CN113862254A

CN113862254A - Non-viral site-directed knock-in method and its use in CAR-T cell therapy

Info

Publication number: CN113862254A
Application number: CN202010607601.4A
Authority: CN
Inventors: 彭作翰; 李玏; 张蕊; 刘瑗; 豆亚丽; 叶亚平
Original assignee: Xi'an Soniser Biomedical Co ltd
Current assignee: Xi'an Soniser Biomedical Co ltd
Priority date: 2020-06-30
Filing date: 2020-06-30
Publication date: 2021-12-31

Abstract

The present invention relates to non-viral site-directed knock-in methods and their use in CAR-T cell therapy. Specifically, the present invention performs PCR amplification of template DNA using a modified primer comprising: i)5 '-Phosphorothioate (PS) modification together with 5' -Biotin-triethylene glycol (Biotin-TEG) modification; or ii) a 5' -Locked Nucleic Acid (LNA) modification. Compared to previous methods, the methods of the invention enable more efficient and precise transduction regimes, simpler CAR-T cell production regimes, safer and more durable expression, and more varied designs.

Description

Non-viral site-directed knock-in method and its use in CAR-T cell therapy

Technical Field

The invention relates to the field of cell therapy, in particular to a non-viral site-directed knock-in method and application thereof in CAR-T cell therapy.

Background

CAR-T therapy

Traditional tumor treatment drugs include chemotherapeutic drugs and targeted drugs, which improve the survival time of cancer patients to a certain extent, but also bring serious side effects, and greatly reduce the life quality of the patients. Even more unfortunately, most patients still relapse after receiving these traditional treatments, and once they do, drug-free rescue is an approximate event.

In recent years, with the development of immunotherapy, the emergence of immune checkpoint inhibitor drugs (such as CTLA-4 and PD-1/PD-L1 antibodies) has drastically changed the mode of tumor therapy, but the effective rate of the drugs in different cancer patients is only 20% -40%, and most cancer patients wait for the emergence of new effective treatment modes.

CAR-T cell therapy technology, namely chimeric antigen receptor T cell therapy technology, is to make human T cells have the capacity of specifically killing tumor cells by transducing an artificial gene recognizing cancer cell surface antigens in vitro onto the human T cells. CAR-T cell therapy belongs to Adoptive Cell Transfer (ACT), which utilizes human immune cells to fight tumors, and is called a "live drug". The ACT therapy has recently been applied in clinical trials, particularly in the effective exploration of various leukemias, and opens up a new way for researchers and physicians to treat tumors. By the end of 2017, two CAR-T drugs have obtained us FDA approval for marketing, however, this ACT therapy is still in its early stages with the following innovation points compared to traditional drugs:

firstly, the tumor is precisely targeted, the side effect is reversible, CAR-T cell therapy has more specific tumor cell killing capacity than chemotherapeutic drugs and targeted drugs, after the CAR-T cell therapy is infused into a patient body, although a cytokine effect can be generated in a short time, the side effect is controllable, and the patient can live basically as a healthy person under the condition of complete remission; secondly, the sustained remission time is long, the CAR-T cell part belongs to a memory T cell, and can exist in the body of a patient for a long time after being infused into the body of the patient, whether tumor cells appear in the body is monitored at any time, and the tumor cells can be killed immediately once new tumor cells appear.

CAR-T cell preparation

The preparation of CAR-T cells can be divided into the following steps: t cell isolation, T cell activation, CAR gene transduction, CAR-T cell expansion, CAR-T cell harvest. Wherein CAR gene transduction is central to the overall manufacturing process. In recent years, a number of CAR transduction approaches have been developed:

1) lentivirus or retrovirus

Lentiviruses or retroviruses can be used to transfect DNA of interest of 3000 bases or less, which has the advantages of: a. the infection efficiency is high, and the positive rate after transfection can reach about 50 percent at most; the virus is secreted into the culture medium more, and the small-scale concentration and purification are relatively easy; c.T the lymphocyte can maintain a good cell state after being infected by virus, and the survival and the expansion are not easy to be affected, which is beneficial to the subsequent transfusion to the patient. The disadvantages are that: a. random insertion into the cell genome is liable to result in exhaustion of the generated CAR-T cells and potential risk of canceration; b. after T cells are infected, the residual virus has strong immunogenicity; c. large-scale preparation of viruses is complicated and the cost is high; d. foreign promoters are susceptible to long-term methylation inactivation.

2) Transposon

The transposon transduction mode has the advantages of simple production process flow and no risk of virus residue. However, since the vector encoding the transposon is plasmid DNA, it has a cytotoxic effect after electroporation into T cells, which is not conducive to the expansion of the resulting CAR-T cells. Likewise, transposon-mediated random insertion of CAR genes into the T cell genome is also prone to the resulting CAR-T cell depletion and potential risk of cancer.

3)mRNA

Transduction of T cells with mRNA encoded CAR genes relies on electroporation, which has the advantage of only transiently expressing the protein of interest in T cells (approximately 7 days of expression), and this approach allows relatively safe human experimentation for CAR-T cells that were previously uncertain for the presence of serious side effects. However, the disadvantage is also apparent, namely that it is not expressed permanently and has a relatively limited duration of action.

CRISPR-Cas9 technology

CRISPR (Clusters of Regularly interleaved Short Palindromic repeats) technology was commonly discovered in 2012 by scientists at the institute of technology, Massachusetts and Burkeley university, California, as a double-stranded DNA endonuclease tool mediated by RNA sequences. The target double-stranded DNA targeted recognition probe is composed of two parts, wherein one part is sgRNA with about 100 bases and used for targeted recognition of target double-stranded DNA, the other part is Cas9 protein with 1369 amino acids, the sgRNA can be combined with the sgRNA, the DNase activity is realized, the artificially designed sgRNA and the Cas9 protein can be specifically cut after forming a complex, when the cut causes mismatch repair, gene frame shifting is caused to achieve the purpose of knockout, and when a repair DNA sequence is added, the target editing can be performed. Since the CRISPR-Cas9 technology is available, the application of the CRISPR-Cas9 technology is available in a plurality of fields.

Large fragment gene knock-in technology

Although the gene knockout technology has been developed, the efficiency of gene knockout for large fragments has been low. Knock-in and knock-out differ by whether a repair template is added. The process is as follows: after a nuclease (such as ZFN, TALEN, CRISPR-Cas9 and the like) cuts a specific DNA, a repair template repairs the damaged DNA according to the homologous recombination principle, and a specific DNA fragment is introduced into a specific site after repair. The repair templates currently used are mainly plasmid DNA, double-stranded DNA, single-stranded DNA and adeno-associated virus vectors. For knocking in single or several bases, the several repairing templates can achieve high efficiency, and for knocking in more than 500 bases, only the adeno-associated virus vector has relatively highest efficiency at present, and about 40% knocking in efficiency can be achieved. However, the preparation cost of the adeno-associated virus vector is high, the adeno-associated virus vector takes long time, and the application of the adeno-associated virus vector in the preparation of CAR-T cells is not facilitated. And the efficiency of gene knock-in by taking plasmid DNA, double-stranded DNA and single-stranded DNA as a repair template is not more than 5 percent. For example, double-stranded DNA as a repair template has the advantage that it can be obtained by a large number of PCRs, which are much cheaper and more readily available than other repair templates; however, previous studies have demonstrated that long fragment gene knock-in efficiency using double-stranded DNA as a repair template is relatively low, reaching a maximum efficiency of about 5%. The single-stranded DNA template has the advantages that: after transduction, the cells were allowed to moderate; however, single-stranded DNA is cumbersome to prepare, costly, not amenable to large-scale application, and the knock-in efficiency is relatively low (comparable to double-stranded DNA templates).

Disclosure of Invention

In order to solve the above technical problems, the present invention provides the following technical solutions.

In one aspect, the present invention provides a non-viral gene site-directed knock-in method, comprising: PCR amplification of the template DNA was performed using the modified primers, and gene site-directed knock-in was performed using the modified template DNA as the donor DNA.

Wherein the modified primer comprises: i)5 '-Phosphorothioate (PS) modification together with 5' -Biotin-triethylene glycol (Biotin-TEG) modification; or ii) a 5' -Locked Nucleic Acid (LNA) modification.

In some embodiments, at least one 5 '-Phosphorothioate (PS) modification and at least one 5' -Biotin-triethylene glycol (Biotin-TEG) modification, or at least one 5 '-Locked Nucleic Acid (LNA) modification, is performed 1 to 15 bases from the 5' end of the upstream and downstream primers. Preferably, 5 '-Phosphorothioate (PS) together with 5' -Biotin-triethylene glycol (Biotin-TEG) modifications include: the 1 st base at the 5' end of the upstream and downstream primers is modified by Biotin-triethylene glycol (Biotin-TEG), and the 2 nd, 3 rd, 4 th, 5 th and 6 th bases are modified by Phosphorothioate (PS). Preferably, 5' -Locked Nucleic Acid (LNA) modifications comprise: the 1 st, 2 nd and 3 rd bases at the 5' end of the upstream and downstream primers are modified by Locked Nucleic Acid (LNA). The length of the modified primer may be 15 to 100 bases in length. Without being limited by theory, the reagents (kits) or associated equipment (PCR amplificators) used for PCR amplification may be from any company or manufacturer.

In some embodiments, the method comprises ligating the template DNA to a plasmid vector prior to PCR amplification. The plasmid vector may be any plasmid vector, such as pUC57, pCDNA3.1, pCMV, or the like. In some embodiments, the method does not include a step of ligating to a plasmid vector.

In some embodiments, the template DNA comprises left homology arm DNA, knock-in DNA, right homology arm DNA, wherein the left homology arm DNA is homologous to a sequence at the 5 'end of a DNA cut targeted by the site-specific knock-in, and the right homology arm DNA is homologous to a sequence at the 3' end of a DNA cut targeted by the site-specific knock-in. In some embodiments, the sequence of the left homology arm 3 'DNA is identical to the sequence of the 5' end 0 to 300 bases from the nicked DNA, preferably the left homology arm DNA has a fragment length of 10 to 5000 bp. In some embodiments, the right homology arm 5 'DNA sequence is identical to the 3' sequence 0 to 300 bases from the nicked DNA, preferably the right homology arm DNA has a fragment length of 10 to 5000 bp.

In some embodiments, wherein the knock-in DNA is any DNA, preferably 0 to 100000 bases in length. Knock-in DNA can be used for "adding" and also for "repairing". In some embodiments, the template DNA may also not comprise knock-in DNA.

In some embodiments, the gene site-directed knock-in method comprises purification of the modified template DNA after PCR amplification. Purification can be performed using a DNA purification kit, DNA-bound magnetic beads, gel chromatography, ion chromatography, affinity chromatography, ultrafiltration tube ultrafiltration, dialysis membrane dialysis, and any combination thereof.

In some embodiments, the gene editing methods used for gene site-directed typing include: CRISPR-Cas9, ZFN, ARCRS, TALEN, and megaTAL. In some embodiments, the gene editing material involved in the gene editing method comprises a plasmid, mRNA, or protein.

In some embodiments, the DNA targeted for gene site-directed knock-in can be any DNA, such as genomic DNA. Specifically, a gene-editing material involved in the gene-editing method is transfected into a cell together with a modified template DNA. Any transfection method can be used, for example, liposome, calcium phosphate, DEAE-dextran, electroporation, microinjection, gene gun, etc. After transfection, the nuclease in the gene editing material cleaves the genomic DNA to nick it into a broken double-stranded DNA, at which time the modified template DNA undergoes homologous recombination with the broken nicked DNA as donor DNA, allowing the knock-in DNA to successfully integrate into the desired genomic site.

The gene site-directed knock-in method of the present invention can be applied to the following cells: cord blood stem cells, bone marrow hematopoietic stem cells, various adult stem cells, embryonic stem cells, T lymphocytes, B lymphocytes, NK cells, NK-92 and NK-92 derived cells, macrophages, DC cells, CHO and CHO derived cells, 293 and 293 derived cells, common cell lines.

The gene site-directed knock-in method of the present invention can be applied to the following fields: CAR-Treg cell therapy, CAR-T cell therapy, TCR-T cell therapy, NK cell therapy, TIL cell therapy, DC cell therapy, macrophage therapy, CIK cell therapy, other immune cell therapy, cord blood stem cell therapy, bone marrow hematopoietic stem cell therapy, adult hematopoietic stem cell therapy, other stem cell therapy, animal model construction, antibody production, fusion protein production, vaccine production, cell line preparation.

The gene site-directed knock-in method of the present invention and products (e.g., cells, antibodies, proteins, etc.) prepared using the method can be used to treat the following diseases: hematologic or cancer (including acute B-lymphocytic leukemia, acute T-lymphocytic leukemia, acute NK-lymphocytic leukemia, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, acute myeloid leukemia, multiple myeloma, Fahrenheit's proteinemia, etc.), solid tumors (including lung cancer, liver cancer, stomach cancer, breast cancer, colorectal cancer, prostate cancer, pancreatic cancer, brain glioma, esophageal cancer, cholangiocarcinoma, endometrial cancer, ovarian cancer, mesothelioma, thymus cancer, etc.), autoimmune diseases (including systemic lupus erythematosus, B-cell-related autoimmune diseases), viral diseases (including AIDS, hepatitis B, hepatitis C, etc.), graft-versus-host disease, type I diabetes, thalassemia, heart failure, Alzheimer's disease, epilepsy, Parkinson's disease, cirrhosis, femoral head necrosis, pulmonary fibrosis, necrosis, Parkinson's disease, etc.), transplantation-versus-host disease, type I diabetes, thalassemia, heart failure, Alzheimer's disease, epilepsy, Parkinson's disease, liver cirrhosis, femoral head necrosis, pulmonary fibrosis, etc, Nerve injury diseases, degenerative diseases, renal failure, uremia, infertility, etc. Other areas of application may also include medical cosmetology.

In another aspect, the invention provides a method of making a CAR-T cell, the method comprising:

1) mixing a Cas9 protein with a targeted sgRNA in vitro to form an RNP complex, or mixing a Cas9 protein-encoding mRNA with a targeted sgRNA in vitro to form a mixture;

2) performing PCR amplification of the template DNA using a modified primer to obtain a modified double-stranded DNA, wherein the modified primer comprises: i)5 '-Phosphorothioate (PS) modification together with 5' -Biotin-triethylene glycol (Biotin-TEG) modification; or ii) a 5' -Locked Nucleic Acid (LNA) modification;

3) transfecting (preferably electrically transferring) the RNP complex or the mixture with the modified double-stranded DNA into a T cell to obtain the CAR-T cell.

