Self-amplifying nucleic acid molecules and uses thereof

Abstract

Relates to an alphavirus RNA replicase coding sequence, an optimized self-amplification nucleic acid molecule, a pharmaceutical composition and application, and the optimized self-amplification nucleic acid molecule is a self-amplification nucleic acid molecule containing the alphavirus RNA replicase coding sequence, a tissue-specific self-amplification nucleic acid molecule or an improved self-amplification mRNA containing a mutated replicase. The mutated replicase comprises a mutated macro domain. The invention also relates to a preparation method of the self-amplification nucleic acid molecule.

Description

Self-amplifying nucleic acid molecules and their applications

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present invention claims priority to Chinese patent applications with application numbers 202310890565.0 filed on July 19, 2023, 202311309636.X filed on October 10, 2023, and 202410268968.6 filed on March 8, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention belongs to the field of biomedicine and relates to an alphavirus RNA replicase coding sequence, an optimized self-amplifying nucleic acid molecule, a pharmaceutical composition and uses. The optimized self-amplifying nucleic acid molecule is a self-amplifying nucleic acid molecule comprising the alphavirus RNA replicase coding sequence, a tissue-specific self-amplifying nucleic acid molecule or a self-amplifying RNA nucleic acid molecule with enhanced exogenous gene expression level. Background Art

[0004] Self-amplifying RNA (saRNA) is a type of RNA that can self-amplify in vivo. It can be used in vaccines to produce more antigens, and due to the immune stimulation brought about by replication, it can stimulate a stronger immune response.

[0005] Typically, in vitro synthesis of mRNA is mainly achieved by in vitro transcription (IVT) using linearized plasmid DNA or PCR amplification products as templates using RNA polymerase. Due to the long length of self-amplified RNA, linearized plasmid DNA is usually selected as the transcription template to ensure sequence accuracy. Therefore, it is necessary to linearize the plasmid using a specific DNA endonuclease (e.g., BspQI or XbaI). However, due to or in part to non-specific digestion of the DNA endonuclease, unexpected linearization products may be produced, resulting in non-target in vitro transcription products, ultimately reducing the integrity and expression efficiency of the self-amplified RNA product. Therefore, one of the optimization directions for self-amplified RNA is to further improve its transcription quality.

[0006] mRNA vaccines are a novel vaccine technology that utilizes synthetic mRNA molecules to encode a target non-endogenous antigen protein. These proteins are then injected into the human body to stimulate an immune response against pathogens or tumors. Currently, mRNA vaccines have been successfully used to prevent the novel coronavirus and hold broad application prospects in research areas such as infectious disease prevention vaccines, tumor treatment, and protein replacement therapy.

[0007] Common mRNA forms include linear mRNA and circular mRNA. Linear mRNA is divided into non-replicating and self-replicating, self-amplifying mRNA. Self-amplifying RNA is considered the next generation of mRNA molecules. Due to its long expression cycle and self-adjuvant effect, it has demonstrated its advantages in effectively inducing specific immunity in infectious disease and tumor vaccines. In addition, self-amplifying RNA also has unique potential in protein replacement therapies with a desired long half-life. In 2023, Japan approved a COVID-19 vaccine based on self-amplifying RNA, further demonstrating the safety of self-amplifying RNA as a vaccine. Compared to non-replicating mRNA, a key advantage of self-amplifying RNA is the high and long-lasting expression levels of exogenous genes, which can significantly reduce the administration dose and minimize side effects. Therefore, further improving expression efficiency has been one of the directions for optimizing self-amplifying RNA sequences.

[0008] For the optimization of non-replicating mRNA, utilizing nucleotide modifications to circumvent mRNA recognition by pattern recognition receptors has proven to be a highly successful strategy aimed at improving translation efficiency by reducing innate immune responses. However, since self-amplifying RNA essentially mimics the viral replication and expression mechanism, it is more complex than non-replicating mRNA. In addition to the presence of unmodified nucleotides, self-amplifying RNA replication products, including double-stranded RNA, progeny RNA with a Cap0 structure, and uncapped 5'-triphosphate progeny RNA, can be recognized by pattern recognition receptors (PRRs) and trigger antiviral immune responses, including but not limited to interferon or NF-κB signaling pathways. The resulting induction of interferon-stimulated factors such as protein kinase R (PKR), 2-5-oligoadenylate synthetase (OAS), and interferon-induced proteins with tetratricopeptide repeats (IFIT), restricts the replication and expression of self-amplifying RNA through host translational repression and apoptosis induction. Therefore, reducing the immunogenicity of the self-amplifying RNA itself has a positive effect on improving the expression efficiency of the self-amplifying RNA, and is therefore the main idea for optimizing the expression efficiency of the self-amplifying RNA.

[0009] The sequence structure of self-amplifying mRNA is relatively complex compared to mRNA, particularly due to the presence of viral replicase and replication-related elements such as RNA promoters. Self-amplifying mRNA utilizes its own RNA replicase, derived from alphaviruses, to enable self-replication. Therefore, injected mRNA can continuously produce the target protein in vivo, thereby enhancing the durability and intensity of the immune response in vaccines or increasing the half-life of protein replacement therapies.

[0010] The current self-amplifying mRNA is an mRNA vector modified from the alphavirus genome. It does not have specific microRNA binding sites, and its replication and expression in different tissues are not subject to microRNA-mediated regulation.

[0011] In addition, after self-amplifying mRNA is encapsulated and delivered into the body via lipid nanoparticles (LNPs), which are currently commonly used for nucleic acid molecule delivery, the inherent physical and chemical properties of lipid nanoparticles may mediate the self-amplifying mRNA to enter parenchymal tissues such as the liver, where it may express the target gene encoded by the self-amplifying mRNA in large quantities, causing potential liver toxicity.

[0012] Therefore, in order to better apply self-amplifying mRNA in various related fields, it is still necessary to develop new self-amplifying mRNA with improved transcription quality, new regulatable and / or low-toxicity self-amplifying mRNA, and new self-amplifying mRNA with enhanced exogenous gene expression levels.

[0013] Summary of the Invention

[0014] Optimized alphavirus RNA replicase coding sequence (translation region) and self-amplifying mRNA backbone sequence

[0015] The present invention obtains an optimized alphavirus RNA replicase coding sequence (translation region) and further obtains an optimized self-amplifying mRNA backbone sequence. Compared with the original sequence, it can significantly reduce the production of non-specific enzyme cleavage products of the DNA endonuclease BspQI, improve the integrity of self-amplifying mRNA in vitro transcription, and maintain the normal replication function and / or expression function of saRNA. The following invention is provided:

[0016] One aspect of the present invention relates to a translation region (coding region) of an mRNA molecule, which encodes the alphavirus RNA replicase shown in SEQ ID NO: 20, wherein the 4500th base of the mRNA molecule is C.

[0017] In some embodiments of the present invention, the translation region of the mRNA molecule is an isolated translation region of the mRNA molecule.

[0018] In some embodiments of the present invention, in the translation region of the mRNA molecule, the 4509th base is T, C or G.

[0019] In some embodiments of the present invention, in the translation region of the mRNA molecule, the 1672nd base is A, the 1673rd base is G, and the 1674th base is C.

[0020] In some embodiments of the present invention, the translation region of the mRNA molecule encodes the alphavirus RNA replicase shown in SEQ ID NO: 20, wherein the 4500th base of the mRNA molecule is C and the 4509th base is T.

[0021] In some embodiments of the present invention, the translation region of the mRNA molecule encodes the alphavirus RNA replicase shown in SEQ ID NO: 20, wherein the 4500th base of the mRNA molecule is C and the 4509th base is C.

[0022] In some embodiments of the present invention, the translation region of the mRNA molecule encodes the alphavirus RNA replicase shown in SEQ ID NO: 20, wherein the 4500th base of the mRNA molecule is C and the 4509th base is G.

[0023] In some embodiments of the present invention, the translation region of the mRNA molecule encodes the alphavirus RNA replicase shown in SEQ ID NO: 20, wherein the 4500th base of the mRNA molecule is C, the 1672nd base is A, the 1673rd base is G, and the 1674th base is C.

[0024] In some embodiments of the present invention, the translation region of the mRNA molecule encodes the alphavirus RNA replicase shown in SEQ ID NO: 20, wherein the 4500th base of the mRNA molecule is C, the 4509th base is T, the 1672nd base is A, the 1673rd base is G, and the 1674th base is C.

[0025] In some embodiments of the present invention, the translation region of the mRNA molecule encodes the alphavirus RNA replicase shown in SEQ ID NO: 20, wherein the 4500th base of the mRNA molecule is C, the 4509th base is C, the 1672nd base is A, the 1673rd base is G, and the 1674th base is C.

[0026] In some embodiments of the present invention, the translation region of the mRNA molecule encodes the alphavirus RNA replicase shown in SEQ ID NO: 20, wherein the 4500th base of the mRNA molecule is C, the 4509th base is G, the 1672nd base is A, the 1673rd base is G, and the 1674th base is C.

[0027] In some embodiments of the present invention, the mRNA molecule translation region, wherein the sequence of the initial mRNA molecule translation region encoding alphavirus RNA replicase is shown as SEQ ID NO:10.

[0028] In some embodiments of the present invention, the sequence of the translation region of the mRNA molecule is shown in any one of SEQ ID NO: 11 to SEQ ID NO: 15.

[0029] Another aspect of the present invention relates to an mRNA molecule comprising, in order:

[0030] 5' untranslated region, RNA replicase coding region, RNA promoter, optional target protein coding sequence, 3' untranslated region and polyadenylation sequence;

[0031] Wherein, the RNA replicase coding region is the mRNA molecule translation region described in any one of the present invention.

[0032] The polyadenylic acid is also called PolyA or PolyA tail, which may contain a small amount of non-adenylic acid (A), such as a small amount of T, C and / or G. Without being bound by theory, the intermediate non-A sequence can increase the stability of DNA template production.

[0033] In some embodiments of the present invention, the mRNA molecule is an isolated mRNA molecule.

[0034] In some embodiments of the present invention, the mRNA molecule, wherein,

[0035] Wherein, the 5' untranslated region is derived from an alphavirus; preferably, it is derived from Venezuelan equine encephalitis virus;

[0036] Preferably, the sequence of the 5' untranslated region is shown as SEQ ID NO: 16.

[0037] In some embodiments of the present invention, the mRNA molecule, wherein,

[0038] Wherein, the 3' untranslated region is derived from an alphavirus; preferably, it is derived from Venezuelan equine encephalitis virus;

[0039] Preferably, the sequence of the 3' untranslated region is as shown in SEQ ID NO: 17.

[0040] In some embodiments of the present invention, the mRNA molecule, wherein the RNA promoter is a subgenomic promoter;

[0041] Preferably, the RNA promoter is a subgenomic promoter derived from an alphavirus;

[0042] Preferably, the RNA promoter is a subgenomic promoter derived from Venezuelan equine encephalitis virus;

[0043] Preferably, the RNA promoter is a 26S promoter;

[0044] Preferably, the sequence of the RNA promoter is shown in SEQ ID NO: 18.

[0045] In some embodiments of the present invention, the mRNA molecule, wherein the sequence of the polyadenylation sequence is as shown in SEQ ID NO:19.

[0046] In some embodiments of the present invention, the mRNA molecule, wherein the sequence encoding the target protein is a sequence encoding a vaccine antigen, a therapeutic protein or an antibody targeting an immune checkpoint.

[0047] In some embodiments of the present invention, the sequence of the mRNA molecule is as shown in SEQ ID NO: 3 and any one of SEQ ID NO: 6 to SEQ ID NO: 9.

[0048] In some embodiments of the invention, the mRNA molecule is a self-amplifying RNA (saRNA).

[0049] Another aspect of the present invention relates to a DNA molecule encoding the translation region of the mRNA molecule according to any one of the present invention or encoding the mRNA molecule according to any one of the present invention.

[0050] Another aspect of the present invention relates to a recombinant vector containing the DNA molecule of the present invention; preferably, the recombinant vector is a recombinant prokaryotic expression vector or a recombinant eukaryotic expression vector.

[0051] Another aspect of the present invention relates to a recombinant host cell, which contains the translation region of the mRNA molecule according to any one of the present invention, ... DNA molecule according to the present invention, or the recombinant vector according to the present invention.

[0052] Another aspect of the present invention relates to a kit comprising the translation region of the mRNA molecule according to any one of the present invention, the mRNA molecule according to any one of the present invention, or the DNA molecule according to the present invention, and a liposome delivery system.

[0053] Another aspect of the present invention relates to a pharmaceutical composition comprising the translation region of the mRNA molecule according to any one of the present invention, the mRNA molecule according to any one of the present invention, or the DNA molecule according to the present invention, and one or more pharmaceutically acceptable excipients; preferably, the excipient is a liposome delivery system.

[0054] Another aspect of the present invention relates to a vaccine preparation comprising the translation region of the mRNA molecule according to any one of the present invention, the mRNA molecule according to any one of the present invention, or the DNA molecule according to the present invention;

[0055] Preferably, the mRNA molecule or DNA molecule is encapsulated by a liposome delivery system;

[0056] Optionally, the vaccine formulation further comprises one or more vaccine adjuvants;

[0057] Preferably, the vaccine preparation is a vaccine preparation for preventing viral infection such as novel coronavirus infection or preventing severe illness caused by novel coronavirus infection.

[0058] Lipid delivery systems include liposomes, lipid nanoparticles (LNPs), and lipid polymer nanocarriers (LPPs).

[0059] Among lipids, LPP is a bilayer structure consisting of a polymer-encapsulated mRNA core and a phospholipid shell. The LPP bilayer membrane effectively encapsulates and protects mRNA, while the LPP core gradually releases mRNA molecules as the polymer degrades. LPP is highly effective in targeting dendritic cells, activating T cell immune responses through antigen presentation and achieving optimal therapeutic effects.

[0060] Without being bound by theory, mRNA can effectively stimulate cellular immunity and humoral immunity. The injected mRNA vaccine is internalized by antigen-presenting cells. After escaping the endosome and entering the cytoplasm, the mRNA is translated into protein by the ribosome. The translated antigen protein can stimulate the immune system in various ways, stimulating the body's cellular immunity and humoral immunity. Compared with traditional inactivated vaccines, subunit vaccines, and genetically engineered vaccines, nucleic acid vaccines have the following advantages: short R&D cycle; simple production process and easy expansion; no adjuvant required and high efficacy; no entry into the cell nucleus and better safety. The mRNA COVID-19 vaccine has verified the applicability of the mRNA technology platform in the vaccine field.

[0061] Another aspect of the present invention relates to the use of the translation region of the mRNA molecule described in any one of the present invention, the mRNA molecule described in any one of the present invention, or the DNA molecule described in any one of the present invention in the preparation of a drug for treating or preventing viral infection, a drug for treating or preventing tumors, or a drug for protein replacement therapy.

