CN107630006B - Method for preparing T cell with double knockout genes of TCR and HLA - Google Patents
Method for preparing T cell with double knockout genes of TCR and HLA Download PDFInfo
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Abstract
The invention discloses a method for preparing T cells with double gene knockout of TCR and HLA, which comprises the steps of transfecting plasmids of CRISPR/Cas9-HLA and CRISPR/Cas9-TCR together with the T cells, carrying out double gene knockout of TCR and HLA, and obtaining the T cells with double gene knockout of TCR and HLA.
Description
Technical Field
The invention relates to the field of biological genes, in particular to a method for preparing T cells with double knockout of TCR and HLA genes.
Background
Tumors are always serious diseases which afflict the whole world, at least hundreds of tumors are needed at present, and the human health is seriously harmed. At present, the treatment means mainly comprise surgical treatment, chemotherapy, radiotherapy, monoclonal antibodies, traditional Chinese medicines and the like, but the treatment effect is limited and the side effect is obvious. In recent years, tumor cell immunotherapy is the most active and promising treatment method, and the immunotherapy comprises immunotherapy drugs and cell immunotherapy. At present, chimeric antigen receptor cell CAR-T therapy brings hope for tumor therapy, the basic principle is to separate cultured T cells from the body of a tumor patient, study and modify the T cells by using gene technology, and the basic process is to perform gene recombination on a single-chain antibody (scFv) for recognizing Tumor Associated Antigen (TAA) and an activation sequence of the T cells in vitro to form recombinant plasmids, and to perform large-scale amplification on the T cells in vitro by using transfection technology. Then the cells modified by the genetic engineering are amplified in vitro and then are infused back into the body of a patient, so that the immune cells have the capacity of specifically recognizing and killing the tumor, thereby achieving the effect of treating the tumor. However, CAR-T has some drawbacks in treating tumors, on the one hand, it causes side effects such as cytokine storm, and on the other hand, the immune system of patients is destroyed after chemotherapy and radiotherapy, and its own T cell number, activity and proliferation capacity are extremely limited, limiting the use of autologous peripheral blood mononuclear cells, which results in the loss of the best treatment for patients with advanced cancer. Therefore, people look to a chimeric antigen receptor cell that can be reinfused by allogeneic sources, on the one hand, a healthy donor has a strong immune system to kill tumor cells effectively, and on the other hand, blood is easy to collect so as to isolate T cells, but allogeneic reinfusion has a strong graft-versus-host reaction.
The T Cell Receptor (TCR) is a molecule on the surface of T cells that specifically recognizes antigens and mediates immune responses, determining how the human immune system adapts to environmental changes. The TCR can be divided into two types of TCR alpha/beta and TCR gamma/and peripheral blood T cells are mainly T cells of TCR alpha/beta and are main cells for mediating specific cellular immune response of organisms.
Human Leukocyte Antigen (HLA), the coding gene is the Major Histocompatibility Complex (MHC) of Human, is located on chromosome 6 (6p21.31), and comprises a series of closely linked loci, which are closely related to the functions of the Human immune system. MHC, in which a portion of the genes encode cell surface antigens, is an indelible "signature" of each individual's cells, and is the basis for the immune system to distinguish between itself and foreign substances. Human leukocyte antigens determine histocompatibility when performing transplantation surgery. The more similar the human leukocyte antigens of the contributor and recipient, the smaller the rejection response. Only the monozygotic twins or the cloned human leukocyte antigens are identical. In addition, many diseases are associated with certain human leukocyte antigens, and thus their potential for certain diseases is relevant. HLA includes class I class II and class III gene portions. The antigens expressed by HLA class I and II genes are located on the cell membrane and are MHC-I (encoded at position A, B, C) and MHC-II (encoded at region D), class I is distributed on the surface of almost all cells in the body, is a heterodimer consisting of a heavy chain (alpha chain) and beta 2 microglobulin (B2M), and class II is mainly glycoproteins located on the surface of macrophages and B lymphocytes.
T cell receptors and human leukocyte antigens are the main causes of allograft-versus-host disease, and therefore, silencing T cell TCR proteins and HLA proteins can achieve allogeneic reinfusion of T cells.
