Tumor drugs based on mRNA and protein coding, preparation method and application

Abstract

The application provides a tumor drug based on mRNA and protein coding, a preparation method and application, the tumor drug comprises self-replicating mRNA, the self-replicating mRNA comprises a codeable self-replicating element and a protein coding region.The protein coding region comprises a therapeutic protein coding region and / or an envelope protein coding region.The structure of the self-replicating mRNA further comprises a subgenomic promoter element located between the codeable self-replicating element and the protein coding region.The structure of the self-replicating mRNA further comprises a cascade expression element located between the therapeutic protein coding region and the envelope protein coding region.The application also realizes embedded expression of multiple therapeutic proteins through the application of multiple cascade protein expression elements in RNA tumor drugs, and realizes the effect of promoting anti-tumor immunity through different mechanisms.

Description

Technical Field

[0001] This invention relates to the application of self-replicating mRNA in the field of tumor drug preparation technology. Background Technology

[0002] Oncology drugs are an important means of treating cancer. Traditional oncology drugs mainly consist of chemotherapy and radiotherapy. With the development of biotechnology, RNA drugs have gradually entered the field of oncology drugs. Oncology vaccines based on RNA technology are gradually becoming a promising anti-tumor treatment method. Through tumor immunotherapy, tumor vaccines are applied directly to the tumor site, thereby inhibiting tumor growth or effectively killing tumor cells.

[0003] A common type of tumor immunotherapy in current technology is LNP-mRNA-based immunotherapy. This immunotherapy involves synthesizing mRNA that can be translated to produce tumor-specific antigens in vitro, encapsulating the mRNA with LNPs to form LNP-mRNA particles, and then administering the drug locally or systemically. This allows for the direct and large-scale expression of tumor-specific antigens or functional proteins within tumor cells or antigen-presenting cells (APCs), increasing tumor-specific antigen presentation and / or stimulating the immune response.

[0004] The biggest challenge facing tumor immunotherapy is that the tumor immune microenvironment is often in a state of immunosuppression, manifested primarily by a lack of immune cell infiltration and a decreased ability of immune cells to respond to tumor cells. For example, 1) tumor cells downregulate their autoimmunogenicity and upregulate the expression of immunosuppressive molecules; 2) the infiltration of CD8+ cytotoxic T lymphocytes, CD4+ helper T cells, mature dendritic cells (DCs), and NK cells decreases; 3) CD4+ regulatory T cells, myeloid suppressor cells, and tumor-associated macrophages accumulate; and 4) cytokines that suppress the immune response increase. Designing tumor therapies requires altering these suppressive factors to enhance the immune response in the tumor microenvironment, thereby significantly improving the effectiveness of tumor treatment. However, questions such as how to modify these factors, whether changes will produce other negative effects, and the effectiveness of altering a single factor still lack comprehensive research and validation. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a tumor drug based on mRNA and protein encoding, a preparation method and application, which can reverse the immunosuppressive state of the tumor microenvironment from multiple aspects, thereby improving the effect of tumor immunotherapy.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides an mRNA- and protein-coded tumor drug, the tumor drug comprising a self-replicating mRNA, the self-replicating mRNA comprising a self-replicating element and a protein-coding region that can be encoded.

[0008] Preferably, the protein-coding region includes a therapeutic protein-coding region and / or a membrane protein-coding region.

[0009] Preferably, the structure of the self-replicating mRNA further includes a subgenetic promoter element located between the self-replicating element and the protein-coding region.

[0010] Preferably, the structure of the self-replicating mRNA further includes a cascade expression element located between the therapeutic protein coding region and the envelope protein coding region.

[0011] Preferably, the gene sequence of the self-replicating mRNA is shown in SEQ NO.1 (SFV-GFP) in the sequence listing.

[0012] Preferably, the gene sequence of the tumor drug is as shown in SEQ NO.2 (SFV-GFP-VSVG), SEQ NO.3 (SFV-pMHC-ova), SEQ NO.4 (SFV-pMHC-ova-VSVG), SEQ NO.5 (SFV-pMHC-ova-aPDL1scFv-VSVG), SEQ NO.6 (SFV-pMHC-ova-CIITA-VSVG), SEQ NO.7 (SFV-pMHCnp), or SEQ NO.8 (SFV-pMHCnp-VSVG) in the sequence listing.

[0013] In a second aspect, the present invention provides a method for preparing a tumor drug based on mRNA and protein encoding, the method being configured for preparing the tumor drug described in the first aspect of the present invention; the method comprising the following steps:

[0014] (1) Construction, amplification and purification of plasmid templates;

[0015] (2) Linearization and purification of plasmid templates;

[0016] (3) In vitro transcription and purification of mRNA tumor drugs.

