WO2018233596A1 - 体外敲除T细胞中靶基因的方法以及该方法中使用的crRNA - Google Patents

体外敲除T细胞中靶基因的方法以及该方法中使用的crRNA Download PDF

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WO2018233596A1
WO2018233596A1 PCT/CN2018/091804 CN2018091804W WO2018233596A1 WO 2018233596 A1 WO2018233596 A1 WO 2018233596A1 CN 2018091804 W CN2018091804 W CN 2018091804W WO 2018233596 A1 WO2018233596 A1 WO 2018233596A1
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gene
crrna
cell
cells
targeting
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PCT/CN2018/091804
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English (en)
French (fr)
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彭作翰
沈连军
陶维康
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Priority to CN201880004422.2A priority Critical patent/CN109963944A/zh
Priority to MX2019014516A priority patent/MX2019014516A/es
Priority to RU2020100919A priority patent/RU2020100919A/ru
Priority to AU2018288048A priority patent/AU2018288048A1/en
Priority to KR1020207000299A priority patent/KR20200018572A/ko
Priority to EP18820610.6A priority patent/EP3650545A4/en
Priority to CA3064807A priority patent/CA3064807A1/en
Priority to US16/623,605 priority patent/US20200181608A1/en
Priority to JP2019570534A priority patent/JP2020528738A/ja
Publication of WO2018233596A1 publication Critical patent/WO2018233596A1/zh

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Definitions

  • the invention belongs to the field of biomedicine. Specifically, it relates to a method for knocking out a target gene in a T cell in vitro, a crRNA used in the method, and a T cell obtained by the method and use thereof.
  • Adoptive cell therapy which involves the transfer of autologous antigen-specific T cells produced ex vivo, is a promising strategy for the treatment of viral infections and cancer.
  • T cells used in adoptive immunotherapy can be generated by amplification of antigen-specific T cells or by genetically designed T cell redirection (Park, Rosenberg et al. Trends Biotechnol. 2011, 29(11): 550-557).
  • CART is an isolated T cell that is genetically engineered into a specific antigen receptor (CAR) to enhance the targeting, killing activity and persistence of T cells, and the recognition of tumor cell surface antigens is not dependent on MHC.
  • CARs consist of extracellular antigen binding regions, transmembrane regions, and signal transduction regions of intracellular T cell receptors (such as CD3 ⁇ and costimulatory molecules).
  • the extracellular antigen binding region is composed of a light chain variable region (VL) and a heavy chain variable region (VH) of a monoclonal antibody, and is hinged to form a single chain fragment variable (scFv), which is capable of recognizing a specific Tumor antigen.
  • CAR has a better therapeutic effect in patients with lymphoma who are ineffective in other treatments.
  • the CART-19 study conducted by Carl June of the University of Pennsylvania showed that in 75 patients with leukemia (including adults and children), 45 patients had complete remission after CART cell therapy.
  • Cellectis has successfully eradicated multiple cases of relapsed acute lymphoblastic leukemia (ALL) by directional knockout of TCR ⁇ gene (reduced GVHD) and CD52 gene (resistance of cells to alemtuzumab) by TALEN technology. child.
  • ALL relapsed acute lymphoblastic leukemia
  • CD52 resistance of cells to alemtuzumab
  • Cellectis knocking out TCR through TALEN requires a cumbersome build process and large-scale sequencing.
  • CRISPR-associated, CRISPR-Cas9 achieves editing of genes by recognizing specific DNA sequences and is simpler and more efficient than TALEN.
  • the object of the present invention is to overcome the problems existing in the prior art in immunotherapy, and to provide a method for constructing TCR-negative T cells, which knocks out TCR by CRISPR/Cas9 gene editing technology, and provides TCR negative T obtained by the method. cell.
  • Another object of the present invention is to provide a TCR, B2M and PD1 triple negative T cell and a method of constructing the same.
  • TCR-negative T cells TCR and PD-1 or B2M double-negative T cells and TCR/B2M/PD1 triple-negative T cells are sorted by magnetic beads, and are used for adoptive cell immunotherapy of tumors and the like.
  • a method of knocking out one or more target genes in a T cell in vitro comprising the steps of:
  • RNP protein RNA complex
  • sgRNA directs the Cas9 protein to a target sequence of the corresponding target gene, respectively, and the target Sequence hybridization wherein the target gene is cleaved, and wherein the cleavage efficiency of the target gene is greater than 75%.
  • the target gene is selected from one or more of the TRAC, TRBC, B2M and PD1 genes,
  • the sgRNA targets a coding sequence of the target gene or a regulatory sequence thereof.
  • the sgRNA is sequentially sequenced from 5' to 3' by a 17-20 nt target target gene of crRNA and Cas9 protein Corresponding tracrRNAs are ligated, wherein the length of the crRNA is preferably 17 nt.
  • the oligodeoxyribonucleic acid in the method of knocking out one or more target genes in T cells in vitro, is double-stranded DNA having a length of 100-250 bp or a length of 100 -250 nt of single-stranded DNA.
  • the crRNA targeting the TRAC gene is selected from the group consisting of the crRNAs shown in SEQ ID NOs: 1-12 Any one or more of the same, the crRNA sequence targeting the B2M gene is set forth in SEQ ID NO: 13, and the crRNA targeting the PD1 gene is selected from any one of the crRNAs shown in SEQ ID NOs: 14-16. Kind or more.
  • the Cas9 protein is a Cas9 protein from Streptococcus pyogenes, the sequence is SEQ ID NO: 18. Shown.
  • the Cas9 protein in the method of knocking out one or more target genes in T cells in vitro, is a Cas9 protein from Streptococcus pyogenes, and the tracrRNA corresponding to the Cas9 protein The sequence is shown in SEQ ID NO: 17.
  • the T cells are selected from the group consisting of helper T cells, cytotoxic T cells, memory T cells, Regulatory T cells, natural killer T cells, ⁇ T cells, CAR-T cells, and TCR-T cells.
  • the present invention provides a target gene knockout T cell obtained according to the above method.
  • the invention provides a crRNA for knocking out a targeted gene, wherein the crRNA comprises one or more sequences selected from the group consisting of SEQ ID NOs: 1-16.
  • the present invention is for knocking out a coding gene of a target gene, a coding sequence of a targeted gene, or a regulatory sequence thereof, wherein the targeted gene is selected from the group consisting of TRAC, TRBC, B2M, and One or more of the PD1 genes.
  • the invention is for knocking out a crRNA of a targeted gene, wherein the targeting gene is a TRAC gene, and the crRNA is selected from one of SEQ ID NOs: 1-12 or Multiple.
  • the invention is for knocking out a crRNA of a targeted gene, wherein the targeting gene is a B2M gene, and the crRNA sequence is set forth in SEQ ID NO: 13.
  • the invention is for knocking out a crRNA of a targeted gene, wherein the targeting gene is a PD1 gene, and the crRNA is selected from one of SEQ ID NOs: 14-16 or Multiple.
  • the present invention also provides an sgRNA for knocking out a targeting gene, the sgRNA being formed by linking a crRNA to a tracrRNA corresponding to a Cas9 protein, wherein the crRNA comprises one or more selected from the group consisting of SEQ ID NO: 1.
  • the sequence of -16 is a sequence of -16.
  • the invention is for knocking out a sgRNA of a targeted gene, wherein the targeting gene is selected from one or more of the TRAC, TRBC, B2M and PD1 genes.
  • the present invention is for knocking out a sgRNA targeting a gene TRAC, which is formed by linking a crRNA to a tracrRNA corresponding to a Cas9 protein selected from the group consisting of SEQ ID NO: 1-12 One or more of the ones shown.
  • the invention is used to knock out a sgRNA targeting the gene B2M, which is formed by the tracrRNA junction of the crRNA and the Cas9 protein, as shown in SEQ ID NO: 13.
  • the present invention is for knocking out an sgRNA targeting the gene PD-1, which is formed by linking a crRNA to a tracrRNA corresponding to a Cas9 protein selected from the group consisting of SEQ ID NO: 14. One or more of the -16.
  • the invention is for knocking out a sgRNA of a targeted gene, wherein the Cas9 protein is a Cas9 protein from S. pyogenes as set forth in SEQ ID NO: 18.
  • the invention is for knocking out a sgRNA targeting a gene, wherein the tracrRNA sequence corresponding to the Cas9 protein is set forth in SEQ ID NO: 17.
  • the invention provides a kit for gene knockout comprising:
  • a one or more crRNAs as described above, or one or more sgRNAs as described above;
  • c oligodeoxyribonucleic acid or fish sperm DNA fragment.
  • the oligodeoxyribonucleic acid in the kit for gene knockout, is double-stranded DNA of 100-250 bp in length or single-stranded DNA of 100-250 nt in length. .
  • the Cas9 protein is a Cas9 protein from Streptococcus pyogenes, and the tracrRNA sequence corresponding to the Cas9 protein is SEQ ID NO: 17. Shown.
  • the invention provides the use of a knockout T cell of the invention for the preparation of an anti-tumor drug.
  • the present invention also provides the use of the knockout T cells of the present invention for the preparation of a medicament for controlling infectious diseases caused by viruses or bacteria.
  • TCR, B2M or PD1 are effectively knocked out using the designed crRNA and method.
  • the in vitro killing activity of CART cells after TCR and B2M and/or PD1 knockout is not affected by TCR, B2M and/or PD1 gene knockout.
  • FIG. 1 Comparison of knockout efficiency for different delivery systems. The results show that the RNP delivery mode has the highest gene knockout efficiency in Jurkat cells.
  • FIG. 2A-2B Effect of N-oligo on the knockdown efficiency of T cell genes based on the CRISPR-Cas9 system.
  • Figure 2A is a comparison of gene knockout efficiencies in T cells;
  • Figure 2B is a comparison of gene knockout efficiencies in CART cells.
  • Figure 3 Effect of fish sperm DNA fragments on T cell gene knockout efficiency.
  • Figure 5 Detection of the knockdown effect of the PD1 gene by the screened crRNA.
  • Figures 6A-6B Analysis of gene mutations caused by RNP and N-Oligo or fish sperm DNA.