[0062] A self-amplifying mRNA backbone that is bound by microRNAs specifically expressed in certain tissues and then regulates the replication of self-amplifying mRNA molecules

[0063] The present invention also provides a self-amplifying mRNA backbone that can be bound by microRNAs specifically expressed in specific tissues and thereby regulate the replication of self-amplifying mRNA molecules. This allows the self-amplifying mRNA molecules to be degraded in tissues that express the specific microRNA, while remaining normally in tissues that lowly express or do not express the specific microRNA, thereby achieving tissue-specific expression of the target gene molecule in the form of self-amplifying mRNA. This provides the following invention:

[0064] Yet another aspect of the present invention relates to an mRNA molecule comprising, in order:

[0065] 5' non-translated sequence, RNA replicase sequence, RNA promoter, optional sequence encoding target protein, 3' non-translated sequence and 3' Poly A tail;

[0066] in,

[0067] A microRNA binding site sequence is contained between nsp1 and nsp2, between nsp2 and nsp3, between nsp3 and nsp4, and / or between the sequence encoding the target protein and the 3'-end non-translated sequence of the RNA replicase.

[0068] In some embodiments of the present invention, the mRNA molecule, wherein,

[0069] The RNA replicase is an RNA replicase derived from an alphavirus; preferably, it is an RNA replicase derived from Venezuelan equine encephalitis virus;

[0070] Preferably, the amino acid sequence of the RNA replicase is shown as SEQ ID NO:41; preferably, the coding sequence of the RNA replicase is shown as SEQ ID NO:42.

[0071] In some embodiments of the present invention, the mRNA molecule, wherein,

[0072] The RNA promoter is a subgenomic promoter;

[0073] Preferably, the RNA promoter is a subgenomic promoter derived from an alphavirus;

[0074] Preferably, the RNA promoter is a subgenomic promoter derived from Venezuelan equine encephalitis virus;

[0075] Preferably, the RNA promoter is the 26S promoter.

[0076] In some embodiments of the present invention, the mRNA molecule, wherein the 5' non-translated sequence and / or the 3' non-translated sequence is derived from an alphavirus; optimally, it is derived from Venezuelan equine encephalitis virus.

[0077] In some embodiments of the present invention, the mRNA molecule, wherein the microRNA binding site sequence is a binding site sequence corresponding to a tissue-specifically expressed microRNA;

[0078] Preferably, the tissue-specifically expressed microRNA is a microRNA that is lowly expressed in tumor tissue;

[0079] Preferably, the microRNA lowly expressed in tumor tissue is selected from miRNA-122, miRNA-143, miRNA-1, miRNA-124, miRNA-217 and miRNA-126.

[0080] In some embodiments of the present invention, the mRNA molecule, wherein the microRNA binding site sequence comprises one or more microRNA mature sequences; preferably, the microRNA binding site sequence comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 microRNA mature sequences that are identical or different in sequence.

[0081] In some embodiments of the present invention, the mRNA molecule, wherein the microRNA mature sequence is shown in any one of SEQ ID NO: 30 to SEQ ID NO: 32.

[0082] Without being bound by theory, the microRNA binding site sequence is theoretically complementary to the microRNA. The length of the binding site sequence can be fine-tuned but must be greater than or equal to 20 nt. For example, if MicroRNA-122-5p is 22 nt long, its binding sequence length can be designed to be 20-24 nt, such as 20 nt, 21 nt, 22 nt, 23 nt, or 24 nt.

[0083] Without being bound by theory, miRNA binding sites are typically located in the 3' untranslated region (3'UTR) of the target mRNA, but can also be located in the 5'UTR or coding region. MicroRNAs bind to target mRNAs, thereby inhibiting translation or degrading the target mRNA, thereby affecting the expression of the target gene.

[0084] The present invention introduces the binding sites corresponding to tissue-specifically expressed microRNA into mRNA, and utilizes the microRNA-mediated post-transcriptional regulatory mechanism to regulate the stability and translation rate of different mRNA molecules, thereby achieving differential expression of mRNA in different tissues, and further achieving tissue-specific expression of mRNA.

[0085] In some embodiments of the present invention, the mRNA molecule contains one or more identical or different internal spacer sequences between each microRNA mature sequence;

[0086] Preferably, the internal spacer sequence is shown in any one of SEQ ID NO: 33 to SEQ ID NO: 34.

[0087] In some embodiments of the present invention, the mRNA molecule, wherein the 5' end and / or 3' end of the microRNA binding site sequence contains one or more identical or different external spacer sequences;

[0088] Preferably, the external spacer sequence at the 5' end is as shown in any one of SEQ ID NO: 35 to SEQ ID NO: 36;

[0089] Preferably, the external spacer sequence at the 3' end is as shown in any one of SEQ ID NO: 37 to SEQ ID NO: 38.

[0090] In some embodiments of the present invention, the mRNA molecule, wherein the 5' end and / or 3' end of the microRNA binding site sequence contains one or more restriction site sequences that can be recognized by alphavirus RNA replicase or nsp2;

[0091] Preferably, it is a restriction site sequence between nsp1 and nsp2 that can be recognized by alphavirus replicase or nsp2;

[0092] Preferably, the restriction enzyme cleavage site sequence is shown in SEQ ID NO: 53.

[0093] Without being bound by theory, nsp2 in the alphavirus replicase is the component that exerts protease activity. It can recognize a specific amino acid sequence between the four components of the alphavirus replicase, thereby cleaving the replicase polyprotein into its individual components to perform replication. Therefore, the cleavage site here should be a protease cleavage site sequence recognized by the alphavirus replicase or nsp2.

[0094] The protease cleavage site sequence is preferably an amino acid sequence between nsp1 and nsp2 that can be recognized by alphavirus replicase or nsp2, and the sequence is: EAGA↓GSVE (SEQ ID NO: 53), where ↓ represents the cleavage site.

[0095] In some embodiments of the present invention, the mRNA molecule, wherein the microRNA binding site sequence is shown as SEQ ID NO:22.

[0096] In some embodiments of the present invention, the mRNA molecule further contains a 5' end cap structure.

[0097] In some embodiments of the present invention, the sequence of the mRNA molecule is shown in SEQ ID NO: 24 or SEQ ID NO: 28.

[0098] In some embodiments of the present invention, the mRNA molecule, wherein the target protein is an antigen.

[0099] The mRNA molecules of the present invention are tissue-specific self-amplifying nucleic acid molecules.

[0100] In some embodiments of the invention, the mRNA molecule is an isolated mRNA molecule.

[0101] Another aspect of the present invention relates to an mRNA molecule combination comprising a first mRNA molecule and a second mRNA molecule, wherein:

[0102] The first mRNA molecule comprises, in order:

[0103] The first 5' untranslated sequence, the RNA replicase sequence, the first 3' untranslated sequence and the 3' Poly A tail;

[0104] The second mRNA molecule comprises, in order:

[0105] The second 5' non-translated sequence, the sequence necessary for alphavirus replication, the RNA promoter, the sequence encoding the target protein, the second 3' non-translated sequence and the 3' PolyA tail;

[0106] Wherein, a microRNA binding site sequence is contained between nsp1 and nsp2, between nsp2 and nsp3, between nsp3 and nsp4, and / or between the sequence encoding the target protein and the second 3'-end non-translated sequence of the RNA replicase.

[0107] In some embodiments of the present invention, the mRNA molecule combination, wherein,

[0108] The first 5' untranslated sequence is the same as or different from the second 5' untranslated sequence; and / or

[0109] The first 3' non-translated sequence is the same as or different from the second 3' non-translated sequence.

[0110] In some embodiments of the present invention, in the mRNA molecule combination, the first 5' non-translated sequence and the first 3' non-translated sequence are not derived from alphavirus.

[0111] In some embodiments of the present invention, the mRNA molecule combination, wherein the second 5' non-translated sequence and the second 3' non-translated sequence are derived from alphavirus; preferably, derived from Venezuelan equine encephalitis virus.

[0112] In some embodiments of the present invention, the mRNA molecule combination, wherein,

[0113] The RNA replicase is an RNA replicase derived from an alphavirus; preferably, it is an RNA replicase derived from Venezuelan equine encephalitis virus;

[0114] Preferably, the amino acid sequence of the RNA replicase is shown as SEQ ID NO:41; preferably, the coding sequence of the RNA replicase is shown as SEQ ID NO:42.

[0115] In some embodiments of the present invention, the mRNA molecule combination, wherein,

[0116] The RNA promoter is a subgenomic promoter;

[0117] Preferably, the RNA promoter is a subgenomic promoter derived from an alphavirus;

[0118] Preferably, the RNA promoter is a subgenomic promoter derived from Venezuelan equine encephalitis virus;

[0119] Preferably, the RNA promoter is the 26S promoter.

[0120] In some embodiments of the present invention, the mRNA molecule combination, wherein the microRNA binding site sequence is a binding site sequence corresponding to a tissue-specifically expressed microRNA;

[0121] Preferably, the tissue-specifically expressed microRNA is a microRNA that is lowly expressed in tumor tissue;

[0122] Preferably, the microRNA lowly expressed in tumor tissue is selected from miRNA-122, miRNA-143, miRNA-1, miRNA-124, miRNA-217 and miRNA-126.

[0123] In some embodiments of the present invention, the mRNA molecule combination, wherein the microRNA binding site sequence comprises one or more microRNA mature sequences; preferably, the microRNA binding site sequence comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 microRNA mature sequences that are identical or different in sequence.

[0124] In some embodiments of the present invention, the mRNA molecule combination, wherein the microRNA mature sequence is shown in any one of SEQ ID NO: 30 to SEQ ID NO: 32.

[0125] In some embodiments of the present invention, the mRNA molecule combination contains one or more identical or different internal spacer sequences between each microRNA mature sequence;

[0126] Preferably, the internal spacer sequence is shown in any one of SEQ ID NO: 33 to SEQ ID NO: 34.

[0127] In some embodiments of the present invention, the mRNA molecule combination, wherein the 5' end and / or 3' end of the microRNA binding site sequence contains one or more identical or different external spacer sequences;

[0128] Preferably, the external spacer sequence at the 5' end is as shown in any one of SEQ ID NO: 35 to SEQ ID NO: 36;

[0129] Preferably, the external spacer sequence at the 3' end is as shown in any one of SEQ ID NO: 37 to SEQ ID NO: 38.

[0130] In some embodiments of the present invention, the mRNA molecule combination, wherein the 5' end and / or 3' end of the microRNA binding site sequence contains one or more restriction site sequences that can be recognized by alphavirus RNA replicase or nsp2;

[0131] Preferably, it is a restriction site sequence between nsp1 and nsp2 that can be recognized by alphavirus replicase or nsp2;

[0132] Preferably, the restriction enzyme cleavage site sequence is shown in SEQ ID NO: 53.

[0133] In some embodiments of the present invention, the mRNA molecule combination, wherein the microRNA binding site sequence is shown as SEQ ID NO:22.

[0134] In some embodiments of the present invention, in the mRNA molecule combination, the first mRNA molecule and / or the second mRNA molecule further contains a 5' end cap structure.

[0135] In some embodiments of the present invention, the mRNA molecule combination, wherein the target protein is an antigen.

[0136] In some embodiments of the present invention, the mRNA molecule combination is as shown in Figure 23.

[0137] Without being bound by theory, the first mRNA molecule encodes nsp1 to nsp4, and the second mRNA molecule encodes the target protein.

[0138] The mRNA molecule combination of the present invention is a tissue-specific self-amplifying nucleic acid molecule combination.

[0139] Another aspect of the present invention relates to a DNA molecule encoding the mRNA molecule described in any one of the present invention, or encoding the first mRNA molecule and the second mRNA molecule described in any one of the present invention, wherein the first mRNA molecule and the second mRNA molecule are on the same DNA molecule.

[0140] In some embodiments of the present invention, the DNA molecule comprises a T7 promoter, an SP6 promoter or a T3 promoter upstream of the sequence encoding the alphavirus RNA replicase.

[0141] In some embodiments of the present invention, the DNA molecule is an isolated DNA molecule.

[0142] Another aspect of the present invention relates to a DNA molecule combination comprising a first DNA molecule and a second DNA molecule, wherein:

[0143] The first DNA molecule encodes the first mRNA molecule of any one of the present invention; and

[0144] The second DNA molecule encodes the second mRNA molecule described in any one of the present invention.

[0145] The mRNA molecule according to any one of the present invention, the mRNA molecule combination according to any one of the present invention, the DNA molecule according to any one of the present invention, or the DNA molecule combination according to any one of the present invention, for use in treating or preventing viral infection, treating or preventing tumors, or protein replacement therapy;

[0146] Preferably, the viral infection refers to novel coronavirus infection;

[0147] Preferably, the tumor is selected from liver cancer, rhabdomyosarcoma, glioma, bladder cancer, colorectal cancer, pancreatic cancer and lung cancer;

[0148] Preferably, the tumor is a tumor with low expression of one microRNA or multiple microRNAs; preferably, the microRNA is one or more selected from miRNA-122, miRNA-143, miRNA-1, miRNA-124, miRNA-217 and miRNA-126.

[0149] MicroRNAs (microRNAs) are a class of non-coding single-stranded RNA molecules of approximately 22 nt in length that are encoded by endogenous genes. They participate in post-transcriptional gene expression regulation in plants and animals. MicroRNAs recognize and bind to the mRNA sequences of target genes, resulting in the inhibition of mRNA translation or the degradation of mRNA, thereby regulating the expression of mRNA-encoding genes. This gene expression regulation mechanism is called microRNA-mediated post-transcriptional regulation. MicroRNAs are tissue-specific, and their expression levels vary in different tissues. For example:

[0150] miRNA-122 is highly expressed in the liver, but is expressed at lower levels in non-hepatic cells (such as muscle cells) and liver cancer tissues;

[0151] miRNA-143 is highly expressed in colorectal tissues but lowly expressed in colorectal cancer tissues;

[0152] miRNA-1 is highly expressed in skeletal muscle and cardiac tissues, but lowly expressed in non-muscle tissues and rhabdomyosarcoma tissues;

[0153] miRNA-124 is highly expressed in neuronal tissues but lowly expressed in glioma and bladder cancer tissues;

[0154] miRNA-217 is highly expressed in pancreatic tissues but lowly expressed in pancreatic cancer tissues;

[0155] miRNA-126 and miRNA-143 were highly expressed in lung tissues but lowly expressed in lung cancer tissues.

[0156] The above specificity makes miRNA a powerful tool for disease diagnosis or treatment.

[0157] Without being bound by theory, any pathogen infection or any tumor type is applicable to this requirement. More specifically, it can also correspond to a tumor type with low expression of a certain specific microRNA. For example, most liver cancers have low expression of microRNA122, and the self-amplifying mRNA of the present invention can be specifically expressed in liver cancer tissue.