[0017] Preferably, the in vitro transcription and purification of the mRNA tumor drug specifically includes the following steps:

[0018] (3.1) Prepare the in vitro mRNA synthesis system at room temperature according to the table below;

[0019] Nuclease-free water 2-6uL NTP Buffer Mix 8-15uL The final concentration of each NTP is 5-15 mM. m7G(5')ppp(5')(2'OMeA)pU 1-5uL Template DNA 1-5uL 0.5-1.5μg T7 RNA Polymerase Mix 1-5uL Total reaction volume 10-30uL

[0020] (3.2) mRNA extraction and incubation;

[0021] (3.3) mRNA determination.

[0022] Preferably, step (3.2) specifically includes the following process:

[0023] (3.2.1) Add the mRNA in vitro synthesis system to the RNA extraction kit, mix thoroughly and centrifuge at low speed to the bottom of the tube;

[0024] (3.2.2) Add enzyme-free water, mix thoroughly, incubate at room temperature, and then centrifuge;

[0025] (3.2.3) Take the supernatant, add an equal volume of isopropanol, mix thoroughly and incubate overnight;

[0026] (3.2.4) After incubation, the sample is centrifuged at room temperature and the supernatant is discarded. The precipitate is washed with anhydrous ethanol, centrifuged at room temperature and the supernatant is discarded. The washing is repeated, and the sample is placed at room temperature to air dry to obtain the mRNA precipitate.

[0027] In a third aspect, the present invention provides the application of the mRNA self-replication process in the preparation of tumor drugs and / or tumor vaccines.

[0028] In a fourth aspect, the present invention provides the use of self-replicating mRNA containing coding regions for therapeutic proteins and envelope proteins to generate virus-like particles in the preparation of tumor drugs and / or tumor vaccines.

[0029] The beneficial effects of this invention are:

[0030] 1. This invention designs an in vitro synthesized self-replicating mRNA, which uses mRNA-LNP technology to generate virus-like particles in vivo and can spread in situ, killing tumor cells through the spread of virus-like particles.

[0031] 2. The present invention designs a virus-like particle generation system that embeds pMHC-I-SCT (SCT, single-chain trimer) therapeutic protein into self-replicating mRNA. This system can achieve tumor killing by dual activation of tumor-specific CD8+ T cells through cell-presented SCT and SCT on the surface of virus-like particles.

[0032] 3. This invention alters the tumor microenvironment and promotes anti-tumor immunity by inducing a cellular innate immune response through self-replicating mRNA.

[0033] 4. This invention also enables the embedded expression of multiple therapeutic proteins through the application of multiple cascade protein expression elements in RNA tumor drugs, thereby promoting anti-tumor immunity through different mechanisms. Attached Figure Description

[0034] Figure 1 This is a structural diagram of the mRNA drug of Example 1 of the present invention.

[0035] Figure 2 This is a flowchart of the mRNA drug model validation process in Example 1 of the present invention.

[0036] Figure 3 This is a schematic diagram of the plasmid template in Embodiment 2 of the present invention.

[0037] Figure 4 This is an image showing the agarose gel electrophoresis results of the circular plasmid template and the linearized template in Example 2 of this invention.

[0038] Figure 5 This is an image showing the agarose gel electrophoresis results of the mRNA drug in Example 2 of this invention.

[0039] Figure 6 This is a schematic diagram of the cell line transfected with mRNA drug to produce viral particles in Example 3 of the present invention.

[0040] Figure 7 This is a verification experiment of TCID50 generated by virus-like particles transfected with mRNA drugs in different cell lines in Example 3 of the present invention.

[0041] Figure 8 This is a fluorescence imaging image of the fluorescent patch pattern formed by the diffusion of mRNA drug virus-like particles in Example 3 of the present invention.

[0042] Figure 9 This is a comparative diagram of the activation experiment of the mRNA drug in the OT-I (Jukart 76) T cell line in Example 3 of this invention.

[0043] Figure 10 This is a diagram showing the results of the activation experiment of the OT-I (Jukart 76) T cell line by virus-like particles in Example 3 of the present invention.

[0044] Figure 11 This is a diagram of the transcriptome sequencing results after cell transfection with mRNA drugs in Example 4 of this invention.

[0045] Figure 12 This is a graph showing the results of the GSEA gene set enrichment analysis in Example 4 of this invention.

[0046] Figure 13 This is the result of real-time fluorescence quantitative PCR verification in Example 4 of the present invention.

[0047] Figure 14 This is a diagram showing the verification results of the mRNA drug acting in mouse tumors and producing virus-like particles in Example 4 of this invention.

