Use of virus 3c protease in prevention or treatment of tumor

Abstract

Provided are a use of a virus 3C protease or a nucleic acid molecule encoding the 3C protease in the prevention and / or treatment of a tumor in a subject (e.g., a human), and a use of the virus 3C protease or the nucleic acid molecule encoding the 3C protease in the preparation of a drug for preventing and / or treating a tumor in a subject (e.g., a human).

Description

Use of viral 3C protease for preventing or treating tumors

[0001] This application is based on the application with CN application number 202311152705.0 and application date September 7, 2023, and claims its priority. The disclosed content of the CN application is hereby introduced as a whole into this application. Technical Field

[0002] The present application relates to the fields of viruses and tumor treatment. Specifically, the present application relates to the use of viral 3C proteases or nucleic acid molecules encoding the 3C proteases for preventing and / or treating tumors in subjects (e.g., humans), as well as the use of the nucleic acid molecules encoding the viral 3C proteases in preparing medicaments for preventing and / or treating tumors in subjects (e.g., humans). Background Art

[0003] Cancer is a disease characterized by uncontrolled cell division and growth in the body. Approximately one-third of all women and half of all men in the United States will develop cancer in their lifetime. Cancer is generally divided into two categories: solid tumors and disseminated cancers. For many cancers, treatment options can include surgery, chemotherapy, radiation therapy, bone marrow / stem cell transplantation, cancer drugs, or immunotherapy, or a combination thereof. While these treatments can be effective, they often have significant side effects. Chemotherapy specifically targets all newly differentiated cells in the body, not just cancer cells. Gene therapy and genetic vaccination are among the most promising and rapidly developing approaches in modern medicine. They offer highly specific and personalized options for treating a wide variety of diseases.

[0004] Compared to DNA and viral vectors, mRNA does not integrate into the genome but only transiently expresses the encoded protein. Therefore, due to its low insertion risk, it offers researchers and pharmaceutical companies an excellent and safe option. mRNA does not need to enter the cell nucleus to function. Therefore, there is no risk of stable integration into the host cell genome. Once in the cytoplasm, mRNA initiates protein translation. mRNA can be easily synthesized through in vitro transcription (IVT). Furthermore, mRNA encapsulated in lipid nanoparticles can achieve targeted delivery to the liver and / or spleen.

[0005] The most common primary brain tumor in adults is glioblastoma, which is also one of the most deadly cancers. For glioblastoma, conventional treatment involves surgical resection followed by radiation and concurrent chemotherapy. Even with this treatment regimen, the median survival of glioblastoma patients is less than 15 months. The poor prognosis is mainly due to tumor recurrence, which is believed to originate from a subgroup of cancer stem cells that survive primary treatment. Recent studies have shown that glioblastoma stem cells survive longer than treatment stress and become more aggressive when they relapse, thereby developing resistance to primary chemotherapy. However, while chemotherapy drugs inhibit and kill tumors, they also have a killing effect on normal cells in the body, especially normal cells that proliferate more rapidly, such as bone marrow hematopoietic cells and gastrointestinal mucosal epithelial cells. Therefore, the toxic side effects of chemotherapy drugs have become a major obstacle to improving their efficacy.

[0006] Current clinical research on gliomas focuses on hormone therapy, immunotherapy, oncolytic virus therapy, and gene therapy. Endogenous ovarian steroid hormones, particularly endogenous estrogen, are also involved in glioma progression. Steroid hormones, particularly endogenous estrogen, play a crucial role in brain development and differentiation. The risks of long-term hormone replacement therapy for gliomas and its side effects remain controversial. While immunotherapies such as those targeting PD-1 and peptide-based vaccines have shown some effectiveness, immunotherapy for glioblastoma presents challenges, such as the induction of secondary brain tumors and autoimmune overactivation. Oncolytic virus therapy is an emerging new treatment approach currently under investigation in preclinical and clinical stages. Several viruses, including herpes simplex virus, adenovirus, poliovirus, and reovirus, are currently in Phase I and II clinical trials for the treatment of glioblastoma and have been shown to improve overall survival. However, most oncolytic viruses fail to overcome numerous obstacles, including neutralization and clearance by antibodies. There is also the potential risk of large amounts of virus being introduced into the bloodstream, triggering a cytokine storm. Currently, these agents have not received FDA approval. Messenger RNA (mRNA) therapy, a form of gene therapy, has been explored as one of the safest and most effective approaches for treating tumors. mRNA is composed of four nucleic acids, abbreviated as the letters A, U, C, and G. Cells interpret the order in which these nucleic acids are arranged, much like the ingredients in a protein recipe. If the mRNA recipe cannot be read, there is no effect, and therefore no side effects. However, if the opposite occurs, the body will produce only the proteins it needs at the right time and in the right place. Therefore, mRNA drug therapy offers a precise and non-toxic approach to treating cancer and viruses.

[0007] Summary of the Invention

[0008] Based on a large number of experimental studies, the inventors of the present application unexpectedly discovered that the 3C protease derived from small RNA viruses (such as enterovirus 71 and coxsackievirus A14) has a broad spectrum and significant ability to kill tumor cells. Based on this discovery, the inventors of the present application have developed a new method for preventing and / or treating tumors based on the 3C protease or the nucleic acid molecule encoding the 3C protease. The therapeutic drugs (for example, nucleic acid drugs) based on the 3C protease or the nucleic acid molecule encoding the 3C protease provided in this application are expected to expand the scope of benefits for cancer patients, improve patient survival rates, and bring more therapeutic benefits to the patients themselves, their families and society. At the same time, it also opens up a new track for the research of anti-tumor gene therapy drugs.

[0009] Therefore, in a first aspect, the present application provides use of a picornavirus 3C protease or a nucleic acid molecule encoding the 3C protease in the preparation of a medicament for preventing and / or treating tumors in a subject.

[0010] In certain embodiments, the picornavirus is an enterovirus, such as a human enterovirus.

[0011] In certain embodiments, the picornavirus is selected from the group consisting of a novel enterovirus, a poliovirus, a coxsackievirus, a rhinovirus, and an echovirus.

[0012] In certain embodiments, the picornavirus is a novel enterovirus (e.g., enterovirus 68 (EV68), enterovirus 69 (EV69), enterovirus 70 (EV70), enterovirus 71 (EV71)), a coxsackievirus (e.g., coxsackievirus A14, coxsackievirus A16, coxsackievirus A6, coxsackievirus B3), or a rhinovirus (e.g., rhinovirus 2, rhinovirus 7, rhinovirus 25).

[0013] In certain embodiments, the picornavirus is enterovirus 71 (EV71) or coxsackievirus A14.

[0014] In certain embodiments, the enterovirus 71 (EV71) is of genotype C4 or C5.

[0015] In certain embodiments, the enterovirus 71 (EV71) is the AH08 / 06 clinical isolate.

[0016] In certain embodiments, the picornavirus is an aphthous virus.

[0017] In certain embodiments, the aphthous ulcer virus is a foot-and-mouth disease virus. In certain embodiments, the foot-and-mouth disease virus is selected from the group consisting of European (A, O, C), Asian (Asia 1), and African (SAT1, SAT2, and SAT3).

[0018] In certain embodiments, the aphthous ulcer virus is selected from the group consisting of strain A10-61, strain HKN / 2002, and strain KEN / 4 / 2013.

[0019] It is easy for those skilled in the art to understand that the "3C protease" described herein can be directly derived from the picornavirus (e.g., wild-type or mutant picornavirus), or can be obtained by artificially modifying (e.g., introducing mutations or modifications) the 3C protease directly derived from the picornavirus, as long as it retains the biological activity (e.g., oncolytic activity) of the 3C protease from which it is derived. In addition, the 3C protease described herein can be a full-length 3C protease or an active fragment thereof (e.g., an active fragment with oncolytic activity).

[0020] In certain embodiments, the 3C protease is derived from a novel enterovirus (e.g., HZ08 strain (Genbank: HQ400942.1), Mahoney strain (Genbank: V01149.1), CHN_HN_2019_214 strain (Genbank: ON755036.1)). In certain embodiments, the 3C protease is as shown in any one of Uniprot: F5A4L9, P03300, and A0A075FJJ1.

[0021] In certain embodiments, the 3C protease is derived from a rhinovirus (e.g., HRV-1A_P25 strain (Genbank: KC894169.1), HRV-1A_P8 strain (Genbank: KC894168.1), RvA1B / KZ / 2021 / 87 strain (Genbank: OP886969.1)). In certain embodiments, the 3C protease is represented by any one of Uniprot: P23008, A0A0F6VY48, and P12916.

[0022] In certain embodiments, the 3C protease is derived from aphthous ulcer virus (e.g., strain A10-61 (Genbank: M31575.1), strain HKN / 2002 (Genbank: AY317098.1), strain KEN / 4 / 2013 (Genbank: OM863583.1)). In certain embodiments, the 3C protease is as shown in any one of Uniprot: P03306, AY317098.1, and F1AW55.

[0023] Those skilled in the art will readily appreciate that, in order to facilitate protein expression, purification and / or tracing, the 3C protease may further contain another polypeptide.

[0024] In certain embodiments, the additional polypeptide is optionally connected to the N-terminus or C-terminus of the 3C protease via a linker (e.g., a peptide linker). Such linkers (e.g., peptide linkers) are well known in the art, and examples include, but are not limited to, peptide linkers comprising one or more (e.g., 1, 2, 3, 4, or 5) amino acids (e.g., Gly or Ser). In certain embodiments, the peptide linker is flexible. In certain embodiments, a flexible peptide linker may be advantageous in that it is able to connect two protein / polypeptide components while maintaining their respective activity and function. Such peptide linkers include, but are not limited to, (GGGGS) n .

[0025] In certain embodiments, the additional polypeptide is selected from a tag, a signal or leader peptide, a detectable marker (eg, luciferase (fluc), green fluorescent protein (GFP)), or any combination thereof.

[0026] In certain embodiments, the 3C protease has an amino acid sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:1.

[0027] In certain embodiments, the 3C protease has an amino acid sequence as shown in SEQ ID NO: 1. The sequence shown here does not contain an amino acid (such as methionine (Met)) encoded by a start codon (such as ATG) at its N-terminus. Those skilled in the art understand that in the process of preparing proteins by genetic engineering, due to the effect of the start codon, the first position of the produced polypeptide chain is often an amino acid (such as Met) encoded by the start codon. The 3C protease of the present application not only encompasses amino acid sequences that do not contain an amino acid (such as Met) encoded by a start codon at its N-terminus, but also encompasses amino acid sequences that contain an amino acid (such as Met) encoded by a start codon at its N-terminus. Therefore, sequences that further contain an amino acid (such as Met) encoded by a start codon at the N-terminus of the above-mentioned amino acid sequence are also within the scope of protection of the present application.

[0028] In certain embodiments, the nucleic acid molecule is a DNA molecule or an RNA molecule.

[0029] In certain embodiments, the nucleic acid molecule is an RNA molecule.

[0030] In certain embodiments, the nucleic acid molecule comprises a coding sequence for the 3C protease.

[0031] In certain embodiments, the nucleic acid molecule further comprises one or more selected from the group consisting of a 5'UTR, a Kozak sequence, a start codon, a stop codon, a 3'UTR, and a poly-A tail.

[0032] In certain embodiments, the nucleic acid molecule comprises, from the 5' end to the 3' end, a 5'UTR, a Kozak sequence, a start codon, a coding sequence for the 3C protease, a stop codon, a 3'UTR, and a poly-A tail.

[0033] According to the codon degeneracy in the art, in certain embodiments, the coding sequence of the 3C protease can be replaced according to the codon degeneracy. In certain embodiments, the coding sequence of the 3C protease is codon-optimized or not codon-optimized according to the codon preference of the host cell (e.g., human cell).

[0034] In certain embodiments, the coding sequence of the 3C protease has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence shown in SEQ ID NO: 3. In certain embodiments, the coding sequence of the 3C protease is shown in SEQ ID NO: 3.

[0035] In certain embodiments, a 5' cap modification (eg, CAP type 0, CAP type 1, CAP type 2) is present at the 5' end of the nucleic acid molecule. In certain embodiments, a 5' Cap type 1 modification is present at the 5' end of the nucleic acid molecule.

[0036] In certain embodiments, the nucleic acid molecule contains one or more N1-methylpseudouridine (m1ψ) modifications. In certain embodiments, all or part of the uracil nucleotides in the nucleic acid molecule are replaced by N1-methyl-pseudouridine nucleotides.

[0037] In certain embodiments, one or more stop codons are present at the 3' end of the coding sequence for the 3C protease.