[0158] Another aspect of the present invention relates to a recombinant vector comprising the DNA molecule described in any one of the present invention; preferably, the recombinant vector is a recombinant prokaryotic expression vector or a recombinant eukaryotic expression vector.

[0159] Another aspect of the present invention relates to a recombinant host cell containing the mRNA molecule according to any one of the present invention, the mRNA molecule combination according to any one of the present invention, the DNA molecule according to any one of the present invention, or the DNA molecule combination according to any one of the present invention.

[0160] Another aspect of the present invention relates to a kit comprising the mRNA molecule described in any one of the present invention, the mRNA molecule combination described in any one of the present invention, the DNA molecule described in any one of the present invention, or the DNA molecule combination described in any one of the present invention, and a liposome delivery system.

[0161] Another aspect of the present invention relates to a pharmaceutical composition comprising the mRNA molecule described in any one of the present invention, the mRNA molecule combination described in any one of the present invention, the DNA molecule described in any one of the present invention, or the DNA molecule combination described in any one of the present invention, and one or more pharmaceutically acceptable excipients; preferably, the excipient is a liposome delivery system.

[0162] Another aspect of the present invention relates to a vaccine formulation comprising an mRNA molecule according to any one of the present invention, a combination of mRNA molecules according to any one of the present invention, a DNA molecule according to any one of the present invention, or a combination of DNA molecules according to any one of the present invention;

[0163] Preferably, the mRNA molecule, combination of mRNA molecules, DNA molecule or combination of DNA molecules is encapsulated by a liposome-based delivery system;

[0164] Optionally, the vaccine formulation further comprises one or more vaccine adjuvants;

[0165] Preferably, the vaccine preparation is a vaccine preparation for preventing viral infection such as novel coronavirus infection or preventing severe illness caused by novel coronavirus infection.

[0166] Lipid delivery systems include liposomes, lipid nanoparticles (LNPs), and lipid polymer nanocarriers (LPPs).

[0167] Among lipids, LPP is a bilayer structure consisting of a polymer-encapsulated mRNA core and a phospholipid shell. The LPP bilayer membrane effectively encapsulates and protects mRNA, while the LPP core gradually releases mRNA molecules as the polymer degrades. LPP is highly effective in targeting dendritic cells, activating T cell immune responses through antigen presentation and achieving optimal therapeutic effects.

[0168] Without being bound by theory, mRNA can effectively stimulate cellular immunity and humoral immunity. The injected mRNA vaccine is internalized by antigen-presenting cells. After escaping the endosome and entering the cytoplasm, the mRNA is translated into protein by the ribosome. The translated antigen protein can stimulate the immune system in various ways, stimulating the body's cellular immunity and humoral immunity. Compared with traditional inactivated vaccines, subunit vaccines, and genetically engineered vaccines, nucleic acid vaccines have the following advantages: short R&D cycle; simple production process and easy expansion; no adjuvant required and high efficacy; no entry into the cell nucleus and better safety. The mRNA COVID-19 vaccine has verified the applicability of the mRNA technology platform in the vaccine field.

[0169] Another aspect of the present invention relates to the use of any of the mRNA molecules, any of the mRNA molecule combinations, any of the DNA molecules, or any of the DNA molecule combinations in the present invention in the preparation of a drug for treating or preventing viral infection, a drug for treating or preventing tumors, or a drug for protein replacement therapy.

[0170] Preferably, the viral infection refers to novel coronavirus infection;

[0171] Preferably, the tumor is one or more selected from liver cancer, rhabdomyosarcoma, glioma, bladder cancer, colorectal cancer, pancreatic cancer and lung cancer;

[0172] Preferably, the tumor is a tumor with low expression of one microRNA or multiple microRNAs; preferably, the microRNA is one or more selected from miRNA-122, miRNA-143, miRNA-1, miRNA-124, miRNA-217 and miRNA-126.

[0173] Another aspect of the present invention relates to a method for treating or preventing viral infection or tumors, or a protein replacement therapy, comprising the step of administering to a subject in need thereof an effective amount of any one of the mRNA molecules of the present invention, any one of the mRNA molecule combinations of the present invention, any one of the DNA molecules of the present invention, or any one of the DNA molecule combinations of the present invention;

[0174] Preferably, the viral infection refers to novel coronavirus infection;

[0175] Preferably, the tumor is one or more selected from liver cancer, rhabdomyosarcoma, glioma, bladder cancer, colorectal cancer, pancreatic cancer and lung cancer;

[0176] Preferably, the tumor is a tumor with low expression of one microRNA or multiple microRNAs; preferably, the microRNA is one or more selected from miRNA-122, miRNA-143, miRNA-1, miRNA-124, miRNA-217 and miRNA-126.

[0177] Self-amplifying mRNA nucleic acid sequences with enhanced expression levels of exogenous genes

[0178] The present invention also provides an improved saRNA construct, and methods for preparing and using the construct. The present invention further provides the use of the saRNA to carry a target gene for therapeutic, preventive and / or diagnostic purposes. Specifically, after in-depth research and creative work, the present invention has obtained an optimized self-amplifying mRNA backbone sequence, which can significantly reduce the intensity of the induced natural immune response, significantly reduce the translation inhibition, and significantly reduce cell apoptosis compared to the original sequence, thereby significantly improving the expression efficiency. The following invention is thus provided:

[0179] One aspect of the present invention provides an mRNA molecule encoding an RNA replicase composed of nonstructural proteins 1, 2, 3, and 4 derived from an alphavirus, wherein the nonstructural protein 3 comprises a macrodomain at its N-terminus as shown in the amino acid sequence of SEQ ID NO: 57 or 59. In some embodiments, the nonstructural proteins 1, 2, 3, and 4 are derived from Venezuelan equine encephalitis virus.

[0180] In some embodiments, the mRNA molecule encodes a nonstructural protein 1 having an amino acid sequence as shown in SEQ ID NO: 71 or at least 90% identical to SEQ ID NO: 71, a nonstructural protein 2 having an amino acid sequence as shown in SEQ ID NO: 72 or at least 90% identical to SEQ ID NO: 72, and a nonstructural protein 4 having an amino acid sequence as shown in SEQ ID NO: 75 or at least 90% identical to SEQ ID NO: 75. Further, the mRNA molecule encodes a nonstructural protein 3 having an amino acid sequence at least 90% identical to SEQ ID NO: 73 or 74 and comprising a macrodomain as shown in the amino acid sequence of SEQ ID NO: 57 or 59 at its N-terminus.

[0181] In some embodiments, the mRNA molecule comprises a nucleotide sequence encoding the macrodomain as shown in SEQ ID NO: 68 or 70.

[0182] In some embodiments, the mRNA molecule comprises or consists of the nucleotide sequence of SEQ ID NO: 67 or 69.

[0183] In some embodiments, the mRNA molecule further comprises a target gene coding sequence and, optionally, an RNA promoter upstream of the target gene coding sequence.

[0184] In some embodiments, the mRNA molecule further comprises:

[0185] 5' untranslated region, RNA promoter, target gene coding sequence, 3' untranslated region and polyadenylation sequence,

[0186] Optionally, the mRNA molecule further comprises a 5' cap sequence, a signal peptide coding sequence, a Kozak sequence and / or a restriction enzyme cleavage site. The Kozak sequence may be connected to the 3' end of the RNA promoter.

[0187] In some embodiments, the mRNA molecule comprises the following operably linked nucleotide sequences from 5' to 3': a 5' untranslated region, coding sequences of nonstructural proteins 1, 2, 3, and 4, an RNA promoter, a target gene coding sequence, a 3' untranslated region, and a polyadenylation sequence.

[0188] Optionally, the nucleotide sequences may be connected via a linker sequence.

[0189] In some embodiments, the coding sequences of nonstructural proteins 1, 2, 3 and 4 comprise a nucleotide sequence encoding nonstructural protein 1, a nucleotide sequence encoding nonstructural protein 2, a nucleotide sequence encoding nonstructural protein 3 and a nucleotide sequence encoding nonstructural protein 4 that are operably linked.

[0190] In some embodiments, the coding sequences of non-structural proteins 1, 2, 3 and 4 comprise, from 5' to 3', a nucleotide sequence encoding non-structural protein 1, a nucleotide sequence encoding non-structural protein 2, a nucleotide sequence encoding non-structural protein 3 and a nucleotide sequence encoding non-structural protein 4 that are operably linked.

[0191] In some embodiments, the 5' untranslated region is derived from an alphavirus; preferably, from a Venezuelan equine encephalitis virus; for example, the 5' untranslated region comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 63.

[0192] In some embodiments, the 3' untranslated region is derived from an alphavirus; preferably, from a Venezuelan equine encephalitis virus; for example, the 3' untranslated region comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 64.

[0193] In some embodiments, the RNA promoter is a subgenomic promoter, such as a subgenomic promoter derived from an alphavirus. Preferably, the RNA promoter is a subgenomic promoter derived from Venezuelan equine encephalitis virus.

[0194] In some embodiments, the RNA promoter is a 26S promoter. For example, the sequence of the RNA promoter is shown in SEQ ID NO:65.

[0195] In some embodiments, the sequence of the poly(A) sequence is as shown in SEQ ID NO:66.

[0196] In some embodiments, the target gene coding sequence is a sequence encoding a therapeutic polypeptide, a prophylactic polypeptide, a diagnostic polypeptide, a reporter gene, an antigen, or a sequence encoding a regulatory structure (eg, a non-coding gene).

[0197] In some embodiments, the mRNA molecule comprises a nucleotide sequence as shown in any one of SEQ ID NO:58, SEQ ID NO:60 to SEQ ID NO:62.

[0198] In one aspect, the invention provides a DNA molecule encoding an mRNA molecule as disclosed herein.

[0199] In one aspect, the present invention provides a recombinant vector comprising a DNA molecule as disclosed herein; preferably, the recombinant vector is a prokaryotic expression vector or a eukaryotic expression vector.

[0200] In one aspect, the present invention provides a recombinant host cell comprising an mRNA molecule, a DNA molecule or a recombinant vector as disclosed herein.

[0201] In one aspect, the present invention provides a pharmaceutical composition comprising an mRNA molecule or a DNA molecule as disclosed herein, and one or more pharmaceutically acceptable carriers; preferably, the carrier is a liposome-based delivery system.

[0202] In one aspect, the present invention provides a vaccine formulation comprising an mRNA molecule or a DNA molecule as disclosed herein; preferably, the mRNA molecule or DNA molecule is encapsulated in a liposome delivery system. The vaccine formulation may further comprise one or more vaccine adjuvants. Accordingly, the target gene encoded by the mRNA may encode a vaccine antigen. For example, the vaccine formulation is a vaccine formulation for preventing viral infection, such as infection with the novel coronavirus, or for preventing severe illness caused by infection with the novel coronavirus. Accordingly, the target gene encoded by the mRNA may encode an immunogenic peptide of the novel coronavirus.

[0203] In one aspect, the present invention provides the use of an mRNA molecule or DNA molecule as disclosed herein in the preparation of a medicament for treating or preventing viral infection, a medicament for treating or preventing tumors, or a medicament for protein replacement therapy. Accordingly, the target gene encoded by the mRNA can be a vaccine antigen gene, a tumor-killing gene, a therapeutic protein gene, an antibody gene, etc.

[0204] In one aspect, the invention provides a kit comprising an mRNA molecule or a DNA molecule as disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0205] Figure 1: Capillary electrophoresis results of in vitro transcribed RNA after modification of positions 4497-4503 in the saRNA-EGFP template plasmid psaRNA-062.

[0206] Figure 2: Overlay of capillary electrophoresis results of RNA transcribed in vitro using different saRNA templates modified from psaRNA-152. saRNA-062 is the original saRNA, and saRNA-183 to 185 are the modified saRNAs.

[0207] Figure 3: Capillary electrophoresis of in vitro transcribed RNA after modification at positions 1680-1676 of psaRNA-183. The black boxes indicate the peaks of the short transcripts before and after backbone optimization.

[0208] Figure 4: Expression levels of optimized luciferase-encoding saRNA-152 and original saRNA-062 after transfection into BHK-21 cells for 24 and 72 hours.

[0209] Figure 5: Expression levels of optimized luciferase-encoding saRNA-243 and original saRNA-062 after transfection into BHK-21 cells for 24 hours.

[0210] Figure 6: Replication results of optimized luciferase-encoding saRNA-152 and original saRNA-062 after transfection of BHK-21 cells 24 and 72 hours after detection. Data were normalized to the results 2 hours after transfection.

[0211] Figure 7: Capillary electrophoresis results for detecting the integrity of S23H02.

[0212] Figure 8: E7-ELISA test results for the expression of secreted HPV16 antigens after S23H02, M22H04 and negative control were transfected into tissue culture containing Lipofectamine 3000.

[0213] Figure 9: Tumor volume data for C57bl / 6 mice challenged with TC1 tumor cells and injected with three doses of therapeutic vaccine. Tumor volume and mouse body weight were recorded. The dose per mouse and per dose was calculated based on the active ingredient.

[0214] Figure 10: Body weight data of C57bl / 6 mice challenged with TC1 tumor cells and injected with three doses of therapeutic vaccine. The dose per mouse per dose is calculated based on the active ingredient.

[0215] Figure 11: Flow cytometry analysis of the number of E7-specific CD8 T cells in the splenocytes of C57bl / 6 mice injected with one dose of therapeutic vaccine. The dose per mouse and per dose was calculated based on the active ingredient.

[0216] Figure 12: Schematic diagram of the miRNA122 binding site cassette. Six microRNA-122-5p sequences are separated by inner spacers, and then outer spacers are added at both ends. The outermost spacer is a protease cleavage site recognized by the replicase.

[0217] Figure 13: Self-amplified mRNA structure. The structural components from left to right are cap structure-5-terminal alphavirus untranslated sequence (5'UTR)-alphavirus replicase sequence-26S promoter sequence-arbitrary target protein sequence X-3-terminal alphavirus untranslated sequence (3'UTR)-polyadenine sequence (polyA sequence).

[0218] Figure 14: Schematic diagram of a self-amplified mRNA structure with microRNA122 binding sites inserted at different locations. Arrows represent the different locations of microRNA122 binding sites. The microRNA122 binding sites are constructed by combining six microRNA122 binding sites in series.

[0219] Figure 15: Denaturing agarose gel electrophoresis results of in vitro transcription of self-amplified mRNA inserted into the microRNA122 binding site at six different locations.

[0220] Figure 16: qPCR detection results of microRNA-122 in different cells.

[0221] Figure 17: 24 hours after Huh7.5.1 and C2C12 cells were transfected with self-amplified mRNA expressing Nluc-EGFP and inserted with microRNA122 binding sites, the expression of fluorescent protein was detected by fluorescence microscopy. The control was self-amplified mRNA without inserted microRNA122 binding sites.