[0048] Figure 15 This is a graph showing the duration of virus-like particle production by the mRNA drug in mouse tumors in Example 4 of this invention.

[0049] Figure 16 is a graph showing the effectiveness test results of the mRNA drug in Example 5 of the present invention.

[0050] Figure 17 shows the effectiveness test results of introducing CIITA protein into the mRNA drug in Example 5 of this invention.

[0051] Figure 18 is a graph showing the effectiveness test results of introducing anti-PDL1 single-chain antibody into the mRNA drug in Example 5 of the present invention.

[0052] Figure 19 shows the results of the effectiveness test of the mRNA drug in inducing systemic immunity in Example 5 of the present invention.

[0053] Figure 20 This is a graph showing the effectiveness test results of the mRNA drug in activating virus-specific bystander CD8+ T cells to induce anti-tumor immunity in Example 6 of this invention.

[0054] Figure 21 This is a diagram showing the results of systemic delivery of mRNA drugs to activate tumor-specific CD8+ T cells in Example 7 of this invention.

[0055] Figure 22 shows the results of the efficacy test of mRNA drugs in inhibiting in situ tumors and metastatic tumors by activating tumor-specific CD8+ T cells through systemic delivery in Example 7 of the present invention. Detailed Implementation

[0056] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Modifications or substitutions made to the methods, steps, or conditions of this invention without departing from the spirit and essence of the invention are all within the scope of this invention.

[0057] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0058] Example 1: Design and model validation of mRNA drugs.

[0059] This embodiment provides an overview of the mRNA drug designed in this invention, as well as its mechanism of action and animal model validation.

[0060] The experimental cell models used in the embodiments of the present invention are as follows:

[0061] 293T, BSR, B16F10, and Jurkat 76 T cells (OT-I).

[0062] The experimental animal model used in the embodiments of the present invention is C57BL / 6.

[0063] The tumor cell model used in the embodiments of the present invention is B16F10-OVA.

[0064] like Figure 1 As shown, the gene structure of the mRNA drug involved in this invention is as follows: Figure 1 As shown, it includes, in sequence, a 5' cap, a 5' non-coding region, a self-replicating element, a subgene promoter element, a therapeutic protein coding region, a cascade expression element, a membrane protein coding region, a 3' non-coding region, and a 3' tail.

[0065] The working mechanisms of each part in this gene structure are as follows:

[0066] a) The protein translation machine recognizes the 5' end cap and the 5' non-coding region and starts the protein translation program.

[0067] b) The self-replicating element is first translated and expressed. In this embodiment, a non-structural protein-coding sequence of the Semliki Forest Virus species of the alphavirus genus is used, which encodes an RNA-dependent RNA polymerase complex. This complex can amplify the entire therapeutic mRNA in its entirety, enhancing efficacy and reducing the dosage. Moreover, the mRNA self-replication process can highly induce the innate immune response of tumor cells, thereby causing changes in the tumor microenvironment and promoting tumor immune effects. In addition, the RNA polymerase complex can also recognize subgenetic initiation elements and synthesize subgenetic mRNAs containing therapeutic protein coding regions, cascade expression elements, and envelope protein coding regions.

[0068] c) Subgenetic mRNAs are recognized by intracellular protein translation machinery, resulting in the expression of a large amount of therapeutic proteins. Through a cascade of expression elements, envelope proteins are also expressed in large quantities simultaneously. Targeting the ability of tumor cells to downregulate MHC-I class molecule expression to achieve immune evasion, we designed the therapeutic protein as a single-chain trimer (pMHC-I-SCT) molecule of tumor-specific antigenic peptides MHC-I class molecule. The highly expressed pMHC-I-SCT molecules are localized to the cell membrane surface, allowing immune cells to more effectively recognize tumor cells and exert their killing function. Furthermore, the high expression of envelope proteins can aggregate on the cell membrane, encapsulating a large number of newly generated self-replicating mRNAs and embedding pMHC-I-SCT molecules, ultimately assembling into virus-like particles containing pMHC-I-SCT molecules on the membrane surface. Tumor cells produce a large number of virus-like particles and release them into the tumor microenvironment, a process that also induces tumor cell death.

[0069] d) Self-replicating mRNA virus-like particles, due to the presence of pMHC-I-SCT molecules on their membrane surface, can directly activate immune cells in the tumor microenvironment. These virus-like particles can also reinfect surrounding bystander tumor cells, and the self-replicating mRNA released into these cells can repeat the above process, creating a cascade amplification effect that further promotes the anti-tumor immune response.

[0070] e) In addition to pMHC-I-SCT, the mRNA in this embodiment can also insert immune-activating molecules such as PD-L1 monoclonal antibody, IL2 or CIITA into the therapeutic protein coding region, which can achieve a combined therapeutic effect with pMHC-I-SCT.