The genomic position knocked in may be an exon region of any gene, an intron region of any gene, a promoter region of any gene, an enhancer region of any gene, or any non-gene coding region. Preferably, the genomic locations knocked in are selected from the group consisting of TRAC, TRBC1, TRBC2, PD-1, LAG-3, TIM-3, TIGIT, CTLA-4, B2M, CTIIA, TET-2, REGNASE-1, GM-CSF, TGFBR2, and NR4A, among others.

Firstly, defining a knock-in genome site, and designing the sgRNA by using a DNA sequence around the site, wherein the PAM region sequence at the 3' end of the targeted genome DNA is NGG (Next Generation group) and N is any one base in A, T, C, G. The sgRNA includes a targeted crRNA sequence and a tracrRNA sequence, where the crRNA can be 17, 18, 19, 20, 21, or 22 bases. The sgRNA may be unmodified sgRNA or chemically modified sgRNA. Chemical modifications include 2 '-O-methylation, 3' -thio, and combinations thereof. Chemical modifications occur in the 5 'and 3' ends of the sgrnas 1 to 10 bases, for example 3 bases at the 5 'and 3' ends with simultaneous 2 '-O-methylation modification and 3' -thio modification. The designed sgRNA can be obtained by T7 in vitro transcription, and can also be directly obtained by in vitro synthesis.

In some embodiments, Cas9 proteins include SpCas9, SaCas9, SpCas9-HF, eSpCas9, xCas9, cpf1 or other Cas9 proteins of different genera, preferably SpCas9, whose amino acid sequence is shown as SEQ ID NO: 62. One or more NLS nuclear signal peptide(s) are coupled at the N end or the C end of the Cas9 protein, and the NLS nuclear signal peptide sequence is shown as SEQ ID NO: 63. The fused Cas9 protein can be obtained by expression purification of bacterial or eukaryotic expression cells.

The RNP complex can be obtained by incubating the Cas9 protein and sgRNA in direct mixing, or by mixing the two in a specific buffer (e.g., an electrotransfer buffer).

In some embodiments, if the CAR gene is driven by a native gene promoter, the template DNA comprises a left homology arm, an optional cleavage peptide or internal ribosome entry site, the CAR gene, polyA, and a right homology arm. If the CAR gene is initiated by an exogenous promoter, the template DNA comprises a left homology arm, an exogenous promoter, a CAR gene, polyA, and a right homology arm.

In some embodiments, the left homology arm is homologous to a sequence at the 5 'end of a DNA nick targeted by a site-directed knock-in, and the right homology arm is homologous to a sequence at the 3' end of a DNA nick targeted by a site-directed knock-in. The 3 'DNA sequence of the left homology arm is identical to the 5' sequence of 0 to 300 bases from the nicked DNA, preferably the fragment length of the left homology arm is 10 to 5000 bp. The DNA sequence at the 5 'end of the right homology arm is identical to the sequence at the 3' end 0 to 300 bases away from the nicked DNA, preferably the fragment length of the right homology arm is 10 to 5000 bp. More preferably, the fragments of the left and right homology arms are 300, 600 or 1000 bases in length, and the sequences are shown in SEQ ID NO 64-69, respectively.

In some embodiments, the cleavage peptide or internal ribosome entry site may be selected from P2A, T2A, IRES, and the like. Preferably, the amino acid sequences of the cleavage peptides P2A and T2A are shown in SEQ ID NO:70-71, respectively, and the DNA sequence of the internal ribosome entry site IRES is shown in SEQ ID NO: 72.

In some embodiments, the exogenous promoter includes EF1, CMV, PGK, MSCV, SFFV, etc., more preferably EF1, whose DNA sequence is shown in SEQ ID NO. 73.

In some embodiments, the polyA may comprise a miniploya or bGHpA structure, among others.

In some embodiments, the CAR gene can be a second or third generation structure, preferably a second generation CAR gene structure. In some embodiments, the target targeted by the CAR gene may be selected from: one or more (i.e., single target, dual target, or multiple target) of CD19, CD22, CD20, CD7, CD123, CD33, CD38, BCMA, PSMA, Her2, Mesothelin, CS1, MUC16, GD2, GPC3, CEA, CD138, EGFR, EGFRVIII, lewis y, DLL3, MG7, and IL13R α 2. For example, the amino acid sequences of the CD19 and the antibody scFv of the BCMA target used in the present invention are shown in SEQ ID NOS: 76 to 77, respectively, and the DNA sequences are shown in SEQ ID NOS: 78 to 79, respectively. The CAR gene may comprise a signal peptide DNA, a hinge region DNA, a transmembrane region DNA, a costimulatory signal region DNA. The signal peptide DNA is selected from signal peptide domains such as CD8, IL-2, GM-CSF and the like, preferably CD8 signal peptide, the amino acid sequence of which is shown as SEQ ID NO. 80, and the DNA sequence of which is shown as SEQ ID NO. 81. The hinge region DNA can be selected from hinge domains such as IgG1, IgG4, IgD or CD8, and preferably is a CD8 hinge structure, the amino acid sequence of the hinge structure DNA is shown as SEQ ID NO. 82, and the DNA sequence of the hinge structure DNA is shown as SEQ ID NO. 83. The transmembrane region DNA can be selected from transmembrane domains such as CD3, CD4, CD5, CD8, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154 and PD1, and more preferably is a CD8 transmembrane structure, the amino acid sequence of the transmembrane region DNA is shown as SEQ ID NO:84, and the DNA sequence is shown as SEQ ID NO: 85. The costimulatory signal region DNA can be selected from costimulatory domains such as CD2, CD7, CD27, CD28, CD30, CD40, CD54, CD83, CD134, CD137, CD150, CD152, CD223, CD270, CD273, CD274, CD278, CARD11, NKD2C, DAP10, LAT, SLP76, ZAP70 and 4-1BB, preferably 4-1BB costimulatory structure, the amino acid sequence of which is shown as SEQ ID NO. 86, and the DNA sequence of which is shown as SEQ ID NO. 87. The activation region DNA can be CD3 zeta activation structural domain, the amino acid sequence of the activation region DNA is shown as SEQ ID NO. 88, and the DNA sequence is shown as SEQ ID NO. 89.

In a preferred embodiment, the genomic site targeted for the sgRNA is the first, second, third or fourth exon, preferably the first exon region, of the TRAC gene. Preferably, the sequence of the TRAC gene (comprising the PAM sequence) targeted by the sgRNA sequence is shown in SEQ ID NO:1-29, more preferably in SEQ ID NO: 7. Preferably, the sgRNA sequence is shown in SEQ ID NO. 30-58, more preferably in SEQ ID NO. 36. Further preferably, the sgRNA of SEQ ID No. 36 comprises a targeted crRNA sequence and a tracrRNA sequence, wherein the crRNA is 17, 18, 19, 20, 21 or 22 bases, preferably 18, 19 or 20 bases, and the sequences thereof are shown in SEQ ID NOs 59-61, respectively.

In a preferred embodiment, if the CAR gene is driven by the TRAC gene promoter, the template DNA sequences of 300, 600, 1000 base homology arms (targeting the CD19CAR gene and carrying miniSolyA) are the template DNAs of SEQ ID NO:90-92, preferably 300 base homology arms, respectively. The template DNA sequence of 300 base homology arm (targeting CD19CAR gene and carrying bGHpA polyA) is SEQ ID NO. 93. The template DNA sequence of 300 base homology arm (targeting BCMA CAR gene with bGHpA polyA) is SEQ ID NO 94. The template DNA sequence of 300 base homology arm (targeting BCMA CAR gene, with miniSolyA) is SEQ ID NO. 95. If the CAR gene is initiated by a foreign promoter, preferably EF1, the template DNA sequence of the 300 base homology arm (targeting CD19CAR gene, with EF1 and miniplolyA) is SEQ ID NO: 96.

In some embodiments, the method comprises purification of the modified double-stranded DNA after PCR amplification. Purification can be performed using a DNA purification kit, DNA-bound magnetic beads, gel chromatography, ion chromatography, affinity chromatography, ultrafiltration tube ultrafiltration, dialysis membrane dialysis, and any combination thereof.

In some embodiments, the method comprises: peripheral Blood Mononuclear Cells (PBMCs) were extracted and T cells were activated. In some embodiments, PBMCs are isolated from human blood, or alternatively PBMCs are apheresis. In some embodiments, PBMC isolation from human blood may be performed using lymphocyte apheresis and in conjunction with common centrifuge tubes, sepnate tubes, or other hardware devices. T lymphocytes may be isolated and purified or not isolated. If T lymphocytes need to be separated and purified, kits of various manufacturers can be adopted, and various negative selection and positive selection methods can be adopted. T cell activation can be performed using the following method: coating with anti-CD 3 antibody alone, coating with anti-CD 3 antibody/anti-CD 28 antibody, directly adding anti-CD 3 antibody alone, directly adding anti-CD 3 antibody/anti-CD 28 antibody, anti-CD 3 antibody/anti-CD 28 antibody magnetic beads, etc. The activation time may be 1 to 8 days. If the magnetic beads of the antibodies are used for activation, magnetic poles are needed to remove the magnetic beads before electrotransfer.

In some embodiments, the cells are enriched by centrifugation after T cell activation is complete.

In some embodiments, the transfection in step 3) is electroporation. The number of T cells per electroporation unit depends on the instrumentation requirements. The configuration of the electric transfer system is determined according to the number of cells in a single electric transfer unit, and the buffer solution of the electric transfer system is usually provided by the equipment manufacturer, and can be replaced by other buffer solutions, such as PBS, MEM or 1640. The prepared RNP complex is mixed with an electrotransfer buffer solution, and then the modified double-stranded DNA is added, wherein the addition amount is also based on the cell number of a single electrotransfer unit. The addition of the double stranded DNA can be incubated with RNP for a period of time varying from 0 to 30 minutes. The prepared electric transfer system is mixed with cells and then added into an electric transfer cup for immediate electric transfer, and the electric transfer condition can be according to the requirements of manufacturers and can also be found by self. The cells after electroporation can be placed in an electroporation cuvette for a period of time and then added to the culture medium.

In some embodiments, the method further comprises expansion culturing of the CAR-T cells. The culture of the T cells after the electrotransformation adopts 1640 culture medium + 10% serum or other serum-free culture medium special for the culture of the T cells, such as X-VIVO-15 and ImmunoCult^TMXF, OpTsizer medium, etc. Can be placed in a culture bottle, a culture dish, a G-rex or a culture bag for culture. In the culture process, certain concentration of cytokine such as IL-2, IL-7, IL-15, etc. is added.

In some embodiments, the method further comprises cryopreserving the CAR-T cells. And (3) freezing and storing the cultured CAR-T cells according to a certain cell concentration, wherein the used freezing and storing liquid can be GMP or non-GMP grade products provided in the market, and can be placed in a special freezing and storing bag or a freezing and storing tube for freezing and storing. After being subjected to gradient cooling by special equipment, the mixture is placed in a refrigerator with eighty degrees below zero or a liquid nitrogen tank for storage.

In some embodiments, the method further comprises performing the following assay prior to CAR-T cell cryopreservation: sterility test, mycoplasma test, endotoxin test, CAR positive proportion, CD4/CD8 proportion, memory T cell proportion, effector T cell proportion, killing effect on target cells, cytokine amount released after killing, anti-tumor effect in mice, off-target effect generated by CAR gene knock-in, and the like.

The CAR-T cells (the CAR-T cells which are knocked into the site-specific integration of TRAC gene loci) prepared by the invention can be used for treating various related diseases, including various leukemias (acute B lymphocyte leukemia, acute T lymphocyte leukemia, acute NK lymphocyte leukemia, various non-Hodgkin lymphomas, chronic lymphocyte leukemia, acute myeloid leukemia, multiple myeloma and the like), various solid tumors (lung cancer, liver cancer, stomach cancer, breast cancer, colorectal cancer, prostate cancer, pancreatic cancer, brain glioma, esophageal cancer, bile duct cancer, endometrial cancer, ovarian cancer, mesothelioma, thymus cancer and the like), AIDS, autoimmune diseases and various diseases which can be treated by the CAR-T cells.

The invention has the beneficial technical effects that:

1. a more efficient accurate transduction mode can meet the application in the preparation of CAR-T cells

The prior non-viral gene site-directed knock-in efficiency is low, especially for long-fragment gene knock-in, the site-directed knock-in efficiency in T cells is not more than 5%, and the damage to cells is extremely large, so the method cannot be applied to the preparation of CAR-T cells. The invention adopts chemically modified double-stranded DNA as a repair template, greatly improves the fixed-point knock-in of long-fragment genes, has the highest knock-in efficiency of 30 percent, has better cell survival rate and can effectively amplify. In addition, the method is used for preparing the CAR-T cells meeting the clinical number in a plurality of volunteers, which fully proves that the method can be completely applied to various CAR-T cell preparations.

2. Simpler CAR-T cell production protocol

In the prior art, lentivirus or a reverse transcription vector is mostly needed for preparing the CAR-T cells to transduce CAR genes, so that the upstream of the CAR-T cells is a viral vector, the upstream plasmid vector is needed for preparing the viral vector, and the plasmid vector and the viral vector need complicated processes for large-scale preparation and have higher requirements on production preparation and instruments and equipment. The non-viral gene site-specific knock-in technology can prepare CAR-T cells without depending on any virus, only needs one section of double-stranded DNA repair template, and bypasses the fussy plasmid and virus preparation process and safety verification.

3. Is safer

The conventional slow virus, retrovirus and transposon technology-mediated CAR gene transfer adopted by CAR-T cell preparation is a random insertion mode, and can cause the canceration of T cells to seriously affect the CAR-T cell treatment safety.

4. More durable expression

The promoter used by lentivirus and retrovirus is influenced by methylation in T cells and is easy to inactivate for a long time so that the CAR gene is not expressed, thus the CAR-T cells are disabled and further disease recurrence is induced.

5. More varied designs

Non-viral gene site-directed knock-in techniques can make the way CAR gene insertion more variable. For example, inserting TRAC and TRBC genomic sites can destroy TCR genes while inserting CAR genes to make universal CAR-T cells, inserting PD-1 genomic sites to be controlled by PD-1 gene promoters can achieve better curative effect and specificity, and inserting HIF-1 genomic sites to be regulated by the promoters can make hypoxia-induced CAR-T cells to be applied to solid tumors more specifically.

Drawings

FIG. 1 is a schematic diagram of a high-efficiency non-viral gene site-directed knock-in technique: a. after template DNA (including left homology arm DNA, knock-in DNA, right homology arm DNA) is connected to a plasmid vector, template DNA PCR is carried out by using a modified primer; b. after the specific nuclease is sheared and broken, the target genome DNA and the modified template DNA are subjected to homologous recombination, so that the target DNA is knocked in.

