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

Abstract

The invention provides a tumor drug based on mRNA and protein coding, a preparation method and application, the tumor drug comprises self-replicating mRNA, and the self-replicating mRNA comprises a self-replicating element capable of being coded and a protein coding region. The protein coding region comprises a treatment protein coding region and / or an envelope protein coding region. The structure of the self-replicating mRNA further comprises a subgene starting element located between the self-replicating element capable of being coded and the protein coding region. The structure of the self-replicating mRNA also comprises a cascade expression element located between the therapeutic protein coding region and the envelope protein coding region. Through application of the multi-cascade protein expression element in RNA tumor drugs, embedding expression of various treatment proteins can be achieved, and the effect of promoting anti-tumor immunity through different mechanisms is achieved.

Description

Technical Field

[0001] The present invention relates to the application of self-replicating mRNA in the technical field of tumor drug preparation. Background Art

[0002] Oncology drugs are an important means of treating cancer. Traditional oncology treatments primarily rely on chemotherapy and radiotherapy. With the advancement of biotechnology, RNA drugs are gradually entering the oncology field. Tumor vaccines based on RNA technology are becoming a promising anti-tumor treatment. Through tumor immunotherapy, tumor vaccines are directly delivered to the tumor site, thereby inhibiting tumor growth or effectively killing tumor cells.

[0003] LNP-mRNA technology is a common tumor immunotherapy in the prior art. This immunotherapy involves synthesizing and translating tumor-specific antigen mRNA in vitro, encapsulating the mRNA in LNPs to form LNP-mRNA particles, and administering them locally or systemically. Administration directly and extensively expresses tumor-specific antigens or functional proteins within tumor cells or antigen-presenting cells (APCs), increasing tumor-specific antigen presentation and / or stimulating an immune response.

[0004] The biggest challenge facing tumor immunotherapy is that the tumor immune microenvironment is often immunosuppressive, manifested primarily by a lack of immune cell infiltration and a reduced ability of immune cells to respond to tumor cells. For example, 1) tumor cells downregulate their own immunogenicity and upregulate the expression of immunosuppressive molecules; 2) infiltration of CD8+ cytotoxic T lymphocytes, CD4+ helper T cells, mature dendritic cells, and natural killer (NK) cells decreases; 3) CD4+ regulatory T cells, myeloid-derived suppressor cells, and tumor-associated macrophages are enriched; and 4) the production of cytokines that suppress the immune response increases. Designing cancer therapies requires modifying these inhibitory factors to enhance the immune response of the tumor microenvironment, significantly improving the effectiveness of cancer treatment. However, how these factors are modified, whether these modifications may have other negative consequences, and the effectiveness of unilateral modification remain largely unresolved. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an mRNA and protein-encoded tumor drug, preparation method and application in response to the problems existing in the background technology, which can reverse the immunosuppressive state of the tumor microenvironment from multiple aspects, thereby improving the effect of tumor immunotherapy.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] In a first aspect of the present invention, the present invention provides an oncology drug based on mRNA and protein coding, wherein the oncology drug comprises a self-replicating mRNA, and the self-replicating mRNA comprises an encodable self-replicating element and a protein coding region.

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

[0009] Preferably, the structure of the self-replicating mRNA further comprises a subgene promoter element located between the self-replicating element and the protein coding region that can be encoded.

[0010] Preferably, 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.

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

[0012] Preferably, the gene sequence of the tumor drug is as shown in the sequence list as 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).

[0013] In a second aspect of the present invention, the present invention provides a method for preparing an oncology drug based on mRNA and protein coding, wherein the method is configured to prepare the oncology drug according to the first aspect of the present invention; the method comprises the following steps:

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

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

[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] Reagent components volume Final concentration Nuclease-free water 2-6uL NTP Buffer Mix 8-15uL Final concentration of each NTP 5-15mM m7G(5')ppp(5')(2'OMeA)pU 1-5uL Template DNA 1-5uL 0.5-1.5 μg T7RNA Polymerase Mix 1-5uL Total reaction volume 10-30uL

[0020] (3.2) mRNA extraction and incubation;

[0021] (3.3) Determination of mRNA.

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

[0023] (3.2.1) Add the in vitro mRNA 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 centrifuge;

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

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

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

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

[0029] The beneficial effects of the present invention are:

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

[0031] 2. The present invention designs virus-like particles that embed pMHC-I-SCT (SCT, single-chain trimer) therapeutic protein in self-replicating mRNA, which can dually activate tumor-specific CD8+ T cells through cell-presented SCT and SCT on the surface of virus-like particles to kill tumors.