  • Fig. 6A is an analysis result for TRAC
  • Fig. 6B is an analysis result for B2M.
  • Figures 7A-7C RNP off-target rate analysis.
  • Fig. 7A shows the results of the off-target analysis of the TRAC gene;
  • Fig. 7B shows the results of the off-target analysis of the B2M gene;
  • Fig. 7C shows the results of the off-target analysis of the PD1 gene.
  • Figures 8A-8B Analysis of CD25 and CD69 activation of TRAC knockout T cells.
  • Figure 8A is a comparison of CD69 activation and
  • Figure 8B is a comparison of CD25 activation.
  • the invention provides a method of altering a target gene in a cell.
  • the studies described herein demonstrate the use of the allele targeting approach of the CRISPR/Cas system to produce mutant cells with efficiencies as high as 80%.
  • the work described herein surprisingly and unexpectedly demonstrates a multiplex guide strategy that provides a method for specifically identifying useful RNA leader sequences, as well as for targeting specific genes (eg, TRAC, TRBC, B2M) , PD1) specific boot sequence.
  • An exemplary method of altering a polynucleotide sequence of a target gene in a cell comprises correlating the polynucleotide sequence with a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) sequence (Cas)
  • the protein is contacted with one or two ribonucleic acids to form an RNP, wherein the ribonucleic acid directs the Cas protein to a target motif of a polynucleotide sequence of the target gene and hybridizes to the target motif, wherein the target gene
  • the polynucleotide sequence was cleaved, and the efficiency of the cells in which the RNP was transformed was 75% or more.
  • the present invention contemplates altering the polynucleotide sequence of a target gene in any manner that is readily available to the skilled artisan using the CRISPR/Cas system of the present invention.
  • Any CRISPR/Cas system capable of altering the polynucleotide sequence of a target gene in a cell can be used.
  • a variety of Cas proteins can be used in this type of CRISPR/Cas system (Haft et al. PLoS Comput Biol. 2005; 1(6) e60).
  • Such Cas proteins allow the CRISPR/Cas system to alter the molecular sequences of the polynucleotide sequences of the target genes in the cell, including RNA binding proteins, endonucleases and exonucleases, helicases, and polymerases.
  • the CRISPR/Cas system is a CRISPR Type I system.
  • the CRISPR/Cas system is a CRISPR Type II system.
  • the CRISPR/Cas system of the invention can be used to alter the polynucleotide sequence of a target gene in a cell.
  • the present invention contemplates altering the polynucleotide sequence of a target gene in a cell for any purpose.
  • the polynucleotide sequence of a target gene in a cell is altered to produce a mutant cell.
  • the Cas9 protein herein may illustratively be a S. pyogenes Cas9 protein or a functional portion thereof.
  • the Cas9 protein is a Cas9 protein from any bacterial species or a functional portion thereof.
  • the Cas9 protein is a member of the Type II CRISPR system, which typically includes a trans-encoded small RNA (tracrRNA), an endogenous ribonuclease 3 (rnc), and a Cas9 protein.
  • CRISPR-derived RNA CRISPR-derived RNA
  • tracrRNA trans-activating RNA
  • the alterations modifies the polynucleotide sequence of the target gene from an undesired sequence to a desired sequence.
  • the CRISPR/Cas system of the invention can be used to correct for any type of mutation or error in the polynucleotide sequence of a target gene.
  • the CRISPR/Cas system of the invention can be used to insert a nucleotide sequence that is missing in the polynucleotide sequence of a target gene due to deletion.
  • the CRISPR/Cas system of the present invention can also be used to delete or excise a nucleotide sequence due to an insertion mutation from a polynucleotide sequence of a target gene.
  • the CRISPR/Cas system of the invention can be used to replace an incorrect nucleotide sequence with a correct nucleotide sequence (eg, to restore a polynucleotide sequence of a target gene that is compromised by loss of a functional mutation)
  • the function ie SNP
  • the CRISPR/Cas system of the present invention can unexpectedly cleave target genes with high efficiency compared to conventional CRISPR/Cas systems.
  • the cleavage efficiency of the target gene is at least about 5%. In certain embodiments, the cleavage efficiency of the target gene is at least about 10%. In certain embodiments, the cleavage efficiency of the target gene is from about 10% to about 80%. In certain embodiments, the cleavage efficiency of the target gene is from about 30% to about 80%. In certain embodiments, the cleavage efficiency of the target gene is from about 50% to about 80%. In some embodiments, the target gene has a cleavage efficiency of greater than or equal to about 75%, or greater than or equal to about 80%.
  • the target gene is a genome. In some embodiments, the target gene is a human genome. In some embodiments, the target gene is a mammalian genome. In some embodiments, the target gene is a vertebrate genome.
  • a polynucleotide sequence or a portion thereof that knocks out a target gene using the CRISPR/Cas system of the present invention can be applied to various applications.
  • a polynucleotide sequence that knocks out a target gene in a cell can be performed in vitro for research purposes.
  • a polynucleotide sequence that knocks out a target gene in a cell can be useful for treating or preventing a disorder associated with expression of a polynucleotide sequence of the target gene (eg, by ex vivo knockout of cells)
  • the allele is mutated and those cells comprising the knockout mutant allele are introduced into the subject).
  • the invention provides a method of treating or preventing a condition associated with expression of a polynucleotide sequence in a subject.
  • the term "contacting" ie, contacting a polynucleotide sequence with a clustered regularly spaced short palindromic repeat (Cas) protein and/or ribonucleic acid
  • contacting is intended to include incubating the Cas protein in vitro and/or RNA or contact cells in vitro.
  • the step of contacting the polynucleotide sequence of the target gene with the Cas protein and/or ribonucleic acid as disclosed herein can be carried out in any suitable manner.
  • the cells can be treated in the form of adherent or suspension culture.
  • cells contacted with Cas protein and/or ribonucleic acid as disclosed herein may also be simultaneously or subsequently contacted with another agent, such as a growth factor or other differentiation agent or environment, to stabilize or render the cells. Further differentiation.
  • the term "treating" or the like includes subjecting the cell to any type of process or condition, or performing any type of operation or procedure on the cell.
  • the term is directed to an individual providing a cell in which the polynucleotide sequence of the target gene has been altered ex vivo according to the methods described herein.
  • the individual is typically ill or injured, or is at an increased risk of illness relative to the average member of the population and requires such attention, care or management.
  • treating refers to administering to a subject an effective amount of a polynucleotide having a polynucleotide sequence that is altered ex vivo according to the methods described herein, such that the subject has the disease.
  • a reduction in at least one symptom or an improvement in the disease for example, a beneficial or desired clinical outcome.
  • beneficial or desired clinical outcomes include, but are not limited to, alleviation of one or more symptoms, reduction in the extent of the disease, stabilization of the disease state (ie, no deterioration), delay or reduction in disease progression. Slow, improved or alleviated disease states, and relief (whether partial or total), whether detectable or undetectable.
  • Treatment may mean prolonging survival as compared to expected survival in the absence of treatment.
  • treatment includes prophylaxis.
  • treatment is "effective” in the event that the progression of the disease is reduced or stopped.
  • Treatment can also mean prolonging survival as compared to expected survival in the absence of treatment.
  • Those in need of treatment include those that have been diagnosed with a disorder associated with expression of a polynucleotide sequence, as well as those that may develop such a disorder due to genetic susceptibility or other factors.
  • mutant cell refers to a cell having a resulting genotype that is different from its original genotype.
  • mutant cells exhibit a mutant phenotype, such as when a functionally normal gene is altered using the CRISPR/Cas system of the invention.
  • mutant cells exhibit a wild-type phenotype, such as when the CRISPR/Cas system of the invention is used to modify a mutant genotype.
  • the polynucleotide sequence of a target gene in a cell is altered to modify or repair the gene mutation (eg, to restore the normal genotype of the cell).
  • the polynucleotide sequence of a target gene in a cell is altered to induce a genetic mutation (eg, to disrupt the function of a gene or genomic element).
  • the alteration is an insertion deletion.
  • Insert deletion refers to a mutation resulting from an insertion, deletion or a combination thereof. As will be understood by those of skill in the art, unless the length of the insertion deletion is a multiple of three, an insertional deletion in the coding region of the genomic sequence will result in a frameshift mutation.
  • the alteration is a point mutation.
  • Point mutation refers to a substitution of one of the alternative nucleotides.
  • the CRISPR/Cas system of the invention can be used to induce insertion or point mutations of any length in a polynucleotide sequence of a target gene.
  • oligodeoxyribonucleic acid or “N-oligo” refers to a deoxyribonucleic acid fragment of a random sequence that is transformed into a cell together with RNP when a gene knockout is performed using an RNP delivery system, preferably a double length of 100-250 bp. Stranded DNA or 100-250 nt single-stranded DNA.
  • “Fish sperm DNA fragment” refers to a small molecule fragment in which a solution containing salmon sperm DNA is mechanically sheared to cut fish sperm DNA. For example, 1% salmon sperm DNA solution is repeatedly beaten with a 7-gauge needle to cut DNA into small molecules, and stored after dispensing.
  • knockout includes deletion of all or a portion of a polynucleotide of the target gene in a manner that interferes with the function of the polynucleotide of the target gene.
  • knockout can be achieved by altering the polynucleotide sequence of the target gene by inducing a functional domain of the polynucleotide sequence of the target gene in the polynucleotide sequence of the target gene (eg, Insertion deletion in the DNA binding domain).
  • cleavage of the target gene results in decreased expression of the target gene.
  • reduced is generally used herein to mean reducing a statistically significant amount.
  • reducing means reducing at least 10% compared to the reference level, for example by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, compared to the reference level, Or at least about 60%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 90%, or up to and including 100% reduction (ie, a level that is not present compared to the reference sample) , or any reduction between 10% and 100%.
  • statically significant refers to statistical significance and generally means two standard deviations (2SD) below or below the normal marker concentration.
  • 2SD standard deviations
  • the term refers to statistical evidence of the difference. It is defined as the probability of making a decision to reject a hypothesis when the hypothesis is actually true. The decision is often expressed using a p value.
  • cleavage of the target gene is cleavage of a homozygous target gene. In some embodiments, cleavage of the target gene is cleavage of a hybrid target gene.