FIG. 2 is a schematic of the CAR-T cell therapeutic application of the gene site-directed knock-in technique of the invention: mixing a Cas9 protein and a targeted sgRNA in vitro to form an RNP complex/mRNA encoding a Cas9 protein and the targeted sgRNA in vitro to form a mixture; b. cloning template DNA to a carrier, then carrying out PCR amplification by using a modified primer, and concentrating and purifying; c. separating and purifying T cells from human body and activating; d. (ii) an electroporated T cell; e. CAR-T cell expansion and functional and phenotypic analysis after electrotransformation.

FIG. 3 is a schematic diagram of knocking-in the CAR gene into a target site in example 1.

Fig. 4 shows the results of flow cytometer (a), agarose gel electrophoresis (b) and LDH killing detection (c) of example 7.

FIG. 5 shows the CAR gene site-directed knock-in different TRAC genomic DNA site comparison experimental procedure (a) and flow assay results (b) in example 8.

Fig. 6 shows the streaming detection result of example 9.

Fig. 7 shows the streaming detection result of example 10.

Fig. 8 shows the streaming detection result of example 11.

FIG. 9 shows a schematic representation (a) and flow assay results (b) of a TRAC-dependent promoter or exogenous promoter comparison experiment after site-specific knock-in of CAR gene in example 12.

FIG. 10 shows TCR expression profile (a) and insertion or deletion mutation analysis results (b) of example 13.

FIG. 11 shows the flow assay results (a-d), LDH killing assay results (e), and cytokine release assay (f) of example 14.

Fig. 12 shows CD3 expression (a), T cell purification results (b), CD19 chimeric antigen receptor CAR expression (c), CAR gene expression assay results (d), CD4/CD8a assay results (e), CD45RO/CD62L assay results (f), and in vitro killing assay (g) of example 15.

FIG. 13 shows CD3 expression (a), T cell purification results (b), CAR expression (c), CAR gene expression assay (d), CD4/CD8a assay (e), CD45RO/CD62L assay (f), and in vitro killing assay (g) of example 16.

Detailed Description

Definition of

CAR-T: a chimeric antigen receptor T cell.

CTLA-4: cytoxic T-lymphocyte associated protein 4, the earliest approved immunodetection point inhibitor target.

PD-1/PD-L1: programmed cell death 1/ligand of PD-1protein, the two best known immunodetection point inhibitor targets.

ACT adoptive cell transfer, which is an adoptive immunotherapy that transfers lymphocytes from a donor to a recipient to enhance the cellular immune function thereof. Adoptive cellular immunity can be classified into specific and non-specific, in which the former is to inject lymphocytes sensitized with known antigens into a receptor to obtain cellular immunity against the antigens, and the latter is to inject normal human lymphocytes not sensitized with specific antigens into a receptor to obtain cellular immunity against various antigens.

CRISPR: clustered regulated short palindromic repeats, a bacterial immune system, have been modified by scientists as the hottest gene editing tool in the year.

ZFN (gene editing technology) is an artificially synthesized restriction enzyme, which is formed by fusing a zinc finger DNA (deoxyribonucleic acid) binding domain and a DNA cutting domain of the restriction enzyme, and researchers can target and locate different DNA sequences by processing and modifying the zinc finger DNA binding domain of the ZFN, so that the ZFN can be combined with a target sequence in a complex genome and is subjected to specific cutting by the DNA cutting domain.

TALEN is a gene editing technology, and a typical TALEN consists of an N-terminal structural domain containing a nuclear localization signal, a central structural domain containing a typical tandem TALE repetitive sequence capable of identifying a specific DNA sequence and a C-terminal structural domain with the function of FokI endonuclease, can be combined with a target sequence in a complex genome, and is specifically cut by a DNA cutting domain.

ARCUS, a gene editing technology, has the advantages that the nuclease used has a small structure and is easy to deliver in vivo.

MegatAL is a gene editing technology, which is characterized in that a homing endonuclease and a DNA binding domain of a transcription activator-like effector are fused together, a target sequence in a complex genome can be bound, and specific cutting is carried out by a DNA cutting domain.

Knockin: the position of the target gene is introduced into a specific mutation or foreign gene by gene editing.

dsDNA-double stranded DNA.

RNP Cas9 protein and sgRNA complex short.

messenger RNA (messenger RNA) is a single-stranded ribonucleic acid which is transcribed by taking one strand of DNA as a template and carries genetic information and can guide protein synthesis. After mRNA is produced by transcription from gene in cell as template based on base complementary pairing principle, the mRNA contains base sequence corresponding to some functional segment in DNA molecule as direct template for protein biosynthesis.

AAV (adeno-associated virus) vector, a high-efficiency and safe gene delivery tool, is mainly used for gene therapy in recent years and can also be used as a repair template for knocking in large-fragment genes.

CAR-Treg is an adoptive cell therapy technology, CAR gene is transferred into Treg cells to be used for inhibiting specific antigen cell activity, and the CAR-Treg is currently in a preclinical research stage and is expected to be used for treating autoimmune diseases in the future.

TCR-T an adoptive cell therapy technique, which is used for treating various refractory tumors by transducing natural or optimized TCR genes screened in a laboratory to T cells, carrying out in-vitro amplification culture and finally returning the T cells to a patient.

sgRNA, an RNA artificially invented in CRISPR application, which fuses crRNA and tracrRNA, can bind Cas9 protein and target DNA of interest.

PolyA-a polyadenylic acid structure, is a post-transcriptional modification of mRNA that helps to stabilize mRNA and improve translation efficiency.

Examples

Example 1: molecular design of repair template DNA

In this example, the CAR gene knocks into the T cell TRAC gene site and a method of co-electrotransformation with modified repair template DNA using a CRISPR-Cas9 element was employed. Firstly, a CRISPR-Cas9 element is utilized to generate a nick on a TRAC gene locus, then the modified repair template DNA is utilized to carry out homologous recombination repair on the nick, and finally, the CAR gene is knocked into a target locus as shown in figure 3.

The repaired template DNA contains two parts:

a. left and right homology arms. The left and right homology arms are used to identify the DNA of interest and to perform recombination exchanges. In order to allow for the correct expression of the knock-in gene, the knock-in gene is "logged in" to the 5' end region of exon 1 of the TRAC gene. According to the length of the base sequence, homologous arms of 300, 600 and 1000 bases are respectively designed, the sequence is shown as SEQ ID NO:64-69, and the following table specifically shows:

b. a knock-in gene. The knocked-in genes comprise a splicing peptide gene, a CAR gene and polyA, depending on the TCR-alpha promoter group; depending on the exogenous promoter set, the knock-in genes include an exogenous promoter, a CAR gene, and polyA. In this example, the cleavage peptide used was P2A and the exogenous promoter used was EF 1. The CAR gene adopts a secondary structure, and the specific structure comprises a targeting CD19 scFv or a targeting BCMA sequence, a CD8 hinge region, a CD8 transmembrane region, a 4-1BB costimulatory region and a CD3 zeta activation region. In this example, the polyA is bGHpA polyA or mini polyA, and the structural sequence of each gene is as follows:

example 2: molecular cloning of repair template DNA

The repair template DNA of SEQ ID NO. 90-98 was synthesized and cloned into a pUC57 plasmid vector. The process comprises the following steps:

1) and (3) preparing a target fragment by PCR. Firstly, a PCR reaction system needs to be prepared:

reagent (cargo number: RO5OA, TAKARA)	Amount used (ul)
		5 XPrimeSTAR GXL buffer solution	10
dNTP Mix	4
		Primer F	1
Primer R	1
		DNA fragment	0.5
Sterilized water	32.5
		PrimeSTAR GXL DNA polymerase	1
Total volume	50

Then, the PCR reaction system is placed in a PCR amplification instrument for amplification according to the following conditions:

2) preparation of vector PCR. Firstly, a PCR reaction system needs to be prepared:

3) and (3) recombining the target fragment with a vector: adding a target gene and a linearized vector into a centrifuge tube on an ice box according to a certain molar ratio for recombination reaction, and reacting for 10 minutes in a water bath kettle at 50 ℃, wherein the reaction system is shown in the following table:

reagent (cat # NR001A, near shore protein)	Amount used (ul)
		Vector pUC57	3.5
DNA fragment 0001	4
		5 × reaction buffer	2
NovoRec Plus recombinase	0.5
		Total volume	10

4) And (3) conversion coating of reaction products: melting a tube of 100. mu.l StbI3 competent cells (Cathaya, DL1046, Withania) on ice, flicking the tube wall to resuspend the cells, adding 10. mu.l of the reaction solution to the competent cells, and ice-cooling for 30 min under flicking; quickly putting the mixture on ice for 5 minutes after heat shock is carried out for 90 seconds in a water bath kettle at 42 ℃; adding 500 mul LB liquid culture medium, and incubating for 45-60 minutes at 37 ℃ by a shaking table; the cells were collected by centrifugation at 5000rpm for 3 minutes, 300. mu.l of the supernatant was discarded, and the remaining cell amount was applied to a plate containing ampicillin and incubated overnight in an incubator at 37 ℃.

5) Single clones were picked, and plasmid miniprep kit (cat No.: DP103-03) was used for plasmid extraction. The column equilibration step comprises: adding 500 μ l of equilibrium liquid BL into adsorption column CP3, centrifuging at 12,000rpm (13,400 Xg) for 1 min, discarding the waste liquid in the collection tube, and replacing the adsorption column in the collection tube; taking 5ml of overnight cultured bacterial liquid, adding the bacterial liquid into a centrifuge tube, centrifuging for 1 minute at 12,000rpm (13,400 Xg) by using a conventional desktop centrifuge, and absorbing the supernatant as much as possible; adding 250 mul of kit solution P1 into the centrifuge tube with the bacterial sediment, and thoroughly suspending the bacterial sediment by using a pipette or a vortex oscillator; adding 250 mul of kit solution P2 into the centrifuge tube, and gently turning the centrifuge tube up and down for 6-8 times to fully crack the thalli; adding 350 mul of kit solution P3 into a centrifuge tube, immediately and gently turning up and down for 6-8 times, and fully mixing uniformly, wherein white flocculent precipitate appears; centrifugation at 12,000rpm (13,400 Xg) for 10 minutes; transferring the supernatant collected in the previous step into adsorption column CP3 (the adsorption column is put into a collection tube) by using a pipette; centrifuging at 12,000rpm (13,400 Xg) for 30-60 s, removing waste liquid from the collecting tube, and placing adsorption column CP3 into the collecting tube; adding 500 μ l deproteinized solution PD into adsorption column CP3, centrifuging at 12,000rpm (13,400 × g) for 30-60 s, discarding the waste liquid in the collection tube, and replacing adsorption column CP3 in the collection tube; adding 600 μ l of rinsing liquid PW into adsorption column CP3, centrifuging at 12,000rpm (13,400 × g) for 30-60 s, pouring off waste liquid in the collection tube, and placing adsorption column CP3 into the collection tube; repeating the previous step; the adsorption column CP3 was placed in a collection tube and centrifuged at 12,000rpm (13,400 Xg) for 2 minutes in order to remove the residual rinse from the adsorption column; the adsorption column CP3 was placed in a clean centrifuge tube, 50-100. mu.l of elution buffer EB was dropped onto the middle portion of the adsorption membrane, and the solution was left at room temperature for 2 minutes and centrifuged at 12,000rpm (13,400 Xg) for 2 minutes to collect the plasmid solution in the centrifuge tube. The resulting solution was re-loaded into the adsorption column, left at room temperature for 2 minutes, centrifuged at 12,000rpm (13,400 Xg) for 2 minutes, and the plasmid solution was collected in a centrifuge tube.

6) And (5) sequencing and confirming.

Example 3: primer design for PCR amplification of repair template DNA

The sequences and modifications of the PCR primers for repairing the template DNA are respectively as follows:

example 4: PCR amplification of repair template DNA

20 general PCR tubes were prepared for each PCR number, and the following PCR reaction mixtures were prepared in each PCR tube:

numbering	Primer F	Primer R	pUC57-SEQ ID
				PCR-1	1F	1R	NO:92
PCR-2	2F	2R	NO:91
				PCR-3	3F	3R	NO:97
PCR-4	4F	4R	NO:98
				PCR-5	5F	5R	NO:90
PCR-6	6F	6R	NO:90
				PCR-7	7F	7R	NO:90
PCR-8	8F	8R	NO:90
				PCR-9	9F	9R	NO:93
PCR-10	10F	10R	NO:94
				PCR-11	11F	11R	NO:95
PCR-12	12F	12R	NO:96

after the PCR is finished, the mixture is collected in a 15ml centrifuge tube.

Example 5: purification of PCR products of repair template DNA

Purification of PCR product of template DNA was carried out by using a large-scale DNA product purification kit (cat # DP205) of Tiangen Biochemical technology (Beijing) Ltd. A100. mu.l PCR system was performed using one column, and 1000. mu.l PCR product required 10 columns for purification, as follows:

1) column equilibration step: adding 500 μ l of equilibrium liquid BL into adsorption column CB3 (the adsorption column is put into the collection tube), centrifuging at 12,000rpm (-13,400 Xg) for 1 min, pouring the waste liquid out of the collection tube, and putting the adsorption column back into the collection tube;

2) estimating the volume of the PCR reaction solution, adding 5 times of the volume of the binding solution PB into the PCR reaction solution, and fully and uniformly mixing;

3) adding the solution obtained in the previous step into an adsorption column CB3 (the adsorption column is placed into a collecting pipe), standing for 2 minutes at room temperature, centrifuging for 30-60 seconds at 12,000rpm (13,400 Xg), pouring the waste liquid in the collecting pipe, and placing an adsorption column CB3 into the collecting pipe;

4) adding 600 mul of rinsing liquid PW into an adsorption column CB3, centrifuging at 12,000rpm (13,400 Xg) for 30-60 seconds, pouring waste liquid in a collecting pipe, and putting the adsorption column CB3 into the collecting pipe;

5) repeating the operation step 4);

6) placing the centrifugal adsorption column CB3 back into the collecting pipe, centrifuging at 12,000rpm (13,400 Xg) for 2 minutes, removing the rinsing liquid as much as possible, placing the adsorption column at room temperature for a plurality of minutes, and completely airing to prevent the residual rinsing liquid from influencing the next experiment;

7) taking out the adsorption column CB3, putting the adsorption column CB3 into a clean centrifugal tube, hanging and dripping a proper amount of elution buffer solution EB into the middle position of an adsorption film, standing the adsorption film for 2 minutes at room temperature, centrifuging the adsorption film for 2 minutes at 12,000rpm (13,400 Xg), and collecting DNA solution;

8) DNA solutions were pooled and collected, and DNA concentration was determined by Nanodrop.