[0032] 3. The present invention can change the tumor microenvironment and promote anti-tumor immunity by innate immune response induced by self-replicating mRNA.

[0033] 4. The present invention also uses multiple cascade protein expression elements in RNA tumor drugs to achieve the embedded expression of multiple therapeutic proteins and promote anti-tumor immunity through different mechanisms. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0035] Figure 2 This is a flow chart of the mRNA drug model verification of Example 1 of the present invention.

[0036] Figure 3 Schematic diagram of the plasmid template in Example 2 of the present invention.

[0037] Figure 4 1 is a diagram showing the agarose gel electrophoresis identification results of the circular plasmid template and the linearized template in Example 2 of the present invention.

[0038] Figure 5 This is a diagram showing the agarose gel electrophoresis identification results of the mRNA drug in Example 2 of the present invention.

[0039] Figure 6 Schematic diagram of the production of viral particles by mRNA drug transfection into a cell line in Example 3 of the present invention.

[0040] Figure 7 This is a TCID 50 verification experiment of the virus-like particles produced by transfecting different cell lines with the mRNA drug in Example 3 of the present invention.

[0041] Figure 8 This is a fluorescence imaging diagram of the mRNA drug virus-like particles diffusing to form a fluorescent plaque pattern in Example 3 of the present invention.

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

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

[0044] Figure 11 This is a graph showing transcriptome sequencing results after cells were transfected with mRNA drugs in Example 4 of the present invention.

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

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

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

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

[0049] FIG16 is a graph showing the effectiveness test results of the mRNA drug in Example 5 of the present invention.

[0050] FIG17 is a graph showing the effectiveness test results of CIITA protein introduction into the mRNA drug in Example 5 of the present invention.

[0051] FIG18 is a graph showing the effectiveness test results of introducing an anti-PDL1 single-chain antibody into the mRNA drug in Example 5 of the present invention.

[0052] FIG19 is a graph showing the effectiveness test results 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 Example 6 of the present invention in inducing anti-tumor immunity by activating virus-specific bystander CD8+ T cells.

[0054] Figure 21 This is a graph showing the results of activating tumor-specific CD8+ T cells through systemic delivery of mRNA drugs in Example 7 of the present invention.

[0055] Figure 22 is a graph showing the effectiveness test results of the mRNA drug in Example 7 of the present invention in activating tumor-specific CD8+ T cells through systemic delivery to inhibit in situ tumors and metastatic tumors. DETAILED DESCRIPTION

[0056] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely for explanation of the present invention and are not intended to limit the present invention. Modifications or replacements made to the method, steps or conditions of the present invention, without departing from the spirit and essence of the present invention, all fall within the scope of the present invention.

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

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

[0059] This example provides an overview of the mRNA drugs designed by the present invention, as well as their mechanism of action and animal model validation.

[0060] The experimental cell model used in the embodiments of the present invention is 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 the present invention is as follows Figure 1 As shown, it includes components such as 5' end cap, 5' non-coding region, self-replication element, sub-gene promoter element, therapeutic protein coding region, cascade expression element, envelope protein coding region, 3' non-coding region and 3' end tail.

[0065] In this gene structure, the working mechanism of each part is as follows:

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

[0067] b) The self-replicating element is first translated and expressed. This example uses a non-structural protein coding sequence from the Semliki Forest virus species of the alphavirus genus, which encodes and expresses an RNA-dependent RNA polymerase complex. This complex can fully amplify the entire therapeutic mRNA, enhancing efficacy and reducing dosage. Furthermore, 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 immunity. Furthermore, the RNA polymerase complex can also recognize subgenomic promoter elements and synthesize subgenomic mRNAs containing the therapeutic protein coding region, cascade expression elements, and envelope protein coding regions.

[0068] c) The subgenomic mRNA is recognized by the intracellular protein translation machinery, leading to the expression of large quantities of the therapeutic protein. Through a cascade of expression elements, the envelope protein is also expressed in large quantities. Targeting the characteristic of tumor cells that downregulate MHC class I expression to achieve immune evasion, we designed the therapeutic protein as a single-chain trimer of a tumor-specific antigen peptide, MHC class I, called SCT (pMHC-I-SCT). The abundantly expressed pMHC-I-SCT molecules localize to the cell membrane, enabling immune cells to more effectively recognize and kill tumor cells. Furthermore, the abundant expression of the envelope protein can accumulate on the cell membrane, encapsulating numerous newly generated self-replicating mRNAs and embedding pMHC-I-SCT molecules. Ultimately, the envelope protein assembles into self-replicating mRNA virus-like particles (VLPs) containing pMHC-I-SCT molecules on the membrane surface. Tumor cells produce large quantities of VLPs and release them into the tumor microenvironment, a process that also leads to tumor cell death.