  • the Cas9 protein (also known as CRISPR-related endonuclease Cas9/Csn1) is a polypeptide comprising 1368 amino acids.
  • An exemplary amino acid sequence of the Cas9 protein is set forth in SEQ ID NO: 18.
  • Cas9 contains two endonuclease domains, including the RuvC-like domain (residues 7-22, 759-766, and 982-989), which cleave target DNA that is not complementary to crRNA; and the HNH nuclease domain (residue) Base 810-872), which cleaves target DNA complementary to the crRNA.
  • T cell receptor is a heterodimeric protein receptor that presents a specific antigenic peptide on the major histocompatibility complex (MHC).
  • MHC major histocompatibility complex
  • APC antigen presenting cells
  • TCR is a glycoprotein on the surface of a cell membrane in the form of a heterodimer formed by an alpha chain/beta chain or a gamma chain/delta chain.
  • the TCR heterodimer consists of alpha and beta chains in 95% of T cells, while 5% of T cells have a TCR consisting of gamma and delta chains.
  • the native ⁇ heterodimeric TCR has an alpha chain and a beta chain, and the alpha chain and the beta chain constitute a subunit of the ⁇ heterodimeric TCR.
  • each of the alpha and beta chains comprises a variable region, a junction region, and a constant region
  • the beta chain typically also contains a short polymorphic region between the variable region and the junction region, but the polymorphic region is often considered as a junction region. a part of.
  • Each variable region comprises three CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, chimeric in framework regions.
  • the CDR regions determine the binding of the TCR to the pMHC complex, wherein the CDR3 is recombined from the variable region and the junction region and is referred to as the hypervariable region.
  • the alpha and beta chains of TCR are generally considered to have two "domains", namely a variable domain and a constant domain, and the variable domain consists of linked variable and linking regions.
  • the sequence of the TCR constant domain can be found in the public database of the International Immunogenetics Information System (IMGT).
  • IMGT International Immunogenetics Information System
  • the constant domain sequence of the TCR molecule ⁇ chain is “TRAC*01”
  • the constant domain sequence of the TCR molecule ⁇ chain is “TRBC1*”. 01" or "TRBC2*01”.
  • the alpha and beta chains of TCR also contain a transmembrane and cytoplasmic regions with a short cytoplasmic region.
  • B2M also known as beta-2 microglobulin
  • B2M is the light chain of MHC class I molecules and is therefore an integral part of the major histocompatibility complex.
  • B2M is encoded by the b2m gene located on chromosome 15 as opposed to other MHC genes located on chromosome 6 as a cluster of genes.
  • the human protein consists of 119 amino acids and has a molecular weight of 11,800 Daltons.
  • a murine model of ⁇ -2 microglobulin deficiency has demonstrated that B2M is required for cell surface expression of MHC class I and stability of peptide binding channels.
  • PD-1 or "PD1” is a 50-55 kDa type I transmembrane receptor originally identified in a T cell line that undergoes activation-induced apoptosis. PD-1 is expressed on top of T cells, B cells and macrophages.
  • the ligand for PD-1 is the B7 family members PD-L1 (B7-H1) and PD-L2 (B7-DC).
  • PD-1 is a member of the immunoglobulin (Ig) superfamily and contains a single IgV-like domain in its extracellular region.
  • the PD-1 cytoplasmic domain contains two tyrosines, of which the membrane closest to tyrosine (VAYEEL in mouse PD-1) is located within ITIM (the inhibitory motif of the immunoreceptor tyrosine).
  • ITIM the inhibitory motif of the immunoreceptor tyrosine
  • the presence of ITIM on PD-1 predicts that this molecule acts by recruiting cytosolic phosphatase to attenuate the signaling of antigen receptors.
  • the human and murine PD-1 proteins share approximately 60% amino acid identity with four potential N-glycosylation sites conserved and residues defining the Ig-V domain.
  • the ITIM-like motif around the ITIM and carboxy terminal tyrosine (TEYATI in humans and mice) in the cytoplasmic region is also conserved between human and murine orthologues.
  • PBMC separation tube Sepmate-50 (STEMCELL Technology), add 15 ml of Ficoll buffer (GE healthcare), and add a mixture of blood PBS.
  • PBS buffer containing 2% fetal bovine serum
  • the pellet was resuspended in PBS after centrifugation. For resuspended cell count, 10 ⁇ l of the suspension was added to 10 ⁇ l of 0.1% trypan blue to mix and count the cell number and survival rate.
  • the cell suspension was added to a 5 ml flow tube and placed in a magnetic pole for 5 minutes.
  • the cell suspension was quickly decanted, PBS buffer was added to the flow tube and resuspended, and repeated 3 times.
  • the obtained cell suspension was centrifuged at 300 g for 5 minutes, the supernatant was discarded, and the cell pellet was resuspended in MONZA VIVO-15 medium, and the density was adjusted to 1 ⁇ 10 6 /ml, and rIL-2 (R&D) was added thereto.
  • the concentration is 100 IU/ml. Then, it was cultured in a 37-degree cell culture incubator.
  • Anti-CD3/anti-CD28 magnetic beads (Life Technology) were resuspended in PBS buffer (containing 2 mM EDTA and 1% fetal bovine serum), and then placed in a magnetic pole for 2 minutes, and the supernatant was discarded. Repeat the above process 4 times. After the magnetic beads were taken, the number of magnetic beads was added to the purified T cells in a ratio of 1:1, mixed, and cultured at 37 degrees for 3 days. After 3 days, the magnetic beads were taken out, and the target cells were first resuspended several times with a pipette. The cell suspension was placed in a magnetic pole, and after standing for two minutes, the magnetic beads on the tube wall were discarded.
  • PBS buffer containing 2 mM EDTA and 1% fetal bovine serum
  • the CD19 CAR structure was constructed from CD19 scFv, Hinge structure, transmembrane structure, 4-1BB and CD3z, CD19 CAR and vector pHR-CAR.
  • the lentiviral plasmid pHR-CAR was ligated with the two helper plasmids dR8.91 and pCMV-VSV-G plasmids using the Tiangen Big Plasmid Kit.
  • Transfection system 1 Transfection system 2 pHR-CAR: 7.5 ⁇ g dR8.91: 5.625 ⁇ g pCMV-VSV-G: 1.875 ⁇ g Opti-MEM (Gibco): 700 ⁇ l Opti-MEM (Gibco): 700 ⁇ l P3000: 30 ⁇ l Lipofectamine: 36 ⁇ l
  • the human primary T cells were resuspended, placed in a magnetic pole for two minutes, and the cell suspension was taken. Cell suspension was performed on the cell suspension. After centrifugation of about 1 ⁇ 10 7 cells at 300 g for 5 minutes, the medium was discarded, and 1 ml of the new medium was added and resuspended. Add concentrated lentivirus to adjust the MOI to 5 and mix. After centrifugation at 2000 g for 90 minutes at 32 ° C, the supernatant was discarded, and the new medium (100 IU/ml of rIL-2) was added to adjust the cell density to 1 ⁇ 10 6 /ml. After resuspension, the newly isolated anti-- CD3/anti-CD28 magnetic beads. The cultivation was continued in a 37 ° C incubator. Obtain CAR-T cells.
  • a suitable target region was selected, and a 17-20 nt crRNA was designed.
  • the crRNA was ligated with the corresponding tracrRNA sequence of the Cas9 protein to form an sgRNA.
  • the crRNA with high knockout efficiency and low target rate was screened by experiment.
  • the selected partial crRNA sequences are as follows:
  • the Cas9 protein is from Cas9 Nuclease NLS (S. pyogenes (BioLabs)), corresponding to the tracrRNA sequence (SEQ ID NO: 17):
  • the amino acid sequence of the Cas9 (including NLS) protein used (SEQ ID NO: 18):
  • the sgRNA linked to the tracrRNA corresponding to the Cas9 protein described above was prepared as the crRNA shown in Table 1, and the crRNA was located at the 5' end of the tracrRNA.
  • DNA is obtained that can be used to transcribe sgRNA in vitro.
  • the obtained sgRNA was purified and detected by spectrophotometer and denaturing agarose gel electrophoresis, and all of them were ready to be dispensed immediately.
  • CAR-T cells which can also be used to knock primary T cells
  • the distribution conversion system 10 ⁇ l of Nucleofector buffer, 30 ⁇ g of Cas9 protein (about 9 ⁇ g/ ⁇ l) and 4 ⁇ g of sgRNA were mixed and incubated at room temperature for 10 minutes. After three days of activation, CAR-T cells were magnetically depleted of anti-CD3/anti-CD28 magnetic beads. 5 x 10 6 cells/tube were taken and centrifuged at 300 g for 5 minutes to completely remove the supernatant. Add the incubated electroporation system to the cell pellet, add 72 ⁇ l of Nucleofector buffer and 18 ⁇ l of Supply buffer, and mix and add to 100 ⁇ l of LONZA electroconversion cup.
  • CAR-T cells were cultured to day 10 after CRISPR-Cas9 knockout of TRAC, and TCR-negative cells were enriched. First centrifuge all cells: 300g for 5 minutes. Wash twice with PBS buffer (containing 2 mM EDTA and 1% fetal bovine serum). The cell density was adjusted to be 1 ⁇ 10 7 /ml, and then 100 ⁇ l/ml of Biotin-TCR antibody (purchased from Meisei, Germany) was added, and incubation was carried out for 10 minutes at 4 ° C in the dark.
  • PBS buffer containing 2 mM EDTA and 1% fetal bovine serum
  • the cell density was adjusted to 1 ⁇ 10 7 cells/ml, and Anti-Biotin Microbeads was added at 50 ⁇ l/ml, and kept at 4 ° C for 15 minutes in the dark.
  • PBS buffer After washing once in PBS buffer, it was resuspended in 500 ⁇ l of buffer.
  • LD column (purchased from Meitian) was placed in a magnetic pole and rinsed with 2 ml of PBS buffer for 1 time. Then, 500 ⁇ l of the cell suspension was added, and the target cells were collected from the bottom of the LD column, and the cells were suspended after the cell suspension was repeated. Add 2 ml of PBS buffer to the LD column. The received cell suspension was centrifuged: 300 g for 5 minutes. Resuspend in pre-warmed medium.
