TNFR1 modified mesenchymal stem cell as well as preparation method and application thereof

Abstract

According to the invention, by designing and optimizing a TNFR1mRNA sequence, rare codons are avoided in a coding region in an mRNA element; in the aspect of selection of the UTR, 5 '-UTR with the length smaller than 100 bp is adopted to avoid the risk of mRNA degradation; meanwhile, 3 '-UTR of globin and actin is used for modifying mRNA so as to further improve the stability of the mRNA. The optimized sequence is cloned into plasmids, high-stability TNFR1mRNA is obtained through in-vitro transcription and purification, the TNFR1mRNA is mixed with a certain proportion of lipidosome to be used for transfecting mesenchymal stem cells, continuous 72-hour stable high expression of the mesenchymal stem cells TNFR1 is achieved, meanwhile, good cell viability is kept, and the TNFR1mRNA is used for treating an inflammatory bowel disease animal model and has good application prospects. The result shows that compared with unmodified mesenchymal stem cells, the TNFR1 modified mesenchymal stem cells have a remarkable treatment effect, and the TNFR1 modified mesenchymal stem cells have a good application prospect in inflammatory diseases.

Description

Technical Field

[0001] The present invention belongs to the field of cell therapy, and in particular relates to TNFR1-modified mesenchymal stem cells and a preparation method and application thereof. Background Art

[0002] Mesenchymal stem cells (MSC) are pluripotent stem cells derived from the early mesoderm of development with high self-renewal ability and multidirectional differentiation potential, and are widely present in various tissues throughout the body. It not only supports the growth of hematopoietic stem cells, but also can differentiate into various tissue cells such as neurons, osteoblasts, chondrocytes, adipocytes, cardiomyocytes, etc. in vitro under different induction conditions. MSC is easy to culture in vitro, has low immunogenicity, and has immunomodulatory effects. It is currently used to treat a variety of autoimmune diseases. In addition, MSC has the function of promoting tissue regeneration and is used to treat diseases such as liver fibrosis, multiple organ failure, and diabetic foot. The results of clinical experimental studies have shown that stem cells can effectively relieve inflammatory bowel disease in the long term, and are gradually being recognized by clinical guidelines.

[0003] Primary unmodified mesenchymal stem cells are heterogeneous, and the infused cells have different subpopulations (and the proportions of each subpopulation are not stable), which leads to unstable functions and inability to fully exert therapeutic effects; in addition, the biological functions of the original unmodified MSCs for a certain disease are not strong enough, and they cannot continuously express biological functional molecules, and the expression level is low, which also leads to differences in therapeutic effects. Modified MSCs can transfer disease-related proteins and factors into MSCs, allowing MSCs to continuously express at high levels, thereby domesticating MSCs and making them uniform, stable, and biologically functional MSC drugs, improving the therapeutic effects of diseases.

[0004] At present, most of the vectors used for mesenchymal stem cell modification are viral vectors, including lentivirus, adenovirus, etc. Although viral gene delivery is highly efficient, viral vectors will randomly integrate the target gene into the host genome, which may interrupt important gene expression and cellular processes; the production of viral vectors usually includes the generation and certification of the original cell bank of the stable production system, which results in high costs in gene and cell therapy. To make up for the shortcomings of viral vectors, non-viral delivery systems have received increasing attention due to their low toxicity, potential for targeted delivery, long-term stability, high DNA loading capacity, controllable chemical structure, low immunogenicity, easy mass preparation and relatively low production cost. With the approval of two mRNA vaccines for vaccination against the new coronavirus, the transfection and modification of mRNA for cells has gradually become possible. Since the modification of cells by mRNA does not need to enter the cell nucleus, the risk of integration into the host genomic DNA is avoided. At the same time, before transfecting mRNA, the structural elements of mRNA are modified to improve its stability and avoid degradation; therefore, choosing mRNA to modify mesenchymal stem cells is a feasible option.