Disclosure of Invention
In order to make up for the defects, the invention provides a method for preparing T cells with double knockout of TCR and HLA genes.
The scheme of the invention is as follows:
a method for preparing T cells with double gene knockout of TCR and HLA is characterized by comprising the following steps:
1) under the enzyme digestion action of BbsI, the expression gene vectors pX330A and pX330S form a linearized skeleton vector pX330A and a linearized skeleton vector pX 330S;
2) the sgRNA of TRAC, TRBC2 and B2M is located in the exon of the gene, the target sequence on the gene is unique, CACCG is added to 5' of the designed TRAC, TRBC2 and B2M sgRNA, C and AAAC are added to 3' and 5' of the reverse oligonucleotide, the sgRNA oligonucleotide double strands are mixed, annealed at 100 ℃ for 5 minutes and then slowly cooled to room temperature, and TRAC-sgRNA, TRBC2-sgRNA and B2M-sgRNA are obtained;
3) connecting the TRAC-sgRNA and TRBC2-sgRNA obtained in the step 2) with the linearized framework vector pX330A and the linearized framework vector pX330S obtained in the step 1) respectively to obtain a recombinant vector pX330A-TRAC containing the TRAC-sgRNA and a recombinant vector pX330S-TRBC2 containing the TRBC 2-sgRNA; constructing a recombinant vector pX330A-TRAC and pX330S-TRBC2 into a vector by BsaI enzyme digestion and T4DNA ligase to obtain a recombinant vector containing two sgRNA genes of TRAC and TRBC2, and naming the recombinant vector as CRISPR/Cas 9-TCR;
4) connecting the B2M-sgRNA obtained in the step 2) with the linearized skeleton vector pX330S obtained in the step 1) to obtain a recombinant vector pX330S-B2M containing the B2M-sgRNA, and converting the recombinant vector into CRISPR/Cas 9-HLA;
5) separating fresh peripheral blood mononuclear cells by using a density gradient, activating by using CD3 and CD28 monoclonal antibodies, culturing IL-2, activating into T cells, then transfecting the T cells by using the CRISPR/Cas9-HLA prepared in the step 3) and the plasmid of the CRISPR/Cas9-TCR prepared in the step 4) together, and carrying out TCR and HLA double gene knockout to obtain the T cells with TCR and HLA double gene knockout.
As a preferred technical scheme, the step 5) is a method for co-transfecting T cells by plasmids: respectively adding plasmids containing CRISPR/Cas9-TCR and CRISPR/Cas9-HLA into an electric shock cup, adding cells into the electric shock cup, inverting the electric shock cup to uniformly mix, putting the electric shock cup into an electric shock groove, performing pulse electric shock once, transferring the cells into a hole containing a culture medium, gently shaking the hole plate, uniformly mixing the cells, and performing electric transfer for 24-48 h to realize instantaneous gene expression.
Preferably, the sgRNA sequence of the TRAC of the targeting gene in step 2) is selected from exon 3 of chr14:22550620, the sgRNA sequence of the TRBC2 of the targeting gene is selected from exon 2 of chr7:142801040, and the sgRNA sequence of the B2M of the targeting gene is selected from exon 1 of chr15: 44711557.
Preferably, the TCR and HLA double-gene knockout T cells prepared in the step 5) are amplified and sequenced, and the transfected cells with knockout effect are sorted out by using a biotin-labeled TCR antibody and a biotin-labeled HLA antibody through magnetic beads.
As a preferred embodiment, the TCR and HLA double knockout T cells can be lentivirally infected with a CAR for the treatment of different types of tumors.
Preferably, the tumor is one of leukemias with high expression of CD19, CD20 and BCMA, or one of solid tumors with expression of mesothelin, GD2, TNFR2, ROR1, EGFR, CEAmAb, Mucin1, HER1 and VEGF.
Compared with the prior art, the invention has the following advantages:
1. the invention simultaneously knocks out the exons of the B2M gene in the coding gene TRAC corresponding to the alpha chain constant region of the TCR, the coding gene TRBC2 corresponding to the beta chain constant region and the class I gene of HLA. A TCR and HLA double-gene knockout universal T cell is constructed based on a CRISPR/Cas9 system, a sgRNA sequence of a targeting gene TRAC is selected from a No.3 exon of chr14:22550620, a sgRNA sequence of a targeting gene TRBC2 is selected from a No.2 exon of chr7:142801040, and a sgRNA sequence of a targeting gene B2M is selected from a No.1 exon of chr15: 44711557.
