A biological preparation for constructing mRNA CAR-T cells, preparation method and application

Abstract

The present invention provides a fusion gene, nucleic acid molecule, recombinant vector, cell and preparation method and application for constructing mRNA CAR T cell biological preparations, and the biological preparations are secreted by engineering cells and internally loaded with microvesicles of specific target CAR mRNA, mainly related to the field of genetic engineering technology. The present invention mainly connects the assembly protein to the C-terminus of the microvesicle membrane protein, inserts the guide binding sequence (a special RNA stem-loop structure) bound to the assembly protein into the 3'-untranslated region (3'-UTR) encoding the target mRNA, to design two fusion gene structures, and constructs a recombinant lentiviral vector and a stable cell line, so as to produce microvesicles. The microvesicles constructed by the present invention have good biocompatibility and a simple preparation method. When the recombinant cell microvesicles are generated, the specific target CAR mRNA can be loaded into the microvesicle by the interaction between the assembly protein and the guide binding sequence. The microvesicles constructed by the present invention can deliver specific target CAR mRNA to cells and can be directly applied in vivo.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and in particular to a biological preparation for constructing mRNA CAR-T cells, a preparation method and an application thereof. Background Art

[0002] Chimeric antigen receptor modified T cell (CAR-T) therapy is one of the most promising tumor immunotherapy methods. It modifies T cells in vitro to give them the ability to target and kill tumor cells in a non-MHC restricted manner. After in vitro amplification and culture, they are then infused back into the patient for treatment. CAR-T therapy targeting CD19 has shown significant efficacy in the treatment of malignant hematological tumors, and a number of CD19 CAR-T products for hematological tumors have been approved by the FDA for marketing. At present, most CAR-T cells are prepared by lentiviral vectors. Although lentivirus can induce permanent expression of CAR, the immunogenicity of the lentiviral vector itself and the uncertainty of the insertion site may cause serious adverse reactions in patients receiving CAR-T treatment. At the same time, due to the long production cycle of lentiviral CAR-T and the unstable transduction efficiency of lentivirus, it is urgent to improve the production method of CAR-T cells.

[0003] Studies have found that using mRNA to induce CAR expression has many potential advantages over viral transduction. First, mRNA can be directly translated and expressed after entering the cell, without being integrated into the genome like lentivirus, which can make CAR expressed for a period of time, reducing the health risks brought by the long-term presence of CAR-T cells in the body, while shortening the preparation cycle of CAR-T, effectively avoiding the best treatment time lost by many patients during the waiting process. In addition, with the large-scale clinical application of mRNA vaccines, mRNACAR-T has the potential to be further used to construct CAR-T in vivo. At present, mRNACAR-T cell therapy has been used in many cancers including leukemia, Hodgkin's lymphoma and melanoma, and it has been proven that it can achieve the same therapeutic effect as virally engineered CAR-T cells in patients.

[0004] To prepare mRNA-based CAR-T cells, it is necessary to efficiently transduce the mRNA expressing CAR into T cells. Since naked mRNA molecules are unstable, easily degraded by RNases, and difficult to pass through the cell membrane into the cytoplasm, it is necessary to rely on suitable delivery vectors or external forces to transduce mRNA molecules into T cells.

[0005] Currently, the most common mRNA cell transduction method is electroporation (EP), which uses high-voltage electric pulses to permeabilize the cell membrane to allow mRNA to enter the cell. However, this permeabilization can cause permanent damage to the cells, including loss of cell contents and changes in gene expression, which can lead to low activity of the obtained CAR-T cells, seriously affecting the expansion of CAR-T cells and their later efficacy.

[0006] Microvesicles are active vesicles secreted by cells, with a diameter of 100-1000nm. They contain a large number of biomolecules, including nucleic acids, proteins and lipids, and play an important role in intercellular communication. Microvesicles have become an ideal carrier for delivering mRNA to cells in vivo and in vitro due to their inherent stability, excellent biocompatibility, low immunogenicity and good tissue penetration. However, the method of loading exogenous mRNA into microvesicles mainly through electroporation has seriously restricted the further application of microvesicle delivery of mRNA. Summary of the invention

[0007] The purpose of the present invention is to address the problems existing in the background technology and propose a biological preparation for constructing mRNA CAR-T cells that can be used in vivo and in vitro. The biological preparation for constructing mRNA CAR-T cells is an engineered microvesicle that is loaded with specific CAR mRNA expressing in the membrane after modification.