[0005] Inflammatory bowel disease (IBD), mainly including ulcerative colitis (UC) and Crohn's disease (CD), is a chronic, nonspecific, recurrent, inflammatory disease that mainly affects the gastrointestinal tract. IBD was rare in China in the past, but in the past 20 years, the incidence of IBD in China has increased rapidly, with an incidence of 1.74 / 100,000 person-years, of which UC has an incidence of 1.18 / 100,000 person-years and CD has an incidence of 0.4 / 100,000 person-years. At present, IBD has become one of the common and difficult diseases of the digestive system in my country, and is the focus, hot spot and difficulty of basic research and clinical diagnosis and treatment of digestive system diseases. IBD often starts in adolescents and is recurrent, progressive and disabling, seriously affecting the patient's growth and development, marriage and childbearing, as well as study, work and daily life. IBD not only affects the digestive tract, but also almost all organs and systems outside the digestive tract. It can also cause complications such as infection and tumors inside and outside the digestive tract. It is a systemic disease involving multiple clinical disciplines.

[0006] In the past 20 years, although domestic and foreign scholars have conducted in-depth research on the pathogenesis and clinical diagnosis and treatment of IBD, most scholars currently believe that the onset of IBD is caused by infection, diet, environment, mental and psychological factors acting on genetically susceptible people. With the participation of intestinal flora, the intestinal immune and non-immune systems are activated, making the intestinal mucosa highly sensitive to antigens and the immune regulation function disordered, which ultimately leads to chronic inflammation of intestinal mucosal cells and persistent damage to tissue cells that is difficult to self-limit. In this process, the interaction between cytokines and inflammatory mediators plays an important role. The immune factors involved in IBD include T cells, cytokines, and autoantibodies.

[0007] There is currently no cure for IBD. Summary of the invention

[0008] In view of the above-mentioned technical problems existing in the prior art, the present invention, based on the treatment of IBD with biomacromolecule preparations such as monoclonal antibodies, modifies MSCs with the mRNA of TNFR1 (tumor necrosis factor receptor 1) using a cationic liposome delivery system targeting the pathogenesis of IBD, thereby reducing the cost of gene-modified MSCs and improving the immunoregulatory ability of MSCs. By developing mRNA-modified MSC drugs, the problem of loss of response after monoclonal antibody treatment can be solved, the response efficiency of IBD treatment can be improved, and clinical needs can be met.

[0009] The technical solution of the present invention is as follows:

[0010] The first object of the present invention is to provide a modified TNFR1 sequence, wherein the modified TNFR1 sequence is the nucleotide sequence shown in SEQ ID NO.1, or the complementary sequence of the nucleotide sequence shown in SEQ ID NO.1:

[0011] taatacgactcactatagggagatcagagagaaaagaagagtaagaagaaatataagagccaccgccaccatggggctaagcactgtcccggatctcctgctccccctggtactgctcgagctcctggtggggatctacccatcaggagttatcgggctagtaccacacctgggggaccgggagaaacgggacagtgtttgcccccagggcaagtatatccatccccagaacaactcgatttgttgtacaaagtgccacaaaggcacttatttgtacaacgactgcccgggccccgggcaggacactgattgcagggagtgcgagtcgggctcgttcaccgcgtcggagaaccacctgcgccattgtctttcctgcagcaaatgcaggaaagagatggggcaggtggagatctcctcctgcaccgtagaccgggacacggtctgcgggtgcaggaagaaccagtacaggcactactggagtgagaacctgtttcagtgttttaactgcagtttgtgcctgaacgggacagtgcacctgtcgtgtcaggagaagcagaacactgtttgtacctgtcacgcagggttcttcctgcgcgagaacgagtgcgtctcgtgctccaactgcaagaagtcgctggagtgcacaaagttgtgcctcccgcaaatcgagaatgttaaggggacggaggatagtggcaccacttgataataggctgccttctgcggggcttgccttctggccatgcccttcttctctcccttgcacctgtacctcttggtctttgaataaagcctgagtaggaaggcggccgctcgagcatgcatctagaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaatgcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa(SEQ ID NO.1)。

[0012] Furthermore, the modified TNFR1 sequence is obtained by the following modification:

[0013] The nucleotide sequence of TNFR1 before modification (NCBI Reference Sequence: NM_001065.4) is:

[0014]

[0015] The 5'-untranslated region, coding region, and 3'-untranslated region of the TNFR1 sequence shown in SEQ ID NO.2 were modified respectively to construct a modified 5'-untranslated region (UTR)-modified TNFR1 mRNA sequence coding region (i.e. Figure 1 TNFR1 (CDS)-modified 3'-untranslated region (UTR) and added with a poly-A tail in the 3'-untranslated region (UTR);

[0016] The nucleotide sequence of the modified TNFR1 mRNA sequence coding region is shown in SEQ ID NO.3;