2. The detection of the invention shows that the knockout of sgRNA corresponding genes has zero off-target effect, and the gene safety can be improved with high efficiency.
3.CRISPR/Cas9 gene editing technology is adopted. The Cas9 protein contains two nuclease domains that can cleave two single strands of DNA, respectively. Cas9 is firstly combined with sgRNA to form a compound, and is identified and combined through a PAM sequence, so that a target DNA double strand is cut, and the aim of knocking out gene expression is finally achieved by utilizing non-homologous recombination repair of cells.
4. Allogeneic source T cells are genetically engineered so that the T cells can be used for xenotransplantation without immune rejection. In addition, the modified T cell is combined with the third generation CAR-T technology to prepare a universal chimeric antigen receptor cell capable of being transplanted by a variant body, so that the CAR-T cell can be simultaneously applied to different individuals to kill tumor cells, and the tumor treatment is facilitated.
5. The universal T cell combined CAR-T therapeutic target can be leukemia highly expressing CD19, CD20 and BCMA, and can also be solid tumors expressing mesothelin, GD2, TNFR2, ROR1, EGFR, CEAmAb, Mucin1, HER1, VEGF and the like.
Drawings
FIG. 1 is a map of a pX330A vector;
FIG. 2 is a map of a pX330S vector;
FIG. 3 shows plasmid sequencing to verify successful insertion of sgRNA of TRA and TRB and B2M genes;
FIG. 4 is a graph of the knockout efficiency of T7E1 enzyme digestion identification cell population;
FIG. 5 shows PCR sequencing verification of successful TRA and TRB and B2M gene knock-outs;
FIG. 6 shows the results of in vitro tumor killing experiments of T cells with double gene knockout of TCR and HLA.
Detailed Description
Example 1CRISPR/Cas9-TCR and CRISPR/Cas9-HLA vector construction
1. Recombinant expression vector
The vector used in the invention is pX330A and pX330S (map is shown in figure 1 and figure 2), the pX330A expression gene vector contains an original replicon, a cytomegalovirus promoter sequence (CMV), restriction endonuclease sites (BbsI, Bsa I and the like), and a resistance screening gene (ampicillin resistance); pX330S is structurally similar to pX330A, differing primarily in cleavage sites and resistance selection genes (spectinomycin). The artificially synthesized sgRNA gene is connected with the linearized framework vector DNA under the action of T4 ligase to form a recombinant vector.
Synthesis of TRAC-sgRNA, TRBC2-sgRNA and B2M-sgRNA
The sgRNA of TRAC, TRBC2 and B2M is located in an exon (SEQ ID NO. 1-3) of a gene, a target sequence on the gene is unique, CACCG is added to 5' of a designed sgRNA forward oligonucleotide, C and AAAC are added to 3' and 5' of a reverse oligonucleotide respectively, sgRNA oligonucleotide double strands are mixed, annealed at 100 ℃ for 5 minutes and then slowly cooled to room temperature, and TRAC-sgRNA, TRBC2-sgRNA and B2M-sgRNA are obtained.
CRISPR/Cas9-TCR vector construction:
and (3) respectively connecting the TRAC-sgRNA and the TRBC2-sgRNA with linearized framework vectors pX330A and pX330S to obtain a recombinant vector pX330A-TRAC containing the TRAC-sgRNA and a recombinant vector pX330S-TRBC2 containing the TRBC 2-sgRNA. The recombinant vector pX330A-TRAC and pX330S-TRBC2 are constructed into a vector by BsaI enzyme digestion and T4DNA ligase, so that the recombinant vector containing two sgRNA genes of TRAC and TRBC2 is obtained, and the recombinant vector is named as CRISPR/Cas 9-TCR.
CRISPR/Cas9-HLA vector construction:
B2M-sgRNA is connected with a linearized framework vector pX330S to obtain a recombinant vector pX330S-B2M containing B2M-sgRNA, and the recombinant vector is called CRISPR/Cas 9-HLA.