[0069] d) Self-replicating mRNA virus-like particles (VLPs) can directly activate immune cells in the tumor microenvironment because they contain pMHC-I-SCT molecules on their membranes. VLPs can also reinfect surrounding tumor cells, where the self-replicating mRNA released by these particles can repeat the aforementioned process, creating a cascade effect that further promotes anti-tumor immune responses.

[0070] e) In addition to pMHC-I-SCT, the mRNA in this embodiment can also insert immune activation molecules such as PD-L1 monoclonal antibody, IL2 or CIITA in 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, in this embodiment, the mRNA drugs that can be provided are as follows:

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

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

[0074] SFV-pMHCova (pMHC-I-SCT protein coding sequence of self-replicating mRNA+ova antigen peptide), the gene sequence of which is shown in SEQ NO.3 in 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), the gene sequence of which is shown in SEQ NO.5 in the sequence listing;

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

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

[0080] like Figure 2 As shown, experiments on tumor cell models confirmed that after self-replicating mRNA containing the envelope protein coding sequence was transfected into the cells, GFP protein (SFV-GFP) could be produced, and virus-like particles could be produced at the same time. The virus-like particles could infect the cells again and form diffuse spots (SFV-GFP-VSVG), proving the ability of virus-like particles to infect bystander cells. Virus-like particles can be produced in different cell lines.

[0081] After self-replicating mRNA is transfected into cells, it can express large amounts of pMHC-I-SCT protein. The cells bind to the corresponding TCR through the 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 the 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 the cell body and increase the expression of corresponding pro-inflammatory molecules.

[0082] In animals, intratumoral administration of mRNA-LNPs can produce virus-like particles in the tumor microenvironment and persist for about 5 days.

[0083] In the B16OVA model, intratumoral injection of SFV-pMHCova-VSVG significantly inhibited tumor growth, enhanced the infiltration and activation of tumor-specific OVA CD8+ T cells, and enhanced DC responses. By incorporating different therapeutic proteins, our mRNA drug can modify the tumor microenvironment. The addition of anti-PD-L1 secretory antibodies can reduce T cell exhaustion, while the addition of CIITA protein can increase the expression and presentation of MHC class II molecules on the tumor surface, enhance immune infiltration of CD4+ helper T cells, and inhibit tumor growth. Furthermore, our mRNA drug can elicit 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 method for preparing an mRNA drug of the present invention, and the preparation method specifically includes the following steps:

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

[0087] First, a DNA sequence encoding an mRNA drug is constructed on a plasmid template (such as Figure 3 ), wherein the DNA sequence encoding the self-replication element (SEQ NO.9) was donated by a university laboratory, and the DNA sequence encoding the therapeutic protein sequence (SEQ NO.10 to 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 the engineered Escherichia coli DH5a (Qingke Biotechnology).

[0089] The plasmid template was purified using a plasmid extraction kit (FastPure EndoFree Plasmid Maxi Kit, Novezan) and the extracted plasmid was identified by agarose gel electrophoresis ( Figure 4 ), the identification results showed that the circular plasmid template was in a supercoiled state, so the molecular size shown on the gel image was less than 10k bp.

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

[0091] A BamHI restriction endonuclease recognition site is added after the DNA sequence encoding the 3' tail, and the circular plasmid template is linearized using a single enzyme digestion reaction.

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

[0093] After the reaction, the linearized template was purified using a DNA purification kit (FastPure Gel DNA Extraction Mini Kit, Novezan), and the purified linearized template was identified by agarose gel electrophoresis ( Figure 4 After treatment with BamHI restriction enzyme, the linearized template restored 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-chain trimer coding sequence + VSVG envelope protein coding sequence).

[0095] mRNA in vitro synthesis kit ( T7 Quick High Yield RNA Synthesis Kit, NEB) was used to co-transcribe and synthesize capped mRNA drugs.