[0222] Figure 18: Nluc luciferase expression 24 hours after transfection of Huh7.5.1 cells and C2C12 cells with self-amplified mRNA expressing Nluc-EGFP and inserted microRNA122 binding sites. The control is self-amplified mRNA without inserted microRNA122 binding sites. The expression values ​​were normalized with firefly luciferase.

[0223] Figure 19: Relative RNA levels of self-amplified mRNA in C2C12 cells 24 hours after transfection with self-amplified mRNA expressing Nluc-EGFP and inserted with microRNA122 binding sites. The control is self-amplified mRNA without inserted microRNA122 binding sites. The relative RNA levels after 24 hours of transfection were normalized with those after 2 hours of transfection. nsp1 represents the relative level of RNA encoding the replicase sequence, and EGFP represents the relative level of RNA encoding the EGFP sequence.

[0224] Figure 20: Relative RNA levels of self-amplified mRNA in Huh7.5.1 cells 24 hours after transfection with self-amplified mRNA expressing Nluc-EGFP and inserted with microRNA122 binding sites. The control is self-amplified mRNA without inserted microRNA122 binding sites. The relative RNA level values ​​24 hours after transfection were normalized with those 2 hours after transfection. nsp1 represents the relative level of RNA encoding the replicase sequence, and EGFP represents the relative level of RNA encoding the EGFP sequence.

[0225] Figure 21: Schematic diagram of self-amplifying mRNAs with different copy numbers of liver-specific microRNA-122 binding sequences inserted.

[0226] FIG22 : Nluc luciferase expression 24 hours after transfection of Huh7.5.1 or C2C12 cells with self-amplified mRNA expressing Nluc-EGFP and inserted with different copy numbers of microRNA122 binding sites.

[0227] Figure 23: Schematic diagram of mRNA molecule assembly.

[0228] Figure 24 shows the positions of amino acid mutations in the saRNA macrodomain and the amino acid sequence alignment of macrodomains from different viruses. Macro represents the macrodomain, AUD represents the alphavirus-unique domain, and HVD represents the hypervariable domain. Different shapes represent different mutations. Amino acids with greater than 50% identity are shaded in the amino acid alignment. The full names of the viruses listed in the figure are as follows: SARS-CoV: Severe Acute Respiratory Syndrome Coronavirus; SARS-CoV-2: Severe Acute Respiratory Syndrome Coronavirus 2; MERS-CoV: Middle East Respiratory Syndrome Coronavirus; SFV: Semliki Forest Virus; CHIKV: Chikungunya Virus; SINV: Sindbis Virus; MAYV: Mayaro virus; EEEV: Eastern Equine Encephalitis Virus; VEEV: Venezuelan Equine Encephalitis Virus.

[0229] Figure 25: Schematic diagram of the protein structure of the saRNA macrodomain. The arrows mark the positions of the macrodomain-bound ADP-ribose, glutamine at position 48 (abbreviated Q), and isoleucine at position 113 (abbreviated I).

[0230] Figure 26: Western blot analysis of the effects of macrodomain amino acid mutations on total protein ADP-ribosylation. Mock represents a mock transfection group, in which only transfection reagent was added without any RNA. GAPDH protein served as an internal control. The bottom value represents the ratio of total protein ADP-ribosylation levels in each group relative to the control.

[0231] Figure 27: Comparison of subgenomic RNA replication capacity of mutant saRNAs in HeLa cells. Significant differences between groups were analyzed using two-way ANOVA. **** and ^^^^ represent P values ​​less than 0.0001 for WT-saRNA versus 190-saRNA or 191-saRNA, respectively.

[0232] Figure 28: Comparison of the double-stranded RNA formation ability of mutant saRNA during HeLa cell replication. Figure 28a is a schematic diagram of the flow cytometry results 24 hours after transfection, and Figure 28b is a bar graph showing the statistical proportion of double-stranded RNA.

[0233] Figure 29: Principal component analysis of transcriptome levels 24 hours after HeLa cells were transfected with different saRNAs.

[0234] FIG30 : Venn diagram of the number of genes upregulated or downregulated in different saRNAs relative to the control group.

[0235] Figure 31: Ridge plot of differentially expressed genes clustered by wild-type saRNA versus control. Figure 31a shows the biological function cluster analysis of differentially expressed genes; Figure 31b shows the molecular function cluster analysis of differentially expressed genes. The horizontal axis represents the log2 value of the fold change, with green representing the adjusted P value. Blue dots represent each downregulated gene, and red dots represent each upregulated gene.

[0236] Figure 32: Heat map of genes at the transcriptome level of the interferon pathway after transfection with different saRNAs.

[0237] Figure 33: Fluorescence quantitative PCR verification results of genes related to the innate immune pathway 2, 6, and 24 hours after HeLa cells were transfected with different saRNAs.

[0238] Figure 34: Luciferase expression levels in HeLa cells 2, 6, 24 and 48 hours after transfection with different saRNAs.

[0239] Figure 35: Western blot analysis of cell translation activity 2, 6, and 24 hours after transfection with different saRNAs. GAPDH was used as an internal control.

[0240] Figure 36: Ribosomal RNA integrity testing of HeLa cells 2, 6, 24, and 48 hours after transfection with different saRNAs. Figure 36a shows the integrity of ribosomal RNA detected by capillary electrophoresis 24 hours after saRNA transfection. Arrows indicate degraded ribosomal RNA. Figure 36b shows the statistical results of ribosomal RNA integrity testing at different times.

[0241] Figure 37: Detection of eIF2α phosphorylation levels 6 and 24 hours after transfection with different saRNAs. peIF2α represents phosphorylated eIF2α, while total eIF2α protein and GAPDH serve as internal controls.

[0242] Figure 38: Fluorescence microscopy images 24 hours after transfection with different saRNAs. Red arrows mark cells with nuclear shrinkage and EGFP expression. DAPI indicates stained nuclei, and EGFP represents cells expressing EGFP.

[0243] Figure 39: Statistical results of the proportion of live cells that did not undergo apoptosis 24 and 48 hours after transfection with different saRNAs.

[0244] Figure 40: Detection of cleaved activated caspase 8 and caspase 3 proteins 24 hours after transfection with different saRNAs. Uncleaved full-length caspase served as a control. GAPDH served as an internal control.

[0245] Figure 41: Detection of cell apoptosis and EGFP expression intensity in HeLa cells transfected with wild-type saRNA and treated with or without caspase inhibitors.

[0246] Figure 42: Schematic diagram of in vivo expression level detection of different mRNA-LNPs. Figure 42a is a schematic diagram of the detection process, with D0 being the time of immunization. Figure 42b is in vivo fluorescence imaging of mice at days 1, 3, and 7 after immunization.

[0247] Figure 43: Time curve of luciferase expression level in mice after mRNA-LNP immunization.

[0248] Figure 44: Statistical results of the area under the curve of in vivo expression levels in mice after mRNA-LNP immunization.

[0249] Detailed Description of the Invention

[0250] As used herein, the terms "self-amplifying RNA" or "saRNA" or "sa-mRNA" are used interchangeably and refer to an mRNA that encodes an RNA replicase domain sequence and thus has the ability to self-replicate and amplify after entering the cell. Similar to mRNA, saRNA typically contains a 5'UTR, a 3'UTR, and a poly (A) tail, but also contains a non-structural protein sequence derived from a virus (such as an alpha virus) and a subgenomic promoter (sgPr) located upstream of the target protein coding sequence. saRNA is a positive-strand RNA molecule that is first translated into several non-structural protein components by the ribosome after entering the cell and assembled into RNA replicase. RNA replicase first uses the saRNA that first enters the cell to synthesize the saRNA negative chain, and then uses the negative chain as a template to synthesize new saRNA copies, thereby achieving self-amplification of saRNA. At the same time, RNA replicase also recognizes sgPr, and then begins to synthesize subgenomic RNA from its downstream, which accumulates in large quantities in the host cell. When the target protein encoded by saRNA is an antigen protein (for example, as a vaccine), the antigen gene encoded by the subgenomic RNA will translate a large number of antigen molecules and trigger cellular antigen presentation. The saRNA described herein includes both saRNA backbone sequences that do not contain target protein coding sequences and saRNAs that operably connect saRNA backbone sequences to target protein coding sequences.

[0251] As used herein, the term "macrodomain" refers to a conserved protein domain encoded by viruses such as alphavirus, coronavirus, and hepatitis E virus, which can recognize and remove ADP ribosylation and is therefore considered an ADP ribosyl hydrolase. Many studies have shown that the weakening of ADP ribose hydrolysis activity in the viral macrodomain will lead to a weakening of the viral replication ability, so the macrodomain is one of the potential hotspots for the recent development of antiviral drugs. In the alphavirus family represented by VEEV, the macrodomain is present at the N-terminus of nonstructural protein 3, as shown in Figure 24. The amino acid sequence of the macrodomain is relatively conserved among different viruses, with a homology of more than 50%.

[0252] As used herein, the term "RNA replicase," also known as RNA-dependent RNA replicase (RdRp), is a type of RNA polymerase that can synthesize RNA using RNA as a template. It is present in most RNA viruses and plays a role in replicating viral RNA and synthesizing mRNA. It is an enzyme essential for the replication of RNA viruses and viroids other than retroviruses. RNA replicases used in saRNA are typically derived from alphaviruses and contain nonstructural proteins 1, 2, 3, and 4 (nsP1, nsP2, nsP3, and nsP4).

[0253] The term "subgenomic promoter" (Subgenomic Promoter, abbreviated as SGP) refers to a nucleic acid sequence upstream (5' end) of a nucleic acid sequence (e.g., a coding sequence) that controls the transcription of the nucleic acid sequence by providing recognition and structural sites for RNA polymerase, typically RNA-dependent RNA polymerase, particularly functional alphavirus nonstructural proteins. SGP may include additional recognition or binding sites for additional factors. Subgenomic promoters are typically genetic elements of positive-strand RNA viruses (e.g., alphaviruses). The subgenomic promoter of an alphavirus is a nucleic acid sequence included in the viral genomic RNA. The general feature of a subgenomic promoter is that it allows transcription (RNA synthesis) to begin in the presence of an RNA-dependent RNA polymerase (e.g., functional alphavirus nonstructural proteins). The RNA (-) chain (i.e., the complementary chain of the alphavirus genomic RNA) serves as a template for the synthesis of (+) chain subgenomic transcripts, and the synthesis of (+) chain subgenomic transcripts typically begins at or near the subgenomic promoter.

[0254] As used herein, the term "subgenomic" refers to a nucleotide sequence (e.g., RNA or DNA) that is smaller in length or size than the genomic nucleotide sequence from which it is derived. For example, a subgenomic region may encode a VEEV structural protein, and subgenomic RNA may be transcribed from the subgenomic region using an internal subgenomic promoter, the sequence of which is located within the genomic viral RNA or its complement. Transcription of the subgenomic region may be mediated by a virally encoded polymerase that is associated with host cell-encoded proteins (e.g., nsP1-4).

[0255] Alphavirus structural proteins (core nucleocapsid protein C, envelope protein E2, and envelope protein E1, all components of the viral particle) are typically encoded by a single open reading frame under the control of a subgenomic promoter (Strauss & Strauss, Microbiol. Rev., 1994, vol. 58, pp. 491-562). The subgenomic promoter is recognized by cis-acting alphavirus nonstructural proteins. In particular, the alphavirus replicase uses the complementary strand of the (-) strand of the genomic RNA as a template to synthesize the (+) strand subgenomic transcript. The (+) strand subgenomic transcript encodes the alphavirus structural proteins (Kim et al., 2004, Virology, vol. 323, pp. 153-163, Vasiljeva et al., 2003, J. Biol. Chem. vol. 278, pp. 41636-41645). The subgenomic RNA transcript serves as a template for translation of an open reading frame encoding a structural protein, which is a polyprotein, and the polyprotein is cleaved to produce the structural proteins. During late stages of alphavirus infection in host cells, a packaging signal located within the nsP2 coding sequence ensures the selective packaging of the genomic RNA into budding virions that are packaged by the structural proteins (White et al., 1998, J. Virol., vol. 72, pp. 4320-4326).

[0256] As used herein, the term "alphavirus replication-essential sequence" refers to a sequence that is recognized by the alphavirus replicase and initiates subsequent RNA transcription and replication, and is derived from the alphavirus replicase sequence. The alphavirus replication-essential sequence must contain a minimum sequence (as shown in SEQ ID NO: 54), and the length of the sequence may be increased or adjusted based on the minimum sequence.

[0257] As used herein, the term "mature microRNA sequence" refers to the microRNA molecule formed after Dicer processing of the hairpin-forming microRNA precursor (pre-miRNA). Each of the two arms of the pre-miRNA produces a functional mature microRNA, each targeting a different site. These are generally designated "-5p" (or 5p) and "-3p" (or 3p), respectively, such as hsa-miR-21-5p and hsa-miR-21-3p, indicating that they are processed from the 5' and 3' arms of the hsa-mir-21 precursor, respectively. Sequence-wise, the 5p and 3p sequences are largely complementary.

[0258] In the present invention, unless otherwise specified, the term "integrity" refers to the percentage of the amount of target RNA product produced after the RNA in vitro transcription reaction is completed (including the target RNA product and unintended RNA products). The amount of target RNA product and the amount of total RNA product can be measured by electrophoresis. For example, if the amount of target saRNA product measured by electrophoresis accounts for 80% of the amount of all RNA products, the saRNA integrity is 80%.

[0259] In the present invention, unless otherwise specified, the "first" (e.g., the first mRNA molecule, the first DNA molecule, etc.) and the "second" (e.g., the second mRNA molecule, the second DNA molecule, etc.) are merely for reference distinction and do not have any particular order meaning.

[0260] In the present invention, the term "effective amount" refers to the amount of the mRNA molecule of any one of the present invention, the mRNA molecule combination of the present invention, the DNA molecule of any one of the present invention, or the saRNA molecule of any one of the present invention that can prevent or treat the indications or symptoms of the present invention, or reduce and / or alleviate the indications or symptoms in a subject.

[0261] mRNA therapy

[0262] Therapies based on mRNA technology deliver in vitro synthesized mRNA to specific cells in the human body, where it is translated into the desired protein in the cytoplasm. As vaccines or drugs, mRNA can be used to prevent infectious diseases, treat tumors, and promote protein replacement therapy. mRNA drugs have a rich target population and are not restricted by the druggability of the target protein or by intracellular or extracellular constraints. mRNA sequences are simple to design, utilizing the patient's own cells to produce molecules, bypassing the challenges of chemical synthesis. Furthermore, self-induced molecules are more potent. The mRNA production platform is highly scalable and reproducible, making it easy to scale up production.