EXAMPLE 2 obtaining T cells with double knockout of TCR and HLA
Fresh peripheral blood mononuclear cells are separated by using a density gradient, activated by CD3 and CD28 monoclonal antibodies, cultured by IL-2 (the final concentration is 300IU/ml), activated into T cells, and then the recombinant vector CRISPR/Cas9-HLA prepared in the embodiment 1 and CRISPR/Cas9-TCR plasmid are used for co-transfecting the T cells to carry out TCR and HLA double gene knockout. Transfection is known as:
plasmids (CRISPR/Cas9-TCR, CRISPR/Cas9-HLA) were added to electroporation cuvettes, respectively, each time the plasmid was 200ng, and cells (1 × 10)7) After the electric shock cup is overturned and mixed evenly, the electric shock cup is placed into an electric shock groove and is subjected to pulse electric shock once (2.0KV, 25uFD), then cells are transferred into holes containing 0.5ml of DMEM culture medium, the hole plate is gently shaken, the cells are mixed evenly, and after the electric shock is carried out for 24h-48h, the gene is expressed instantly.
Further, after plasmid transfection was completed, T cells continued to be cultured and further expanded. After 72h, T cells transfected with pX330A, pX330S, CRISPR/Cas9-TCR and CRISPR/Cas9-HLA are collected respectively, genomic DNA is extracted by using a kit, then T7E1 enzyme is used for detecting knockout efficiency, and PCR products are subjected to TA clone sequencing to further verify whether TCR and HLA genes are knocked out or not, as shown in figures 4-5.
Further, transfected cells with knockout effect are detected, and T cells with TCR and HLA double gene knockout are separated by using biotin-labeled TCR antibodies and biotin-labeled HLA antibodies through magnetic beads.
Example 3TCR and HLA double knockout T cell binding CAR treatment of tumors
The CAR for treating different types of tumors is infected by lentivirus into the isolated TCR and HLA double-knockout T cell, and the specific process is as follows: synthesizing Leader-scFv-CD8-CD137-CD3 zeta-T2A-HSV-TK nucleotide, inserting the gene fragment of the synthesized Leader-scFv-CD8-CD137-CD3 zeta-T2A-HSV-TK into the pLent-C-GFP vector NotI-AsiSI site, constructing a plasmid after the sequencing is correct, transfecting 293T cells with the plasmid, packaging into a lentivirus carrying an encoding gene, and carrying L carrying the encoding gene1mL of lentivirus of eader-scFv-CD8-CD137-CD3 zeta-T2A-HSV-TK coding gene is added into T cells for culturing double gene knockout of TCR and HLA (1 × 10)7) The culture dish is evenly mixed, and fresh culture solution is replaced after 24 hours.
Further, after infection is completed, the TCR expressing the CAR and the HLA double knockout T cells are expanded in vitro on a large scale, the cells are collected, and finally washed three times with phosphate buffer solution for foreign body reinfusion to treat different tumors.
Example 4 results of T cell in vitro tumor killing experiments by double knockout of TCR and HLA
The experiment uses LDH release method to measure cell killing activity, and uses patient autologous T cells as comparison. Taking 100ul of target cells and effector cells respectively, and setting different effective target ratios as 5: 1. 10: 1. 20: 1. 40:1, adding the mixture into a U-shaped 96-well culture plate, wherein the natural release hole of the target cell and the culture solution are 100ul respectively, the maximum release hole of the target cell and 2.5 percent Triton are 100ul respectively, the three holes are provided with three parallel holes, and culturing for 4 hours in a 5 percent CO2 incubator at 37 ℃. And then centrifuging the 96-well plate for 5min at 1500rpm of a multi-well plate centrifuge, respectively taking 100ul of supernatant from each well, adding the supernatant into corresponding wells of the flat-bottom 96-well plate, respectively adding 100ul of Lactate Dehydrogenase (LDH) matrix solution into each well, reacting for 10min, adding 30ul of HCl (1mol/L) into each well, and measuring the absorbance (A) at 490nm of an enzyme labeling instrument. Cell killing activity was calculated according to the following formula: the killing activity (%) [ (reaction well a value-natural release well a value)/(maximum release well a value-natural release well a value) ] × 100%.
