expressed in the tissues of all higher vertebrates, and are called HLA antigens in human cells. The response plays an important role, with rejection mediated by T cells that respond to histocompatibility antigens on the surface of the implanted tissue. MHC proteins play a vital role in T cell stimulation. Antigen-presenting cells (usually dendritic cells) display peptides that are degradation products of foreign proteins on the cell surface on MHC in the presence of co-stimulation signals T cells are activated and act on target cells that also display the same peptide / MHC complex.
  • stimulated T helper cells will target macrophages that display antigens that bind to their MHC, or cytotoxic T cells (CTL) will act on virus-infected cells that display foreign viral peptides.
  • MHC antigens are divided into NHC class I antigens and MHC class II antigens.
  • the class I HLA gene cluster includes three major loci, HLA-A, HLA-B, and HLA-C, and several minor loci.
  • Class II HLA clusters also include three major loci: HLA-DP, HLA-DQ, and HLA-DR,
  • HLA Human leukocyte antigen
  • HLA Human leukocyte antigen
  • MHC-I encoded for HLA-A, HLA-B, HLA-C sites
  • MHC-II encoded for HLA-D regions
  • Type II is mainly a glycoprotein located on the surface of macrophages and B lymphocytes.
  • B2M is ⁇ -2 microglobulin, also known as B2M, and is the light chain of MHC class I molecules.
  • B2M is encoded by the b2m gene located on chromosome 15 as opposed to other MHC genes located on chromosome 6 as a gene cluster.
  • a mouse model of ⁇ -2 microglobulin deficiency indicates that B2M is necessary for cell surface expression of MHC class I and stability of peptide-binding grooves.
  • the T cells provided by the present invention include T cells that inactivate or mutate one TCR gene and one HLA gene.
  • TCR is not active means that the endogenous TCR inactivates at least one subunit gene, especially the TCR ⁇ and / or TCR ⁇ gene, and more preferably the TCR ⁇ gene.
  • MHC is not active refers to an endogenous MHC gene that inactivates at least one subunit, particularly a gene that inactivates MHC I, and more preferably, a B2M gene.
  • T-cell antigen coupler (TELL, ANTIGEN, COUPLER, TAC)” includes three functional domains: tumor targeting domains, including single-chain antibodies, designed ankyrin repeat protein (DARPin) or Other targeting groups 2 are extracellular domain domains, single-chain antibodies that bind to CD3, so that the TAC receptor is close to other TCR receptors; the transmembrane region and the intracellular region of the CD4 co-receptor, where the cell
  • the internal domain of the protein kinase LCK catalyzes the phosphorylation of the immune receptor tyrosine activation motif (ITAMs) of the TCR complex as an initial step in T cell activation.
  • ITAMs immune receptor tyrosine activation motif
  • activation and “activation” are used interchangeably, and they and their grammatical other forms can refer to the process by which a cell changes from a resting state to an active state.
  • the process may include a response to a phenotypic or genetic change in the state of antigen, migration, and / or functional activity.
  • activation may refer to a process in which T cells are gradually activated.
  • T cells may require at least two signals to be fully activated. The first signal may occur after the TCR is joined by the antigen-MHC complex, and the second signal may occur through the engagement of a co-stimulatory molecule (see the co-stimulatory molecules listed in Table 1).
  • anti-CD3 can mimic the first signal and anti-CD28 can mimic the second signal.
  • engineered T cells can be activated by an expressed CAR.
  • T cell activation or T cell triggering as used herein may refer to the state of T cells that have been sufficiently stimulated to induce detectable cell proliferation, cytokine production, and / or detectable effector function.
  • chimeric receptor refers to a fusion molecule composed of DNA fragments of different sources or corresponding cDNAs of a protein using genetic recombination technology, and includes an extracellular domain, a transmembrane domain, and an intracellular domain.
  • Chimeric receptors include but are not limited to: chimeric antigen receptor (CAR), modified T cell (antigen) receptor (TCR), T cell fusion protein (TFP), T cell antigen coupler (TAC).
  • co-stimulatory ligand includes molecules on antigen-presenting cells (e.g., aAPC, dendritic cells, B cells, etc.) that specifically bind to an identical costimulatory molecule on a T cell, thereby providing a signal, such as by The first signal provided by the binding of the TCR / CD3 complex and the peptide-loaded MHC molecule together mediates T cell responses, including but not limited to proliferation, activation, differentiation, and the like.
  • antigen-presenting cells e.g., aAPC, dendritic cells, B cells, etc.
  • Co-stimulatory ligands can include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L) , Intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, binding Toll ligand receptor Agonist or antibody and a ligand that specifically binds B7-H3.
  • Co-stimulatory ligands also specifically include antibodies that specifically bind to co-stimulatory molecules present on T cells, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, and lymphocyte function.
  • co-stimulatory molecule refers to an identity-binding partner on a T cell that specifically binds to a co-stimulatory ligand, thereby mediating the T-cell's co-stimulatory response, such as, but not limited to, proliferation.
  • Co-stimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors.
  • co-stimulatory signal refers to a signal that, in combination with a cell-stimulating signal molecule, such as TCR / CD3, results in T cell proliferation and / or up- or down-regulation of key molecules.
  • CAR chimeric antigen receptor
  • immune cells including, but not limited to, T cells.
  • CAR is expressed in T cells and can redirect T cells to induce killing of target cells with specificity determined by artificial receptors.
  • the extracellular binding domain of CAR can be derived from a murine, humanized or fully human monoclonal antibody. When it is in an immune effector cell, the cell is given specificity for a target cell (usually a cancer cell) and has intracellular signal production.
  • a CAR typically includes at least one extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain"), which includes stimulating molecules derived from and / Or a costimulatory domain of a costimulatory molecule.
  • the polypeptide groups are adjacent to each other.
  • a polypeptide group includes a dimerization switch that can couple polypeptides to each other in the presence of a dimerization molecule, for example, can couple an antigen binding domain to an intracellular signaling domain.
  • the stimulatory molecule is a zeta chain that binds to a T cell receptor complex.
  • the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below.
  • the costimulatory molecule is selected from the costimulatory molecules described herein, such as 4-1BB (ie, CD137), CD27, and / or CD28.
  • the CAR includes a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule.
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and a functional signaling domain derived from a co-stimulatory molecule and functionality derived from a stimulatory molecule An intracellular signaling domain of a signaling domain.
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and two functional signalings derived from one or more costimulatory molecules.
  • signaling domain refers to the functional portion of a protein that functions by transmitting information within a cell, and is used to regulate a cell via a defined signaling pathway by generating a second messenger or by responding to such a messenger as an effector Of activity.
  • cell and other grammatical forms may refer to cells of human or non-human animal origin. Engineered cells can also refer to cells that express CAR.
  • transfection refers to the introduction of exogenous nucleic acid into a eukaryotic cell. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, Liposomal fusion, lipid transfection, protoplast fusion, retroviral infection, and biolistics.
  • stable transfection or “stable transfection” refers to the introduction and integration of exogenous nucleic acid, DNA or RNA into the genome of a transfected cell.
  • stable transfectant refers to a cell that stably integrates foreign DNA into genomic DNA.
  • nucleic acid molecule encoding refers to the order or sequence of deoxyribonucleotides along a DNA strand. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. Thus, the nucleic acid sequence encodes an amino acid sequence.
  • the term "individual” refers to any animal, such as a mammal or marsupial. Individuals of the invention include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and any kind of poultry.
  • non-human primates e.g., rhesus monkeys or other types of macaques
  • mice pigs, horses, donkeys, cattle, sheep, rats, and any kind of poultry.
  • peripheral blood mononuclear cells refers to cells with a single nucleus in peripheral blood, including lymphocytes, monocytes, and the like.
  • T cell activation or “T cell activation” and other grammatical forms thereof may refer to the state of a T cell that is sufficiently stimulated to induce detectable cell proliferation, cytokine production, and / or detectable effector function. In some cases, “full T cell activation” may be similar to triggering cytotoxicity of T cells.
  • Various assays known in the art can be used to measure T cell activation.
  • the assay may be an ELISA for measuring cytokine secretion, ELISPOT, a flow cytometry assay (CD107) for measuring intracellular cytokine expression, a flow cytometry assay for measuring proliferation, and for determining target cell elimination Cytotoxicity assay (51Cr release assay).
  • the assays typically use controls (non-engineered cells) and engineering cells (CAR T) to compare to determine the relative activation of engineered cells compared to controls.
  • the assay can be compared to engineered cells that are incubated or contacted with target cells that do not express the target antigen.
  • the comparison may be a comparison of GPC3-CART cells incubated with target cells that do not express GPC3.
  • sequence When used in reference to a nucleotide sequence, the term "sequence" and other grammatical forms thereof used herein may include DNA or RNA, and may be single-stranded or double-stranded. Nucleic acid sequences can be mutated. The nucleic acid sequence can be of any length.
  • an effective amount refers to an amount that provides a therapeutic or prophylactic benefit.
  • expression vector refers to a vector comprising a recombinant polynucleotide, which comprises an expression control sequence operably linked to a nucleotide sequence to be expressed.
  • Expression vectors contain sufficient cis-acting elements for expression; other elements for expression can be provided by host cells or in vitro expression systems.
  • Expression vectors include all those known in the art, such as cosmids, plasmids (eg, naked or contained in liposomes), and viruses (eg, lentivirus, retrovirus, adenovirus, and adeno-associated virus).
  • lentivirus refers to a genus of the retroviridae family. Retroviruses are unique among retroviruses in being able to infect non-dividing cells; they can deliver large amounts of genetic information to the DNA of the host cell, so they are one of the most effective methods of gene delivery vectors. HIV, SIV and FIV are all examples of lentiviruses. Lentivirus-derived vectors provide a means to achieve significant levels of gene transfer in vivo.
  • vector is a composition comprising an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
  • vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • vector includes autonomously replicating plasmids or viruses.
  • the term should also be interpreted to include non-plasmid and non-viral compounds, such as polylysine compounds, liposomes, and the like, that facilitate the transfer of nucleic acids into cells.
  • examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, and the like.
  • sequence identity determines the percent identity by comparing two best-matched sequences over a comparison window (e.g., at least 20 positions), where a portion of the polynucleotide or polypeptide sequence in the comparison window may contain Additions or deletions (ie gaps), e.g. 20% or less gaps (e.g. 5 to 15%, or 10 to 12) compared to the reference sequence (which does not contain additions or deletions) for the two sequences that best match %).
  • Additions or deletions ie gaps
  • e gaps e.g. 20% or less gaps (e.g. 5 to 15%, or 10 to 12) compared to the reference sequence (which does not contain additions or deletions) for the two sequences that best match %).
  • the percentage is usually calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to produce the number of correctly matched positions, dividing the number of correctly matched positions by the total number of positions in the reference sequence ( Window size) and multiply the result by 100 to produce a percentage of sequence identity.
  • exogenous refers to a nucleic acid molecule or polypeptide that does not have endogenous expression in a cell, or that the level of expression is insufficient to achieve the function it has when overexpressed.
  • exogenous includes recombinant nucleic acid molecules or polypeptides expressed within a cell, such as exogenous, heterologous, and overexpressed nucleic acid molecules and polypeptides.
  • a chimeric receptor of the invention is a chimeric antigen receptor.
  • CAR Chimeric Antigen Receptor
  • Chimeric antigen receptors typically contain (fine) extracellular antigen-binding regions.
  • the extracellular antigen binding region can be fully human. In other cases, extracellular antigen binding regions can be humanized. In other cases, the extracellular antigen binding region may be of mouse origin, or the chimera in the extracellular antigen binding region consists of amino acid sequences from at least two different animals. In some embodiments, the extracellular antigen binding region may be non-human.
  • a variety of antigen-binding regions can be designed. Non-limiting examples include a single chain variable fragment (scFv) derived from an antibody, a fragment antigen binding region (Fab) selected from a library, a single domain fragment, or a natural ligand associated with its cognate receptor.
  • scFv single chain variable fragment
  • Fab fragment antigen binding region
  • the extracellular antigen binding region may comprise scFv, Fab or a natural ligand, as well as any derivatives thereof.
  • An extracellular antigen-binding region may refer to a molecule other than an intact antibody, which may comprise a portion of an intact antibody and may bind to an antigen to which the intact antibody binds.
  • antibody fragments may include, but are not limited to, Fv, Fab, Fab ', Fab'-SH, F (ab') 2; bifunctional antibodies, linear antibodies; single chain antibody molecules (e.g., scFv); and those formed from antibody fragments Multispecific antibodies.
  • Extracellular antigen-binding regions such as scFv, Fab, or natural ligands, can be part of a CAR that determines antigen specificity.
  • the extracellular antigen-binding region can bind any complementary target.
  • the extracellular antigen binding region may be derived from an antibody of known variable region sequence.
  • Extracellular antigen-binding regions can be obtained from antibody sequences obtained from available mouse hybridomas. Alternatively, extracellular antigen-binding regions can be obtained from total excision sequencing of tumor cells or primary cells such as tumor infiltrating lymphocytes (TIL).
  • TIL tumor infiltrating lymphocytes
  • the binding specificity of an extracellular antigen-binding region can be determined by a complementarity determining region or CDR, such as a light chain CDR or a heavy chain CDR.
  • CDR complementarity determining region
  • binding specificity can be determined by light chain CDRs and heavy chain CDRs.
  • a given combination of heavy chain CDRs and light chain CDRs can provide a given binding pocket compared to other reference antigens, which can confer greater affinity and / or specificity to an antigen (eg, GPC3).
  • CDRs specific to phosphatidylinosin-3 can be expressed in the extracellular binding region of CAR, so that GPC3-targeting CAR can target T cells to tumor cells expressing GPC3.
  • an extracellular antigen binding region such as a scFv
  • a light chain CDR can comprise a light chain CDR specific for an antigen.
  • the light chain CDR may be the complementarity determining region of an scFv light chain of an antigen binding unit such as CAR.
  • a light chain CDR may comprise a sequence of consecutive amino acid residues, or two or more sequences of consecutive amino acid residues separated by non-complementarity determining regions (e.g., framework regions).
  • a light chain CDR may comprise two or more light chain CDRs, which may be referred to as light chain CDR-1, CDR-2, and the like.
  • a light chain CDR may comprise three light chain CDRs, which may be referred to as light chain CDR-1, light chain CDR-2, and light chain CDR-3, respectively.
  • a set of CDRs present on a common light chain may be collectively referred to as a light chain CDR.
  • an extracellular antigen binding region such as a scFv
  • an extracellular antigen binding region can comprise an antigen-specific heavy chain CDR.
  • the heavy chain CDR may be a heavy chain complementarity determining region of an antigen binding unit such as scFv.
  • the heavy chain CDR may comprise a continuous sequence of amino acid residues, or a continuous sequence of two or more amino acid residues separated by a non-complementarity determining region (eg, a framework region).
  • a heavy chain CDR may comprise two or more heavy chain CDRs, which may be referred to as heavy chain CDR-1, CDR-2, and the like.
  • a heavy chain CDR may include three heavy chain CDRs, which may be referred to as heavy chain CDR-1, heavy chain CDR-2, and heavy chain CDR-3, respectively.
  • a group of CDRs present on a common heavy chain may be collectively referred to as a heavy chain CDR.
  • extracellular antigen-binding regions can be modified in various ways.
  • the extracellular antigen-binding region can be mutated so that the extracellular antigen-binding region can be selected to have a higher affinity for its target.
  • the affinity of an extracellular antigen-binding region for its target can be optimized for a target that can be expressed at low levels on normal tissue. This optimization can be performed to minimize potential toxicity.
  • the cloning of an extracellular antigen-binding region that has a higher affinity for the membrane-bound form of the target may be superior to its soluble form counterpart. This modification can be made because different levels of soluble forms of the target can also be detected and their targeting can cause undesired toxicity.
  • the extracellular antigen-binding region includes a hinge or a spacer.
  • the terms hinge and spacer are used interchangeably.
  • the hinge can be thought of as part of a CAR that provides flexibility to extracellular antigen binding regions.
  • hinges can be used to detect CAR on the cell's cell surface, especially when antibodies that detect extracellular antigen-binding regions are ineffective or available.
  • the length of the hinge derived from an immunoglobulin may need to be optimized, depending on where the extracellular antigen-binding region targets the epitope on the target.
  • the hinge may not belong to the immunoglobulin, but belong to another molecule, such as the natural hinge of the CD8 ⁇ molecule.
  • the CD8 ⁇ hinge may contain cysteine and proline residues known to play a role in the interaction of CD8 co-receptors and MHC molecules.
  • the cysteine and proline residues can affect the performance of the CAR.
  • the CAR hinge can be adjustable in size. This morphology of the immune synapse between T cells and target cells also limits the distance that cannot be functionally bridged by CAR due to the epitope of the membrane on the cell surface of the target molecule, even using short hinge CAR cannot make the synapse The touch distance reaches an approximate value that the signal can conduct.
  • the hinge can be adjusted according to the extracellular antigen binding region used.
  • the hinge can be of any length.
  • a transmembrane domain can anchor CAR to the plasma membrane of a cell.
  • the natural transmembrane portion of CD28 can be used for CAR.
  • the natural transmembrane portion of CD8 ⁇ can also be used in CAR.
  • CD8 may be a protein that has at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to the NCBI reference number: NP_001759 or a fragment having stimulating activity.
  • the "CD8 nucleic acid molecule” may be a polynucleotide encoding a CD8 polypeptide.
  • the transmembrane region may be a natural transmembrane portion of CD28.
  • CD28 may refer to the NCBI reference number: NP_006130 or a stimulatory A fragment has a protein that is at least 85, 90, 95, 96, 97, 98, 99, or 100% identical.
  • a "CD28 nucleic acid molecule” may be a polynucleotide encoding a CD28 polypeptide.
  • the transmembrane portion may contain a CD8 ⁇ region.
  • the intracellular signal domain of the CAR may be responsible for activating at least one of the effector functions of the T cells in which the CAR has been placed.
  • CAR can induce effector functions of T cells, for example, the effector function is cytolytic activity or auxiliary activity, including secretion of cytokines.
  • the term "intracellular signal domain" refers to the portion of the protein that transduces effector function signals and directs cells to perform specific functions.
  • the entire intracellular signaling region can often be used, in many cases it is not necessary to use the entire chain of signal domains. In some cases, a truncated portion of the intracellular signaling region is used. In some cases, the term intracellular signaling domain is therefore intended to include any truncated portion of the intracellular signaling region sufficient to transduce effector functional signals.
  • Preferred examples of signaling domains used in CAR may include the cytoplasmic sequence of T cell receptors (TCRs) and co-receptors that act synergistically to initiate signal transduction after target-receptor binding, as well as any of their derivatives or Variant sequences and any synthetic sequences of these sequences that have the same functionality.
  • TCRs T cell receptors
  • co-receptors that act synergistically to initiate signal transduction after target-receptor binding
  • the intracellular signaling domain may contain a signal motif of a known immune receptor tyrosine activation motif (ITAM).
  • ITAM immune receptor tyrosine activation motif
  • Examples of ITAM containing cytoplasmic signaling sequences include functional signaling domains of proteins derived from DAP10, or DAP12 of TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, CD66d.
  • the intracellular signaling domain is derived from the CD3 ⁇ chain.
  • An example of a T cell signaling domain containing one or more ITAM motifs is the CD3 ⁇ domain, also known as the T cell receptor T3 ⁇ chain or CD247.
  • CD3 [zeta] refers primarily to human CD3 [zeta] and its isoforms, as known from the Swissprot entry P20963, and includes proteins with substantially the same sequence.
  • CD3 [zeta] refers primarily to human CD3 [zeta] and its isoforms, as known from the Swissprot entry P20963, and includes proteins with substantially the same sequence.
  • the chimeric antigen receptor it is reiterated that no full T cell receptor T3 ⁇ chain is required, and that any derivative that contains the signal domain of the T cell receptor T3 ⁇ chain is suitable, including any functional equivalents thereof .
  • the intracellular signaling domain may be selected from any one of the domains of Table 1.
  • the domain can be modified so that the identity to the reference domain can be about 50% to about 100%.