[0008] In order to achieve the above invention object, the present invention provides the following technical solutions:

[0009] In the first aspect, a fusion gene of a membrane-loaded specific CAR mRNA microvesicle is provided, comprising a first fusion gene and a second fusion gene, wherein the first fusion gene comprises a transmembrane region gene and an assembly protein gene of a membrane protein enriched in the microvesicle; and the second fusion gene comprises a sequence of a target CAR mRNA and a guide binding sequence of an assembly protein.

[0010] Microvesicle membrane proteins include Lamp2b, CD63, CD9, Alix, Tsg101, Flotillin, CD81, CD82, CD151, tetraspanin, etc.

[0011] Preferably, the transmembrane region gene of Lamp2b is SEQ ID NO.1, and the transmembrane region gene of CD63 is SEQ ID NO.2.

[0012] The assembly protein can bind to a specific RNA stem-loop structure, such as phage envelope protein MS2, ribosome binding protein L7Ae, transcription transactivator protein Tat, etc. The present invention utilizes the assembly protein to bind to its guide binding sequence to introduce the target mRNA into the microvesicle.

[0013] Preferably, the sequence of the bacteriophage envelope protein MS2 is SEQ ID NO.3; the sequence of the ribosome binding protein L7Ae is SEQ ID NO.4, and the sequence of the transcription transactivator protein Tat is SEQ ID NO.5.

[0014] The target CAR mRNA is the messenger RNA that needs to be delivered into cells and can be translated into protein or peptide in the recipient cells.

[0015] In some embodiments, the CAR-T target is selected from any one or more combinations of B7H3, Mesothelin, Claudin18.2, GPC3, PD-L1, MUC1, Her2, EGFR, PSMA, CEA, GD2, EpCAM, EGFRvⅢ, CD70, CD20, CD133, CD177, AFP, AXL, CD171, CD117, C-MET, FAP, MUC16, NKG2D, NY-ESO-1, PSCA, VEGFR-2, Lewis-Y, Gp100, FAP or EPHA2.

[0016] Preferably, when the target is B7H3, the nucleotide sequence of its CAR is shown as SEQ ID NO.6; when the target is Mesothelin, the nucleotide sequence of its CAR is shown as SEQ ID NO.7.

[0017] The guide binding sequence of the assembly protein is a special type of sequence that can bind to the assembly protein to introduce the target CAR mRNA into the microvesicle.

[0018] Preferably, the sequence binding to MS2 is MS2bs, and its nucleotide sequence is shown in SEQ ID NO.8; the sequence binding to L7Ae is C / D box, and its nucleotide sequence is shown in SEQ ID NO.9; the sequence binding to Tat is TAR-RNA, and its nucleotide sequence is shown in SEQ ID NO.10.

[0019] In a second aspect, a recombinant vector is provided, comprising nucleotide sequences of a first fusion gene and a second fusion gene, wherein the framework of the recombinant vector is a eukaryotic cell expression vector.

[0020] In some embodiments, the eukaryotic cell expression vector is a pLVX-Puro vector;

[0021] Through double restriction digestion with XhoI and EcoRI and ligation, the nucleotide sequences of the first fusion gene and the second fusion gene are inserted into the pLVX-Puro vector separately or simultaneously for parallel expression or tandem expression.

[0022] When expressed in tandem, the first fusion gene and the second fusion gene are linked by IRES, and the nucleotide sequence of IRES is shown in SEQ ID NO.11.

[0023] In a third aspect, a stable cell line for secreting microvesicles loaded with specific CAR mRNA in the membrane is provided, and the stable cell line is constructed using the recombinant vector described in the second aspect.

[0024] In some embodiments, a stably transfected cell line for secreting membrane-bound microvesicles loaded with specific CAR mRNA comprises 293T cells transformed with the above-mentioned recombinant vector.

[0025] In a fourth aspect, a method for preparing membrane-loaded microvesicles with specific CAR mRNA is provided, which comprises the following steps: inoculating the stably transfected cell line in a serum-free complete culture medium and culturing for 24-72 hours, and the supernatant contains the microvesicles.

[0026] The present invention also provides a method for preparing a membrane-loaded specific CAR mRNA microvesicle, comprising the following steps: inoculating the above-mentioned stable cell line in serum-free complete medium and culturing for 2 days, and the supernatant contains the membrane-expressed membrane-loaded specific CAR mRNA microvesicle. The obtained supernatant is centrifuged at 250-450g for 10-20min, 2500-4500g for 10-30min to remove cells and cell debris, and then centrifuged at 6000-10000g for 20-40min to remove smaller cell fragments, and finally centrifuged at 100000-150000g for 90-120min, and the resulting precipitate is the microvesicle, and the microvesicle can also be enriched by commercial reagents.