[0017] Specifically, the coding region is modified by optimizing the gene sequence by designing the coding region shown at 263bp to 895bp of SEQ ID NO.2 without changing its amino acid sequence through mammalian cell codon preference. The specific operation method can be optimized online at https: / / www.vectorbuilder.cn to select the sequence with the highest codon adaptation index (CAI). The optimized coding region sequence is:

[0018] (SEQ ID NO.3);

[0019] The modified 5'-untranslated region nucleotide sequence is shown in SEQ ID NO.4;

[0020] The 5'-untranslated region is modified by replacing the 5'-untranslated region shown at 1 bp to 262 bp of SEQ ID NO.2 with a nucleotide sequence shorter than 100 bp to avoid the risk of mRNA degradation; in a certain embodiment, the nucleotide sequence shorter than 100 bp is taatacgactcactatagggagatcagagagaaaagaagagtaagaagaaatataagagccaccgccacc (SEQ ID NO.4); the modified 3'-untranslated region nucleotide sequence is shown in SEQ ID NO.5;

[0021] The 3'-untranslated region modification method is to select globin as the 3'-untranslated region shown at 1631 bp to 2171 bp of SEQ ID NO.2, and the sequence is:

[0022] gctgccttctgcggggcttgccttctggccatgcccttcttctctcccttgcacctgtacctcttggtctttgaataaagcctgagtaggaaggc ggccgctcgagcatgcatctaga (SEQ ID NO. 5).

[0023] The sequence to add a poly-A tail at the end is:

[0024] aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaatgcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa (SEQ ID NO. 6).

[0025] The second object of the present invention is to provide a modified TNFR1 mRNA sequence, which is obtained by transcribing the modified TNFR1 sequence and adding a cap at the 5' end.

[0026] Furthermore, the modified TNFR1 mRNA sequence is shown in SEQ ID NO.7:

[0027] uaauacgacucacuauagggagaucagagagaaaagaagaguaagaagaaauauaagagccaccgccaccauggggcuaagcacugucccggaucuccugcucccccugguacugcucgagcuccugguggggaucuacccaucaggaguuaucgggcuaguaccacaccugggggaccgggagaaacgggacaguguuugcccccagggcaaguauauccauccccagaacaacucgauuuguuguacaaagugccacaaaggcacuuauuuguacaacgacugcccgggccccgggcaggacacugauugcagggagugcgagucgggcucguucaccgcgucggagaaccaccugcgccauugucuuuccugcagcaaaugcaggaaagagauggggcagguggagaucuccuccugcaccguagaccgggacacggucugcgggugcaggaagaaccaguacaggcacuacuggagugagaaccuguuucaguguuuuaacugcaguuugugccugaacgggacagugcaccugucgugucaggagaagcagaacacuguuuguaccugucacgcaggguucuuccugcgcgagaacgagugcgucucgugcuccaacugcaagaagucgcuggagugcacaaaguugugccucccgcaaaucgagaauguuaaggggacggaggauaguggcaccacuugauaauaggcugccuucugcggggcuugccuucuggccaugcccuucuucucucccuugcaccuguaccucuuggucuuugaauaaagccugaguaggaaggcggccgcucgagcaugcaucuagaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaugcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa(SEQ ID NO.7)。

[0028] The third object of the present invention is to provide a method for constructing the modified TNFR1 mRNA sequence, which comprises transcribing the modified TNFR1 sequence to obtain the mRNA sequence shown in SEQ ID NO.7 and then capping the 5' end to obtain the modified TNFR1 mRNA sequence.

[0029] Furthermore, the method comprises the following steps:

[0030] 1) Plasmid linearization: Take 1-10 μg of pUC57-Kan plasmid containing the modified TNFR1 sequence, the plasmid vector is pUC57-Kan, add 1-5 μl of EcoRI endonuclease, 5-25 μl of EcoRI endonuclease buffer and double distilled water to a final system of 50-200 μl, and incubate at 37°C for 30 min-3 h;

[0031] 2) Purification of linearized plasmid: Use FastPure Gel DNA Extraction Mini Kit to purify the linearized plasmid. The purified product is subjected to gel electrophoresis to determine the size and integrity of the product and then used for subsequent in vitro transcription experiments;

[0032] Use FastPure Gel DNA Extraction Mini Kit to purify the linearized plasmid. Take 50μl linearization system as an example. Add 50μl RNase-free water to each system, then add 500μl Buffer GDP, mix well and transfer to the adsorption column. Centrifuge and discard the filtrate. Then add eluent to collect the eluate, which is the purified linear plasmid. Finally, use gel electrophoresis to determine the size and integrity of the product before using it in subsequent in vitro transcription experiments.