[0263] (1) Preventive vaccines for infectious diseases

[0264] mRNA vaccines targeting infectious diseases encode antigens of related pathogens. After injection, they can express specific antigens in the body, induce cellular immunity and humoral immunity at the same time, and stimulate the production of corresponding antibodies and immune cells to prevent the corresponding pathogens.

[0265] (2) Tumor therapeutic vaccines / drugs

[0266] The mRNA encoding tumor-specific antigen targets is delivered into the body in a specific manner, so that these tumor-specific antigens are translated and presented on the cell surface, thereby activating the immune system, enabling it to specifically identify and kill tumor cells.

[0267] (3) Protein replacement therapy

[0268] Typically, protein replacement therapy is used to treat rare monogenic diseases and aims to restore enzyme function. Protein synthesis is difficult, administration presents many challenges and is expensive, and using mRNA to turn the human body into its own protein processing factory is theoretically an economical and efficient approach. Currently, mRNA-based protein replacement therapy mainly focuses on inherited metabolic diseases. mRNA can theoretically synthesize any protein and can be used as a protein supplement or replacement therapy to treat a variety of diseases. However, due to the need for targeted expression of mRNA and repeated administration, or even systemic administration, higher safety requirements are placed on it.

[0269] Self-amplifying RNA

[0270] The present invention constructs an improved saRNA, which includes a specific mutation in the macrodomain of the encoded replicase, so that the saRNA has reduced ADP ribose hydrolysis activity. At the same time, the mutation causes the replication ability of the replicase encoded by the sgRNA to be moderately weakened, thereby causing the proportion of double-stranded RNA formed in the saRNA replication to decrease. Since double-stranded RNA is a by-product in the saRNA replication process and is often recognized by the pattern recognition receptors in the cell to stimulate the innate immune response of the cell, the reduction of double-stranded RNA will reduce the activation of the natural immune receptor, thereby causing the intensity of the natural immune response induced by saRNA to be appropriately reduced. As verified in the examples, compared to the original wild-type saRNA, the translation inhibition of the sgRNA containing the mutation is significantly weakened, and cell apoptosis is significantly reduced, so the saRNA expression efficiency is significantly improved after the mutation. Accordingly, this improved sgRNA may include nucleotide sequences encoding various target proteins (such as antigenic proteins), and saRNA that effectively induces specific immunity in vivo can be obtained, and prepared into infectious diseases and tumor vaccines.

[0271] In another aspect, the present application is to carry out amino acid mutation in the macro domain in the replicase protein domain encoded by saRNA, weakening ADP ribose hydrolysis activity, thereby reducing the replication efficiency of saRNA, and then reducing immunogenicity compared to conventional saRNA, and achieving the improvement of expression efficiency. Therefore, the present invention provides a mutation macro domain and a nucleic acid molecule (such as an mRNA molecule or a DNA molecule) encoding the mutation macro domain. The present invention also provides a replicase comprising the mutation macro domain and a nucleic acid molecule (such as an mRNA molecule or a DNA molecule) encoding the replicase. The present invention further provides a nucleic acid molecule (such as a saRNA molecule or a DNA molecule) constructed by combining the nucleic acid sequence encoding the replicase with the sequence encoding the target gene.

[0272] In some embodiments, the saRNA disclosed herein comprises a nucleotide sequence encoding an RNA replicase, wherein the RNA replicase comprises a mutant macrodomain having a Q48P or I113F substitution (numbering according to SEQ ID NO: 55) compared to the wild-type macrodomain from VEEV (as shown in SEQ ID NO: 55). Specifically, the mutant macrodomain encoded by the saRNA may comprise an amino acid sequence as shown in SEQ ID NO: 57 or SEQ ID NO: 59. More specifically, the saRNA may comprise a nucleotide sequence encoding a mutant macrodomain as shown in SEQ ID NO: 68 or SEQ ID NO: 70. The replicase may be an alphavirus replicase, for example, comprising alphavirus nonstructural proteins 1, 2, 3, and 4.

[0273] In some embodiments, the saRNA disclosed herein comprises nucleotide sequences encoding nsP1, nsP2, nsP3, and nsP4, components of the replicase, derived from an alphavirus. The four replicase components are capable of assembling into an RNA replicase complex, wherein the mutant macrodomain is located at the N-terminus of nsP3. The four nonstructural protein components, nsP1, nsP2, nsP3, and nsP4, assemble into an RNA replicase complex in the form of a polyprotein. Each of nsP1-4 has its own unique function, wherein nsP4 plays the role of an RNA polymerase using RNA as a template. The sequences of the replicase components encoded by the saRNA can be derived from, for example, Venezuelan equine encephalitis virus (VEEV) and forest encephalitis virus (SFV). In some embodiments, the domain sequence of at least one nonstructural replicase comprises a sequence selected from Group IV RNA viruses, including Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus, Mucambo virus, Pixuna virus, Western equine encephalitis virus (WEE), Sindbis virus, forest encephalitis virus (SFV), and the like. In some embodiments, the multiple non-structural protein sequences that form the replicase are derived from an alphavirus, such as VEEV. Specifically, the saRNA may comprise a nucleotide sequence encoding a replicase component protein as shown in SEQ ID NO: 67 or SEQ ID NO: 69.

[0274] Although native alphavirus genomes encode structural proteins in addition to non-structural replicases, in some embodiments, the alphavirus-based saRNA of the present invention does not encode alphavirus structural proteins.

[0275] In some embodiments, in addition to the nucleotide sequences encoding the four replicase proteins nsP1, nsP2, nsP3, and nsP4, the saRNA further comprises a 5' untranslated region, a 3' untranslated region, and a poly(A) tail, wherein the nucleotide sequences encoding the four replicase proteins nsP1, nsP2, nsP3, and nsP4 are located between the 5' untranslated region and the 3' untranslated region. It will be readily understood by those skilled in the art that the 5' UTR, 3' UTR, and poly(A) sequences are not limited to those exemplified herein, and sequences conventionally used for mRNA construction can be used for the saRNA of the present invention. The 5' untranslated region can be derived from a variety of viruses, such as alphaviruses and coronaviruses. In some embodiments, the 5' untranslated region is derived from Venezuelan equine encephalitis virus. Specifically, the 5' untranslated region may comprise the nucleotide sequence shown in SEQ ID NO: 63. The 3' untranslated region can be derived from a variety of viruses, such as alphaviruses and coronaviruses. In some embodiments, the 3' untranslated region is derived from Venezuelan equine encephalitis virus. Specifically, the 3' untranslated region may comprise the nucleotide sequence shown in SEQ ID NO: 64. In some embodiments, the poly(A) sequence comprises 20-200 adenylate nucleotides. The poly(A) sequence may contain a restriction enzyme site within or at the 3' end.

[0276] In some embodiments, the saRNA further comprises a nucleotide sequence encoding a protein of interest or a target gene (GOI) coding sequence, wherein the protein of interest is or the target gene encodes a gene selected from the group consisting of a therapeutic polypeptide, a prophylactic polypeptide, a diagnostic polypeptide, a reporter gene, an antigen, or a gene encoding a regulatory structure. For example, the protein of interest can be an infectious disease antigen, an allergic antigen, or a tumor antigen. Alternatively, the target gene can encode a non-coding gene, such as an siRNA, a microRNA, a gRNA, or the like.

[0277] In some embodiments, the saRNA further comprises a nucleotide sequence encoding one or more antigenic proteins for use as a vaccine. The antigenic protein can be a viral antigenic protein.

[0278] In some embodiments, the saRNA further comprises a nucleotide sequence encoding one or more therapeutic proteins or immunomodulators for use as disease therapeutic agents. The immunomodulators can be cytokines, chemokines or other immunostimulants or inhibitors.

[0279] In some embodiments, the saRNA disclosed herein comprises two expression units, wherein the first expression unit encodes multiple nonstructural domain sequences of an RNA replicase, and the second expression unit encodes a protein of interest operably linked to a subgenomic promoter, the first and second expression units are operably linked, and the first expression unit is located 5' to the second expression unit. In some embodiments, the subgenomic promoter is a 26S promoter, for example, as set forth in SEQ ID NO: 65.

[0280] Therefore, the saRNA of the present disclosure has at least two coding regions, the first coding region encoding multiple non-structural replicase domain sequences; and the second coding region encoding a gene of interest operably linked to a subgenomic promoter.

[0281] More specifically, the saRNA disclosed herein may comprise the following 5' to 3' operably linked nucleic acid sequence: 5'UTR-nsP-SGP-GOI-3'UTR-Poly A,

[0282] More specifically, the saRNA disclosed herein may comprise the following nucleic acid sequences operably linked from 5' to 3': 5'UTR-nsP1-nsP2-nsP3-nsP4-SGP-GOI-3'UTR-Poly A,

[0283] Wherein, 5'UTR is 5' untranslated region, nsP (including nsP1, nsP2, nsP3, nsP4) are multiple non-structural protein sequences capable of forming replicase, SGP is a subgenomic promoter, GOI is one or more target protein encoding genes, 3'UTR is 3' untranslated region, Poly-A is 3' polyadenylation tail, and wherein nsP comprises a mutated macrodomain. When multiple GOIs are present, each GOI can be operably linked to its own SGP. In some embodiments, the SGP is a 26S promoter, for example, as shown in SEQ ID NO:65.

[0284] In some embodiments, the saRNA disclosed herein comprises a 5' untranslated sequence that is at least 85% identical to SEQ ID NO: 63, a non-structural protein coding sequence that is at least 85% identical to SEQ ID NO: 68 or 70 and comprises a nucleotide sequence encoding a macrodomain as set forth in SEQ ID NO: 57 or 59, and / or a 3' untranslated sequence that is at least 85% identical to SEQ ID NO: 64. The at least 85% identity may be at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, and encompasses functional variants that retain the corresponding functions of these sequences.

[0285] In some embodiments, the 5'UTR, nsP, SGP and 3'UTR sequences are all derived from alphaviruses. The alphavirus genome encodes 4 nonstructural proteins and 6 structural proteins (capsid, E3, E2, 6K, TF and E1). Alphavirus structural proteins (core nucleocapsid protein C, envelope protein E2 and envelope protein E1, all components of the virus particle) are usually encoded by a single open reading frame under the control of a subgenomic promoter (Strauss & Strauss, Microbiol. Rev., 1994, vol. 58, pp. 491-562). The subgenomic promoter is recognized by cis-acting alphavirus nonstructural proteins. In particular, the alphavirus replicase uses the (-) chain complementary chain of the genomic RNA as a template to synthesize the (+) chain subgenomic transcript. (+) strand subgenomic transcripts encode alphavirus structural proteins (Kim et al., 2004, Virology, vol. 323, pp. 153-163, Vasiljeva et al., 2003, J. Biol. Chem. vol. 278, pp. 41636-41645). Subgenomic RNA transcripts serve as templates for translation of open reading frames encoding structural proteins, which are polyproteins, and the polyprotein is cleaved to produce the structural proteins.

[0286] The saRNA disclosed herein can be prepared by in vitro transcription using a DNA plasmid template that is linearized. To this end, a restriction enzyme site can be added to the 3' end of the saRNA to facilitate cleavage. In addition, the saRNA can further contain other coding sequences, such as a signal peptide coding sequence at the 5' end, other sequences compatible with the replicase coding sequence, a Kozak sequence downstream of the SGP promoter, and additional downstream coding regions, such as for encoding other desired gene products.

[0287] In some specific embodiments, the saRNA disclosed herein comprises a saRNA backbone sequence as shown in SEQ ID NO: 61 or 62 (i.e., without the addition of a target protein coding sequence or target gene sequence). In some specific embodiments, the saRNA disclosed herein comprises a nucleic acid sequence as shown in SEQ ID NO: 58 or 60, wherein luciferase and green fluorescent fusion protein coding sequences are used as exemplary target proteins.

[0288] Methods for preparing saRNA

[0289] saRNA is typically obtained by in vitro transcription (IVT) using an RNA polymerase. In one aspect, the present disclosure relates to a method for obtaining self-amplified mRNA, comprising: using a DNA molecule (e.g., a plasmid) encoding the saRNA of the present disclosure as a template and an RNA polymerase to produce saRNA by in vitro transcription. For example, saRNA can be prepared by in vitro transcription of DNA encoding saRNA using a suitable DNA-dependent RNA polymerase, such as T7 phage RNA polymerase, SP6 phage RNA polymerase, T3 phage RNA polymerase, T5 phage RNA polymerase, RNA polymerase III, RNA polymerase II, Taq polymerase, etc., or mutants of these polymerases.

[0290] The transcription reaction will contain nucleotides and other components that support the activity of the selected polymerase, such as a suitable buffer and suitable salts. In addition, nucleotide analogs can also be incorporated into saRNA to, for example, alter the stability of such RNA molecules, increase resistance to ribonucleases, establish replication after introduction into appropriate host cells, and / or induce or reduce innate and adaptive immune responses.

[0291] Applications of saRNA

[0292] mRNA-based therapies involve delivering in vitro synthesized mRNA to specific cells in the human body, where it is translated into the desired protein in the cytoplasm. As vaccines or drugs, mRNA can be used to prevent infectious diseases, treat tumors, and promote protein replacement therapy. mRNA drugs offer a rich array of targets and are not restricted by the druggability of the target protein or by intracellular or extracellular constraints. mRNA sequences are simple to design, utilizing the patient's own cells to produce molecules, bypassing the challenges of chemical synthesis. Furthermore, self-induced molecules are more potent. The mRNA production platform is highly scalable and reproducible, making it easy to scale up production.

[0293] In one aspect, the present disclosure relates to a pharmaceutical composition, such as a vaccine composition comprising a saRNA of the present disclosure and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be a saRNA delivery system, preferably a nanoparticle composition. In some aspects, the nanoparticle composition comprises a cationic lipid, a PEG-modified lipid, a sterol, and a non-cationic lipid.

[0294] In one aspect, the sa-mRNA disclosed herein can be used for vaccination. The vaccine can be a preventive vaccine in the field of infectious diseases. For example, the target protein encoded in the saRNA is an antigen encoding a pathogen related to an mRNA vaccine for an infectious disease. After injection, the specific antigen can be expressed in the body, which can simultaneously induce cellular immunity and humoral immunity, stimulate the production of corresponding antibodies and immune cells to prevent the corresponding pathogen. The vaccine can be a tumor therapeutic vaccine. For example, mRNA encoding tumor-specific antigen targets is delivered into the body in a specific manner, so that these tumor-specific antigens are translated and presented on the cell surface, thereby activating the immune system so that it can specifically recognize and kill tumor cells.

[0295] In one aspect, the saRNA disclosed herein can be used to treat diseases, for example by providing therapeutic or immunomodulatory proteins targeting a disease. Protein replacement therapy is typically used to treat rare monogenic diseases, aiming to restore enzyme function. Protein synthesis is difficult, administration presents numerous challenges, and is expensive. However, using mRNA to transform the human body into its own protein processing plant is theoretically an economically viable and efficient approach. The saRNA disclosed herein can theoretically synthesize any target protein and can be used as a protein supplement or replacement therapy to treat a variety of diseases.