[0038] In certain embodiments, the 5'UTR is selected from the 5'UTR derived from human α-globin mRNA.

[0039] In certain embodiments, the 3'UTR is selected from 3'UTRs based on mtRNR1 (mitochondrial 12Sribosomal RNA) and AES (Amino-terminal enhancer of split).

[0040] In certain embodiments, the 5'UTR has a nucleotide sequence that has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 4. In certain embodiments, the 5'UTR has the nucleotide sequence set forth in SEQ ID NO: 4.

[0041] In certain embodiments, the 3'UTR has a nucleotide sequence that has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 6. In certain embodiments, the 3'UTR has the nucleotide sequence set forth in SEQ ID NO: 6.

[0042] In certain embodiments, the nucleic acid molecule has a nucleotide sequence that has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 2. In certain embodiments, the nucleic acid molecule has the nucleotide sequence set forth in SEQ ID NO: 2.

[0043] In certain embodiments, the Kozak sequence is shown as SEQ ID NO:5.

[0044] In certain embodiments, the poly-A tail comprises one or more poly(A) sequences, each of which independently consists of 20-120 consecutive adenylate nucleotides. In certain embodiments, the poly-A tail comprises multiple poly(A) sequences, and adjacent poly(A) sequences are connected by spacer sequences comprising non-A.

[0045] In certain embodiments, the medicament is for use in a subject:

[0046] (1) Inhibit tumor cell proliferation;

[0047] (2) inhibiting tumor cell migration and / or invasion;

[0048] (3) Promote tumor cell apoptosis;

[0049] (4) downregulating the upstream protein p38, downstream protein PSF and / or heterogeneous nuclear ribonucleoprotein (hnRNP) A1 in the MNK1 / 2 signaling pathway in tumor cells;

[0050] (5) Upregulation of caspase-3, an apoptosis execution protein in tumor cells; and / or

[0051] (6) Inhibit the activity of tumor cells.

[0052] In certain embodiments, the tumor is selected from a solid tumor or a hematological tumor (eg, leukemia, lymphoma, myeloma).

[0053] In certain embodiments, the tumor is selected from glioma (e.g., glioblastoma), liver cancer, kidney cancer, melanoma, prostate cancer, colon cancer, cervical cancer, gastric cancer, lung cancer, colorectal cancer, bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, gastrointestinal cancer, pancreatic cancer, skin cancer, leukemia, sarcoma (e.g., osteosarcoma).

[0054] In certain embodiments, the 3C protease or a nucleic acid molecule encoding the 3C protease is administered in combination with another pharmaceutically active agent, eg, simultaneously, separately, or sequentially.

[0055] In certain embodiments, the additional pharmaceutically active agent is a drug with anti-tumor activity.

[0056] In certain embodiments, the additional pharmaceutically active agent is selected from: EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-MET or VEGF inhibitors, antibody drugs, biological drugs, chemotherapy drugs or any combination thereof.

[0057] In a second aspect, the present application provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a 3C protease; wherein the 3C protease is as defined in the first aspect.

[0058] In certain embodiments, the isolated nucleic acid molecule is a nucleic acid molecule as defined in the first aspect.

[0059] In certain embodiments, the isolated nucleic acid molecule encodes the nucleic acid molecule defined in the first aspect.

[0060] In certain embodiments, the isolated nucleic acid molecule is a DNA molecule or an RNA molecule.

[0061] In certain embodiments, the isolated nucleic acid molecule is an RNA molecule.

[0062] In certain embodiments, the isolated nucleic acid molecule comprises a coding sequence for the 3C protease.

[0063] In certain embodiments, the isolated nucleic acid molecule further comprises one or more selected from the group consisting of a 5'UTR, a Kozak sequence, a start codon, a stop codon, a 3'UTR, and a poly-A tail.

[0064] In certain embodiments, the isolated nucleic acid molecule comprises, from 5' end to 3' end, a 5'UTR, a Kozak sequence, a start codon, a coding sequence for the 3C protease, a stop codon, a 3'UTR, and a poly-A tail.

[0065] In certain embodiments, the coding sequence of the 3C protease is codon-optimized or non-optimized according to the codon preference of the host cell (eg, human cell).

[0066] In certain embodiments, the coding sequence of the 3C protease has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence shown in SEQ ID NO: 3. In certain embodiments, the coding sequence of the 3C protease is shown in SEQ ID NO: 3.

[0067] In certain embodiments, the isolated nucleic acid molecule has a 5' cap modification (eg, CAP type 0, CAP type 1, CAP type 2) at its 5' end. In certain embodiments, the isolated nucleic acid molecule has a 5' Cap type 1 modification at its 5' end.

[0068] In certain embodiments, the isolated nucleic acid molecule contains one or more N1-methyl pseudouridine (m1ψ) modifications. In certain embodiments, all or part of the uracil nucleotides in the isolated nucleic acid molecule are replaced by N1-methyl-pseudouracil nucleotides.

[0069] In certain embodiments, one or more stop codons are present at the 3' end of the coding sequence for the 3C protease.

[0070] In certain embodiments, the 5'UTR is selected from the 5'UTR derived from human α-globin mRNA.

[0071] In certain embodiments, the 3'UTR is selected from 3'UTRs based on mtRNR1 (mitochondrial 12S ribosomal RNA) and AES (Amino-terminal enhancer of split).

[0072] In certain embodiments, the 5'UTR has a nucleotide sequence that has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 4. In certain embodiments, the 5'UTR has the nucleotide sequence set forth in SEQ ID NO: 4.

[0073] In certain embodiments, the 3'UTR has a nucleotide sequence that has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 6. In certain embodiments, the 3'UTR has the nucleotide sequence set forth in SEQ ID NO: 6.

[0074] In certain embodiments, the isolated nucleic acid molecule has a nucleotide sequence that has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 2. In certain embodiments, the isolated nucleic acid molecule has the nucleotide sequence set forth in SEQ ID NO: 2.

[0075] In certain embodiments, the Kozak sequence is shown as SEQ ID NO:5.

[0076] In certain embodiments, the poly-A tail comprises one or more poly(A) sequences, each of which independently consists of 20-120 consecutive adenylate nucleotides. In certain embodiments, the poly-A tail comprises multiple poly(A) sequences, and adjacent poly(A) sequences are connected by spacer sequences comprising non-A.

[0077] In a third aspect, the present application provides a vector comprising the isolated nucleic acid molecule of the second aspect.

[0078] In certain embodiments, the vector of the present application is, for example, a plasmid, a cosmid, a phage, a lentivirus, or the like.

[0079] In certain embodiments, the vector is a cloning vector or an expression vector.

[0080] In certain embodiments, the vector is capable of expressing the 3C protease described in the first aspect in a subject (eg, a mammal, such as a human).

[0081] In certain embodiments, the vector is a DNA vector or an RNA vector.

[0082] In a fourth aspect, the present application provides a delivery composition comprising a delivery vector and one or more selected from the following: 3C protease, the isolated nucleic acid molecule of the second aspect, the vector of the third aspect;

[0083] Wherein, the 3C protease is as defined in the first aspect.

[0084] In certain embodiments, the delivery vehicle is a particle.

[0085] In certain embodiments, the delivery vehicle is selected from lipid particles, sugar particles, metal particles, protein particles, liposomes, exosomes, microvesicles, a gene gun, or a viral vector (e.g., a replication-defective retrovirus, a lentivirus, an adenovirus, or an adeno-associated virus).

[0086] In certain embodiments, the delivery vehicle is a lipid nanoparticle.

[0087] In certain embodiments, the lipid nanoparticles comprise an ionizable cationic lipid SM-102, a helper lipid DSPC, cholesterol, and a pegylated lipid PEG2000-DMG.

[0088] In certain embodiments, the molar concentration ratio of the ionizable cationic lipid SM-102 to the helper lipid DSPC in the lipid nanoparticles is in the range of 2: 1 to 10: 1 (e.g., 2: 1 to 5: 1, 2: 1 to 8: 1, 3: 1 to 5: 1, 3: 1 to 8: 1, 3: 1 to 10: 1, 5: 1 to 8: 1, 5: 1 to 10: 1). In certain embodiments, the molar concentration ratio of the ionizable cationic lipid SM-102 to the helper lipid DSPC in the lipid nanoparticles is 5: 1.

[0089] In certain embodiments, the molar concentration ratio of the ionizable cationic lipid SM-102 to cholesterol in the lipid nanoparticles is in the range of 1:1 to 2:1 (e.g., 1:1 to 1.3:1, 1:1 to 1.5:1, 1.3:1 to 1.5:1, 1.3:1 to 2:1). In certain embodiments, the molar concentration ratio of the ionizable cationic lipid SM-102 to the helper lipid DSPC in the lipid nanoparticles is 50:38.5.

[0090] In certain embodiments, the molar concentration ratio of the ionizable cationic lipid SM-102 to the PEGylated lipid PEG2000-DMG in the lipid nanoparticles is in the range of 20: 1 to 50: 1 (e.g., 20: 1 to 30: 1, 20: 1 to 40: 1, 30: 1 to 40: 1, 30: 1 to 50: 1). In certain embodiments, the molar concentration ratio of the ionizable cationic lipid SM-102 to the helper lipid DSPC in the lipid nanoparticles is 50: 1.5.

[0091] In certain embodiments, in the lipid nanoparticles, the molar concentration ratio of the ionizable cationic lipid SM-102, the helper lipid DSPC, cholesterol, and the PEGylated lipid PEG2000-DMG is 50:10:38.5:1.5.

[0092] In a fifth aspect, the present application provides a pharmaceutical composition comprising 3C protease, the isolated nucleic acid molecule of the second aspect, the vector of the third aspect and / or the delivery composition of the fourth aspect, and a pharmaceutically acceptable carrier and / or excipient;

[0093] Wherein, the 3C protease is as defined in the first aspect.

[0094] In certain embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent.

[0095] In certain embodiments, the additional pharmaceutically active agent is a drug with anti-tumor activity.

[0096] In certain embodiments, the additional pharmaceutically active agent is selected from: EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-MET or VEGF inhibitors, antibody drugs, biological drugs (e.g., biological macromolecule drugs), chemotherapy drugs, cell therapy drugs or any combination thereof.

[0097] In certain embodiments, the additional pharmaceutically active agent is provided as a separate component or as a mixed component with the 3C protease, isolated nucleic acid molecule, vector and / or delivery composition.

[0098] In a sixth aspect, the present application provides a method for inhibiting tumor cells, comprising: contacting 3C protease, the isolated nucleic acid molecule of the second aspect, the vector of the third aspect, the delivery composition of the fourth aspect, or the pharmaceutical composition of the fifth aspect with the tumor cell, or delivering the 3C protease into the tumor cell;

[0099] Wherein, the 3C protease is as defined in the first aspect.

[0100] In certain embodiments, the methods are used to:

[0101] (1) inhibiting the proliferation of the tumor cells;

[0102] (2) inhibiting the migration and / or invasion of the tumor cells;

[0103] (3) promoting apoptosis of the tumor cells;

[0104] (4) downregulating the upstream protein p38, downstream protein PSF and / or heterogeneous nuclear ribonucleoprotein (hnRNP) A1 in the MNK1 / 2 signaling pathway in the tumor cells;

[0105] (5) upregulating the apoptosis execution protein caspase-3 in the tumor cells; and / or

[0106] (6) Inhibiting the activity of the tumor cells.

[0107] In certain embodiments, the tumor is selected from a solid tumor or a hematological tumor (eg, leukemia, lymphoma, myeloma).

[0108] In certain embodiments, the tumor is selected from glioma (e.g., glioblastoma), liver cancer, kidney cancer, melanoma, prostate cancer, colon cancer, cervical cancer, gastric cancer, lung cancer, colorectal cancer, bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, gastrointestinal cancer, pancreatic cancer, skin cancer, leukemia, sarcoma (e.g., osteosarcoma).

[0109] In certain embodiments, the method is an in vitro method or an in vivo method. In certain embodiments, the method is an in vitro method.

[0110] In a seventh aspect, the present application provides a method for preventing and / or treating tumors in a subject, the method comprising administering to a subject in need thereof an effective amount of 3C protease, the isolated nucleic acid molecule of the second aspect, the vector of the third aspect, the delivery composition of the fourth aspect, or the pharmaceutical composition of the fifth aspect.

[0111] In certain embodiments, the methods are used in a subject:

[0112] (1) Inhibit tumor cell proliferation;

[0113] (2) inhibiting tumor cell migration and / or invasion;

[0114] (3) Promote tumor cell apoptosis;

[0115] (4) downregulating the upstream protein p38, downstream protein PSF and / or heterogeneous nuclear ribonucleoprotein (hnRNP) A1 in the MNK1 / 2 signaling pathway in tumor cells;

[0116] (5) Upregulation of caspase-3, an apoptosis execution protein in tumor cells; and / or

[0117] (6) Inhibit the activity of tumor cells.