  • Test Example 1 Choosing the best crRNA to CRISPR-Cas9 knockout TRAC
  • the test was compared to the crRNA sequence designed for TRAC shown in Example 5.
  • Cas9 protein was electroporated into activated primary T cells. After 48 hours, the expression of extracellular TCR protein was detected by flow cytometry. The results showed that crRNA can knock out TRAC gene to varying degrees. Among them, crRNA-11 has the highest knockout efficiency.
  • plasmid Three delivery systems: plasmid, mRNA and RNP (protein RNA complex), crRNA for TRAC, plasmid extraction in large quantities.
  • Example 4 for in vitro transcription of Cas9 mRNA.
  • PCR with T7 primer to obtain DNA template containing T7 promoter.
  • the Cas9 mRNA was then transcribed in vitro using Ambion's T7 in vitro transcription kit.
  • the sgRNA and Cas9 protein complexes were obtained in the same manner as in Example 5.
  • Jurkat cells were centrifuged to remove 5 ⁇ 10 6 cells, respectively, and electrotransformed on Invitrogen's electrotransfer system Neon MPK5000 using three different delivery materials.
  • Test Example 3 Random N-oligo or fish sperm DNA increases the efficiency of CRISPR-Cas9 knockout TRAC
  • RNP When a gene knockout is performed using an RNP delivery system, RNP is simultaneously electrotransformed by mixing with a random sequence of N-oligo (oligodeoxyribonucleic acid) or fish sperm DNA.
  • N-oligo sequence An exemplary N-oligo sequence:
  • Example 5 On the basis of Example 5 (3), 100-200 nM of N-oligo DNA was further added to the RNP complex, and the N-oligo DNA was Page grade.
  • the effect of N-oligo on the efficiency of CRISPR-Cas9 knockout TRAC is shown in Figure 2A-2B. The results show that N-oligo can effectively enhance the CRISPR-Cas9 knockout TRAC gene for both T cells and CAR-T cells. s efficiency.
  • Example 5 (3) 100-200 nM fish sperm DNA fragment was further added to the RNP complex, and the effect of the fish sperm DNA fragment on the knockout TRAC efficiency was as shown in Fig. 3, and the result showed that the fish sperm DNA fragment was Increasing the efficiency of TRAC gene knockout is more efficient than N-oligo.
  • Test Example 4 T cell knockout B2M, PD1 efficiency test
  • a number of crRNAs were also designed, and the knockdown of the B2M gene was performed by comparing the crRNA with the highest knockout efficiency and the lowest off-target rate.
  • the B2M and/or PD1 genes of T cells were knocked out using the RNP delivery system and N-oligo based on the same method as in Example 5 (3).
  • the B2M gene expression was closely related to the display of HLA-ABC on the cell membrane, and the knockdown efficiency of the B2M gene was detected using the APC-HLA-ABC antibody (eBioscience).
  • the results show that the knockdown efficiency of the B2M gene is greater than 80%.
  • the RNP and N-oligo mixture were electrotransformed for 48 hours, and 1 ⁇ 10 6 cells were taken separately. After washing twice with PBS buffer, the supernatant was completely aspirated, and the reference kit was used.
  • the Genomic Cleavage Detection Kit (Thermo Fisher) performed the T7E1 experiment. The results (shown in Figure 5) showed that the three crRNAs of the selected PD1 could effectively knock out the PD1 gene, and the knockout efficiency was more than 80%.
  • Test Example 5 Analysis of gene mutations caused by CRISPR-Cas9
  • TRAC TRAC
  • the obtained PCR product DNA fragment was ligated with a T-terminal vector (pEASY-Blunt Simple Cloning Kit, Beijing Quanjin Biotechnology Co., Ltd.). After ligation, TOP10 competent cells were transformed and Amp-resistant solid plates were coated. The next day, clones will be sequenced and at least 30 clones per plate will be tested.
  • Test Example 7 Analysis of the effect of TCR knockdown on cell signaling pathway and killing activity
  • a CD3 antibody solution (5 ⁇ g/ml) was placed in a 96-well plate, and a volume of 100 ⁇ l was added to each well, and coated at 37 ° C for two hours, and after taking out, it was washed twice with PBS. TCR-negative T cells and normal T cells were separately added, and the cell density was 1 ⁇ 10 6 /ml. After incubation at 37 ° C for 24 hours, the flow-through antibodies CD25 and CD69 were taken out, and the results were as shown in Fig. 8A and Fig. 8B. This indicates that T cells cannot be induced to express CD25 and CD69 by CD3 antibodies after TRAC gene knockout.

Abstract

提供了一种基于CRISPR-Cas9系统的体外敲除T细胞中靶基因的方法。还提供了针对靶基因TRAC、B2M和PD1的crRNA，以及包含该crRNA与Cas9蛋白对应的tracrRNA连接形成的的sgRNA、Cas9蛋白、寡聚脱氧核糖核酸(N-oligo)/或鱼精DNA片段的试剂盒，该试剂盒用于敲除T细胞中的TCR、B2M和/或PD1基因。还提供了根据本发明的方法所获得的基因敲除的T细胞及其用途。