[0071] Based on the above structure and mechanism of action, the mRNA drugs that can be provided in this embodiment are as follows:

[0072] SFV-GFP (self-replicating mRNA + GFP protein coding sequence), its gene sequence is shown in SEQ NO.1 of the sequence listing;

[0073] SFV-GFP-VSVG (self-replicating mRNA + GFP protein coding sequence + VSVG envelope protein coding sequence), its gene sequence is shown in SEQ NO.2 of the sequence listing;

[0074] SFV-pMHCova (the pMHC-I-SCT protein encoding sequence of self-replicating mRNA + ova antigen peptide), its gene sequence is shown in SEQ NO.3 of the sequence listing;

[0075] SFV-pMHCova-VSVG (self-replicating mRNA + pMHC-I-SCT protein coding sequence of ova antigen peptide + VSVG envelope protein coding sequence), the gene sequence of which is shown in SEQ NO.4 in the sequence listing;

[0076] SFV-pMHCova-aPDL1scFv-VSVG (self-replicating mRNA + pMHC-I-SCT protein coding sequence of ova antigen peptide + anti-PDL1 single-chain antibody coding sequence + VSVG envelope protein coding sequence), its gene sequence is shown in SEQ NO.5 of the sequence listing;

[0077] SFV-pMHCova-CIITA-VSVG (self-replicating mRNA + pMHC-I-SCT protein-coding sequence of ova antigen peptide + CIITA protein-coding sequence + VSVG envelope protein-coding sequence), its gene sequence is shown in SE Q NO.6 of the sequence listing;

[0078] SFV-pMHCnp (the pMHC-I-SCT protein encoding sequence of self-replicating mRNA + influenza np antigen peptide), the gene sequence of which is shown in SEQ NO.7 of the sequence listing;

[0079] SFV-pMHCnp-VSVG (self-replicating mRNA + pMHC-I-SCT protein coding sequence of influenza np antigen peptide + VSVG envelope protein coding sequence), the gene sequence of which is shown in SEQ NO.8 of the sequence listing;

[0080] like Figure 2 As shown, experiments on tumor cell models have confirmed that self-replicating mRNA containing envelope protein coding sequences can produce GFP protein (SFV-GFP) after transfection into cells, and can also produce virus-like particles. These virus-like particles can reinfect cells and form spreading spots (SFV-GFP-VSVG), demonstrating the ability of virus-like particles to infect bystander cells. These virus-like particles can be produced in different cell lines.

[0081] After self-replicating mRNA is transfected into cells, it can express a large amount of pMHC-I-SCT protein. Cells bind to the corresponding TCR through pMHC-I-SCT protein and stimulate the expression of the Jurkat 76 T cell (OT-I) reporter gene GFP protein. Virus-like particles embedded with pMHC-I-SCT protein can directly activate the expression of the Jurkat 76 T cell (OT-I) reporter gene GFP protein. Self-replicating mRNA can induce a strong innate immune response in cells and increase the expression of corresponding pro-inflammatory molecules.

[0082] In animals, intratumoral injection of mRNA-LNP can generate virus-like particles in the tumor microenvironment, which last for about 5 days.

[0083] Using the B16OVA model, intratumoral injection of SFV-pMHCova-VSVG significantly inhibited tumor growth, increased the infiltration and activation of tumor-specific OVA CD8+ T cells, and enhanced the response of dendritic cells (DCs). By incorporating different therapeutic proteins, our designed mRNA drug can alter the tumor microenvironment. The addition of an anti-PD-L1 secretory antibody reduces T cell exhaustion, while the addition of CIITA protein increases the expression and presentation of MHC class II molecules on the tumor surface, enhances the immune infiltration of CD4+ helper T cells, and inhibits tumor growth. Simultaneously, our designed mRNA drug can induce a systemic immune response and also inhibit the growth of distant tumors.

[0084] Example 2: Preparation of mRNA drugs.

[0085] This embodiment provides a specific scheme for preparing an mRNA drug according to the present invention, and the preparation method specifically includes the following process:

[0086] 1. Construction, amplification, and purification of plasmid templates.

[0087] First, the DNA sequence that can encode the mRNA drug is constructed in a plasmid template (e.g., Figure 3 As shown, the DNA sequence encoding the self-replicating element (SEQ NO.9) was provided by a university laboratory, and the DNA sequences encoding the therapeutic protein (SEQ NO.10-SEQ NO.14) and the envelope protein (SEQ NO.15) were from the NCBI database.

[0088] The constructed plasmid template was amplified in large quantities using engineered Escherichia coli DH5a (Qingke Biotechnology).