[0033] 3) In vitro transcription and capping: Use the HiSynth T7 Co-transcription RNA Synthesis Kit to perform in vitro capping and co-transcription, take 1-5 μg of linear plasmid and add it to an appropriate amount of nuclease-free water, then add 2-10 μl of 10× reaction buffer, 2-10 μl of 2×NTP / cap analog and 1-5 μl of T7 RNA Polymerase in sequence, mix well and incubate at 37°C for 2-3 hours to obtain the capped mRNA shown in SEQ ID NO.7;

[0034] 4) mRNA purification: use RNA Cleanup Kit was used for purification. 200 μl of the concentrated binding solution in the kit was added to every 100 μl, and then 300 μl of anhydrous ethanol was added to mix well. The mixture was centrifuged at high speed and the filtrate was discarded. After washing twice with 0.1-1 ml of washing solution and centrifuged at high speed, the capped mRNA was dissolved with 1-100 μl of elution solution and eluted by high-speed centrifugation, and the filtrate was collected.

[0035] 5) Final purification of mRNA: Add 0.1-1 ml of clearing binding buffer, mix well, then add 0.1-1 ml of anhydrous ethanol and mix well, centrifuge at high speed to pass through the column and discard the filtrate; wash twice with 0.1-1 ml of washing solution by high-speed centrifugation, add 10-50 μl of preheated nuclease-free water to the center of the central filter membrane of the filter column to dissolve the capped mRNA and elute by high-speed centrifugation, collect the filtrate to complete the preparation of TNFR1 mRNA with high translation stability.

[0036] The third object of the present invention is to provide an mRNA liposome complex, which uses liposomes to encapsulate the aforementioned modified TNFR1 mRNA sequence, and the molar ratio of liposomes to the modified TNFR1 mRNA sequence is 1:3-1:6. The liposomes include ionizable cationic lipids, cholesterol (Chol), neutral auxiliary phospholipids, and PEG lipids. The ionizable cationic lipids are Dlin-MC3-DMA or DOTAP, the neutral auxiliary phospholipids are DSPC or DOPE, and the PEG lipids are DSPE-PEG2000.

[0037] Furthermore, the molar ratio of the ionizable cationic lipid, cholesterol (Chol), neutral auxiliary phospholipid and PEG lipid in the mRNA liposome complex is preferably one of 40:40:10:5, 40:40:10:10 and 40:40:10:15.

[0038] The fourth object of the present invention is to provide TNFR1-modified mesenchymal stem cells, wherein the TNFR1-modified mesenchymal stem cells are transfected with the aforementioned mRNA liposome complex.

[0039] The fifth object of the present invention is to provide the use of the aforementioned modified TNFR1 sequence or the aforementioned modified TNFR1 mRNA sequence or the aforementioned mRNA liposome complex or the aforementioned TNFR1 modified mesenchymal stem cells in the preparation of a drug for treating inflammatory bowel disease.

[0040] The beneficial effects of the present invention are

[0041] 1) The present invention designs and optimizes the TNFR1 mRNA structure to obtain an optimized stable TNFR1 mRNA sequence;

[0042] 2) The present invention establishes an mRNA-liposome complex that can efficiently transfect mesenchymal stem cells.

[0043] 3) Transfecting mRNA into MSC to obtain empowered stem cells: The mRNA liposome complex of the present invention can safely and efficiently empower MSC, achieving a balance between cell activity and enhanced function. Specifically, through mRNA transfection, mRNA does not need to enter the cell nucleus, avoiding the safety risks brought by integration into the host genomic DNA; the optimized mRNA and cationic liposome complex delivery system achieves efficient mRNA transfection, and TNFR1 is stably expressed at a high level in MSC while maintaining cell viability.

[0044] 4) Conduct animal research on mesenchymal stem cells modified with mRNA genes: establish an IBD disease animal model and complete animal efficacy trials of TNFR1-modified mesenchymal stem cells for the treatment of IBD.

