Universal CART cell homologous recombination construction method

Abstract

The invention relates to the technical field of genetic engineering and synthetic biology, and particularly provides a universal CART cell homologous recombination construction method, the universal CART cell comprises but is not limited to EGFRvIII / Mesothelin / CLDN6 targeted universal CART cell and an allogenic T cell with a chimeric antigen receptor, and a T cell receptor alpha chain, HLA class I, HLA class II and PD1 molecules of the universal CART cell are knocked out; according to the knockout, TRAC, B2M, CIITA and PD1 genes are specifically and simultaneously knocked out in allogenic T cells by utilizing a CRISPR (clustered regularly interspaced short palindromic repeats) technology; according to the invention, four knockout of TRAC, B2M, CIITA and PD1 molecules and knockin of large fragments of EGFRvIII / Mesothelin / CLDN6CAR are realized, targeting of tumor specific antigens is generated, GvHD is eliminated through knockout of TCR and HLA class I and class II, allogenic T cells are prevented from being rejected, and the allogenic T cells knocked out by PD1 molecules can more effectively exert tumor killing activity due to down-regulation of immunosuppression.

Description

Technical Field

[0001] The present invention relates to the technical fields of genetic engineering and synthetic biology, and particularly relates to a method for constructing a universal CART cell by homologous recombination. Background Art

[0002] Chimeric antigen receptor T cells (CART) are one of the most promising tumor immunotherapies at present. Its basic principle is mainly to extract the patient's own T cells, and through genetic and cell engineering means, make them express specific chimeric antigen receptors, so that they can recognize and bind to tumor cell surface antigens, thereby playing a role in targeting and killing tumor cells. CAR-T has achieved remarkable curative effects in the treatment of leukemia and lymphoma, and its curative effect in solid tumors is the focus of current research.

[0003] Currently, the first echelon of CART, including Novartis, Kite Pharma, and Juno, all adopt autologous transplantation methods to prepare CART cells. This individualized treatment method consumes a large amount of manpower and material resources, so the price is high. This makes it difficult for some patient families to bear, and at the same time greatly limits its market application. Using allogeneic T cells to prepare CART / TCRT cells for treatment provides another idea, but due to the graft-versus-host reaction (GvHD) of donor T cells to the host and the rejection of allogeneic T cells by the host immune system, its application is greatly limited.

[0004] For CART cell therapy, strategies to reduce the GvHD problem include depletion of alloreactive T cells after stimulation with recipient antigen-presenting cells; induction of allo-deactivation in donor CAR-T cells by culturing with allogeneic stimulants and CD80 / 86 blocking antibodies; or using third-party T cells with defined antigen specificity for viral antigens. Conversely, mismatched donor T cells can be recognized and rejected by the host immune system. Therapies for allogeneic transplantation, such as alemtuzumab and fingolimod, limit the rejection of allogeneic CAR-T cells. However, these strategies require long-term suppression of the host immune system, which may not be ideal in the context of cancer immunotherapy.

[0005] With the emergence of highly efficient gene editing techniques in primary human T cells, knocking out endogenous TCR and HLA represents a viable approach that has the potential to completely and permanently eliminate these allogeneic immune responses. The target gene on CART cells can be effectively knocked out by means of CRISPR. As a latest genome editing tool, the CRISPR / Cas system is capable of accomplishing RNA-guided specific DNA recognition and editing. The nuclease Cas9, with the help of short guide RNA (gRNA), recognizes the target DNA sequence and introduces double-strand breaks (DSBs) in the genome. DSBs are mainly repaired in cells by non-homologous end joining (NHEJ), which generates short deletions or insertions (indels) that can be used to knock out genes. The development of the CRISPR / Cas system provides a brand-new platform for constructing more efficient site-directed gene modification techniques.

[0006] As an immunotherapy for treating tumors, CAR-T cells are one of the hot research areas in current cancer immunotherapy. As an immunosuppressive molecule, PD1 (Programmed death 1) has become an effective target molecule for important cancer treatments. In 2016, a study reported that knocking out the PD1 molecule on CART cells using CRISPR technology could significantly improve the therapeutic effect of CART cells on solid tumors.

[0007] The efficiency of homologous directed repair (HDR) is lower than that of NHEJ and requires a template (donor) DNA. The exogenous donor DNA delivered into cells can insert the nucleotide sequence of interest into the genome, and this method is called knock-in. Currently, low efficiency still characterizes most CRISPR / Cas9-mediated knock-ins and decreases with the increase in the length of the knock-in fragment, restricting the clinical application of this technology. In existing homologous recombination protocols, although the overall concentration of donor DNA molecules is very high, they are free throughout the cytoplasm and have a low local density at the DSB site.

