Exosome loaded with FAP CAR mRNA and preparation method and application thereof

Abstract

The invention provides an exosome loaded with FAP CAR mRNA and a preparation method and application thereof, and belongs to the technical field of biological medicine. The nucleotide sequence of the mRNA loaded by the exosome and used for expressing the FAP CAR structure is shown as SEQ ID NO. 1. The prepared exosome loaded with the FAP CAR mRNA can deliver the FAP CAR mRNA into T cells through fusion with the T cells or endocytosis, and after the T cells uptake the FAP CAR mRNA, the FAP CAR mRNA is transcribed and translated into FAP CAR protein in the cells, so that the FAP CAR-T cells are formed. The FAP CAR-T cells circulate in vivo and are targeted to the atherosclerosis lesion site, and the FAP CAR-T cells recognize and combine with the activated fibroblasts expressing the FAP.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to exosomes loaded with FAP CAR mRNA, and a preparation method and application thereof. Background Art

[0002] Atherosclerosis (AS) is a major cause of cardiovascular disease and poses a serious threat to human health. Traditional treatments, including medication, surgery, and Traditional Chinese Medicine (TCM), are limited in efficacy, have significant side effects, and are complex procedures. In recent years, the rapid development of gene therapy and cell therapy has provided new avenues for the treatment of atherosclerosis.

[0003] Chimeric antigen receptor T cell (CAR-T) therapy, a cutting-edge cancer treatment, has demonstrated remarkable clinical efficacy. However, the application of CAR-T cell therapy to non-cancerous conditions, such as atherosclerosis, still faces numerous challenges. Among these challenges is how to safely and effectively generate CAR-T cells in situ in vivo and precisely target them to diseased tissues, a current research hotspot and challenge.

[0004] Exosomes, small vesicles secreted by cells, have recently attracted widespread attention as potential drug carriers due to their low immunogenicity, high stability, and ability to cross biological barriers. Loading mRNA into exosomes enables targeted delivery and cellular uptake, potentially enabling the in situ generation of CAR-T cells. Summary of the Invention

[0005] The purpose of the present invention is to provide an exosome loaded with FAP CAR mRNA and its preparation method and application.

[0006] Currently, there is no complete cure for atherosclerosis, as most hardened arteries are irreversible. Surgical and medical treatments primarily aim to promote blood circulation, alleviate arterial stenosis, and alleviate symptoms, thereby controlling the progression of the disease. Fibroblast proliferation and activation can promote intimal hyperplasia and fibrosis, leading to thickening of the arterial wall and the formation of atherosclerotic plaques. Inhibiting fibroblast activity is an important potential strategy for treating atherosclerosis.

[0007] Therefore, in this invention, the inventors targeted the activated adventitial fibroblasts in atherosclerosis and designed a novel CAR-T cell therapy based on mRNA technology. This approach uses mRNA to reprogram T cell receptors to target FAP in adventitial fibroblasts, and then delivers them using exosomes. The resulting exosomes can specifically target T cells, generating FAP-CAR-T cells in situ to inhibit fibroblast activation, thereby achieving treatment and intervention for atherosclerotic vascular fibrosis.

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

[0009] The present invention provides an mRNA expressing a FAP CAR structure, wherein the nucleotide sequence of the mRNA sequence is shown as SEQ ID NO.1.

[0010]

[0011] The present invention provides an exosome loaded with the mRNA expressing the FAP CAR structure.

[0012] The present invention provides a method for preparing the exosomes, comprising the following steps:

[0013] (1) Adding a recombinant sequence homologous to the plasmid vector before and after the mRNA sequence described in claim 1 to obtain the FAP-CAR-Ms2bs target gene fragment;

[0014] (2) adding recombinant sequences homologous to the plasmid vector before and after the LAMP-2B gene to obtain the LAMP-2B-MS2 target gene fragment; the nucleotide sequence of the LAMP-2B-MS2 target gene fragment is shown in SEQ ID NO.4;

[0015] (3) The FAP-CAR-Ms2bs target gene fragment and the LAMP-2B-MS2 target gene fragment were recombined with the enzyme-digested plasmid vector to obtain the FAP-CAR-Ms2bs plasmid and the LAMP-2B-MS2 plasmid;

[0016] (4) co-transfecting the FAP-CAR-Ms2bs plasmid and the LAMP-2B-MS2 plasmid into exosome-secreting cells to obtain exosome-secreting cells expressing FAP CAR mRNA;

[0017] (5) Exosomes were isolated from exosome-secreting cells expressing FAP CAR mRNA to obtain exosomes loaded with FAP CAR mRNA.