[0118] In certain embodiments, the tumor is selected from a solid tumor or a hematological tumor (eg, leukemia, lymphoma, myeloma).

[0119] In certain embodiments, the tumor is selected from glioma (e.g., glioblastoma), liver cancer, kidney cancer, melanoma, prostate cancer, colon cancer, cervical cancer, gastric cancer, lung cancer, colorectal cancer, bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, gastrointestinal cancer, pancreatic cancer, skin cancer, leukemia, sarcoma (e.g., osteosarcoma).

[0120] In certain embodiments, the method further comprises administering to the subject a second therapy selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof; optionally, the second therapy can be applied simultaneously, separately, or sequentially.

[0121] In an eighth aspect, the present application provides the use of the isolated nucleic acid molecule of the second aspect, the vector of the third aspect, the delivery composition of the fourth aspect, or the pharmaceutical composition of the fifth aspect for preparing an anti-tumor drug, or the use of the 3C protease, the isolated nucleic acid molecule of the second aspect, the vector of the third aspect, the delivery composition of the fourth aspect, or the pharmaceutical composition of the fifth aspect for inhibiting tumor cells; wherein, the 3C protease is as defined in the first aspect.

[0122] In certain embodiments, the tumor is selected from a solid tumor or a hematological tumor (eg, leukemia, lymphoma, myeloma).

[0123] In certain embodiments, the tumor is selected from glioma (e.g., glioblastoma), liver cancer, kidney cancer, melanoma, prostate cancer, colon cancer, cervical cancer, gastric cancer, lung cancer, colorectal cancer, bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, gastrointestinal cancer, pancreatic cancer, skin cancer, leukemia, sarcoma (e.g., osteosarcoma).

[0124] The pharmaceutical composition of the present application can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical composition of the present application should be sterile and stable under production and storage conditions. A preferred dosage form is an injection. Such an injection can be a sterile injectable solution. For example, a sterile injectable solution can be prepared by the following method: the pharmaceutical composition of the present application is mixed with the necessary dose in an appropriate solvent, and optionally, other desired ingredients (including but not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof) are mixed at the same time, followed by filtration sterilization. In addition, the sterile injectable solution can be prepared as a sterile lyophilized powder (for example, by vacuum drying or freeze drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable vehicle, such as sterile pyrogen-free water, before use.

[0125] In addition, the 3C protease, the nucleic acid molecule encoding the 3C protease, the vector or the delivery composition of the present application can be present in a pharmaceutical composition in the form of a unit dosage for easy administration.

[0126] The pharmaceutical composition of the present application can be used by any suitable method known in the art, including but not limited to, oral, oral, sublingual, eyeball, local, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, intravesical, local (such as, powder, ointment or drops), or nasal route. But, for many therapeutic uses, preferred route of administration / mode is parenteral administration (such as intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection, intratumoral injection). Technicians should understand that route of administration and / or mode will change according to intended purpose. In part preferred embodiments, the pharmaceutical composition of the present application is given by intravenous infusion or injection.

[0127] The pharmaceutical compositions of the present application may include a "therapeutically effective amount" or a "prophylactically effective amount" of the 3C protease of the present application, a nucleic acid molecule encoding the 3C protease, a vector or a delivery composition. A "prophylactically effective amount" refers to an amount sufficient to prevent, prevent, or delay the onset of a disease. A "therapeutically effective amount" refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount of the 3C protease of the present application, a nucleic acid molecule encoding the 3C protease, a vector or a delivery composition may vary depending on factors such as the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently.

[0128] In the present application, the dosage regimen can be adjusted to obtain the best intended response (e.g., therapeutic or preventive response). For example, a single dose can be administered, multiple doses can be administered over a period of time, or the dosage can be proportionally reduced or increased according to the urgency of the treatment situation.

[0129] In the present application, the subject may be a mammal, such as a human. In certain embodiments, the subject (eg, a human) suffers from a tumor, or has a risk of suffering from the above-mentioned disease.

[0130] Definition of terms

[0131] In this application, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the virology, biochemistry, and immunology laboratory procedures used herein are conventional procedures widely used in the respective fields. To facilitate a better understanding of this application, definitions and explanations of relevant terms are provided below.

[0132] When the terms "for example," "such as," "including," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0133] The terms "a" and "an" and "the" and similar referents in the context of describing this application (especially in the context of the following claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0134] As used herein, the term "and / or" should be considered as a specific disclosure of each of two or more specified features or elements, as well as any combination of two or more features or elements. Thus, the term "and / or" used in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone).

[0135] As used herein, the term "picornavirus" refers to a virus belonging to the Picornaviridae family, a family of non-enveloped, icosahedral capsid RNA viruses that includes the genera Enterovirus, Aphthousvirus, and others.

[0136] As used herein, the term "enterovirus" refers to viruses belonging to the genus Enterovirus, which includes poliovirus, coxsackievirus, rhinovirus, enterocytopathic human orphan virus (ECHO virus) and novel enterovirus. Among them, the serotyping of enterovirus can be found in, T, Hovi T, Knowles NJ, Stanway G. Classification of enteroviruses based on molecular and biological properties. J Gen Virol. 1997; 78(Pt 1): 1-11., which is incorporated herein by reference in its entirety.

[0137] As used herein, the term "oral virus" refers to a virus belonging to the genus Oral Virus, which includes foot-and-mouth disease virus, bovine rhinitis type A virus, and bovine rhinitis type B virus. For example, see: Schoch, Conrad L et al. "NCBI Taxonomy: a comprehensive update on curation, resources and tools." Database: the journal of biological databases and curation vol. 2020(2020): baaa062., the entire text of which is incorporated herein by reference.

[0138] As used herein, the term "p38 protein" refers to p38 mitogen-activated protein kinase, which has a meaning generally understood by those skilled in the art. In certain embodiments, the Uniprot number of the p38 protein described herein is Q15759. In certain embodiments, the p38 protein described herein has: (i) an amino acid sequence corresponding to the Uniprot number, or (ii) an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to (i).

[0139] As used herein, the term "PSF" refers to a PTB (polypyrimidine tract binding protein)-related splicing factor, which has the meaning generally understood by those skilled in the art. In certain embodiments, the PSF protein described herein has a Uniprot number of P23246. In certain embodiments, the PSF protein described herein has: (i) an amino acid sequence corresponding to the Uniprot number, or (ii) an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to (i).

[0140] As used herein, the term "caspase-3" refers to cysteine-aspartic acid protease 3, which has the meaning generally understood by those skilled in the art. In certain embodiments, the caspase-3 described herein has the Uniprot number P42574. In certain embodiments, the caspase-3 described herein has: (i) an amino acid sequence corresponding to the Uniprot number, or (ii) an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to (i).

[0141] As used herein, the term "identity" refers to the match between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match). Typically, two sequences are compared when they are aligned for maximum identity. Such an alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0142] As used herein, the term "mtRNR1 (mitochondrial 12S ribosomal RNA) and AES (Amino-terminal enhancer of split)-based 3'UTR" has the meaning commonly understood by those skilled in the art, and generally refers to a 3'UTR comprising a mitochondrial 12S rRNA-related sequence and an AES (Amino-terminal enhancer of split) mRNA sequence. For example, exemplary 3'UTRs can be found in Orlandini von Niessen AG, et al. Improving mRNA-Based Therapeutic Gene Delivery by Expression-Augmenting 3'UTRs Identified by Cellular Library Screening. Mol Ther. 2019 Apr 10; 27(4): 824-836. doi: 10.1016 / j.ymthe.2018.12.011., the entire text of which is incorporated herein by reference.

[0143] The twenty conventional amino acids referred to herein are compiled according to conventional usage. See, for example, Immunology—A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this application, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in this application, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0144] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc. Stabilizers have the meanings generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), etc.

[0145] As used herein, the term "prevention" refers to the method implemented in order to prevent or delay the occurrence of disease or illness or symptom (for example, tumor) in a subject. As used herein, the term "treatment" refers to the method implemented in order to obtain beneficial or required clinical results. For the purpose of the application, beneficial or required clinical results include but are not limited to, alleviating symptoms, narrowing the scope of the disease, stabilizing (that is, no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease and alleviating symptoms (no matter in part or in whole), no matter it is detectable or undetectable. In addition, "treatment" can also refer to, compared with the expected survival (if not receiving treatment), extending the survival.

[0146] The terms "cancer" and "tumor" are used interchangeably to refer to a broad category of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division can lead to the formation of malignant tumors, or cells that invade neighboring tissues and may spread to distant parts of the body via the lymphatic system or bloodstream. Cancer includes both benign and malignant cancers, as well as dormant tumors or micrometastases. Cancer also includes hematologic malignancies.

[0147] Advantageous Effects of the Invention

[0148] The method for preventing and / or treating tumors provided in this application based on a small RNA virus (such as enterovirus type 71, coxsackievirus A14) 3C protease or a nucleic acid molecule encoding the 3C protease has a broad spectrum and significant tumor cell killing ability and has great clinical application value.

[0149] The embodiments of the present application will be described in detail below with reference to the accompanying drawings and examples, but those skilled in the art will understand that the following drawings and examples are only used to illustrate the present application and are not intended to limit the scope of the present application. Based on the following detailed description of the drawings and preferred embodiments, various objects and advantages of the present application will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0150] FIG1 shows the vector sequence of EV-3C-mRNA and the in vitro transcription vector.

[0151] FIG2 shows that EV-3C-mRNA can inhibit the proliferation of glioblastoma cells.

[0152] FIG3 shows that EV-3C-mRNA inhibits the migration and invasion of glioblastoma cell lines.

[0153] FIG4 shows that EV-3C protein induces the upregulation of Cleaved Caspase-3 and activates the apoptosis signaling pathway.

[0154] FIG5 shows that EV-3C-mRNA can induce apoptosis of human glioblastoma cells.

[0155] Figure 6 shows that EV-3C protein downregulated multiple proteins in the MNK1 / 2 signaling pathway.

[0156] FIG7 shows that EV-3C-mRNA can inhibit the cell viability of glioblastoma cells.

[0157] FIG8 shows a schematic diagram and physical characterization of EV-3C-mRNA lipid nanoparticle (LNP) encapsulation.

[0158] FIG9 shows the in vivo delivery capability evaluation of lipid nanoparticles.

[0159] FIG10 shows that EV-3C-mRNA-LNP can inhibit the growth of orthotopic brain tumors in mice and prolong the survival time of mice.

[0160] FIG11 shows the in vivo safety evaluation of EV-3C-mRNA lipid nanoparticles.

[0161] FIG12 shows the evaluation of EV-3C-mRNA on cell viability of other tumor cell lines.

[0162] Figure 13 shows the effect of EV-3C-mRNA on the IC of different cell lines 50 Inhibition rate and therapeutic index.

[0163] Sequence information

[0164] A description of the sequences involved in this application is provided in the table below.

[0165] Table 1: Sequence information DETAILED DESCRIPTION

[0166] The present application will now be described with reference to the following examples which are intended to illustrate the present application (but not to limit the present application).

[0167] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in this application are basically based on the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Ausubel et al., Molecular Biology: A Laboratory Manual, 3rd edition, John Wiley & Sons, Inc., 1995. The use of restriction endonucleases follows the conditions recommended by the product manufacturers. It is understood by those skilled in the art that the examples describe this application by way of example and are not intended to limit the scope of protection claimed in this application.

[0168] Example 1 In vitro transcription and synthesis of EV-3C-mRNA

[0169] Experimental materials: SapI restriction endonuclease used in the experiment was purchased from NEB (catalog number: R0569S), T7 RNA polymerase was purchased from Thermo (catalog number: EP0111), ATP was purchased from Thermo (catalog number: R0441), CTP was purchased from Thermo (catalog number: 18331017), GTP was purchased from Thermo (catalog number: 18332015), UTP was purchased from Thermo (catalog number: R1471), Nuclease-free water was purchased from Thermo (catalog number: 10977015), Capping buffer was purchased from Thermo (catalog number: J62446.AP), S-adenosyl methionine (SAM) was purchased from NEN (catalog number: B9003S), Vaccinia mRNA capping enzyme was purchased from NEB (catalog number: M2080S), DNase I was purchased from Thermo (catalog number: 18047019), Dynabeads 1.5xRNA Binding Buffer was purchased from Yisheng (catalog number: A33562), Dynabeads Myone Carboxylic Acid was purchased from Yisheng (catalog number: 65011), and Ambion TM RNA storage solution was purchased from Thermo Fisher Scientific (Cat. No. AM7000). The gene thermal cycler was purchased from Hangzhou Boheng Technology Co., Ltd. (model: AUTO-96). The ultra-micro-volume UV-Vis spectrophotometer was purchased from Thermo Scientific (Cat. No. 840-317400). The thermomixer (refrigerated) was purchased from Lindbergh Instruments (Cat. No. BE-3200). The refrigerated centrifuge was purchased from Eppendorf (Model: 5424R). The dry block was purchased from Hangzhou Aosheng Instrument Co., Ltd. (Model: K30).