  • Any one of the domains of Table 1 may be modified such that the modified form may contain about 50, 60, 70, 80, 90, 95, 96, 97, 98, 99 or up to about 100% identity.
  • the intracellular signaling region of the CAR may further include one or more costimulatory domains.
  • the intracellular signaling region may contain a single co-stimulatory domain, such as the zeta chain (first generation CAR) or with CD28 or 4-1BB (second generation CAR).
  • the intracellular signaling region may comprise two costimulatory domains, such as CD28 / OX40 or CD28 / 4-1BB (third generation).
  • co-stimulatory domains can generate downstream activation of the kinase pathway, thereby supporting gene transcription and functional cellular responses.
  • the co-stimulatory domain of CAR can activate CD28 (phosphatidylinositol-4,5-bisphosphate 3-kinase) or 4-1BB / OX40 (TNF-receptor related factor adapter protein) pathways as well as MAPK and Akt activation Proximal signaling protein.
  • signals generated by CAR may be combined with auxiliary or co-stimulatory signals.
  • chimeric antigen receptor-like complexes can be designed to contain several possible co-stimulatory signal domains.
  • individual junctions of T cell receptors are not sufficient to induce complete activation of T cells into cytotoxic T cells.
  • Full production T cell activation requires a second costimulatory signal.
  • receptors have been reported to provide co-stimulation to T cell activation, including but not limited to CD28, OX40, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a / CD18), 4-1BBL, MyD88, and 4- 1BB.
  • the signal transduction pathways used by these costimulatory molecules all work synergistically with primary T cell receptor activation signals.
  • the signals provided by these co-stimulatory signaling regions can synergize with the main effect activation signals derived from one or more ITAM motifs (such as the CD3zeta signal transduction domain), and can fulfill the requirements of T cell activation.
  • adding a co-stimulatory domain to a chimeric antigen receptor-like complex can enhance the efficacy and durability of engineered cells.
  • the T cell signaling domain and the co-stimulatory domain are fused to each other to form a signaling region.
  • modulation refers to a positive or negative change. Examples of adjustments include 1%, 2%, 10%, 25%, 50%, 75%, or 100% changes.
  • treatment refers to a clinical intervention in an attempt to alter an individual or to treat a disease caused by a cell, both for prevention and for intervention in a clinical pathological process.
  • Therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, reducing symptoms, reducing the direct or indirect pathological consequences of any disease, preventing metastasis, slowing the progress of the disease, improving or alleviating the condition, and alleviating or improving the prognosis.
  • the T cell described herein refers to a T cell engineered by the method of the present invention, and the T cell has an endogenous TCR gene and / or MHC gene silenced.
  • the T cells can be dry memory TSCM cells consisting of CD45RO (-), CCR7 (+), CD45RA (+), CD62L + (L-selectin), CD27 +, CD28 +, and / or IL-7R ⁇ +
  • the stem memory cells can also express CD95, IL-2R ⁇ , CXCR3, and / or LFA-1, and show many functional attributes that are different from the stem memory cells.
  • the immunoreactive cells may also be central memory TCM cells comprising L-selectin and CCR7, wherein the central memory cells may secrete, for example, IL-2, but not IFN ⁇ or IL-4.
  • the immunoreactive cells may also be effector memory TEM cells containing L-selectin or CCR7, and produce, for example, effector cytokines such as IFN ⁇ and IL-4.
  • the delivery vehicle is typically delivered systemically (e.g., intravenously, intraperitoneally, intramuscularly, subcutaneously, or intracranially) by topical administration to an individual patient, as described below.
  • the vector may be delivered to cells ex vivo, such as cells removed from an individual patient (e.g., lymphocytes, T cells, bone marrow aspirate, tissue biopsy), and then the cells are typically re-selected after selecting cells that incorporate the vector. Implanted in the patient. Cells can be expanded before or after selection.
  • the T cells can be obtained from many sources, including PBMC, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, and tissue from infected sites, ascites, pleural effusion, spleen tissue, and tumors.
  • T cells can be obtained from blood collected from an individual using any number of techniques known to those skilled in the art, such as FicollTM separation.
  • cells from a subject's circulating blood are obtained by apheresis.
  • Apheresis products usually contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • the cells collected by apheresis harvesting can be washed to remove the plasma fraction and placed in a suitable buffer or medium for subsequent processing steps.
  • cells can be derived from a healthy donor, from a patient diagnosed with cancer.
  • the cells can be part of a mixed cell population with different phenotypic characteristics.
  • Cell lines can also be obtained from transformed T cells according to the aforementioned methods.
  • Cells can also be obtained from cell therapy libraries.
  • suitable primary cells include peripheral blood mononuclear cells (PBMC), peripheral blood lymphocytes (PBL), and other subpopulations of blood cells such as, but not limited to, T cells, natural killer cells, monocytes, natural Killer T cells, monocyte precursor cells, hematopoietic stem cells or non-pluripotent stem cells.
  • the cells can be any T cells such as tumor infiltrating cells (TIL), such as CD3 + T cells, CD4 + T cells, CD8 + T cells, or any other type of T cell.
  • T cells can also include memory T cells, memory stem T cells, or effector T cells.
  • T cells can also be selected from a large population, such as from whole blood. T cells can also be expanded from a large population.
  • T cells may also tend to specific populations and phenotypes.
  • T cells can be tilted to a phenotype comprising CD45RO (-), CCR7 (+), CD45RA (+), CD62L (+), CD27 (+), CD28 (+), and / or IL-7R ⁇ (+).
  • Suitable cells can be selected from one or more of the following lists: CD45RO (-), CCR7 (+), CD45RA (+), CD62L (+), CD27 (+), CD28 (+), and / or IL-7R ⁇ (+).
  • Suitable cells also include stem cells, such as, for example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neuronal stem cells, and mesenchymal stem cells.
  • Suitable cells may include any number of primary cells, such as human cells, non-human cells, and / or mouse cells.
  • Suitable cells may be progenitor cells.
  • Suitable cells may be derived from a subject (eg, a patient) to be treated.
  • the amount of therapeutically effective cells required in a patient may vary depending on the viability of the cells and the efficiency with which the cells are genetically modified (e.g., the efficiency with which a transgene is integrated into one or more cells, or the expression level of a protein encoded by the transgene ).
  • the genetically modified product of cell viability (eg, doubling) and the efficiency of transgene integration may correspond to the amount of treatment available to the cells of the subject.
  • an increase in cell viability after genetic modification may correspond to a decrease in the amount of necessary cells that is effective for the patient given the treatment.
  • an increase in the efficiency of integration of the transgene into one or more cells may correspond to a decrease in the number of cells necessary to administer a treatment effective in a patient.
  • determining the amount of therapeutically effective cells required may include determining functions related to changes in cells over time.
  • determining the amount of cells that need to be therapeutically effective can include determining functions that correspond to changes in the efficiency of integrating the transgene into one or more cells based on time-dependent variables (e.g., cell culture time, electroporation time, cells Stimulation time).
  • a therapeutically effective cell may be a population of cells that contains about 30% to about 100% expression of a chimeric receptor on the cell surface.
  • therapeutically effective cells can express the chimeric receptor on the cell surface for approximately 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or More than about 99.9%.
  • the T cells of the present invention can be used to prepare a pharmaceutical composition.
  • the pharmaceutical composition may further include a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means that when the molecular body and composition are properly administered to an animal or human, they do not cause adverse, allergic or other adverse reactions.
  • antioxidants include antioxidants; preservatives; pyrogen-free water; isotonic saline solutions; and phosphate buffers and the like.
  • composition of the present invention can be made into various dosage forms according to needs, and can be administered by a physician according to the patient's type, age, weight, general disease status, administration mode and other factors.
  • the method of administration may be, for example, parenteral administration (such as injection) or other treatment methods.
  • Parenteral administration of the composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.) or intrasternal injection or infusion techniques.
  • a T cell population-containing formulation administered to an individual comprises a plurality of T cells effective to treat and / or prevent a particular indication or disease.
  • a therapeutically effective population of immunoreactive cells can be administered to an individual.
  • a formulation comprising about 1 ⁇ 10 4 to about 1 ⁇ 10 10 immunoreactive cells is administered.
  • the formulation will contain about 1 ⁇ 10 5 to about 1 ⁇ 10 9 immunoreactive cells, about 5 ⁇ 10 5 to about 5 ⁇ 10 8 immunoreactive cells, or about 1 ⁇ 10 6 to About 1 ⁇ 10 7 immunoreactive cells.
  • the number of CAR immunoreactive cells administered to the individual will vary between a wide range. The doctor will ultimately determine the appropriate dose to use.
  • a chimeric antigen receptor is used to stimulate an immune cell-mediated immune response.
  • a T cell-mediated immune response is an immune response involving T cell activation.
  • Activated antigen-specific cytotoxic T cells are capable of inducing apoptosis in target cells that display foreign epitopes on the surface, such as cancer cells that display tumor antigens.
  • a chimeric antigen receptor is used to provide antitumor immunity in a mammal. Due to the T cell-mediated immune response, the subject will develop anti-tumor immunity.
  • a method of treating a subject with cancer may involve administering one or more T cells described herein to a subject in need of treatment.
  • the T cells can bind tumor target molecules and induce cancer cell death.
  • the invention also provides a method of treating a pathogen infection in an individual, comprising administering to said individual a therapeutically effective amount of a T cell of the invention.
  • the T cells of the invention can be administered in combination with another therapeutic agent.
  • the another therapeutic agent is a chemotherapeutic agent.
  • Chemotherapy drugs that can be used in combination with T cells of the present invention include, but are not limited to, mitotic inhibitors (vinca alkaloids), including vinblastine, vinblastine, vinblastine, and novibin (TM) (vinorelbine, 5'-dehydrosulfide); topoisomerase I inhibitors, such as camptothecin compounds, including CamptosarTM (Irinotecan HCL), HycamtinTM (Topotecan HCL) and derivatives derived from camptothecin and its analogs Other compounds; podophyllotoxin derivatives, such as etoposide, teniposide, and midazoxid; alkylating agents cisplatin, cyclophosphamide, nitrogen mustard, trimethylenethiophosphoramid, carmo Statin, busulfan, chlorambuci
  • anti-angiogenic agents including anti-VEGF antibodies (including humanized and chimeric antibodies, anti-VEGF aptamers, and antisense oligos Glycosides) and other angiogenesis inhibitors, such as tissue inhibitors of angiostatin, endostatin, interferon, retinoic acid, and metalloproteinases-1 and -2.
  • kits comprising a T cell of the invention.
  • the kit can be used to treat or prevent cancer, pathogen infection, immune disorder, or allograft.
  • a kit can include a therapeutic or prophylactic composition comprising an effective amount of T cells comprising one or more unit dosage forms.
  • the kit comprises a sterile container that can contain a therapeutic or prophylactic composition.
  • the kit can include about 1 ⁇ 10 4 cells to about 1 ⁇ 10 6 cells. In some cases, the kit can include at least about 1 ⁇ 10 5 cells, at least about 1 ⁇ 10 6 cells, at least about 1 ⁇ 10 7 cells, at least about 4 ⁇ 10 7 cells, at least about 5 ⁇ 10 7 cells, at least about 6 ⁇ 10 7 cells, at least about 6 ⁇ 10 7 cells, 8 ⁇ 10 7 cells, at least about 9 ⁇ 10 7 cells, at least about 1 ⁇ 10 8 cells, at least about 2 ⁇ 10 8 cells, at least about 3 ⁇ 10 8 cells, at least about 4 ⁇ 10 8 cells, at least about 5 ⁇ 10 8 cells, at least about 6 ⁇ 10 8 cells, at least about 6 ⁇ 10 8 cells, At least about 8 ⁇ 10 8 cells, at least about 9 ⁇ 10 8 cells, at least about 1 ⁇ 10 9 cells, at least about 2 ⁇ 10 9 cells, at least about 3 ⁇ 10 9 cells, at least about 4 ⁇ 10 9 Cells, at least about 5 ⁇ 10 8
  • the kit may include allogeneic cells. In some cases, the kit can include cells that can include genomic modifications. In some cases, the kit may contain "off-the-shelf" cells. In some cases, the kit can include cells that can be expanded for clinical use. In some cases, the kit may contain content for research purposes.
  • Gene editing according to the method of the present invention has not only high editing efficiency but also good cell survival rate.
  • T cells are selected to describe the method of the present invention.
  • PBMC Human peripheral blood mononuclear cells
  • TRAC T cell receptor ⁇ constant locus
  • SEQ ID NO: 1 the nucleotide sequence is shown in SEQ ID NO: 2
  • sg-TRAC-2 SEQ ID NO: 3
  • sg-TRAC-3 SEQ ID NO: 4
  • sg-TRAC-4 SEQ ID NO: 5
  • sg-TRAC-5 SEQ ID NO: 32
  • sg-TRAC-6 SEQ ID NO: 33
  • sg-TRAC-7 SEQ ID NO: 39
  • sg-TRAC-8 (SEQ ID NO: 40).
  • sg-TRAC-1 SEQ ID NO: 2
  • sg-TRAC-2 SEQ ID NO: 3
  • sg-TRAC-3 SEQ ID NO: 4
  • sg-TRAC-5 SEQ ID NO: 32
  • sg-TRAC-6 SEQ ID NO: 33
  • sg-TRAC-7 SEQ ID NO: 39
  • sg-TRAC-8 SEQ ID NO: 40
  • gRNA transcription kit purchased from Thermo Fisher
  • sg-TRAC-7 was transcribed and amplified
  • primers shown in SEQ ID NO: 43 and 44 were synthesized in vitro
  • in vitro gRNA transcription kit purchased from Thermo Fisher
  • transcription and Sg-TRAC-8 was amplified.
  • TRAC-exon 1 sequence (SEQ ID NO: 1):
  • sg-TRAC-1 (SEQ ID NO: 2): AGAGTCTCTCAGCTGGTACA
  • sg-TRAC-2 (SEQ ID NO: 3): TCTCTCAGCTGGTACACGGC
  • sg-TRAC-3 (SEQ ID NO: 4): GAGAATCAAAATCGGTGAAT
  • sg-TRAC-4 (SEQ ID NO: 5): CTCTCAGCTGGTACACGGCA
  • sg-TRAC-5 (SEQ ID NO: 32): GTCTCTCAGCTGGTACA
  • sg-TRAC-6 (SEQ ID NO: 33): AGTCTCTCAGCTGGTACA
  • sg-TRAC-7 (SEQ ID NO: 39): TTAGAGTCTCTCAGCTGGTACA
  • sg-TRAC-8 (SEQ ID NO: 40): TTTAGAGTCTCTCAGCTGGTACA
  • Example 2 Effect of different ratios of Cas9 enzyme and sg-TRAC on knockout efficiency
  • Sg-TRAC-1 SEQ ID NO: 2 was selected as the sgRNA.
  • a BTX electrorotator (Harvard, USA) was used to introduce the RNP complex into T cells.
  • the electrorotation parameters were 250V and 5ms.
  • T cells were taken for CD3 antibody (BD Biosciences) flow staining to verify the efficiency of TCR knockout.
  • Flow cytometry results are shown in Figure 2 and Table 1: When the molar ratio of Cas 9 enzyme to sg-TRAC-1 is 1: 3 to 1: 5, the knockout efficiency is more than 70%. When the molar ratio of Cas 9 enzyme and sg-TRAC-1 was 1: 4, the knockout efficiency was the highest, reaching 87.2%. It showed that when the molar ratio of Cas 9 enzyme and gRNA was 1: 4, it had the best gene knockout effect.
  • sgRNAs targeting the TRAC gene sg-TRAC-1, sg-TRAC-2, sg-TRAC-3,
  • TRAC gene knockout was examined.
  • Three sg-TRAC-1, sg-TRAC-2, and sg-TRAC-3 synthesized in Example 1 were mixed with Cas9 enzyme (0.5 ⁇ M) to form an RNP complex at a ratio of 4: 1, and a Maxcyte electrometer was used.
  • Maxcyte Electroporation was performed based on the parameters set by the instrument to introduce the RNP complex into T cells. On day 5 after transfection, T cells were taken for CD3 antibody (BD Biosciences) flow staining to verify the efficiency of TCR knockout. Flow cytometry results are shown in Figure 3 and Table 2.
  • the knockout effect of sg-TRAC-1 is significantly better than that of sg-TRAC-2 and sg-TRAC-3, indicating that sg-TRAC-1 has the best knockdown. In addition to the effect.
  • the effect of different lengths of sg-TRAC-1 on knockout efficiency was tested, and sg-TRAC-1 (-3bp) (sg-TRAC-5), sg-TRAC-1 (-2bp) (sg-TRAC- 6), four sgRNAs, sg-TRAC-1 (+ 3bp) (sg-TRAC-7), sg-TRAC-1 (+ 2bp) (sg-TRAC-8), and the Cas9 enzyme (0.5 ⁇ M) in a molar ratio
  • the RNP complex was formed at a ratio of 4: 1, and introduced into T cells under the above conditions.
  • T cells were taken for CD3 antibody flow staining to verify the efficiency of TCR knockout.
  • the experimental results are shown in Figure 3b.
  • the truncated 2 or 3 base sg-TRAC-1 has a small effect on the TCR knockout efficiency, and the addition of 2 or 3 bases will reduce the TCR knockout efficiency. It shows that the sgRNA length design for this site can be changed to a certain extent, especially the truncated bases within 3 can also achieve a relatively high knock-out effect.
  • sg-TRAC-1 (SEQ ID NO: 2) as the sgRNA.
  • the molar ratio of Cas 9 and sg-TRAC-1 is 1: 4
  • set different concentrations of Cas 9 (0.0625 ⁇ M, 0.125 ⁇ M, 0.25 ⁇ M, 0.5 ⁇ M), to detect the effect on TRAC gene knockout.
  • the RNP complex was incubated at room temperature for 10 minutes, the RNP complex was introduced into T cells using a Maxcyte electrorotator (Maxcyte) based on the conditions set by the instrument. On day 5 after transfection, T cells were taken for CD3 antibody (BD Biosciences) flow staining to verify the efficiency of TCR knockout.
  • Maxcyte Maxcyte electrorotator
  • the TCR knockout efficiency can reach more than 70%. For example, at 0.125 ⁇ M, the TCR knockout efficiency can reach 75.
  • the knockout efficiency of TCR can reach more than 90%, especially when it is larger than 0.2 ⁇ M.
  • the knockout efficiency of TCR can reach more than 94.5%; when the concentration of Cas9 enzyme is 0.3-0.5 ⁇ M, It can reach more than 95%.
  • the concentration of Cas9 enzyme is 0.5 ⁇ M
  • the knockout efficiency of TCR can reach 97.4%, and the cell survival rate is more than 90%.
  • the nucleotide sequence is shown in SEQ ID NO: 10, and four sgRNA sequences targeting the B2M gene sg-B2M-1 (SEQ ID ID NO : 11), sg-B2M-2 (SEQ ID NO: 12), sg-B2M-3 (SEQ ID NO: 13), sg-B2M-4 (SEQ ID NO: 14).
  • Sg-B2M-1, sg-B2M-2, sg-B2M-3 were selected for experiments, and the primers shown in SEQ ID NO: 26 and 27 were synthesized in vitro, and an in vitro gRNA transcription kit (purchased from Thermo Fisher) was used for transcription and amplification Sg-B2M-1 was added; primers shown in SEQ ID Nos: 28 and 29 were synthesized in vitro, and an in vitro gRNA transcription kit (purchased from Thermo Fisher) was used to transcribe and amplify sg-B2M-2; SEQ ID was synthesized in vitro : The primers shown in 30 and 31, in vitro gRNA transcription kit (purchased from Thermo Fisher), transcribed and amplified sg-B2M-3.
  • sg-B2M-1 (SEQ ID NO: 11): GGCCACGGAGCGAGACATCT
  • sg-B2M-2 (SEQ ID NO: 12): GAGTAGCGCGAGCACAGCTA
  • sg-B2M-3 (SEQ ID NO: 13): CGCGAGCACAGCTAAGGCCA
  • Example 6 Effects of different sgRNA sequences on B2M gene knockout.