[0296] In one aspect, the present disclosure provides a method of delivering a protein of interest to a subject, comprising administering to the subject a pharmaceutical composition containing one or more self-amplifying mRNAs of the present disclosure.

[0297] Advantageous Effects of the Invention

[0298] In one aspect, the present invention achieves one or more of the following technical effects (1) to (3):

[0299] (1) The present invention improves the integrity of saRNA transcription in vitro.

[0300] (2) The present invention has no negative impact on the replication function of saRNA.

[0301] (3) The present invention has no negative impact on the expression function of saRNA.

[0302] In addition, the current self-amplifying mRNA is an mRNA carrier modified from the alphavirus genome and lacks specific microRNA binding sites. After being encapsulated and delivered into the body by currently commonly used lipid nanoparticles (LNPs), the inherent physical and chemical properties of the nanoparticles may mediate the large-scale entry of self-amplifying mRNA into parenchymal tissues such as the liver for expression, potentially causing liver toxicity in certain application examples. Therefore, in another aspect, the technical effect achieved by the present invention also includes the use of microRNAs that are specifically highly expressed in liver tissue to degrade self-amplifying mRNA, thereby reducing liver toxicity.

[0303] In yet another aspect, the saRNA of the present invention has advantages over conventional mRNA, namely, it can achieve the same protein expression levels as conventional mRNA at much lower doses and prolong the presence of antigenic proteins in the body, potentially enhancing immune responses. Furthermore, lower effective doses can reduce saRNA production costs. As a therapeutic, the dose and number of injections used in mRNA therapy can be reduced, thereby prolonging the therapeutic effect while reducing potential toxic side effects of mRNA and delivery vectors.

[0304] Furthermore, the saRNA of the present invention is improved to have one or more of the following characteristics:

[0305] (1) The replication efficiency is reduced, and the dsRNA formed during saRNA replication is significantly reduced;

[0306] (2) Reduced the intensity of the innate immune response induced by saRNA;

[0307] (3) Reduced translational inhibition of saRNA;

[0308] (4) Reduced saRNA-induced cell apoptosis;

[0309] (5) Improved the expression efficiency of saRNA.

[0310] The saRNA of the present disclosure contains a mutated macrodomain, which weakens the ADP ribose hydrolysis activity, thereby weakening the ability of viral replication and stimulating the innate immune system. Therefore, the saRNA of the present disclosure may have a reduced cytotoxic effect on host cells or subjects, providing enhanced safety. For example, the saRNA of the present disclosure can produce a high expression level of the encoded gene product while reducing the risk of undesirable effects (such as injection site irritation and / or pain). In addition, since the ability of the saRNA of the present disclosure to stimulate the innate immune system is reduced, it is particularly suitable for vaccine preparation to provide appropriate immunity to the host.

[0311] In addition, the saRNA of the present invention does not contain nucleotide sequences encoding viral structural proteins and therefore cannot lead to the production of alphavirus particles containing RNA. The inability to produce these viral particles means that the saRNA cannot self-perpetuate in an infectious form. The alphavirus structural proteins required for perpetuation in wild-type viruses are not present in the saRNA, and their position is replaced by the GOI, so that the saRNA encodes the desired gene products rather than the structural proteins of the alphavirus virion.

[0312] More specifically, the saRNAs containing mutated macrodomains of the present invention significantly reduce the amount of dsRNA formed during replication. Human cells contain sensors that detect invading viruses, known as pattern recognition receptors. One of the signals they recognize is double-stranded RNA (dsRNA) present in the cytoplasm, which may indicate viral RNA replication within the cell. During replication, saRNAs form double-stranded RNA, which closely resembles replicating viral RNA and therefore may stimulate the cell's innate immune response. This may further enhance the effectiveness of vaccines. However, while this stimulated immune response may promote immune responses as a vaccine, it may also cause side effects as a therapy. If the stimulated innate immune response is too strong, it may suppress mRNA expression, negatively impacting the efficacy of both the therapy and the vaccine. Furthermore, the double-stranded RNA produced during saRNA replication is key to activating the interferon or NF-κB signaling pathways, thereby inducing a large number of downstream antiviral genes to inhibit saRNA replication. Therefore, the immunogenicity of saRNAs requires precise design and adjustment to appropriately reduce their replication capacity and double-stranded RNA production. The sgRNAs disclosed herein achieve this goal.

[0313] At the same time, the translation inhibition and induced apoptosis of the saRNA containing the mutant macro domain of the present invention are significantly reduced. mRNA usually triggers intrinsic receptors in cells, such as toll-like receptors (TLRs), which lead to the production of type I interferon and the inhibition of translation. However, the saRNA of the present invention is subject to reduced translation inhibition due to reduced immunogenicity, so although its replication ability is reduced, its expression efficiency in the cell is ultimately improved compared to the control saRNA encoding the wild-type macro domain.

[0314] The sequences involved in the present invention are summarized as follows (when it is an RNA sequence, T is replaced by U): Sequence 1: BspQI endonuclease recognition site

[0315] Sequence 2: saRNA-062 sequence

[0316] Sequence 3: saRNA-152 sequence

[0317] Sequence 4: saRNA-153 sequence

[0318] Sequence 5: saRNA-154 sequence

[0319] Sequence 6: saRNA-183 sequence

[0320] Sequence 7: saRNA-184 sequence

[0321] Sequence 8: saRNA-185 sequence

[0322] Sequence 9: saRNA-243 sequence

[0323] In the above sequence 2-9:

[0324] "NNNNNNNNNNNNNNNN" indicates exogenous genes, including but not limited to: vaccine antigen genes, tumor-killing genes, therapeutic protein genes, and antibody genes; exogenous genes can be adjusted according to different purposes;

[0325] Here, "N" can be any base among A, T, C, and G, and the number of bases is not particularly limited; the 16 Ns here are only for illustration and are not limited to 16 bases.

[0326] Sequence 10:062 replicase coding sequence (SEQ ID NO:10)

[0327] Sequence 11:152 replicase coding sequence (SEQ ID NO:11)

[0328] Sequence 12:183 replicase coding sequence (SEQ ID NO:12)

[0329] Sequence 13:184 replicase coding sequence (SEQ ID NO:13)

[0330] Sequence 14:185 replicase coding sequence (SEQ ID NO:14)

[0331] Sequence 15:243 replicase coding sequence (SEQ ID NO:15)

[0332] Sequence 16: 5' untranslated sequence (SEQ ID NO: 16)

[0333] Sequence 17: 3' untranslated sequence (SEQ ID NO: 17)

[0334] Sequence 18: Subgenomic promoter sequence (SEQ ID NO: 18)

[0335] Sequence 19: Polyadenylation sequence (SEQ ID NO: 19)

[0336] Sequence 20: Amino acid sequence of alphavirus RNA replicase (SEQ ID NO: 20)

[0337] Sequence 21: S23H02 sequence

[0338] Sequence 22: Six microRNA-122 binding site tandem cassette sequences

[0339] Sequence 23: saRNA-EGFP-miRNA-1 sequence (SEQ ID NO: 23)

[0340] Sequence 24: saRNA-EGFP-miRNA-2 (SEQ ID NO: 24)

[0341] Sequence 25: saRNA-EGFP-miRNA-3 (SEQ ID NO: 25)

[0342] Sequence 26: saRNA-EGFP-miRNA-4 (SEQ ID NO: 26)

[0343] Sequence 27: saRNA-EGFP-miRNA-5 (SEQ ID NO: 27)

[0344] Sequence 28: saRNA-EGFP-miRNA-6 (SEQ ID NO: 28)

[0345] Sequence 29: saRNA-EGFP sequence (SEQ ID NO: 29)

[0346] Sequence 30: microRNA-122-5p binding site sequence (SEQ ID NO: 30)

[0347] Sequence 31: microRNA-1-5p binding site sequence (SEQ ID NO: 31)

[0348] Sequence 32: microRNA-124-5p binding site sequence (SEQ ID NO: 32)

[0349] Sequence 33: Inner spacer sequence 1

[0350] AGCGCGA (SEQ ID NO: 33)

[0351] Sequence 34: Inner spacer sequence 2

[0352] ACGCGAA (SEQ ID NO: 34)

[0353] Sequence 35: 5' outer spacer sequence 1 (SEQ ID NO: 35)

[0354] Sequence 36: 5' outer spacer sequence 2 (SEQ ID NO: 36)

[0355] Sequence 37: 3' outer spacer sequence 1 (SEQ ID NO: 37)

[0356] Sequence 38: 3' outer spacer sequence 2 (SEQ ID NO: 38)

[0357] Sequence 39: 5' replicase recognition protease cleavage site 1 (SEQ ID NO: 39)

[0358] Sequence 40: 3' replicase recognition protease cleavage site 1 (SEQ ID NO: 40)

[0359] Sequence 41: Amino acid sequence of alphavirus RNA replicase (SEQ ID NO: 41)

[0360] Sequence 42: Coding sequence of alphavirus RNA replicase (SEQ ID NO: 42)

[0361] Sequence 54: The shortest sequence contained in the essential sequence for alphavirus replication: (SEQ ID NO: 54)

[0362] Sequence 55: Wild-type macrodomain amino acid sequence from VEEV (SEQ ID NO: 55)

[0363] Sequence 57: amino acid sequence of the macrodomain corresponding to saRNA-190 (introducing the Q48P mutation, changing the corresponding RNA coding sequence from CAG to CCG) (SEQ ID NO: 57)

[0364] Sequence 59: amino acid sequence of the macrodomain corresponding to saRNA-191 (introducing the I113F mutation, the corresponding RNA coding sequence changed from ATC to TTC) (SEQ ID NO: 59)

[0365] Sequence 56: Wild-type saRNA sequence

[0366] Single underline: 5' and 3' UTR; Double underline: replicase domain sequence (shaded part is macro domain coding sequence); Box: 26S promoter and Kozak sequence; Italic: luciferase and green fluorescent fusion protein coding sequence, but can be replaced by any exogenous gene sequence; Dotted underline: BsQI restriction enzyme sequence

[0367] Sequence 58: saRNA-190 sequence (differs from sequence 57 only by the Q48P mutation introduced into the macrodomain) (SEQ ID NO: 58)

[0368] Sequence 60: saRNA-191 sequence (differs from sequence 57 only by the I113F mutation introduced into the macrodomain) (SEQ ID NO: 60)

[0369] Sequence 61: saRNA-190 backbone sequence

[0370] Sequence 62: saRNA-191 backbone sequence

[0371] In the sequence 61-62 above:

[0372] "NNNNNNNNNNNNNNNN" indicates exogenous genes, including but not limited to: vaccine antigen genes, tumor-killing genes, therapeutic protein genes, and antibody genes; exogenous genes can be adjusted according to different purposes;

[0373] Here, "N" can be any base among A, T, C, and G, and the number of bases is not particularly limited; the 16 Ns here are only for illustration and are not limited to 16 bases.

[0374] Sequence 63: 5' untranslated sequence (SEQ ID NO: 63)

[0375] Sequence 64: 3' untranslated sequence (SEQ ID NO: 64)

[0376] Sequence 65: Subgenomic promoter sequence (SEQ ID NO: 65)

[0377] Sequence 66: Polyadenylation sequence (SEQ ID NO: 66)

[0378] Sequence 67: saRNA-190 replicase coding sequence (SEQ ID NO: 67)

[0379] Sequence 68: saRNA-190 macrodomain encoding sequence (SEQ ID NO: 68)

[0380] Sequence 69: saRNA-191 replicase coding sequence (SEQ ID NO: 69)

[0381] Sequence 70: saRNA-191 macrodomain encoding sequence (SEQ ID NO: 70)

[0382] Sequence 71: Nonstructural protein 1 amino acid sequence (SEQ ID NO: 71)

[0383] Sequence 72: Nonstructural protein 2 amino acid sequence (SEQ ID NO: 72)

[0384] Sequence 73: Nonstructural protein 3 amino acid sequence (corresponding to saRNA-190, with Q48P mutation introduced) (SEQ ID NO: 73)

[0385] Sequence 74: Nonstructural protein 3 amino acid sequence (corresponding to saRNA-191, with I133F mutation introduced) (SEQ ID NO: 74)

[0386] Sequence 75: Nonstructural protein 4 amino acid sequence (SEQ ID NO: 75) DETAILED DESCRIPTION

[0387] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0388] Huh7.5.1 cells were purchased from Mingzhou bio, catalog number MZ-2129.

[0389] C2C12 cells were purchased from Mingzhou bio, catalog number MZ-0034.

[0390] Lipofectamine transfection reagent -mRNA Transfection Kit was purchased from Mirus, catalog number MIR 2225.

[0391] Example 1: Modification and integrity detection of positions 4497-4503 in the saRNA-EGFP template plasmid psaRNA-062

[0392] 1. Optimize the position: Modify positions 4497-4503 (5'-ACTCTTC-3') in the saRNA template plasmid psaRNA-062 (such as SEQ ID NO: 2) (the A base in the ATG start codon sequence of the alphavirus replicase is marked as the first position, the same below).

[0393] 2. Optimization method: By using the sequence-directed mutagenesis method, the T base at the 4500th bp position was mutated to the C base or the A base, or the TCA base at bp 4501-4503 was mutated to the AGC base. The three template plasmids thus obtained were named psaRNA-152 (SEQ ID NO: 3), psaRNA-153 (SEQ ID NO: 4), and psaRNA-154 (SEQ ID NO: 5).

[0394] 3. Preparation of modified saRNA by in vitro transcription:

[0395] a. Linearization of in vitro transcription template plasmid: Specifically, add 8 μg of in vitro transcription template plasmid (psaRNA-062, psaRNA-152, psaRNA-153, and psaRNA-154), 4 μL of BspQ I, and 4 μL of 10× Digestion Buffer in sequence. Finally, add DEPC water to 40 μL and incubate at 50°C for 2 h to linearize the plasmid.

[0396] b. In vitro transcription to obtain modified saRNA: Specifically, add 10× T7 polymerase buffer, 7.5 mM ATP, 7.5 mM GTP, 7.5 mM UTP, 7.5 mM CTP, 1 μg of linearized in vitro transcription template, 400 U T7 RNA polymerase, 20 U RNase I inhibitor, and 6 mM cap analog, bring the volume up to 20 μL, and incubate at 37°C for 2 hours. After the reaction, add 2 U DNase I and incubate at 37°C for 15 minutes. The reaction mixture is brought up to 50 μL, and 25 μL of 7.5 M lithium chloride solution is added and mixed. After incubation at -20°C for 30 minutes, centrifuge at 13,000 g for 10 minutes at 4°C. Discard the supernatant, wash the pellet with 70% ethanol, centrifuge at 13,000 g for 2 minutes at 4°C, discard the supernatant, and dissolve in 30 μL of water. After measuring the concentration using a spectrophotometer, an appropriate volume of RNA was taken and the RNA integrity was tested using a Qsep400 capillary electrophoresis device. The test results are shown in Figure 1. The results show that only the integrity of the optimized saRNA-152 was improved, from 76.7% to 81.3%.