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

[0097] 3.2. Prepare the in vitro transcription system at room temperature as follows:

[0098] Reagent components volume Final concentration Nuclease-free water 4 μL NTP Buffer Mix 10 μL Final concentration of each NTP: 10 mM m7G(5')ppp(5')(2'OMeA)pU 2μL Template DNA 2μL 1 μg T7RNA Polymerase Mix 2μL Total reaction volume 20 μL

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

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

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

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

[0105] 3.9. Centrifuge at room temperature at high speed (13,000 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 high speed (13000 rpm, 5424R high-speed refrigerated centrifuge, Eppendorf) at room temperature for 5 minutes, and discard the supernatant.

[0107] 3.11. Repeat the ethanol washing step once.

[0108] 3.12. Leave to dry at room temperature.

[0109] 3.13. Dissolve the mRNA precipitate in enzyme-free water and measure the concentration and purity using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific).

[0110] It has been determined that each 20 μL of mRNA in vitro transcription system can produce approximately 100 μg of high-purity mRNA drug (260 / 280>2.0, 260 / 230>2.3). Agarose gel electrophoresis was also used to identify that in vitro mRNA transcription initiated from the T7 promoter, transcribed from the replication mRNA element, the pMHC-ova single-chain trimer element, the protein cascade expression element, and the envelope protein element, and finally terminated at the single enzyme cut point. The total length is approximately 12 kb, and the position shown on the gel image is greater than 10 kb ( Figure 5 ).

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

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

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

[0114] SFV-GFP-VSVG mRNA was transfected into 293T, BSR and B16F10 cell lines (such as Figure 6 As shown), it was found that the transfected cells could not only produce GFP reporter protein, but also 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 addition of envelope protein sequence could not produce virus-like particles, indicating that the mRNA drug designed in this embodiment not only acts directly on cells, but also can produce virus-like particles to carry out a new round of infection in the surrounding area. Afterwards, different cell lines (293T, BSR, B16F10, MC38, LLC, 4T1 and CT26) were transfected with SFV-GFP-VSVGmRNA, and the TCID50 experiment was used to verify that ( Figure 7 ), the mRNA drug designed by the present invention can play a role in different tumor cell lines and produce virus-like particles, indicating that the mRNA drug designed by the present invention has the potential to be applied to different tumor models.

[0115] In order to verify that mRNA drugs can produce virus-like particles and continue to infect new recipient cells, fluorescence imaging of transfected BSR cells was performed at different time points ( Figure 8), fluorescence imaging revealed that cells transfected with SFV-GFP-VSVG mRNA could release virus-like particles, which further infect surrounding cells and produce a new round of virus-like particles, leading to a cascade reaction and forming a fluorescent plaque pattern that spread over time. However, no such phenomenon occurred with SFV-GFP mRNA designed without the addition of an envelope protein sequence, demonstrating that the mRNA drug designed in the present invention has the potential to produce a cascade-amplified therapeutic effect in tumor tissue.

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

[0117] In order to verify the ability of the mRNA drug designed by the present invention to activate T cells, BSR cells were infected with SFV-pMH C-ova-VSVG mRNA and SFV-pMHC-ova mRNA respectively. After 24 hours, the transfected BSR cells were incubated with OT-I (Jukart 76) T cell line, and the activation of T cells was detected after 24 hours ( Figure 9 ), from the activation situation, it can be found that mRNA drugs designed with envelope protein sequences can produce higher activation efficacy. At the same time, we tested 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) produced by SFV-pMHC-ova-VSVG mRNA can directly activate the OT-I (Jukart 76) T cell line. The second-generation virus-like particles (SFV-pMHC-ova-VSVG VLV-2) produced by a new round of cell infection with the first-generation virus-like particles can also activate the OT-I (Jukart 76) T cell line. However, the mRNA drug designed without the envelope protein sequence failed to produce virus-like particles and failed to further activate the OT-I (Jukart 76) T cell line. These results demonstrate that the mRNA drug designed by the present invention that can produce virus-like particles can effectively amplify the benefits of T cell activation.

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

[0119] In order to verify that the mRNA drug designed by the present invention can have a wider range of effects and influences on tumor cells, this example uses transcriptome sequencing technology RNA-seq (RNA sequencing) to analyze B16F10 cells transfected with mRNA drugs ( Figure 11), analysis revealed that transfection of mRNA carrying self-replicating elements can cause extensive changes in the transcriptome of B16F10 cells. The insertion of self-replicating elements can induce strong innate immune responses in cells, especially stimulating the type I interferon signaling pathway, and also promoting the production of cytokines ( Figure 12 ), however, transfection of mRNA without self-replication elements did not induce these changes, which were similar to the blank control. Real-time fluorescence quantitative PCR (RT-PCR) was further used to verify the relevant pathways, and the results were as follows Figure 13 shown.