  • the knock-out effect of sg-B2M-1 and sg-B2M-2 reached more than 90%, which was significantly better than sg-B2M-3, indicating that sg-B2M-1 and sg- B2M-2 has a good knockout effect.
  • gRNA sg-B2M-1 sg-B2M-2 sg-B2M-3 KO efficiency (Day 5) 95.0% 90.0% 67.0%
  • the knockout rate of B2M can be greatly improved, which can reach 95%.
  • the knock-out efficiency can reach more than 70%. For example, at 0.25 ⁇ M, the knock-out efficiency is 72.2%.
  • the concentration of Cas9 enzyme is not less than 1 ⁇ M
  • the knock-out efficiency can reach more than 90%. For example, it shows good knock-out efficiency at 1 ⁇ M-3 ⁇ M, especially at 1 ⁇ M-2 ⁇ M, the knock-out efficiency is about 93%.
  • Genomic DNA of TRAC, B2M single genes and two gene knockouts were extracted from T cells, and gene fragments containing knockout site fragments were amplified by PCR.
  • the PCR products were purified and recovered after gel electrophoresis, and then sequenced.
  • the sequencing results of the TRAC and B2M genes in the PCR products of the control group were single peaks. In the knockout group, the sequencing results of the TRAC and B2M genes would correspond. Multiple sets of peaks appeared, indicating that the TRAC and B2M genes were mutated.
  • TRAC B2M single gene and two knockout genomic DNAs were extracted from T cells, and PCR was used to amplify the gene fragments containing the knockout site fragments.
  • the PCR products were purified and recovered after gel electrophoresis, ligated to a T vector, transformed, and monoclonal colonies were randomly picked for sequencing and identification. As shown in Figure 11, the picked clone
  • the sequences in the knockout group showed deletion or insertion of bases, which indicated that the TCR and B2M genes were mutated.

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Abstract

提供了一种基于CRISPR/Cas系统对细胞进行基因编辑的方法，其中Cas酶为酶活力为0.1-1nmol的Cas9酶。还提供了一种构建通用型T细胞的方法，制备得到的T细胞及其用途，其中通过基因编辑技术对T细胞的TCR基因和MHC基因进行基因编辑。还提供了一种gRNA构建体。

Description

基于CRISPR/Cas系统对细胞进行基因编辑的方法 技术领域
本发明涉及基因编辑的方法。更具体地，涉及采用CRISPR/Cas系统对细胞进行基因编辑的方法。
背景技术
基因编辑包括通过缺失、插入、突变或置换特定的核酸序列改变基因组。CRISPR-Cas系统由规律成簇的间隔短回文重复序列(CRISPR)和所缔合的Cas蛋白构成。RNA-指导的Cas内切核酸酶以序列依赖性方式特异性地靶向和切割DNA(Jinek,M.等,“A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,”Science 337,816–821(2012)；Sternberg,S.H.等,“DNA interrogation by the CRISPR RNA-guided endonuclease Cas9,”Nature 507,62(2014))，并且已广泛用于在各种生物和模型系统中的基因编辑。
但基因编辑过程中依然存在着基因编辑效率不高的问题，如CRISPR-Cas9对T细胞进行编辑时，由于T细胞是一种终末分化的原代细胞，体外扩增的时间窗口有限，基因转染效率较低，如Clin Cancer Res；23(9)May 1,2017公开的单独对编码TCR受体的基因或者编码HLA蛋白的基因进行敲除的效率，最高能达到80％左右，而两者同时敲除的效率只有60％左右。
因此，如何快速，高效的对细胞进行基因敲除、或快速，高效的一次性敲除多个基因成为了该领域的难点。
发明内容
本发明的目的在于提供一种快速，高效的对细胞进行基因敲除，特别是能够快速，高效的一次性敲除多个基因的方法。
在本发明的第一方面，提供了一种基于CRISPR/Cas系统对细胞进行基因编辑的方法，将Cas酶和gRNA的复合物导入所述细胞中进行基因编辑，其中，所述复合物中Cas酶和gRNA的比例为1:3～1:5。
在一具体实施方式中，所述Cas酶为Cas9酶。
在一具体实施方式中，所述Cas9酶的酶活力为0.1～1nmol，优选为0.2～0.7nmol，进一步优选为0.3～0.5nmol，最优选为0.37nmol。
在一具体实施方式中，所述Cas酶为Cas9酶，在所述复合物中，Cas9酶和gRNA的摩尔比比例为1:1～1:10，优选为1:3～1:5，进一步优选为1:4。
在本发明中，例如可以使用来自NEB公司的Cas9酶，当然本领域技术人员可以选择具有相同或相似功能的其他Cas9酶。
在一具体实施方式中，所述Cas9酶可以实现的功能是，在30μl的反应的Cas9酶反应体系(该反应体系包括：20mM HEPES、100mM NaCl、5mM MgCl 2、0.1mM EDTA且在25℃时，pH为6.5)中，当含有1nM PvuII线性化的pBR322 DNA(一个目标位点CGCTTGTTTCGGCGTGGGTA)，40nM sgRNA和20nM Cas9酶的情况下，在37℃温育1小时的情况下，通过琼脂糖凝胶电泳确认有90％的pBR322 DNA被降解。在这个反应体系中，1分钟催化1nmol底物(PvuII线性化的pBR322 DNA)完全转化成产物的Cas9酶量为0.37nmol，Cas9酶克数为59.57ng。所述Cas9酶的酶活力为0.37nmol(1分钟催化1nmol底物转化成产物的酶量)。在本发明中，如果以NEB的酶为例，该酶的酶活力为0.37nmol。
本领域技术人员可以理解，在本文中是基于具有上述Cas9酶活性来计算Cas9酶和希望导入的gRNA的摩尔比比例，以及确认Cas9酶在导入复合物中的浓度，当Cas9酶的活性发生变化时，本领域技术人员可以基于不同的酶的说明书中对于活性的描述来基于本文确定的比例来进行换算以选取Cas9酶的使用浓度，及其与gRNA的摩尔比。
本领域技术人员也可以理解，上述酶的酶活力为0.37nmol的Cas酶仅仅是一个列举，对于其他Cas9酶，如果酶的酶活力与所述Cas酶不同时，本领域技术人员可以酶的酶活力来进行计算，以确认Cas9酶的使用量，及其与gRNA的摩尔比。
在一具体实施方式中，本发明涉及对两种基因进行编辑的方法，具体来说，将Cas9酶和第一gRNA的复合物一以及Cas9酶和第二gRNA的复合物二导入所述细胞中进行基因编辑。
在一具体实施方式中，将Cas9酶、第一gRNA和第二gRNA的复合物三同时导入所述细胞中进行基因编辑。
在一具体实施方式中，将复合物一和复合物二先后导入所述细胞中进行基因编辑。
在所述复合物一或复合物二或复合物三中，Cas9酶和gRNA的摩尔比比例为1:1～1:10，优选为1:3～1:5，进一步优选为1:4。
例如在复合物一Cas9酶和gRNA的摩尔比比例为1:1～1:10，优选为1:3～1:5，进一步优 选为1:4，在复合物二中Cas9酶和gRNA的摩尔比比例为1:1～1:10，优选为1:3～1:5，进一步优选为1:4。在复合物三中，Cas9酶和第一gRNA和第二gRNA总和的摩尔比比例为1:1～1:10，优选为1:3～1:5，进一步优选为1:4。
在本文中，所述摩尔比是指Cas9酶和gRNA物质的量之间的比例，其中Cas9酶的量或者酶活力是基于制造商提供的Cas9酶的说明书计算得出，而对应gRNA的量按照RNA碱基组成以及体外转录的浓度计算得出。
在一具体实施方式中，所述Cas酶和gRNA的比例为1:4。
在一具体实施方式中，所述细胞为真核细胞；在具体实施方式中，所述真核细胞是免疫效应细胞；在具体实施方式中，所述免疫效应细胞是T细胞。
在一具体实施方式中，所述Cas酶和gRNA的复合物中，所述Cas酶的浓度约为0.1μM～3μM；优选的，约为0.125μM～3μM；更优选的，约为0.2μM～3μM；更优选的，约为0.25μM～3μM；更优选的，约为0.5μM～3μM。
在一具体实施方式中，在所述Cas9酶和gRNA形成的复合物或复合物一或复合物二或复合物三中，所述Cas9酶的浓度约为0.1μM～3μM；优选的，约为0.125μM～3μM；更优选的，约为0.2μM～3μM；更优选的，约为0.25μM～3μM；更优选的，约为0.5μM～3μM。
在一具体实施方式中，所述Cas酶和gRNA的复合物中，所述Cas酶的浓度为约0.1μM～2μM；优选的，约为0.125μM～2μM；更优选的，约为0.5μM～2μM；更优选的，约为0.5μM～2μM；更优选的，约为0.5μM～2μM。
在具体实施方式中，所述细胞是T细胞，所述CRISPR/Cas系统对所述T细胞的基因进行编辑；在一具体实施方式中，对所述T细胞的TCR的α链和β链中的任意一条或者两条链的基因进行基因编辑；在具体实施方式中，对TRAC进行基因编辑；在具体实施方式中，对TRAC的恒定区进行基因编辑；在具体实施方式中，对TRAC中包含有SEQ ID NO：1所示的序列进行基因编辑。
在具体实施方式中，所述细胞为T细胞，利用所述CRISPR/Cas9系统对所述T细胞的基因进行编辑；包括：
利用所述CRISPR/Cas9系统对所述T细胞的TCR的α链和β链中的任意一条或者两条链的基因进行基因编辑；优选对TRAC进行基因编辑；进一步优选对TRAC的恒定区进行基因编辑；进一步优选对TRAC中如SEQ ID NO：45所示的序列进行基因编辑；进一步优选对TRAC中包含有SEQ ID NO：1所示的序列进行基因编辑，和/或
利用所述CRISPR/Cas9系统对所述T细胞的MHC基因进行基因编辑，优选对B2M基因进行基因编辑，进一步优选对B2M基因中如SEQ ID NO：38所示的序列进行基因编辑，进 一步优选对B2M基因中包含有SEQ ID NO：10所示的序列进行基因编辑。
在一具体实施方式中，依据SEQ ID NO：1所示的序列中的PAM序列，来设计gRNA。
在一具体实施方式中，所述gRNA约为15-50bp，优选的，约为15-30bp，更优选的为约17-21bp；更优选为20bp。
在一具体实施方式中，对TRAC的进行编辑采用的gRNA为包含有SEQ ID NO：2、3、4、或5所示的序列；优选的，采用的gRNA为包含有SEQ ID NO：2所示的序列。
在一具体实施方式中，对TRAC的进行编辑采用的gRNA为包含有SEQ ID NO：2、3、4、5、32、33、39或40所示的序列；优选的，采用的gRNA为包含有SEQ ID NO：2、32或33所示的序列。
在一具体实施方式中，对TRAC的进行编辑采用的gRNA为如SEQ ID NO：2、3、4、5、32、33、39或40所示的序列；优选的，采用的gRNA为如SEQ ID NO：2、32或33所示的序列。
具体来说，上述第一gRNA可以是包含有SEQ ID NO：2、3、4、5、32、33、39或40所示的序列。
在一具体实施方式中，所述的Cas酶的浓度约为0.1μM～0.5μM；优选的，约为0.125μM～0.5μM，更优选的，约为0.25μM～0.5μM。
在具体实施方式中，所述细胞是T细胞，所述CRISPR/Cas系统对所述T细胞的B2M基因进行基因编辑；在具体实施方式中，对B2M基因中包含有SEQ ID NO：10所示的序列进行基因编辑；在具体实施方式中，依据SEQ ID NO：10所示的序列中的PAM序列，来设计gRNA。
在一具体实施方式中，对B2M基因进行编辑采用的gRNA包含有SEQ ID NO：11、12、13、或14所示的序列；优选的，采用的gRNA包含有SEQ ID NO：12所示的序列。
在一具体实施方式中，对B2M基因进行编辑采用的gRNA为如SEQ ID NO：11、12、13、或14所示的序列；优选的，采用的gRNA为如SEQ ID NO：12所示的序列。
具体来说，上述第二gRNA可以是包含有SEQ ID NO：11、12、13、或14所示的序列。
下文中，针对复合物一、复合物二或复合物三的描述与上文一致，针对第一gRNA和第二gRNA的描述也与上文一致，应当理解复合物、复合物一、复合物二或复合物三意在表示不同的复合物，对其编号没有任何优先顺序，第一gRNA和第二gRNA也是同样，意在表示两种不同的gRNA，也可以用一种gRNA和另一种gRNA表示，即一种gRNA可以是包含有SEQ ID NO：2、3、4、5、32、33、39或40所示的序列，另一种gRNA可以是包 含有SEQ ID NO：11、12、13、或14所示的序列。
在一具体实施方式中，所述的Cas酶的浓度约为0.25μM～3μM，优选的约为0.5μM～3μM，更优选的约为1μM～3μM。
在具体实施方式中，所述细胞是T细胞，所述CRISPR/Cas系统对所述T细胞的TRAC和B2M基因进行基因编辑；在具体实施方式中，对TRAC和B2M基因的第一外显子进行基因编辑。
在一具体实施方式中，TRAC和/或B2M基因进行基因编辑，TRAC和/或B2M基因被沉默。
在一具体实施方式中，对TRAC的进行编辑采用的gRNA包含有SEQ ID NO：2、3、4、或5所示的序列，对B2M基因进行编辑采用的gRNA包含有SEQ ID NO：11、12、13、或14所示的序列；优选的，对TRAC的进行编辑采用的gRNA包含有SEQ ID NO：2所示的序列，对B2M基因进行编辑采用的gRNA包含有SEQ ID NO：12所示的序列。
在具体实施方式中，所述gRNA约为15-50bp，优选的，约为15-30bp，更优选的为约20bp；在一具体实施方式中，为20bp。
在一具体实施方式中，对TRAC和B2M基因进行基因编辑时，采用的编辑B2M的gRNA和编辑TRAC的gRNA的比例约为1.5:1～0.5:1；优选的，约为1:1。在一具体实施方式中，所述的Cas酶的浓度约为1μM～3μM。
在具体实施方式中，所述的T细胞还表达有嵌合受体、外源性的细胞因子、抑制性/激活性受体或配体，共刺激因子；在具体实施方式中，所述T细胞还表达有嵌合抗原受体受体。
在本发明的第二方面，提供了一种基于CRISPR/Cas系统对T细胞的TRAC基因进行基因编辑的方法，将Cas酶和gRNA的复合物导入所述细胞中进行基因编辑，其中，Cas酶和gRNA的比例为1:3～1:5；在一具体实施方式中，所述Cas酶为Cas9酶。
在具体实施方式中，对所述T细胞的TCR的α链和β链中的任意一条或者两条链的基因进行基因编辑；在具体实施方式中，对所述T细胞的TRAC进行基因编辑；在具体实施方式中，对所述T细胞的TRAC的恒定区进行基因编辑；在具体实施方式中，对所述T细胞的TRAC中的包含有SEQ ID NO：1所示的序列进行基因编辑；在具体实施方式中，依据SEQ ID NO：1所示的序列中的PAM序列，来设计gRNA。
在具体实施方式中，所述Cas酶和gRNA的比例为1:4。
在具体实施方式中，所述Cas酶的浓度约为0.1μM～0.5μM；优选的，约为0.125μM～0.5μM，更优选的，约为0.25μM～0.5μM。
在具体实施方式中，对TRAC的进行编辑采用的gRNA包含有SEQ ID NO：2、3、4、或5所示的序列；优选的，采用的gRNA为包含有SEQ ID NO：2所示的序列。
在具体实施方式中，对TRAC的进行编辑时，所述Cas酶和gRNA的比例为1:4；所述Cas酶的浓度为0.25μM～0.5μM；采用的gRNA为包含有SEQ ID NO：2所示的序列。
在本发明的第三方面，提供了一种基于CRISPR/Cas系统对T细胞的B2M基因进行基因编辑的方法，将Cas酶和gRNA的复合物导入所述细胞中进行基因编辑，其中，Cas酶和gRNA的比例为1:3～1:5；在一具体实施方式中，所述Cas酶为Cas9酶。
在具体实施方式中，对B2M基因中包含有SEQ ID NO：10所示的序列进行基因编辑。
在具体实施方式中，依据SEQ ID NO：10所示的序列中的PAM序列，来设计gRNA。在具体实施方式中，所述Cas酶和gRNA的比例为1:4。
在具体实施方式中，所述的Cas酶的浓度约为0.25μM～3μM，优选的，约为0.5μM～3μM，更优选的，约为1μM～3μM。
在具体实施方式中，对B2M基因进行编辑采用的gRNA包含有SEQ ID NO：11、12、13、或14所示的序列；优选的，采用的gRNA包含有SEQ ID NO：12所示的序列。
在具体实施方式中，对B2M基因的进行编辑时，所述Cas酶和gRNA的比例为1:4；所述Cas酶的浓度为1μM～3μM；采用的gRNA为包含有SEQ ID NO：12所示的序列。
在本发明的第四方面，提供了一种基于CRISPR/Cas系统对T细胞的TRAC基因和B2M基因进行基因编辑的方法，将Cas酶和gRNA的复合物导入所述细胞中，其中，Cas酶和总gRNA的比例为1:3～1:5；在一具体实施方式中，所述Cas酶为Cas9酶。
在具体实施方式中，对B2M基因中包含有SEQ ID NO：10所示的序列进行基因编辑；在具体实施方式中，在具体实施方式中，依据SEQ ID NO：10所示的序列中的PAM序列，来设计gRNA。