[0008] For successful knock-in, the donor DNA should be located locally at the DSB at the moment of DSB formation. When using non-viral donor delivery methods, there is no specific mechanism to transfer the donor to the DSB local area. In proliferating transfected cells, the donor DNA enters the nucleus through the cyclic assembly / disassembly of the nuclear membrane. Variants of covalently linking the donor to gRNA or Cas9 have been proposed to improve the delivery efficiency of the donor to the DSB site and ensure the spatial proximity of the donor and the Cas9 / gRNA complex. All these methods usually impair the target cleavage activity of the complex. Therefore, the reported increase in HDR modification above is actually an increase in the HDR / NHEJ ratio, while the absolute HDR level decreases. Summary of the Invention

[0009] In view of the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide a general method for homologous recombination construction of CART cells, which is used to solve the technical problems of graft-versus-host disease (GvHD) caused by allogeneic T cells and low gene editing knock-in efficiency in the prior art.

[0010] To achieve the above object and other related objects, the present invention provides a general method for homologous recombination construction of CART cells, including but not limited to knock-in targeting EGFRvIII / Mesothelin / CLDN6; the general CART cells are allogeneic T cells with chimeric antigen receptors, and the T cell receptor alpha chain, HLA class I and II, and PD1 molecules of the general CART cells are knocked out;

[0011] The knockout uses CRISPR technology to specifically knockout the TRAC, B2M, CIITA, and PD1 genes in allogeneic T cells at the same time; it includes destroying the expression of endogenous TCR by targeting the TCRα (TRAC) chain, eliminating HLA class I expression by targeting the gRNA-B2M gene, and eliminating HLA class II expression by targeting the human class II major histocompatibility complex transactivator (CIITA) gene.

[0012] In an embodiment of the present invention, the avidin-RNA sequence used for the knock-in is:

[0013] T7-2×λN22-Avidin-SV40polyA

[0014] TAATACGACTCACTATAGGGAATACAAGCTACTTGTTCTTTTTGCAGGATCTGCCACCGGTACCGCCACCATGGACGCCCAGACCAGAAGGCGCGAGCGCAGAGCCGAGAAGCAGGCCCAGTGGAAGGCCGCCAACCCTCCCCTCGATGGCGCCGGTGCCGGCGCTGGCGCTGGAGCAGGCGCCGGCGGACTGGCCACAATGGACGCCCAGACCAGAAGGCGCGAGCGCAGAGCCGAGAAGCAGGCCCAGTGGAAGGCCGCCAACCCTCCCCTCGATGGCGCCGGTGCCGGCGCTGGCGCTGGAGCAGGCGCCGGCGGACTGGCCACAAGCGCTGGAGGAGGTGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGTAGCGGACCTAAGAAAAAGAGGAAGGTGGCTGCCGCTGGATCCATGGTGCACGCAACCTCCCCGCTGCTGCTGCTGCTGCTGCTCAGCCTGGCTCTGGTGGCTCCCGGCCTCTCTGCCAGAAAGTGCTCGCTGACTGGGAAATGGACCAACGATCTGGGCTCCAACATGACCATCGGGGCTGTGAACAGCAGAGGTGAATTCACAGGCACCTACATCACAGCCGTAACAGCCACATCAAATGAGATCAAAGAGTCACCACTGCATGGGACACAAAACACCATCAACAAGAGGACCCAGCCCACCTTTGGCTTCACCGTCAATTGGAAGTTTTCAGAGTCCACCACTGTCTTCACGGGCCAGTGCTTCATAGACAGGAATGGGAAGGAGGTCCTGAAGACCATGTGGCTGCTGCGGTCAAGTGTTAATGACATTGGTGATGACTGGAAAGCTACCAGGGTCGGCATCAACATCTTCACTCGCCTGCGCACACAGAAGGAGTAATCTAGAACTATAGTGAGTCGTATTACGTAGATCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAATTCGC(SEQ ID NO.26)

[0015] The sequence of the gRNA used in the CRISPR technology is as follows:

[0016] gRNA-TRAC is selected from the following sequences:

[0017] gRNA-TRAC-1: AGAGCAACAGTGCTGTGGCC(SEQ ID NO.1)

[0018] gRNA-TRAC-2: CTTCAAGAGCAACAGTGCTG(SEQ ID NO.2)