[0170] The experimental plan is as follows:

[0171] (1) DNA template generation

[0172] The template sequence was designed based on codon bias, GC content, and the thermodynamic stability of the RNA secondary structure. The DNA sequence of the EV-3C coding region (whose amino acid sequence is shown in SEQ ID NO:1 and is derived from EV71 or Coxsackievirus A14) was then cloned into the in vitro transcription vector pmRVac to generate the DNA template for in vitro transcription. The pmRVac vector contains a T7 promoter sequence, a 5' untranslatable region (UTR), a 3' untranslatable region (UTR), a 110 nt poly A segment, and a SapI restriction endonuclease site. The insertion site is located between the UTRs. The reaction product was transformed into Escherichia coli and plated on LB plates containing kanamycin. The resulting colonies were screened by PCR to confirm positive insertion events. PCR-positive clones were further evaluated by restriction enzyme digestion and Sanger sequencing. Correct plasmids were cultured in LB medium and purified using an endotoxin-free plasmid Midprep kit. Plasmid DNA concentration and purity were determined using a UV-visible spectrophotometer and then linearized with the SapI restriction endonuclease.

[0173] (2) mRNA synthesis

[0174] Cap1 mRNA was generated by T7 in vitro transcription followed by enzymatic capping and methylation. Uncapped RNA transcripts were prepared by in vitro transcription by mixing a linear DNA template with T7 RNA polymerase, nucleotide triphosphates (NTPs), and a magnesium-containing buffer. The reaction mixture was incubated at 37°C for 2 hours. For the synthesis of N1-methyl pseudouridine (m1ψ)-modified transcripts, m1ψTP (N1-methyl-pseudouridine triphosphate) was used in place of UTP in the in vitro transcription.

[0175] Cap1 mRNA was prepared using the vaccinia capping system. After heat denaturation of the uncapped transcript, the capping reaction was performed by adding vaccinia virus capping enzyme, 2'-O methyltransferase, GTP, S-adenosylmethionine (SAM), and capping buffer. The reaction mixture was incubated at 37°C for 1 hour. Subsequently, the template DNA was removed by DNase I treatment. The capped transcript was further purified using magnetic beads. The reaction mixture was incubated at 37°C for 1 hour. Subsequently, the template DNA was removed by DNase I treatment. The capped transcript was further purified using magnetic beads. The isolated mRNA was eluted with an acidic buffer and stored at -80°C. The mRNA concentration and purity were determined using a UV-visible spectrophotometer. The mRNA integrity was determined by denaturing agarose gel analysis. Finally, mRNA sequencing was used to identify the mRNA molecules. The nucleotide sequence of the resulting mRNA molecule is shown in SEQ ID NO:2, where the coding sequence of EV-3C is shown in SEQ ID NO:3.

[0176] The sequence of the in vitro transcription vector is shown in Figure 1A. Figure B is a schematic diagram of the in vitro transcription (IVT) vector backbone. Sanger sequencing confirmed the nucleic acid sequence, demonstrating 100% identity between the plasmid backbone insert and the reference sequence. mRNA concentration was quantified using a UV-visible spectrophotometer and was found to be 1553 ng / μl. The OD260 / 280 ratio obtained using the UV-visible spectrophotometer was 2.06.

[0177] Example 2 Detection of the effect of EV-3C-mRNA on cell proliferation based on EdU method

[0178] This example is based on the EV-3C-mRNA prepared in Example 1.

[0179] Experimental materials: The human glioblastoma cells U87 MG used in the experiment were purchased from ATCC (catalog number: HTB-14), A172 were purchased from ATCC (catalog number: CRL-1620) and human embryonic lung fibroblasts MRC-5 were purchased from ATCC (catalog number: CCL-171). The above cells were cultured in a cell culture incubator at 37°C and 5% CO2. The complete culture medium used for cell growth was DMEM high glucose medium (purchased from Gibco, catalog number: 11995065) and MEM medium (purchased from Gibco, catalog number: 10370021) supplemented with 10% fetal bovine serum (purchased from Gibco, catalog number: 16000044) and penicillin-streptomycin double antibody (purchased from Gibco, catalog number: 2321152). BeyoClick TM EdU-488 cell proliferation detection kit (BeyoClick TM EdU Cell Proliferation Kit with Alexa Fluor 488) was purchased from Bio-Tech (Product No.: C0071S). 2000 (Thermo Fisher Scientific catalog number: 11668-019).

[0180] The experimental plan is as follows:

[0181] The detection of cell proliferation ability is a basic method for evaluating cell activity, genotoxicity and anti-tumor drug effects. The most recognized and accurate method for detecting cell proliferation is to directly detect DNA synthesis in cells. TMEdU-488 Cell Proliferation Detection Kit, EdU (5-ethynyl-2'-deoxyuridine), is 5-ethynyl-2'-deoxyuridine. EdU is a thymidine analog that can replace T in the period of cell proliferation and infiltrate into the replicating DNA molecule. The specific reaction of fluorescent dyes detects DNA replication activity, and the detection of EdU labeling can accurately reflect the cell proliferation.

[0182] 5 × 10 5 After the cells were cultured overnight and returned to normal state, the medium was changed and EV-3C-mRNA transfection was performed. U87 MG, A172, and MRC5 cells were transfected with 2.5 μg, 3 μg, and 3.5 μg of 3C-mRNA using 2000 transfection reagent. EdU labeling was performed by preheating 2x EdU solution and then adding it to an equal volume of culture medium containing experimental cells to obtain a 1x EdU solution. Treated cells were incubated at 37°C for 2 hours before fixation and permeabilization. The click reaction was prepared in a 500 μl volume per well of a 6-well plate, containing 430 μl of Click Reaction Buffer, 20 μl of CuSO₄, 1 μl of Azide 488, and 50 μl of Click Additive Solution. Incubate at room temperature in the dark for 30 minutes. Aspirate the click reaction solution and wash three times with wash buffer for 3-5 minutes each. To monitor cell proliferation, use Hoechst 33342 for nuclear staining. Prepare a 1X Hoechst 33342 solution by diluting Hoechst 33342 (1000X) in PBS at a 1:1000 ratio. Add 1 ml of 1X Hoechst 33342 solution to each well and incubate in the dark for 10 minutes at room temperature. Aspirate the 1X Hoechst 33342 solution. Wash the wells three times with wash buffer for 3-5 minutes each. Fluorescence detection is then performed. Hoechst 33342 emits blue fluorescence with a maximum excitation wavelength of 346 nm and a maximum emission wavelength of 460 nm.

[0183] Data Analysis: Statistical significance was calculated using analysis of variance (ANOVA). Data are presented as mean ± standard error. A p < 0.05 indicated statistical significance.

[0184] The results of EdU cell proliferation assays of different cell lines treated with EV-3C mRNA are shown in Figure 2 and Table 2. Figure 2, Panel A, shows that the EdU / DAPI ratio of cell proliferation in U87 MG cells decreased significantly with increasing EV-3C mRNA transfection doses. Panel B shows that the EdU / DAPI ratio of cell proliferation in A172 cells decreased in a dose-dependent manner with increasing EV-3C mRNA transfection doses. Panel C shows that the EdU / DAPI ratio of cell proliferation in MRC5 cells did not change with increasing EV-3C mRNA transfection doses.

[0185] In summary, EV-3C-mRNA can inhibit the proliferation of various glioblastomas, and the inhibitory effect is dose-dependent.

[0186] Table 2. Results of EdU cell proliferation assay of different cell lines using EV-3C-mRNA

[0187] Example 3 Transwell assay to detect the effect of EV-3C-mRNA on the migration and invasion ability of human glioblastoma cell lines

[0188] This example is based on the EV-3C-mRNA prepared in Example 1.

[0189] Experimental Materials: Human glioblastoma cells U87 MG were purchased from ATCC (catalog number: HTB-14), A172 cells were purchased from ATCC (catalog number: CRL-1620), and MRC-5 human embryonic lung fibroblasts were purchased from ATCC (catalog number: CCL-171). These cells were cultured in a cell culture incubator at 37°C and 5% CO2. The complete culture media used for cell growth were DMEM high-glucose medium (purchased from Gibco, catalog number: 11995065) and MEM medium (purchased from Gibco, catalog number: 10370021) supplemented with 10% fetal bovine serum (purchased from Gibco, catalog number: 16000044) and penicillin-streptomycin (purchased from Gibco, catalog number: 2321152). (Cat. No. 07200174); Corning Matrix (Article No.: 356234).

[0190] The experimental plan is as follows:

[0191] For the cell migration and invasion assay, a Transwell chamber was placed in a culture plate. The inner chamber was called the upper chamber, and the inner chamber of the culture plate was called the lower chamber. The upper and lower culture media were separated by a polycarbonate membrane. Cells were seeded in the upper chamber. Since the polycarbonate membrane is permeable, cells transfected with EV-3C-mRNA and the control group were seeded in the upper chamber. Cells that migrated to the lower membrane were stained and photographed to assess their effects on cell migration and invasion.

[0192] 1. U87 MG and A172 cells were transfected with different concentrations of EV-3C mRNA and incubated overnight for 12 hours. The cells were harvested and resuspended in FBS-free medium.

[0193] 2. Cells (1×10 per well) 4 ~2×10 4 Cells were seeded in the upper chamber of a Transwell plate (the upper chamber of the Transwell for cell invasion was coated with Matrigel). 0.6 ml of DMEM + 10% fetal bovine serum was added to the plate wells (lower chamber) as an attractant and incubated at 37° C. for 16 hours.

[0194] 3. Fix the cells with methanol for 15 minutes and then stain with 1% crystal violet for 20 minutes. Wash the membrane in PBS for a few seconds to remove excess dye.

[0195] 4. Allow the membrane to dry completely and count the cells under a microscope. Randomly select different views and take the average count.

[0196] The results of EV-3C-mRNA inhibition of glioblastoma cell migration and invasion are shown in Figure 3 and Table 3. Panel A in Figure 3 shows that with increasing transfection doses, the number of cells migrating from the upper chamber to the lower chamber of U87 MG and A172 cells gradually decreased. Panels B and C show the cell counts under microscopic fields of view for U87 MG and A172 cells, respectively. Panel D shows that with increasing transfection doses, the number of cells invading from the upper chamber to the lower chamber of U87 MG and A172 cells gradually decreased. Panels E and F show the cell counts under microscopic fields of view for U87 MG and A172 cells, respectively.

[0197] In summary, EV-3C-mRNA can inhibit the migration and invasion of various glioblastoma cell lines, and the inhibitory effect is dose-dependent.

[0198] Table 3. Transwell assay to verify the inhibition of EV-3C-mRNA on migration and invasion of different cell lines

[0199] Example 4 Immunofluorescence assay to detect that EV-3C-mRNA can induce apoptosis of human glioblastoma cells

[0200] Experimental Materials: Human glioblastoma cells U87 MG (catalog number: HTB-14) and A172 (catalog number: CRL-1620) were purchased from ATCC during the experiment. These cells were cultured in a cell culture incubator at 37°C and 5% CO2. The complete culture media used for cell growth were DMEM high-glucose medium (purchased from Gibco, catalog number: 11995065) and MEM medium (purchased from Gibco, catalog number: 10370021) supplemented with 10% fetal bovine serum (purchased from Gibco, catalog number: 16000044) and penicillin-streptomycin (purchased from Gibco, catalog number: 2321152). Enterovirus 71 3C Antibody (B3) was purchased from Gene Tex (Cat. No. GTX630191); Cleaved Caspase-3 (Asp175) Antibody was purchased from CST (Cat. No. #9661); Anti-Rabbit IgG (H+L) Alexa Flour was purchased from Invitrogen (Cat. No. A16098); Anti-Mouse IgG (H+L) Alexa Flour was purchased from Invitrogen (Cat. No. A16013).