[0045] TNFR1 mRNA-modified MSCs were used in DSS (dextran sulfate sodium)-modeled IBD mice. Compared with the model group and unmodified MSCs, the modified MSCs showed significant differences in individual body weight changes, disease activity index, and colon length, initially showing good therapeutic effects and laying a solid foundation for the treatment of other immune diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Modified mRNA elements;

[0047] Figure 2 Preparation and application process of mRNA-modified mesenchymal stem cells;

[0048] Figure 3 TNFR1 expression at different times after mRNA transfection;

[0049] Figure 4 Disease activity index of mice in different groups (#P<0.05);

[0050] Figure 5 Changes in individual body weight of mice in different groups (#P<0.05);

[0051] Figure 6 Changes in colon length among different groups (#P<0.05). DETAILED DESCRIPTION

[0052] The present invention is further explained below with reference to the examples, but the examples do not limit the present invention in any form.

[0053] Example 1 Obtaining Modified TNFR1 mRNA Sequence

[0054] The modified TNFR1 sequence is shown as SEQ ID NO.1, or is the complementary sequence of the sequence shown as SEQ ID NO.1.

[0055] The modified TNFR1 sequence can be artificially synthesized or obtained by the following modification methods:

[0056] The nucleotide sequence of TNFR1 (NCBI Reference Sequence: NM_001065.4) before modification is shown in SEQ ID NO.2.

[0057] The 5'-untranslated region, coding region, and 3'-untranslated region of the TNFR1 mRNA sequence shown in SEQ ID NO.2 were modified respectively to construct a modified 5'-untranslated region (UTR)-modified TNFR1 mRNA sequence coding region (i.e. Figure 1 TNFR1 (CDS)-modified 3'-untranslated region (UTR) and added with a poly-A tail in the 3'-untranslated region (UTR);

[0058] Specifically, the coding region is modified by optimizing the gene sequence by designing the coding region shown at 263 to 895 bp of SEQ ID NO.2 without changing its amino acid sequence through mammalian cell codon preference. The specific operation method can be optimized online at https: / / www.vectorbuilder.cn to select the sequence with the highest codon adaptation index (CAI). The optimized coding region sequence is shown in SEQ ID NO.3.

[0059] The 5'-untranslated region is modified by replacing the 5'-untranslated region shown at bp 1 to 262 of SEQ ID NO.2 with a nucleotide sequence shorter than 100 bp to avoid the risk of mRNA degradation; in a certain embodiment, the nucleotide sequence shorter than 100 bp is shown in SEQ ID NO.4.

[0060] The 3'-untranslated region is modified by selecting globin as the 3'-untranslated region shown at 1631 to 2171 bp of SEQ ID NO.2, and the sequence is shown in SEQ ID NO.5.

[0061] The sequence of the poly-A tail is shown in SEQ ID NO.6.

[0062] Finally, the modified TNFR1 sequence shown in SEQ ID NO.1 was obtained.

[0063] The modified TNFR1 mRNA sequence is obtained by transcribing the modified TNFR1 sequence (ie, the sequence shown in SEQ ID NO.1 or its complementary sequence) and adding a cap at the 5' end. The modified TNFR1 mRNA sequence is shown in SEQ ID NO.7.

[0064] The modified TNFR1 mRNA sequence is prepared by the following method:

[0065] 1) Plasmid linearization: Take 5 μg of pUC57-Kan plasmid containing the modified TNFR1 sequence (as shown in SEQ ID NO.1), add 3 μl of EcoRI endonuclease, 15 μl of EcoRI endonuclease buffer and double distilled water to a final volume of 300 μl, and incubate at 37°C for 1 h;

[0066] The pUC57-Kan plasmid containing the modified TNFR1 sequence is constructed by inserting the modified TNFR1 sequence between the HindIII and EcoRI restriction sites of the pUC57-Kan plasmid.

[0067] 2) Purification of linearized plasmid: Use FastPure Gel DNA Extraction Mini Kit to purify the linearized plasmid. Take 50μl linearization system as an example, add 50μl RNase-free water to each system, then add 500μl Buffer GDP, mix well and transfer to the adsorption column, centrifuge and discard the filtrate, then add eluent to collect the eluate, which is the purified linear plasmid. Finally, use gel electrophoresis to determine the size and integrity of the product before using it in subsequent in vitro transcription experiments;

[0068] 3) In vitro transcription and capping: Use the HiSynth T7 Co-transcription RNA Synthesis Kit to perform in vitro capping and co-transcription, take 5 μg of linear plasmid and add it to an appropriate amount of nuclease-free water, then add 10 μl of 10× reaction buffer, 10 μl of 2×NTP / cap analog and 5 μl of T7 RNA Polymerase in sequence, mix well and incubate at 37°C for 3 hours to obtain the capped mRNA shown in SEQ ID NO.7;