[0019] gRNA-TRAC-3: AGAGTCTCTCAGCTGGTACA(SEQ ID NO.3)

[0020] gRNA-B2M is selected from the following sequences:

[0021] gRNA-B2M-1: GAGTAGCGCGAGCACAGCTA(SEQ ID NO.4)

[0022] gRNA-B2M-2: GGCCACGGAGCGAGACATCT(SEQ ID NO.5)

[0023] gRNA-B2M-3: CACAGCCCAAGATAGTTAAG(SEQ ID NO.6)

[0024] gRNA-B2M-4: GCTGGATCTCGGGGAAGCGG(SEQ ID NO.7)

[0025] gRNA-B2M-5: TAGTCCAGGGCTGGATCTCG(SEQ ID NO.8)

[0026] gRNA-B2M-6: TATAAGTGGAGGCGTCGCGC (SEQ ID NO.9)

[0027] gRNA-CIITA is selected from the following sequences:

[0028] gRNA-CIITA-1: GATATTGGCATAAGCCTCCC (SEQ ID NO.10)

[0029] gRNA-CIITA-2: GCTGAACTGGTCGCAGTTGA (SEQ ID NO.11)

[0030] gRNA-CIITA-3: TGGAAGGTGATGAAGAGACC (SEQ ID NO.12)

[0031] gRNA-CIITA-4: AGCCAGGCAACGCATTGTGT (SEQ ID NO.13)

[0032] gRNA-CIITA-5: ATTGTGTAGGAATCCCAGCC (SEQ ID NO.14)

[0033] gRNA-CIITA-6: AGCTTCCCCAAGGATGCCTT (SEQ ID NO.15)

[0034] gRNA-CIITA-7: GGAAGGTGATGAAGAGACCA (SEQ ID NO.16)

[0035] gRNA-CIITA-8: TCAACTGCGACCAGTTCAGC (SEQ ID NO.17)

[0036] gRNA-CIITA-9: CATCGCTGTTAAGAAGCTC (SEQ ID NO.18)

[0037] gRNA-CIITA-10: GGTCCATCTGGTCATAGAAG (SEQ ID NO.19)

[0038] gRNA-CIITA-11: CCATTGCTTGAACCGTCCGG (SEQ ID NO.20)

[0039] gRNA-CIITA-12: GAGAAGACAAAGTCGTACTG (SEQ ID NO.21)

[0040] The gRNA-PD1 is selected from the following sequences:

[0041] gRNA-PD1-1: ACAGGCGCCCTGGCCAGTCG (SEQ ID NO.22)

[0042] gRNA-PD1-2: GGGCGGTGCTACAACTGGGC (SEQ ID NO.23)

[0043] gRNA-PD1-3: AACTGGGCTGGCGGCCAGGATGG (SEQ ID NO.24)

[0044] gRNA-PD1-4: GTCTGGGCGGTGCTACAACT (SEQ ID NO.25).

[0045] In one embodiment of the present invention, the targeting target sequence of the gRNA-TRAC includes the sequence shown in SEQ ID No:3; the targeting target sequence of the gRNA-B2M includes the sequence shown in SEQ ID No:4; the targeting target sequence of the gRNA-CIITA includes the sequence shown in SEQ ID No:21; the targeting target sequence of the gRNA-PD1 includes the sequence shown in SEQ ID No:25.

[0046] In one embodiment of the present invention, the knock-in includes the steps of binding the avidin protein to the gRNA designed for the targeting target sequence and the ligand of the biotin-binding protein modified at the end, and introducing the homologous recombination fragment containing the exogenous DNA molecule into the receptor, so that the BOXb Loop structure binding to the λN22 protein is carried on the gRNA designed for the target sequence, and the protein binds to the Avidin-loop bind RNA and the homologous recombination fragment modified by the ligand of the protein at the end and containing the exogenous DNA molecule is introduced into the receptor.

[0047] In one embodiment of the present invention, the homologous recombination fragment includes an upstream homologous arm, an exogenous DNA molecule, and a downstream homologous arm arranged in sequence.

[0048] In one embodiment of the present invention, the homologous recombination fragment is single-stranded DNA.

[0049] In one embodiment of the present invention, the Cas9 protein, the homologous recombination fragment, and at least one gRNA are introduced into the cell.