As shown in fig. 6, the tumor killing ability of the TCR and HLA double knockout T cell prepared by the present invention is significantly stronger than that of the patient autologous T cell, and when the effective target ratio is 40, the tumor killing ability of the TCR and HLA double knockout T cell of the present invention is about 56%, while the tumor killing ability of the patient autologous T cell is only about 37%.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
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Claims (3)
1. A method for preparing T cells with double gene knockout of TCR and HLA is characterized by comprising the following steps:
1) expressing gene vectors pX330A and pX330S, and forming a linearized skeleton vector pX330A and a linearized skeleton vector pX330S under the enzyme digestion action of BbsI;
2) the sgRNA of TRAC, TRBC2 and B2M is located in the exon of the gene, the target sequence on the gene is unique, CACCG is added to 5' of the designed TRAC, TRBC2 and B2M sgRNA, C and AAAC are added to 3' and 5' of the reverse oligonucleotide, the sgRNA oligonucleotide double strands are mixed, annealed at 100 ℃ for 5 minutes and then slowly cooled to room temperature, and TRAC-sgRNA, TRBC2-sgRNA and B2M-sgRNA are obtained;
3) connecting the TRAC-sgRNA and TRBC2-sgRNA obtained in the step 2) with the linearized framework vector pX330A and the linearized framework vector pX330S obtained in the step 1) respectively to obtain a recombinant vector pX330A-TRAC containing the TRAC-sgRNA and a recombinant vector pX330S-TRBC2 containing the TRBC 2-sgRNA; constructing a recombinant vector pX330A-TRAC and pX330S-TRBC2 into a vector by BsaI enzyme digestion and T4DNA ligase to obtain a recombinant vector containing two sgRNA genes of TRAC and TRBC2, and naming the recombinant vector as CRISPR/Cas 9-TCR;
the sgRNA sequence of the TRAC is a nucleotide sequence shown as SEQ ID NO.1 in a sequence table; the sgRNA sequence of the TRBC2 is a nucleotide sequence shown as SEQ ID NO.2 in a sequence table; the sgRNA sequence of B2M is a nucleotide sequence shown as SEQ ID NO.3 in the sequence table;
4) connecting the B2M-sgRNA obtained in the step 2) with the linearized skeleton vector pX330S obtained in the step 1) to obtain a recombinant vector pX330S-B2M containing the B2M-sgRNA, and converting the recombinant vector into CRISPR/Cas 9-HLA;
5) separating fresh peripheral blood mononuclear cells by using a density gradient, activating by using CD3 and CD28 monoclonal antibodies, culturing IL-2, activating into T cells, then transfecting the T cells by using the CRISPR/Cas9-HLA prepared in the step 3) and the plasmid of the CRISPR/Cas9-TCR prepared in the step 4) together, and carrying out TCR and HLA double gene knockout to obtain the T cells with TCR and HLA double gene knockout.
2. A method for preparing a TCR and HLA double knockout T cell according to claim 1 wherein the plasmid of step 5) co-transfects the T cell: respectively adding plasmids containing CRISPR/Cas9-TCR and CRISPR/Cas9-HLA into an electric shock cup, adding cells into the electric shock cup, inverting the electric shock cup to uniformly mix, putting the electric shock cup into an electric shock groove, performing pulse electric shock once, transferring the cells into a hole containing a culture medium, gently shaking the hole plate, uniformly mixing the cells, and performing electric transfer for 24-48 h to realize instantaneous gene expression.
3. A method according to claim 1, wherein the T cell comprises a double knockout TCR and HLA: and (3) amplifying and sequencing the T cells subjected to double gene knockout of the TCR and the HLA prepared in the step 5), and sorting the T cells subjected to double gene knockout of the TCR and the HLA by using the TCR antibody marked by the biotin and the HLA antibody marked by the biotin for the transfected cells with knockout effects after sequencing.
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| CN105647871A (en) * | 2016-01-27 | 2016-06-08 | 苏州佰通生物科技有限公司 | Chimeric antigen receptor T cell capable of conducting allograft and preparation method |
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