[0069] 4) mRNA purification: use RNA Cleanup Kit was used for purification. 200 μl of the concentrated binding buffer (Cleanup Binding Buffer) in the kit was added to every 100 μl, and then 0.5 ml of anhydrous ethanol was added to mix well. The mixture was centrifuged at high speed and the filtrate was discarded. After washing twice with 1 ml of washing buffer and centrifuged at high speed, the capped mRNA was dissolved with 100 μl of elution buffer and eluted by high-speed centrifugation, and the filtrate was collected.

[0070] 5) Final purification of mRNA: Add 0.5 ml of clearing binding buffer, mix well, then add 0.5 ml of anhydrous ethanol and mix well, centrifuge at high speed to pass through the column and discard the filtrate; after washing twice with 1 ml of washing solution by high-speed centrifugation, add 30 μl of preheated nuclease-free water to the center of the central filter membrane of the filter column to dissolve the capped / tailed mRNA and elute by high-speed centrifugation, collect the filtrate to complete the preparation of TNFR1 mRNA with high translation stability.

[0071] Example 2 Cell culture and transfection

[0072] (1) MSC subculture

[0073] Human umbilical cord MSCs were 1×10 4 / cm 2 The cell culture flask was inoculated at a density of 1.50 μg / cm2 in a culture flask. The cell culture flask was placed in an incubator at 37°C and 5% CO2 saturated humidity, and the medium was changed every three to four days. Subculture was performed one day before transfection of MSCs.

[0074] (2) Transfect MSCs using mRNA transfection kit

[0075] 18-24 hours before transfection, 0.8-3.0×10 4 cells / cm 2 Cells should be passaged at a density of 1:1 to ensure that the cells reach an appropriate cell density (usually ≥80% confluence) before transfection.

[0076] Preparation of liposomes: The liposomes are composed of ionizable cationic lipid Dlin-MC3-DMA, cholesterol (Chol), neutral auxiliary phospholipid DSPC, and PEG lipid DSPE-PEG2000, and the molar ratio between the four is 40:40:10:5.

[0077] The corresponding lipid components were mixed in anhydrous ethanol as an organic phase to a total lipid concentration of 10 mM, and the mRNA shown in SEQ ID NO.7 was diluted to 150 μg / ml with a pH = 4.5 acetic acid-sodium acetate buffer as an aqueous phase; the aqueous phase and the organic phase were mixed and emulsified using a microfluidic preparation system to obtain liposome particles encapsulating mRNA. The molar ratio of liposome to modified TNFR1 mRNA was 1:3.

[0078] Then dialyze with RNase-free 1x PBS buffer at 4°C for 18 hours; then concentrate with a 10KD ultrafiltration tube, and finally filter and sterilize the LNP with a 0.22μm membrane, and then add about 100μL of it into a T75 culture flask where cells are cultured. After 6 hours of culture, the mRNA liposome complex is obtained for subsequent sample preparation.

[0079] (3) Preparation of TNFR1-modified MSCs

[0080] After 6 hours of transfection, the cells were free of culture medium and washed twice with PBS. 5 ml of digestion solution was added to digest for 5 min, and then 5 ml of stop solution was added to stop digestion. The cells were made into a cell suspension by pipetting and collected into a centrifuge tube. The cells were centrifuged at 270 g for 5 min. The supernatant was discarded and the cells were washed several times with resuspension solution. The mRNA liposome complex prepared in step (2) was added according to the number of mesenchymal cells. After transfection, the MSC-TNFR1 preparation was obtained.

[0081] Example 3 Analysis of TNFR1 mRNA transfection efficiency in MSCs

[0082] In the example of transfection efficiency detection, TNFR1 mRNA was synthesized by the preparation method of the present invention, and MSCs were transfected as in Example 2 (2). After 6 hours of transfection, the preparation of MSC-TNFR1 preparation was completed. The expression level of TNFR1 was significantly higher than that of untransfected MSCs at different times (0-96 hours) after the preparation was prepared. Figure 3 shown.