[0027] The present invention also provides membrane-loaded specific CAR mRNA microvesicles prepared by the above method.

[0028] The present invention provides a biological preparation that can be used in vivo or in vitro, wherein the active ingredient of the biological preparation is a microvesicle loaded with a specific CAR mRNA in the membrane, and a pharmaceutical composition, wherein the pharmaceutical composition comprises the above-mentioned fusion gene, the above-mentioned nucleic acid molecule, the above-mentioned recombinant vector or recombinant virus, and a pharmaceutically acceptable carrier.

[0029] The present invention also provides the use of the above-mentioned membrane-loaded specific CAR mRNA microvesicles in vivo and in vitro, and specifically relates to the use of the above-mentioned fusion gene, the above-mentioned nucleic acid molecule, the above-mentioned recombinant vector or recombinant virus, and biological preparation in the preparation of therapeutic drugs.

[0030] Compared with the prior art, the present invention has the following beneficial technical effects:

[0031] The present invention can specifically identify and bind to a specific RNA stem-loop structure by assembling proteins, connect the assembly to the C-terminus of the membrane protein on the microvesicle, and insert the guide binding sequence of the assembly protein into the 3'-untranslated region (3'-UTR) encoding the target CAR mRNA fusion protein sequence mRNA. Due to the high expression of membrane proteins on microvesicles, the target CAR mRNA can be actively encapsulated into the microvesicles by the mutual binding between the guide binding sequence of the 3'-UTR and the assembly protein at the C-terminus of the membrane protein on the microvesicle. The microvesicles thus obtained can deliver the loaded target CAR mRNA to the receptor cell, inducing it to produce the protein or polypeptide expressed by the target CAR mRNA. The microvesicles of the present invention can load the target CAR mRNA into the microvesicles when the microvesicles are generated, effectively avoiding the damage to the microvesicle structure caused by the electrotransduction method, and at the same time, due to the good biocompatibility of the microvesicles, it can be directly applied to the delivery of CAR mRNA drugs in vivo, which is of great significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the preparation and characterization of microvesicles CARM-MVS in an embodiment of the present invention;

[0033] Figure 2 Schematic diagram of the detection results of CARM-MVS loaded with CAR mRNA in an embodiment of the present invention;

[0034] Figure 3 In the embodiment of the present invention, CARM-MVS is used to prepare CAR-T flow analysis in vitro;

[0035] Figure 4 In the embodiment of the present invention, CARM-MVS was used to prepare CAR-T Western blot test in vitro;

[0036] Figure 5 B7H3 CAR-T-MVS cytotoxicity experimental analysis in the embodiments of the present invention;

[0037] Figure 6 Detection of cytotoxic cytokine secretion by B7H3 CAR-T-MVS in the embodiments of the present invention;

[0038] Figure 7 In vivo preparation of CAR-T flow analysis by CARM-MVS in the embodiment of the present invention;

[0039] Figure 8 Schematic diagram of preparing CAR-T using microvesicles (CARM-MVS) in an embodiment of the present invention. DETAILED DESCRIPTION

[0040] The embodiments of the present invention are described in detail below in conjunction with the accompanying drawings so that the technical solutions and advantages of the present invention can be more easily understood by those skilled in the relevant art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0041] Example 1 Acquisition and Characterization of Microvesicles CAR-MVS

[0042] 1. The fusion gene SEQ ID NO.12: Lamp2b-MS2; SEQ ID NO.13: B7H3-CAR-MS2bs sequences were commissioned to Nanjing Qingke Biotechnology for synthesis.

[0043] 2. Through double restriction digestion with XhoI and EcoRI and ligation, SEQ ID NO.12 and SEQ ID NO.13 were respectively inserted into the lentiviral vector pLVX-Puro to construct a recombinant lentiviral vector.

[0044] 3. During tandem expression, SEQ ID NO. 12 and SEQ ID NO. 13 were connected by IRES (SEQ ID NO. 11) through homologous recombination and inserted into the lentiviral vector pLVX-Puro.

[0045] 3. Use transfection reagent lipo2000 (purchased from Thermo) to transfect 293 cells with the recombinant lentiviral vector, packaging plasmids pGag / Pol, pRev and pVSV-G (purchased from Addgene) in a certain ratio, and package and collect the lentivirus.