在具体实施方式中，对TCR的α链和β链中的任意一条或者两条链进行基因编辑；在具体实施方式中，对TRAC进行基因编辑；
在具体实施方式中，对TRAC的恒定区进行基因编辑；
在具体实施方式中，对TRAC中的包含有SEQ ID NO：1所示的序列进行基因编辑；在具体实施方式中，在具体实施方式中，依据SEQ ID NO：1所示的序列中的PAM序列，来设计gRNA。
在具体实施方式中，所述Cas酶和总gRNA的比例为1:4。在具体实施方式中，所述Cas酶的浓度为1μM～3μM。
在具体实施方式中，编辑B2M基因和编辑TRAC采用的gRNA的比例是0.5:1～1.5:1，优选的，为1:1。
在具体实施方式中，对B2M基因进行编辑采用的gRNA包含有SEQ ID NO：11、12、13、或14所示的序列；优选的，采用的gRNA包含有SEQ ID NO：12所示的序列。在具体实施方式中，对TRAC的进行编辑采用的gRNA包含有SEQ ID NO：2、3、4、或5所示的序列；优选的，采用的gRNA为包含有SEQ ID NO：2所示的序列。
在具体实施方式中，所述Cas酶和总gRNA的比例为1:4；所述Cas酶的浓度为1μM～3μM；采用的gRNA为包含有SEQ ID NO：12所示的序列和包含有SEQ ID NO：2所示的序列。
在具体实施方式中，上述第二方面、第三方面、第四方面所述的T细胞还表达有识别肿瘤抗原或病原体抗原的嵌合受体，该嵌合受体具有胞外抗原结合域、跨膜域、和胞内域，所述胞外抗原结合域特异性识别靶抗原。
在具体实施方式中，所述靶抗原为肿瘤抗原，该肿瘤抗原选自：促甲状腺激素受体(TSHR)；CD171；CS-1；C型凝集素样分子-1；神经节苷脂GD3；Tn抗原；CD19；CD20；CD 22；CD 30；CD 70；CD 123；CD 138；CD33；CD44；CD44v7/8；CD38；CD44v6；B7H3(CD276)，B7H6；KIT(CD117)；白介素13受体亚单位α(IL-13Rα)；白介素11受体α(IL-11Rα)；前列腺干细胞抗原(PSCA)；前列腺特异性膜抗原(PSMA)；癌胚抗原(CEA)；NY-ESO-1；HIV-1Gag；MART-1；gp100；酪氨酸酶；间皮素；EpCAM；蛋白酶丝氨酸21(PRSS21)；血管内皮生长因子受体；路易斯(Y)抗原；CD24；血小板衍生生长因子受体β(PDGFR-β)；阶段特异性胚胎抗原-4(SSEA-4)；细胞表面相关的粘蛋白1(MUC1)，MUC6；表皮生长20因子受体家族及其突变体(EGFR，EGFR2，ERBB3，ERBB4，EGFRvIII)；神经细胞粘附分子(NCAM)；碳酸酐酶IX(CAIX)；LMP2；肝配蛋白A型受体2(EphA2)；岩藻糖基GM1；唾液酸基路易斯粘附分子(sLe)；邻乙酰基GD2神经节苷脂(OAcGD2)；神经节苷脂GM3(aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer；TGS5；高分子量黑素瘤相关抗原(HMWMAA)；叶酸受体；肿瘤血管内皮标记25 1(TEM1/CD248)；肿瘤血管内皮标记7相关的(TEM7R)；Claudin6、Claudin18.2(CLD18A2)、Claudin18.1；ASGPR1；CDH16；5T4；8H9；αvβ6整合素；B细胞成熟抗原(BCMA；CA9；κ轻链(kappa light chain)；CSPG4；EGP2，EGP40；FAP；FAR；FBP；胚胎型AchR；HLA-A1，HLA-A2；MAGEA1，MAGE3；KDR；MCSP；NKG2D配体；PSC1；ROR1；Sp17；SURVIVIN；TAG72；TEM1；纤连蛋白；腱生蛋白；肿瘤坏死区的癌胚变体；G蛋白偶 联受体C类5组-成员D(GPRC5D)；X染色体开放阅读框61(CXORF61)；CD97；CD179a；间变性淋巴瘤激酶(ALK)；聚唾液酸；胎盘特异性1(PLAC1)；globoH glycoceramide的己糖部分(GloboH)；乳腺分化抗原(NY-BR-1)；uroplakin 2(UPK2)；甲型肝炎病毒细胞受体1(HAVCR1)；肾上腺素受5体β3(ADRB3)；pannexin 3(PANX3)；G蛋白偶联受体20(GPR20)；淋巴细胞抗原6复合物基因座K9(LY6K)；嗅觉受体51E2(OR51E2)；TCRγ交替阅读框蛋白(TARP)；肾母细胞瘤蛋白(WT1)；ETS易位变异基因6(ETV6-AML)；精子蛋白17(SPA17)；X抗原家族成员1A(XAGE1)；血管生成素结合细胞表面受体2(Tie2)；黑素瘤癌睾丸抗原-1(MAD-CT-1)；黑素瘤癌睾丸抗原-2(MAD-CT-2)；Fos相关抗原1；p53突变10体；人端粒酶逆转录酶(hTERT)；肉瘤易位断点；细胞凋亡的黑素瘤抑制剂(ML-IAP)；ERG(跨膜蛋白酶丝氨酸2(TMPRSS2)ETS融合基因)；N-乙酰葡糖胺基转移酶V(NA17)；配对盒蛋白Pax-3(PAX3)；雄激素受体；细胞周期蛋白B1；V-myc鸟髓细胞瘤病病毒癌基因神经母细胞瘤衍生的同源物(MYCN)；Ras同源物家族成员C(RhoC)；细胞色素P4501B1(CYP1B1)；CCCTC结合因子(锌指蛋白)样(BORIS)；由T细胞识别的鳞状细胞癌抗原3(SART3)；配对盒蛋白Pax-5(PAX5)；proacrosin结合蛋白sp32(OYTES1)；淋巴细胞特异性蛋白酪氨酸激酶(LCK)；A激酶锚定蛋白4(AKAP-4)；滑膜肉瘤X断点2(SSX2)；CD79a；CD79b；CD72；白细胞相关免疫球蛋白样受体1(LAIR1)；IgA受体的Fc片段(FCAR)；白细胞免疫球蛋白样受体亚家族成员2(LILRA2)；CD300分子样家族成员f(CD300LF)；C型凝集素结构域家族12成员A(CLEC12A)；骨髓基质细胞抗原2(BST2)；含有EGF样模块粘蛋白样激素受体样2(EMR2)；淋巴细胞抗原75(LY75)；磷脂酰肌醇蛋白聚糖-3(GPC3)；Fc受体样5(FCRL5)；免疫球蛋白λ样多肽1(IGLL1)；
在具体实施方式中，所述靶抗原为病原体抗原，该病原体抗原选自：病毒、细菌、真菌、原生动物、或寄生虫的抗原；在一实施例中，所述病毒抗原选自：巨细胞病毒抗原、爱泼斯坦-巴尔病毒抗原、人类免疫缺陷病毒抗原或流感病毒抗原。
在具体实施方式中，所述嵌合受体选自嵌合抗原受体(CAR)或T细胞抗原耦合器(TAC)。
在具体实施方式中，所述嵌合受体为嵌合抗原受体。在具体实施方式中，所述嵌合抗原受体包括：
(i)特异性结合肿瘤抗原的抗体、CD28或者CD8的跨膜区、CD28的共刺激信号结构域、和CD3ζ；或
(ii)特异性结合肿瘤抗原的抗体、CD28或者CD8的跨膜区、CD137的共刺激信号结构域、和CD3ζ；或
(iii)特异性结合肿瘤抗原的抗体、CD28或者CD8的跨膜区、CD28的共刺激信号结构域、CD137的共刺激信号结构域、和CD3ζ。
在具体实施方式中，所述嵌合受体为TAC，包括：
(a)胞外结构域:所述胞外结构域包括具有抗原结合结构域的抗体结构域，和与CD3结合的单链抗体；
(b)跨膜区；
(c)胞内结构域，所述胞内结构域连接蛋白激酶LCK。
在具体实施方式中，所述嵌合抗原受体的特异性结合肿瘤抗原的抗体为全长抗体、scFv、Fab、(Fab’)、或单域抗体。
在本发明的第五方面，提供了上述第二方面、第三方面、第四方面所述的T细胞的用途，用于制备表达有嵌合受体的T细胞，所述嵌合受体具有胞外抗原结合域、跨膜域、和胞内域，所述胞外抗原结合域特异性识别靶抗原。
在具体实施方式中，所述靶抗原为肿瘤抗原或病原体抗原。
在具体实施方式中，所述靶抗原为肿瘤抗原，该肿瘤抗原选自：促甲状腺激素受体(TSHR)；CD171；CS-1；C型凝集素样分子-1；神经节苷脂GD3；Tn抗原；CD19；CD20；CD 22；CD 30；CD 70；CD 123；CD 138；CD33；CD44；CD44v7/8；CD38；CD44v6；B7H3(CD276)，B7H6；KIT(CD117)；白介素13受体亚单位α(IL-13Rα)；白介素11受体α(IL-11Rα)；前列腺干细胞抗原(PSCA)；前列腺特异性膜抗原(PSMA)；癌胚抗原(CEA)；NY-ESO-1；HIV-1Gag；MART-1；gp100；酪氨酸酶；间皮素；EpCAM；蛋白酶丝氨酸21(PRSS21)；血管内皮生长因子受体；路易斯(Y)抗原；CD24；血小板衍生生长因子受体β(PDGFR-β)；阶段特异性胚胎抗原-4(SSEA-4)；细胞表面相关的粘蛋白1(MUC1)，MUC6；表皮生长20因子受体家族及其突变体(EGFR，EGFR2，ERBB3，ERBB4，EGFRvIII)；神经细胞粘附分子(NCAM)；碳酸酐酶IX(CAIX)；LMP2；肝配蛋白A型受体2(EphA2)；岩藻糖基GM1；唾液酸基路易斯粘附分子(sLe)；邻乙酰基GD2神经节苷脂(OAcGD2)；神经节苷脂GM3(aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer；TGS5；高分子量黑素瘤相关抗原(HMWMAA)；叶酸受体；肿瘤血管内皮标记25 1(TEM1/CD248)；肿瘤血管内皮标记7相关的(TEM7R)；Claudin6、Claudin18.2(CLD18A2)、Claudin18.1；ASGPR1；CDH16；5T4；8H9；αvβ6整合素；B细胞成熟抗原(BCMA)；CA9；κ轻链(kappa light chain)；CSPG4；EGP2，EGP40；FAP；FAR；FBP；胚胎型AchR；HLA-A1，HLA-A2；MAGEA1，MAGE3；KDR；MCSP；NKG2D配体；PSC1；ROR1；Sp17；SURVIVIN； TAG72；TEM1；纤连蛋白；腱生蛋白；肿瘤坏死区的癌胚变体；G蛋白偶联受体C类5组-成员D(GPRC5D)；X染色体开放阅读框61(CXORF61)；CD97；CD179a；间变性淋巴瘤激酶(ALK)；聚唾液酸；胎盘特异性1(PLAC1)；globoH glycoceramide的己糖部分(GloboH)；乳腺分化抗原(NY-BR-1)；uroplakin 2(UPK2)；甲型肝炎病毒细胞受体1(HAVCR1)；肾上腺素受5体β3(ADRB3)；pannexin3(PANX3)；G蛋白偶联受体20(GPR20)；淋巴细胞抗原6复合物基因座K9(LY6K)；嗅觉受体51E2(OR51E2)；TCRγ交替阅读框蛋白(TARP)；肾母细胞瘤蛋白(WT1)；ETS易位变异基因6(ETV6-AML)；精子蛋白17(SPA17)；X抗原家族成员1A(XAGE1)；血管生成素结合细胞表面受体2(Tie2)；黑素瘤癌睾丸抗原-1(MAD-CT-1)；黑素瘤癌睾丸抗原-2(MAD-CT-2)；Fos相关抗原1；p53突变10体；人端粒酶逆转录酶(hTERT)；肉瘤易位断点；细胞凋亡的黑素瘤抑制剂(ML-IAP)；ERG(跨膜蛋白酶丝氨酸2(TMPRSS2)ETS融合基因)；N-乙酰葡糖胺基转移酶V(NA17)；配对盒蛋白Pax-3(PAX3)；雄激素受体；细胞周期蛋白B1；V-myc鸟髓细胞瘤病病毒癌基因神经母细胞瘤衍生的同源物(MYCN)；Ras同源物家族成员C(RhoC)；细胞色素P4501B1(CYP1B1)；CCCTC结合因子(锌指蛋白)样(BORIS)；由T细胞识别的鳞状细胞癌抗原3(SART3)；配对盒蛋白Pax-5(PAX5)；proacrosin结合蛋白sp32(OYTES1)；淋巴细胞特异性蛋白酪氨酸激酶(LCK)；A激酶锚定蛋白4(AKAP-4)；滑膜肉瘤X断点2(SSX2)；CD79a；CD79b；CD72；白细胞相关免疫球蛋白样受体1(LAIR1)；IgA受体的Fc片段(FCAR)；白细胞免疫球蛋白样受体亚家族成员2(LILRA2)；CD300分子样家族成员f(CD300LF)；C型凝集素结构域家族12成员A(CLEC12A)；骨髓基质细胞抗原2(BST2)；含有EGF样模块粘蛋白样激素受体样2(EMR2)；淋巴细胞抗原75(LY75)；磷脂酰肌醇蛋白聚糖-3(GPC3)；Fc受体样5(FCRL5)；免疫球蛋白λ样多肽1(IGLL1)；
在具体实施方式中，所述靶抗原为病原体抗原，该病原体抗原选自：病毒、细菌、真菌、原生动物、或寄生虫的抗原；在一实施例中，所述病毒抗原选自：巨细胞病毒抗原、爱泼斯坦-巴尔病毒抗原、人类免疫缺陷病毒抗原或流感病毒抗原。
在具体实施方式中，所述嵌合受体选自嵌合抗原受体(CAR)或T细胞抗原耦合器(TAC)。
在具体实施方式中，所述嵌合受体为嵌合抗原受体(CAR)；
在具体实施方式中，所述嵌合抗原受体包括：
(i)特异性结合肿瘤抗原的抗体、CD28或者CD8的跨膜区、CD28的共刺激信号结构域、和CD3ζ；或
(ii)特异性结合肿瘤抗原的抗体、CD28或者CD8的跨膜区、CD137的共刺激信号 结构域、和CD3ζ；或
(iii)特异性结合肿瘤抗原的抗体、CD28或者CD8的跨膜区、CD28的共刺激信号结构域、CD137的共刺激信号结构域、和CD3ζ。
在具体实施方式中，所述嵌合受体为TAC，包括：
(a)胞外结构域:所述胞外结构域包括具有抗原结合结构域的抗体结构域，和与CD3结合的单链抗体；
(b)跨膜区；
(c)胞内结构域，所述胞内结构域连接蛋白激酶LCK。
在具体实施方式中，所述嵌合抗原受体的特异性结合肿瘤抗原的抗体为全长抗体、scFv、Fab、(Fab’)、或单域抗体。
在本发明中，对于电转条件没有具体限定，例如所述电转条件例如可以为150-600V，0.5ms-20ms，例如可以优选为150V-300V，2ms-15ms。
在一具体的实施方式中，对TCR基因进行基因编辑gRNA和对MHC基因进行基因编辑的gRNA的摩尔比比例约为1:5～5:1，优选为1:2～2:1；进一步优选约为1:1。
在一具体的实施方式中，所述T细胞如上述方面所示。
在一具体的实施方式中，所述嵌合受体为嵌合抗原受体(CAR)，所述嵌合抗原受体如上述方面所示。
在本发明的第七方面，涉及一种通用型T细胞，其是通过上述本发明的方法构建的。
在本发明的第八方面，涉及一种通用型T细胞，其TRAC和/或B2M基因被沉默。
在一具体实施方式中，TRAC基因被沉默是对包含有SEQ ID NO：1所示的序列进行基因编辑而实现的，进一步优选TRAC基因被沉默是对包含有SEQ ID NO：1所示的序列中的如SEQ ID NO：45所示的序列进行基因编辑而实现的；
B2M基因被沉默是对包含有SEQ ID NO：10所示的序列进行基因编辑而实现的，进一步优选B2M基因被沉默是对包含有SEQ ID NO：10所示的序列中的如SEQ ID NO：38所示的序列进行基因编辑而实现的。
在一具体实施方式中，TRAC基因被沉默是利用如SEQ ID NO：2、32或33所示的序列的gRNA对TRAC基因进行基因编辑而实现的，B2M基因被沉默是利用如SEQ ID NO：12所示的序列的gRNA对B2M基因进行基因编辑而实现的。
在一具体实施方式中，所述T细胞还表达有嵌合抗原受体，优选所述T细胞还表达有识别肿瘤抗原或病原体抗原的嵌合受体，该嵌合受体具有胞外抗原结合域、跨膜域、和胞内域，所述胞外抗原结合域特异性识别靶抗原。
所述T细胞如上述方面所示。所述嵌合抗原受体如上述方面所示。
在本发明的第九方面，涉及一种gRNA构建体，其包含选自SEQ ID NO：2、3、4、5、32、33、39、40、11、12、13或14之一的核苷酸序列。
在一具体的实施方式中，本发明的gRNA构建体，其包含：选自SEQ ID NO：2、3、4、5、32、33、39或40之一的核苷酸序列，以及选自SEQ ID NO：11、12、13或14之一的核苷酸序列。
在一具体的实施方式中，本发明的gRNA构建体，其包含：选自SEQ ID NO：2、32或33所示的序列，和/或SEQ ID NO：12所示的序列。
本发明涉及利用基因编辑技术对T细胞进行修饰，通过多基因的敲除能够有效抑制T细胞抗原受体(TCR)和主要组织相容性复合体(MHC)在T细胞中的功能；编码TCR的基因为TRAC，编码MHC I的基因为B2M。基于Cas9/CRISPR基因技术，以及对RNP(RNA核酸和蛋白复合物)电转方式的改进和优化，实现了在短时间内一次性、高效地对TRAC和B2M双基因进行敲除，敲除效率高达90％以上。
附图说明
图1是sgRNA与TRAC基因结合位点示意图；
图2显示了不同组成比例的RNP对TRAC敲除效果的影响；
图3显示了不同gRNA序列对TRAC敲除效果的影响；
图4显示了不同浓度Cas9酶对TRAC敲除效果的影响；
图5显示了gRNA与B2M基因结合位点示意图；
图6显示了不同gRNA对B2M基因敲除效果的影响；
图7显示了不同浓度Cas9酶对B2M基因敲除效果的影响；
图8显示了同时敲除TRAC和B2M时，不同gRNA组成成分对对TRAC和B2M双敲除的影响；
图9显示了靶向TRAC和B2M基因组成的gRNA混合物与Cas9酶之间形成的RNP复合物浓度对敲除效率的影响；
图10(a)-(d)显示了Tide在线软件预测TRAC和B2M基因突变效率；
图11显示了克隆测序验证TRAC和B2M基因突变结果。
图12显示了靶向BCMA的CAR T细胞中TRAC和B2M基因敲除效率
具体实施方式
发明人研究发现使用CRISPR/Cas9系统进行基因编辑时，gRNA的选择，Cas9酶和gRNA的比例等对编辑效率的影响很大，在此基础上完成了本发明。
术语
除非专门定义，本文所用的所有技术和科学术语具有在基因治疗，生物化学、遗传学和分子生物学领域内的技术人员通常理解的相同含义。类似或等效于本文中描述的所有方法和材料都可以在本发明的实践或测试中使用，其中，本文描述的是合适的方法和材料。本文提及的所有出版物、专利申请、专利和其他参考文献都以其全部内容结合于本文中作为参考。在冲突的情况下，以本说明书，包括定义为准。此外，除非另有规定，材料、方法和实施例仅是说明性的，而并非旨在进行限制。