[0201] The experimental plan is as follows:

[0202] On the first day, U87 MG and A172 cells were plated at 1.0×10 4Cells / well / 100 μL were seeded in a black-bottomed 96-well plate and placed in a 37°C 5% CO2 cell culture incubator overnight for adherence culture. The next day, the pre-seeded black-bottomed 96-well plate was removed from the cell culture incubator and the medium was replaced with 200 μL. The experimental group was transfected with EV-3C-mRNA (n=6; 100 ng / well) and cultured in a cell culture incubator overnight. The samples were fixed with formaldehyde at a final concentration of 4% for 0.5 h, with 100 μL per well. The solution was discarded and washed three times with 200 μL / well PBS. The solution was discarded and a solution containing a final concentration of 1 μM was added. Hoechst33342 (nuclear dye) (blue fluorescence) in PBS, 200 μL / well, incubate at 37°C for 20-30 min; discard the solution, add 100 μL / well 1× membrane permeabilization solution (PBS solution containing 0.1% TritonX-100), incubate at room temperature in the dark for 0.5 h; discard the solution, wash 3 times with 200 μL / well PBS; discard the solution, add 100 μL / well 1× blocking solution (PBS solution containing 5% BSA) and incubate at 37°C for 0.5 h; prepare EV-3C antibody and Cleaved Caspase-3 antibody, dilute the antibody 400 times with 1× blocking solution, mix thoroughly; discard the solution, add 40 μL / well of the above-diluted primary antibody working solution, be careful to avoid bubbles, incubate at room temperature in the dark for 2 h, or incubate at 37°C in the dark for 1 h; recover the primary antibody, add 100 μL / well 1× blocking solution and wash 3 times; prepare sufficient secondary antibody, and Dilute IgG (H+L) Alexa Flour series secondary antibodies and Anti-Mouse IgG (H+L) Alexa Flour series secondary antibodies 500-fold with 1× blocking buffer and mix thoroughly; discard the solution and add 50 μL / well secondary antibody working solution. Be careful not to overlap the fluorescent color of the selected secondary antibody with other colors in this experiment and avoid bubbles. Incubate in the dark at room temperature for 1 hour or at 37°C for 0.5 hour; discard the solution, wash three times with 200 μL / well PBS, and add 200 μL / well PBS; analyze the cells.

[0203] The results, shown in Figure 4, demonstrate that EV-3C mRNA transfection induced upregulation of Cleaved Caspase-3 and activated the apoptotic signaling pathway. Figure A shows immunofluorescence staining of U87 MG cells 24 hours after transfection with EV-3C mRNA. EV-3C protein fluoresces green, while Cleaved Caspase-3 fluoresces red. Furthermore, after merging, 3C and Cleaved Caspase-3 proteins overlap. Figure B shows fluorescence staining of A172 cells. The significant contrast between the EV-3C mRNA-transfected and PBS control groups in both cell lines indicates that 3C can induce apoptosis and activate Cleaved Caspase-3.

[0204] In summary, EV-3C-mRNA promotes apoptosis of glioblastoma by inducing upregulation of Cleaved Caspase-3.

[0205] Example 5 Annexin V-FITC apoptosis assay detected that EV-3C-mRNA can promote apoptosis of human glioblastoma cells

[0206] This example is based on the EV-3C-mRNA prepared in Example 1.

[0207] Experimental Materials: Human glioblastoma cells used in the experiments were purchased from ATCC (catalog number: HTB-14), A172 cells were purchased from ATCC (catalog number: CRL-1620), and U118MG cells were purchased from ATCC (catalog number: HTB-15). These cells were cultured in a cell culture incubator at 37°C with 5% CO₂. The complete culture media used for cell growth were DMEM high-glucose medium (Gibco, catalog number: 11995065) and MEM medium (Gibco, catalog number: 10370021) supplemented with 10% fetal bovine serum (Gibco, catalog number: 16000044) and penicillin-streptomycin (Gibco, catalog number: 2321152). Annexin V-FITC apoptosis detection kit (BYT product number: C1062M) was used.

[0208] The experimental plan is as follows:

[0209] Phosphatidylserine is primarily distributed on the inner side of the cell membrane, adjacent to the cytoplasm. Early in apoptosis, different cell types externalize phosphatidylserine to the cell surface, the outer side of the cell membrane. Exposed phosphatidylserine on the cell surface promotes coagulation and inflammation. Annexin V binds to externalized phosphatidylserine on the cell surface and blocks its procoagulant and proinflammatory activities. Annexin V labeled with the green fluorescent probe FITC (Annexin V-FITC) allows for straightforward and simple detection of phosphatidylserine externalization, a key hallmark of apoptosis, using flow cytometry or fluorescence microscopy.

[0210] Cells were cultured in black, transparent 96-well plates. The experimental group was transfected with EV-3C-mRNA (n=3; 100ng / well). After apoptosis induction, the cells were centrifuged at 1000g for 5 minutes. The cell culture medium was aspirated, and the cells were washed once with PBS. The cells were centrifuged at 1000g for 5 minutes before aspirating the PBS. 195μl of Annexin V-FITC conjugate solution was added. 5μl of Annexin V-FITC was added and the cells were gently mixed. 10μl of propidium iodide staining solution was added and the cells were gently mixed. Incubate at room temperature (20-25°C) in the dark for 10-20 minutes, then place in an ice bath. Be sure to use aluminum foil to protect the cells from light. Observe the cells under a fluorescence microscope. Annexin V-FITC will emit green fluorescence, and propidium iodide (PI) will emit red fluorescence. Note: The cells should be tested as soon as possible after staining, usually within 1 hour.

[0211] Annexin V-FITC and propidium iodide (PI) staining results are shown in Figure 5. Figures A to C show the staining results for U87 MG, A172, and U118 MG cells, respectively. Cells stained only with green fluorescence indicate apoptotic cells, cells stained with both green and red fluorescence indicate necrotic cells, and cells unstained with fluorescence indicate normal cells. In Figures A, B, and C, both Annexin V-FITC and propidium iodide staining in the Ctrl group were very weak, while U87 MG, A172, and U118 MG cells appeared normal. In contrast, the 3C mRNA-treated group showed significant apoptotic and necrotic cell counts.

[0212] In summary, EV-3C-mRNA can promote apoptosis of various glioblastoma cells.

[0213] Example 6: Western blotting verifies that EV-3C can downregulate proteins related to the MAPK signaling pathway in tumor cells

[0214] This example is based on the EV-3C-mRNA prepared in Example 1.

[0215] Experimental Materials: Human glioblastoma cells U87 MG (catalog number: HTB-14) and A172 (catalog number: CRL-1620) were purchased from ATCC. These cells were cultured at 37°C in a cell culture incubator with 5% CO2. The complete culture media used for cell growth were DMEM high-glucose medium (Gibco, catalog number: 11995065) and MEM medium (Gibco, catalog number: 10370021) supplemented with 10% fetal bovine serum (Gibco, catalog number: 16000044) and penicillin-streptomycin (Gibco, catalog number: 2321152). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody was purchased from abcam (catalog number: ab205719). Blocking skim milk powder was purchased from Prilai (catalog number: P1622), BCA protein quantification kit was purchased from Prilai (catalog number: P1511), RIPA lysis buffer was purchased from Prilai (catalog number: C1053), ECL chemiluminescence solution P1000 was purchased from Prilai (catalog number: P1000-25), Enterovirus 71 3C Antibody (B3) was purchased from NB (catalog number: NBP3-13501), Caspase-3 (D3R6Y) Rabbit mAb was purchased from CST (catalog number: #14220), hnRNP A1 (K350) Antibody was purchased from CST (catalog number: #4296), p38 MAPK Antibody was purchased from CST (catalog number: #9212), and PSF antibody (D-8) was purchased from Santa Cruz Biotechnology (sc-271796).

[0216] Preparation of reagents: SDS-PAGE gel electrophoresis buffer solution: accurately weigh 3.03g Tris base, 18.7g glycine, 1g SDS, and add ultrapure water to make up to 1L; TBST solution: accurately weigh 3g Tris base, 8g NaCl, and add ultrapure water to make up to 1L, which is TBS solution. Add 0.1% Tween-20 to prepare a TBST solution. Western Blot blocking buffer: Accurately weigh 2.5g BSA and add it to 50ml TBST to create a blocking buffer containing 5% BSA. 10x Transfer Buffer: Accurately weigh 30.3g tris-base and 144g glycine and dilute to 1L with ultrapure water. 1x Transfer Buffer: Add 200ml methanol and 700ml ultrapure water to 100ml of 10x Transfer Buffer. Separating Gel: Mix HO, 30% Acr-Bis (29:1), and Resolving Gel Buffer (pH 8.8), then add 10% APS and TEMED and mix thoroughly. Stacking Gel: Mix HO, 30% Acr-Bis (29:1), and Stacking Gel Buffer (pH 6.8), then add 10% APS and TEMED and mix thoroughly.

[0217] The experimental plan is as follows:

[0218] The MAPK pathway has four main branch routes: ERK, JNK, p38 / MAPK and ERK5. Among them, JNK and p38 have similar functions and are related to inflammation, apoptosis and growth; ERK is mainly responsible for cell growth and differentiation, and its upstream signal is the famous Ras / Raf protein. The mitogen-activated protein kinase MAPK signaling pathway is a very important information transmission chain in cells. It is composed of Ser / Thr protein kinases widely expressed in cells and participates in a series of cell physiological activities such as cell growth, development, differentiation, and apoptosis. It is an associated factor in inducing tumor formation. ERK and p38 intersect at MNK1 / 2, and PSF, eIF4E, hnRNPA1, cPLA2, Spry2 and other proteins downstream of MNK1 / 2 are all highly upregulated proteins in tumor cells. The detection method for EV-3C downregulation of MAPK signaling pathway-related proteins in tumor cells is as follows:

[0219] Treat cells with EV-3C mRNA and set up corresponding blank controls. After 12 hours of incubation, remove the culture medium, wash once with PBS, and add 150-250 μl of lysis buffer to each well of a 6-well plate. Pipet several times with a pipette to ensure thorough contact between the lysate and cells. After complete lysis, centrifuge at 10,000-14,000 g for 3-5 minutes. Remove the supernatant and proceed with subsequent PAGE, Western blotting, and immunoprecipitation. Add 3 μl of the protein collected in the previous step to 57 μl of PBS and dilute 20-fold. Mix BCA Reagent and Cu Reagent (50:1). Add 200 μl of this BCA mixture to a 96-well plate and add 25 μl of the protein sample. Set up a replicate well and incubate at 37°C for 0.5 hour. Analyze using a multi-function microplate reader and calculate the protein concentration based on the standard curve. After calculation, add lysis buffer to the corresponding protein to be tested to the same concentration. Then add 5× Loading Buffer for subsequent loading.

[0220] Install the vertical electrophoresis glass plate and prepare the SDS-PAGE separating gel and stacking gel according to the recipe.

[0221] Add the prepared separation gel to the glass plate to 1.5 cm from the upper edge of the short plate. Gently add reverse osmosis water to the upper edge of the short plate and polymerize at room temperature for 25 to 30 minutes. After the separation gel is polymerized, slowly pour off the reverse osmosis water on the separation gel and carefully absorb it with absorbent paper. Add the prepared stacking gel, insert a suitable sample comb, and polymerize at room temperature for 25 to 30 minutes. After the stacking gel is polymerized, install the Mini-PROTEAN vertical electrophoresis device. Add freshly prepared 1× electrophoresis buffer to immerse the upper edge of the inner tank glass short plate. Carefully load 25μg protein marker and sample protein in each well. Replenish the electrophoresis buffer, check the electrophoresis device, and connect the circuit. First, perform constant voltage electrophoresis at 60V. When the bromophenol blue dye leaves the stacking gel, increase the voltage to 80V constant voltage electrophoresis and stop electrophoresis when the bromophenol blue dye reaches the bottom of the separation gel.