[0046] 4. The obtained lentivirus was used to infect 293 cells, and a stable cell line stably expressing the fusion genes SEQ ID NO.12 and SEQ ID NO.13 was obtained by puromycin screening.

[0047] 5. The obtained stable cell line was cultured in an incubator at 37°C containing 5% CO2, with serum-free complete medium, and the supernatant was collected after 48 hours;

[0048] 6. The supernatant was centrifuged at 350 g for 13 min to remove cells, 3500 g for 10 min to remove cell debris, 6000 g for 20 min to remove small cell debris, and then centrifuged at 100,000 g for 120 min. The resulting precipitate is the microvesicle (CARM-MVS).

[0049] 7. The microvesicles were separated by ultracentrifugation in 20 ml of culture medium, stained with tungsten phosphate, and images were recorded using TEM-2100 (see Figure 1 ) characterization.

[0050] Example 2 Analysis of the efficiency of loading CAR mRNA in CARM-MVS

[0051] The loading efficiency of CAR mRNA in microvesicles CARM-MVS was analyzed by fluorescence quantitative PCR. RNA was extracted from each group of microvesicles, and cDNA was obtained after reverse transcription. Then primers were designed for fluorescence quantitative PCR. The results showed (see Figure 2 ), the amount of CAR mRNA in CARM-MSV was higher than that in other groups, indicating that a large amount of CAR mRNA was loaded into the microvesicles through the action of MS2-MS2bs.

[0052] Example 3: Preparation of CAR-T Detection by Microvesicle CARM-MVS in vitro

[0053] CARM-MVS was added to the activated T cells of healthy people. Two days later, the expression of B7H3 CAR protein on T cells was detected by flow cytometry using APC anti-mouse F(ab)2 antibody. The results showed (see Figure 3 ) The mean fluorescence intensity (MFI) value of the CARM-MVS group was 3468, which was significantly higher than that of the control group. In terms of transduction efficiency, the CAR positive rate of T cells in the CARM-MVS group was approximately 11.23±3.5%. Subsequently, the total protein of T cells in each treatment group was extracted, separated by gel electrophoresis, transferred to a membrane, and the expression of B7H3 CAR protein in T cells was further detected by western blot using anti-human CD3ζ antibody. The results showed (see Figure 4 ) In addition to the endogenous CD3ζ (15KD) band detected in each group of cells, the B7H3 CAR protein (64KD) band was clearly observed in the CARM-MVS group and the positive control group CAR-T-LV (CAR-T cells constructed using lentivirus), but it was difficult to detect in the control group. The above results confirm that the addition of microvesicles CARM-MVS to activated T cells successfully constructed mRNA CAR-T cells targeting B7H3.

[0054] Example 4 Preparation of CAR-T (CAR-MVS) microvesicles and detection of in vitro killing function

[0055] Using luciferase-labeled target cells, the luciferase method was used to detect the specific killing function of B7H3 CAR-T (B7H3CAR-T-MVS) prepared by microvesicles on target cells in vitro.

[0056] The detection formula is as follows:

[0057]

[0058] The killing efficacy of B7-H3 CAR-T-MVS or B7-H3 CAR-T-LV at effector-target ratios (E:T) of 10:1, 5:1, and 1:1 was tested and compared. The results showed (see Figure 5 ), compared with untreated T cells, both B7-H3 CAR-T-MVS and B7-H3CAR-T-LV cells can effectively kill SGC cells with high expression of B7-H3 (B7-H3-SGC), and with the increase of effector-target ratio, the killing ratio of target cells B7-H3-SGC also increases continuously. However, no obvious killing effect was observed when the two CAR-T cells were co-cultured with B7-H3-negative SGC cells.

[0059] The secretion of inflammatory factors IL-2 and IFN-γ in each culture system was detected by ELISA when the effector-target ratio was 5:1. The results showed (see Figure 6 ), the secretion of inflammatory factors was consistent with the results of the cell killing experiment. Compared with the untreated group of T cells, the secretion of inflammatory factors IL-2 and IFN-γ in the culture system of B7-H3 CAR-T-MVS and B7-H3 CAR-T-LV increased significantly after co-culture with target cells B7-H3-SGC, while this phenomenon was not detected in the SGC cell group. These results show that CAR-T cells constructed using microvesicles can exert specific killing function on target cells in vitro, just like CAR-T cells constructed normally by lentivirus.