Description

体外敲除T细胞中靶基因的方法以及该方法中使用的crRNA 技术领域
本发明属于生物医药领域。具体地说涉及一种体外敲除T细胞中靶基因的方法、该方法中使用的crRNA以及该方法所获得的T细胞及其用途。
背景技术
过继性细胞免疫疗法(adoptive cell therapy,ACT)，涉及离体(ex vivo)产生的自体抗原特异性T细胞的转移，是治疗病毒感染和癌症的有前途的策略。可以通过抗原特异性T细胞的扩增或通过遗传设计的T细胞重定向产生过继性免疫疗法使用的T细胞(Park，Rosenberg et al.Trends Biotechnol.2011,29(11):550-557)。
CART是将分离的T细胞，通过基因工程嵌入特定的抗原受体(CAR)，使得T细胞的靶向性、杀伤活性和持久性增强，并且对肿瘤细胞表面抗原的识别不依赖MHC的限制性。CARs由胞外抗原结合区、跨膜区、以及胞内的T细胞受体的信号转导区(如CD3ζ和共刺激分子)组成。胞外抗原结合区由单克隆抗体的轻链可变区(VL)和重链可变区(VH)组成，中间由铰链连接形成单链抗体(single chain fragment variable，scFv)，能够识别特定的肿瘤抗原。有相关临床试验表明，CAR在其它治疗方法无效的淋巴癌患者身上具有较好的治疗效果。美国宾夕法尼亚大学Carl June进行的CART-19研究表明在75名白血病患者(包括成人和儿童患者)，有45人经过CART细胞治疗之后病情完全缓解(complete remission)。
然而，现有的CART疗法通常是利用来自肿瘤患者自身的淋巴细胞，经体外修饰、培养、激活和扩增之后回输给患者。除了会导致细胞因子风暴等副作用外，还存在3大问题：首先，对于一些因淋巴细胞数量较低或质量较差的晚期患者失去CART治疗的机会；其次，CART疗法在实体瘤中的疗效仍不显著，可能因为免疫抑制检验点信号通路的影响，导致免疫细胞在肿瘤组织内的存活率较差、活性不高；最后，由于CART是个体化治疗，成本昂贵，增加了病人负担。因此开发同种异体来源的通用CAR-T细胞(UCART)能够推广其应用。
早期曾利用同源重组打靶载体、或RNAi技术对基因进行敲除，但均存在操作繁琐，效率低等问题。Cellectis公司则通过TALEN技术开发的异体CAR-T疗法UCART19定向敲除TCRα基因(降低GVHD)和CD52基因(使细胞对alemtuzumab耐药)已经成功治愈了多例复发性急性淋巴细胞白血病(ALL)患儿。但是Cellectis通过TALEN敲除TCR需要繁琐的构建过程和大规模测序。当前，规律成簇间隔短回文重复系统(clustered regularly interspaced short palindromic repeat；CRISPR-associated,CRISPR-Cas9)通过识别特定的DNA序列实现对基因的编辑，并且比TALEN更简单、高效。
目前，已存在基于CRISPR/Cas9系统进行基因敲除的实例，如CN104395463A、CN105518146A和CN106191062A。但仍存在目的基因敲除率低、或需要多次转染或转化，操作繁琐，且对T细胞造成更多伤害，或者存在脱靶率高等问题。因此，仍需要优化基于CRISPR/Cas9系统的TCR等基因敲除方法和更高准确性的crRNA。
发明内容
本发明的目的在于克服现有技术在免疫治疗中存在的问题，提供一种TCR-阴性T细胞的构建方法，通过CRISPR/Cas9基因编辑技术敲除TCR，并提供利用该方法获得的TCR阴性T细胞。
本发明的目的在于提供一种TCR和PD1或B2M双阴性T细胞及其构建方法。
本发明的另一目的在于提供一种TCR、B2M和PD1三阴性T细胞及其构建方法。
更进一步地，通过磁珠分选出上述TCR阴性T细胞、TCR和PD-1或B2M双阴性T细胞和TCR/B2M/PD1三阴性T细胞，用于肿瘤的过继细胞免疫治疗等方面。
在第一实施方案中，提供一种体外敲除T细胞中一个或多个靶基因的方法，所述方法包括如下步骤：
1)使用靶向所述T细胞中的一个或多个靶基因的sgRNA分别与Cas9蛋白接触，形成蛋白RNA复合体(RNP)；
2)将RNP与寡聚脱氧核糖核酸(N-oligo)或鱼精DNA片段混合后转化所述T细胞，其中所述sgRNA将Cas9蛋白分别引导至相应靶基因的靶序列，并且与所述靶序列杂交，其中所述靶基因被裂解，并且其中所述靶基因的裂解效率大于75％。
在一种优选的实施方式中，在本发明的体外敲除T细胞中一个或多个靶基因的方法中，所述靶基因选自TRAC、TRBC、B2M和PD1基因中的一个或多个，所述sgRNA靶向所述靶基因的编码序列或其表达的调控序列。
进一步地，在本发明的体外敲除T细胞中一个或多个靶基因的方法中，所述sgRNA从5’到3’依次由长度为17-20nt的靶向靶基因的crRNA和与Cas9蛋白对应的tracrRNA连接而成，其中所述crRNA的长度优选为17nt。
在一种优选的实施方式中，在本发明的体外敲除T细胞中一个或多个靶基因的方法中，所述寡聚脱氧核糖核酸是长度为100-250bp的双链DNA或长度为100-250nt的单链DNA。
在一种优选的实施方式中，在本发明的体外敲除T细胞中一个或多个靶基因的方法中，所述靶向TRAC基因的crRNA选自SEQ ID NO:1-12所示crRNA中的任意一种或多种，所述靶向B2M基因的crRNA序列如SEQ ID NO:13所示，所述靶向PD1基因的crRNA选自SEQ ID NO:14-16所示crRNA中的任意一种或多种。
在一种优选的实施方式中，在本发明的体外敲除T细胞中一个或多个靶基因 的方法中，所述Cas9蛋白为来自酿脓链球菌的Cas9蛋白，序列如SEQ ID NO：18所示。
在一种优选的实施方式中，在本发明的体外敲除T细胞中一个或多个靶基因的方法中，所述Cas9蛋白为来自酿脓链球菌的Cas9蛋白，所述Cas9蛋白对应的tracrRNA序列如SEQ ID NO：17所示。
在一种优选的实施方式中，在本发明的体外敲除T细胞中一个或多个靶基因的方法中，所述T细胞选自辅助性T细胞、细胞毒性T细胞、记忆性T细胞、调节性T细胞、自然杀伤T细胞、γδT细胞、CAR-T细胞和TCR-T细胞。
另一方面，本发明还提供根据上述方法获得的靶基因敲除的T细胞。
另一方面，本发明还提供用于敲除靶向基因的crRNA，其中所述crRNA包含一种或多种选自SEQ ID NO:1-16的序列。
在一种优选的实施方式中，本发明用于敲除靶向基因的crRNA靶向靶向基因的编码序列或其表达的调控序列，其中所述的靶向基因选自TRAC、TRBC、B2M和PD1基因中的一个或多个。
在一种优选的实施方式中，本发明用于敲除靶向基因的crRNA，其中所述的靶向基因为TRAC基因，所述的crRNA选自SEQ ID NO:1-12所示的一个或多个。
在一种优选的实施方式中，本发明用于敲除靶向基因的crRNA，其中所述的靶向基因为B2M基因，所述的crRNA序列如SEQ ID NO:13所示。
在一种优选的实施方式中，本发明用于敲除靶向基因的crRNA，其中所述的靶向基因为PD1基因，所述的crRNA选自SEQ ID NO:14-16所示的一个或多个。
另一方面，本发明还提供用于敲除靶向基因的sgRNA，所述的sgRNA由crRNA与Cas9蛋白对应的tracrRNA连接形成，其中所述crRNA包含一种或多种选自SEQ ID NO:1-16的序列。
在一种优选的实施方式中，本发明用于敲除靶向基因的sgRNA，其中所述的靶向基因选自TRAC、TRBC、B2M和PD1基因中的一个或多个。
在一种优选的实施方式中，本发明用于敲除靶向基因TRAC的sgRNA，所述的sgRNA由crRNA与Cas9蛋白对应的tracrRNA连接形成，所述的crRNA选自SEQ ID NO:1-12所示的一个或多个。
在一种优选的实施方式中，本发明用于敲除靶向基因B2M的sgRNA，所述的sgRNA由crRNA与Cas9蛋白对应的tracrRNA连接形成，所述的crRNA如SEQ ID NO:13所示。
在一种优选的实施方式中，本发明用于敲除靶向基因PD-1的sgRNA，所述的sgRNA由crRNA与Cas9蛋白对应的tracrRNA连接形成，所述的crRNA选自SEQ ID NO:14-16所示的一个或多个。
在一种优选的实施方式中，本发明用于敲除靶向基因的sgRNA，其中所述Cas9蛋白为来自酿脓链球菌的Cas9蛋白如SEQ ID NO：18所示。
在一种优选的实施方式中，本发明用于敲除靶向基因的sgRNA，其中所述与Cas9蛋白对应的tracrRNA序列如SEQ ID NO：17所示。
在另一方面，本发明提供了一种用于基因敲除的试剂盒，其包含：
a：一种或多种如前所述的crRNA，或一种或多种如前所述的sgRNA；
b：Cas9蛋白；
c：寡聚脱氧核糖核酸或鱼精DNA片段。
在一种优选的实施方式中，在所述的用于基因敲除的试剂盒中，所述寡聚脱氧核糖核酸是长度为100-250bp的双链DNA或长度为100-250nt的单链DNA。
在一种优选的实施方式中，在所述用于基因敲除的试剂盒中，所述Cas9蛋白为来自酿脓链球菌的Cas9蛋白，所述Cas9蛋白对应的tracrRNA序列如SEQ ID NO：17所示。
在一些实施方式中，本发明提供本发明所述的基因敲除的T细胞在制备抗肿瘤药物中的用途。
在一些实施方式中，本发明还提供本发明所述的基因敲除的T细胞在制备防治病毒或细菌引起的感染性疾病药物中的用途。
在一些实施方式中，利用所设计的crRNA及方法，TCR、B2M或PD1均被有效敲除。TCR及B2M和/或PD1敲除后的CART细胞的体外杀伤活性不受TCR、B2M和/或PD1基因敲除的影响。
附图说明
图1：不同递送系统敲除效率比较。结果显示RNP递送方式在Jurkat细胞中基因敲除效率最高。
图2A-2B：N-oligo对基于CRISPR-Cas9系统对T细胞基因敲除效率的影响。图2A是在T细胞中基因敲除效率的比较；图2B是在CART细胞中基因敲除效率的比较。
图3：鱼精DNA片段对T细胞基因敲除效率的影响。
图4：B2M基因敲除效率的检测。
图5：筛选的crRNA对PD1基因敲除效果的检测。
图6A-图6B：RNP与N-Oligo或鱼精DNA造成的基因突变分析。图6A是针对TRAC的分析结果，图6B是针对B2M的分析结果。
图7A-7C：RNP脱靶率分析。图7A为TRAC基因脱靶率分析结果；图7B为B2M基因脱靶率分析结果；图7C为PD1基因脱靶率分析结果。
图8A-8B：TRAC基因敲除T细胞的CD25和CD69激活情况分析。图8A为CD69激活情况比较，图8B为CD25激活情况比较。