[0050] In one embodiment of the present invention, the molar ratio of the Cas9 protein to the at least one gRNA is 1:(1 - 10); the Cas9 protein and the at least one gRNA form a complex through incubation; the temperature of the incubation is 20 - 50 °C, and the time of the incubation is 2 - 30 minutes; the dosage ratio of the Cas9 protein to cells is 2 - 20 μg Cas9:(1×10 6 - 5×10 6 cells); the mass ratio of the homologous recombination fragment to the gRNA is 1:(0.5 - 3); the dosage ratio of the Avidin - loop bind RNA to cells is 1 - 10 μg Avidin - loop bind RNA:(1×10 6 - 5×10 6 cells); the electroporation density of the cells is (5×10 7 - 2×10 8 cells / mL).

[0051] The present invention also provides a gRNA composition for use in the general CART cell homologous recombination construction method according to any one of claims 1 - 9, characterized in that: the gRNA composition includes any one or more of the gRNAs defined above for gene editing.

[0052] As described above, the general CART cell homologous recombination construction method of the present invention has the following beneficial effects:

[0053] The EGFRvIII / Mesothelin - targeted general CART cells of the present invention achieve quadruple knockout of the TRAC, B2M, CIITA, and PD1 molecules and the knock - in of a large - fragment EGFRvIII / Mesothelin CAR, which not only generates the targeting of tumor - specific antigens but also eliminates GvHD through the knockout of TCR, HLA class I, and class II, thereby preventing allogeneic T cells from being rejected. The allogeneic T cells with PD1 molecule knockout can more effectively exert tumor - killing activity because of the down - regulation of immunosuppression; the general CART cells constructed by the present invention can be used as a general high - activity CART cell preparation. By recruiting the homologous recombination fragment to the double - strand DNA break cut by the Cas9 protein, the present invention enables the homologous recombination fragment to be highly enriched at this site, and its local density at this site is significantly increased. Therefore, the difficulty for the homologous recombination mechanism to search for donor DNA molecules is greatly reduced. Based on the homologous recombination mechanism of the present invention, the precise insertion efficiency of the donor DNA sequence is greatly improved, thereby greatly improving the efficiency of producing homologous recombination CART cells, and further reducing the technical threshold of CRISPR - produced CART cells, which has obvious innovation and superiority compared with traditional methods. Brief Description of the Drawings

[0054] Figure 1 It shows a schematic diagram of homologous recombination of an exogenous DNA molecule into a target sequence in Example 2. Detailed Embodiments

[0055] The following specific embodiments illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

[0056] Example 1 provides a method for constructing a universal CART cell by homologous recombination, including knock-in targeting EGFRvIII / Mesothelin; the universal CART cell is an allogeneic T cell with a chimeric antigen receptor, and the T cell receptor α chain, HLA class I and class II, and PD1 molecule of the universal CART cell are knocked out;

[0057] The knockout uses CRISPR technology to specifically knockout the TRAC, B2M, CIITA, and PD1 genes in allogeneic T cells at the same time; it includes disrupting the expression of endogenous TCR by targeting the TCRα (TRAC) chain, eliminating HLA class I expression by targeting the gRNA-B2M gene, and eliminating HLA class II expression by targeting the human class II major histocompatibility complex transactivator (CIITA) gene.

[0058] The avidin-RNA sequence used for the knock-in is:

[0059] T7-2×λN22-Avidin-SV40polyA

[0060] TAATACGACTCACTATAGGGAATACAAGCTACTTGTTCTTTTTGCAGGATCTGCCACCGGTACCGCCACCATGGACGCCCAGACCAGAAGGCGCGAGCGCAGAGCCGAGAAGCAGGCCCAGTGGAAGGCCGCCAACCCTCCCCTCGATGGCGCCGGTGCCGGCGCTGGCGCTGGAGCAGGCGCCGGCGGACTGGCCACAATGGACGCCCAGACCAGAAGGCGCGAGCGCAGAGCCGAGAAGCAGGCCCAGTGGAAGGCCGCCAACCCTCCCCTCGATGGCGCCGGTGCCGGCGCTGGCGCTGGAGCAGGCGCCGGCGGACTGGCCACAAGCGCTGGAGGAGGTGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGTAGCGGACCTAAGAAAAAGAGGAAGGTGGCTGCCGCTGGATCCATGGTGCACGCAACCTCCCCGCTGCTGCTGCTGCTGCTGCTCAGCCTGGCTCTGGTGGCTCCCGGCCTCTCTGCCAGAAAGTGCTCGCTGACTGGGAAATGGACCAACGATCTGGGCTCCAACATGACCATCGGGGCTGTGAACAGCAGAGGTGAATTCACAGGCACCTACATCACAGCCGTAACAGCCACATCAAATGAGATCAAAGAGTCACCACTGCATGGGACACAAAACACCATCAACAAGAGGACCCAGCCCACCTTTGGCTTCACCGTCAATTGGAAGTTTTCAGAGTCCACCACTGTCTTCACGGGCCAGTGCTTCATAGACAGGAATGGGAAGGAGGTCCTGAAGACCATGTGGCTGCTGCGGTCAAGTGTTAATGACATTGGTGATGACTGGAAAGCTACCAGGGTCGGCATCAACATCTTCACTCGCCTGCGCACACAGAAGGAGTAATCTAGAACTATAGTGAGTCGTATTACGTAGATCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAATTCGC(SEQ ID NO.26)