[0222] Pre-activate the PVDF membrane by immersing it in anhydrous methanol for 1 minute. Then, equilibrate it with the transfer sponge and thick and thin filter papers in 1× transfer buffer for 15 minutes. Meanwhile, place the Mini Trans-Blot transfer chamber in an ice bath and pre-chill with an appropriate amount of freshly prepared 1× transfer buffer. After electrophoresis, dismantle the vertical electrophoresis apparatus. Use a gel scraper to pry open the glass plates and carefully transfer the gel to a bubble-free sponge-thick filter paper-thin filter paper structure. Gently roll the gel surface with a glass rod to remove bubbles, creating a transfer sandwich. Place the prepared transfer sandwich (from positive to negative, in the order of filter paper: membrane: gel: filter paper) into the Mini Trans-Blot transfer chamber and fill it with 1× transfer buffer to cover the sandwich. Check the electrotransfer apparatus, set the appropriate current and transfer time, connect the circuit, and begin electrotransfer at 4°C, 300 mA, for 1.5 hours. After electrotransfer, turn off the power and dismantle the transfer apparatus. Carefully unwrap the transfer sandwich, position the PVDF membrane protein-side up, mark the membrane, and transfer it to a wash tray containing an appropriate amount of 1× TBST buffer. Rinse with agitation at room temperature for 5 minutes. Pour off the buffer from the wash tray and add an appropriate amount of fresh blocking buffer. Block with agitation at room temperature for 1 hour. After blocking, rinse three times with an appropriate amount of 1× TBST buffer at room temperature for 5 minutes each. Transfer the PVDF membrane to an incubation tray containing an appropriate amount of primary antibody working solution diluted 1:1 (refer to the antibody data sheet for 1000-2000 dilutions) and incubate at 4°C with agitation overnight (>12 hours). After incubation with the primary antibody, rinse three times with an appropriate amount of 1× TBST buffer at room temperature for 5 minutes each. Transfer the PVDF membrane to an incubation tray containing an appropriate amount of secondary antibody working solution diluted 1:4000 (refer to the antibody data sheet for 1000-2000 dilutions) and incubate with agitation at 4°C overnight (>12 hours). After incubation with the primary antibody, rinse three times with an appropriate amount of 1× TBST buffer at room temperature for 5 minutes each. Transfer the PVDF membrane to an incubation tray containing an appropriate amount of secondary antibody working solution diluted 1:4000 (refer to the antibody data sheet for 1000-2000 dilutions) and incubate with agitation at room temperature for 1 hour. After secondary antibody incubation, rinse three times with 1× TBST buffer at room temperature for 5 minutes each wash. Transfer the PVDF membrane to an ECL luminescence incubation chamber. Trim the transferred PVDF membrane to an appropriate size and place it in the antibody incubation chamber. Apply an appropriate amount of ECL luminescence reagent to the protein side of the PVDF membrane and place it in the ChemiDoc XRS+ imaging system for luminescence detection.

[0223] The experimental results are shown in Figure 6, where Figure A shows the Western blotting results of the U87 MG cell line and Figure B shows the Western blotting results of the A172 cell line. The results showed that EV-3C protein downregulated the upstream protein p38 and the downstream proteins PSF (PTB (polypyrimidine tract binding protein)-related splicing factor) and hnRNPA1 in the MNK1 / 2 signaling pathway, and promoted the upregulation of the apoptosis execution protein caspase-3 downstream of hnRNPA1. Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is an RNA-binding protein that shuttles between the nucleus and cytoplasm and participates in various RNA metabolic processes, such as pre-mRNA splicing and RNA transport. hnRNP A1 is also an IRES trans-acting factor (ITAF). Binding to the IRES of apaf-1 can prevent its translation, thereby inhibiting apoptosis. Apaf-1, also known as apoptotic protease activating factor-1 (APAF-1), plays a key role in the mitochondrial apoptosis pathway. This protein combines cytochrome c with deoxyadenosine triphosphate (dATP) to form an oligomeric apoptosome. This apoptosome manifests its mature activation by binding to or cleaving the apoptotic protein 9 (CP9) preproprotein. Activated CP9 stimulates subsequent CPs to induce apoptosis.

[0224] In summary, EV-3C broadly cleaves multiple key proteins in the MNK1 / 2 signaling pathway.

[0225] Example 7 EV-3C-mRNA can inhibit the cell viability of glioblastoma cells

[0226] This example is based on the EV-3C-mRNA prepared in Example 1.

[0227] Experimental Materials: Human glioblastoma cells U87 MG (catalog number: HTB-14) and A172 (catalog number: CRL-1620) were purchased from ATCC during the experiment. These cells were cultured in a 37°C, 5% CO2 cell culture incubator. The complete culture media used for cell growth were DMEM high-glucose medium (Gibco, catalog number: 11995065) and MEM medium (Gibco, catalog number: 10370021) supplemented with 10% fetal bovine serum (Gibco, catalog number: 16000044) and penicillin-streptomycin (Gibco, catalog number: 2321152). Rupintrivir (AG7088) was purchased from MCE (catalog number: HY-106161). Cell-Titer Luminescent Cell Viability Assay Solution was purchased from Promega (Cat. No. G7572). Microplate reader was purchased from Molecular Devices (model: SpectraMax M5).

[0228] The experimental plan is as follows:

[0229] (1) Evaluation of EV-3C-mRNA on the viability of U87 MG and A172 cells

[0230] U87 MG cells and A172 cells were cultured at 1.0×10 4 The cells were inoculated with a density of 100 μg / ml in a 96-well plate with a white wall and a transparent bottom and cultured at 37°C and 5% CO2 for 24 hours. EV-3C-mRNA was then diluted with Opti-MEM to a final concentration of 998.4 ng / ml, 499.2 ng / ml, 249.6 ng / ml, 124.8 ng / ml, 62.4 ng / ml, 31.2 ng / ml, and 15.6 ng / ml and transfected into the cells using Lipo2000 liposomes. After 48 hours, the supernatant was discarded and the diluted Cell-Titer Luminescent Cell Viability assay solution, shake and lyse for 5 minutes in the dark, let it stand for 3 minutes, and finally measure the luminescence value of each well using a microplate reader. Cell viability (%) = experimental group / average value of cell control group × 100

[0231] (2) Rupintrivir (AG7088) can prevent EV-3C-mRNA from inhibiting the viability of U87 MG and A172 cells

[0232] Rupintrivir (AG7088) is a potent, selective and irreversible 3C protease inhibitor. U87 MG cells and A172 cells were cultured at 1.0×10 4 The cells were inoculated at a density of 100 μg / ml in a 96-well plate with a white wall and a transparent bottom and cultured at 37 °C and 5% CO2 for 24 hours. EV-3C-mRNA was then diluted with Opti-MEM to a final concentration of 998.4 ng / ml, 499.2 ng / ml, 249.6 ng / ml, 124.8 ng / ml, 62.4 ng / ml, 31.2 ng / ml, and 15.6 ng / ml and transfected into the cells using Lipo2000 liposomes. Compound AG7088 was diluted in 2% cell maintenance solution and then added to a 96-well plate to a final concentration of 10 μM, 1 μM, and 0 μM, respectively. After 48 hours, the supernatant was discarded and the diluted Cell-Titer Luminescent Cell Viability Assay Solution was added, the cells were shaken and lysed for 5 minutes in the dark, and allowed to stand for 3 minutes. Finally, the luminescence value of each well was measured using a microplate reader.

[0233] Cell viability (%) = experimental group / average value of cell control group × 100

[0234] Data analysis: Origin9.0 software was used to fit the inhibition rate-concentration S-shaped curve and calculate the half-maximal effective concentration (EC) of AG7088 on 3C-mRNA. 50 The same method was used to calculate the half-cytotoxic concentrations (CC 50 ).

[0235] The experimental results are shown in Figure 7 and Tables 4-5. Figure 7 A shows the effect of EV-3C-mRNA on the viability of U87MG cells. The results show that the viability of U87MG cells gradually decreases with the increase of 3C-mRNA transfection dose. Figure B shows the effect of EV-3C-mRNA on the viability of A172 cells. The results show that the viability of A172 cells gradually decreases with the increase of 3C-mRNA transfection dose. Figure C shows that AG7088 can inhibit the decrease in cell viability caused by 3C-mRNA transfection in U87MG and inhibit the activity of 3C protein in U87MG cells. 50 The CC50 value of AG7088 for U87 MG cells was 1.149±0.19μM. At the same time, the CC50 value of AG7088 for U87 MG cells was >100μM. Figure D shows that AG7088 can inhibit the decrease in cell viability caused by 3C-mRNA transfection in A172 cells and inhibit the activity of 3C protein in A172 cells. 50 The CC of A172 cells was 0.439±0.03μM. 50 The values ​​were >100 μM.

[0236] In summary, the antitumor effect of EV-3C-mRNA depends on 3C protease activity.

[0237] Table 4. Inhibition of U87 MG and A172 cell viability by EV-3C-mRNA

[0238] Table 5. Rupintrivir (AG7088) can prevent EV-3C-mRNA from inhibiting the viability of U87 MG and A172 cells

[0239] Example 8 EV-3C-mRNA lipid nanoparticle (LNP) encapsulation

[0240] Experimental materials: SM-102 used in the experiment was purchased from MCE (catalog number: HY-134541), DSPC was purchased from Aladdin (catalog number: 816-94-4), Cholesterol was purchased from Sigma (catalog number: 57-88-5), PEG2000-DMG was purchased from MCE (catalog number: HY-145411), sodium citrate acidification buffer, RNase-free was purchased from Thermo (catalog number: J63888.AP), Tris-HCl buffer, RNase-free, 1 M, pH 7.4 was purchased from MacLean's reagent (catalog number: 1185-53-1), Sucrose (≥99.5%, BioReagent) was purchased from Beyotime (catalog number: ST1672), sodium acetate solution, RNase-free, pH 5.2, 3 M was purchased from MacLean's reagent (catalog number: 6131-90-4), sodium hydroxide solution, RNase free, 2M was purchased from Macklin reagent (catalog number: 1310-73-2), Acetic acid was purchased from Sigma (catalog number: 64-19-7), Quant-iT TM The RiboGreen RNA quantification kit was purchased from Thermo (Cat. No. R11490). Ignite manufacturer is Precision Nanosystems (PNI) (model: NIN0001), Zetasizer Ultra manufacturer is Malvern Panalytical (model: ZSU3305), Fluoroskan TM FL Microplate Fluorometer and Luminometer (Thermo Fisher Scientific, Inc., catalog number 5200222).

[0241] The experimental plan is as follows:

[0242] Lipids (ionizable cationic lipid SM-102, helper lipid DSPC, cholesterol, and PEG2000-DMG) were dissolved in ethanol at an optimal molar ratio. The LNP component formulation was: SM-102:DSPC:Cholesterol:DMG-PEG = 50:10:38.5:1.5. mRNA was diluted to the desired concentration in sodium citrate acidified buffer. LNPs were produced by mixing the lipids with mRNA using a microfluidic mixer. Subsequently, the LNPs were dialyzed against sucrose against Tris-HCl buffer in a dialysis cassette. After dialysis, the LNPs were filtered through a 0.22 μm filter and stored at −80°C. mRNA in the LNPs was quantified using the RiboGreen assay. Particle size and surface charge were determined by dynamic light scattering and zeta potential using a Zetasizer. Endotoxin levels were detected using a kinetic colorimetric TAL assay. LNP dispersions were prepared in 20 mM Tris, 87 mg / mL sucrose, 10.7 mM sodium acetate, pH 7.5.

[0243] The experimental results are shown in Figure 8 and Table 6. Panel A in Figure 8 shows the LNP component. The mRNA concentration, as determined by the RiboGreen assay, was 120 μg / mL. Particle size parameters were measured using a Zetasizer Advance potentiometer. The encapsulated LNP had a diameter of 82.76 nm. Transmission electron microscopy results are shown in Figure B, and the particle size distribution is shown in Figure C.

[0244] Functional testing showed that the average zeta potential of the mRNA-carrying LNPs (LNP-mRNA) was -3.26 mV, the encapsulation efficiency was 90.77%, and the average endotoxin content of the mRNA-carrying LNPs (LNP-mRNA) was less than 10 EU / ml.

[0245] Table 6. EV-3C-mRNA-LNP nanoparticle size distribution

[0246] Example 9 Evaluation of in vivo delivery ability of lipid nanoparticles

[0247] Experimental Materials: 6- to 8-week-old female BALB / c nude mice (n = 6) were purchased from Spectrum Biotechnology (Beijing) Co., Ltd. of proven proven quality. D-Luciferin potassium salt was purchased from Bio-Tech (product number: ST196-100mg). In vivo imaging was performed using the PerkinElmer IVIS Spectrum CT Small Animal In Vivo 3D Multimodal Imaging System. FLuc mRNA-LNPs were purchased from Yunzhou Biotechnology.