Example 6: sgRNA design and Synthesis

The sgRNA used in the invention is synthesized in vitro, and has two types of chemical modification and non-chemical modification, wherein 3 bases of the chemically modified sgRNA at the 5 'end and 3' end are subjected to 3 '-thio and 2' -O-methylation modification, so that the sgRNA is more stable and is not easily and rapidly degraded by nuclease after being transduced into cells. After chemically synthesizing corresponding sgrnas, HLPC is used for purification, and the following are chemically synthesized sgRNA sequences used in the present invention:

example 7: effect of different chemically modified repair template DNAs on CAR Gene knock-in efficiency

The fixed-point knock-in efficiency of double-stranded DNA as a repair template is always low, so that the double-stranded DNA cannot be applied to the transduction of CAR genes in CAR-T cell preparation. The invention innovatively adopts several chemically modified double-stranded DNAs as a repair template, greatly improves CAR gene transduction efficiency, and comprises the following specific steps:

1) cell preparation

PBMC extraction: healthy volunteers were recruited, who did not have symptoms of cold and fever, and 100ml of blood was drawn from the median vein of the elbow by medical professionals and connected to an anticoagulation vessel (cat No. 367886, Becton Dickinson); after the blood collection, the blood was mixed with an equal amount of PBS buffer (containing 2% fetal bovine serum (cat # SE100-B, Vistech)); taking a PBMC separation tube Sepmate-50 (cat # 86450, STEMCELL), carefully adding 15ml of Ficoll buffer solution, then adding the mixed solution of blood PBS, and carefully adding about 30ml of the mixed solution into each tube; centrifuging 1200g for 10 minutes, quickly pouring the supernatant into a new 50ml tube, centrifuging 200g for 8 minutes, discarding the supernatant, adding 10ml PBS buffer solution for resuspension and precipitation, discarding the supernatant, adding 10ml PBS buffer solution for resuspension, centrifuging and discarding the supernatant, and then resuspending the cell precipitation by 10ml PBS buffer solution; the resuspended cells were counted, 10. mu.l of the suspension was added to 10. mu.l of 0.1% trypan blue (cat # 15250061, Gibco) and mixed, and the cell count and viability were counted on the machine.

T cell purification: taking a small amount of PBMC cells obtained, and calculating the proportion of CD3 positive T by a flow cytometer; taking out according to the proportion of the CD3 positive cells to the experimentThe required PBMC cells should be 70% of the cells taken (lost during the procedure); sorting was performed using a kit Release Human CD3 Positive Selection Cocktail (CatCELL, cat: 17751), and total PBMC cells were first resuspended in Positive Selection buffer to a total cell concentration of 1X 10⁸Per ml; each 1 × 10⁸Cells were added with 100 μ l of CD3 antibody; incubation for 3 minutes at room temperature; the magnetic beads (reusable Rapid Sphenes) were mixed together in advance by a vortex mixer for 30 seconds at 1X 10 intervals⁸Cells were added to 100. mu.l of magnetic beads and incubated at room temperature for 3 minutes; transferring the cell suspension into a special sorting tube, metering the volume to 2.5ml by using positive sorting buffer solution, and placing the cell suspension on a magnetic frame for incubation for 5 minutes at room temperature; carefully grasping the magnetic frame, dumping the sorting tube, discarding the unbound cell suspension, re-suspending the attached magnetic beads and cells with 2.5ml of positive selection buffer solution, placing on the magnetic frame again, incubating at room temperature for 3 minutes, and discarding the unbound cell suspension again; taking down the sorting tube from the magnetic frame, adding a 'Release buffer solution' into the suspension, and incubating for 3 minutes at room temperature; placing on a magnetic frame and incubating for 5 minutes at room temperature; carefully grasping the magnetic frame, pouring the sorting tube, and collecting the unbound cell suspension in a clean sterile 15ml centrifuge tube, wherein the cell is the extracted T cell.

Activation of T cells: taking the purified T cells 1.5X 10⁷Activation with anti-CD 3/anti-CD 28 magnetic beads (cat # 40203D, Thermo): anti-CD 3/anti-CD 28 magnetic beads 1X 10 are taken⁷Resuspend with PBS buffer (containing 2mM EDTA and 1% fetal calf serum), add to the magnetic pole, stand for 2 minutes, and carefully discard the supernatant; repeating the above process; taking the washed magnetic beads, adding the magnetic beads into T cells, uniformly mixing, and culturing for two days at 37 ℃; taking out the magnetic beads after two days, firstly re-suspending the T cells for multiple times by using a pipette, then placing the cell suspension in a magnetic pole, standing for two minutes, and then removing the magnetic beads on the tube wall; cell number and survival rate were measured on the machine.

Electric conversion: each taking 3X 10⁶Placing the cell suspension in a centrifuge tube (5 tubes in total) for centrifugation at the rotation speed of 200g for 5 minutes, and completely removing the culture medium for later use after centrifugation; lonza electrotransfer buffer (cat # V4XP-3024, Lonza) was prepared, 15. mu.g spCas9 protein (cat # A36499, Thermo) and sgRNA-00011. mu.g of the designed and synthesized protein were added,after mixing evenly for 10 minutes at room temperature, 12 mu g of modified and unmodified SEQ ID NO 20 repair template DNA is added, wherein sgRNA and the corresponding repair template DNA in the example are shown in the following table:

after 10 minutes, the cell pellet was mixed well and added to an electric rotor (cat # V4XP-3024, Lonza), and EH115 was programmed to shock, and 400. mu.l of the medium was added immediately after completion of the shock and left in a 37 ℃ incubator for 15 minutes, and then added to 5ml of the preheated cell medium.

2) Flow assay

After the completion of electrotransformation, the control T cells and the electrotransformed CAR-T cells were incubated once every 1 day, and the cell density was adjusted to 1X 10 after incubation⁶Each ml of the culture medium was supplemented with 10ng/ml of recombinant IL-2 (cat # 200-02-1000, PeproTech), and the cells were cultured in well plates throughout the culture. Cells were harvested at 1X 10 days after completion of electroporation⁶After one time of PBS washing, CD19-FITC (cat # CD9-HF251, Poppsies) is added for staining for 25 minutes, PBS is washed again, cells are finally resuspended by PBS, and the cells are analyzed by an up-flow cytometer, and the result is shown in FIG. 4a, and from the flow result, the two modifications, namely 5 '-Phosphorothioate (PS) combined with 5' -Biotin-triethylene glycol (Biotin-TEG) and 5 '-Locked Nucleic Acid (LNA), can improve the knock-in efficiency of the CAR gene, wherein the 5' -Locked Nucleic Acid (LNA) modification is most significant.

3) Targeted genomic locus PCR detection

Cell collection: counting the cell samples to be extracted:

taking each sample 3X 10⁶(ii) individual cells; after centrifugation, the cells were washed once with PBS and the cell pellet was retained for DNA extraction.

DNA extraction: extracting with DNA extraction kit (cat # DP349-02, Tiangen Biochemical), adding 750 μ l/tube cell lysate CLA into cell precipitate, and reversing and mixing for 20 times; centrifuging at 12000rpm for 1 min, discarding the supernatant, placing the centrifuge tube upside down on clean absorbent paper and staying for 2 min to ensure the precipitation in the tube; preparing a mixed solution (200. mu.l: 1.5. mu.l) of a buffer solution FGA and proteinase K; 3000 mul buffer solution FGA +22.5 mul proteinase K, and mixing evenly; adding 200 mul/tube buffer solution FGA and proteinase K mixed solution, immediately mixing by vortex, adding 30 mul/tube buffer solution FGA and proteinase K mixed solution because colloidal precipitation is difficult to mix uniformly, and mixing by vortex again until the solution has no lumps; water bath at 65 deg.c for 10 min while reversing and mixing for several times; adding 200 mul/tube of isopropyl ketone, reversing the mixture up and down, and uniformly mixing the mixture for 50 times until filiform or clustered genome DNA appears; centrifuging at 12000rpm for 5 min, discarding the supernatant, inverting the centrifuge tube, and adding onto clean absorbent paper to ensure that the precipitate is in the tube; adding 300 mul/tube of 70% ethanol, vortexing and shaking for 5 seconds, centrifuging at 12000rpm for 2 minutes, and pouring off the supernatant; inverting the centrifuge tube over clean absorbent paper and allowing it to stand for 2 minutes to ensure settling in the tube; air dry the DNA pellet until all liquid was evaporated clean (15 min); 200 mul/tube buffer TB was added, vortexed for 5 seconds, heated at 65 ℃ for 20 minutes to dissolve DNA, during which the solution was made by flicking several times, and the incubation time was extended to 1h due to the presence of insoluble material.

And (3) DNA concentration detection: the resulting DNA concentration was measured using a microspectrophotometer as follows:

and (3) PCR: firstly, preparing a PCR reaction system:

reagent	Amount used (ul)
		5 XPrimeSTAR GXL buffer solution	4
dNTP Mix	1.6
		9F(5’-CAAACCTCTGTTTCCTCCTCAAAAG-3’)	0.5
9R(5’-TGCTGCTCTTCTCCTTTCTCATTG-3’)	0.5
		The above DNA extracted from cells	5
Sterilized water	17.9
		PrimeSTAR GXL DNA polymerase	0.5
Total volume	20

DNA agarose gel electrophoresis: taking out after the PCR operation is finished, loading agarose gel, and adding 2 mul of loading buffer solution into 8 mul of sample; the size of the bands was observed by a gel imaging instrument after the running of the gel was completed, as shown in fig. 4b, and the result shows that wild type T cell genomic DNA can PCR amplify one DNA band close to 2000 bases, and all CAR knock-in groups have one more DNA band above the wild type, especially compared with the chemically unmodified group, the combination of 5' -Phosphorothioate (PS) with 5' -Biotin-triethylene glycol (Biotin-TEG) and 5' -Locked Nucleic Acid (LNA) modified group is most significant, indicating that the gene knock-in was successful. In theory, the CAR gene knock-in successful cells have only one more DNA band on the wild type, but probably because double-stranded DNA forms polymers, different forms of repair template DNA polymerization are knocked in during DNA recombination, but the 5 '-Locked Nucleic Acid (LNA) chemical modification group has only one more DNA band on the wild type, which suggests that template repair double-stranded DNA polymerization can be inhibited by 5' -Locked Nucleic Acid (LNA) chemical modification.

4) And (3) LDH killing detection: cell killing experiments were performed using the Lactate Dehydrogenase (LDH) cytotoxicity assay. After the cells are cultured to the 10 th day, respectively sucking cell suspensions of effector cells and target cells into a 1.5ml EP tube, wherein the target cells are constructed by transferring a lentiviral vector containing CD19 protein into a K562 cell line which does not express CD19 protein originally in a high-speed centrifugation mode and then carrying out expanded culture; then, 300g of the above cells were centrifuged for 5 minutes while preparing a 1640 medium (Cat. No.: 61870-; discarding the supernatant in the cells as much as possible after centrifugation, suspending the cells by 200 mul of prepared culture medium, and counting the cells after fully and uniformly mixing; configuring a system according to the counting result; sequentially adding the materials into a V-bottom 96-hole plate according to the following design scheme; the ratio of the number of effector cells to the number of target cells in this experiment was 5: 1; after the addition is finished, putting the cell plate into a centrifuge, centrifuging the cell plate for 2 minutes at 100g, and after the centrifugation is finished, putting the cell plate into an incubator; the following morning, 20. mu.l of lysate from LDH cell killing assay detection kit (cat # G1782, Promega) was added to only 9 wells of "volume control" and "K562-CD 19 maximum release" and then the 96-well plate was placed in an incubator for 45 minutes while the substrates in the kit were left to dissolve at room temperature; after incubation is finished, sucking 50 mu l of liquid out of each hole, transferring the liquid into a 96-hole flat bottom plate, quickly adding 50 mu l of substrate in the kit into each hole by using a line gun, reacting the liquid to be detected and the substrate at room temperature for about 3 minutes to obviously see the color change of the hole with maximum release, and quickly adding 50 mu l of termination reaction liquid in the kit into each hole; then after waiting about 3 minutes, the color of each well stabilized, the air bubbles were punctured with a clean needle, the values of each well were measured at 490nm with a microplate reader (Thermo Fisher Multiskan FC), and the raw data were processed according to the formula of the kit instructions. The results of the detection of the killing effect of the control T cell and the CAR-T cell on the cancer cell K562-CD19 are shown in FIG. 4c, and the results show that all CAR knock-in groups can kill the K562-CD19 cell effectively, wherein compared with the chemically unmodified group, the killing efficiency is improved significantly by combining 5 '-Phosphorothioate (PS) with 5' -Biotin-triethylene glycol (Biotin-TEG) and 5 '-Locked Nucleic Acid (LNA) modification, and particularly the improvement of the 5' -Locked Nucleic Acid (LNA) modification group is more obvious.

And (4) conclusion: the template DNA modified by 5 '-Locked Nucleic Acid (LNA) and 5' -thiophosphoric acid (PS) combined with 5 '-Biotin-triethylene glycol (Biotin-TEG) can obviously improve the knocking-in efficiency of the template DNA compared with an unmodified group, wherein the 5' -Locked Nucleic Acid (LNA) modification can not only improve the knocking-in efficiency of the template DNA, but also inhibit the template DNA from forming polymers to knock in target genomic DNA.

Example 8: CAR gene site-specific knock-in different TRAC genomic DNA site alignment

Researchers in "reproducing human T cell function and specificity with non-viral genome targeting" knock NY-ESO-1TCR genes into TRAC genomic sites through unmodified double-stranded DNA templates in combination with CRISPR-Cas9 technology, respectively, although feasibility has been demonstrated, efficiency is still not high. The TRAC genomic site targeted and knocked-in this example is not in agreement with the above reference and shows that this site will be more efficient for CAR gene knock-in. FIG. 5a is a comparison of the knockin TRAC genomic locus of the target of this example with that of the above-mentioned reference. In fig. 5a, the base sequence is TRAC genomic DNA, wherein the underdrawn bases are intron regions, the underdrawn bases are the first exon regions of the TRAC gene, the large font bases are PAM sequences, and the triangular cusps point to the corresponding CRISPR-Cas9 cleavage sites. FIG. 5a shows the comparative experimental procedure for site-directed knock-in of the CAR gene into the genomic DNA of different TRACs.

1) Cell preparation

PBMC extraction: same as in example 7.

T cell purification: same as in example 7.

Activation of T cells: taking the purified T cells 1X 10⁷Activation with anti-CD 3/anti-CD 28 magnetic beads was performed in the same manner as in example 7.