[0120] Based on the in vitro experimental results, this example further conducted in vivo animal experiments on mRNA drugs. First, mice (C57BL / 6) were given a unilateral tumor (B16F10 cell line). After the tumor successfully grew, the mRNA drug was injected into the tumor to test its ability to produce virus-like particles in the animals. 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 produce virus-like particles, while mRNA without envelope protein sequences does not produce virus-like particles in vivo. At the same time, we tested the duration of continuous production of virus-like particles after a single mRNA drug intratumoral injection. We collected tumor interstitial fluid at different time points after intratumoral injection of mRNA drugs and used 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 test results show that the virus-like particles produced on the first day are the highest. As time goes by, the abundance of virus-like particles drops to a very low level until the fifth day, indicating that the mRNA drug designed by the present invention can play a role in the body, but will not cause the same pathogenic risk as the real virus.

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

[0122] Based on the functional verification results of the mRNA drugs in the above examples, the effect of mRNA drugs on tumor treatment was further tested in this example. In the verification experiment of this example, mice (C57BL / 6) were first unilaterally tumor-bearing (B16F10 cell line). After the tumor successfully grew, the tumor was injected with mRNA drugs (SFV-pMMHC-ova, SFV-pMMHC-ova-VSVG) for a total of three injections, each with an interval of one day. The tumor size was measured at intervals of one day, and a control group (control) was set for comparison. The results were recorded as follows: Figure 16A 、 Figure 16BAs shown in the figure, it can be seen that the mRNA drug designed with the envelope protein sequence can effectively inhibit tumor growth. At the same time, we further analyzed the immune cells infiltrating the tumor using flow cytometry and found that the mRNA drug that can produce virus-like particles can induce stronger tumor-specific T cell infiltration and can also induce stronger dendritic cell activation ( Figure 16C ).

[0123] Based on the mRNA drug design in Example 1, this example further utilized the protein cascade coding sequence to insert the coding sequences of other therapeutic proteins (CIITA and anti-PDL1 single-chain antibody) into the mRNA drug, thereby finding that the introduction of 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 and B), indicating that the mRNA drug of the present invention can be added into a flexible treatment combination to achieve a stronger therapeutic effect.

[0124] In order to verify the function of mRNA drugs in inducing systemic immunity, mice (C57BL / 6) were first bilaterally loaded with tumors (B16F10 cell line). After the tumors successfully grew, the mRNA drugs were injected into the tumors unilaterally. A total of three injections were made, with one day between each injection. The tumor size was measured one day after each injection, and the results were recorded as follows: Figure 19A 、 Figure 19B and Figure 19C As shown in the figure, it can be seen that the mRNA drug can not only effectively inhibit the growth of the tumor on the injected side, but also the growth of the tumor on the opposite side is correspondingly inhibited, indicating that the mRNA drug designed by the present invention has the potential to activate 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 unilaterally tumor-bearing (B16F10 cell line). After the tumor successfully grew, the tumor was injected with mRNA drugs (SFV-pMHC-np and SFV-pMHC-np-VSVG) intratumorally for a total of one injection. Tumor size was measured every other day, and the endpoint tumor weight measurement results were as follows: 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 is described above by way of example in conjunction with the accompanying drawings, but the scope of protection of the present invention is not limited thereto. As long as various improvements are made using the method concepts and technical solutions of the present invention, or the concepts and technical solutions of the present invention are directly applied to other occasions without improvement, they are all within the scope of protection of the present invention.

[0128] Example 7: Verification that systemic delivery of mRNA drugs stimulates anti-tumor immunity and significantly inhibits in situ tumors and metastatic tumors.

[0129] To verify that systemic delivery of mRNA drugs can induce a systemic immune response, this example subcutaneously injected mRNA drugs into mice (C57BL / 6). Analysis of various immune cells in the spleen revealed an increase in both total CD8+ T cell and tumor-specific CD8+ T cell populations, and an increase in the proportion of effector cells in both populations ( Figure 21 At the same time, it was also found that the ratio and activation status of macrophages and dendritic cells in the spleen increased ( Figure 21 ).