[0061] The sequence of the gRNA used in the CRISPR technology is:

[0062] gRNA-TRAC is selected from the following sequences:

[0063] gRNA-TRAC-1: AGAGCAACAGTGCTGTGGCC(SEQ ID NO.1)

[0064] gRNA-TRAC-2: CTTCAAGAGCAACAGTGCTG(SEQ ID NO.2)

[0065] gRNA-TRAC-3: AGAGTCTCTCAGCTGGTACA(SEQ ID NO.3)

[0066] gRNA-B2M is selected from the following sequences:

[0067] gRNA-B2M-1: GAGTAGCGCGAGCACAGCTA(SEQ ID NO.4)

[0068] gRNA-B2M-2: GGCCACGGAGCGAGACATCT(SEQ ID NO.5)

[0069] gRNA-B2M-3: CACAGCCCAAGATAGTTAAG(SEQ ID NO.6)

[0070] gRNA-B2M-4: GCTGGATCTCGGGGAAGCGG(SEQ ID NO.7)

[0071] gRNA-B2M-5: TAGTCCAGGGCTGGATCTCG(SEQ ID NO.8)

[0072] gRNA-B2M-6: TATAAGTGGAGGCGTCGCGC(SEQ ID NO.9)

[0073] gRNA-CIITA is selected from the following sequences:

[0074] gRNA-CIITA-1: GATATTGGCATAAGCCTCCC (SEQ ID NO.10)

[0075] gRNA-CIITA-2: GCTGAACTGGTCGCAGTTGA (SEQ ID NO.11)

[0076] gRNA-CIITA-3: TGGAAGGTGATGAAGAGACC (SEQ ID NO.12)

[0077] gRNA-CIITA-4: AGCCAGGCAACGCATTGTGT (SEQ ID NO.13)

[0078] gRNA-CIITA-5: ATTGTGTAGGAATCCCAGCC (SEQ ID NO.14)

[0079] gRNA-CIITA-6: AGCTTCCCCAAGGATGCCTT (SEQ ID NO.15)

[0080] gRNA-CIITA-7: GGAAGGTGATGAAGAGACCA (SEQ ID NO.16)

[0081] gRNA-CIITA-8: TCAACTGCGACCAGTTCAGC (SEQ ID NO.17)

[0082] gRNA-CIITA-9: CATCGCTGTTAAGAAGCTC (SEQ ID NO.18)

[0083] gRNA-CIITA-10: GGTCCATCTGGTCATAGAAG (SEQ ID NO.19)

[0084] gRNA-CIITA-11: CCATTGCTTGAACCGTCCGG (SEQ ID NO.20)

[0085] gRNA-CIITA-12: GAGAAGACAAAGTCGTACTG (SEQ ID NO.21)

[0086] gRNA-PD1 is selected from the following sequences:

[0087] gRNA-PD1-1: ACAGGCGCCCTGGCCAGTCG (SEQ ID NO.22)

[0088] gRNA-PD1-2: GGGCGGTGCTACAACTGGGC (SEQ ID NO.23)

[0089] gRNA-PD1-3: AACTGGGCTGGCGGCCAGGATGG (SEQ ID NO.24)

[0090] gRNA-PD1-4: GTCTGGGCGGTGCTACAACT (SEQ ID NO.25)

[0091] The sequence of the EGFRvIII / Mesothelin CAR is as follows:

[0092]

[0093]

[0094] The homologous fragment sequence of the CAR is as follows:

[0095] 31bp - 5’: TCTGGGCGGTGCTACAACTGGGCTGGCGGCC(SEQ ID NO.29)

[0096] 33bp - 3’: GGATGGTTCTTAGGTAGGTGGGGTCGGCGGTCA(SEQ ID NO.30)