Electric conversion: each taking 3X 10⁶The cell suspension is placed in a centrifuge tube (3 tubes in total) for centrifugation at the rotation speed of 200g for 5 minutes, and the culture medium is completely removed for standby after centrifugation. A Lonza electrotransfer buffer solution is prepared, 15 mu g of spCas9 protein (cargo number: A36499, Thermo) and 1 mu g of sgRNA designed and synthesized are added, and after the mixture is mixed for 10 minutes at room temperature, 12 mu g of repair template DNA is added, wherein the sgRNA and the corresponding repair template DNA in the example are shown in the following table:

after 10 minutes, the cell pellet is mixed evenly and added into an electric rotating cup, electric shock is carried out by an EH115 program, 400 mul of culture medium is added immediately after the electric shock is finished, the culture medium is placed in a 37-degree incubator for 15 minutes, and then the culture medium is added into 5ml of preheated cell culture medium.

2) Flow assay

The procedure for flow-based detection was the same as in example 7, and the results are shown in FIG. 5b, which shows that in both groups, the targeted and knocked-in TRAC genomic sites and their sgRNAs in this example were used for CAR gene knock-in with higher efficiency than the report on copying human T cell function and specificity with non-viral genome targeting.

Example 9: comparison of CAR Gene knock-in efficiency after mRNA or RNP form CRISPR-Cas9 Combined with chemically modified template DNA for T cell electrotransformation

CRISPR-Cas9 can be delivered into cells in different forms, including: plasmid vectors, mRNA, RNP, lentiviral or retroviral vectors, adeno-associated viral vectors. Among these, the most widely used in T cells are the mRNA and RNP forms. The mRNA form refers to that in vitro transcribed mRNA encodes Cas9 protein, is jointly transferred into a T cell by combining with sgRNA, and after being transferred into the T cell, the mRNA is translated into Cas9 protein and acts on target site DNA after forming a complex RNP with the sgRNA in a cell nucleus. The RNP form is directly formed in vitro and then transduced into T cells, and the RNP form directly acts after introduction into T cells. Both mRNA and RNP can theoretically be knocked in CAR gene after being combined with the inventive modified template DNA to electroporate cells, and this example will compare the effect of the two delivery forms of CRISPR-Cas9 on CAR gene knock-in, with the following specific steps:

1) cell preparation

PBMC extraction: same as in example 7.

T cell purification: same as in example 7.

Electric conversion: each taking 3X 10⁶Placing the cell suspension in a centrifuge tube (2 tubes in total) for centrifugation at the rotation speed of 200g for 5 minutes, and completely removing the culture medium for later use after centrifugation; two tubes of Lonza electrotransfer buffer were prepared and then added: a.5 μ g spCas9 mRNA (obtained by in vitro transcription with T7-spCas9 DNA as a template), 1 μ g of synthetic sgRNA and 12 μ g of repair template DNA are designed; b. 15 μ g of spCas9 protein (cat # A36499, Thermo) and 1 μ g of designed and synthesized sgRNA were added, mixed for 10 minutes at room temperature, and then 12 μ g of repair template DNA was added. Sgrnas and corresponding repair template DNAs in this example are as follows:

group of	sgRNA	Repair template DNA	Number of cells transferred by electroporation
				mRNA	sgRNA-0001	PCR-8 product	3×10⁶
RNP	sgRNA-0001	PCR-8 product	3×10⁶

2) Flow assay

The procedure for flow detection was the same as in example 7, and the results are shown in FIG. 6. The results show that both mRNA and RNP groups knock-in the targeted CD19CAR gene, but RNP group knock-in efficiency is relatively higher and cell viability is better.

Example 10: effect of different polyA on efficiency of knocking-in of CAR Gene into T cells

In this example, two polyAs, miniPolyA and bGHpA, whose sequences are SEQ ID NO 74 and SEQ ID NO 75, were used, respectively. The repair template DNA sequences adopted in this example are SEQ ID NO:90 and SEQ ID NO:93, respectively, with miniPolyA and bGHpA, and the CAR gene is a targeting CD19CAR, and this example compares the effects of these two polyAs on the expression of the knocked-in gene, and the specific implementation steps are as follows:

1) cell preparation

PBMC extraction: same as in example 7.

T cell purification: same as in example 7.

Electric conversion: each taking 3X 10⁶Placing the cell suspension in a centrifuge tube (2 tubes in total) for centrifugation at the rotation speed of 200g for 5 minutes, and completely removing the culture medium for later use after centrifugation; a Lonza electrotransfer buffer solution is prepared, 15 mu g of spCas9 protein (cargo number: A36499, Thermo) and 1 mu g of sgRNA designed and synthesized are added, and after the mixture is mixed for 10 minutes at room temperature, 12 mu g of repair template DNA is added, wherein the sgRNA and the corresponding repair template DNA in the example are shown in the following table:

group of	sgRNA	Repair template DNA	Number of cells transferred by electroporation
				miniPolyA	sgRNA-0001	PCR-8 product	3×10⁶
bGHpA	sgRNA-0001	PCR-9 product	3×10⁶

2) Flow assay

The procedure for flow detection was the same as in example 7, and the results are shown in FIG. 7. The results show that the two polyA groups of miniPolyA and bGHpA have the same expression efficiency of the knock-in targeting CD19CAR gene, indicating that both polyas can be used as polyA structures of knock-in genes.

Example 11: effect of different sgRNAs on CAR Gene knockin T cell efficiency

In this example, three sgrnas were designed for the same cleavage site, wherein the lengths of the grnas were 18, 19, and 20 bases, and the sequences thereof were sgRNA-0001, sgRNA-0009, and sgRNA-0010, respectively. In this example, the effect of the three sgrnas on CAR gene knock-in was compared, specifically as follows:

1) cell preparation

PBMC extraction: same as in example 7.

T cell purification: same as in example 7.

Electric conversion: each taking 3X 10⁶Placing the cell suspension in a centrifuge tube (3 tubes in total) for centrifugation at the rotation speed of 200g for 5 minutes, and completely removing the culture medium for later use after centrifugation; a Lonza electrotransfer buffer solution is prepared, 15 mu g of spCas9 protein (cargo number: A36499, Thermo) and 1 mu g of sgRNA designed and synthesized are added, and after the mixture is mixed for 10 minutes at room temperature, 12 mu g of repair template DNA is added, wherein the sgRNA and the corresponding repair template DNA in the example are shown in the following table:

group of	sgRNA	Repair template DNA	Number of cells transferred by electroporation
				sgRNA-0001	sgRNA-0001	PCR-8 product	3×10⁶
sgRNA-0009	sgRNA-0009	PCR-8 product	3×10⁶
				sgRNA-0010	sgRNA-0010	PCR-8 product	3×10⁶

2) Flow assay

After the completion of electrotransformation, the control T cells and the electrotransformed CAR-T cells were incubated once every 1 day, and the cell density was adjusted to 1X 10 after incubation⁶Each ml of recombinant IL-2 was added at 10ng/ml during the culture and the cells were cultured in well plates throughout. Cells were harvested at 1X 10 days after completion of electroporation⁶After one PBS wash, CD19-FITC protein and PE-TCR-. alpha./beta.antibody were added for 25 min, PBS was washed again, and finally the cells were resuspended in PBS and analyzed by flow cytometry, and the results are shown in FIG. 8. The results showed that sgRNA-0009 and sgRNA-0010 had higher TCR gene knock-out efficiency than sgRNA-0001, but only sgRNA-0010 had higher CAR gene knock-in efficiency than sgRNA-0001. The results indicate that sgrnas of 20bp in length are likely to be knocked out and knocked in more efficiently.

Example 12: contrast experiment of TRAC gene promoter or exogenous promoter dependent after CAR gene site-specific knock-in

According to the invention, the CAR gene knock-in TRAC genomic site can start CAR gene expression by utilizing a TCR-alpha gene promoter, and can also start CAR gene expression by adding an exogenous promoter in front of the CAR gene, wherein the CAR gene expression is started by adopting an EF1 exogenous promoter in the embodiment, and a related schematic diagram is shown in figure 9 a.

The repair template DNAs adopted in this example are shown in SEQ ID NO. 90 and SEQ ID NO. 96, respectively, and the specific steps are as follows:

1) cell preparation

PBMC extraction: same as in example 7.

T cell purification: same as in example 7.

2) Flow assay

The procedure for flow detection was the same as in example 7, and the results are shown in FIG. 9 b. The results show that both TCR gene promoter and EF1 promoter were able to drive knockin CAR gene expression.

Example 13: CRISPR-Cas9 off-target TIDE assay

Utilizing a website https:// design. synthgo.com/analysis sgRNA-0010 off-target site, selecting an encoded protein region off-target site therein, and obtaining the following results:

primers are respectively designed according to DNA sequences of the sites, the size of PCR fragments of the primers is about 750 bases, and specific primer sequences are as follows:

t cells knock out TCR-alpha genes after sgRNA-0010 and spCas9 protein electrotransformation, and TCR-alpha negative homozygous cells are obtained by purifying with a kit, and the method comprises the following specific steps: according to the number of magnetic beads: TCR positive cell number 2: 1, taking out a proper amount of sorted magnetic beads (cargo number: 11151D, Thermo) and placing the sorted magnetic beads into a 1.5ml EP tube, adding 1ml of washing buffer solution (1 XPBS + 0.1% FBS +2mM EDTA), slightly reversing, uniformly mixing, placing on a magnetic frame, standing for 3 minutes, and then sucking liquid in the tube by using a pipette and discarding the liquid to achieve the purpose of washing the magnetic beads; collecting all experimental groups in cultured cells into 115 ml centrifuge tube, placing the centrifuge tube into a centrifuge for 300g, centrifuging for 5 minutes, discarding supernatant, fully mixing the cells and the magnetic beads prepared in the previous step by using 1ml of serum-free T cell amplification culture medium, re-suspending, transferring into 1 new 1.5ml EP tube, and placing the EP tube on a rotary incubator for slow rotary incubation for 30 minutes at room temperature; after the incubation is finished, placing the EP tube on a magnetic frame and standing for 3 minutes, sucking the liquid in the tube by using a pipette and transferring the liquid into a new 1.5ml EP tube, wherein the cells contained in the cell suspension are the cells with negative TCR expression after sorting and purification; and (3) taking out about 30 mu l of cell suspension obtained after sorting and purification from the EP tube, washing the cells, then incubating Anti-TCR-PE antibody, washing away the unbound antibody, finally re-suspending the cells, and detecting the TCR expression condition by an up-flow cytometer. The detection result is shown in fig. 10a, and the result shows that 96% of the sorted and purified living cells are negative for TCR expression.

Genomic DNA of T cell control and sorted TCR-negative homozygous cells were extracted separately (same procedure as in example 7); the off-target primers are respectively used for PCR amplification, and the prepared PCR system and the prepared PCR program are respectively as follows:

after PCR is finished, running DNA electrophoresis; cutting the gel and recovering DNA; sequencing with primers F respectively; performing insertion or deletion mutation analysis on the sequencing result of the obtained ab1 file in pairs respectively by using a website https:// ice.synthego.com/#/; the alignment result is shown in fig. 10b, and the result shows that no insertion or deletion mutation occurs in the potential 10 off-target sites, whereas 95% of the targeted TRAC genomic sites have insertion or deletion mutation, which proves that the sgRNA and the corresponding method used in the present invention are precise gene editing, and the off-target caused by the precise gene editing is theoretically very limited.

Example 14: targeting CD19 CAR-T cells by comparison of non-viral gene site-directed knock-in with Lentiviral transduction

The preparation of CAR-T cells by lentivirus transduction is already a relatively mature technology, and this example compares the process and species product differences of preparing targeted CD19 CAR-T cells by non-viral gene site-directed knock-in and lentivirus transduction, and the specific steps are as follows:

1) cell preparation

PBMC extraction: same as in example 7.

T cell purification: same as in example 7.

Activation of T cells: taking the purified T cells 2X 10⁷Activation with anti-CD 3/anti-CD 28 magnetic beads: 2X 10 magnetic beads of anti-CD 3/anti-CD 28 were used⁷The rest of the procedure was the same as in example 7.

Lentivirus transfection: 48 hours after activation, 1X 10⁷Putting the T cells into a 15ml tube, centrifuging to remove the culture medium, adding the targeting CD19CAR gene lentivirus according to MOI (equal to 5), and centrifuging for 90 minutes at 2000 g; after the centrifugation is finished, removing the lentivirus supernatant, and adding a culture medium for continuous culture.

Electric conversion: each taking 3.3 × 10⁶Placing the cell suspension in a centrifuge tube (3 tubes in total) for centrifugation at the rotation speed of 200g for 5 minutes, and completely removing the culture medium for later use after centrifugation; a Lonza electrotransfer buffer solution is prepared, 15 mu g of spCas9 protein (cargo number: A36499, Thermo) and 1 mu g of sgRNA designed and synthesized are added, and after the mixture is mixed for 10 minutes at room temperature, 12 mu g of repair template DNA is added, wherein the sgRNA and the corresponding repair template DNA in the example are shown in the following table:

sgRNA	repair template DNA	Number of cells transferred by electroporation
			sgRNA-0001	PCR-8 product	3.3×10⁶
sgRNA-0001	PCR-8 product	3.3×10⁶
			sgRNA-0001	PCR-8 product	3.3×10⁶

2) Flow assay

After the completion of the electrotransformation, the control T cells, the lentivirus-transfected cells and the electrotransformed CAR-T cells were subjected to fluid exchange every 1 day, after which the cell density was adjusted to 1X 10⁶Adding 10ng/ml recombinant IL-2 in the culture process every milliliter, and placing the cells in a pore plate for culture in the whole process; cells were harvested at 1X 10 days after completion of electroporation⁶After one wash with PBS, flow antibody staining for 25 min, a further wash with PBS, and finally resuspension of the cells with PBS, CAR gene/CD 4/CD8, CD45RO/CD62L and CD69/Lag-3 were analyzed on an up-flow cytometer with the antibodies: CD4 (cat # 12-0048-42, Thermo), CD8 (cat # 17-0088-42, Thermo), CD45RO (cat # 12-0457-42, Thermo), CD62L (cat # 25-0629-42, Thermo), CD69 (cat # 17-0699-42, Thermo), Lag-3 (cat # 369205, Biolegend). The results of the tests are shown in FIGS. 11 a-d. The results show that site-directed knockin CAR-T cells (GT CAR-T) and lentiviral transduced CAR-T cells (lenti-CAR-T) both expressed the CAR gene well, with a CD4/8 ratio, CD45RO/CD62L ratio comparable to control T cells, while the lentiviral transduced CAR-T positive cells had a significantly higher CD69 positive ratio than the site-directed knockin CAR-T cells, suggesting that the lentiviral transduced CAR-T cells were always in an activated state and that the lentiviral transduced CAR-T positive cells had a significantly higher bag-3 positive ratio than the site-directed knockin CAR-T cells, suggesting that many depleted cells were generated.