[0130] In order to verify the efficacy of systemic delivery of mRNA drugs in two subcutaneous orthotopic tumor models, we carried out unilateral tumor-bearing (B16F10 or MC38 cell lines) on mice. After the tumors successfully grew, the mice were injected with mRNA drugs (SFV-pMHC-ova-aPDL1scFv-VSVG) subcutaneously for a total of three times, with one day between each injection. The tumor size was measured one day after each injection, and the results were recorded as follows: Figure 22A As shown. 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 increased significantly.

[0131] We also tested the efficacy of systemic delivery of mRNA drugs in a lung metastasis model. We injected tumor cells (B16F10 cell line) into mice through the tail vein to form a lung metastasis model. We injected the mRNA drug (SFV-pMHC-ova-aPDL1scFv-VSVG) subcutaneously into the mice for a total of three injections, with one day between each injection. The final test results are as follows: Figure 22B As shown. Systemic delivery of mRNA drugs can effectively inhibit the growth of lung metastases, and the number of tumor spots and tumor area are significantly smaller than those in the control group ( Figure 22C). This indicates that the mRNA drug designed by the present invention can be delivered systemically to activate systemic anti-tumor immune response and has potential in clinical application.

[0132] The present invention is described above by way of example in conjunction with the accompanying drawings, but the scope of protection of the present invention is not limited thereto. As long as various improvements are made using the method concepts and technical solutions of the present invention, or the concepts and technical solutions of the present invention are directly applied to other occasions without improvement, they are all within the scope of protection of the present invention.

Claims

1. An oncology drug based on mRNA and protein coding, characterized in that: The tumor drug includes a self-replicating mRNA, and the self-replicating mRNA includes an encoded self-replicating element and a protein coding region.

2. The tumor drug based on mRNA and protein coding according to claim 1, characterized in that: The protein coding region includes a therapeutic protein coding region and / or an envelope protein coding region.

3. The tumor drug based on mRNA and protein coding according to claim 1 or 2, characterized in that The structure of the self-replicating mRNA further includes a subgene promoter element located between the self-replicating element and the protein coding region that can be encoded.

4. The tumor drug based on mRNA and protein coding according to claim 1 or 2, characterized in that The structure of the self-replicating mRNA also includes a cascade expression element located between the therapeutic protein coding region and the envelope protein coding region.

5. The tumor drug based on mRNA and protein coding according to claim 1, characterized in that The gene sequence of the self-replicating mRNA is shown in SEQ NO.1 (SFV-GFP) or SEQ NO.3 (SFV-pMHCova) in the sequence list.

6. The tumor drug based on mRNA and protein coding according to claim 1, characterized in that The gene sequence of the tumor drug is shown in the sequence list as SEQ NO.2 (SFV-GFP-VSVG), SEQ NO.4 (SFV-pMHCova-VSVG), SEQ NO.5 (SFV-pMHCova-aPDL1scFv-VSVG), SEQ NO.6 (SFV-pMHCova-CIITA-VSVG), SEQ NO.7 (SFV-pMHCnp) or SEQ NO.8 (SFV-pMHCnp-VSVG).

7. A method for preparing an oncology drug based on mRNA and protein coding, characterized in that: The preparation method is configured to prepare the tumor drug according to any one of claims 1 to 6; the preparation method comprises the following steps: (1) Construction, amplification and purification of plasmid templates; (2) Linearization and purification of plasmid template; (3) In vitro transcription and purification of mRNA tumor drugs.

8. The method for preparing an oncology drug based on mRNA and protein coding according to claim 7, 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) Determination of mRNA.

9. The method for preparing an oncology drug based on mRNA and protein coding according to claim 7, characterized in that: The step (3.2) is specifically The following processes are included: (3.2.1) Add the in vitro mRNA 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 centrifuge; (3.2.3) Aspirate the supernatant, add an equal volume of isopropanol, mix thoroughly, and incubate overnight; (3.2.4) After incubation, the sample was centrifuged at high speed at room temperature, the supernatant was discarded, and the precipitate was washed with anhydrous ethanol. The sample was centrifuged at high speed at room temperature, the supernatant was discarded, and the washing was repeated. The sample was allowed to dry at room temperature to obtain the mRNA precipitate.

10. Application of mRNA self-replication in the preparation of tumor drugs and / or tumor vaccines.

11. Use of virus-like particles generated by self-replicating mRNA containing envelope protein and therapeutic protein coding regions in the preparation of tumor drugs and / or tumor vaccines.

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