[0097] 800bp - 5': GTGGAAAGATCTGGAACTGTGGCCATGGTGTGAGGCCATCCACAAGGTGGAAGCTTTGAGGGGGAGCCGATTAGCCATGGACAGTTGTCATT CAGTAGGGTCACCTGTGCCCCAGCGAAGGGGGATGGGCCGGGAAGGCAGAGGCCAGGCACCTGCCCCCAGCAGGGGCAGAGGCTGTGGGCAGCCGGGAGGCTCCCAGAGGCTCCGACAGAATGGGAGTGGGGTTGAGCCCACCCCTCACTGCAGCCCAGGAACCTGAGCCCAGAGGGGGCCACCCACCTTCCCCAGGCAGGGAGGCCCGGCCCCCAGGGAGATGGGGGGGATGGGGGAGGAGAAGGGCCTGCCCCCACCCGGCAGCCTCAGGAGGGGCAGCTCGGGCGGGATATGGAAAGAGGCCACAGCAGTGAGCAGAGACACAGAGGAGGAAGGGGCCCTGAGCTGGGGAGACCCCCACGGGGTAGGGCGTGGGGGCCACGGGCCCACCTCCTCCCCATCTCCTCTGTCTCCCTGTCTCTGTCTCTCTCTCCCTCCCCCACCCTCTCCCCAGTCCTACCCCCTCCTCACCCCTCCTCCCCCAGCACTGCCTCTGTCACTCTCGCCCACGTGGATGTGGAGGAAGAGGGGGCGGGAGCAAGGGGCGGGCACCCTCCCTTCAACCTGACCTGGGACAGTTTCCCTTCCGCTCACCTCCGCCTGAGCAGTGGAGAAGGCGGCACTCTGGTGGGGCTGCTCCAGGCATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCA(SEQ ID NO.31)

[0098] 800bp-3’: GGATGGTTCTTAGGTAGGTGGGGTCGGCGGTCAGGTGTCCCA GAGCCAGGGGTCTGGAGGGACCTTCCACCCTCAGTCCCTGGCAGGTCGGGGGGTGCTGAGGCGGGCCTGGCCCTGGCAGCCCAGGGGTCCCGGAGCGAGGGGTCTGGAGGGACCTTTCACTCTCAGTCCCTGGCAGGTCGGGGGGTGCTGTGGCAGGCCCAGCCTTGGCCCCCAGCTCTGCCCCTTACCCTGAGCTGTGTGGCTTTGGGCAGCTCGAACTCCTGGGTTCCTCTCTGGGCCCCAACTCCTCCCCTGGCCCAAGTCCCCTCTTTGCTCCTGGGCAGGCAGGACCTCTGTCCCCTCTCAGCCGGTCCTTGGGGCTGCGTGTTTCTGTAGAATGACGGGTCAGGCTGGCCAGAACCCCAAACCTTGGCCGTGGGGAGTCTGCGTGGCGGCTCTGCCTTGCCCAGGCATCCTTGGTCCTCACTCGAGTTTTCCTAAGGATGGGATGAGCCCCATGTGGGACTAACCTTGGCTTTACGACGTCAAAGTTTAGATGAGCTGGTGATATTTTTCTCATTATATCCAAAGTGTACCTGTTCGAGTGAGGACAGTTCTTCTGTCTCCAGGATCCCTCCTGGGTGGGGATTGTGCCCGCCTGGGTCTCTGCCCAGATTCCAGGGCTCTCCCCGAGCCCTGTTCAGACCATCCGTGGGG GAGGCCTTGGCCTCACTCTCCCGGATCGAGGAGAGAGGGAGCCTCTTCCTGGGCTGCCCGTGACCCTGGGCCCTCTGTGTACACTGTGACCACAGCCCGCT(SEQ ID NO.32)

[0099] The targeting target sequence of the gRNA-TRAC includes the sequence shown in SEQ ID No: 3; the targeting target sequence of the gRNA-B2M includes the sequence shown in SEQ ID No: 4; the targeting target sequence of the gRNA-CIITA includes the sequence shown in SEQ ID No: 21; the targeting target sequence of the gRNA-PD1 includes the sequence shown in SEQ ID No: 25.

[0100] The EGFRvIII / Mesothelin-targeted universal CART cells of the present invention achieve quadruple knockout of TRAC, B2M, CIITA, and PD1 molecules and knock-in of large-fragment EGFRvIII / Mesothelin CAR, which not only generates the targeting of tumor-specific antigens but also eliminates GvHD through the knockout of TCR, HLA class I, and class II, thereby preventing allogeneic T cells from being rejected. Moreover, allogeneic T cells with PD1 molecule knockout can more effectively exert tumor-killing activity due to downregulated immunosuppression; the universal CART cells constructed in the present invention can be used as a universal high-activity CART cell preparation.