3) LDH killing detection

In this example we used the Lactate Dehydrogenase (LDH) cytotoxicity assay to perform the cell killing assay. After the cells had been cultured to day 15, the medium in the upper layer of the G-rex flask was removed, and the remaining CAR-T cell suspension was mixed well and aspirated to approximately 1.5X 10 each⁶The corresponding cell suspension volume for each cell was transferred to a 1.5ml EP tube and similarly aspirated at approximately 5X 10⁶The corresponding cell suspension volume for each control cell was transferred to a 1.5ml EP tube and an additional aspiration of approximately 5X 10⁵Transferring the cell suspension volume corresponding to the K562-CD19 cells into a 1.5ml EP tube, wherein the tube cells are target cells in an experiment, and the target cells are constructed by transferring a lentiviral vector containing CD19 protein into a K562 cell line which does not express CD19 protein originally in a high-speed centrifugation mode and then carrying out expanded culture; then, the above three cells were centrifuged at 300g for 5 minutes while preparing 1640 medium containing 1% bovine serum, and only this medium was used in this experiment; discarding the supernatant in the cells as much as possible after centrifugation, suspending the cells by 200 mul of prepared culture medium, and counting the cells after fully and uniformly mixing; configuring a system according to the counting result; sequentially adding the materials into a V-bottom 96-hole plate according to the following design scheme; the ratio of the number of effector cells to the number of target cells in this experiment was: 1:1, 5:1 and 10: 1; after the addition is finished, putting the cell plate into a centrifuge, centrifuging the cell plate for 2 minutes at 100g, and after the centrifugation is finished, putting the cell plate into an incubator; the next morning, 20 μ l of lysate from the LDH cell killing assay detection kit was added to only 9 wells of the "volume control" and "K562-CD 19 maximum release" and the 96-well plate was incubated in the incubator for 45 minutes while the substrates in the kit were left to dissolve at room temperature; after incubation is finished, sucking 50 mu l of liquid out of each hole, transferring the liquid into a 96-hole flat bottom plate, quickly adding 50 mu l of substrate in the kit into each hole by using a line gun, reacting the liquid to be detected and the substrate at room temperature for about 3 minutes to obviously see the color change of the hole with maximum release, and quickly adding 50 mu l of termination reaction liquid in the kit into each hole; then after waiting for about 3 minutes, the color of each well stabilized and was cleanedThe needle of the kit punctures the bubbles, then the value of each hole is detected at 490nm by using an enzyme-labeling instrument, and finally the original data is processed according to the formula of the kit specification to make a bar chart; the results of the detection of the killing effect of the control T cells and the CAR-T cells on the target cells K562-CD19 are shown in FIG. 11 e. The results show that both lentiviral transduced CAR-T cells (lenti-CAR-T) and site-directed knockin CAR-T cells (GT CAR-T) can significantly kill the target cells.

4) Cytokine release assay: the detection is carried out by using an IFN-gamma detection kit (cargo number: 1110002) of Dake corporation, and the specific steps are as follows: fully and uniformly mixing all reagents to avoid generating foams; determining the number of required laths according to the number of experimental holes (blank and standard); sample adding: adding the diluted cytokine standard substance to a standard substance hole at 100 mu L/hole, adding the sample to a sample hole at 100 mu L/hole, and adding a dilution buffer solution R (1X) to a blank control hole at 100 mu L/hole; adding a detection antibody: adding 50 mu L/hole of biotinylated antibody working solution; after mixing, covering a sealing plate membrane, and incubating for 2 hours at room temperature (18-25 ℃); washing the plate: deducting liquid in the hole, adding 1 multiplied washing buffer solution working solution into 300 mu L/hole, staying for 1 minute, and then discarding liquid in the hole; repeating for 3 times, and drying on the filter paper each time; adding an enzyme: add Streptavidin-horse radish peroxidase (Streptavidin-HRP) working solution to 100. mu.L/well, cover the sealing plate membrane, incubate for 20 minutes at room temperature (18-25 ℃); repeating the plate washing step; color development: adding TMB into each hole with the volume of 100 mu L, incubating for 5-30 minutes at room temperature (18-25 ℃) in the dark, judging to stop reaction according to the shade (dark blue) of the color in the holes, and usually developing for 10-20 minutes to achieve good effect; and (3) terminating the reaction: adding stop solution into 100 mu L/hole to stop reaction; reading a plate: reading the value with the detection wavelength (measurement wavelength) of 450nm within 10 minutes after termination; calculated IFN- γ cytokines released by both lentiviral transduced and non-viral site-directed knock-in targeted CD19 CAR-T killed target cells as shown in figure 11f, the results show that significant killing of target cells by both lentiviral transduced CAR-T cells (lenti-CAR-T) and site-directed knock-in CAR-T cells (GT CAR-T) resulted in a massive release of the cytokines IFN- γ.

Example 15: preparation of non-viral site-directed knock-in targeting CD19 CAR-T cells from single-sampling volunteers

1) Obtaining T cells in peripheral blood of volunteers

Obtaining about 30ml of single blood collected by a volunteer 1, and transferring the single blood collected by a 50ml sterile syringe into a 50ml centrifuge tube; slowly pouring 1 XPBS into the blood to dilute the single blood in a ratio of 1:1 for further use; a new 50ml centrifuge tube was prepared, and a human peripheral blood lymphocyte separation medium (cat # 17-5442-03, GE Healthcare) was added thereto; slowly adding the diluted blood sample obtained in the previous step along the wall gently to ensure that the layering state of the separation liquid and the blood sample is not damaged; the volume ratio of the final separation liquid to the single blood sample is about 3: 7; then carefully placing the added centrifuge tube into a centrifuge, centrifuging for 15 minutes at 1200g, and adjusting the brake to be minimum; carefully taking out the centrifuge tube after centrifugation is finished and observing the position of a leucocyte layer, wherein the leucocyte layer is the position of peripheral blood mononuclear cells, sucking yellow clear liquid at the upper layer by using a pipette and discarding the yellow clear liquid, carefully sucking the leucocyte layer into a new 50ml centrifuge tube by using a pipette gun, adding 1 multiplied by PBS into the centrifuge tube to 50ml, then 1200g, centrifuging for 5 minutes to clean the leucocyte layer, and if the obtained leucocyte layer has a larger volume, separating the obtained leucocyte layer into multiple tubes for cleaning; repeating the washing step once, wherein the parameter of the second centrifugation is 300g and the time is 5 minutes; after washing, resuspending the cells with 30ml of 1 × PBS, uniformly mixing, detecting the concentration and the survival rate of the cells, and calculating the number of the living cells, wherein the specific operation steps are shown in a later part of cell quality inspection; the specific data of the total number of peripheral blood mononuclear cells and the cell viability of the volunteers collected in the example are as follows:

according to the counting result, taking out the cell suspension volume corresponding to a proper amount of cells, and averagely dividing the cell suspension into two EP tubes with the volume of 1.5 ml; adding 2 μ l of CD3 flow antibody to one tube, mixing well, standing at room temperature in the dark for 15 min, and using the other tube as control cell without adding antibody; after the antibody incubation is finished, resuspending cells for detection, wherein the specific operation steps are shown in the 'cell quality detection' part below; collecting offers using streaming machines1×10⁴Detecting the expression condition of CD3 of each cell, and calculating the number of T cells in the peripheral blood mononuclear cell suspension of the current volunteer according to the finally calculated CD3 positive rate in the total cells, namely the proportion of the T cells in the peripheral blood mononuclear cells of the volunteer; the results of the test of the expression of CD3 in the PBMC cells of the volunteers in this example are shown in FIG. 12a and the results of the T cell count calculation are as follows:

	total number of PBMC cells	Positive rate of CD3	Total number of T cells
				Volunteers	5.40×10⁸	16％	8.60×10⁷

Taking out 3.5 × 10 according to the calculation result of flow detection⁷The cell suspension volume corresponding to each T cell is selected by using a human T cell positive sorting kit, and finally, the sorted T cell suspension obtained by the experiment is counted to calculate the actual T cell number, and considering the loss of the operation process, about 2.8 multiplied by 10 can be generally obtained⁷(ii) a T cell; the positive rate of CD3 was again determined from the counting results, and if the positive rate was almost 100%, the T cell purification was successful, and the verification of the T cell purification results in this example is shown in FIG. 12b, which indicates that the positive rate is close to 100%, and that the T cells are close to 100%The number calculation results are as follows:

after the purification was successful, 2.5X 10 cells were removed according to the counting results⁷Putting the cell suspension volume corresponding to each T cell into a centrifuge tube for 300g, and centrifuging for 5 minutes; also according to the number of cells: number of magnetic beads 1: 3 preparing T cell activation magnetic beads; after centrifugation, the supernatant was discarded, and the cells and magnetic beads were resuspended in 20ml of serum-free T cell expansion medium containing 0.1ng/ml IL-2, mixed well and cultured in six-well plates on average.

2) Electric converter

After the T cells are activated for 48 hours, fully adsorbing the magnetic beads by using a magnetic frame, transferring the activated T cell suspension into a new 50ml tube by using a pipette, centrifuging for 5 minutes at 300g, removing supernatant, and adding about 5ml of 1 XPBS (phosphate buffer solution) into the supernatant to resuspend cell precipitates; simultaneously counting the uniformly mixed cell suspension, and taking out 5 multiplied by 10 according to the counting result⁵Individual T cells were cultured as control cells without electrotransfection in 1ml serum-free T cell expansion medium supplemented with 0.01ng/ml IL-2; then according to 5 multiplied by 10 of each tube⁶Evenly dividing the cell suspension into a plurality of 1.5ml EP tubes by a plurality of T cells, centrifuging at 300g for 5 minutes, removing the supernatant as much as possible, and waiting for the next step; the calculation results of the total number of electrically transfectable cells obtained after the demagnetization operation in this example are as follows:

in addition, while centrifuging, serum-free T cell amplification culture medium is added into a six-hole cell culture plate, and then the culture plate is placed in a cell culture box for preheating, so that the thin cells knocked in at fixed points are finally incubatedReducing the stimulation of low temperature to the cell when the cell is in the middle of the period; every 5X 10⁶Each T cell to be transfected corresponds to 5ml of serum-free T cell amplification medium and is placed in one hole of a six-hole cell culture plate for incubation and culture; preparing cells, and configuring an electrotransformation system by using the electrotransfection kit; adding 18 mu l of supplementary solution into a 1.5ml EP tube, adding 82 mu l P3 primary cell sap into the supplementary solution, gently and fully mixing the supplementary solution and the primary cell sap, and standing the mixture for 2 minutes at room temperature; then, 15 μ g of spCas9 protein (cat # A36499, Thermo) and 1ug of chemically modified 0001sgRNA were sequentially added to the mixed buffer; after the addition is finished, the mixture is soft and fully mixed, and is kept stand for 10 minutes at room temperature; then adding 6 mu g of chemically modified template DNA SEQ ID NO:93 into the mixture, softly and fully mixing the mixture evenly, and standing the mixture for 10 minutes at room temperature; preheating an electrotransfection instrument (LONZA 4D-Nucleofector) in the standing process; immediately after standing, using the mixed buffer solution obtained in the previous step to re-suspend the T cell sediment of the volunteer 1 to be used, slowly injecting the re-suspended and uniformly mixed cell suspension into an electric transfer cup along the wall by using a liquid transfer gun, and taking care not to generate bubbles; after the cover of the electric rotating cup is opened, the electric rotating cup is quickly and lightly shaken left and right to enable the front liquid level and the rear liquid level of the electric rotating cup to be equal in height; immediately placing the electric rotating cup into an electric transfectance instrument for electric rotation, wherein the electric rotating mode is EH 115; after the electric shock process is successfully completed, carefully opening the electric rotating cup, slightly adding 400 mu l of the preheated serum-free T cell amplification culture medium to the edge wall of the electric rotating cup, covering the electric rotating cup with a cover, and slightly shaking the electric rotating cup left and right to fully mix the culture medium and the original cell suspension in the electric rotating cup; immediately mixing, vertically placing the electric rotating cup in a cell culture box for standing and incubating for 15 minutes; after the incubation is finished, immediately transferring the cell suspension in the electrotransfection cups to a six-hole cell culture plate which is prepared before and contains a preheated culture medium by using a siphon tube in the electrotransfection kit, and transferring the cell suspension in each electrotransfection cup to one hole of the six-hole cell culture plate; immediately supplementing the IL-2 cytokine after the transfer is finished, so that the final concentration of the cytokine is 0.1 ng/ml; finally, the cell culture plate is placed in a cell culture box.

3) Preliminary examination of the expression Effect of Targeted CD19CAR Gene site-directed knock-in

Selecting any one hole of a six-hole cell culture plate to lightly blow and uniformly mix when the electrotransfection cell culture reaches 72 hours, and taking out a proper amount of CAR-T cells to carry out preliminary detection on the expression effect of CAR gene fixed-point knocking-in; the specific steps of flow sample treatment and detection are described in the section "cell quality testing" below; the results of a preliminary test of the expression of the chimeric antigen receptor CAR of CD19 on the surface of the electrically transfected cells of volunteer 1 in this example are shown in fig. 12c, suggesting that targeted CD19CAR expression is beginning.

4) Expanded culture of CAR-T cells

After the primary detection is qualified (namely the expression of CAR gene is more than 5% in general), all cells in a six-hole culture plate are gently blown and uniformly mixed, the cell suspension is directly injected into a G-rex cell culture bottle (product number: 81100, Wilson Wolf Manufacturing), the volume of a culture medium in the culture bottle is added to 100ml, and a serum-free T cell amplification culture medium with 0.01ng/ml IL-2 is added into a culture medium for amplification culture; after 48h, the cells in the flask were mixed well and then taken out about 1.8X 10⁶The individual cells were placed in a 1.5ml EP tube and were awaited for in vitro level killing detection; then supplementing the culture medium in the G-rex cell culture bottle to 500 ml; after the culture medium is added into 500ml, the culture medium is not added, IL-2 is added every other day, the growth condition of cells is observed, and the large shaking is avoided during observation; the culture was terminated by day 13 from the day of cell activation.