除非另有说明，本发明的实践将采用细胞生物学、细胞培养、分子生物学、转基因生物学、微生物学、重组DNA和免疫学的传统技术，这都属于本领域的技术范围。这些技术充分解释于文献中。参见，例如，Current Protocols in Molecular Biology(FrederickM.AUSUBEL，2000，Wileyand sonInc，Library of Congress，USA)；Molecular Cloning:A Laboratory Manual，Third Edition，(Sambrooketal，2001，Cold Spring Harbor，NewYork:Cold Spring Harbor Laboratory Press)；Oligonucleotide Synthesis(M.J.Gaited.，1984)；Mullis et al.U.S.Pat.No.4,683,195；Nucleic Acid Hybridization(B.D.Harries&S.J.Higginseds.1984)；Transcription And Translation(B.D.Hames&S.J.Higginseds.1984)；Culture Of Animal Cells(R.I.Freshney，Alan R.Liss，Inc.，1987)；Immobilized Cells And Enzymes(IRL Press，1986)；B.Perbal，A Practical Guide To Molecular Cloning(1984)；the series，Methods In ENZYMOLOGY(J.Abelson和M.Simon，eds.-in-chief，Academic Press，Inc.，New York)，尤其是Vols.154和155(Wuetal.eds.)和Vol.185，“Gene Expression Technology”(D.Goeddel，ed.)；Gene Transfer Vectors For Mammalian Cells(J.H.Miller和M.P.Caloseds.，1987，Cold Spring Harbor Laboratory)；Immunochemical Methods In Cell And Molecular Biology(Mayer和Walker，eds.，Academic Press，London，1987)；Hand book Of Experimental Immunology，卷I-IV(D.M.Weir和C.C.Blackwell，eds.，1986)；和Manipulating the Mouse Embryo(Cold Spring Harbor Laboratory Press，Cold Spring Harbor，N.Y.，1986)。公开内容中，请求保护 的主题的各个方面均以范围形式呈现。应当理解，范围形式的描述仅仅是为了方便和简洁，并且不应被解释为对所要求保护的主题的范围的硬性限制。因此，范围的描述应当被认为已经具体公开了所有可能的子范围以及该范围内的单个数值。例如，在提供值的范围的情况下，应当理解，在该范围的上限和下限之间的每个中间值以及在所述范围内的任何其他所述的或中间的值均被包括在要求保护的主题内，所述范围的上下限也属于请求保护的主题的范围。所述较小范围内可独立地包含这些较小范围的上下限，它们也属于请求保护的主题的范围，除非明确地排除所述范围的上下限。设定范围包含一个或两个限值时，请求保护的主题也包括排除所述限值之一个或两个的范围。这适用而无关范围的宽度。
本文使用的术语约是指本技术领域技术人员容易知晓的各值的通常误差范围。本文中述及“约”值或参数，包括(并描述)指向该值或参数本身的实施方式。例如，关于“约X”的描述包括“X”的描述。例如，“约”或“包含”可意指按照在该领域中的实际的标准偏差在1以内或多于1。或者“约”或“包含”可意指至多10％(即±10％)的范围。例如，约5μM可包括在4.5μM与5.5μM之间的任何数目。当在申请案与申请专利范围中提供特定值或组成时，
除非另外指出，否则“约”或“包含”应假定为在该特定值或组成的可接受误差范围内。
本文中所述任何浓度范围、百分比范围、比例范围或整数范围应理解为包括在所述范围内的任何整数，以及在合适情况下，其分数(例如整数的十分之一与百分之一)的数值，除非另外指出。
为便于更好地理解本发明，对相关术语定义如下：
术语“基因编辑”，指能够让人类对目标基因进行“编辑”，实现对特定DNA片段的敲除、加入等。
术语“分子沉默”或“基因沉默”，是指由于各种原因，在不损伤原有DNA的情况下使基因不表达或低表达的现象。基因沉默发生在两种水平上，一种是由于DNA甲基化、异染色质化以及位置效应等引起的转录水平的基因沉默，另一种是转录后基因沉默，即在基因转录后的水平上通过对靶标RNA进行特异性抑制而是基因失活，包括反义RNA、共抑制、基因压抑、RNA干扰和微小RNA介导的翻译抑制等。CRISPR(Clustered regularly interspaced short palindromicrepeats)规律成簇间隔短回文重复；Cas9(CRISPRassociated nuclease)是CRISPR相关核酸酶，CCRISPR/Cas9是最新出现的一种由RNA指导的，利用Cas9核酸酶对靶向基因进行编辑的技术。
CRISPER/Cas9系统”统称为转录物和涉及Cas9酶基因的表达或指导其活性的其他元件，包括编码Cas9基因的序列、tracr(反式激活CRISPR)序列(例如tracrRNA或活性部分tracrRNA)、tracr配对序列(涵盖“同向重复”和在内源CRISPR系统背景下的tracrRNA 加工的部分同向重复)、指导序列(在内源CRISPR系统背景下也称为“间隔子(spacer)”，即gRNA)、或来自CRISPR座位的其他序列和转录物。一般而言，CRISPR系统的特征为促进在靶序列的位点处的CRISPR复合物(在内源CRISPR系统的背景下也称为前间区)的形成的元件。
术语“靶序列”是指与指导序列具有互补性的序列，靶序列与指导序列之间互补配对促进CRISPR复合物的形成。完全互补性不是必需的，条件是存在足够互补性以引起杂交并且促进一种CRISPR复合物的形成。一个靶序列可以包含任何多核苷酸，如DNA或RNA多核苷酸。在一些实施例中，靶序列位于细胞的细胞核或细胞质中。
一般而言，指导序列(gRNA)是与靶多核苷酸序列具有足够互补性以便与该靶序列杂交并且指导CRISPR复合物与该靶序列的序列特异性结合的任何多核苷酸序列。在一些实施例中，当使用适合的比对算法进行最佳比对时，在指导序列与其相应的靶序列之间的互补程度是约或多于约50％、60％、75％、80％、85％、90％、95％、97.5％、99％、或更多。可以使用用于比对序列的任何适合的算法来确定最佳比对，其非限制性实例包括史密斯-沃特曼(Smith-Waterman)算法、尼德曼-翁施(Needleman-Wunsch)算法、基于伯罗斯-惠勒变换(Burrows-Wheeler Transform)的算法(例如伯罗斯-惠勒比对工具(Burrows Wheeler Aligner))、ClustalW、Clustal X、BLAT、Novoalign(Novocraft技术公司)、ELAND(亿明达公司(Illumina)，圣地亚哥，加利福尼亚州)、SOAP(在soap.genomics.org.cn可获得)、以及Maq(在maq.sourceforge.net可获得)。
在一些实施例中，该CRISPR酶是包含一个或多个异源蛋白结构域(例如除了该CRISPR酶之外的约或多于约1、2、3、4、5、6、7、8、9、10个或更多个结构域)的融合蛋白的一部分。CRISPR酶融合蛋白可以包含任何其他蛋白质，以及任选地在任何两个结构域之间的连接序列。可以融合到CRISPR酶上的蛋白质结构域的实例包括但不限于，表位标签、报告基因序列、以及具有下列活性的一个或多个的蛋白质结构域：甲基酶活性、脱甲基酶活性、转录激活活性、转录阻抑活性、转录释放因子活性、组蛋白修饰活性、RNA切割活性和核酸结合活性。表位标签的非限制性实例包括组氨酸(His)标签、V5标签、FLAG标签、流感病毒血凝素(HA)标签、Myc标签、VSV-G标签、和硫氧还蛋白(Trx)标签。报告基因的实例包括，但不限于，谷胱甘肽-S-转移酶(GST)、辣根过氧化物酶(HRP)、氯霉素乙酰转移酶(CAT)、β-半乳糖苷酶、β-葡糖醛酸糖苷酶、萤光素酶、绿色荧光蛋白(GFP)、HcRed、DsRed、青荧光蛋白(CFP)、黄色荧光蛋白(YFP)、以包括蓝色荧光蛋白(BFP)的自发荧光蛋白。CRISPR酶可以融合到编码一种蛋白质或蛋白质片段的基因序列上，所述蛋白质或蛋白质片段结合DNA分子或结合其他细胞分子，其包括但不限于，麦芽糖结合蛋白(MBP)、S-tag、 Lex A DNA结合结构域(DBD)融合物、GAL4DNA结合结构域融合物、以及单纯疱疹病毒(HSV)BP16蛋白融合物。可以形成包含CRISPR酶的融合蛋白的一部分的另外的结构域描述于US 20110059502中，通过引用将其并入本文。
术语“Cas9酶”可以是野生型Cas9或Cas9任何修改版本，包括任何天然存在的细菌Cas9以及任何嵌合体、突变体、同源物或直向同源物。Cas9酶可以包含一个或多个突变，并且可以用作具有或不具有与功能结构域融合的通用DNA结合蛋白。这些突变可以是人工引入的突变或获得性和丢失性功能突变。这些突变可以包括但不限于分别在RuvC和HNH催化结构域中的催化结构域(D10和H840)之一中的突变。
在本发明中，例如可以使用来自NEB公司的Cas9酶，当然本领域技术人员可以选择具有相同或相似功能的其他Cas9酶。在本文中，Cas9酶可以实现的功能是，在30μl的反应的Cas9酶反应体系(该反应体系包括：20mM HEPES、100mM NaCl、5mM MgCl 2、0.1mM EDTA且在25℃时，pH为6.5)中，当含有1nM PvuII线性化的pBR322 DNA(一个目标位点CGCTTGTTTCGGCGTGGGTA)，40nM sgRNA和20nM Cas9酶的情况下，在37℃温育1小时的情况下，通过琼脂糖凝胶电泳确认有90％的pBR322 DNA被降解。在这个反应体系中，1分钟催化1nmol底物(PvuII线性化的pBR322 DNA)完全转化成产物的Cas9酶量为0.37nmol，Cas9酶克数为59.57ng。所述Cas9酶的酶活力为0.37nmol(1分钟催化1nmol底物转化成产物的酶量)。
本领域技术人员可以理解，在本文中是基于具有上述Cas9酶活性来计算Cas9酶和希望导入的gRNA的摩尔比比例，以及确认Cas9酶在导入复合物中的浓度，当Cas9酶的活性发生变化时，本领域技术人员可以基于不同的酶的说明书中对于活性的描述来基于本文确定的比例来进行换算以选取Cas9酶的使用浓度，及其与gRNA的摩尔比。
在一个方面，该Cas酶是切口酶。在一个优选的实施例中，该Cas9以mRNA的形式递送到该细胞中。这允许该酶的瞬时表达，由此降低毒性。Cas9还可以在编码且表达Cas9酶的核苷酸构建体中递送至细胞中。另外，还可以在诱导型启动子的控制下表达Cas9。
术语CRISPR和Cas酶在本文中通常可互换地使用，除非另外说明。如上提及的，在本文中使用的许多残基编号是指来自化脓链球菌中的II型CRISPR座位的Cas9酶。然而，应当理解的是，本发明包括更多的来自其他微生物物种的Cas9，如SpCas9、SaCa9、St1Cas9等等。本领域技术人员将能够通过比较相关氨基酸序列而确定在除了SpCas9之外的Cas9酶中的适当的相应残基。术语sgRNA指短小的gRNA。在进行基因编辑时，給予的gRNA、tracr配对序列、及tracr序列可以单独给予，也可以一条完整的RNA序列。Cas9蛋白与gRNA结合能够实现在特异位点处切割DNA，来源于Streptococcus pyogenes的CRISPR/Cas系统识别 序列为23bp，并能靶向20bp，其识别位点最末3位NGG序列被称作PAM(protospacer adjacent motif)序列。
除非另外表明，否则术语Cas酶、CRISPR酶、CRISPR蛋白、Cas蛋白和CRISPRCas通常是可以互换使用的。
Cas转基因可以通过载体(例如AAV、腺病毒、慢病毒)、和/或粒子和/或纳米粒子、和/或电转来递送。
在一实施例中，对在TCR的α和β链中的一种或两种链的恒定区域的相应编码基因的外显子用CRISPER/Cas技术敲除，使内源性TCR不具有活性，优选为定点敲除内源性TCRα链恒定区的第一外显子。
“抑制”或“遏制”B2M或TCR的表达是指细胞中B2M或TCR的表达减少至少1％、至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或100％。更具体而言，“抑制”或“遏制”B2M的表达是指细胞中B2M的含量降低至少1％、至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或100％。可以通过本领域内已知的任何合适的方法，如ELISA、免疫组织化学、免疫印迹(Western Blotting)或流式细胞术使用B2M或TCR的特异性抗体测定细胞中蛋白的表达或含量。
本发明所使用的“修饰”一词是指本发明的蛋白或多肽的状态或结构的改变。修饰的方式可以是化学的、结构的和功能上的。T细胞受体(TCR)是响应于抗原的呈递参与T细胞活化的细胞表面受体。TCR通常由α和β两个链构成，其可组装以形成异源二聚体并与所述CD3转导性亚基相关联以形成存在于细胞表面上的T细胞受体复合物。TCR的α链和β链是由以下各项组成的：免疫球蛋白样的N-末端可变区(V)和恒定区(C)、疏水跨膜结构域和短胞质区域。对于免疫球蛋白分子，α链和β链的可变区是由V(D)J重组产生的，使得在T细胞的群体内产生大量的多样化的抗原特异性。然而，与识别完整抗原的免疫球蛋白相反，T细胞是由与MHC分子相关联的加工后的肽片段激活，通过T细胞将额外维度引入抗原识别，称为MHC限制。通过T细胞受体识别供体和受体之间的MHC差异导致细胞增殖和GVHD的潜在发展。已经表明，TCR的正常表面表达依赖于复合物的所有七种组分的协同合成和组装(Ashwell and Klusner 1990)。TCRα或TCRβ的失活可以导致TCR从T细胞表面的消除，从而防止识别同种异体抗原和因此导致的GVHD。
术语“MHC”为组织相容性复合物，是所有编码生物相容复合体抗原的基因群一种统称，MHC抗原表达于所有高等脊椎动物的组织，在人类细胞中称为HLA抗原，在移植 反应中发挥重要作用，由对所植入的组织的表面上的组织相容性抗原产生反应的T细胞介导排异。MHC蛋白质在T细胞刺激中发挥至关重要的作用，抗原呈递细胞(通常是树突状细胞)展示属于MHC上的细胞表面上的外源蛋白的降解产物的肽，在共刺激信号的存在下，T细胞被活化并作用于也展示相同肽/MHC复合体的靶细胞。例如，刺激的T辅助细胞会靶向巨噬细胞，该巨噬细胞展示与其MHC结合的抗原，或细胞毒性T细胞(CTL)会作用于展示外源病毒肽的病毒感染的细胞。MHC抗原分为NHC I类抗原和MHC II类抗原。人类中，I类的HLA基因簇包括三个主要的基因座HLA-A、HLA-B、和HLA-C，以及几个次要基因座。II类HLA簇也包括三种主要基因座：HLA-DP、HLA-DQ和HLA-DR，
术语“人类白细胞抗原”(Human leukocyte antigen，HLA)是人类的主要组织相容性复合体的编码基因，位于6号染色体上(6p21.31)，包括一系列紧密连锁的基因座，与人类的免疫系统功能密切相关。HLA包括有I类、II类和III类基因部分。HLA的I类和II类基因所表达的抗原位于细胞膜上，为MHC-I(HLA-A、HLA-B、HLA-C位点编码)和MHC-II(HLA-D区编码)，I类几乎分布于身体全部细胞表面，是一个异二聚体，由重链(α链)与β2微球蛋白组成(B2M)，II类主要是定位于巨噬细胞和B淋巴细胞表面的糖蛋白。
术语“B2M”为β-2微球蛋白，也称为B2M，是MHC I类分子的轻链。在人类中，B2M由位于15号染色体上的b2m基因编码，与6号染色体上的作为基因簇定位的其他MHC基因相对。β-2微球蛋白缺陷的小鼠模型表明，B2M对于MHC I类的细胞表面表达和肽结合沟槽的稳定性是必要的。进一步表明，由于β-2微球蛋白基因中的靶向突变，来自缺乏正常细胞表面MHC I表达的小鼠的造血移植物被正常小鼠中的NK1.1+细胞排斥，表明MHC I分子的缺陷性表达使骨髓细胞易于被宿主免疫系统排斥(Bix et al.1991)。
因此，为了提供较低同种异体反应活性的T细胞，本发明所提供的T细胞包括使一个TCR基因和一个HLA基因的失活或突变的T细胞。
所述“TCR不具有活性”是指内源性的TCR失活至少一个亚基的基因，尤其是失活了TCRα和/或TCRβ基因，更佳的，是TCRα基因。
所述“MHC不具有活性”是指内源性的MHC失活至少一个亚基的基因，尤其是失活了MHC I的基因，更佳的，是B2M基因。
术语“T细胞抗原耦合器(T CELL ANTIGEN COUPLER,TAC)”，包括三个功能结构域：肿瘤靶向结构域，包括单链抗体、设计的锚蛋白重复蛋白(designed ankyrin repeat protein，DARPin)或其他靶向基团2；为胞外区结构域，与CD3结合的单链抗体，从而使得 TAC受体与其他TCR受体靠近；跨膜区和CD4共受体的胞内区，其中，胞内区连接蛋白激酶LCK，催化TCR复合物的免疫受体酪氨酸活化基序(ITAMs)磷酸化作为T细胞活化的初始步骤。
本文所用的术语“激活”和“活化”可互换使用，它们以及其语法上的其他形式可以指细胞从静止状态转变为活性状态的过程。该过程可以包括对抗原、迁移和/或功能活性状态的表型或遗传变化的响应。例如，术语“激活”可以指T细胞逐步活化的过程。例如，T细胞可能需要至少两个信号才能完全激活。第一信号可以在由抗原-MHC复合物接合TCR之后发生，而第二信号可以通过共刺激分子(参见表1中所列举的共刺激分子)的接合发生。在体外，抗CD3可以模拟所述第一信号，抗CD28可以模拟所述第二信号。例如，工程化T细胞可以被表达的CAR激活。本文所用的T细胞激活或T细胞触发可以指已经被充分刺激以诱导可检测的细胞增殖、细胞因子产生和/或可检测的效应物功能的T细胞的状态。
术语“嵌合受体”，即用基因重组技术将不同来源的DNA片段或蛋白质相应的cDNA连接而成的融合分子，包括胞外域、跨膜域和胞内域。嵌合受体包括但不限于：嵌合抗原受体(CAR)、修饰的T细胞(抗原)受体(TCR)、T细胞融合蛋白(TFP)、T细胞抗原耦合器(TAC)。
术语“共刺激配体”包括特异性结合T细胞上的同一性共刺激分子的抗原呈递细胞(例如，aAPC、树突状细胞、B细胞等)上的分子，由此提供信号，与由例如TCR/CD3复合物与加载有肽的MHC分子的结合提供的第一信号共同介导T细胞应答，包括但不限于增殖、激活、分化等。共刺激配体可以包括但不限于CD7、B7-1(CD80)、B7-2(CD86)、PD-L、PD-L2、4-1BBL、OX40L、诱导型共刺激配体(ICOS-L)、细胞间粘附分子(ICAM)、CD30L、CD40、CD70、CD83、HLA-G、MICA、MICB、HVEM、淋巴毒素β受体、3/TR6、ILT3、ILT4、HVEM、结合Toll配体受体的激动剂或抗体以及与B7-H3特异性结合的配体。共刺激配体还特别包括与T细胞上存在的共刺激分子特异性结合的抗体，例如但不限于CD27、CD28、4-1BB、OX40、CD30、CD40、PD-1、ICOS、淋巴细胞功能相关抗原-1(LFA-1)、CD2、CD7、LIGHT、NKG2C、B7-H3和与CD83特异性结合的配体。
术语“共刺激分子”是指与共刺激配体特异性结合的T细胞上的同一性结合配偶体，从而介导T细胞的共刺激应答，例如但不限于增殖。共刺激分子包括但不限于MHC I类分子、BTLA和Toll配体受体。
术语“共刺激信号”指与细胞刺激信号分子，例如TCR/CD3结合，组合导致T细胞增殖和/或关键分子的上调或下调的信号。