[0018] Preferably, the nucleotide sequence of the FAP-CAR-Ms2bs target gene fragment in step (1) is shown as SEQ ID NO.2, and the plasmid vector is a pcDNA3.1-EGFP plasmid vector.

[0019]

[0020]

[0021] Preferably, the enzymes used in the enzymatic digestion in step (3) are MluI enzyme and XhoI enzyme.

[0022] Preferably, the exosome-secreting cells in step (4) are 293T cells.

[0023] Preferably, the extraction system used for isolating exosomes in step (5) is the Exodus H600 fully automatic exosome extraction system.

[0024] The present invention provides a use of the mRNA expressing the FAP CAR structure, the exosomes, or the exosomes prepared according to the method in the preparation of a drug for treating atherosclerosis and / or cardiac fibrosis.

[0025] Preferably, the atherosclerosis is atherosclerosis caused by abnormal proliferation and activation of fibroblasts.

[0026] The exosomes loaded with FAP CAR mRNA prepared by the present invention can deliver FAP CAR mRNA to T cells by fusion or endocytosis with T cells. When used, the exosomes are injected into the patient's body through an appropriate route of administration (such as intravenous injection, an injection volume of 10 to 20 μg). After the T cells take up FAP CAR mRNA, they are transcribed and translated into FAP CAR protein in the cells, thereby forming FAP CAR-T cells. FAP CAR-T cells circulate in the body and target atherosclerotic lesions, recognizing and binding to activated fibroblasts expressing FAP. In addition, FAP CAR-T cells can also induce apoptosis of activated fibroblasts by releasing substances such as perforin and granzymes, thereby reducing cardiac fibrosis and improving cardiac function.

[0027] The beneficial effects of the present invention are mainly reflected in:

[0028] 1. Improve treatment efficiency: The present invention uses exosomes as carriers of mRNA, achieving targeted delivery and cellular uptake of mRNA, thereby improving the generation efficiency and targeting of CAR-T cells.

[0029] 2. Reduce immunogenicity: Exosomes have low immunogenicity, which reduces the occurrence of immune rejection reactions and improves the safety of treatment.

[0030] 3. Reduce side effects: The in situ generation of CAR-T cells in vivo avoids the risk of cell damage and infection during in vitro transformation and reinfusion, reducing the occurrence of side effects.

[0031] 4. Broaden the scope of application: The exosomes prepared by the present invention are not only suitable for the treatment of atherosclerosis, but can also provide new ideas and methods for the treatment of other non-cancerous diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0033] Figure 1 is the structure diagram of FAP-CAR-Ms2bs plasmid;

[0034] Figure 2 is the structure diagram of LAMP-2B-MS2 plasmid;

[0035] Figure 3 is the transfection efficiency of exosomes in T cells in different treatment groups in Example 2;

[0036] Figure 4 The results show the killing rate of exosomes on fibroblasts in different treatment groups in Example 3. DETAILED DESCRIPTION

[0037] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0038] Example 1

[0039] A method for preparing exosomes loaded with FAP CAR mRNA, comprising the following steps:

[0040] 1. Design, optimize, and synthesize an mRNA sequence expressing the FAP CAR structure. The nucleotide sequence of the optimized mRNA sequence expressing the FAP CAR structure is shown in SEQ ID NO. 1.

[0041] 2. Synthesize FAP-CAR-MS2bs plasmid and LAMP-2B-MS2 plasmid. The specific method is as follows:

[0042] According to the principle of seamless cloning, recombinant sequences homologous to the plasmid vector were added before and after the target gene (FAP CAR mRNA), and the FAP-CAR-Ms2bs target gene fragment (SEQ ID NO. 2) was chemically synthesized. The synthesized PCR fragment was recovered by agarose gel excision.

[0043] Similarly, according to the principle of seamless cloning, recombinant sequences homologous to the plasmid vector were added before and after the LAMP-2B gene, and the LAMP-2B-MS2 target gene fragment (SEQ ID NO. 4) was chemically synthesized. The synthesized PCR fragment was recovered by agarose gel excision.

[0044] Afterwards, the pcDNA3.1-EGFP plasmid vector was double-digested with MluI / XhoI. The enzyme digestion system is shown in Table 1. The digestion product was recovered after 0.5 h at 37°C. The digested vector was then recovered by cutting on a 1.5% agarose gel.

[0045] The target gene fragment was recombined into the pcDNA3.1-EGFP restriction enzyme-digested vector. The recombination system was shown in Table 2. After mixing, the recombination reaction was carried out at 37°C for 30 minutes. After the reaction, the recombination reaction solution was immediately placed in an ice bath for 5 minutes.