5) Cell harvesting

After the cell amplification culture is finished, sucking about 400ml of culture medium in a G-rex culture bottle by using a 50ml pipette, and discarding; attention is paid to ensure that the cell layer at the bottom of the culture bottle is almost still when the culture medium is sucked, so that the cell layer is prevented from entering the culture medium to be discarded due to blowing off, and the loss of cells is avoided; then gently and uniformly blowing and beating the residual about 100ml of cell suspension at the bottom of the culture bottle, then evenly dividing the cell suspension into 350 ml centrifuge tubes, demagnetizing the centrifuge tubes again by using a magnetic rack, and ensuring that no magnetic beads are left in the finally harvested CAR-T cells; then, the demagnetized cells were centrifuged at 300g for 5 minutes, the supernatant was discarded, and 30ml of 1 XPBS was added thereto forWashing the cell sediment, then centrifuging for 5 minutes at 300g, and discarding the supernatant; then adding 30ml of 1 XPBS, then sucking 10 mul of cell suspension from the uniformly mixed cell suspension for cell counting, and simultaneously obtaining the value of the cell viability, wherein the specific operation method is described in the section of 'cell quality inspection' in the following text; then, based on the number of cells measured, 4X 10 cells were taken out⁵Individual CAR-T cells were ready for quality testing experiments; the results of the total number of site-directed knockins of the chimeric antigen receptor CAR into CAR-T cells of the volunteers CD19 harvested at the end of this example were calculated as described in the "cell quality test" section below; the remaining cells were centrifuged at 300g for 5 min, the supernatant discarded and the cells were centrifuged at 2X 10⁸The frozen stock solution (cat # 07930, STEMCELL) was added to the cell concentration per ml, and the volume of each frozen tube was 1 ml. Gradient cooling, and storing in-80 deg.C refrigerator.

Volunteer 1 cell quality test:

1) CAR-T cell number and viability assays:

before the finally harvested cells are frozen and stored in a warehouse, the number and the survival rate of the cells need to be determined. The specific operation method comprises the following steps: resuspending the finally harvested cell pellet with 30ml of 1 XPBS, mixing uniformly, sucking 10 mul of cell suspension, mixing the cell suspension with 10 mul of trypan blue staining solution fully, then laterally injecting the mixture into a counting plate, standing at room temperature for 1-2 minutes, and then inserting the counting plate into an automatic cell counter (Countstar BioMed Model IM1200) for detection; selecting three visual fields with uniformly distributed cells through a visual interface of matched software on a computer, setting the diameter range of the cells, and then counting the cells in sequence; the counting software can automatically display the average value result of cell counting of three fields, namely the cell concentration value of the cell suspension, the average value of the cell survival rate and other parameters. The results of the calculation of the total number of the finally harvested volunteer 1 CAR-T cells and the specific values of the cell viability in this example are as follows:

	viable T cell concentration	Survival rate of T cells	Total number of T cells
				Sample
1	5.31×10⁶	83.01％	1.59×10⁸
				Sample 2	5.34×10⁶	83.52％	1.60×10⁸
Sample 3	5.28×10⁶	85.14％	1.58×10⁸
				Mean value of	5.31×10⁶	83.89％	1.59×10⁸

2) Flow assay

And (3) CAR gene expression detection: based on the counting result, 2X 10 is taken out⁵Cell suspension volumes corresponding to individual CAR-T cells and dividing the cell suspension into two 1.5ml EP tubes on average; one of the tubes is not contaminated with antibody and is arranged to exclude the electric shock process from affecting the cell stateThe other tube was filled with 2. mu.l of CD19-FITC protein to examine the expression effect of the CAR gene, and about 2X 10 of the protein was removed⁵The number of control cells not transfected was divided on average into 2 1.5ml EP tubes, one tube of cells not stained with antibody and the other tube of cells supplemented with 2. mu.l of CD19-FITC protein, the tube being set up to exclude false positive results with antibody itself; after the cell suspension is well taken, because the volume of the cell suspension is usually small, 1ml of 1 XPBS (phosphate buffer solution) is directly added into the cell suspension, 300g of the cell suspension is obtained, and after centrifugation for 3 minutes, the supernatant is discarded, so that the purpose of cleaning cells is achieved; then adding 30. mu.l of 1 XPBS into all groups needing antibody staining, adding 2. mu.l of CD19-FITC protein into the groups needing antibody staining, fully mixing the whole system, then placing the system for 15 minutes at room temperature in a dark place, and resuspending the cells in other groups without antibody staining by using 200. mu.l of 1 XPBS for preparing the cells for use on a computer; after the incubation of the CD19-FITC protein is finished, 1ml of 1 XPBS (phosphate buffered saline) and 300g are directly added into all tubes, and after 3 minutes of centrifugation, the supernatant is discarded so as to achieve the effect of removing the antibody which is not combined with the cells; finally resuspend the cells with 200. mu.l of 1 XPBS for use on the machine; harvesting of about 1X 10 Using a flow cytometer⁴The individual cells were examined for CAR gene expression and the results are shown in figure 12 d. Calculating the number of the CAR positive cells prepared for the volunteer at present, namely the effective cell number according to the positive rate of the CAR expression in the displayed total cells, namely the proportion of the CAR positive cells in the total CAR-T cells of the volunteer; the calculation results of the effective cell number finally obtained in this example are as follows:

	total number of CAR-T cells	CAR positivity rate	Effective total number of cells
				Volunteers	1.59×10⁸	14％	2.23×10⁷

CD4/CD8a assay: based on the counting result, 1 × 10 samples are taken⁵The volume of cell suspension corresponding to each CAR-T cell was transferred to a 1.5ml EP tube, which is the test group to be tested, and about 2X 10 cells were removed⁵The individual untransfected control cells were divided on average into 2 1.5ml EP tubes, one of which was not stained with antibody, and the other was added with 2. mu.l of CD4 antibody and 2. mu.l of CD8a antibody at the same time as the experimental group; after the supernatant is taken, 1ml of 1 XPBS (1 XPBS) and 300g are directly added into the supernatant, and the supernatant is discarded after 3 minutes of centrifugation; then adding 30 mu l of 1 XPBS into all groups needing antibody staining, adding 2 mu l of CD4 antibody and 2 mu l of CD8a antibody into the groups, fully mixing the whole system, and then placing the system for 15 minutes at room temperature in a dark place; other antibody-free groups resuspended cells in 200. mu.l of 1 XPBS for use on the machine; after the incubation of the CD4 and CD8a antibodies is finished, 1ml of 1 XPBS (phosphate buffered saline) is directly added into all tubes, 300g of the mixture is centrifuged for 3 minutes, the supernatant is discarded, and then 200 mu l of the 1 XPBS is used for resuspending the cells for preparing on-machine use; harvesting of about 1X 10 Using a flow cytometer⁴Detecting the expression conditions of CD4 and CD8a proteins by using each cell, and finally displaying the positive rate of CD4 and the positive rate of CD8a in the total cells, namely the proportion of CD4-T cells and CD8-T cells in the total CAR-T cells respectively; the results of the measurements of the proportion of the volunteer CAR-T cells CD4-T cells and CD8-T cells in this example are shown in FIG. 12 e.

CD45RO/CD62L assay: based on the counting result, 1 × 10 samples are taken⁵The volume of cell suspension corresponding to each CAR-T cell was transferred to a 1.5ml EP tube, which is the test group to be tested, and about 2X 10 cells were removed⁵An average of 2 untransfected control cells were divided into 1.5ml EP tubes, one of which was not stained with antibodyA body; the other tube of cells was supplemented with 2. mu.l of CD45RO antibody and 2. mu.l of CD62L antibody at the same time as the experimental group. After the supernatant is taken, 1ml of 1 XPBS (1 XPBS) and 300g are directly added into the supernatant, and the supernatant is discarded after 3 minutes of centrifugation; then adding 30 mu l of XPBS into all groups needing antibody staining, adding 2 mu l of CD45RO antibody and 2 mu l of CD62L antibody into the XPBS, fully mixing the whole system, and then placing the XPBS for 15 minutes at room temperature in a dark place; other antibody-free groups resuspended cells in 200. mu.l of 1 XPBS for use on the machine; after the incubation of the CD45RO and CD62L antibodies is finished, 1ml of 1 XPBS (phosphate buffered saline) is directly added into all tubes, 300g of the mixture is centrifuged for 3 minutes, the supernatant is discarded, and then 200 mu l of the 1 XPBS is used for resuspending the cells for preparing the cells for use on a machine; harvesting of about 1X 10 Using a flow cytometer⁴Detecting the expression conditions of CD45RO and CD62L proteins of each cell, and finally displaying that the double positive rate of CD45RO and CD62L in the total cells is the proportion of memory T cells in the total CAR-T cells; the results of the detection of the volunteer CAR-T cell CD45RO protein and CD62L protein in this example are shown in figure 12 f.

3) In vitro killing assay

The strength of the killing effect of the T cells on the cancer cells is an important index for reflecting the performance of the T cells, the cell killing experiment is an important method for detecting the killing effect of the T cells on the cancer cells in vitro, and in the example, a Lactate Dehydrogenase (LDH) cytotoxicity detection method is adopted for carrying out the cell killing experiment. In the afternoon of 9 days after cell activation, after removal of the supernatant medium from the G-rex flask, the remaining CAR-T cell suspension was mixed well and aspirated to approximately 1.5X 10⁶The corresponding cell suspension volume for each cell was transferred to a 1.5ml EP tube and similarly aspirated at about 1.5X 10⁶The volume of cell suspension corresponding to each control cell was transferred to 1.5ml EP tube, which was the effector cell in the experiment, and about 5X 10 cells were aspirated⁵Transferring the cell suspension volume corresponding to the K562-CD19 cells into a 1.5ml EP tube, wherein the tube cells are target cells in an experiment, and the target cells are constructed by transferring a lentiviral vector containing CD19 protein into a K562 cell line which does not express CD19 protein originally in a high-speed centrifugation mode and then carrying out expanded culture; then, the three cells were centrifuged at 300g for 5 minutes while preparing a solution containing 1% bovine bloodClear 1640 medium, only this medium was used in this experiment; discarding the supernatant in the cells as much as possible after centrifugation, suspending the cells by 200 mul of prepared culture medium, and counting the cells after fully and uniformly mixing; configuring a system according to the counting result; the cells were sequentially added to a V-bottom 96-well plate according to the following design, in which the ratio of the number of effector cells to the number of target cells was 5: 1; after the addition is finished, putting the cell plate into a centrifuge, centrifuging the cell plate for 2 minutes at 100g, and after the centrifugation is finished, putting the cell plate into an incubator; the next morning, 20 μ l of lysate from the LDH cell killing assay detection kit was added to only 9 wells of the "volume control" and "K562-CD 19 maximum release" and the 96-well plate was incubated in the incubator for 45 minutes while the substrates in the kit were left to dissolve at room temperature; after incubation is finished, sucking 50 mu l of liquid out of each hole, transferring the liquid into a 96-hole flat bottom plate, quickly adding 50 mu l of substrate in the kit into each hole by using a line gun, reacting the liquid to be detected and the substrate at room temperature for about 3 minutes to obviously see the color change of the hole with maximum release, and quickly adding 50 mu l of termination reaction liquid in the kit into each hole; then after waiting for about 3 minutes, the color of each hole is stabilized, the bubbles are punctured by a clean needle head, then the value of each hole is detected at 490nm by using an enzyme-labeling instrument (Thermo Fisher Multiskan FC type), and finally, the original data is processed according to the formula of the kit specification to make a bar chart; the results of the detection of the killing effect of the control T cells and the CAR-T cells on the cancer cells K562-CD19 are shown in FIG. 12 g.

To summarize: the quality test results above show that the targeted CD19 spot-tap CAR-T cells prepared for volunteer 1 in this example had a higher survival rate; the CAR gene is obviously and stably expressed on the cell surface; the CD4-T and CD8-T subtypes are obviously grouped in total cells; compared with a control T cell which does not express the CAR, the CAR-T cell has a remarkable killing effect on a cancer cell K562-CD19 at an in vitro level, so that the preparation quality is good.

Example 16: preparation of non-viral site-directed knock-in-targeted BCMA CAR-T cells by Single-sampling volunteers

1) T cells in peripheral blood of volunteer 2 were obtained and the procedure was the same as in example 15.

The specific data of the total number of peripheral blood mononuclear cells and the cell viability of the volunteers collected in this example are as follows:

the results of the test of the expression of CD3 in the PBMC cells of the volunteers in this example are shown in FIG. 13a, and the results of the T cell count calculation are as follows:

the verification and detection of the T cell purification result in this example is shown in fig. 13b, the positive rate of CD3 is close to 100%, and the calculation result of the T cell number is as follows:

	viable T cell concentration	Survival rate of T cells	Total number of T cells
				Sample
1	1.19×10⁷	99.19％	2.98×10⁷
				Sample 2	1.23×10⁷	98.87％	3.08×10⁷
Sample 3	1.11×10⁷	99.02％	2.78×10⁷
				Mean value of	1.18×10⁷	99.03％	2.95×10⁷

2) Electric conversion: the procedure was the same as in example 15.

The calculation results of the total number of electrically transfectable cells obtained after the demagnetization operation in this example are as follows:

the subsequent steps were also the same as in example 15 except that 6. mu.g of chemically modified template DNA SEQ ID NO:94 was added and T cells of volunteer 2 were used.

3) Preliminary examination of the expression Effect of anti-BCMA CAR Gene site-directed knock-in

When the electrotransfection cell culture reaches 72 hours, selecting any one hole in a six-hole cell culture plate, gently and uniformly blowing and beating the selected hole, taking out a proper amount of CAR-T cells, and carrying out primary detection on the expression effect of CAR gene fixed-point knocking-in, wherein the specific flow type sample treatment and detection steps are described in the section of 'cell quality detection' in the following text. The preliminary test results for the expression of the target BCMA CAR after electroporation in volunteer 2 of this example are shown in fig. 13c, suggesting that target BCMA CAR expression is beginning.

4) Expanded culture of CAR-T cells: same as in example 15.

5) Cell harvesting: same as in example 15.

Volunteer 2 cell quality test:

1) CAR-T cell number and viability assays: the procedure was the same as in example 15.

The results of the calculation of the total number of volunteer 2CAR-T cells harvested finally and the specific values of cell viability in this example are as follows:

2) Flow assay

The procedure for CAR gene expression detection was the same as in example 15 except that CD19-FITC protein was replaced with BCMA-FITC protein (cat # BCA-HF254, Popses), and the results are shown in FIG. 13d, and the effective cell number calculation results finally obtained in this example were as follows:

CD4/CD8a assay: the procedure was the same as in example 15; the results of the measurements of the proportion of the volunteer CAR-T cells CD4-T cells and CD8-T cells in this example are shown in FIG. 13 e.

CD45RO/CD62L assay: the procedure was the same as in example 15; the results of the detection of the volunteer CAR-T cell CD45RO protein and CD62L protein in this example are shown in figure 13 f.

3) In-vitro killing detection: the procedure was the same as in example 15, except that: another suction of about 5X 10⁵Transferring the cell suspension volume corresponding to the K562-BCMA cells into a 1.5ml EP tube, wherein the tube cells are target cells in an experiment, and the target cells are constructed by transferring a lentiviral vector containing the BCMA proteins into a K562 cell line which does not express the BCMA proteins originally in a high-speed centrifugation mode and then carrying out expanded culture; FIG. 13g shows the results of the detection of the killing effect of the control T cells and the CAR-T cells on the cancer cells K562-BCMA, respectively.