术语“嵌合抗原受体”或“CAR”是指可以由包括但不限于T细胞的免疫细胞表达的工程化分子。CAR在T细胞中表达并且可以重定向T细胞以诱导以由人造受体决定的特异性杀死 靶细胞。CAR的细胞外结合结构域可以衍生自鼠、人源化或完全人单克隆抗体。当其在免疫效应细胞中时，给所述的细胞提供针对靶细胞(通常是癌细胞)的特异性，并且具有细胞内信号产生。CAR通常包括至少一个细胞外抗原结合结构域、跨膜结构域和细胞质信号传导结构域(本文中也称为“胞内信号传导结构域”)，其包括来源于如下定义的刺激性分子和/或共刺激性分子的功能信号传导结构域。在某些方面，多肽组彼此邻接。多肽组包括在存在二聚化分子时可以使多肽彼此偶联的二聚化开关，例如，可以使抗原结合结构域偶联至胞内信号传导结构域。在一个方面，刺激性分子为与T细胞受体复合体结合的ζ链。在一个方面，细胞质信号传导结构域进一步包括一种或多种来源于至少一个如下定义的共刺激性分子的功能性信号传导结构域。在一个方面，共刺激性分子选自本文所述共刺激性分子，例如4-1BB(即CD137)、CD27和/或CD28。在一个方面，CAR包括嵌合融合蛋白，该融合蛋白包含细胞外抗原结合结构域、跨膜结构域和包含来源于刺激性分子的功能性信号传导结构域的胞内信号传导结构域。在一个方面，CAR包含嵌合融合蛋白，该融合蛋白包含细胞外抗原结合结构域、跨膜结构域和包含来源于共刺激性分子的功能性信号传导结构域和来源于刺激性分子的功能性信号传导结构域的胞内信号传导结构域。在一个方面中，CAR包含嵌合融合蛋白，该融合蛋白包含细胞外抗原结合结构域、跨膜结构域和包含来源于一个或更多个共刺激性分子的两个功能性信号传导。
术语“信号传导结构域”是指通过在细胞内传递信息而起作用的蛋白质的功能性部分，用来通过产生第二信使或通过响应这样的信使起效应物作用经由确定的信号传导途径调节细胞的活性。
术语“细胞”及其语法上的其他形式可以指人或非人动物来源的细胞。工程细胞也可以指表达CAR的细胞。
术语“转染”是指将外源核酸引入真核细胞。转染可以通过本领域已知的各种手段来实现，包括磷酸钙-DNA共沉淀、DEAE-葡聚糖介导的转染、聚凝胺介导的转染、电穿孔、显微注射、脂质体融合、脂质转染、原生质体融合、逆转录病毒感染和生物弹道技术(biolistics)。
术语“稳定转染”或“稳定地转染”是指将外源核酸、DNA或RNA引入和整合到转染细胞的基因组中。术语“稳定转染体”(stable transfectant)是指将外来DNA稳定地整合到基因组DNA中的细胞。
术语“核酸分子编码”、“编码DNA序列”和“编码DNA”是指沿着脱氧核糖核酸链的脱氧核糖核苷酸的顺序或顺序。这些脱氧核糖核苷酸的顺序决定了沿着多肽(蛋白质)链的氨基酸的顺序。因此，核酸序列编码氨基酸序列。
术语“个体”是指任何动物，例如哺乳动物或有袋动物。本发明的个体包括但不限于人 类、非人类灵长类动物(例如恒河猴或其他类型的猕猴)、小鼠、猪、马、驴、牛、绵羊、大鼠和任何种类的家禽。
术语“外周血单个核细胞”(peripheral blood mononuclear cell,PBMC)是指外周血中具有单个核的细胞，包含淋巴细胞、单核细胞等。
术语“T细胞活化”或“T细胞激活”及其语法上的其他形式可以指被充分刺激以诱导可检测的细胞增殖、细胞因子产生和/或可检测的效应物功能的T细胞的状态。在一些情况下，“完全T细胞活化”可以类似于触发T细胞的细胞毒性。可以使用本领域已知的各种测定来测量T细胞活化。所述测定可以是测量细胞因子分泌的ELISA、ELISPOT、用于测量细胞内细胞因子表达的流式细胞术测定(CD107)、用于测量增殖的流式细胞术测定、和用于确定靶细胞消除的细胞毒性测定(51Cr释放测定)。所述测定通常使用对照(非工程细胞)与工程细胞(CAR T)进行比较，以确定与对照相比，工程细胞的相对激活。此外，所述测定可以与未表达靶抗原的靶细胞孵育或接触的工程细胞进行比较。例如，所述比较可以是与不表达GPC3的靶细胞孵育的GPC3-CART细胞进行的比较。
当用于指核苷酸序列时，本文所用的术语“序列”及其语法上的其他形式可以包括DNA或RNA，并且可以是单链或双链。核酸序列可以突变。核酸序列可以具有任何长度。
本文所用的术语“有效量”是指提供治疗或预防益处的量。
本文所用的术语“表达载体”是指包含重组多核苷酸的载体，其包含与待表达的核苷酸序列有效连接的表达调控序列。表达载体包含用于表达的足够的顺式作用元件(cis-acting elements)；用于表达的其它元件可以由宿主细胞或体外表达系统提供。表达载体包括本领域所有已知的那些，例如粘粒、质粒(例如裸露或包含在脂质体中的)和病毒(例如，慢病毒、逆转录病毒、腺病毒和腺相关病毒)。
本文所用的术语“慢病毒”是指逆转录病毒科的属。逆转录病毒在能够感染非分裂细胞方面是逆转录病毒中独特的；它们可以将大量的遗传信息递送到宿主细胞的DNA中，因此它们是基因递送载体最有效的方法之一。HIV、SIV和FIV都是慢病毒的实例。源自慢病毒的载体提供了在体内实现显著水平的基因转移的手段。
本文使用的术语“载体”是包含分离的核酸并可用于将分离的核酸递送至细胞内部的组合物。在本领域中已知许多载体，包括但不限于线性多核苷酸、与离子或两亲化合物相关的多核苷酸、质粒和病毒。因此，术语“载体”包括自主复制的质粒或病毒。该术语还应被解释为包括促进核酸转移到细胞中的非质粒和非病毒化合物，例如聚赖氨酸化合物、脂质体等。病毒载体的实例包括但不限于腺病毒载体、腺相关病毒载体、逆转录病毒载体等。
本文使用的术语序列“同一性”通过在比较窗口(例如至少20个位置)上比较两个经最佳 匹配的序列来确定同一性百分比，其中比较窗口中多核苷酸或多肽序列的部分可以包含添加或缺失(即间隙)，例如对于最佳匹配的两个序列而言与参考序列(其不包含添加或缺失)相比20％或更少的间隙(例如5至15％、或10至12％)。通常通过确定在两个序列中发生相同的核酸碱基或氨基酸残基的位置的数目来计算百分比，以产生正确匹配的位置的数目，将正确匹配位置的数目除以参考序列中的位置总数(即窗口大小)，并将结果乘以100，以产生序列同一性的百分比。
本文所用的术语“外源性”指的是一个核酸分子或多肽，其在细胞内没有内源性表达，或表达水平不足以实现过表达时具有的功能。因而，“外源性”包括在细胞内表达的重组核酸分子或多肽，如外源性、异源性和过表达的核酸分子和多肽。
术语“内源性”是指一个核酸分子或多肽来自生物体自身基因组内的基因。在一些实施方案中，本发明嵌合受体是嵌合抗原受体。本文所用的术语“嵌合抗原受体(Chimeric Antigen Receptor，CAR)”指一种融合到细胞内信号转导域的肿瘤抗原结合结构域，能激活T细胞。常见地，CAR的胞外结合结构域来源于小鼠或人源化或人的单克隆抗体。
嵌合抗原受体通常包含(细)胞外抗原结合区。在一些实施方案中，胞外抗原结合区可以是完全人的。在其他情况下，胞外抗原结合区域可以被人源化。在其他情况下，胞外抗原结合区可以是鼠源的，或者所述胞外抗原结合区中的嵌合体由来自至少两种不同动物的氨基酸序列组成。在一些实施方案中，所述胞外抗原结合区可以是非人的。可以设计多种抗原结合区域。非限制性实例包括衍生自抗体的单链可变片段(scFv)、选自文库的片段抗原结合区(Fab)、单结构域片段或与接合其同源受体的自然配体。在一些实施方案中，胞外抗原结合区域可以包含scFv、Fab或天然配体，以及它们的任何衍生物。细胞外抗原结合区可以指除完整抗体之外的分子，其可以包含完整抗体的一部分并且可以与完整抗体所结合的抗原结合。抗体片段的实例可以包括但不限于Fv、Fab、Fab'、Fab'-SH、F(ab')2；双功能抗体、线性抗体；单链抗体分子(例如scFv)；和由抗体片段形成的多特异性抗体。细胞外抗原结合区，例如scFv、Fab或天然配体，可以是确定抗原特异性的CAR的一部分。细胞外抗原结合区可以结合任何互补靶。细胞外抗原结合区可以衍生自已知可变区序列的抗体。细胞外抗原结合区可以从获自可获得的小鼠杂交瘤的抗体序列中得到。或者，可以从肿瘤细胞或原代细胞例如肿瘤浸润淋巴细胞(TIL)的全外切割测序获得细胞外抗原结合区。
在一些情况下，细胞外抗原结合区的结合特异性可以通过互补决定区或CDR，如轻链CDR或重链CDR来确定。在许多情况下，结合特异性可以通过轻链CDR和重链CDR来确定。与其他参考抗原相比，给定的重链CDR和轻链CDR的组合可以提供给定的结合袋，其可以赋予抗原(例如GPC3)更大的亲和力和/或特异性。例如，特异于磷脂酰肌醇蛋白聚糖-3的 CDR可以在CAR的细胞外结合区域中表达，使得靶向GPC3的CAR可以将T细胞靶向表达GPC3的肿瘤细胞。
在本文公开的任何实施方案的某些方面，细胞外抗原结合区，例如scFv可以包含对抗原特异性的轻链CDR。轻链CDR可以是抗原结合单元例如CAR的scFv轻链的互补决定区。轻链CDR可以包含连续的氨基酸残基序列，或由非互补决定区(例如框架区)隔开的两个或更多个连续的氨基酸残基序列。在一些情况下，轻链CDR可以包含两个或更多个轻链CDR，其可以被称为轻链CDR-1，CDR-2等。在一些情况下，轻链CDR可以包含三个轻链CDR，其可分别称为轻链CDR-1，轻链CDR-2和轻链CDR-3。在一些实例中，存在于普通轻链上的一组CDR可统称为轻链CDR。
在本文公开的任何实施方案的某些方面，细胞外抗原结合区，例如scFv可以包含对抗原特异的重链CDR。重链CDR可以是抗原结合单元例如scFv的重链互补决定区。重链CDR可以包含氨基酸残基的连续序列，或由非互补决定区(例如框架区)隔开的两个或更多个氨基酸残基的连续序列。在一些情况下，重链CDR可以包含两个或更多个重链CDR，其可以称为重链CDR-1，CDR-2等。在一些情况下，重链CDR可以包含三个重链CDR，其可分别称为重链CDR-1，重链CDR-2和重链CDR-3。在一些情况下，存在于共同重链上的一组CDR可统称为重链CDR。
通过使用基因工程，可以以各种方式修饰细胞外抗原结合区。在一些情况下，可以突变细胞外抗原结合区域，从而可以选择细胞外抗原结合区域以对其靶标具有更高的亲和力。在一些情况下，细胞外抗原结合区域对其靶标的亲和力可针对可在正常组织上以低水平表达的靶标进行优化。可以进行此优化，以尽量减少潜在的毒性。在其他情况下，对靶标的膜结合形式具有更高亲和力的细胞外抗原结合区域的克隆可以优于其可溶形式的对应物。可以进行这种修饰，因为也可以检测到不同水平的可溶形式的靶标，并且它们的靶向可引起不期望的毒性。
在一些情况下，细胞外抗原结合区域包括铰链或间隔区。术语铰链和间隔区可以互换使用。铰链可以被认为是用于向细胞外抗原结合区提供柔性的CAR的一部分。在一些情况下，铰链可用于检测细胞的细胞表面上的CAR，特别是当检测细胞外抗原结合区的抗体不起作用或可用时。例如，衍生自免疫球蛋白的铰链的长度可能需要优化，这取决于细胞外抗原结合区域靶向靶上的表位的位置。
在一些情况下，铰链可能不属于免疫球蛋白，而是属于另一种分子，如CD8α分子的天然铰链。CD8α铰链可以含有已知在CD8辅助受体和MHC分子的相互作用中起作用的半胱氨酸和脯氨酸残基。所述半胱氨酸和脯氨酸残基可影响所述CAR的性能。CAR铰链可以是尺 寸可调的。T细胞和靶细胞之间的免疫突触的这种形貌还限定了由于细胞表面靶分子上的膜远端表位而不能由CAR进行功能桥接的距离，即使用短铰链CAR也不能使突触距离达到信号能够传导的近似值。同样，膜近端CAR靶抗原表位仅在长铰链CAR的背景下观察到信号输出。可以根据所使用的细胞外抗原结合区域来调节铰链。铰链可以是任何长度的。跨膜结构域可以将CAR锚定在细胞的质膜上。CD28的天然跨膜部分可用于CAR。在其他情况下，也可以在CAR中使用CD8α的天然跨膜部分。“CD8”可以是与NCBI参考号：NP_001759或其具有刺激活性的片段具有至少85、90、95、96、97、98、99或100％同一性的蛋白质。“CD8核酸分子”可以是编码CD8多肽的多核苷酸，在某些情况下，跨膜区可以是CD28的天然跨膜部分，“CD28”可以指与NCBI参考号：NP_006130或其具有刺激活性的片段具有至少85、90、95、96、97、98、99或100％同一性的蛋白质。“CD28核酸分子”可以是编码CD28多肽的多核苷酸。在一些情况下，跨膜部分可以包含CD8α区域。CAR的胞内信号域可以负责活化CAR已经置于其中的T细胞的效应子功能中的至少一种。CAR可以诱导T细胞的效应子功能，例如，所述效应子功能是细胞溶解活性或辅助活性，包括细胞因子的分泌。因此，术语“细胞内信号域”是指转导效应子功能信号并引导细胞进行特异功能的蛋白质部分。虽然通常可以使用整个细胞内信号传导区域，但是在许多情况下，不必使用信号结构域的整个链。在一些情况下，使用细胞内信号传导区的截短部分。在一些情况下，术语细胞内信号域因此意在包括足以转导效应子功能信号的细胞内信号传导区的任何截短部分。
在CAR中使用的信号结构域的优选实例可以包括T细胞受体(TCR)的细胞质序列和协同作用以在靶-受体结合之后启动信号转导的共同受体，以及它们的任何衍生物或变体序列和这些序列的具有相同功能性的任何合成序列。
在一些情况下，所述细胞内信号域可以含有已知的免疫受体酪氨酸激活基序(ITAM)的信号基序。含有细胞质信号传导序列的ITAM的实例包括衍生自TCRζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、CD66d的DAP10、或DAP12的蛋白质的功能性信号传导结构域。然而，在优选的实施方案中，细胞内信号结构域衍生自CD3ζ链。含有一个或多个ITAM基序的T细胞信号结构域的实例是CD3ζ结构域，也称为T细胞受体T3ζ链或CD247。该结构域是T细胞受体-CD3复合物的一部分，并且在将几种细胞内信号转导途径的抗原识别与T细胞的主效应激活相结合方面起重要作用。如本文所用，CD3ζ主要是指人类CD3ζ及其同种型，如从Swissprot条目P20963所知的，包括具有基本相同序列的蛋白质。作为嵌合抗原受体的一部分，再次重申，不需要全T细胞受体T3ζ链，并且其包含T细胞受体T3ζ链的信号结构域的任何衍生物都是合适的，包括其任何功能等同物。
细胞内信号传导结构域可以选自表1的任何一个结构域。在一些情况下，可以修饰结 构域，使得与参考结构域的同一性可以为约50％至约100％。可以修饰表1的任何一个结构域，使得修饰形式可以包含约50、60、70、80、90、95、96、97、98、99或至多约100％的同一性。CAR的细胞内信号传导区可以进一步包含一个或多个共刺激结构域。细胞内信号传导区可以包含单个共刺激结构域，例如ζ链(第一代CAR)或其与CD28或4-1BB(第二代CAR)。在其他实例中，细胞内信号传导区可以包含两个共刺激结构域，例如CD28/OX40或CD28/4-1BB(第三代)。
与细胞内信号结构域如CD8一起，这些共刺激结构域可以产生激酶途径的下游激活，从而支持基因转录和功能性细胞反应。CAR的共刺激结构域可以激活与CD28(磷脂酰肌醇-4,5-二磷酸3-激酶)或4-1BB/OX40(TNF-受体相关因子衔接蛋白)途径以及MAPK和Akt激活相关的近端信号蛋白。
在某些情况下，通过CAR产生的信号可能与辅助或共刺激信号相结合。对于共刺激信号结构域，嵌合抗原受体样复合物可被设计成包含若干可能的共刺激信号结构域。如本领域众所周知的，在幼稚T细胞中，T细胞受体的单独接合不足以诱导T细胞的完全活化为细胞毒性T细胞。完整的生产性T细胞激活需要第二共刺激信号。已经报道对T细胞活化提供共刺激的几种受体，包括但不限于CD28、OX40、CD27、CD2、CD5、ICAM-1、LFA-1(CD11a/CD18)、4-1BBL、MyD88和4-1BB。这些共刺激分子使用的信号传导途径均能与主T细胞受体激活信号协同作用。这些共刺激信号传导区域提供的信号可以与源自一个或多个ITAM基序(例如CD3zeta信号转导域)的主效应激活信号协同作用，并且可以完成T细胞激活的要求。
在一些情况下，向嵌合抗原受体样复合物添加共刺激结构域可以增强工程细胞的功效和耐久性。在另一个实施方案中，T细胞信号结构域和共刺激结构域彼此融合从而构成信号传导区。
表1.共刺激结构域
Figure PCTCN2019107374-appb-000001
本文所用的术语“调节”是指正向或负向改变。调节范例包括1％、2％、10％、25％、50％、75％、或100％变化。
本文所用的术语“治疗”是指在试图改变个人或处理细胞引起的的疾病过程中的临床干预，既可以进行预防也可以在临床病理过程干预。治疗效果包括但不限于，防止疾病的发生或复发、减轻症状、减少任何疾病直接或间接的病理后果、防止转移、减慢疾病的进展速度、改善或缓解病情、缓解或改善预后等。
T细胞
本文所述的T细胞是指通过本发明的方法改造的T细胞，该T细胞的内源性的TCR基因和/或MHC基因沉默。
在一些情况下，T细胞可以是由CD45RO(-)、CCR7(+)、CD45RA(+)、CD62L+(L-选择素)、CD27+、CD28+和/或IL-7Rα+组成的干记忆TSCM细胞，所述干记忆细胞还可以表达CD95、IL-2Rβ、CXCR3和/或LFA-1，并且显示出与所述干记忆细胞不同的许多功能属性。或者，免疫反应性细胞还可以是包含L-选择素和CCR7的中枢记忆体TCM细胞，其中中枢记忆细胞可以分泌例如IL-2，但不分泌IFNγ或IL-4。免疫反应性细胞还可以是包含L-选择蛋白或CCR7的效应记忆TEM细胞，并产生例如效应细胞因子如IFNγ和IL-4。
通常通过全身给药(例如静脉内、腹膜内、肌内、皮下或颅内输注)或局部应用，通过给予个体患者体内递送载体，如下所述。或者，载体可以离体递送到细胞，例如从个体患者(例如，淋巴细胞、T细胞、骨髓抽吸物、组织活检)移出的细胞，然后通常在选择并入了该载体的细胞后将细胞再植入患者体内。在选择之前或之后，可以扩增细胞。
所述T细胞可以从许多来源获得，包括PBMC、骨髓、淋巴结组织、脐带血、胸腺组织和来自感染部位、腹水、胸腔积液、脾组织和肿瘤的组织。在某些情况下，可以使用任何数量的本领域技术人员已知的技术，例如FicollTM分离，从个体收集的血液获得T细胞。在一个实施方案中，通过单采血获得来自个体的循环血液的细胞。单采制品通常含有淋巴细胞，包括T细胞、单核细胞、粒细胞、B细胞、其他有核白细胞、红细胞和血小板。在一个实施方案中，可以洗涤通过单采采集收集的细胞以除去血浆级分并将细胞置于合适的缓冲液或培养基中用于随后的加工步骤。或者，可以从健康供体，来自诊断患有癌症的患者衍生细胞。
在一些实施方案中，细胞可以是具有不同表型特征的混合细胞群体的一部分。还可以根据前述方法从转化的T细胞获得细胞系。还可以从细胞治疗库获得细胞。
在一些情况下，合适的原代细胞包括外周血单核细胞(PBMC)、外周血淋巴细胞(PBL)和其它血液细胞亚群，例如但不限于T细胞、天然杀伤细胞、单核细胞、天然杀伤剂T细胞、单核细胞前体细胞、造血干细胞或非多能干细胞。在一些情况下，细胞可以是任何T细胞如肿瘤浸润细胞(TIL)，如CD3+T细胞、CD4+T细胞、CD8+T细胞或任何其他类型的T细胞。T细胞还可以包括记忆T细胞、记忆干T细胞或效应T细胞。也可以从大量群体中选择T细胞，例如从全血中选择T细胞。T细胞也可以从大量群体中扩增。T细胞也可能倾向于特定种群和表型。例如，T细胞可以倾斜于表型包含CD45RO(-)、CCR7(+)、CD45RA(+)、CD62L(+)、CD27(+)、CD28(+)和/或IL-7Rα(+)。合适的细胞可以选自以下列表中的一种或多种标志物： CD45RO(-)、CCR7(+)、CD45RA(+)、CD62L(+)、CD27(+)、CD28(+)和/或IL-7Rα(+)。合适的细胞还包括干细胞，例如，例如胚胎干细胞、诱导的多能干细胞、造血干细胞、神经元干细胞和间充质干细胞。合适的细胞可以包含任何数量的原代细胞，例如人细胞、非人细胞和/或小鼠细胞。合适的细胞可以是祖细胞。合适的细胞可以衍生自要治疗的受试者(例如，患者)。