[0046] 100 μL of E. coli Top10 competent cells were added to the recombinant product, mixed well, heat-shocked at 42°C for 60 seconds, and then placed in an ice-water bath for 120 seconds. The transformed mixture was evenly spread on an LB plate containing ampicillin resistance and cultured at 37°C overnight. Single clones were picked for PCR identification reaction, and positive clones were sent to Beijing Qingke Company for sequencing to obtain the FAP-CAR-Ms2bs plasmid (nucleotide sequence shown in SEQ ID NO.3, structure shown in Figure 1 shown) and LAMP-2B-MS2 plasmid ( Figure 2 ).

[0047] Table 1 Enzyme digestion system

[0048] Element Dosage pcDNA3.1-EGFP 10 μL MluI 2μL XOt 2μL CutSmartBuffer 10 μL <![CDATA[ddH2O]]> 76μL total 100 μL

[0049] Table 2 Recombination system

[0050] Element Dosage pcDNA3.1-EGFP digestion product 1 μL Target gene fragment 1 μL ExnaseII 2μL 5×CEIIBuffer 4 μL <![CDATA[ddH2O]]> 12 μL total 20 μL

[0051] 3. Transfect the synthesized FAP-CAR-MS2bs plasmid and LAMP-2B-MS2 plasmid into 293T cells. The specific method is as follows:

[0052] a. Prepare DNA dilution buffer by adding 1500 μL of Opti-MEM I ReLipced Serum Medium and 24 μg of DNA to an Eppendorf tube and gently mixing.

[0053] b. Add 1500 μL of Opti-MEMI RelLipced Serum Medium and 60 μL of Transfection Reagent (mix before use) to another eppendorf tube, mix gently to make Transfection Reagent dilution solution, and let it stand at room temperature for 5 minutes.

[0054] c. Combine the DNA diluent and Transfection Reagent diluent, mix gently, and let stand at room temperature for 20 minutes to form a DNA-Transfection Reagent complex. The DNA-Transfection Reagent complex is stable at room temperature for 6 hours.

[0055] d. Add the DNA-Transfection Reagent complex to the seeded cells and gently rock the 10 cm culture plate back and forth to evenly distribute the complex. Culture in a 37°C, CO2 incubator for 5 hours, then replace the medium and continue culturing for 36 hours to obtain 293T cells expressing the FAP-CAR-MS2bs plasmid.

[0056] 4. Isolate exosomes loaded with CAR mRNA from 293T cells expressing FAP-CAR-MS2bs plasmid and LAMP-2B-MS2 plasmid. The specific method is as follows:

[0057] (1) Centrifuge the culture medium of 293T cells at 4°C and 2000g for 30 minutes and collect the supernatant.

[0058] (2) Centrifuge the supernatant at 4°C and 12,000 g for 30 min and collect the supernatant.

[0059] (3) Repeat step (2) until no obvious precipitation occurs.

[0060] (4) Filter the supernatant using a 0.22 μm filter membrane into a 50 mL centrifuge tube to obtain a cell supernatant sample.

[0061] (5) Exosomes were extracted using the Exodus H600 fully automatic exosome extraction system. The buffer used was PBS. After the extraction was completed, the chip was removed and PBS was blown onto the membranes on both sides of the chip about 30 times each and then aspirated into a 1.5 mL centrifuge tube to obtain exosomes loaded with FAP CAR mRNA, which were then stored at -80°C.

[0062] Example 2 Transfection efficiency of exosomes loaded with FAP CAR mRNA on T cells

[0063] 1. Isolate and collect mouse peripheral blood mononuclear cells (PBMC). The specific method is:

[0064] (1) Centrifuge 20 mL of EDTA-anticoagulated fresh peripheral blood sample at 2400 rpm for 10 min, aspirate the upper plasma, and aliquot and freeze.

[0065] (2) The plasma obtained in step (1) was mixed with an equal volume of PBS and mixed by inverting the tube to obtain diluted plasma.

[0066] (3) Add 6 mL of Ficoll separation solution to a centrifuge tube (SepMate tube), use a pipette to draw up the diluted plasma and gently spread it on the Ficoll separation solution (keeping a clear boundary between the two liquid surfaces), with 5 mL of diluted plasma spread on each SepMate tube.

[0067] (4) Centrifuge the SepMate tube from step (3) at 22°C and 1800 g for 12 min, discard the upper plasma layer, pour the middle buffy coat layer into a new centrifuge tube, add PBS to 11-12 mL, centrifuge at 2400 rpm for 10 min, remove the residual Ficoll separation solution, and resuspend the cells in PBS to obtain PBMCs.

[0068] (5) The collected mouse PBMC cells were used to activate T cells using CD3 / CD28 magnetic beads.