具体实施方式
一方面，本发明提供一种改变细胞中的靶基因的方法。
本文所述的研究展示了使用CRISPR/Cas系统的等位基因靶向方法以高达80％的效率产生突变细胞。具体地说，本文所述的工作出人意料地且意外地证明一种多重引导策略，提供特异性地鉴定有用的RNA引导序列的方法，连同适用于靶向特异性基因(例如，TRAC、TRBC、B2M、PD1)的特定引导序列。
CRISPR系统用于本发明所提供的方法和组合物还包括那些国际公开号为WO2013142578A1和WO2013098244A1所描述的CRISPR系统，其内容在此全部并入本发明。
一种改变细胞中的靶基因的多核苷酸序列的示例性方法包括使所述多核苷酸序列与CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats，成簇规律间隔的短回文重复)序列相关(Cas)蛋白质和一至两种核糖核酸相接触，形成RNP，其中所述核糖核酸将Cas蛋白引导至所述靶基因的多核苷酸序列的靶基序并且与所述靶基序杂交，其中所述靶基因的多核苷酸序列被裂解，并且其中转化RNP的细胞的改变效率是75％以上。
本发明考虑以任何方式改变靶基因的多核苷酸序列，所述方式是熟练的技术人员利用本发明的CRISPR/Cas系统可获得的。可以使用能够改变细胞中的靶基因的多核苷酸序列的任何CRISPR/Cas系统。这类CRISPR/Cas系统可采用多种Cas蛋白(Haft等PLoS Comput Biol.2005；1(6)e60)。这类Cas蛋白允许CRISPR/Cas系统改变细胞中的靶基因的多核苷酸序列的分子包括RNA结合蛋白、核酸内切酶和核酸外切酶、解旋酶以及聚合酶。在一些实施方案中，所述CRISPR/Cas系统是CRISPR I型系统。在一些实施方案中，所述CRISPR/Cas系统是CRISPR II型系统。
本发明的CRISPR/Cas系统可用于改变细胞中的靶基因的多核苷酸序列。本发明考虑出于任何目的改变细胞中的靶基因的多核苷酸序列。在一些实施方案中，改变细胞中的靶基因的多核苷酸序列以产生突变细胞。
本文中Cas9蛋白示例性地可以是酿脓链球菌Cas9蛋白或其功能部分。在一些实施方案中，所述Cas9蛋白是来自任何细菌物种的Cas9蛋白或其功能部分。Cas9蛋白是II型CRISPR系统的成员，所述II型CRISPR系统通常包括反式编码的小RNA(tracrRNA)、内源性核糖核酸酶3(rnc)以及Cas9蛋白。
通过基因工程手段对crRNA(CRISPR-derived RNA，CRISPR衍生RNA)和tracrRNA(trans-activating RNA，反式激活RNA)进行改造，将二者连接在一起得到sgRNA(single guide RNA，sgRNA，单导RNA)。最终再通过将表达sgRNA的序列与Cas9形成复合物，转化至细胞，便能够对目的基因进行敲除。
在一些实施方案中，所述改变使得所述靶基因的多核苷酸序列从不想要的序列修正为所需序列。本发明的CRISPR/Cas系统可用于修正靶基因的多核苷酸序列 中的任何类型的突变或错误。例如，本发明的CRISPR/Cas系统可用于插入由于缺失而在靶基因的多核苷酸序列中缺少的核苷酸序列。本发明的CRISPR/Cas系统还可用于从靶基因的多核苷酸序列中缺失或切除由于插入突变所致的核苷酸序列。在一些实例中，本发明的CRISPR/Cas系统可用于用正确的核苷酸序列替代不正确的核苷酸序列(例如，以恢复靶基因的多核苷酸序列的由于功能突变的损失而受损的功能，即SNP)。
与常规CRISPR/Cas系统相比，本发明的CRISPR/Cas系统可以出人意料地高效率裂解靶基因。在某些实施方案中，所述靶基因的裂解效率是至少约5％。在某些实施方案中，所述靶基因的裂解效率是至少约10％。在某些实施方案中，所述靶基因的裂解效率是约10％至约80％。在某些实施方案中，所述靶基因的裂解效率是约30％至约80％。在某些实施方案中，所述靶基因的裂解效率是约50％至约80％。在一些实施方案中，所述靶基因的裂解效率大于或等于约75％，或大于或等于约80％。
在一些实施方案中，所述靶基因是基因组。在一些实施方案中，所述靶基因是人基因组。在一些实施方案中，所述靶基因是哺乳动物基因组。在一些实施方案中，所述靶基因是脊椎动物基因组。
使用本发明的CRISPR/Cas系统敲除靶基因的多核苷酸序列或其部分可适用于多种应用。例如，敲除细胞中的靶基因的多核苷酸序列可出于研究目的在体外进行。出于离体目的，敲除细胞中的靶基因的多核苷酸序列可适用于治疗或预防与所述靶基因的多核苷酸序列的表达相关的病症(例如，通过离体敲除细胞中的突变等位基因并且将包含敲除的突变等位基因的那些细胞引入受试者中)。
另一方面，本发明提供一种治疗或预防受试者的与多核苷酸序列的表达相关的病症的方法。
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。
一.术语
如本文所用，术语"接触"(即，使多核苷酸序列与成簇规律间隔的短回文重复序列相关(Cas)蛋白质和/或核糖核酸相接触)意图包括在体外孵育Cas蛋白和/或核糖核酸或离体接触细胞。如本文所公开使靶基因的多核苷酸序列与Cas蛋白和/或核糖核酸相接触的步骤可以任何适合的方式进行。例如，可以贴壁培养或悬浮培养的形式处理所述细胞。应了解如本文所公开与Cas蛋白和/或核糖核酸相接触的细胞还可同时或随后与另一种试剂，如生长因子或其他分化剂或环境相接触以稳定所述细胞或使所述细胞进一步分化。
当应用于分离的细胞时，术语"处理"等包括使所述细胞经受任何类型的过程或 条件，或对所述细胞进行任何类型的操作或工序。当应用于受试者时，所述术语是指向个体提供细胞，在所述细胞中己根据本文所述的方法离体改变靶基因的多核苷酸序列。所述个体通常是生病的或受伤的，或相对于群体的平均成员处于增加的生病风险并且需要这种注意、护理或管理。
如本文所用，术语"治疗"是指向受试者施用有效量的具有根据本文所述的方法离体改变的靶基因的多核苷酸序列的细胞，以使得所述受试者具有所述疾病的至少一种症状的减少或所述疾病的改善，例如，有益的或所需的临床结果。出于本发明的目的，有益的或所需的临床结果包括但不限于一种或多种症状的减轻、疾病程度的减小、疾病状态的稳定(即不恶化)、疾病进展的延迟或减慢、疾病状态的改善或缓和，以及缓解(无论是部分缓解还是全部缓解)，无论是可检测的或是不可检测的。治疗可指与未接受治疗情况下的预期存活期相比，延长存活期。因此，本领域的技术人员意识到治疗可改善疾病状况，但可能不是疾病的完全治愈。如本文所用，术语"治疗"包括预防。或者，治疗在疾病的进展减少或停止的情况下是"有效的"。“治疗”还可意指与在未接受治疗情况下的预期存活期相比，延长存活期。需要治疗的那些包括已经被诊断具有与多核苷酸序列的表达相关的病症的那些，以及由于遗传易感性或其他因素可能发展这种病症的那些。
如本文所用"突变细胞"是指具有与其原始基因型不同的所得基因型的细胞。在一些实例中"突变细胞"表现出突变表型，例如当使用本发明的CRISPR/Cas系统改变功能正常的基因时。在其他实例中"突变细胞"表现出野生型表型，例如当本发明的CRISPR/Cas系统用于修正突变基因型时。在一些实施方案中，改变细胞中的靶基因的多核苷酸序列以修正或修复基因突变(例如，以恢复所述细胞的正常基因型)。在一些实施方案中，改变细胞中的靶基因的多核苷酸序列以诱导基因突变(例如，以破坏基因或基因组元件的功能)。
在一些实施方案中，所述改变是插入缺失。如本文所用"插入缺失"是指由插入、缺失或其组合产生的突变。如本领域的技术人员将理解，除非插入缺失的长度是三的倍数，否则基因组序列的编码区中的插入缺失将导致移码突变。在一些实施方案中，所述改变是点突变。如本文所用"点突变"是指替代核苷酸中的一个的取代。本发明的CRISPR/Cas系统可用于诱导靶基因的多核苷酸序列中的任何长度的插入缺失或点突变。
“寡聚脱氧核糖核酸”或“N-oligo”是指在利用RNP递送系统进行基因敲除时，与RNP一同转化至细胞内的随机序列的脱氧核糖核酸片段，优选长度为100-250bp的双链DNA或100-250nt的单链DNA。
“鱼精DNA片段”是指含鲑鱼精DNA的溶液经机械剪切，将鱼精DNA剪切成的小分子片段。如1％鲑鱼精DNA溶液用7号针头反复抽打以剪切DNA成为小分子，分装后贮藏。
如本文所用"敲除"包括以干扰靶基因的多核苷酸的功能的方式缺失所述靶基 因的多核苷酸的全部或一部分。例如，敲除可通过改变靶基因的多核苷酸序列来实现，所述改变是通过在所述靶基因的多核苷酸序列中诱导所述靶基因的多核苷酸序列的功能结构域(例如，DNA结合结构域)中的插入缺失来进行的。基于本文所述的细节，本领域的技术人员将容易地理解如何使用本发明的CRISPR/Cas系统来敲除靶基因的多核苷酸或其部分。
在一些实施方案中，所述靶基因的裂解导致所述靶基因的表达降低。术语"降低"在本文中通常都用于意指降低统计上显著的量。然而，为避免疑惑"降低"意指与参考水平相比降低至少10％，例如与参考水平相比降低至少约20％、或至少约30％、或至少约40％、或至少约50％、或至少约60％、或至少约70％、或至少约75％、或至少约80％、或至少约90％，或多达且包括100％降低(即与参考样品相比不存在的水平)，或10％-100％之间的任何降低。
术语"统计上显著的"或"显著地"是指统计显著性并且通常意指在正常标记物浓度以下或低于标记物浓度的两个标准偏差(2SD)。所述术语是指存在差异的统计证据。它被定义为当假设实际上为真实时做出拒绝假设的决定的概率。所述决定经常使用p值表示。
在一些实施方案中，所述靶基因的裂解是纯合靶基因的裂解。在一些实施方案中，所述靶基因的裂解是杂合靶基因的裂解。
Cas9蛋白(还被称为CRISPR相关的核酸内切酶Cas9/Csn1)是包含1368个氨基酸的多肽。Cas9蛋白的示例性氨基酸序列如SEQ ID NO:18所示。Cas9含有2个核酸内切酶结构域，包括RuvC样结构域(残基7-22、759-766和982-989)，其裂解与crRNA非互补的靶DNA；和HNH核酸酶结构域(残基810-872)，其裂解与crRNA互补的靶DNA。
T细胞受体(TCR)，是呈递在主组织相容性复合体(MHC)上的特异性抗原肽的异源二聚体蛋白受体。在免疫系统中，通过抗原特异性的TCR与pMHC复合物的结合引发T细胞与抗原呈递细胞(APC)直接的物理接触，然后T细胞及APC两者的其他细胞膜表面分子就发生相互作用，这就引起了一系列后续的细胞信号传递和其他生理反应，从而使得不同抗原特异性的T细胞对其靶细胞发挥免疫效应。