[0083] This example studies the difference in the TNFR1 expression ability of the MSC samples prepared according to the method of Example 2 96 hours after preparation compared with the non-transfected control group. The specific method is as follows:

[0084] a After transfection, discard the culture medium of the cells, wash twice with PBS, add 5 ml of digestion solution for 5 min, then add 5 ml of stop solution to stop digestion, pipette to make cell suspension and collect into centrifuge tube, centrifuge at 270g for 5 min, discard the supernatant and add resuspension solution for washing several times, add appropriate amount of auxiliary material solution according to the number of cells, and the MSC-TNFR1 preparation is prepared.

[0085] The MSC-TNFR1 preparation obtained in step (3) of Example 2 was sampled every 24 hours, and the cell viability of MSC in the preparation was detected by AO / PI staining. The obtained samples were rotated at 270g for 5 minutes, and the cell precipitate was discarded. The supernatant was used to detect the content of TNFR1 in the preparation by TNFR1 ELISA kit. The standard was set to 8 concentration gradients, and the test sample was taken out. 1) HD1-7 solution was added at 50 μl / well, 2) standard and test solution were added at 200 μl / well, and duplicate wells were made. At the same time, blank control (diluent) was added to 2 wells, and the plate was sealed and incubated at room temperature for 2 hours. 3) Human TNFR1 conjugate was added at 200 μl / well, and a new sealing film was covered. After incubation at room temperature for 2 hours, the liquid was shaken off. 4) 1× washing buffer was added at 400 μl / well and washed 3 times, and each time it was left for 30 seconds and then shaken off. 5) Substrate solution was added at 200 μl / well, and incubated at room temperature in the dark for 20 minutes. 6) Stop solution was added at 50 μl / well, and the plate was gently tapped to mix the liquid. The absorbance was measured at a wavelength of 450 nm using an ELISA reader, and the concentration was calculated based on the fitted standard curve.

[0086] The results showed that the expression level of TNFR1 in the transfected group was still at a high level 96 hours after transfection, reaching a maximum of about 3500pg / ml. The concentration of TNFR1 at different time points (24h, 48h, 96h) was greater than 1500pg / ml, while no obvious expression was observed in the control group (untransfected mesenchymal stem cells MSC). Figure 3 )

[0087] Example 4 Animal Model Study

[0088] 1) Establishment of an inflammatory bowel disease animal model: Except for the blank group, the mice in other groups were allowed to drink freely by adding 3.5% dextran sulfate sodium (DSS) in drinking water to establish an inflammatory bowel disease (IBD) model;

[0089] 2) Grouping: The day when mice drank DSS was recorded as Day 0. On Day 2, they were randomly divided into model group ip (intraperitoneal injection) and control process ip group (1×10 6 cells / cell), transfected MSC ip group (MSC-TNFR1 preparation, 1×10 6 cells / ). See the table below.

[0090]

[0091] 3) Drug administration and observation: On Day 2, each experimental group was given the corresponding drug according to the above-labeled drug administration method. On Day 7, the experimental mice in each group were euthanized, and the intestinal segment of the mouse was cut from the anus to the cecum of the mouse, and the length from the anus to the cecum of the mouse was measured. The weight of the mice in each group was weighed every day during the experiment, and the DAI (Disease Activity Index) score was performed.

[0092] 4) Result analysis:

[0093] The disease activity index (DAI) and weight loss percentage of the transfected MSC intraperitoneal injection group (transfected MSC ip group) were superior to those of the model group (P<0.05). Figure 4 and Figure 5 ;

[0094] The length of the colon and rectum in the MSC transfected intraperitoneal injection group (MSC transfected ip group) was significantly different from that in the model group (P<0.05). Figure 6 .

[0095] The above experimental results suggest that TNFR1-modified MSCs have a therapeutic effect on DSS-induced inflammatory bowel disease.

[0096] 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A modified TNFR1 sequence, characterized in that The modified TNFR1 sequence is the nucleotide sequence shown in SEQ ID NO.1, or the complementary sequence of the nucleotide sequence shown in SEQ ID NO.

1.

2. The modified TNFR1 sequence according to claim 1, characterized in that The modified TNFR1 sequence is obtained by the following modification: The 5'-untranslated region, coding region, and 3'-untranslated region of the TNFR1 sequence shown in SEQ ID NO.2 are modified respectively to construct a modified 5'-untranslated region-modified TNFR1 mRNA sequence coding region-modified 3'-untranslated region, and a poly-A tail is added to the 3'-untranslated region; The nucleotide sequence of the modified TNFR1 mRNA sequence coding region is shown in SEQ ID NO.3; The modified 5'-untranslated region nucleotide sequence is shown in SEQ ID NO.4; The modified 3'-untranslated region nucleotide sequence is shown in SEQ ID NO.5; The sequence of the poly-A tail is shown in SEQ ID NO.