[0397] Example 2: Integrity detection of different saRNAs constructed using the optimized psaRNA-152 template plasmid as the backbone

[0398] Using the psaRNA-152 plasmid as a template, the A base at position 4509 was mutated to T, C, and G, respectively. The resulting template plasmids were named psaRNA-183 (SEQ ID NO: 6), psaRNA-184 (SEQ ID NO: 7), and psaRNA-185 (SEQ ID NO: 8). The specific site-directed mutagenesis and detection methods were the same as in Example 1. As shown in the capillary electrophoresis results in Figure 2, the integrity of the different saRNAs constructed using psaRNA-152 as the backbone was significantly improved compared to the original backbone psaRNA-062.

[0399] Example 3: Modification and integrity detection of positions 1680-1676 in the psaRNA-183 template plasmid

[0400] The optimized saRNA template plasmid psaRNA-183 was modified at positions 1680-1676 (5'-GCTCTTA-3'), specifically by mutating the TCT bases at positions 1672-1674 to AGC bases. The resulting template plasmid was named psaRNA-243 (SEQ ID NO: 9). The specific site-directed mutagenesis and detection methods were the same as in Example 1. The results of capillary electrophoresis after optimization are shown in Figure 3. The results show that the proportion of short transcripts in the optimized saRNA is significantly reduced, and the integrity of saRNA-243 is significantly improved.

[0401] Example 4: Verification of luciferase gene saRNA replication expression efficiency after skeleton optimization

[0402] 1. Luciferase Assay

[0403] a. Cell seeding: An appropriate number of Huh7.5.1 cells were seeded into a 24-well cell culture plate and cultured overnight at 37°C in a 5% CO2 atmosphere.

[0404] b. Self-amplified mRNA transfection into Huh7.5.1 cells: 0.5 μg of backbone-optimized luciferase gene-expressing saRNA-152, saRNA-243, or non-optimized saRNA-062 was transfected into Huh7.5.1 cells using a lipofectamine transfection reagent and cultured overnight at 5% CO2 and 37°C for 72 hours.

[0405] c. Luciferase detection: 24 and 72 hours after transfection, Nano-Glo Luciferase Assay System detection reagent was used. 25 μl of Nano-Glo Luciferase Assay Reagent was transferred to a 96-well white opaque bottom plate. 25 μl of supernatant sample was added to the well and mixed evenly. After waiting for 3 minutes, the signal was detected using a microplate reader. The test results are shown in Figures 4 and 5. The luciferase expression levels of saRNA-152 and saRNA-243 after backbone optimization were not significantly different from those of the unoptimized backbone at different time points, indicating that backbone optimization did not affect saRNA expression.

[0406] 2. Detection of self-amplification mRNA replication efficiency

[0407] Cell seeding and self-amplified mRNA transfection: Same as fluorescent protein and luciferase detection.

[0408] Treatment of cell samples: 2 hours, 24 hours, and 72 hours after transfection, discard the supernatant, rinse the cells once with 500 μl PBS, discard the supernatant, add 500 μl cell lysis buffer and collect the cells by pipetting into EP tubes, and store in a -80°C refrigerator.

[0409] Reverse transcription fluorescence quantitative PCR detection of saRNA replication: After total RNA was extracted from cell samples using the RNeasy Mini Kit, RNA was quantified using a UV spectrophotometer. After reverse transcription was completed using the HiScript III 1st Strand cDNA Synthesis Kit, fluorescence quantitative PCR experiments were performed using 2×ChamQ Universal SYBR qPCR Master Mix. The detection primers are as follows: alphavirus replicase sequence (nsp1) upstream primer F: 5'-GACGGACCGACAAGTCTCTA-3' (SEQ ID NO: 22), alphavirus replicase sequence (nsp1) downstream primer R: 5'-GGTGGTGTCAAAGCCTATCCA-3' (SEQ ID NO: 23), EGFP sequence upstream primer F: 5'-AGCTGGAGTACAACTACA-3' (SEQ ID NO: 24), EGFP downstream primer R: 5'-CTGATCTTGAAGTTCACC-3' (SEQ ID NO: 25), GAPDH sequence upstream primer F: 5'-GGTATCGTGGAAGGACTC-3' (SEQ ID NO: 26), GAPDH downstream primer R: 5'-GTAGAGGCAGGGATGATG-3' (SEQ ID NO: 27). The reaction cycle conditions were: 95°C for 30 seconds - (95°C for 10 seconds - 60°C for 30 seconds) × 40 cycles. The test results are shown in Figure 6. The results show that there is no significant difference between the optimized saRNA-152 and the unoptimized backbone at different time points, indicating that backbone optimization also does not affect the replication ability of saRNA.

[0410] Example 5: Preparation of optimized backbone saRNA expressing human papillomavirus antigen gene Ag9.1

[0411] 1. Construction of in vitro transcription template plasmid

[0412] Using saRNA-243 as a template plasmid, the luciferase coding sequence in it was replaced with the human papillomavirus (HPV) antigen gene Ag9.1 coding sequence through homologous recombination method to obtain a self-amplified mRNA in vitro transcription template plasmid expressing Ag9.1, named L23H02.

[0413] 2. Preparation of in vitro transcription of saRNA expressing Ag9.1:

[0414] a. Linearization of in vitro transcription template plasmid

[0415] 8 μg of in vitro transcription template plasmid L23H04, 4 μL of BspQI, and 4 μL of 10× Digestion Buffer were added in sequence, and finally the system was supplemented to 40 μL with double distilled water. The plasmid was linearized at 50°C for 2 hours. After agarose gel electrophoresis to detect the complete linearization of the plasmid, it was directly used as an in vitro transcription template without purification.

[0416] b. Obtain L23H02 self-amplified mRNA by in vitro transcription

[0417] 10× T7 polymerase buffer, 7.5 mM ATP, 7.5 mM GTP, 7.5 mM UTP, 7.5 mM CTP, 1 μg of linearized in vitro transcription template, 400 U T7 RNA polymerase, 20 U RNase I inhibitor, and 6 mM cap analog were added sequentially. The volume was brought to a total of 20 μL and incubated at 37°C for 2 hours. After the reaction, 2 U DNase I was added and incubated at 37°C for 15 minutes. The reaction system was brought to 50 μL and mixed with 25 μL of 7.5 M lithium chloride solution. After incubation at -20°C for 30 minutes, the reaction system was centrifuged at 13,000 g for 10 minutes at 4°C. The supernatant was discarded and the pellet was washed with 70% ethanol. The pellet was centrifuged at 13,000 g for 2 minutes at 4°C and the supernatant was discarded. The wash was repeated once. The supernatant was aspirated and dissolved in 30 μL of water to obtain saRNA expressing Ag9.1, designated S23H02 (sequence shown in SEQ ID NO: 21). After measuring the concentration with a spectrophotometer, the integrity was tested using Qsep400. The capillary electrophoresis results are shown in Figure 7 , which showed that the size of the in vitro transcribed mRNA was consistent with expectations, the integrity was 82%, and there were no obvious short transcripts.

[0418] Example 6: Preparation of saRNA lipid nanoparticle formulation expressing Ag9.1

[0419] The S23H02 prepared in Example 5 was encapsulated in four lipid components to form mRNA lipid nanoparticles. Specifically, a lipid mixture (ionizable lipid ALC-0315: distearoylphosphatidylcholine: cholesterol: PEG lipid ALC-0159) dissolved in ethanol was mixed with S23H02 dissolved in citrate buffer using a commercial microfluidic device, NanoAssembler. After mixing, the lipid nanoparticles were mixed with phosphate-buffered saline, and residual ethanol was removed by ultrafiltration. Finally, the lipid nanoparticles were stored at -80°C with a cryoprotectant to obtain the lipid nanoparticle formulation S23H02-LNP, which was used in the following experiments.

[0420] Example 7: Comparison of tumor therapeutic effects of optimized backbone saRNA expressing HPV antigens and mRNA expressing HPV antigens

[0421] The drugs used in this example are shown in Table 1 below.

[0422] Table 1

[0423] S23H02, M22H04, and a negative control were transfected into tissue culture media containing Lipofectamine 3000. Supernatants were collected at the time points indicated in Figure 8 , and the expression of secreted HPV16 antigens was detected using an ELISA assay that detects the E7 domain. The results are shown in Figure 8 . S23H02 showed stronger humoral immunity against specific antigens than M22H04.

[0424] C57bl / 6 mice were challenged with TC1 tumor cells on day 0, followed by three doses of therapeutic vaccine injected into the hind leg muscles on days 5, 8, and 11.

[0425] The tumor volume and mouse body weight were recorded, and the results are shown in Figures 9 and 10, respectively.

[0426] Splenocytes were collected from C57bl / 6 mice that had received one dose of the therapeutic vaccine. After labeling, the number of E7-specific CD8 T cells in the spleen was measured using flow cytometry. The results are shown in Figure 11. S23H02 demonstrated significantly stronger cellular immunity against specific antigens than M22H04.

[0427] The above experimental results show that compared with traditional mRNA vaccines, the optimized saRNA vaccine can achieve better therapeutic effects at a lower dose, showing higher antigen expression levels, better durability and safety.

[0428] Preparation Example 1: Preparation of self-amplified mRNA with liver-specific microRNA-122 binding sequences inserted at six different locations

[0429] 1. MicroRNA binding site design: Different numbers of identical or different microRNA mature sequences (5p or 3p) are separated by an inner spacer sequence and then external spacers are added at both ends. The outermost site is a protease cleavage site that can be recognized by the replicase, as shown in Figure 12.

[0430] 2. Self-amplifying mRNA structure design: This mRNA was modified from the genome of the Venezuelan equine encephalitis virus strain TC-83, a member of the alphavirus family. Specifically, the nonstructural protein, namely the viral replicase sequence, and the non-translated sequence, including the non-translated sequences at the 5' and 3' ends of the viral genome and the 26S promoter following the non-structural protein, were retained. The alphavirus structural protein coding sequence was replaced with an arbitrary target protein sequence, X. Furthermore, a T7 polymerase promoter site was added before the alphavirus non-translated sequence at the 5' end, and a polyadenylation sequence (polyA sequence) was added after the alphavirus non-translated sequence at the 3' end. The specific structure is shown in Figure 13.

[0431] 3. Preparation of self-amplifying mRNA in vitro transcription templates with microRNA122 sequences inserted at six different locations: T7 polymerase promoter site, 5-terminal alphavirus non-translated sequence, alphavirus replicase sequence, 26S promoter sequence, Nluc-EGFP reporter gene sequence, 3-terminal alphavirus non-translated sequence, polyadenylation sequence and restriction endonuclease BspQI cleavage site sequence were constructed into pUC57-Kan plasmid by gene synthesis to obtain self-amplifying mRNA plasmid psaRNA-Nluc (fluorescein)-EGFP (green fluorescent protein), named saRNA-EGFP (SEQ ID NO: 29); then, six microRNA-122 binding sequence cassettes (SEQ ID NO: 30) were inserted into the pUC57-Kan plasmid by seamless cloning technology. NO:22) were respectively inserted between the 5-terminal alphavirus non-translated sequence and the alphavirus replicase sequence nsP1, between the alphavirus replicase sequence nsP1 and nsP2, between the alphavirus replicase sequence nsP2 and nsP3, between the alphavirus replicase sequence nsP3 and nsP4, between the 26S promoter sequence and the EGFP reporter gene sequence, and between the EGFP reporter gene sequence and the 3-terminal alphavirus non-translated sequence to obtain six self-amplified mRNA in vitro transcription template plasmids with different microRNA insertion positions, respectively named N21091, N21092, N21093, N21094, N21095 and N21096. The corresponding self-amplified mRNA structures are shown in Figure 14, and the corresponding sequences are as SEQ ID NO:23 to SEQ ID NO:28.

[0432] 4. Preparation of self-amplified mRNA in vitro transcription with microRNA-122 binding sequence cassettes inserted into six different locations:

[0433] a. Linearize the in vitro transcription template plasmid: Specifically, add 8 μg of the in vitro transcription template plasmid psaRNA-EGFP-microRNA, 4 μL of BspQI, and 4 μL of 10× Digestion Buffer, and finally add ddH2O to 40 μL. Incubate at 50°C for 2 hours to linearize the plasmid. After completion, add ddH2O to 100 μL of the enzyme digestion mixture and purify the linearized in vitro transcription template using a large-scale agarose gel DNA recovery kit.

[0434] b. In vitro transcription to obtain amplified mRNA: Specifically, add 10× T7 polymerase buffer, 7.5 mM ATP, 7.5 mM GTP, 7.5 mM UTP, 7.5 mM CTP, 1 μg of linearized in vitro transcription template, 400 U T7 RNA polymerase, 20 U RNase I inhibitor, and 6 mM cap analog, bring the volume to 20 μL, and incubate at 37°C for 2 hours. After the reaction, add 2 U DNase I and incubate at 37°C for 15 minutes. Make up the volume to 50 μL, add 25 μL of 7.5 M lithium chloride solution, mix, and incubate at -20°C for 30 minutes. Centrifuge at 13,000 g for 10 minutes at 4°C, discard the supernatant, and wash the pellet with 70% ethanol. Centrifuge at 13,000 g for 2 minutes at 4°C, discard the supernatant, repeat the wash cycle, aspirate the supernatant, and dissolve in 30 μL of water. After measuring the concentration using a spectrophotometer, 500 ng of the self-amplified mRNA obtained by in vitro transcription was diluted to 2 μL and mixed with 2 μL of 2× RNA Loading Dye. After incubation at 70°C for 10 minutes, it was immediately placed on ice for 2 minutes and detected by denaturing agarose gel electrophoresis. The size of the in vitro transcribed mRNA was consistent with expectations. The in vitro transcription preparation of self-amplified mRNA with microRNA122 sequences inserted in six different locations was completed. The electrophoresis detection results are shown in Figure 15, indicating that the self-amplified mRNA was successfully prepared.

[0435] Example 8: Detection of microRNA-122 expression in different cells

[0436] Detection Method: Reverse transcription fluorescence quantitative PCR was used to detect miRNA122 expression in C2C12 and huh7.5.1 cells: Total RNA was extracted from cell samples using RNA-easy Isolation Reagent, and RNA was quantified using a UV spectrophotometer. After reverse transcription using the miRNA 1st Strand cDNA Synthesis Kit (by stem-loop), quantitative PCR was performed using the miRNA Universal SYBR qPCR Master Mix. Detection primers are as follows:

[0437] miRNA122 upstream primer F: 5'-CGCGTGGAGTGTGACAATGG-3' (SEQ ID NO: 43),

[0438] miRNA122 downstream primer R: 5'-AGTGCAGGGTCCGAGGTATT-3' (SEQ ID NO: 44),

[0439] U6 promoter upstream primer F: 5'-CTCGCTTCGGCAGCACAT-3' (SEQ ID NO: 45),

[0440] U6 promoter downstream primer R: 5′-TTTGCGTGTCATCCTTGCG-3′ (SEQ ID NO: 46).