TCR是由α链/β链或者γ链/δ链以异质二聚体形式存在的细胞膜表面的糖蛋白。在95％的T细胞中TCR异质二聚体由α和β链组成，而5％的T细胞具有由γ和δ链组成的TCR。天然αβ异质二聚TCR具有α链和β链，α链和β链构成αβ异源二聚TCR的亚单位。广义上讲，α和β各链包含可变区、连接区和恒定区，β链通常还在可变区和连接区之间含有短的多变区，但该多变区常视作连接区的一部分。各可变区包含嵌合在框架结构(framework regions)中的3个CDR(互补决定区)，CDR1、CDR2和CDR3。CDR区决定了TCR与pMHC复合物的结合，其中 CDR3由可变区和连接区重组而成，被称为超变区。TCR的α和β链一般看作各有两个“结构域”即可变域和恒定域，可变域由连接的可变区和连接区构成。TCR恒定域的序列可以在国际免疫遗传学信息系统(IMGT)的公开数据库中找到，如TCR分子α链的恒定域序列为“TRAC*01”，TCR分子β链的恒定域序列为“TRBC1*01”或“TRBC2*01”。此外，TCR的α和β链还包含跨膜区和胞质区，胞质区很短。
B2M，也称为β-2微球蛋白，是MHC I类分子的轻链，并因此是主要组织相容性复合体的不可缺少的部分。在人类中，由位于15号染色体上、与在6号染色体上以基因簇定位的其他MHC基因相对的b2m基因编码B2M。人源蛋白由119个氨基酸组成，并具有11800道尔顿的分子量。β-2微球蛋白缺陷的鼠模型已经证明，B2M是MHC I类的细胞表面的表达和肽结合槽的稳定性所必需的。
“PD-1”或“PD1”为50-55kDa的I型跨膜受体，其最初是在经历激活诱导的细胞凋亡的T细胞系中鉴定的。PD-1表达于T细胞、B细胞和巨噬细胞之上。PD-1的配体为B7家族成员PD-L1(B7-H1)和PD-L2(B7-DC)。
PD-1是免疫球蛋白(Ig)超家族成员，在其胞外区含有单个IgV-样结构域。PD-1胞浆结构域含有两个酪氨酸，其中最接近于膜的酪氨酸(小鼠PD-1中的VAYEEL)位于ITIM(免疫受体酪氨酸的抑制基序)之内。PD-1上ITIM的存在预示着该分子通过募集胞浆磷酸酶发挥作用以削弱抗原受体的信号传导。人和鼠PD-1蛋白共有大约60％的氨基酸同一性，具有保守的四个潜在的N-糖基化位点以及限定Ig-V结构域的残基。胞浆区中的ITIM以及羧基末端酪氨酸(人和小鼠中的TEYATI)周围的ITIM-样基序在人和鼠直系同源物(orthologue)之间也是保守的。
二.实施例与测试例
以下结合实施例用于进一步描述本发明，但这些实施例并非限制本发明的范围。
本发明实施例或测试例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例
实施例1：PBMC提取
招募健康志愿者，无感冒发烧症状，签署知情同意书。由专业医务人员于静脉取血100ml到BD抗凝血管。血液与等量的PBS缓冲液(含2％的胎牛血清)混合。取PBMC分离管Sepmate-50(STEMCELL Technology)，加入15ml的Ficoll缓冲液(GE healthcare)，再加入血液PBS的混合液。离心后PBS重悬沉淀细胞。 对重悬细胞计数，取10μl混悬液加入10μl 0.1％的台盼蓝混匀，计细胞数和存活率。
实施例2：T细胞纯化
300g离心PBMC 5分钟后，弃去上清，加入相应量的PBS缓冲液(含2mM的EDTA和1％的胎牛血清)重悬细胞使细胞密度调整为5×10 7个/ml。用STEMCELL Technology公司的试剂盒EasySep TM Human T Cell Enrichment Kit来纯化人T细胞，首先加入50μl/ml的Cooktail到PBMC混悬液中，混匀后室温静置10分钟。然后加入50μl/ml的EasySep TM D Magnetite Particles混匀，室温静置5分钟。将细胞悬液加入到5ml流式管中，再放入磁极中5分钟。快速倒出细胞悬液，补充PBS缓冲液到流式管中并重悬，重复3次。将得到的细胞悬液300g离心5分钟，弃上清，细胞沉淀用LONZA公司的VIVO-15培养基重悬，并调整密度为1×10 6个/ml，再加入rIL-2(R&D)使之浓度为100IU/ml。然后放入37度细胞培养箱中培养。
实施例3：T细胞激活
抗-CD3/抗-CD28磁珠(Life Technology)用PBS缓冲液(含2mM的EDTA和1％的胎牛血清)重悬，后加入磁极中静置2分钟后弃去上清。重复4次上述过程。取洗后磁珠，磁珠数量按1:1加入纯化好的T细胞中，混匀，放入37度培养3天。3天后取出磁珠，首先将目的细胞用移液器重悬多次。将细胞悬液置于磁极中，静置两分钟后，弃去管壁上的磁珠。
实施例4：CAR病毒感染T细胞
CAR慢病毒质粒构建：
CD19 CAR结构由外到内依次为CD19 scFv,Hinge结构,跨膜结构，4-1BB和CD3z，CD19 CAR和载体pHR-CAR构建表达载体。慢病毒质粒pHR-CAR与两个辅助质粒dR8.91和pCMV-VSV-G质粒用天根公司的大提质粒试剂盒大提质粒。
CAR慢病毒包装和浓缩：
293T细胞(购自ATCC)在转染前一天在75cm 2培养皿中长满，按1:3传代，每个培养皿培养基为15ml。转染按照Lipo3000的步骤进行，先配转染体系：
转染体系1 转染体系2
pHR-CAR:7.5μg  
dR8.91:5.625μg  
pCMV-VSV-G:1.875μg  
Opti-MEM(Gibco):700μl Opti-MEM(Gibco):700μl
P3000:30μl Lipofectamine:36μl
混匀体系1和2，静置5分钟后将二者混匀，再静置10分钟。小心加入293T细胞中。6小时后换新鲜培养基。48小时后收培养基存于4℃，重新加入新培养基15ml，过24小时再收上清。将得到的病毒上清用0.45μm的滤膜过滤，装入超速离心管 中。在4℃条件下50000g离心2小时45分钟，将上清小心彻底去除，剩下肉眼可见的白色病毒沉淀用百分之一上清体积的PBS缓冲液重悬，病毒重悬后于4℃溶解30分钟左右。溶解完成后分装成小份冻存于-80℃冰箱。
CAR慢病毒感染T细胞：
人原代T细胞在抗-CD3/抗-CD28磁珠激活一天后，重悬细胞，置于磁极中静置两分钟，取细胞悬液。对细胞悬液进行细胞计数。取约1×10 7个细胞300g离心5分钟后弃去培养基，加入新培养基1ml重悬。加入浓缩的慢病毒调整MOI为5，混匀。32℃条件下2000g离心90分钟，弃去上清，加入新培养基(100IU/ml的rIL-2)使之细胞密度调整为1×10 6个/ml，重悬后加入刚分离的抗-CD3/抗-CD28磁珠。37℃培养箱中继续培养。获得CAR-T细胞。
实施例5：TCR，B2M，PD1基因敲除
(1)crRNA的设计
基于TRAC、B2M和PD1的核苷酸序列选取适当的靶区域，设计长度为17-20nt的crRNA，crRNA与所使用Cas9蛋白相应的tracrRNA序列连接形成sgRNA。通过实验比较筛选敲除效率高、脱靶率低的crRNA。所选取部分crRNA序列如下：
表1 针对靶基因的crRNA
Figure PCTCN2018091804-appb-000001
Cas9蛋白来自酿脓链球菌(Cas9 Nuclease NLS,S.pyogenes(BioLabs))，所对应的tracrRNA序列(SEQ ID NO：17)：
Figure PCTCN2018091804-appb-000002
所应用的Cas9(含NLS)蛋白的氨基酸序列(SEQ ID NO：18)：
Figure PCTCN2018091804-appb-000003
制备表1所示crRNA与上述与Cas9蛋白对应的tracrRNA连接的sgRNA，crRNA位于tracrRNA的5’端。
(2)sgRNA体外转录：
先进行sgRNA的模板PCR扩增：
Figure PCTCN2018091804-appb-000004
Figure PCTCN2018091804-appb-000005
再进行PCR产物回收：
参考Tiangen普通DNA产物纯化试剂盒DP214说明书。获得可用于体外转录sgRNA的DNA。利用Ambion体外转录试剂盒MEGAshortscript TM Kit Cat#AM1354)，转录sgRNA。参考Ambion MEGAclear TM Kit说明书cat#AM1908。纯化所获得的sgRNA，经分光光度计和变性琼脂糖凝胶电泳检测，均达到要求立即进行分装，备用。
(3)电转化CRISPR-Cas9敲除T细胞中TRAC基因：
我们电转化所获得的CAR-T细胞(该方法也可用于敲除原代T细胞)，主要利用LONZA 4D电转化仪，采用的试剂盒为：P3 Primary Cell 4D-Nucleofector TM X kit。
首先，配电转化体系：10μl的Nucleofector缓冲液、30μg的Cas9蛋白(约9μg/μl)再加4μg的sgRNA在室温混合孵育10分钟。CAR-T细胞在激活了三天以后，用磁极去除抗-CD3/抗-CD28磁珠。取5×10 6个细胞/管，300g离心5分钟，彻底去除上清。加入孵育好的电转化体系到细胞沉淀中，另外再加72μl的Nucleofector缓冲液和18μl的Supplement缓冲液，混匀加入到100μl的LONZA电转化杯中。放入LONZA-4D电穿孔仪中按E0-115程序电转化。电转化完成后，电转化杯室温静置5分钟。将电转化杯中细胞移入预热的VIVO-15培养基中，调成细胞密度1×10 6个/ml，37℃继续培养。
实施例6：TCR阴性细胞筛选
CAR-T细胞在CRISPR-Cas9敲除TRAC后培养至第10天，做TCR阴性细胞富集。首先把所有细胞离心：300g离5分钟。用PBS缓冲液(含2mM的EDTA和1％胎牛血清)洗两遍。调细胞密度为：1×10 7个/ml，然后加入100μl/ml的Biotin-TCR抗体(购自德国美天旎公司)，4℃避光孵育10分钟。用PBS缓冲液洗一遍后重新调细胞密度为：1×10 7个/ml，按50μl/ml加入Anti-Biotin Microbeads,放4℃避光15分钟。PBS缓冲液洗一遍后用500μl的缓冲液重悬。LD column(购自美天旎)放置于磁极中用2ml的PBS缓冲液润洗1遍后，加入500μl的细胞悬液，目的细胞从LD柱底下流出收集，待细胞悬液流完后反复2次加入2ml PBS缓冲液于LD柱上。接收的细胞悬液离心：300g离5分钟。重悬于预热的培养基中。
测试例
测试例1：选取最优的crRNA来CRISPR-Cas9敲除TRAC
对实施例5所示针对TRAC设计的crRNA序列进行试验比较。体外转录得到sgRNA后，和Cas9蛋白被电转入激活的原代T细胞，48小时后用流式细胞仪检测胞外TCR蛋白的表达，结果显示，crRNA均能不同程度地敲除TRAC基因，其中crRNA-11的敲除效率最高。