6.

3. A modified TNFR1 mRNA sequence, characterized in that The modified TNFR1 sequence of claim 1 is transcribed and capped at the 5' end.

4. The modified TNFR1 mRNA sequence according to claim 3, characterized in that The modified TNFR1 mRNA sequence obtained by transcription is shown in SEQ ID NO.

7.

5. The method for constructing the modified TNFR1 mRNA sequence according to claim 1, characterized in that: The modified TNFR1 sequence of claim 1 is transcribed to obtain the mRNA sequence shown in SEQ ID NO. 7 and the modified TNFR1 mRNA sequence is obtained after capping at the 5' end.

6. The construction method according to claim 4, characterized in that: The method comprises the following steps: 1) Plasmid linearization: Take 1-10 μg of the pUC57-Kan plasmid containing the modified TNFR1 sequence of claim 1, add 1-10 μl of EcoRI endonuclease, 5-50 μl of EcoRI endonuclease buffer and double distilled water to a final volume of 50-500 μl, and incubate at 37° C. for 30 min-3 h; 2) Purification of linearized plasmid: The linearized pUC57-Kan plasmid containing the TNFR1 sequence was purified using the FastPure Gel DNA Extraction Mini Kit. The purified product was subjected to gel electrophoresis to determine the size and integrity of the product and then used for subsequent in vitro transcription experiments; 3) In vitro transcription and capping: Use the HiSynth T7 Co-transcription RNA Synthesis Kit to perform in vitro capping and co-transcription. Take 1-5 μg of the linear plasmid pUC57-Kan containing the TNFR1 sequence and add it to nuclease-free water. Then add 2-10 μl of 10× reaction buffer, 2-10 μl of 2×NTP / cap analog and 1-5 μl of T7 RNA. Polymerase, mix well and incubate at 37°C for 2-3h to obtain the capped mRNA shown in SEQ ID NO.7; 4) mRNA purification: use RNA Cleanup Kit was used for purification. 200 μl of the concentrated binding solution in the kit was added to every 100 μl, and then 300 μl of anhydrous ethanol was added to mix well. The filtrate was discarded after high-speed centrifugation through the column. After washing twice with 0.1-1 ml of washing solution and high-speed centrifugation, the capped mRNA was dissolved with 1-100 μl of elution solution and eluted by high-speed centrifugation, and the filtrate was collected. 5) Final purification of mRNA: Add 0.1-1 ml of clearing binding buffer, mix well, then add 0.1-1 ml of anhydrous ethanol and mix well, centrifuge at high speed to pass through the column and discard the filtrate; wash twice with 0.1-1 ml of washing solution by high-speed centrifugation, add 10-50 μl of preheated nuclease-free water to the center of the central filter membrane of the filter column to dissolve the capped mRNA and elute by high-speed centrifugation, collect the filtrate to complete the preparation of TNFR1 mRNA with high translation stability.

7. mRNA liposome complex, characterized in that Liposomes are used to encapsulate the modified TNFR1 mRNA sequence according to claim 3 or 4, the molar ratio of liposomes to the modified TNFR1 mRNA sequence is 1:3-1:6, the liposomes include ionizable cationic lipids, cholesterol, neutral auxiliary phospholipids, and PEG lipids, the ionizable cationic lipids are Dlin-MC3-DMA or DOTAP, the neutral auxiliary phospholipids are DSPC or DOPE, and the PEG lipids are DSPE-PEG2000.

8. The mRNA liposome complex according to claim 7, characterized in that The molar ratio of ionizable cationic lipid, cholesterol, neutral auxiliary phospholipid and PEG lipid is one of 40:40:10:5, 40:40:10:10 and 40:40:10:

15.

9. TNFR1-modified mesenchymal stem cells, characterized in that: The TNFR1-modified mesenchymal stem cells are transfected with the mRNA liposome complex described in claim 7.

10. Use of the modified TNFR1 sequence according to claim 1 or 2, or the modified TNFR1 mRNA sequence according to claim 3 or 4, or the mRNA liposome complex according to claim 7, or the TNFR1 modified mesenchymal stem cell according to claim 9 in the preparation of a medicament for treating inflammatory bowel disease.

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