[0101] Example 2, based on Example 1, the knock-in includes the steps of binding the avidin protein to the gRNA designed for the target sequence, modifying the end with the ligand of the streptavidin-binding protein, and introducing the homologous recombination fragment containing the exogenous DNA molecule into the receptor, such that the gRNA designed for the target sequence has a BOXb Loop structure that binds to the λN22 protein, and the protein binds to the Avidin-loop bind RNA and the homologous recombination fragment modified with the ligand of the protein at the end and containing the exogenous DNA molecule is introduced into the receptor. Please refer to Figure 1 , Figure 1Among them, λN22 is an arginine-rich peptide segment derived from the N protein of phage λ; Boxb is an RNA stem-loop structure that can specifically bind to λN22; λN22-Streptavidin is an RNA transcript of a flexible-linked λN22-avidin fusion protein; the ligand of the protein is biotin, and the protein is a biotin-binding protein. The step of binding the biotin-binding protein to the gRNA designed for the target sequence and modifying the end with the ligand of the biotin-binding protein and introducing the homologous recombination fragment containing the exogenous DNA molecule into the receptor makes the gRNA designed for the target sequence carry the Boxb RNA Loop that binds to Avidin-λN22. The protein binds to the Avidin-loop bind RNA and the homologous recombination fragment modified at the end with the ligand of the protein and containing the exogenous DNA molecule is introduced into the receptor, reducing the influence of the direct ligation of the fusion protein on the gRNA's recognition of the target sequence. The Boxb RNA structure and the λN22 protein are a pair of naturally occurring high-affinity interacting molecules in phage λ. Avidin was initially discovered in ovalbumin and can form a tetramer, mainly used for signal cascade amplification reactions in antibody labeling. Avidin can bind tightly to biotin, with an equilibrium coefficient Kd value of 10 to the -16th power. If it is necessary to dissociate the two, very extreme heating and strong acid conditions are required, which are sufficient to denature the protein. Currently, a series of derivatives of Avidin have been developed, by changing individual bases and then modifying the isoelectric point PI to reduce non-specific binding, such as Neutravidin, Streptavidin, etc.; by changing the protein sequence, converting the easily tetramerized Avidin into a dimerized Avidin or monomeric Avidin; and in other species, Avidin-like proteins with similar functions and strong binding to biotin have also been found. By placing the two pairs of interactions of Boxb and biotin on the sgRNA and ssDNA respectively and adding a fusion protein with an intermediate secondary link, the ssDNA is pulled close to the knockout site through the two pairs of interactions of λN22-Boxb and biotin-avidin. The setting of this step can reduce the influence of the direct ligation of the fusion protein on the gRNA's recognition of the target sequence and improve the efficiency of gene knock-in. See the principle schematic diagram.

[0102] More specifically, the homologous recombination fragment includes an upstream homologous arm, an exogenous DNA molecule, and a downstream homologous arm arranged in sequence; the homologous recombination fragment is single-stranded DNA.

[0103] The mechanism of homologous recombination of the avidin protein includes:

[0104] 1) Modify the 5' end of the homologous recombination fragment to be integrated with biotin;

[0105] 2) Under the guidance of gRNA, the Cas9 protein with the ability to cleave DNA performs double-strand cleavage at specific positions of the targeted genome and excises several bases;

[0106] 3) The BOXb RNA Loop at the sgRNAtetraloop end can specifically bind to the λ phage protein λN22 conjugated with streptavidin, and then recruit the homologous recombination fragment modified with biotin at the 5' or 3' end to the vicinity of the double-strand DNA break for homologous recombination. The knock-in process is to add four molecules, namely Cas9 protein, optimized sgRNA sequence, single-stranded RNA fragment of λN22 tandem avidin, and ssDNA homologous recombination template with CAR structure modified with biotin at the 5' / 3' end, into the in vitro transfection buffer system and mix them together to transfer into the cell interior. After entering the cell, the single-stranded RNA fragment of λN22 tandem avidin is translated into a protein form. The λN22 region at one end recognizes and binds to the BOXb structure on the sgRNA, and the avidin at the other end binds to the biotin on the homologous recombination template, pulling the three molecules together to form a complex, and performing large-fragment homologous recombination insertion at the position where the Cas9 protein edits the genome to form a gap.

[0107] Among them: A segment of the original stem-loop structure of sgRNA is replaced with the BOXb sequence.