患者中需要的治疗有效的细胞的量可以根据细胞的存活力和细胞被遗传修饰的效率而变化(例如，转基因被整合到一个或多个细胞中的效率，或者由转基因编码的蛋白质的表达水平)。在一些情况下，遗传修饰后细胞存活力的产物(例如，倍增)和转基因整合的效率可以对应于可用于给予受试者的细胞的治疗量。在一些情况下，遗传修饰后细胞存活力的增加可能对应于给予治疗对患者有效的必需细胞量的减少。在一些情况下，转基因整合到一个或多个细胞中的效率的增加可以对应于给予在患者中治疗有效的必需的细胞数量的减少。在一些情况下，确定所需的治疗有效的细胞的量可以包括确定与细胞随时间变化相关的功能。在一些情况下，确定需要治疗有效的细胞的量可以包括确定与根据时间相关变量将转基因整合到一个或多个细胞中的效率变化相对应的功能(例如，细胞培养时间、电穿孔时间、细胞刺激时间)。在一些情况下，治疗有效的细胞可以是细胞群，其包含在细胞表面上约30％至约100％的嵌合受体的表达。在一些情况下，通过流式细胞术测量，治疗有效的细胞可以在细胞表面上表达所述嵌合受体约30％、35％、40％、45％、50％、55％、60％、65％、70％、75％80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％、99.9％或超过约99.9％。
药物组合物
本发明的T细胞可以应用于制备药物组合物。所述的药物组合物除了包括有效量的T细胞，还可包含药学上可接受的载体。术语“药学上可接受的”是指当分子本体和组合物适当地给予动物或人时，它们不会产生不利的、过敏的或其它不良反应。
可作为药学上可接受的载体或其组分的一些物质的具体例子是抗氧化剂；防腐剂；无热原水；等渗盐溶液；和磷酸盐缓冲液等。
本发明的组合物可根据需要制成各种剂型，并可由医师根据患者种类、年龄、体重和大致疾病状况、给药方式等因素确定对病人有益的剂量进行施用。给药方式例如可以采用肠胃外给药(如注射)或其它治疗方式。
组合物的“肠胃外”施用包括例如皮下(s.c.)、静脉内(i.v.)、肌内(i.m.)或胸骨内注射或输注技术。
给予个体的包含T细胞群体的制剂包含有效治疗和/或预防特定适应症或疾病的多个T 细胞。因此，可以向个体施用免疫反应性细胞的治疗有效群体。通常，施用包含约1×10 4至约1×10 10个免疫反应性细胞的制剂。在大多数情况下，制剂将包含约1×10 5至约1×10 9个免疫反应性细胞、约5×10 5至约5×10 8个免疫反应性细胞、或约1×10 6至约1×10 7个免疫反应性细胞。然而，根据癌症的位置、来源、身份、程度和严重程度、待治疗的个体的年龄和身体状况等，对个体施用的CAR免疫反应性细胞的数量将在宽的范围之间变化。医生将最终确定要使用的适当剂量。
在一些实施方案中，使用嵌合抗原受体来刺激免疫细胞介导的免疫应答。例如，T细胞介导的免疫应答是涉及T细胞活化的免疫应答。活化的抗原特异性细胞毒性T细胞能够在表面上显示外源抗原表位的靶细胞中诱导细胞凋亡，例如显示肿瘤抗原的癌细胞。在另一个实施方案中，使用嵌合抗原受体在哺乳动物中提供抗肿瘤免疫。由于T细胞介导的免疫应答，受试者将产生抗肿瘤免疫。
在某些情况下，治疗患有癌症的受试者的方法可以涉及向需要治疗的受试者施用一种或多种本发明所述的T细胞。所述T细胞可结合肿瘤靶分子并诱导癌细胞死亡。
如前文所述，本发明还提供治疗个体中的病原体感染的方法，包括向所述个体施用治疗有效量的本发明的T细胞。
与抗肿瘤药物联合
在一些实施方案中，本发明的T细胞可以与另一治疗剂联合给药。在一些实施方案中，所述另一治疗剂是化疗药。可以与本发明的T细胞联合应用的化疗药物包括但不限于有丝分裂抑制剂(长春花生物碱)，包括长春新碱、长春花碱、长春地辛和诺维宾(TM)(长春瑞滨，5'-去氢硫化氢)；拓扑异构酶I抑制剂，例如喜树碱化合物，包括CamptosarTM(伊立替康HCL)、HycamtinTM(托泊替康HCL)和衍生自喜树碱及其类似物的其它化合物；鬼臼毒素衍生物，例如依托泊苷、替尼泊苷和米多昔佐兹；烷基化剂顺铂、环磷酰胺、氮芥、三亚甲基硫代磷酰胺、卡莫司汀、白消安、苯丁酸氮芥、布列喹嗪、尿嘧啶芥末、氯洛芬和达卡巴嗪；抗代谢物，包括阿糖胞苷、氟尿嘧啶、甲氨蝶呤、巯嘌呤、硫唑嘌呤和丙卡巴肼；抗生素，包括但不限于多柔比星、博来霉素、更生霉素、柔红霉素、霉素霉素、丝裂霉素、肉瘤霉素C和道诺霉素；以及其它化疗药物，包括但不限于抗肿瘤抗体、达卡巴嗪、氮胞苷、阿姆沙康、美法仑、异环磷酰胺和米托蒽醌。
在一些实施方案中，可以与本发明的T细胞联合应用的化疗药物包括但不限于抗血管生成剂，包括抗VEGF抗体(包括人源化和嵌合抗体、抗VEGF适体和反义寡核苷酸)以及其他血管发生抑制剂，例如血管抑素、内皮抑制素、干扰素、视黄酸和金属蛋白酶-1和-2的组 织抑制剂。
试剂盒
本发明还提供了包含本发明T细胞的试剂盒。所述试剂盒可用于治疗或预防癌症、病原体感染、免疫病症或同种异体移植。在一个实施方案中，试剂盒可以包括含有有效量的包含一种或多种单位剂型的T细胞的治疗或预防组合物。
在一些实施方案中，试剂盒包含可含有治疗或预防性组合物的无菌容器。
在一些情况下，试剂盒可以包括约1×10 4个细胞至约1×10 6个细胞。在一些情况下，试剂盒可以包括至少约1×10 5个细胞，至少约1×10 6个细胞，至少约1×10 7个细胞，至少约4×10 7个细胞，至少约5×10 7个细胞，至少约6×10 7个细胞，至少约6×10 7个细胞，8×10 7个细胞，至少约9×10 7个细胞，至少约1×10 8个细胞，至少约2×10 8个细胞，至少约3×10 8个细胞，至少约4×10 8个细胞，至少约5×10 8个细胞，至少约6×10 8个细胞，至少约6×10 8细胞，至少约8×10 8个细胞，至少约9×10 8细胞，至少约1×10 9个细胞，至少约2×10 9个细胞，至少约3×10 9个细胞，至少约4×10 9个细胞，至少约5×10 9个细胞，至少约6×10 9个细胞，至少约8×10 9个细胞，至少约9×10 9个细胞，至少约1×10 10个细胞，至少约2×10 10个细胞，至少约3×10 10个细胞，至少约4×10 10个细胞，至少约5×10 10个细胞，至少约6×10 10个细胞，至少约9×10 10个细胞，至少约9×10 10个细胞，至少约1×10 11个细胞，至少约2×10 11个细胞，至少约3×10 11个细胞，至少约4×10 11个细胞，至少约5×10 11个细胞，至少约8×10 11个细胞，至少约9×10 11个细胞，或至少约1×10 12个细胞。例如，可以在试剂盒中包括大约5×10 10个细胞。
在一些情况下，试剂盒可以包括同种异体细胞。在一些情况下，试剂盒可以包括可以包含基因组修饰的细胞。在一些情况下，试剂盒可以包含“现成的”细胞。在一些情况下，试剂盒可以包括可以扩展用于临床使用的细胞。在某些情况下，试剂盒可能包含用于研究目的的内容物。
本发明的优点：
依据本发明的方法进行基因编辑，不仅编辑效率高，细胞存活率也很好。
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J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
示例性的，在下述实施例中，选择T细胞对本发明的方法进行说明。
T细胞的获得：由健康供者采集的外周血分离得到人外周血单个核细胞(PBMC)，加入偶联CD3/CD28抗体的beads进行活化，并进行培养和扩增，得到T细胞。
实施例一、靶向TRAC基因的sgRNA的设计和合成
针对TRAC(TCRαC,T细胞受体α恒定基因座)基因的第一个外显子(核苷酸序列如SEQ ID NO:1所示)，如图1所示，设计获得靶向TRAC基因的八条sgRNA序列sg-TRAC-1(SEQ ID NO:2)、sg-TRAC-2(SEQ ID NO:3)、sg-TRAC-3(SEQ ID NO:4)、sg-TRAC-4(SEQ ID NO:5)、sg-TRAC-5(SEQ ID NO:32)、sg-TRAC-6(SEQ ID NO:33)、sg-TRAC-7(SEQ ID NO:39)以及sg-TRAC-8(SEQ ID NO:40)。
选取sg-TRAC-1(SEQ ID NO:2)，sg-TRAC-2(SEQ ID NO:3)，sg-TRAC-3(SEQ ID NO:4)、sg-TRAC-5(SEQ ID NO:32)、sg-TRAC-6(SEQ ID NO:33)、sg-TRAC-7(SEQ ID NO:39)以及sg-TRAC-8(SEQ ID NO:40)进行实验。体外合成SEQ ID NO：20和21所示的引物，体外gRNA转录试剂盒(购自Thermo Fisher)，转录和扩增出sg-TRAC-1；体外合成SEQ ID NO：22和23所示的引物，体外gRNA转录试剂盒(购自Thermo Fisher)，转录和扩增出sg-TRAC-2；体外合成SEQ ID NO：24和25所示的引物，体外gRNA转录试剂盒(购自Thermo Fisher)，转录和扩增出sg-TRAC-3，体外合成SEQ ID NO：34和35所示的引物，体外gRNA转录试剂盒(购自Thermo Fisher)，转录和扩增出sg-TRAC-5，体外合成SEQ ID NO：36和37所示的引物，体外gRNA转录试剂盒(购自Thermo Fisher)，转录和扩增出sg-TRAC-6，体外合成SEQ ID NO：41和42所示的引物，体外gRNA转录试剂盒(购自Thermo Fisher)，转录和扩增出sg-TRAC-7，体外合成SEQ ID NO：43和44所示的引物，体外gRNA转录试剂盒(购自Thermo Fisher)，转录和扩增出sg-TRAC-8。
TRAC-exon 1序列(SEQ ID NO:1)：
Figure PCTCN2019107374-appb-000002
sg-TRAC-1(SEQ ID NO:2)：AGAGTCTCTCAGCTGGTACA
sg-TRAC-2(SEQ ID NO:3)：TCTCTCAGCTGGTACACGGC
sg-TRAC-3(SEQ ID NO:4)：GAGAATCAAAATCGGTGAAT
sg-TRAC-4(SEQ ID NO:5)：CTCTCAGCTGGTACACGGCA
sg-TRAC-5(SEQ ID NO:32)：GTCTCTCAGCTGGTACA
sg-TRAC-6(SEQ ID NO:33)：AGTCTCTCAGCTGGTACA
sg-TRAC-7(SEQ ID NO:39)：TTAGAGTCTCTCAGCTGGTACA
sg-TRAC-8(SEQ ID NO:40)：TTTAGAGTCTCTCAGCTGGTACA
实施例二、不同的Cas 9酶和sg-TRAC的比例对敲除效率的影响
取活化T细胞进行细胞计数，调整至细胞密度为2*10^7/ml。选取sg-TRAC-1(SEQ ID NO:2)作为sgRNA。
Cas 9酶(购自NEB)和sg-TRAC-1的摩尔比分别按1：2、1：3、1：4和1：5比例进行混合，形成RNP复合物。室温孵育10分钟后，加入到1*10^6T细胞中(Cas 9酶的终浓度为0.3μM)。其中，sg-TRAC-1的摩尔数基于gRNA的碱基组成和浓度为4.03μg/μl(OD260/OD280＝1.98)来计算。
利用BTX电转仪(美国哈佛仪器)将RNP复合物导入到T细胞中，电转参数为250V，5ms。转染后第5天，取T细胞进行CD3抗体(BD Biosciences)流式染色，以验证TCR敲除的效率。流式细胞术检测结果如图2和表1所示：Cas 9酶和sg-TRAC-1的摩尔比在1:3～1:5时，敲除效率都在70％以上。在Cas 9酶和sg-TRAC-1的摩尔比在1:4时，敲除效率最高，达到87.2％。表明当Cas 9酶和gRNA的摩尔为1:4时，具有最佳的基因敲除效果。
表1.不同组成比例的RNP对TCR敲除的结果统计
Figure PCTCN2019107374-appb-000003
实施例三、不同sgRNA对TRAC基因敲除的影响
选取靶向TRAC基因的3条不同sgRNA：sg-TRAC-1，sg-TRAC-2，sg-TRAC-3，
检测对TRAC基因敲除的影响。将实施例一合成的sg-TRAC-1、sg-TRAC-2、sg-TRAC-3三条sgRNA，分别与Cas9酶(0.5μM)按照4:1的比例混合形成RNP复合物，利用Maxcyte电转仪(Maxcyte公司)基于仪器设定参数进行电转，将RNP复合物导入T细胞中。转染后第5天，取T细胞进行CD3抗体(BD Biosciences)流式染色，以验证TCR敲除的效率。流式细胞术检测结果如图3和表2所示，sg-TRAC-1的敲除效果显著优于sg-TRAC-2和sg-TRAC-3，表明sg-TRAC-1具有最佳的敲除效果。同时测试不同长度sg-TRAC-1对敲 除效率的影响，分别合成了sg-TRAC-1(-3bp)(sg-TRAC-5)，sg-TRAC-1(-2bp)(sg-TRAC-6)，sg-TRAC-1(+3bp)(sg-TRAC-7)，sg-TRAC-1(+2bp)(sg-TRAC-8)四条sgRNA，分别与Cas9酶(0.5μM)按照摩尔比4:1的比例混合形成RNP复合物，采用上述条件导入T细胞中。转染后第5天，取T细胞进行CD3抗体流式染色，验证TCR敲除的效率。实验结果如图3b所示，截短2个或者3个碱基的sg-TRAC-1对TCR敲除效率的影响较小，而增加2个或者3个碱基会降低TCR的敲除效率，表明针对该位点的sgRNA长度设计可以有一定的变化，特别是截短3个以内的碱基同样能够达到比较高的敲除效果。
表2.不同gRNA序列对TCR敲除的结果统计
sgRNA sg-TRAC-1 sg-TRAC-2 sg-TRAC-3
KO效率(Day 5) 98.1％ 48.3％ 48.2％
需要指出的是，虽然在公开发表的文章中，sg-TRAC-1也能使TCR敲除效率达到90％，但该文章中采用的是经过优化，两次电转的敲除方法，过程相对较繁琐(参考Clin Cancer Res.2017 May 1；23(9):2255-2266，Fig1A中显示TCR敲除率95.7％)。而我们一次电转就能达到90％以上的敲除率，具有明显的优势。
实施例四、Cas 9酶的浓度对TRAC基因敲除的影响
选择sg-TRAC-1(SEQ ID NO:2)作为sgRNA，Cas 9酶和sg-TRAC-1的摩尔比在1:4时，分别设置不同浓度的Cas 9酶(0.0625μM,0.125μM,0.25μM,0.5μM)，检测对TRAC基因敲除的影响。
RNP复合物室温孵育10分钟后，利用Maxcyte电转仪(Maxcyte公司)基于仪器设定条件，将RNP复合物导入T细胞中。转染后第5天，取T细胞进行CD3抗体(BD Biosciences)流式染色，以验证TCR敲除的效率。
流式细胞术检测结果如图4和表3所示，当Cas9酶的浓度大于0.1μM时，TCR的敲除效率可以达到70％以上，如在0.125μM时，TCR的敲除效率可以达到75％以上；特别是在大于0.2μM时，TCR的敲除效率可以达到90％以上，如在0.25μM时，TCR的敲除效率可以达到94.5％以上；Cas9酶的浓度在0.3-0.5μM时，可以达到95％以上，Cas9酶的浓度在0.5μM时，TCR的敲除效率可以达到97.4％，而细胞存活率都在90％以上。
表3.不同浓度Cas9酶对TCR敲除的结果统计
Figure PCTCN2019107374-appb-000004
实施例五、靶向B2M基因的sgRNA的设计和合成
如图5所示，根据B2M基因的第一个外显子B2M-exon 1，核苷酸序列见SEQ ID NO：10，获取靶向B2M基因的四条sgRNA序列sg-B2M-1(SEQ ID NO：11)、sg-B2M-2(SEQ ID NO：12)、sg-B2M-3(SEQ ID NO：13)、sg-B2M-4(SEQ ID NO：14)。
选取sg-B2M-1、sg-B2M-2、sg-B2M-3进行实验，体外合成SEQ ID NO：26和27所示的引物，体外gRNA转录试剂盒(购自Thermo Fisher)，转录和扩增出sg-B2M-1；体外合成SEQ ID NO：28和29所示的引物，体外gRNA转录试剂盒(购自Thermo Fisher)，转录和扩增出sg-B2M-2；体外合成SEQ ID NO：30和31所示的引物，体外gRNA转录试剂盒(购自Thermo Fisher)，转录和扩增出sg-B2M-3。
B2M-exon 1序列(SEQ ID NO：10)：
Figure PCTCN2019107374-appb-000005
sg-B2M-1(SEQ ID NO：11)：GGCCACGGAGCGAGACATCT
sg-B2M-2(SEQ ID NO：12)：GAGTAGCGCGAGCACAGCTA
sg-B2M-3(SEQ ID NO：13)：CGCGAGCACAGCTAAGGCCA
实施例六、不同sgRNA序列对B2M基因敲除的影响。
针对实施例五得到的靶向B2M基因的sgRNA序列sg-B2M-1、sg-B2M-2、sg-B2M-3、对比对B2M基因敲除的影响。
Cas9酶(0.5μM)与gRNA按摩尔比1：4混合形成RNP复合物后，利用Maxcyte电转仪(Maxcyte公司)，基于仪器的设定条件，将RNP复合物导入T细胞中。转染后第5天，取T细胞进行β-微球蛋白抗体(BD Biosciences)流式染色，以验证B2M敲除的效率。流式结果如图6和表4所示，sg-B2M-1和sg-B2M-2的敲除效果达到90％以上，显著优于sg-B2M-3，表明sg-B2M-1和sg-B2M-2均具有很好的敲除效果。
表4.不同gRNA序列对B2M敲除的结果统计
gRNA sg-B2M-1 sg-B2M-2 sg-B2M-3
KO效率(Day 5) 95.0％ 90.0％ 67.0％
使用相同的sg-B2M-1序列，公开报道的敲除率只能达到50-60％(Nature.2017 Mar 2；543(7643):113-117，Fig3c显示B2M敲除率为55％)，经过我们的方法优化后，大大提高B2M的敲除率，能够达到95％。
实施例七、Cas 9酶的浓度对敲除效率的影响
选取sg-B2M-2作为sgRNA，在Cas 9酶和sgRNA的比例为1：4的情况下，分别设置不同浓度的Cas 9酶(0.125μM,0.25μM,0.5μM，1.0μM,2.0μM,3.0μM,)，检测对B2M基因敲除的影响。RNP复合物室温孵育10分钟后，利用Maxcyte电转仪(Maxcyte公司)基于仪器的电转条件，将RNP复合物导入T细胞中，在转染后第5天，取T细胞进行B2M抗体(BD Biosciences)流式染色，以验证B2M敲除的效率。流式细胞术检测
结果如图7和表5所示，当Cas9酶的浓度大于0.2μM时，敲出效率可达70％以上，如在0.25μM，敲出效率为72.2％；当Cas9酶的浓度不低于1μM时，敲除效率可达90％以上，如在1μM-3μM时均显示出好的敲除效率，特别是在1μM-2μM时，敲除效率都在93％左右。
表5.不同浓度Cas9酶对B2M敲除的结果统计
Figure PCTCN2019107374-appb-000006
实施例八、T细胞中同时高效地敲除TRAC和B2M基因
1、gRNA组成成分的比例的影响