[0089] Plasmid templates were purified using a FastPure EndoFree Plasmid Maxi Kit (Novozymes), and the extracted plasmids were identified by agarose gel electrophoresis. Figure 4 The identification results showed that the cyclic plasmid template was in a supercoiled state, so the molecular size was less than 10 kbp as shown on the gel image.

[0090] 2. Linearization and purification of plasmid templates.

[0091] A BamHI restriction endonuclease recognition site was added after the DNA sequence that could encode the 3' end, and the circular plasmid template was linearized using a single enzyme digestion reaction.

[0092] The reaction system (taking a total volume of 20 μL as an example) includes: 7 μg plasmid template, 1 μL SwiftCut BamHI (Novitamin), and 2 μL SwiftCut Buffer (10×); the reaction is carried out at 37 °C for 5 hours (ProFlex). TM PCR system (Thermo Fisher Scientific).

[0093] After the reaction, the linearized template was purified using a DNA purification kit (FastPure Gel DNA Extraction Mini Kit, Novizon). The purified linearized template was then identified by agarose gel electrophoresis. Figure 4 After treatment with BamHI restriction endonuclease, the linearized template recovered its original molecular size (>10 kbp) on the gel image. The concentration of the linearized template was determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific), and was approximately 0.5 μg / μL.

[0094] 3. In vitro transcription and purification of the mRNA drug SFV-pMHC-ova-VSVG mRNA (self-replicating mRNA sequence + pMHC-ova single-stranded trimeric coding sequence + VSVG envelope protein coding sequence).

[0095] mRNA in vitro synthesis kit ( The T7 Quick High Yield RNA Synthesis Kit (NEB) co-transcribes and synthesizes capped mRNA drugs.

[0096] 3.1 Melt the components in the kit, mix thoroughly, centrifuge at low speed (4000 rpm, 5424R high-speed refrigerated centrifuge, Eppendorf) until the bottom of the tube is reached, and place on ice.

[0097] 3.2. The in vitro transcription system was prepared at room temperature as follows:

[0098] Nuclease-free water 4μL NTP Buffer Mix 10μL Final concentration of each NTP: 10 mM m7G(5')ppp(5')(2'OMeA)pU 2μL Template DNA 2μL 1μg T7 RNA Polymerase Mix 2μL Total reaction volume 20μL

[0099] 3.3. Mix thoroughly and centrifuge at low speed (4000 rpm, 5424R high-speed refrigerated centrifuge, Eppendorf) until the bottom of the tube is reached. React at 37°C for 4 hours (ProFlex). TM PCR system (Thermo Fisher Scientific).

[0100] 3.4. Add 1 μL of DNase I to the reaction system, mix thoroughly, and centrifuge at low speed (4000 rpm, 5424R high-speed refrigerated centrifuge, Eppendorf) until the bottom of the tube is reached. React at 37 degrees Celsius for 15 minutes (ProFlex). TM PCR system (Thermo Fisher Scientific).

[0101] 3.5 Add 500 μL of RNA-easy Isolation Reagent (Novozymes) to the mRNA in vitro synthesis system, mix thoroughly and centrifuge at low speed (4000 rpm, 5424R high-speed refrigerated centrifuge, Eppendorf) until the bottom of the tube is reached.

[0102] 3.6 Add 200 μL of enzyme-free water, mix thoroughly, and incubate at room temperature for 5 minutes.

[0103] 3.7 Centrifuge at room temperature at high speed (13000 rpm, 5424R high-speed refrigerated centrifuge, Eppendorf) for 15 minutes.

[0104] 3.8. Transfer the supernatant (700 μL) into a clean EP tube, add an equal volume of isopropanol, mix thoroughly, and incubate overnight at -20°C.

[0105] 3.9 Centrifuge at room temperature at high speed (13000 rpm, 5424R high-speed refrigerated centrifuge, Eppendorf) for 15 minutes, and discard the supernatant.

[0106] 3.10 Wash the mRNA precipitate with 80% ethanol, centrifuge at room temperature at high speed (13000 rpm, 5424R high-speed refrigerated centrifuge, Eppendorf) for 5 minutes, and discard the supernatant.

[0107] 3.11 Repeat the ethanol cleaning step once.

[0108] 3.12. Let it air dry at room temperature.

[0109] 3.13. Add enzyme-free water to dissolve the mRNA precipitate, and determine the concentration and purity using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific).