[0069] 2. The exosomes loaded with FAP CAR mRNA obtained in Example 1 (experimental group, pLAMP + pFAP CAR group) were co-incubated with the activated mouse T cells described above, and exosomes not loaded with FAP CAR mRNA were set as blank controls (pLAMP + pGFP group). Flow cytometry was used to observe and count the uptake rates of exosomes by T cells in different treatment groups. The results are shown in Tables 3 and Figure 3 As shown in the figure, compared with the blank control group, the transfection efficiency of the experimental group was significantly improved, indicating that the exosomes carrying FAP CAR mRNA successfully improved the transfection efficiency of FAP CAR in mouse T cells.

[0070] Table 3 T cell uptake rate of exosomes in different treatment groups

[0071] Treatment group Uptake rate% Experimental group 17±1.54 Blank control group 2.54±0.26

[0072] Example 3

[0073] The exosomes loaded with FAP CAR mRNA and the exosomes not loaded with FAP CAR mRNA obtained in Example 1 were injected into mice respectively. The exosomes delivered FAP CAR mRNA into T cells by fusion with T cells or endocytosis, and the modified CAR-T cells were isolated and collected.

[0074] The modified CAR-T cells were co-cultured with fibroblasts (purchased from Zhejiang Meisen Cell Technology Co., Ltd. and labeled with CFSE dye) in 96-well plates at different effector-target ratios (1:1, 5:1, and 10:1), with three replicate wells, and cultured for 24 hours.

[0075] The number of surviving fibroblasts was observed by real-time imaging, and the killing rate of fibroblasts was calculated.

[0076]

[0077] The results are as follows Figure 4 As shown in Table 4, it can be seen that the killing efficiency of the experimental group (exosomes loaded with FAP CAR mRNA) at each effector-target ratio was significantly higher than that of the control group (exosomes not loaded with FAP CAR mRNA), and as the proportion of effective cells increased, that is, the proportion of CAR-T cells incubated with exosomes increased, the killing efficiency was significantly enhanced.

[0078] Table 4 Fibroblast killing rate results

[0079] Treatment group 1：1 5：1 10：1 Experimental group 22.3％ 44.2％ 73.6％ control group 0.43％ 1.41％ 3.69％

[0080] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An mRNA expressing a FAP CAR structure, characterized in that: The nucleotide sequence of the mRNA sequence is shown as SEQ ID NO.

1.

2. An exosome loaded with the mRNA expressing the FAP CAR structure according to claim 1.

3. A method for preparing exosomes according to claim 2, characterized in that: The steps include: (1) Adding a recombinant sequence homologous to the plasmid vector before and after the mRNA sequence described in claim 1 to obtain the FAP-CAR-Ms2bs target gene fragment; (2) adding recombinant sequences homologous to the plasmid vector before and after the LAMP-2B gene to obtain the LAMP-2B-MS2 target gene fragment; the nucleotide sequence of the LAMP-2B-MS2 target gene fragment is shown in SEQ ID NO.4; (3) The FAP-CAR-Ms2bs target gene fragment and the LAMP-2B-MS2 target gene fragment were recombined with the enzyme-digested plasmid vector to obtain the FAP-CAR-Ms2bs plasmid and the LAMP-2B-MS2 plasmid; (4) co-transfecting the FAP-CAR-Ms2bs plasmid and the LAMP-2B-MS2 plasmid into exosome-secreting cells to obtain exosome-secreting cells expressing FAP CAR mRNA; (5) Exosomes were isolated from exosome-secreting cells expressing FAP CAR mRNA to obtain exosomes loaded with FAP CAR mRNA.

4. The method according to claim 3, wherein The nucleotide sequence of the FAP-CAR-Ms2bs target gene fragment in step (1) is shown in SEQ ID NO.2, and the plasmid vector is a pcDNA3.1-EGFP plasmid vector.

5. The method according to claim 3, wherein The enzymes used in the enzymatic digestion in step (3) are MluI enzyme and XhoI enzyme.

6. The method according to claim 3, wherein The exosome-secreting cells in step (4) are 293T cells.

7. The method according to claim 3, wherein The extraction system used for isolating exosomes in step (5) is the Exodus H600 fully automatic exosome extraction system.

8. Use of the mRNA expressing the FAP CAR structure according to claim 1, the exosomes according to claim 2, or the exosomes prepared according to the method of any one of claims 3 to 7 in the preparation of a medicament for treating atherosclerosis and / or cardiac fibrosis.

9. The use according to claim 8, characterized in that The atherosclerosis is caused by abnormal proliferation and activation of fibroblasts.

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