[0060] Although the CAR-T cells prepared by the method of the present invention have similar target cell killing functions as those prepared by conventional methods, the present invention only utilizes mRNA to express the CAR molecular chain in T cells, and thus the prepared CAR-T cells (B7-H3CAR-T-MVS) avoid the potential safety hazards caused by the random insertion of the CAR molecular chain into the T cell genome in the conventional method for preparing CAR-T cells (B7-H3 CAR-T-LV), and are particularly suitable for direct preparation of CAR-T in vivo.

[0061] Example 5: Preparation of microvesicles CARM-MVS for CAR-T detection in vivo

[0062] Compared with lentiviral particles, microvesicles have the advantages of good stability and excellent biocompatibility, making CARM-MVS expected to be further used in the preparation of CAR-T in vivo.

[0063] CARM-MVS and control groups were injected into the lymph nodes of mice, respectively. After 24 hours, T cells were extracted from the lymph nodes of mice in each group. The expression of B7H3 CAR protein on T cells in each group was detected by flow cytometry using APC anti-mouse F(ab)2antibody. The results showed (see Figure 7 ) The average fluorescence intensity and transduction efficiency of the CARM-MVS group were significantly higher than those of the other control groups, indicating that the constructed microvesicle CARM-MVS can be used for the preparation of CAR-T in vivo. These results show that the CARM-MVS prepared by the method of the present invention using the assembly protein contains more CARmRNA than the CAR-MV prepared without the assembly protein, and therefore shows a higher preparation efficiency when preparing CAR-T cells in vivo.

[0064] Example 6 Experiment and effect verification of preparing CARM-MVS loaded with CAR mRNA B7H3-CAR using Lamp2b transmembrane region and assembly protein L7Ae and its guide binding sequence C / D box;

[0065] 1. The fusion gene SEQ ID NO.14: Lamp2b-L7Ae; SEQ ID NO.15: B7H3-CAR-C / Dbox sequence was commissioned to Nanjing Qingke Biotechnology for synthesis.

[0066] 2. The experimental operation and inspection steps are the same as Example 1-4.

[0067] 3. The results are shown in Table 1:

[0068] Table 1 shows the killing efficiency (%) of B7-H3 CAR-T-MVS cells on target cells at different effector-target ratios (E:T)

[0069]

[0070] The results showed that the preparation of CARM-MSV by the method of assembling protein to guide mRNA in the present invention can introduce CAR mRNA into T cells, and the B7-H3 CAR-T-MVS prepared in this way can kill target cells with high expression of B7-H3.

[0071] Example 7 Experiment and effect verification of preparing CARM-MVS loaded with CAR mRNA Meso-CAR using CD63 transmembrane region and assembly protein Tat and its guide binding sequence TAR-RNA;

[0072] 1. The fusion gene SEQ ID NO.16: CD63-Tat; SEQ ID NO.17: Meso-CAR-TAR RNA sequence was commissioned to Nanjing Qingke Biotechnology for synthesis.

[0073] 2. The experimental operation and inspection steps are the same as Example 1-4.

[0074] 3. The results are shown in Table 2:

[0075] Table 2 shows the killing efficiency (%) of Meso CAR-T-MVS cells on target cells at different effector-target ratios (E:T)

[0076]

[0077] The results showed that the preparation of CARM-MSV by the method of assembling protein to guide mRNA in the present invention can introduce CAR mRNA into T cells, and the Meso CAR-T-MVS prepared in this way can effectively kill target cells with high expression of Mesothelin.

[0078] In summary, the present invention can specifically recognize and bind to a specific RNA stem-loop structure by assembling proteins, connect the assembly to the C-terminus of the membrane protein on the microvesicle, and insert the guide binding sequence of the assembly protein into the 3'-untranslated region (3'-UTR) encoding the target CAR mRNA fusion protein sequence mRNA. Due to the high expression of membrane proteins on microvesicles, the target CAR mRNA can be actively encapsulated into the microvesicles by the mutual binding between the guide binding sequence of the 3'-UTR and the assembly protein at the C-terminus of the membrane protein on the microvesicle. The microvesicles thus obtained can deliver the loaded target CAR mRNA to the receptor cells to induce the production of proteins or polypeptides expressed by the target CAR mRNA. The microvesicles of the present invention can load the target CAR mRNA into the microvesicles when the microvesicles are generated, effectively avoiding the damage to the microvesicle structure caused by the electrotransduction method, and at the same time, due to the good biocompatibility of the microvesicles, it can be directly applied to the delivery of CARmRNA drugs in vivo, which is of great significance.