[0110] It was determined that each 20 μL mRNA in vitro transcription system could produce approximately 100 μg of high-purity mRNA drug (260 / 280 > 2.0, 260 / 230 > 2.3). Simultaneously, agarose gel electrophoresis confirmed that the in vitro transcription of mRNA began at the T7 promoter, transcribed from replicating mRNA elements, pMHC-ova single-stranded trimeric elements, protein cascade expression elements, and envelope protein elements, finally terminating at a single enzyme cleavage site, with a total length of approximately 12 kb, and the position shown on the gel image being greater than 10 kb. Figure 5 ).

[0111] Example 3: Functional validation of mRNA drugs.

[0112] Following the design of the mRNA drug described in Example 1, in the verification process of this example, the coding element of the pMHC-ov a single-stranded trimer was replaced with the coding element of the GFP reporter protein. SFV-GFP-VS VG mRNA (self-replicating mRNA sequence + GFP protein coding sequence + VSVG envelope protein coding sequence, its gene sequence is shown in SEQ NO.2 in the sequence listing) was synthesized in vitro. At the same time, a plasmid template without the envelope protein coding element was constructed, and SFV-GFP mRNA (self-replicating mRNA sequence + GFP protein coding sequence, as shown in SE Q NO.1 in the sequence listing) and SFV-pMHC-ova mRNA (self-replicating mRNA sequence + pMHC-ova single-stranded trimer coding sequence, as shown in SEQ NO.3 in the sequence listing) were synthesized in vitro and used for experimental reference.

[0113] 1. Verification of the generation of virus-like particles.

[0114] SFV-GFP-VSVG mRNA was transfected into 293T, BSR, and B16F10 cell lines (e.g., ...). Figure 6 As shown in the figure, it was found that transfected cells could not only produce GFP reporter protein, but also generate virus-like particles for a new round of infection of receiving cells. Cells infected with the first generation of virus-like particles could also express GFP reporter protein. However, SFV-GFP mRNA designed without the inclusion of envelope protein sequence could not produce virus-like particles, indicating that the mRNA drug designed in this example not only acts directly on cells, but can also generate virus-like particles to infect surrounding cells in a new round. Subsequently, different cell lines (293T, BSR, B16F10, MC38, LLC, 4T1, and CT26) were transfected with SFV-GFP-VSVG mRNA, and the results were verified using the TCID50 assay. Figure 7 The mRNA drugs designed in this invention can exert their effects and produce virus-like particles in different tumor cell lines, indicating that the mRNA drugs designed in this invention have the potential to be applied in different tumor models.

[0115] To verify that the mRNA drug can generate virus-like particles and continuously infect new recipient cells, fluorescence imaging was performed on transfected BSR cells at different time points. Figure 8Fluorescence imaging revealed that cells transfected with SFV-GFP-VSVG mRNA could release virus-like particles. These newly released virus-like particles further infected surrounding cells and generated a new round of virus-like particles, resulting in a cascade reaction that formed fluorescent patch patterns that diffused over time. However, no such phenomenon occurred with SFV-GFP mRNA designed without the inclusion of envelope protein sequences. This demonstrates that the mRNA drug designed in this invention has the potential to form a cascade amplification of therapeutic effects within tumor tissue.

[0116] 2. Verification of T cell activation capacity.

[0117] To verify the ability of the mRNA drug designed in this invention to activate T cells, BSR cells were infected with SFV-pMH-ova-VSVG mRNA and SFV-pMHC-ova mRNA, respectively. After 24 hours, the transfected BSR cells were incubated with the OT-I (Jukart 76) T cell line, and the activation status of T cells was detected after another 24 hours. Figure 9 The activation data revealed that mRNA drugs with added envelope protein sequences exhibited higher activation potency. Simultaneously, we examined the effect of virus-like particles on T cell activation. Figure 10 ),from Figure 10 It was found that the first-generation virus-like particles (SFV-pMHC-ova-VSVG VLV-1) generated from SFV-pMHC-ova-VSVG mRNA can directly activate the OT-I (Jukart 76) T cell line. A second round of infection with these first-generation virus-like particles produces second-generation virus-like particles (SFV-pMHC-ova-VSVG VLV-2), which also activate the OT-I (Jukart 76) T cell line. However, mRNA drugs designed without the inclusion of envelope protein sequences cannot generate virus-like particles and cannot further activate the OT-I (Jukart 76) T cell line. These results demonstrate that the mRNA drugs designed in this invention, capable of generating virus-like particles, can effectively amplify the benefits of T cell activation.

[0118] Example 4: Verification of the broader effects and influences of mRNA drugs on tumor cells.