目前已有的报道中，TCR和B2M双敲除的效率最高只有60％左右(参考Clin Cancer Res.2017 May 1；23(9):2255-2266中的图3b和Oncotarget,2017,Vol.8,(No.10),pp:17007-17011中的Fig3a)。因此，在本实施例中希望进一步利用上述优化的方法筛选出高效双敲除B2M和TCR的组合。
为了检测出sg-TRAC-1和sg-B2M-2之间的比例对TRAC和B2M基因双敲除的影响，按照Cas9酶与总gRNA摩尔比为1：4时，分别设定sg-B2M-2和sg-TRAC-1比例为1.5:1，1:1和0.5:1，检测对基因敲除的影响。利用Maxcyte电转仪(Maxcyte公司)将RNP复合物导入T 细胞后，于第5天进行CD3抗体和B2M抗体(BD Biosciences)流式染色。流式细胞术检测结果如图8和表6所示，当sg-B2M-2和sg-TRAC-1比例为1:1时，TRAC和B2M基因双敲除的效果最佳。
表6.不同gRNA组成成分对TRAC和B2M双敲除的影响
gRNA比例 1.5:1 1:1 0.5:1
KO效率(Day 5) 74.3％ 93.0％ 82.7％
2、RNP浓度的优化。
为了摸索靶向TRAC和B2M基因组成的gRNA混合物与Cas9酶之间形成的RNP复合物浓度，按照优化好的Cas9酶与gRNA摩尔比为1:4时，分别设定不同浓度的Cas 9酶(0.25μM,0.5μM,1.0μM,2.0μM，3.0μM)，检测对基因敲除的影响。利用Maxcyte电转仪(Maxcyte公司，)将RNP复合物导入T细胞后，于第5天进行CD3抗体和B2M抗体(BD Biosciences)流式染色。流式细胞术检测结果如构图9和表7所示，在Cas 9酶的终浓度不低于1μM时，TRAC和B2M双敲除的效果均能达到90％以上，且在3μM时达到93.4％。
表7.不同浓度Cas9酶对TRAC和B2M双敲除的结果统计
Figure PCTCN2019107374-appb-000007
实施例九、T细胞中同时敲除TRAC和B2M基因的分子水平的验证
1.Tide方法验证TRAC和B2M基因敲除。
T细胞中分别提取TRAC、B2M单个基因及两个基因敲除的基因组DNA，利用PCR扩增包含敲除位点片段的基因片段。PCR产物经凝胶电泳后进行纯化和回收，进行测序，对照组PCR产物中TRAC和B2M基因的测序结果为单一的峰，而进行了敲除组中，TRAC和B2M基因的测序结果会对应的出现多条套峰，表明TRAC和B2M基因发生了突变。
将测序结果提交https://tide.deskgen.com/网站进行分析，得到预测的突变效率，结果如图10所示，表明TCR和B2M得到了有效敲除。
2、克隆测序验证TRAC和B2M基因敲除。
T细胞中分别提取TRAC、B2M单个基因及两个基因敲除的基因组DNA，利用PCR扩 增包含敲除位点片段的基因片段。PCR产物经凝胶电泳后进行纯化和回收，连接至T载体，转化，随机挑取单克隆菌落进行测序鉴定。如图11所示，挑取的克隆
经测序比对，相对于TRAC和B2M的原始序列，敲除组的序列均出现了碱基的缺失或者插入，表明TCR和B2M基因均发生了突变。
实施例十：BCMA CAR-T细胞高效敲除TRAC和B2M基因
进一步地，我们利用制备的BCMA CAR-T细胞测试了TRAC和B2M双基因敲除的效果。
1.靶向BCMA-CAR-T细胞的制备。参考中国发明专利201810065525.1，设计并构建包含抗BCMA的嵌合抗原受体，T细胞共刺激因子41-BB，T细胞激活因子CD3ζ的CAR载体，并包装慢病毒，命名为PRRL-BCMA-BBZ(TM)。T细胞活化与扩增48小时后，调整细胞密度至2*10^6/mL，按MOI＝4的比例加入PRRL-BCMA-BBZ(TM)慢病毒，24小时后进行换液，得到靶向BCMA CAR-T细胞。
2.靶向BCMA CAR-T细胞中TCR和B2M基因的敲除。体外扩增CAR-T细胞48小时后，调整细胞密度至2*10^7/mL。分别将sg-TRAC-1，sg-B2M-2以及sg-TRAC-1/sg-B2M-2混合物，按Cas 9酶和gRNA按1：4比例进行室温孵育10分钟，将1*10^6细胞与RNP进行混合(Cas 9酶的终浓度为3μM),利用maxcyte电转仪将RNP复合物导入到CAR-T细胞中。分别于24小时，48小时以及72小时检测细胞存活状态(表8)，电转后CAR-T细胞恢复良好。于电转后第5天，利用流式细胞术检测TRAC和B2M基因的敲除情况，TRAC和B2M单基因以及双基因敲除均达到90％以上，表明高效实现了TCR和B2M双敲除(参见图12)。
表8.CAR-T细胞电转后细胞活率
Figure PCTCN2019107374-appb-000008
本文所使用的序列如下:
Figure PCTCN2019107374-appb-000009
Figure PCTCN2019107374-appb-000010

Claims (30)

  1. 一种基于CRISPR/Cas系统对细胞进行基因编辑的方法，其特征在于，将Cas酶和gRNA的复合物导入所述细胞中进行基因编辑，其中，所述Cas酶为Cas9酶，所述Cas9酶的酶活力为0.1～1nmol，优选为0.2～0.7nmol，进一步优选为0.3～0.5nmol；
    进一步优选在所述复合物中，Cas9酶和gRNA的摩尔比比例为1:1～1:10，优选为1:3～1:5，进一步优选为1:4。
  2. 如权利要求1所述的方法，其特征在于，
    将Cas9酶和第一gRNA的复合物一以及Cas9酶和第二gRNA的复合物二导入所述细胞中进行基因编辑，
    优选将Cas9酶、第一gRNA和第二gRNA的复合物三同时导入所述细胞中进行基因编辑，
    其中，在所述复合物一或复合物二或复合物三中，Cas9酶和gRNA的摩尔比比例为1:1～1:10，优选为1:3～1:5，进一步优选为1:4。
  3. 如权利要求2所述的方法，其特征在于，所述第一gRNA和第二gRNA的摩尔比比例约为1:5～5:1，优选为1:2～2:1；进一步优选约为1:1。
  4. 如权利要求1-3任一所述的方法，其特征在于，在所述Cas9酶和gRNA形成的复合物或复合物一或复合物二或复合物三中，所述Cas9酶的浓度约为0.1μM～3μM；优选的，约为0.125μM～3μM；更优选的，约为0.2μM～3μM；更优选的，约为0.25μM～3μM；更优选的，约为0.5μM～3μM；更优选的，约为1μM～3μM。
  5. 如权利要求1-4任一所述的方法，其特征在于，所述细胞为真核细胞；优选所述真核细胞是免疫效应细胞；优选所述免疫效应细胞是T细胞。
  6. 如权利要求5所述的方法，其特征在于，所述细胞为T细胞，利用所述CRISPR/Cas9系统对所述T细胞的基因进行编辑；包括：
    利用所述CRISPR/Cas9系统对所述T细胞的TCR的α链和β链中的任意一条或者两条链的基因进行基因编辑；优选对TRAC进行基因编辑；进一步优选对TRAC的恒定区进行基因编辑；进一步优选对TRAC中如SEQ ID NO：45所示的序列进行基因编辑；进一步优选对TRAC中包含有SEQ ID NO：1所示的序列进行基因编辑，和/或
    利用所述CRISPR/Cas9系统对所述T细胞的MHC基因进行基因编辑，优选对B2M基因进行基因编辑，进一步优选对B2M基因中如SEQ ID NO：38所示的序列进行基因编辑，进 一步优选对B2M基因中包含有SEQ ID NO：10所示的序列进行基因编辑。
  7. 如权利要求1-6任一所述的方法，其特征在于，所述gRNA约为15-50bp，优选的，约为15-30bp，更优选的为约17-21bp；更优选为20bp。
  8. 如权利要求7所述的方法，其特征在于，对TRAC的进行编辑采用的gRNA为包含有SEQ ID NO：2、3、4、5、32、33、39或40所示的序列；优选的，采用的gRNA为包含有SEQ ID NO：2、32或33所示的序列。
  9. 如权利要求7所述的方法，其特征在于，对B2M基因进行编辑采用的gRNA包含有SEQ ID NO：11、12、13、或14所示的序列；优选的，采用的gRNA包含有SEQ ID NO：12所示的序列。
  10. 如权利要求5-9任一所述的方法，其特征在于，所述的T细胞还表达有嵌合受体、外源性的细胞因子、抑制性/激活性受体或配体，共刺激因子；优选所述T细胞还表达有嵌合抗原受体。
  11. 一种构建通用型T细胞的方法，包括：
    通过基因编辑技术对T细胞的TCR基因和MHC基因进行基因编辑，其中，
    对T细胞的TCR基因进行基因编辑优选对T细胞的TCR的α链和β链中的任意一条或者两条链的基因进行基因编辑，优选对TRAC基因进行基因编辑，进一步优选对TRAC的恒定区进行基因编辑，进一步优选对TRAC中如SEQ ID NO：45所示的序列进行基因编辑，进一步优选对TRAC中包含有SEQ ID NO：1所示的序列进行基因编辑；以及
    对T细胞的MHC基因进行基因编辑，优选对T细胞的B2M基因进行基因编辑，进一步优选对B2M基因中如SEQ ID NO：38所示的序列进行基因编辑，进一步优选对B2M基因中包含有SEQ ID NO：10所示的序列进行基因编辑。
  12. 如权利要求11所述的方法，其中，所述基因编辑技术为CRISPR/Cas9基因编辑技术。
  13. 如权利要求11或12所述的方法，其中，
    将包含选自SEQ ID NO：2、3、4、5、32、33、39或40所示的序列的gRNA导入所述T细胞以实现对T细胞的TRAC基因进行基因编辑，优选将包含有SEQ ID NO：2、32或33所示的序列的gRNA导入所述T细胞以实现对T细胞的TRAC基因进行基因编辑。
  14. 如权利要求12或13所述的方法，其中，
    将包含选自SEQ ID NO：11、12、13、或14所示的序列的gRNA导入所述T细胞以实现对T细胞的MHC基因进行基因编辑，优选将包含有SEQ ID NO：12所示的序列的gRNA导入所述T细胞以实现对T细胞的MHC基因进行基因编辑。
  15. 如权利要求11-14中任一项所述的方法，其中，
    通过基因编辑技术对T细胞的TCR基因进行基因编辑通过将Cas9酶和gRNA的第一复合物一导入所述细胞中进行基因编辑，
    通过基因编辑技术对T细胞的MHC基因进行基因编辑通过将Cas9酶和gRNA的第二复合物导入所述细胞中进行基因编辑，
    优选，复合物一和复合物二同时以复合物三的形式导入所述细胞中进行基因编辑。
  16. 如权利要求15所述的方法，其中，
    在所述复合物一或复合物二中Cas9酶和gRNA的摩尔比比例为1:1～1:10，优选为1:3～1:5，进一步优选Cas9酶和gRNA的摩尔比比例为1:4。
  17. 如权利要求15所述的方法，其中，
    在所述复合物一或复合物二或复合物三中，所述Cas9酶的浓度约为0.1μM～3μM；优选的，约为0.125μM～3μM；更优选的，约为0.2μM～3μM；更优选的，约为0.25μM～3μM；更优选的，约为0.5μM～3μM，更优选地约为1μM～3μM，更优选地约为0.125μM～0.5μM，更优选的，约为0.25μM～0.5μM。
  18. 如权利要求11-17任一所述的方法，其中，
    对TCR基因进行基因编辑gRNA和对MHC基因进行基因编辑的gRNA的摩尔比比例约为1:5～5:1，优选为1:2～2:1；进一步优选约为1:1。
  19. 如权利要求11-18任一所述的方法，其特征在于，所述T细胞还表达有嵌合抗原受体，优选所述T细胞还表达有识别肿瘤抗原或病原体抗原的嵌合受体，该嵌合受体具有胞外抗原结合域、跨膜域、和胞内域，所述胞外抗原结合域特异性识别靶抗原；
    优选所述靶抗原为肿瘤抗原，该肿瘤抗原选自：促甲状腺激素受体(TSHR)；CD171；CS-1；C型凝集素样分子-1；神经节苷脂GD3；Tn抗原；CD19；CD20；CD 22；CD 30；CD 70；CD 123；CD 138；CD33；CD44；CD44v7/8；CD38；CD44v6；B7H3(CD276)，B7H6；KIT(CD117)；白介素13受体亚单位α(IL-13Rα)；白介素11受体α(IL-11Rα)；前列腺干细胞抗原(PSCA)；前列腺特异性膜抗原(PSMA)；癌胚抗原(CEA)；NY-ESO-1；HIV-1Gag；MART-1；gp100；酪氨酸酶；间皮素；EpCAM；蛋白酶丝氨酸21(PRSS21)；血管内皮生长因子受体；路易斯(Y)抗原；CD24；血小板衍生生长因子受体β(PDGFR-β)；阶段特异性胚胎抗原-4(SSEA-4)；细胞表面相关的粘蛋白1(MUC1)，MUC6；表皮生长20因子受体家族及其突变体(EGFR，EGFR2，ERBB3，ERBB4，EGFRvIII)；神经细胞粘附分子(NCAM)；碳酸酐酶IX(CAIX)； LMP2；肝配蛋白A型受体2(EphA2)；岩藻糖基GM1；唾液酸基路易斯粘附分子(sLe)；邻乙酰基GD2神经节苷脂(OAcGD2)；神经节苷脂GM3；TGS5；高分子量黑素瘤相关抗原(HMWMAA)；叶酸受体；肿瘤血管内皮标记25 1(TEM1/CD248)；肿瘤血管内皮标记7相关的(TEM7R)；Claudin6，Claudin18.2(CLD18A2)、Claudin18.1；ASGPR1；CDH16；5T4；8H9；αvβ6整合素；B细胞成熟抗原(BCMA)；CA9；κ轻链(kappa light chain)；CSPG4；EGP2，EGP40；FAP；FAR；FBP；胚胎型AchR；HLA-A1，HLA-A2；MAGEA1，MAGE3；KDR；MCSP；NKG2D配体；PSC1；ROR1；Sp17；SURVIVIN；TAG72；TEM1；纤连蛋白；腱生蛋白；肿瘤坏死区的癌胚变体；G蛋白偶联受体C类5组-成员D(GPRC5D)；X染色体开放阅读框61(CXORF61)；CD97；CD179a；间变性淋巴瘤激酶(ALK)；聚唾液酸；胎盘特异性1(PLAC1)；globoH glycoceramide的己糖部分(GloboH)；乳腺分化抗原(NY-BR-1)；uroplakin 2(UPK2)；甲型肝炎病毒细胞受体1(HAVCR1)；肾上腺素受5体β3(ADRB3)；pannexin 3(PANX3)；G蛋白偶联受体20(GPR20)；淋巴细胞抗原6复合物基因座K9(LY6K)；嗅觉受体51E2(OR51E2)；TCRγ交替阅读框蛋白(TARP)；肾母细胞瘤蛋白(WT1)；ETS易位变异基因6(ETV6-AML)；精子蛋白17(SPA17)；X抗原家族成员1A(XAGE1)；血管生成素结合细胞表面受体2(Tie2)；黑素瘤癌睾丸抗原-1(MAD-CT-1)；黑素瘤癌睾丸抗原-2(MAD-CT-2)；Fos相关抗原1；p53突变10体；人端粒酶逆转录酶(hTERT)；肉瘤易位断点；细胞凋亡的黑素瘤抑制剂(ML-IAP)；ERG(跨膜蛋白酶丝氨酸2(TMPRSS2)ETS融合基因)；N-乙酰葡糖胺基转移酶V(NA17)；配对盒蛋白Pax-3(PAX3)；雄激素受体；细胞周期蛋白B1；V-myc鸟髓细胞瘤病病毒癌基因神经母细胞瘤衍生的同源物(MYCN)；Ras同源物家族成员C(RhoC)；细胞色素P4501B1(CYP1B1)；CCCTC结合因子(锌指蛋白)样(BORIS)；由T细胞识别的鳞状细胞癌抗原3(SART3)；配对盒蛋白Pax-5(PAX5)；proacrosin结合蛋白sp32(OYTES1)；淋巴细胞特异性蛋白酪氨酸激酶(LCK)；A激酶锚定蛋白4(AKAP-4)；滑膜肉瘤X断点2(SSX2)；CD79a；CD79b；CD72；白细胞相关免疫球蛋白样受体1(LAIR1)；IgA受体的Fc片段(FCAR)；白细胞免疫球蛋白样受体亚家族成员2(LILRA2)；CD300分子样家族成员f(CD300LF)；C型凝集素结构域家族12成员A(CLEC12A)；骨髓基质细胞抗原2(BST2)；含有EGF样模块粘蛋白样激素受体样2(EMR2)；淋巴细胞抗原75(LY75)；磷脂酰肌醇蛋白聚糖-3(GPC3)；Fc受体样5(FCRL5)；免疫球蛋白λ样多肽1(IGLL1)；
    优选所述靶抗原为病原体抗原，该病原体抗原选自：病毒、细菌、真菌、原生 动物、或寄生虫的抗原；在一实施例中，所述病毒抗原选自：巨细胞病毒抗原、爱泼斯坦-巴尔病毒抗原、人类免疫缺陷病毒抗原或流感病毒抗原。
  20. 如权利要求19所述的方法，其特征在于，所述嵌合受体为嵌合抗原受体(CAR)。
  21. 如权利要求20所述的方法，其特征在于，所述嵌合抗原受体包括：
    (i)特异性结合肿瘤抗原的抗体或其片段、CD28或者CD8的跨膜区、CD28的共刺激信号结构域、和CD3ζ；或
    (ii)特异性结合肿瘤抗原的抗体或其片段、CD28或者CD8的跨膜区、CD137的共刺激信号结构域、和CD3ζ；或
    (iii)特异性结合肿瘤抗原的抗体或其片段、CD28或者CD8的跨膜区、CD28的共刺激信号结构域、CD137的共刺激信号结构域、和CD3ζ。
  22. 如权利要求20所述的方法，其特征在于，所述嵌合抗原受体的特异性结合肿瘤抗原的抗体为全长抗体、scFv、Fab、(Fab’)、或单域抗体。
  23. 根据权利要求11-22任一所述的方法制备得到的T细胞的用途，用于制备表达有嵌合受体的T细胞，所述嵌合受体具有胞外抗原结合域、跨膜域、和胞内域，所述胞外抗原结合域特异性识别靶抗原。
  24. 一种通用型T细胞，其是通过权利要求11-22中任一项所述的方法构建的。
  25. 一种通用型T细胞，其TRAC和/或B2M基因被沉默。
  26. 如权利要求25所述的T细胞，其中，TRAC基因被沉默是对包含有SEQ ID NO：1所示的序列进行基因编辑而实现的，进一步优选TRAC基因被沉默是对包含有SEQ ID NO：1所示的序列中的如SEQ ID NO：45所示的序列进行基因编辑而实现的；
    B2M基因被沉默是对包含有SEQ ID NO：10所示的序列进行基因编辑而实现的，进一步优选B2M基因被沉默是对包含有SEQ ID NO：10所示的序列中的如SEQ ID NO：38所示的序列进行基因编辑而实现的。
  27. 如权利要求25所述的T细胞，其中，TRAC基因被沉默是利用如SEQ ID NO：2、32或33所示的序列的gRNA对TRAC基因进行基因编辑而实现的，
    B2M基因被沉默是利用如SEQ ID NO：12所示的序列的gRNA对B2M基因进行基因编辑而实现的。
  28. 如权利要求24-27任一所述的T细胞，其中，所述T细胞还表达有嵌合抗原受体，优选所述T细胞还表达有识别肿瘤抗原或病原体抗原的嵌合受体，该嵌合受体具有胞外抗原结合域、跨膜域、和胞内域，所述胞外抗原结合域特异性识别靶抗原。
  29. 一种gRNA构建体，其包含选自SEQ ID NO：2、3、4、5、32、33、39、40、11、12、13、或14之一的核苷酸序列。
  30. 根据权利要求20所述的gRNA构建体，其包含：
    选自SEQ ID NO：2、3、4、5、32、33、39或40之一的核苷酸序列，以及
    选自SEQ ID NO：11、12、13、或14之一的核苷酸序列。
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