[0108] In a specific embodiment of the present invention, avidin is used as the biotin-binding protein. The interaction between biotin and avidin is the strongest non-covalent interaction known so far, which is 10,000 times higher than the affinity between antigen and antibody; by pulling the donor DNA to the double-strand DNA break instead of floating in the vast space of the cell like the traditional method, the technical solution of the present invention greatly improves the homologous recombination efficiency of exogenous DNA.

[0109] In the best implementation scheme of this Example 1, Cas9 protein, homologous recombination fragment and at least one gRNA are introduced into the cell, and the electrotransformation technology is used to introduce a complex containing Cas9 protein, homologous recombination fragment and at least one gRNA into the cell. The molar ratio of the Cas9 protein to the at least one gRNA is 1:(1-3), preferably 1:2. The Cas9 protein and the at least one gRNA form a complex through incubation; preferably, the incubation temperature is 20-50 °C, more preferably 25-37 °C; preferably, the incubation time is 2-30 minutes, preferably 5-20 minutes, more preferably 15 minutes. The dosage ratio of the Cas9 protein to the cell is 2-20 μg Cas9:(1×10 6 -5×10 6 cells), preferably 3.3 μg Cas9:1×106 cells. The mass ratio of the homologous recombination fragment to the gRNA is 1:(0.5 - 3), preferably 1:1. The dosage ratio of the Avidin-loop bind RNA to the cells is 1 - 10 μg Avidin-loop bind RNA:(1×10 6 -5×10 6 cells), preferably 2 μg Avidin-loop bind RNA:1×10 6 cells. The electroporation density of the cells is (5×10 7 -2×10 8 cells / mL), preferably 1×10 8 cells / mL.

[0110] In the present invention, by recruiting the homologous recombination fragment to the double-stranded DNA break cleaved by the Cas9 protein, the homologous recombination fragment is highly enriched here, and its local density here is significantly increased. Therefore, the difficulty of the homologous recombination mechanism in searching for donor DNA molecules is greatly reduced. Based on the homologous recombination mechanism of the present invention, the precise insertion efficiency of the donor DNA sequence is greatly improved, thereby greatly improving the efficiency of producing homologous recombinant CART cells, and further reducing the technical threshold of CRISPR for producing CART cells, showing obvious innovation and superiority compared with traditional methods.

[0111] Example 3. This example provides a gRNA composition for gene editing including any one or more of the gRNAs defined above.

[0112] Example 4. This example provides a detection method. The electroporated cells are cultured in an IL2 + IL7 + IL15 + XVIVO15 system for 7 days, and the genomic DNA of the cells obtained in the above steps is extracted for genotype identification to determine the knockout and knock-in efficiencies. At 3 - 4 days of culture, an appropriate amount of cells are collected, and Protein-L, CD3, HLA-ABC, and HLA-DR are detected by flow cytometry staining to further determine the knockout and knock-in efficiencies. See the following table for details:

[0113] Flow knockout efficiency TIDE knockout efficiency TRAC 88.50％ 72.40％ B2M 96.80％ 84.80％ CIITA 57.20％ 50％ PD1 / 90.6％

[0114] Since TRAC knockout will reduce the CD3 positive rate, B2M knockout will disrupt the HLA-ABC protein, and CIITA knockout will disrupt the HLA-DR protein, in this example, these proteins are stained by flow cytometry to detect the knockout rate.

[0115] The inventors of the present invention have confirmed through experiments that the avidin-based aggregation system of the present invention can produce large-fragment gene-knocked-in T cells with high efficiency; in contrast, the efficiency of producing large-fragment gene-knocked-in T cells using the traditional method without homologous recombination fragment aggregation is less than 5%, which cannot meet the requirements of production and clinical applications. Therefore, the homologous recombination fragment aggregation system of the present invention has made significant progress compared with the prior art.

[0116] In summary, the present invention uses CRISPR technology to perform quadruple knockout of the TRAC, B2M, CIITA, and PD1 genes in T cells of allogeneic CART cells, and the resulting negative cell populations reach 88%, 96%, 57%, and 90% respectively. This cell population simultaneously loses GvHD activity and enhances the anti-tumor activity of CART cells; when treating patients, the allogeneic CART cells with quadruple knockout can treat tumors more effectively and for a longer time without causing GvHD, thereby greatly reducing the production and treatment costs. Therefore, the present invention effectively overcomes various disadvantages in the prior art and has high industrial utilization value.

[0117] The above embodiments are only illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed by the present invention should still be covered by the claims of the present invention.

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