测试例2：不同递送系统比较分析
三种递送系统：质粒、mRNA和RNP(蛋白RNA复合体)，crRNA针对的是TRAC，质粒大量提取参照实施例4实施，Cas9的mRNA体外转录首先用T7引物PCR得到含T7启动子的DNA模板，然后利用Ambion的T7体外转录试剂盒体外转录得到Cas9的mRNA。sgRNA和Cas9蛋白复合物同实施例5得到。Jurkat细胞分别取5×10 6个细胞离心弃去上清，分别用三种不同递送物质在Invitrogen的电转系统Neon MPK5000上电转。48小时后取0.5×10 6个细胞，用PBS缓冲液洗2遍后用100μl的缓冲液重悬，加入10μl的PE-TCR抗体(eBioscience)，混匀后4℃孵育30分钟。PBS缓冲液洗一遍后加入500μl缓冲液重悬细胞，上流式细胞仪检测TCR通道，结果图1所示。
测试例3：随机N-oligo或鱼精DNA增加CRISPR-Cas9敲除TRAC效率
在利用RNP递送系统进行基因敲除时，RNP与随机序列N-oligo(寡聚脱氧核糖核酸)或鱼精DNA混匀后同时电转化。
示例性的N-oligo序列：
Figure PCTCN2018091804-appb-000006
在实施例5(3)的基础之上，向RNP复合物中再加入100-200nM的N-oligo DNA，N-oligo DNA为Page级。N-oligo对CRISPR-Cas9敲除TRAC效率影响如图2A-图2B所示，结果显示N-oligo无论是对T细胞或是CAR-T细胞，都能有效的提高CRISPR-Cas9敲除TRAC基因的效率。
在实施例5(3)的基础之上，向RNP复合物中再加入100-200nM的鱼精DNA片段，鱼精DNA片段对敲除TRAC效率影响如图3所示，结果显示鱼精DNA片段提高TRAC基因敲除效率，其效率较N-oligo效率更高。
测试例4：T细胞敲除B2M，PD1效率检测
同样设计多条crRNA，通过实验比较，筛选出敲除效率最高，脱靶率最低的crRNA进行B2M基因的敲除。基于实施例5(3)相同的方法，利用RNP递送系统和N-oligo，对T细胞的B2M和/或PD1基因进行敲除。
对于B2M蛋白，因B2M基因表达与HLA-ABC在细胞膜上的展示呈紧密联系，利用APC-HLA-ABC抗体(eBioscience)对B2M基因的敲除效率进行检测。结果(如图4所示)显示B2M基因的敲除效率大于80％。
对于PD1基因，RNP和N-oligo混合物电转化细胞后48小时，分别取1×10 6个细胞，用PBS缓冲液洗2遍后彻底吸去上清，参照试剂盒
Figure PCTCN2018091804-appb-000007
Genomic Cleavage Detection Kit(Thermo Fisher)进行T7E1实验，结果(如图5所示)显示所选取的PD1的三个crRNA能有效的敲除PD1基因，敲除效率均大于80％。
测试例5：CRISPR-Cas9造成的基因突变分析
首先在TRAC，B2M和PD1基因的打靶位点附近设计引物。T细胞基于CRISPR-Cas9系统，利用RNP+N-oligo或鱼精DNA片段敲除TRAC，B2M和PD1后，分别取正常T和基因敲除T细胞各1×10 6个提取基因组DNA。将得到的PCR产物DNA片段和T平端载体(pEASY-Blunt Simple Cloning Kit，北京全式金生物技术有限公司)连接。连接后转化TOP10感受态细胞，涂Amp抗性的固体培养板。第二天将得到克隆进行测序，每板至少测30个以上克隆。将得到的测序结果与野生型序列对比分析，结果(如图6A至图6B所示，PD1突变分析结果未示出)显示CRISPR-Cas9分别造成了三个基因在crRNA对应的基因组DNA处发生了基因突变。
测试例6：脱靶分析
在http://crispr.mit.edu/网站上，基于所设计crRNA对可能出现的脱靶位点进行预测，TRAC，B2M和PD1分别选取8或9个潜在的脱靶位点(OT1-OT9)，针对这些潜在的脱靶位点设计引物进行PCR扩增并测序。敲除细胞基因组DNA脱靶位点与对照(目标基因TRAC、B2M或PD1)的峰图测序结果在网站https://tide.nki.nl/上进行TIDE比对分析，结果(如图7A至图7C所示)显示，所选取的crRNA及敲除方法的脱靶率极低。
测试例7：TCR敲除对细胞信号通路及杀伤活性的影响分析
配置CD3抗体溶液(5μg/ml)包被96孔板，每孔加100μl体积，37℃包被两个小时，取出后，用PBS洗两遍。分别加入TCR阴性T细胞和普通T细胞，细胞密度为1×10 6个/ml，37℃培养24小时后，取出染流式抗体CD25和CD69，结果(如图8A和图8B所示)，表明T细胞在TRAC基因敲除后无法被CD3抗体诱导表达CD25和CD69。通过针对CD19的TCR阳性CAR-T和TCR阴性(如TRAC敲除)CAR-T细胞对CD19阳性的肿瘤细胞系(K562-CD19)的杀伤作用比较显示，TCR的敲除对CAR-T的杀伤作用无显著影响。
虽然为了清楚的理解，已经借助于附图和实例详细描述了上述发明，但是描述和实例不应当解释为限制本发明的范围。本文中引用的所有专利和科学文献的公开内容通过引用完整地清楚结合。

Claims (26)

  1. 一种体外敲除T细胞中一个或多个靶基因的方法，所述方法包括如下步骤：
    1)使用靶向所述T细胞中的一个或多个靶基因的sgRNA分别与Cas9蛋白接触，形成蛋白RNA复合体；
    2)将所述蛋白RNA复合体与寡聚脱氧核糖核酸或鱼精DNA片段混合后转化所述T细胞，其中所述sgRNA将Cas9蛋白分别引导至相应靶基因的靶序列，并且与所述靶序列杂交，其中所述靶基因被裂解，并且其中所述靶基因的裂解效率大于75％。
  2. 如权利要求1所述的体外敲除T细胞中一个或多个靶基因的方法，其中所述靶基因选自TRAC、TRBC、B2M和PD1基因中的一个或多个，所述sgRNA靶向所述靶基因的编码序列或其表达的调控序列。
  3. 如权利要求1或2所述的体外敲除T细胞中一个或多个靶基因的方法，其中所述sgRNA从5’到3’依次由长度为17-20nt的靶向靶基因的crRNA和与Cas9蛋白对应的tracrRNA连接而成。
  4. 如权利要求1-3任一项所述的体外敲除T细胞中一个或多个靶基因的方法，其中所述寡聚脱氧核糖核酸是长度为100-250bp的双链DNA或长度为100-250nt的单链DNA。
  5. 如权利要求1-4任一项所述的体外敲除T细胞中一个或多个靶基因的方法，其中靶向TRAC基因的crRNA选自SEQ ID NO:1-12所示crRNA中的任意一种或多种，靶向B2M基因的crRNA序列如SEQ ID NO:13所示，靶向PD1基因的crRNA选自SEQ ID NO:14-16所示crRNA中的任意一种或多种。
  6. 如权利要求1-5任一项所述的体外敲除T细胞中一个或多个靶基因的方法，其中所述Cas9蛋白为来自酿脓链球菌的Cas9蛋白如SEQ ID NO：18所示。
  7. 如权利要求1-6任一项所述的体外敲除T细胞中一个或多个靶基因的方法，其中所述与Cas9蛋白对应的tracrRNA序列如SEQ ID NO：17所示。
  8. 如权利要求1-7任一项所述的体外敲除T细胞中一个或多个靶基因的方法，其中所述T细胞选自辅助性T细胞、细胞毒性T细胞、记忆性T细胞、调节性T细胞、自然杀伤T细胞、γδT细胞、CAR-T细胞和TCR-T细胞。
  9. 如权利要求1-8任一项所述的方法获得的靶基因敲除的T细胞。
  10. 用于敲除靶向基因的crRNA，其中所述crRNA包含一种或多种选自SEQ ID NO:1-16的序列。
  11. 如权利要求10所述的用于敲除靶向基因的crRNA，其中所述的靶向基因选自TRAC、TRBC、B2M和PD1基因中的一个或多个。
  12. 如权利要求10或11所述的用于敲除靶向基因的crRNA，其中所述的靶向基因为TRAC基因，所述的crRNA选自SEQ ID NO:1-12所示的一个或多个。
  13. 如权利要求10或11所述的用于敲除靶向基因的crRNA，其中所述的靶向基因为B2M基因，所述的crRNA序列如SEQ ID NO:13所示。
  14. 如权利要求10或11所述的用于敲除靶向基因的crRNA，其中所述的靶向基因为PD1基因，所述的crRNA选自SEQ ID NO:14-16所示的一个或多个。
  15. 用于敲除靶向基因的sgRNA，由crRNA与Cas9蛋白对应的tracrRNA连接形成，其中所述crRNA包含一种或多种选自SEQ ID NO:1-16的序列。
  16. 如权利要求15所述的用于敲除靶向基因的sgRNA，其中所述的靶向基因选自TRAC、TRBC、B2M和PD1基因中的一个或多个。
  17. 如权利要求15或16所述的用于敲除靶向基因的sgRNA，其中所述的靶向基因为TRAC基因，所述的crRNA选自SEQ ID NO:1-12所示的一个或多个。
  18. 如权利要求15或16所述的用于敲除靶向基因的sgRNA，其中所述的靶向基因为B2M基因，所述的crRNA如SEQ ID NO:13所示。
  19. 如权利要求15或16所述的用于敲除靶向基因的sgRNA，其中所述的靶向基因为PD1基因，所述的crRNA选自SEQ ID NO:14-16所示的一个或多个。
  20. 如权利要求15所述的用于敲除靶向基因的sgRNA，其中所述Cas9蛋白为来自酿脓链球菌的Cas9蛋白如SEQ ID NO：18所示。
  21. 如权利要求15所述的用于敲除靶向基因的sgRNA，其中所述与Cas9蛋白对应的tracrRNA序列如SEQ ID NO：17所示。
  22. 一种用于基因敲除的试剂盒，其中所述试剂盒包含：
    a：一种或多种选自权利要求9-14中任一项所述的crRNA，或一种或多种选自权利要求15-21中任一项所述的sgRNA；
    b：Cas9蛋白；
    c：寡聚脱氧核糖核酸或鱼精DNA片段。
  23. 如权利要求22所述的用于基因敲除的试剂盒，其中所述寡聚脱氧核糖核酸是长度为100-250bp的双链DNA或长度为100-250nt的单链DNA。
  24. 如权利要求22所述的用于基因敲除的试剂盒，其中所述Cas9蛋白为来自酿脓链球菌的Cas9蛋白如SEQ ID NO：18所示。
  25. 如权利要求9所述的靶基因敲除的T细胞在制备抗肿瘤药物中的用途。
  26. 如权利要求9所述的靶基因敲除的T细胞在制备防治病毒或细菌引起的感染性疾病药物中的用途。
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