[0119] To verify that the mRNA drug designed in this invention can have a broader effect on tumor cells, this embodiment uses RNA-seq (RNA sequencing) technology to analyze B16F10 cells transfected with the mRNA drug. Figure 11Analysis revealed that transfection with mRNA carrying self-replication elements induced widespread changes in the transcriptome of B16F10 cells. The insertion of these self-replication elements elicited a strong innate immune response, particularly stimulating the type I interferon signaling pathway, and also promoting cytokine production. Figure 12 However, transfection with mRNA lacking self-replication elements did not induce these changes, remaining similar to the blank control. Further validation of the relevant pathways was performed using real-time quantitative PCR (RT-PCR), with results as follows: Figure 13 As shown.

[0120] Based on the in vitro experimental results, this embodiment further conducted in vivo animal experiments on the mRNA drug. First, mice (C57BL / 6) were given unilateral tumor-bearing tumors (B16F10 cell line). After successful tumor growth, the mRNA drug was injected into the tumor, and its ability to produce virus-like particles in vivo was tested. The results are as follows: Figure 14 As shown, from Figure 14 It can be seen that mRNA drugs designed with envelope protein sequences can effectively generate virus-like particles, while mRNAs without envelope protein sequences do not generate virus-like particles in vivo. Simultaneously, we tested the duration of virus-like particle generation after a single intratumoral injection of mRNA drug, collected tumor interstitial fluid at different time points after intratumoral injection, and used the TCID50 assay to detect the abundance of virus-like particles in the tumor interstitial fluid. The results are as follows: Figure 15 As shown, the detection results show that the highest number of virus-like particles were produced on the first day. As time went on, the abundance of virus-like particles dropped to a very low level by the fifth day. This indicates that the mRNA drug designed in this invention can work in the body without causing the same pathogenic risk as a real virus.

[0121] Example 5: Validation of the effectiveness of mRNA drugs.

[0122] Based on the functional validation results of the mRNA drugs performed in the above embodiments, this embodiment further tests the efficacy of the mRNA drugs in tumor treatment. In the validation experiment of this embodiment, mice (C57BL / 6) were first given unilateral tumor-bearing tumors (B16F10 cell line). After the tumors successfully grew, intratumoral mRNA drugs (SFV-pMMHC-ova, SFV-pMMHC-ova-VSVG) were injected into the tumors three times in total, with each injection one day apart. Tumor size was measured one day apart, and a control group was set up for comparison. The results were recorded as follows. Figure 16A , Figure 16BAs shown in the figure, the mRNA drug designed with the added envelope protein sequence can effectively inhibit tumor growth. Furthermore, we used flow cytometry to analyze the infiltrating immune cells within the tumor and found that the mRNA drug, which can produce virus-like particles, can induce stronger tumor-specific T cell infiltration and also induce stronger dendritic cell activation. Figure 16C ).

[0123] Based on the mRNA drug design in Example 1, this example further utilizes protein cascade coding sequences to insert the coding sequences of other therapeutic proteins (CIITA and anti-PDL1 single-chain antibody) into the mRNA drug. It was found that the introduction of the CIITA protein can effectively inhibit tumor growth and simultaneously induce stronger CD4+ T cell infiltration. Figure 17A And B), while the introduction of anti-PDL1 single-chain antibody can reduce the exhaustion state of tumor-specific CD8 positive T cells while inhibiting tumor growth. Figure 18A (A) and (B) illustrate that the mRNA drugs involved in this invention can be added to flexible treatment combinations to achieve stronger therapeutic effects.

[0124] To verify the systemic immune response induced by the mRNA drug, mice (C57BL / 6) were first bilaterally tumor-bearing (B16F10 cell line). After successful tumor growth, the tumors were unilaterally injected with the intratumoral mRNA drug. A total of three injections were administered, each one day apart. Tumor size was measured one day apart, and the results were recorded as follows: Figure 19A , Figure 19B and Figure 19C As shown in the figure, the mRNA drug can not only effectively inhibit the growth of the tumor on the injection side, but also inhibit the growth of the tumor on the contralateral side, indicating that the mRNA drug designed in this invention has the potential to activate a systemic anti-tumor immune response.

[0125] Example 6: Validation of mRNA drugs using non-tumor-specific virus-specific CD8+ bystander T cells to stimulate anti-tumor immunity.

[0126] To verify that mRNA drugs can utilize non-tumor-specific virus-specific CD8+ bystander T cells to stimulate anti-tumor immune responses, mice (C57BL / 6) were first given unilateral tumor-bearing tumors (B16F10 cell line). After successful tumor growth, intratumoral injections of mRNA drugs (SFV-pMHC-np and SFV-pMHC-np-VSVG) were administered, with a total of one injection. Tumor size was measured one day apart, and the terminal tumor weight measurement results are shown below. Figure 20As shown, mRNA drugs can significantly inhibit tumor growth, indicating that mRNA drugs can also be widely used to activate anti-tumor immunity by utilizing virus-specific CD8+ T cells.