[0248] The experimental plan is as follows:

[0249] Before conducting in vivo experiments, the in vivo delivery and protein expression capabilities of the nanoparticles must be evaluated. First, luciferase reporter gene nanoparticles (FLuc mRNA-LNPs) were constructed (the FLuc mRNA structure is shown in Figure 9, Panel A). This was used to examine the in vivo distribution of the mRNA-LNPs and the duration of mRNA titer in vivo. Female BALB / c nude mice aged 6-8 weeks were inoculated with 12 μg of FLuc mRNA-LNPs (120 μg / ml, i.e., 100 μl per mouse) via tail vein injection or intranasal drip. IVIS imaging was performed at 6 and 24 hours p.i. Prior to imaging, each mouse was intraperitoneally injected with 10 μl / g of luciferin potassium salt. Imaging analysis was performed 10-20 minutes after injection, and after the fluorescence signal reached a peak and stable plateau, analysis was performed. Imaging was performed on three intranasal and three tail vein-injected mice. Imaging was performed on one mouse with the strongest signal in each group, and the brain and viscera were removed and placed in a culture dish for imaging.

[0250] The experimental results are shown in Figure 9. Six hours after tail vein injection, the fluorescence intensity was highest in vivo, with the signal becoming weaker after 24 hours, as shown in Figure B. In contrast, intranasal administration resulted in virtually no detectable signal in vivo. Six hours later, an autopsy of a mouse from the tail vein injection group revealed that the nanoparticles were concentrated primarily in the spleen and liver, as shown in Figure C.

[0251] In summary, both the tail vein and nasal drops cannot penetrate the blood-brain barrier and target brain tissue, so the in situ brain administration route should be selected for mice with orthotopic brain tumors.

[0252] Example 10 EV-3C-mRNA-LNP can inhibit the growth of in situ brain tumors in mice and prolong their survival time

[0253] This example is based on the EV-3C-mRNA-LNP prepared in Example 8.

[0254] Experimental Materials: 6- to 8-week-old female BALB / c nude mice (n = 30) were purchased from Spefox (Beijing) Biotechnology Co., Ltd. of proven proven origin and qualified after inspection. U87 MG-luc, a human glioma cell line expressing luc, was purchased from Fenghui Biotechnology; D-luciferin potassium salt was purchased from Beyotime (product number: ST196-100mg); in vivo imaging was performed using a PerkinElmer IVIS Spectrum CT small animal in vivo 3D multimodal imaging system; FLuc mRNA-LNPs were purchased from Yunzhou Biotechnology; and Hamilton microsyringes were purchased from the Shanghai Representative Office of Hamilton Bonatus AG, Switzerland. 27-G syringe needles were purchased from BD (catalog number: 20200520); slides (purchased from Servicebio); upright optical microscope (manufacturer: Nikon, Japan; model: Nikon EclipseE100); imaging system (manufacturer: Nikon, Japan; model: Nikon DS-U3); anhydrous ethanol was purchased from Sinopharm Chemical Reagent Co., Ltd., catalog number: 100092683; xylene was purchased from Sinopharm Chemical Reagent Co., Ltd., catalog number: 10023418; HE stain was purchased from Servicebio, catalog number: G1005; differentiation solution was purchased from Servicebio, catalog number: G1005-3.

[0255] The experimental plan is as follows:

[0256] (1) Establishment of an orthotopic brain tumor model in mice and evaluation of the in vivo efficacy of EV-3C-mRNA-LNP

[0257] ① 6-8 week old female BALB / c nude mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (0.1 mL / 10 g body weight).

[0258] ② After disinfecting the top of the head with 75% ethanol, make a vertical incision along the midline of the skull to expose the anterior fontanelle. Inoculation site: 0 mm anterior to the midpoint of the anterior fontanelle, 3 mm to the right of the sagittal suture, and 3 mm deep.

[0259] ③ Use a microinjector loaded with 5 μL of 1*10^5 U87 MG-luc cell suspension to vertically enter the striatum through the 27-G needle hole, and slowly inject the tumor cell suspension at an injection rate of 0.5 μL·min-1. Stay for 2 minutes before removing the needle, then slowly withdraw it, suture the scalp, and finally disinfect it with iodine.

[0260] ④ After the operation, place the mouse in a new cage and place the cage on a 37℃ electric blanket to keep it warm. After the mouse wakes up, put the cage back on the cage rack.

[0261] ⑤ Observe the living conditions of the mice daily, including their mental state, diet, activity, and body weight. Perform IVIS imaging weekly.

[0262] ⑥ After tumor implantation, the mice were divided into two groups and administered EV-3C mRNA-LNP orthotopically injected into the brain once a week (1.2 μg) and twice a week (2.4 μg).

[0263] ⑦Record the survival time and weight changes of mice, and monitor IVIS signals weekly.

[0264] (2) HE staining of different groups of in situ brain tumors

[0265] On the 21st day after tumor inoculation, brain tissues of mice in the PBS group, EV-3C mRNA-LNP (1.2 μg) group, and EV-3C mRNA-LNP (2.4 μg) group were collected for HE staining and section analysis.

[0266] ① Deplasticize the slices to water: place the slices in ethylene glycol ether acetate I for 6 hours at 37°C, ethylene glycol ether acetate II overnight at 37°C, ethylene glycol ether acetate III at room temperature for 10-15 minutes, ethylene glycol ether acetate IV at room temperature for 10-15 minutes, 100% ethanol I for 10 minutes, 100% ethanol II for 10 minutes, 95% ethanol for 10 minutes, 90% ethanol for 10 minutes, 80% ethanol for 10 minutes, and wash with tap water.

[0267] ②Hematoxylin staining: Stain the sections in hematoxylin solution for 8-10 minutes, wash with tap water, differentiate with differentiation solution, wash with tap water, blue with bluing solution, and rinse with running water.

[0268] ③Eosin staining: Dehydrate the sections in 85% and 95% gradient alcohol for 5 minutes each, and then stain them in eosin solution for 8-10 minutes.

[0269] ④ Dehydration and sealing: The sections were sequentially placed in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, xylene I for 5 min, and xylene II for 5 min until transparent, and then sealed with neutral gum.

[0270] ⑤ Microscopic examination, image acquisition and analysis. The cell nucleus appears blue and the cytoplasm appears red.

[0271] Data Analysis: Log-Rank was used to calculate statistical significance of survival curves. Data are presented as mean ± standard error. A p < 0.05 indicated statistical significance.

[0272] The experimental results are shown in Figure 10 and Tables 7-8. Figure A in Figure 10 is the overall timeline of the in vivo experimental design. Figure B shows the in vivo imaging and fluorescence value quantification of mice in the first week (W1), second week (W2), third week (W3), fourth week (W4), and fifth week (W5). The results of the PBS group show that the construction of the orthotopic brain tumor model was very successful, and the tumor signal in the brain continued to grow. Compared with the PBS group, the signal of the drug-treated group with a single dose per week gradually weakened, while the signal of the group given the drug twice a week decreased more significantly or even disappeared. Figure C shows the signal statistics corresponding to Figure B. Figure D shows the statistics of the weight change trend. The survival curve in Figure E shows that the drug-treated group significantly prolonged the survival time of the mice. The HE staining results in Figure F show that the tumor in the drug-treated group has basically disappeared.

[0273] In summary, EV-3C-mRNA-LNP can effectively inhibit the growth of in situ brain tumors in mice and prolong the survival time of mice.

[0274] Table 7. IVIS signal monitoring of different groups of mice with orthotopic brain tumors

[0275] Table 8. Body weight changes (g) after orthotopic brain tumor implantation in mice

[0276] (2) In vivo safety evaluation of EV-3C-mRNA-LNP

[0277] Experimental Materials: 6- to 8-week-old female BALB / c nude mice (n = 6) were purchased from Spefox (Beijing) Biotechnology Co., Ltd. of proven origin and qualified after inspection. Hamilton microsyringes were purchased from the Shanghai Representative Office of Hamilton Bonatus AG, Switzerland; 27-G syringe needles were purchased from BD (catalog number: 20200520); glass slides were purchased from Servicebio; upright optical microscope (manufacturer: Nikon, Japan; model: Nikon Eclipse E100); imaging system (manufacturer: Nikon, Japan; model: Nikon DS-U3); anhydrous ethanol was purchased from Sinopharm Chemical Reagent Co., Ltd., catalog number: 100092683; xylene was purchased from Sinopharm Chemical Reagent Co., Ltd., catalog number: 10023418; HE staining solution was purchased from Servicebio, catalog number: G1005; and differentiation medium was purchased from Servicebio, catalog number: G1005-3.

[0278] The experimental plan is as follows:

[0279] Parallel to the in vivo efficacy evaluation of EV-3C-mRNA-LNP, an in vivo safety study was conducted in 6-8 week-old female BALB / c nude mice without orthotopic tumor implantation. EV-3C-mRNA-LNP was injected orthotopically twice weekly into the brain, the same frequency as in the brain tumor model group. Mouse body weights were recorded. After one month of observation, tissues from the heart, liver, spleen, lung, kidney, and brain of three mice in each group were analyzed for pathological sections.

[0280] 1. Deplasticize the slices to water: Place the slices in ethylene glycol ether acetate I for 6 hours at 37°C, ethylene glycol ether acetate II overnight at 37°C, ethylene glycol ether acetate III at room temperature for 10-15 minutes, ethylene glycol ether acetate IV at room temperature for 10-15 minutes, 100% ethanol I for 10 minutes, 100% ethanol II for 10 minutes, 95% ethanol for 10 minutes, 90% ethanol for 10 minutes, 80% ethanol for 10 minutes, and wash with tap water.

[0281] 2. Hematoxylin staining: Stain the sections in hematoxylin solution for 8-10 minutes, wash with tap water, differentiate with differentiation solution, wash with tap water, blue with bluing solution, and rinse with running water.

[0282] 3. Eosin staining: Dehydrate the sections in 85% and 95% gradient alcohol for 5 minutes each, and then stain them in eosin solution for 8-10 minutes.

[0283] 4. Dehydration and sealing: The sections were sequentially placed in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, xylene I for 5 min, and xylene II for 5 min until transparent, and then sealed with neutral gum.

[0284] 5. Microscopic examination, image acquisition and analysis. The cell nucleus appears blue and the cytoplasm appears red.

[0285] The results are shown in Figure 11. Figure A shows the weight change trend of the mice, which showed a steady increase in weight, with no decrease. Figures B and C are comparisons of tissue sections from mice in the PBS group and the 3C group.

[0286] In the heart tissue, compared with the PBS group, the 3C group showed no obvious abnormalities in the myocardial fibers within the visual field; the myocardial fibers within the visual field were evenly colored, with clear cell boundaries and consistent shapes, clear myocardial cell striations, alternating light and dark, and no abnormalities in the interstitium; no obvious inflammatory cell infiltration was observed; in the liver tissue, compared with the PBS group, a large number of hepatocyte granular degeneration, loose cytoplasm, and eosinophilic granules were observed in the 3C group within the visual field, and occasional extramedullary hematopoietic foci were seen in the liver lobules, but no obvious inflammatory cell infiltration was observed; in the spleen tissue, compared with the PBS group, a small number of extramedullary hematopoietic cells were seen in the red pulp within the visual field, the boundaries of splenic nodules were unclear, and a small number of scattered granulocytes were seen in the red pulp, but no obvious abnormalities were observed; in the lung tissue, in the PBS group, mild thickening of the alveolar wall and a small amount of granulocyte infiltration were observed in the alveolar wall in some areas within the visual field, hemorrhage foci were observed in some areas, and a large number of red blood cells were seen in the alveolar cavity, but no obvious abnormalities were observed in the bronchial structure. Compared with the PBS group, the 3C group showed widespread mild thickening of the alveolar wall and a small amount of granulocyte infiltration in the alveolar wall. There was no obvious abnormality in the bronchial structure and no obvious bleeding. In the kidney tissue, compared with the PBS group, the 3C group showed no obvious abnormality in the kidney tissue. The glomeruli were evenly distributed in the field of view, with uniform cell number and matrix in the glomeruli. The renal tubular epithelial cells were round and plump, with neat and regular brush border arrangement. There was no obvious proliferation of the interstitium and no obvious inflammatory cell infiltration. In the brain tissue, there was no significant difference between the 3C group and the PBS group. The neurons in the field of view were abundant, evenly distributed, and regular in morphology. There was no obvious glial cell proliferation.

[0287] In summary, EV-3C-mRNA-LNP has good in vivo safety via in situ brain administration.

[0288] Table 9 Body weight changes of mice after in situ brain administration in the safety evaluation group (g)

[0289] Example 11 Effect of EV-3C-mRNA on cell viability of other tumor cell lines

[0290] This example is based on the EV-3C-mRNA prepared in Example 1.