To summarize: the quality test results above show that the targeted BCMA spot knock-in CAR-T cells prepared for volunteer 2 in this example are more viable; the CAR gene is obviously and stably expressed on the cell surface; the CD4-T and CD8-T subtypes are obviously grouped in total cells; compared with a control T cell which does not express the CAR, the CAR-T cell has a remarkable killing effect on cancer cells K562-BCMA at an in vitro level, so that the preparation quality is good.

Claims

1. A non-viral gene site-directed knock-in method, comprising: PCR amplification of template DNA is performed using the modified primer, and gene site-directed knock-in is performed using the modified template DNA as donor DNA,

2. The method of claim 1, wherein at least one 5 '-Phosphorothioate (PS) modification and at least one 5' -Biotin-triethylene glycol (Biotin-TEG) modification, or at least one 5 '-Locked Nucleic Acid (LNA) modification, is performed 1 to 15 bases from the 5' end of the upstream and downstream primers; preferably, the modified primer comprises: biotin-triethylene glycol (Biotin-TEG) modification at the 1 st base at the 5' end of the upstream and downstream primers and Phosphorothioate (PS) modification at the 2 nd, 3 rd, 4 th, 5 th and 6 th bases; preferably, the modified primer comprises: locked Nucleic Acid (LNA) modifications at 1, 2 and 3 bases at the 5' end of the upstream and downstream primers; preferably, the length of the modified primer is 15 to 100 bases in length.

3. The method of claim 1 or 2, wherein the template DNA comprises left homology arm DNA, knock-in DNA, and right homology arm DNA, wherein the left homology arm DNA is homologous to a sequence at the 5 'end of a DNA nick targeted by a gene site-directed knock-in, and the right homology arm is homologous to a sequence at the 3' end of the DNA nick; more preferably, the sequence of the DNA at the 3 'end of the left homology arm is identical to the sequence at the 5' end which is 0 to 300 bases away from the nicked DNA, preferably the fragment length of the DNA of the left homology arm is 10 to 5000 bp; more preferably, the DNA sequence of the 5 'end of the right homology arm is identical to the sequence of the 3' end which is 0 to 300 bases away from the nicked DNA, preferably the fragment length of the DNA of the right homology arm is 10 to 5000 bp; further preferably, the knock-in DNA has a length of 0 to 100000 bases.

4. The method according to claim 1 or 2, wherein the method further comprises: ligating the template DNA to a plasmid vector prior to the PCR amplification; preferably, the plasmid vector is selected from the group consisting of pUC57, pCDNA3.1, and pCMV.

5. The method according to claim 1 or 2, wherein the method further comprises: purifying the modified template DNA after the PCR amplification; preferably, the purification is performed using a DNA purification kit, DNA-bound magnetic beads, gel chromatography, ion chromatography, affinity chromatography, ultrafiltration tube ultrafiltration, dialysis membrane dialysis, and any combination thereof.

6. The method according to claim 1 or 2, wherein the gene editing method used for gene site-directed typing comprises: CRISPR-Cas9, ZFN, ARCRS, TALEN, and megaTAL; preferably, the gene editing material involved in the gene editing method comprises a plasmid, mRNA or protein; preferably, the DNA targeted for gene site-directed knock-in is genomic DNA; more preferably, the gene-editing substance is transfected into the cell together with the modified template DNA using the following transfection method: liposomes, calcium phosphate, DEAE-dextran, electroporation, microinjection or gene gun.

7. The method of claim 1 or 2, wherein the method is used to perform gene site-directed knock-in: cord blood stem cells, bone marrow hematopoietic stem cells, adult stem cells, embryonic stem cells, T lymphocytes, B lymphocytes, NK cells, NK-92 and NK-92 derived cells, macrophages, DC cells, CHO and CHO derived cells, 293 and 293 derived cells and common cell lines.

8. A method of making a CAR-T cell, the method comprising:

1) mixing a Cas9 protein with a targeted sgRNA in vitro to form an RNP complex, or a Cas9 protein-encoding mRNA with a targeted sgRNA in vitro to form a mixture;

9. The method of claim 8, wherein at least one 5 '-Phosphorothioate (PS) modification and at least one 5' -Biotin-triethylene glycol (Biotin-TEG) modification, or at least one 5 '-Locked Nucleic Acid (LNA) modification, is performed 1 to 15 bases from the 5' end of the upstream and downstream primers; preferably, the modified primer comprises: biotin-triethylene glycol (Biotin-TEG) modification at the 1 st base at the 5' end of the upstream and downstream primers and Phosphorothioate (PS) modification at the 2 nd, 3 rd, 4 th, 5 th and 6 th bases; preferably, the modified primer comprises: locked Nucleic Acid (LNA) modifications at 1, 2 and 3 bases at the 5' end of the upstream and downstream primers; preferably, the length of the modified primer is 15 to 100 bases in length.

10. The method of claim 8, wherein in the CAR-T cell, the genomic location of the knock-in is an exonic region, an intronic region, a promoter region, an enhancer region, or a non-gene coding region of a gene; preferably, the genomic location of the knock-in is selected from the group consisting of TRAC, TRBC1, TRBC2, PD-1, LAG-3, TIM-3, TIGIT, CTLA-4, B2M, CTIIA, TET-2, REGNASE-1, GM-CSF, TGFBR2 and NR4A sites.

11. The method of claim 8, wherein the sgRNA targets genomic DNA having a sequence of the 3' PAM region that is NGG, wherein N is any one of A, T, C, G; preferably, the sgRNA is a chemically modified sgRNA; more preferably, the chemical modification of the sgRNA includes 2 '-O-methylation, 3' -thio, and combinations thereof; more preferably, chemical modification of the sgRNA occurs at 1 to 10 bases from the 5 'and 3' ends of the sgRNA, preferably 3 bases from the 5 'and 3' ends are simultaneously 2 '-O-methylated modified and 3' -thio modified.

12. The method of claim 8, wherein the Cas9 protein comprises SpCas9, SaCas9, SpCas9-HF, eSpCas9, xCas9, cpf1 or a Cas9 protein of a different genus, preferably SpCas9, the amino acid sequence of which is shown in SEQ ID NO: 62; preferably, one or more NLS nuclear signal peptide(s) is/are coupled to the N-terminus or C-terminus of the Cas9 protein, wherein the NLS nuclear signal peptide sequence is shown in SEQ ID NO: 63.

13. The method of claim 8, wherein the template DNA comprises a left homology arm, a cleavage peptide or an internal ribosome entry site, a CAR gene, polyA and a right homology arm, wherein the CAR gene is driven by a native gene promoter (preferably a TRAC gene promoter); or the template DNA comprises a left homology arm, an exogenous promoter, a CAR gene, polyA, and a right homology arm, wherein the CAR gene is driven by the exogenous promoter; preferably, the left homology arm DNA is homologous with the 5 'end sequence of the DNA cut targeted by the gene site-directed knock-in, and the right homology arm DNA is homologous with the 3' end sequence of the DNA cut; more preferably, the sequence of the DNA at the 3 'end of the left homology arm is identical to the sequence at the 5' end which is 0 to 300 bases away from the nicked DNA, preferably the fragment length of the DNA of the left homology arm is 10 to 5000 bp; more preferably, the DNA sequence of the 5 'end of the right homology arm is identical to the sequence of the 3' end which is 0 to 300 bases away from the nicked DNA, preferably the fragment length of the DNA of the right homology arm is 10 to 5000 bp; further preferably, the fragments of the left and right homology arms are 300, 600 or 1000 bases in length, for example, the sequences are shown in SEQ ID NO 64-69, respectively.

14. The method of claim 13, wherein the cleavage peptide or internal ribosomal entry site is P2A, T2A, or IRES; preferably, the amino acid sequences of the cleavage peptides P2A and T2A are shown as SEQ ID NO 70-71, respectively, and the DNA sequence of the internal ribosome entry site IRES is shown as SEQ ID NO 72; preferably, the exogenous promoter is EF1, CMV, PGK, MSCV or SFFV, more preferably EF1, and the DNA sequence thereof is shown as SEQ ID NO. 73; preferably, polyA is a minipoly A or a bGHpA structure, and more preferably, the DNA sequences of the polyA and the bGHpA are respectively shown in SEQ ID NO. 74-75; preferably, the CAR gene can be a second or third generation structure, more preferably a second generation CAR gene structure; preferably, the CAR gene is directed against a target selected from the group consisting of: CD19, CD22, CD20, CD7, CD123, CD33, CD38, BCMA, PSMA, Her2, Mesothelin, CS1, MUC16, GD2, GPC3, CEA, CD138, EGFR, EGFRVIIII, Lewis Y, DLL3, MG7 and IL13R alpha 2, more preferably CD19 and BCMA targets, the scFv amino acid sequences of the antibodies are shown as SEQ ID NO:76-77, respectively, and the DNA sequences are shown as SEQ ID NO:78-79, respectively.

15. The method of claim 13, wherein the CAR gene comprises a signal peptide DNA, a hinge region DNA, a transmembrane region DNA, a costimulatory signal region DNA, and an activation region DNA; preferably, the signal peptide DNA is selected from the group consisting of CD8, IL-2 or GM-CSF signal peptide domains, more preferably CD8 signal peptide, the amino acid sequence of which is shown in SEQ ID NO:80 and the DNA sequence of which is shown in SEQ ID NO: 81; preferably, the hinge region DNA is selected from the group consisting of IgG1, IgG4, IgD or CD8 hinge domain, more preferably CD8 hinge structure, the amino acid sequence of which is shown in SEQ ID NO. 82 and the DNA sequence of which is shown in SEQ ID NO. 83; preferably, the transmembrane region DNA is selected from CD3, CD4, CD5, CD8, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154 or PD1 transmembrane domain, more preferably CD8 transmembrane structure, the amino acid sequence of which is shown in SEQ ID NO:84, and the DNA sequence of which is shown in SEQ ID NO: 85; preferably, the costimulatory signal region DNA is selected from the group consisting of CD2, CD7, CD27, CD28, CD30, CD40, CD54, CD83, CD134, CD137, CD150, CD152, CD223, CD270, CD273, CD274, CD278, CARD11, NKD2C, DAP10, LAT, SLP76, ZAP70 or 4-1BB costimulatory domain, more preferably 4-1BB costimulatory structure, the amino acid sequence of which is shown in SEQ ID NO:86, and the DNA sequence of which is shown in SEQ ID NO: 87; preferably, the activation region DNA is a CD3 zeta activation domain, the amino acid sequence of which is shown in SEQ ID NO. 88, and the DNA sequence of which is shown in SEQ ID NO. 89.

16. The method according to claim 8, wherein the knock-in genomic site is a TRAC gene, preferably the first, second, third or fourth exon of a TRAC gene, more preferably the first exon region; preferably, the sequence of TRAC gene (including PAM sequence) aimed by sgRNA sequence is shown in SEQ ID NO. 1-29, more preferably in SEQ ID NO. 7; preferably, the sgRNA sequence is shown in SEQ ID NO 30-58, more preferably in SEQ ID NO 36; further preferably, the sgRNA of SEQ ID No. 36 comprises a targeted crRNA sequence and a tracrRNA sequence, wherein the crRNA is 17, 18, 19, 20, 21 or 22 bases, preferably 18, 19 or 20 bases, and the sequences thereof are shown in SEQ ID NOs 59-61, respectively.

17. The method according to claim 8, wherein when the CAR gene is driven by a native TRAC gene promoter, the template DNA of 300, 600 or 1000 base homology arms comprises a template DNA targeting the CD19CAR gene and miniplolya, the sequences of which are shown in SEQ ID NOs 90-92, more preferably 300 base homology arms, respectively; preferably, the template DNA of 300 base homology arm comprises targeting CD19CAR gene and bGHpA polyA, the sequence of which is shown in SEQ ID NO: 93; preferably, the template DNA of 300 base homology arm comprises targeting BCMA CAR gene and bGHpA polyA, the sequence of which is shown in SEQ ID NO: 94; preferably, the template DNA of the 300-base homology arm comprises a targeting BCMA CAR gene and miniSolyA), and the sequence of the template DNA is shown as SEQ ID NO. 95; or

When the CAR gene is started by an exogenous promoter EF1, the template DNA of the 300-base homology arm comprises a targeting CD19CAR gene, EF1 promoter DNA and miniplolyA, and the sequences of the targeting CD19CAR gene, the EF1 promoter DNA and the miniplolyA are shown as SEQ ID NO: 96.

18. The method of claim 8, wherein the method further comprises: ligating the template DNA to a plasmid vector prior to the PCR amplification; preferably, the plasmid vector is selected from the group consisting of pUC57, pCDNA3.1, and pCMV.

19. The method of claim 8, wherein the method further comprises: purifying the modified template DNA after the PCR amplification; preferably, the purification is performed using a DNA purification kit, DNA-bound magnetic beads, gel chromatography, ion chromatography, affinity chromatography, ultrafiltration tube ultrafiltration, dialysis membrane dialysis, and any combination thereof.

20. The method of claim 8, wherein the method comprises extracting Peripheral Blood Mononuclear Cells (PBMCs), and activating T cells; preferably, the source of Peripheral Blood Mononuclear Cells (PBMCs) is venous blood draw or apheresis of mononuclear cells; preferably, T cell activation is performed using anti-CD 3 antibody coating alone, anti-CD 3 antibody/anti-CD 28 antibody coating, direct addition of anti-CD 3 antibody alone, direct addition of anti-CD 3 antibody/anti-CD 28 antibody, direct addition of anti-CD 3 antibody/anti-CD 28 antibody/CD 2 antibody or anti-CD 3 antibody/anti-CD 28 antibody magnetic beads; more preferably, the activation time is from 1 to 8 days.

21. The method of claim 8, further comprising expanded culture of the transfected CAR-T cells, assay analysis of the CAR-T cells, and cryopreservation of the CAR-T cells.

22. A CAR-T cell made by the method of any one of claims 8-21.

23. Use of the cell of claim 22 for the preparation of a medicament for the treatment of leukemia (such as acute B-lymphocytic leukemia, acute T-lymphocytic leukemia, acute NK-lymphocytic leukemia, multiple non-hodgkin's lymphomas, chronic lymphocytic leukemia, acute myeloid leukemia, multiple myeloma), solid tumors (lung cancer, liver cancer, stomach cancer, breast cancer, colorectal cancer, prostate cancer, pancreatic cancer, brain glioma, esophageal cancer, cholangiocarcinoma, endometrial cancer, ovarian cancer, mesothelioma, thymus cancer), aids, autoimmune diseases and other CAR-T cell treatable diseases.