[0079] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. For those skilled in the relevant technical field, without departing from the principle of the present invention, any improvement and modification of the above-mentioned technical solution shall be included in the patent protection scope of the present invention.

Claims

1. A biological preparation for constructing mRNA CAR-T cells, with a membrane-loaded specific target CAR mRNA microvesicle as an effective ingredient, wherein the microvesicle contains a fusion gene, characterized in that: The fusion gene includes a transmembrane region gene and an assembly protein gene of a membrane protein that is rich in microvesicles, as well as a sequence of a target CAR mRNA and a guide binding sequence of the assembly protein; The transmembrane regions of the membrane proteins enriched in the microvesicles include the transmembrane regions of the molecules Lamp2b and CD63; The assembly protein is selected from L7Ae and Tat; The target of CAR-T is selected from B7H3 and Mesothelin; The target CAR mRNA is translated into protein in the recipient cell; The guide binding sequence of the assembly protein is used to bind to the assembly protein to introduce the target CAR mRNA into the microvesicle; The fusion gene is synthesized by Lamp2b-L7Ae with a nucleotide sequence of SEQ ID NO.14 and B7H3-CAR-C / Dbox with a nucleotide sequence of SEQ ID NO.15; wherein the transmembrane region is Lamp2b, the assembly protein is the ribosome binding protein L7Ae, the target is B7H3, and the guide binding sequence of the assembly protein is C / D box; or, The fusion gene is synthesized from CD63-Tat with a nucleotide sequence of SEQ ID NO.16 and Meso-CAR-TARRNA with a nucleotide sequence of SEQ ID NO.17; wherein the transmembrane region is CD63, the assembly protein is the transcription transactivator protein Tat, the target is Mesothelin, and the guide binding sequence of the assembly protein is TAR-RNA.

2. A biological preparation for constructing mRNA CAR-T cells according to claim 1, characterized in that: The ribosome binding protein L7Ae, the nucleotide sequence of which is shown in SEQ ID NO.

4.

3. A biological preparation for constructing mRNA CAR-T cells according to claim 1 or 2, characterized in that: The guide binding sequence C / D box of the assembly protein has a nucleotide sequence as shown in SEQ ID NO.

9.

4. A biological preparation for constructing mRNA CAR-T cells according to claim 1, characterized in that: The nucleotide sequence of the transcriptional transactivator protein Tat is shown in SEQ ID NO.

5.

5. A biological preparation for constructing mRNA CAR-T cells according to claim 1 or 4, characterized in that: The guide binding sequence TAR-RNA of the assembly protein has a nucleotide sequence as shown in SEQ ID NO.

10.

6. A nucleic acid molecule, characterized in that Encodes the fusion gene according to any one of claims 1 to 5.

7. A carrier, characterized in that The vector comprises the nucleic acid molecule of claim 6.

8. A recombinant virus, characterized in that The recombinant virus comprises the vector of claim 7.

9. The method for constructing a recombinant virus according to claim 8, characterized in that: The method comprises: synthesizing the fusion gene and then introducing it into a plasmid vector, amplifying the fusion gene coding sequence through PCR primers containing homology arms, and inserting the fusion gene coding sequence into a virus vector through a homologous recombination method.

10. A recombinant cell, characterized in that The recombinant cell expresses the fusion gene described in any one of claims 1 to 5, the nucleic acid molecule described in claim 6, the vector described in claim 7, or the recombinant virus described in claim 8; Recombinant cells include modified 293T cells.

11. A method for preparing microvesicles loaded with specific target CAR mRNA in the membrane, characterized in that: The preparation is carried out using the recombinant cells described in claim 10, comprising the following steps: inoculating the recombinant cells in a culture medium and culturing for 24-72 hours, the supernatant contains microvesicles, the obtained supernatant is centrifuged at 250-450g for 10-20 minutes, 2500-4500g for 10-30 minutes to remove cells and cell debris, and then centrifuged at 6000-10000g for 20-40 minutes to remove smaller cell debris, and finally centrifuged at 100000-150000g for 90-120 minutes, and the obtained precipitate is microvesicles.

12. A pharmaceutical composition, characterized in that It comprises the fusion gene according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6, the vector according to claim 7 or the recombinant virus according to claim 8, and a pharmaceutically acceptable carrier.

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