[0291] Experimental Materials: The experimental materials for this section are the same as above. Additional experimental materials required are as follows: human venous endothelial cells (HUVECs) were purchased from Zhejiang Meisen Cell Technology Co., Ltd. (catalog number: CTCC-0804-PC), U2OS cells were purchased from ATCC (catalog number: HTB-96), CHL-1 cells were purchased from ATCC (catalog number: CRL-9446), DU145 cells were purchased from ATCC (catalog number: HTB-81), HCT-116 cells were purchased from ATCC (catalog number: CCL-247), HeLa cells were purchased from ATCC (catalog number: CCL-2), HepG2 cells were purchased from ATCC (catalog number: HB-8065), AGS cells were purchased from ATCC (catalog number: CRL-1739), A549 cells were purchased from ATCC (catalog number: CCL-185), ACHN cells were purchased from ATCC (catalog number: CRL-1611), and Huh7 cells were purchased from the National Laboratory Cell Resource Sharing Platform. The cells were cultured in a cell culture incubator at 37°C and 5% CO2. The complete culture medium used for cell growth was DMEM high-glucose medium (purchased from Gibco, catalog number: 11995065) and MEM medium (purchased from Gibco, catalog number: 10370021) supplemented with 10% fetal bovine serum (purchased from Gibco, catalog number: 16000044) and penicillin-streptomycin (purchased from Gibco, catalog number: 2321152). Endothelial-specific culture medium was purchased from Zhejiang Meisen Cell Technology Co., Ltd. (catalog number: M22SN3004). Luminescent Cell Viability Assay Solution was purchased from Promega (Cat. No. G7572). Microplate reader was purchased from Molecular Devices (model: SpectraMax M5).

[0292] The experimental plan is as follows:

[0293] HUVEC cells, A375 cells, U2OS cells, CHL-1 cells, DU145 cells, HCT-116 cells, Hela cells, HepG2 cells, AGS cells, A549 cells, ACHN cells, and Huh7 cells were cultured at a density of 1.0 × 10 cells per well. 4The cells were inoculated at a density of 100 μg / ml in a 96-well plate with a white wall and a transparent bottom and cultured at 37°C and 5% CO2 for 24 hours. EV-3C-mRNA was then diluted with Opti-MEM to a final concentration of 998.4 ng / ml, 499.2 ng / ml, 249.6 ng / ml, 124.8 ng / ml, 62.4 ng / ml, 31.2 ng / ml, and 15.6 ng / ml and transfected into the cells using Lipo2000 liposomes. The final concentrations of EV-3C-mRNA transfected into HUVEC cells were: 10000 ng / ml, 5000 ng / ml, 2500 ng / ml, 1250 ng / ml, 625 ng / ml, 312.5 ng / ml, 156.25 ng / ml, 78.125 ng / ml, and 39.06 ng / ml. After 48 hours, the supernatant was discarded and diluted Cell-Titer Luminescent Cell Viability assay solution, shake and lyse for 5 minutes in the dark, let it stand for 3 minutes, and finally measure the luminescence value of each well using a microplate reader. Cell viability (%) = experimental group / average value of cell control group × 100

[0294] Origin9.0 software was used to fit the inhibition rate-concentration S-shaped curve and calculate the half-maximal effective concentration (EC) of EV-3C-mRNA on cells. 50 ).

[0295] The results are shown in Figures 12-13 and Tables 10-11. EV-3C-mRNA had no significant inhibitory effect on normal HUVEC cells at the highest concentration of 10,000 ng / ml, indicating that EV-3C-mRNA has good safety. At the same time, the results in Table 11 and Figure 12 indicate that EV-3C-mRNA can inhibit the cell viability of various human tumor cell lines and has good broad-spectrum anti-tumor activity. Figure 13 shows the effects of EV-3C-mRNA on the IC of different cell lines. 50 Statistical summary of inhibition rate and therapeutic index.

[0296] In summary, EV-3C-mRNA has broad-spectrum anti-tumor activity in vitro.

[0297] Table 10. Effects of EV-3C-mRNA on the viability of normal HUVEC cells

[0298] Table 11. Evaluation of cell viability of other tumor cell lines by EV-3C-mRNA

[0299] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are within the scope of protection of the present application. The entire application is given by the attached claims and any equivalents thereof.

Claims

1. Use of a 3C protease of a picornavirus or a nucleic acid molecule encoding the 3C protease in the preparation of a drug for preventing and / or treating a tumor in a subject.

2. The use of claim 1, wherein The picornavirus is an enterovirus, such as a human enterovirus; Preferably, the picornavirus is selected from the group consisting of novel enterovirus, poliovirus, coxsackievirus, rhinovirus and echovirus; Preferably, the picornavirus is a novel enterovirus (e.g., enterovirus 68 (EV68), enterovirus 69 (EV69), enterovirus 70 (EV70), enterovirus 71 (EV71)), a coxsackievirus (e.g., coxsackievirus A14, coxsackievirus A16, coxsackievirus A6, coxsackievirus B3) or a rhinovirus (e.g., rhinovirus 2, rhinovirus 7, rhinovirus 25); Preferably, the picornavirus is enterovirus 71 (EV71) or coxsackievirus A14.

3. The use of claim 1 or 2, wherein The small RNA virus is aphthous virus; Preferably, the aphthous ulcer virus is foot-and-mouth disease virus; Preferably, the foot-and-mouth disease virus is selected from the group consisting of European type (type A, type O, type C), Asian type (Asian type 1), and African type (SAT1 type, SAT2 type, SAT3 type).

4. The use according to any one of claims 1 to 3, wherein The 3C protease has an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 1; Preferably, the 3C protease has an amino acid sequence as shown in SEQ ID NO:

1.

5. The use according to any one of claims 1 to 4, wherein The nucleic acid molecule is a DNA molecule or an RNA molecule.

6. The use of claim 5, wherein The nucleic acid molecule is an RNA molecule; Preferably, the nucleic acid molecule comprises the coding sequence of the 3C protease; Preferably, the nucleic acid molecule further comprises one or more selected from 5'UTR, Kozak sequence, start codon, stop codon, 3'UTR, poly-A tail; Preferably, the nucleic acid molecule comprises, from the 5' end to the 3' end, 5'UTR, Kozak sequence, start codon, coding sequence of the 3C protease, stop codon, 3'UTR, poly-A tail.

7. The use according to claim 6, which has one or more characteristics selected from the following: (1) The coding sequence of the 3C protease is codon-optimized or unoptimized according to the codon preference of the host cell (e.g., human cell); (2) the coding sequence of the 3C protease has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:3; preferably, the coding sequence of the 3C protease is as shown in SEQ ID NO:3; (3) The 5' end of the nucleic acid molecule has a 5' cap modification (e.g., CAP type 0, CAP type 1, CAP type 2); preferably, the 5' end of the nucleic acid molecule has a 5' Cap type 1 modification; (4) The nucleic acid molecule contains one or more N1-methyl pseudouridine (m1ψ) modifications; preferably, all or part of the uracil nucleotides in the nucleic acid molecule are replaced by N1-methyl-pseudouracil nucleotides; (5) There are one or more stop codons at the 3' end of the coding sequence of the 3C protease; (6) the 5'UTR is selected from the 5'UTR derived from human α-globin mRNA; (7) the 3'UTR is selected from 3'UTR based on mtRNR1 (mitochondrial 12S ribosomal RNA) and AES (Amino-terminal enhancer of split); (8) the 5'UTR has a nucleotide sequence that has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence shown in SEQ ID NO:4; preferably, the 5'UTR has the nucleotide sequence shown in SEQ ID NO:4; (9) the 3'UTR has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at 98%, at least 99%, or 100% sequence identity; preferably, the 3'UTR has the nucleotide sequence shown in SEQ ID NO: 6; (10) The Kozak sequence is shown in SEQ ID NO: 5; (11) The poly-A tail comprises one or more poly(A) sequences, each of which independently consists of 20-120 consecutive adenylic acids; preferably, the poly-A tail comprises multiple poly(A) sequences, and adjacent poly(A) sequences are connected by a non-A spacer sequence; (12) The nucleic acid molecule has a nucleotide sequence that has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence shown in SEQ ID NO:2; preferably, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:

2.

8. The use according to any one of claims 1 to 7, wherein The drug is used in a subject: (1) Inhibit tumor cell proliferation; (2) inhibiting tumor cell migration and / or invasion; (3) Promote tumor cell apoptosis; (4) downregulating the upstream protein p38, downstream protein PSF and / or heterogeneous nuclear ribonucleoprotein (hnRNP) A1 in the MNK1 / 2 signaling pathway in tumor cells; (5) Upregulation of caspase-3, an apoptosis-executing protein in tumor cells; and / or (6) Inhibit the activity of tumor cells.

9. The use according to any one of claims 1 to 8, wherein The tumor is selected from a solid tumor or a blood tumor (e.g., leukemia, lymphoma, myeloma); Preferably, the tumor is selected from glioma (e.g., glioblastoma), liver cancer, kidney cancer, melanoma, prostate cancer, colon cancer, cervical cancer, gastric cancer, lung cancer, colorectal cancer, bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, gastrointestinal cancer, pancreatic cancer, skin cancer, lymphoma, leukemia, sarcoma (e.g., osteosarcoma).

10. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a 3C protease; wherein The 3C protease is as defined in any one of claims 1 to 4; Preferably, the isolated nucleic acid molecule is a nucleic acid molecule as defined in any one of claims 5 to 7. Alternatively, the isolated nucleic acid molecule encodes a nucleic acid molecule as defined in any one of claims 5 to 7.

11. A vector comprising the isolated nucleic acid molecule of claim 10.

12. A delivery composition comprising a delivery vector, and one or more selected from the following: 3C protease, the isolated nucleic acid molecule of claim 10, the vector of claim 11; in, The 3C protease is as defined in any one of claims 1 to 4; Preferably, the delivery vehicle is a particle; Preferably, the delivery vehicle is selected from lipid particles, sugar particles, metal particles, protein particles, liposomes, exosomes, microvesicles, gene guns or viral vectors (e.g., replication-defective retroviruses, lentiviruses, adenoviruses or adeno-associated viruses).

13. The delivery composition of claim 12, wherein The delivery vehicle is a lipid nanoparticle; Preferably, the lipid nanoparticles comprise ionizable cationic lipid SM-102, auxiliary lipid DSPC, cholesterol and PEGylated lipid PEG2000-DMG.

14. A pharmaceutical composition comprising a 3C protease, an isolated nucleic acid molecule according to claim 10, a vector according to claim 11 and / or a delivery composition according to claim 12 or 13, and a pharmaceutically acceptable carrier and / or excipient; in, The 3C protease is as defined in any one of claims 1 to 4; Preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent; Preferably, the additional pharmaceutically active agent is a drug having anti-tumor activity; Preferably, the additional pharmaceutically active agent is selected from: EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-MET or VEGF inhibitors, chemotherapeutic drugs, antibody drugs, biomacromolecule drugs, cell therapy drugs or any combination thereof; Preferably, the additional pharmaceutically active agent is provided as a separate component or as a mixed component with the 3C protease, isolated nucleic acid molecule, vector and / or delivery composition.

15. A method for inhibiting tumor cells, comprising: contacting the tumor cell with, or delivering into the tumor cell, a 3C protease, an isolated nucleic acid molecule of claim 10, a vector of claim 11, a delivery composition of claim 12 or 13, or a pharmaceutical composition of claim 14; Wherein, the 3C protease is as defined in any one of claims 1-4.

16. The method of claim 15, wherein The method is used to: (1) inhibiting the proliferation of the tumor cells; (2) inhibiting the migration and / or invasion of the tumor cells; (3) promoting apoptosis of the tumor cells; (4) down-regulating the upstream protein p38, the downstream protein PSF and / or heterogeneous nuclear ribonucleoprotein (hnRNP) A1 in the MNK1 / 2 signaling pathway in the tumor cells; (5) upregulating the apoptosis execution protein caspase-3 in the tumor cells; and / or (6) Inhibiting the activity of the tumor cells.

17. The method of claim 15 or 16, wherein The tumor is selected from a solid tumor or a blood tumor (e.g., leukemia, lymphoma, myeloma); Preferably, the tumor is selected from glioma (e.g., glioblastoma), liver cancer, kidney cancer, melanoma, prostate cancer, colon cancer, cervical cancer, gastric cancer, lung cancer, colorectal cancer, bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, gastrointestinal cancer, pancreatic cancer, skin cancer, leukemia, sarcoma (e.g., osteosarcoma).

18. Use of the isolated nucleic acid molecule of claim 10, the vector of claim 11, the delivery composition of claim 12 or 13, or the pharmaceutical composition of claim 14 for preparing an anti-tumor drug, or use of 3C protease, the isolated nucleic acid molecule of claim 10, the vector of claim 11, the delivery composition of claim 12 or 13, or the pharmaceutical composition of claim 14 for inhibiting tumor cells; wherein, The 3C protease is as defined in any one of claims 1-4.

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