[0127] The present invention has been described above with reference to the accompanying drawings, but the scope of protection of the present invention is not limited thereto. Any improvements made using the inventive concept and technical solution of the present invention, or the direct application of the inventive concept and technical solution to other situations without modification, are all within the scope of protection of the present invention.

[0128] Example 7: Validation of the significant inhibition of in situ tumors and metastatic tumors by systemic delivery of mRNA drugs to stimulate anti-tumor immunity.

[0129] To verify that systemic delivery of mRNA drugs can induce a systemic immune response, this embodiment involved subcutaneous injection of mRNA drugs into mice (C57BL / 6). Analysis of various immune cells in the spleen revealed elevated levels of both total CD8+ T cells and tumor-specific CD8+ T cells, with an increased proportion of effector cells within both populations. Figure 21 Simultaneously, it was also observed that the ratio of macrophages to dendritic cells and their activation status were increased in the spleen. Figure 21 ).

[0130] To validate the efficacy of systemic mRNA drug delivery in two subcutaneous orthotopic tumor models, we unilaterally implanted tumors (B16F10 or MC38 cell lines) in mice. After successful tumor growth, the mice were subcutaneously injected with the mRNA drug (SFV-pMHC-ova-aPDL1scFv-VSVG) three times, one day apart. Tumor size was measured one day apart, and the results were recorded as follows: Figure 22A As shown. From Figure 22A It can be seen that systemic delivery of mRNA drugs can effectively inhibit the growth of both tumor models, and the number of tumor-specific CD8+ T cells in the tumor also increases significantly.

[0131] We also tested the efficacy of systemic mRNA drug delivery in a lung metastasis model. We established a lung metastasis model in mice by tail vein injection of tumor cells (B16F10 cell line). We then administered subcutaneous mRNA drug (SFV-pMHC-ova-aPDL1scFv-VSVG) to the mice three times, one day apart. The final results are shown below. Figure 22B As shown, systemic delivery of mRNA drugs effectively inhibited the growth of lung metastases, with significantly smaller tumor numbers and tumor size compared to the control group. Figure 22CThis indicates that the mRNA drug designed in this invention can be delivered systematically to activate a systemic anti-tumor immune response, and has potential for clinical application.

[0132] The present invention has been described above with reference to the accompanying drawings, but the scope of protection of the present invention is not limited thereto. Any improvements made using the inventive concept and technical solution of the present invention, or the direct application of the inventive concept and technical solution to other situations without modification, are all within the scope of protection of the present invention.

Claims

1. A tumor drug based on mRNA and protein encoding, characterized in that, The tumor drug comprises a self-replicating mRNA, which includes a self-replicating element and a protein-coding region; the protein-coding region includes a therapeutic protein-coding region and a capsule protein-coding region; the structure of the self-replicating mRNA further includes a subgenetic promoter element located between the self-replicating element and the protein-coding region; the structure of the self-replicating mRNA further includes a cascade expression element located between the therapeutic protein-coding region and the capsule protein-coding region; the gene sequence of the tumor drug is shown in SEQ NO.4, SEQ NO.5, and SEQ NO.6 in the sequence listing.

2. A method for preparing tumor drugs based on mRNA and protein encoding, characterized in that, The preparation method is configured to prepare the tumor drug according to claim 1; the preparation method includes the following steps: (1) Construction, amplification, and purification of plasmid templates; (2) Linearization and purification of plasmid templates; (3) In vitro transcription and purification of mRNA tumor drugs.

3. The method for preparing a tumor drug based on mRNA and protein encoding according to claim 2, characterized in that, The in vitro transcription and purification of the mRNA tumor drug specifically includes the following steps: (3.1) Prepare an in vitro mRNA synthesis system at room temperature; (3.2) mRNA extraction and incubation; (3.3) mRNA determination.

4. The method for preparing a tumor drug based on mRNA and protein encoding according to claim 3, characterized in that, Step (3.2) specifically includes the following process: (3.2.1) Add the mRNA in vitro synthesis system to the RNA extraction kit, mix thoroughly and centrifuge at low speed to the bottom of the tube; (3.2.2) Add enzyme-free water, mix thoroughly, incubate at room temperature, and then centrifuge; (3.2.3) Take the supernatant, add an equal volume of isopropanol, mix thoroughly and incubate overnight; (3.2.4) After incubation, the sample is centrifuged at room temperature and the supernatant is discarded. The precipitate is washed with anhydrous ethanol, centrifuged at room temperature and the supernatant is discarded. The washing is repeated, and the sample is air-dried at room temperature to obtain the mRNA precipitate.

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