[0441] After normalizing the microRNA-122 CT values ​​of C2C12 and huh7.5.1 cells with the CT value of the cellular RNA internal reference U6, the deltaCT detection results of microRNA-122 in different cells are shown in Figure 16. The deltaCT value of Huh7.5.1 cells is approximately 15 higher than that of C2C12 cells, indicating that the expression level of microRNA-122 in Huh7.5.1 cells is much higher than that in C2C12 cells.

[0442] Example 9: In vitro expression and replication detection of self-amplified mRNA with liver-specific microRNA-122 binding sequences inserted at six different locations

[0443] 1. Fluorescent protein and luciferase detection

[0444] a. Cell seeding: Huh7.5.1 cells with high expression of microRNA-122 and C2C12 cells with low expression of microRNA-122 were seeded in 75 cm 2 In a cell culture flask, maintain a medium consisting of DMEM high glucose medium + 10% fetal bovine serum + 1% double-antibody. Once the cells reach 80% or greater confluency, trypsinize and count the cells. Plate an appropriate number of cells into a 24-well cell culture plate and incubate overnight at 37°C in a CO2 incubator.

[0445] b. Self-amplified mRNA transfection of Huh7.5.1 cells and C2C12 cells: 0.5 μg of six self-amplified mRNAs and a control self-amplified mRNA without microRNA-122 binding sites (SEQ ID NO: 29) were mixed with 50 ng of linear mRNA expressing firefly luciferase and then transfected using liposomes. -mRNA Transfection Kit was used to transfect Huh7.5.1 cells and C2C12 cells, and the cells were cultured at 37°C in a CO2 incubator for 24 hours.

[0446] c. Verification of fluorescent protein expression: 24 hours after transfection, the cell culture plate was placed under a fluorescence microscope for fluorescence imaging. The results are shown in Figure 17.

[0447] The results showed that, during the same transfection period, N21091, N21093, and N21094 self-amplified mRNAs were not significantly expressed in C2C12 cells with low microRNA-122 expression, while N21092 and N21096 were expressed normally, at levels comparable to the control self-amplified mRNA. In contrast, in Huh7.5.1 cells with high microRNA-122 expression, the expression of N21092 and N21096 was significantly reduced, indicating that the N21092 and N21096 self-amplified mRNAs were regulated by intracellular microRNAs as expected, demonstrating good cellular expression specificity. N21095 self-amplified mRNA showed similar expression to the control self-amplified mRNA, suggesting that placing the microRNA-122 binding site at the corresponding site did not affect the normal expression of the self-amplified mRNA, but also failed to be effectively regulated by the microRNA.

[0448] d. Luciferase Assay: 24 hours after transfection, Nano-Glo Luciferase Assay System detection reagent was used. 25 μl of Nano-Glo Luciferase Assay Reagent was transferred to a 96-well white opaque-bottom plate. 25 μl of supernatant sample was added to each well and mixed evenly. After waiting for 3 minutes, the signal was detected using a microplate reader. The detection results are shown in Figure 18, which also shows that the expression levels of self-amplified mRNAs N21092 and N21096 in Huh7.5.1 cells with high microRNA-122 expression and C2C12 cells with low microRNA-122 expression were significantly different, indicating good cell expression specificity.

[0449] 2. Detection of self-amplification mRNA replication efficiency

[0450] a. Cell seeding and self-amplified mRNA transfection: Same as fluorescent protein and luciferase detection.

[0451] b. Cell sample processing: 2 hours and 24 hours after transfection, aspirate and discard the supernatant, rinse the cells once with 500 μl PBS, discard the supernatant, add 500 μl cell lysis buffer and pipette to collect the cells into EP tubes, and store in a -80°C refrigerator.

[0452] c. Reverse transcription quantitative PCR detection of saRNA replication: Total RNA was extracted from cell samples using the RNeasy Mini Kit, and RNA was quantified using a UV spectrophotometer. After reverse transcription using the HiScript III 1st Strand cDNA Synthesis Kit, quantitative PCR was performed using 2× ChamQ Universal SYBR qPCR Master Mix. Detection primers are as follows:

[0453] Alphavirus replicase sequence (nsp1) upstream primer F: 5'-GACGGACCGACAAGTCTCTA-3' (SEQ ID NO: 47),

[0454] Alphavirus replicase sequence (nsp1) downstream primer R: 5'-GGTGGTGTCAAAGCCTATCCA-3' (SEQ ID NO: 48),

[0455] EGFP sequence upstream primer F: 5'-AGCTGGAGTACAACTACA-3' (SEQ ID NO: 49),

[0456] EGFP downstream primer R: 5'-CTGATCTTGAAGTTCACC-3' (SEQ ID NO: 50),

[0457] GAPDH sequence upstream primer F: 5'-GGTATCGTGGAAGGACTC-3' (SEQ ID NO: 51),

[0458] GAPDH downstream primer R: 5′-GTAGAGGCAGGGATGATG-3′ (SEQ ID NO: 52).

[0459] The reaction cycle conditions were: 95°C for 30 s-(95°C for 10 s-60°C for 30 s)×40 cycles.

[0460] The detection results are shown in Figures 19 and 20.

[0461] The results showed significant differences in the relative RNA expression of N21092 and N21096 self-amplified mRNAs between Huh7.5.1 cells, which overexpress microRNA-122, and C2C12 cells, which underexpress microRNA-122. In Huh7.5.1 cells, the relative expression of N21092 and N21096 self-amplified RNAs was significantly downregulated compared to C2C12 cells, indicating that microRNA-122 can regulate gene expression by effectively degrading self-amplified RNA levels.

[0462] Example 10: In vitro expression and replication detection of self-amplified mRNAs inserted with different copy numbers of liver-specific microRNA-122 binding sequences

[0463] 1. The structure of self-amplified mRNA is shown in Figure 21. 1, 2, 3 or 6 copies of the microRNA-122 binding sequence are respectively placed into the Nsp1 / 2 or 3'UTR site to obtain self-amplified mRNAs with different copy numbers of liver-specific microRNA-122 binding sequences inserted. The preparation method is similar to Preparation Example 1.

[0464] 2. Fluorescent protein and luciferase detection methods are the same as those in Example 8. The relative expression levels of the self-amplified mRNA to be tested and the control self-amplified mRNA without the microRNA-122 binding sequence in different cells are shown in FIG22 .

[0465] The results showed that in Huh7.5.1 cells with high expression of microRNA-122, the relative expression level of self-amplifying mRNA binding to microRNA-122 was inversely proportional to the number of copies of the inserted microRNA-122 binding sequence. At the same insertion position, the expression level of self-amplifying RNA with 1, 2, 3 or 6 copies of the microRNA-122 binding sequence inserted gradually decreased, among which the expression level of self-amplifying RNA with 6 copies of the microRNA-122 binding sequence inserted decreased the most, which decreased by more than 90% compared with the wild type. In addition, the relative expression level of self-amplifying mRNA with 6 microRNA-122 binding sequences placed in the 3'UTR in C2C12 cells with low expression of microRNA-122 was still more than 70%.

[0466] The results also showed that the expression levels of self-amplified RNAs with 1, 2 or 3 copies of microRNA-122 binding sequences inserted in Huh7.5.1 cells decreased to a certain extent compared with the wild type, and maintained a high expression level in C2C12 cells.

[0467] In summary, the regulation of self-amplified mRNA expression is based on microRNA-122 in a dose-dependent manner, and the 3'UTR is the optimal site for the placement of the microRNA binding sequence.

[0468] Example 11: Design of macrodomain mutations encoded by saRNA and detection of ADP ribose hydrolase activity

[0469] The present invention selected saRNA derived from Venezuelan Equine Encephalitis Virus (VEEV) for subsequent design. In the alphavirus family represented by VEEV, the macrodomain is present at the N-terminus of nonstructural protein 3, as shown in Figure 24. The amino acid sequence of the macrodomain is relatively conserved between different viruses, with a homology of more than 50%. Therefore, we selected amino acids 113 close to the ADP ribose hydrolysis active center and 48 away from the ADP ribose hydrolysis active center for amino acid mutation. The specific positions are shown in Figure 25. In coronaviruses and alphaviruses, position 113 encodes isoleucine (I) or valine (V), which is highly conserved. We infer that it is involved in the ADP ribose hydrolysis function. However, position 48 is quite different and is only encoded as glutamine (Q) in VEEV, so it may not be involved in the ADP ribose hydrolysis function. Since the side chains of phenylalanine and prolineamide are both cyclic structures, they have obvious structural differences from glutamine, which has only an amino group on its side chain, or isoleucine, which has one methyl group and one ethyl group on its side chain. Therefore, we constructed the following three saRNAs through homologous recombination and in vitro transcription:

[0470] Next, 200 ng of saRNA-190, saRNA-191, and wild-type saRNA were transfected into HeLa cells. After 24 hours, the level of ADP-ribosylation of total intracellular proteins was detected using an anti-mono-ADP-ribosylation antibody. As shown in Figure 26, 24 hours after transfection, the ADP-ribosylation of cellular proteins in 190 / 191-saRNA was significantly stronger than that in the wild-type transfection group, indicating that the ADP-ribosyl hydrolase activity of saRNA-190 and saRNA-191 was significantly reduced relative to that of wild-type saRNA. This data shows that the mutation design of amino acids 113 and 48 in the macrodomain successfully reduced the ADP-ribosyl hydrolase activity.

[0471] Example 12: Detection of replication levels of mutant saRNA with impaired ADP ribose hydrolase activity

[0472] After verifying that the ADP-ribose hydrolase activity of the two mutant saRNAs was reduced, we examined whether the mutant saRNAs' replication capacity was altered. RNA was extracted 2, 6, 24, and 48 hours after transfection of saRNA-190, saRNA-191, and wild-type saRNA into HeLa cells, and quantitative PCR was performed for the EGFP gene encoded by the saRNAs.

[0473] As shown in Figure 27, 190 / 191-saRNA significantly reduced the level of RNA encoding EGFP compared to wild-type saRNA. At the same time, we used anti-double-stranded RNA antibodies to perform flow cytometry on double-stranded RNA in Hela cells 6 hours and 24 hours after transfection with wild-type saRNA, saRNA-190 and saRNA-191. As shown in Figure 28, compared with wild-type saRNA, saRNA-190 and saRNA-191 had a lower proportion of double-stranded RNA formed during replication 6 hours after transfection, and this difference was more significant 24 hours after transfection. 24 hours after transfection, more than half of the cells transfected with wild-type saRNA had dsRNA detected, which was much higher than the other two groups. There was no significant difference between 190-saRNA and 191-saRNA. In summary, the above results show that 190 / 191-saRNA with macrodomain amino acid mutations has a weakened ability to replicate on cells in vitro.

[0474] Example 13: Detection of Innate Immunity Differences Induced by Mutant saRNA

[0475] Given that double-stranded RNA can significantly trigger innate immune responses, it is necessary to examine the changes in innate immunity levels induced by mutant saRNA and wild-type saRNA. We transfected HeLa cells with 190 / 191-saRNA and wild-type saRNA for 24 hours, extracted total cellular RNA, and performed RNA sequencing to analyze differences in transcriptome levels.

[0476] According to the results of principal component analysis, compared with the control group, the transcriptome difference of the group transfected with 190-saRNA was the smallest, while the wild-type saRNA group induced very significant transcriptome changes (see Figure 29). Further, we counted the number of genes that were differentially expressed in different saRNA groups relative to the control group. We found that in all saRNA groups, the number of upregulated genes was far greater than the number of downregulated genes, while most of the upregulated genes in the 190 and 191 groups were almost the same as those in the wild-type saRNA group. However, we found that the number of genes that were commonly downregulated between different groups was relatively small (see Figure 30), suggesting that the biological functions involved in the upregulated genes of 190 / 191-saRNA and wild-type saRNA may be similar. Therefore, a cluster analysis of the differentially expressed genes in the wild-type saRNA group relative to the control group was performed. The results showed that the 10 groups with the most significant differences in biological processes (GO:BP) were almost all related to immune response, and almost all of the genes were highly upregulated (see Figure 31a), indicating that wild-type saRNA induced a strong antiviral innate immune response in cells. Further analysis of the molecular function (GO:MF) of the differentially expressed genes revealed that the functions of the upregulated genes were related to cytokine / chemokine / growth factor / receptor binding and dsRNA / ssRNA binding (see Figure 31b). Given that the proportion of cells forming double-stranded RNA after 190 / 191-saRNA transfection is significantly lower than that of wild-type RNA, it is not difficult to imagine that the difference in the proportion of double-stranded RNA formed during the replication of different saRNAs may affect the induction of innate immune responses.

[0477] Next, we examined upregulated genes in double-stranded RNA / single-stranded RNA binding proteins. We observed significant upregulation of genes involved in the intracellular RNA sensors RIG-I and MDA-5, the endosomal RNA sensor TLR3, and interferon-stimulated genes downstream of the IFN pathway, such as the OAS family and EIF2AK2 (see Figure 32). This suggests that double-stranded RNA generated during saRNA replication is key to activating the interferon or NFκB signaling pathway, thereby inducing a large number of downstream antiviral genes to inhibit saRNA replication. A heat map of interferon pathway genes across different groups reveals that compared to the control group, all cells transfected with saRNA showed significant upregulation of interferon pathway gene transcript levels. In addition to the aforementioned RNA-binding proteins, interferon beta, transcription factors IRF7 / 9, STAT1, and numerous downstream interferon-stimulated genes were also upregulated. Cytokine upregulation was also observed, suggesting that the NF-κB pathway was also effectively activated. As expected, the transcript levels of interferon signaling pathway genes induced by saRNA in different groups varied significantly. Compared to the control group, 190 induced the weakest IFN immune response, 191 induced a slightly stronger response, and the wild-type strain induced the strongest response. As shown in Figure 33, the mRNA levels of interferon pathways and cytokines were verified by fluorescent quantitative PCR 2, 6, and 24 hours after transfection. It was found that within 2 hours, the RNA transcription levels of each group did not change compared with the control group. However, at 6 hours, the RNA levels of IFN-beta, CXCL10, CCL5, and IFIT2 in the wild-type saRNA group were significantly upregulated compared with the control group. CXCL10, IFIT2, and PKR were also upregulated in the 190 and 191 groups, but there was no difference between 190 and 191. 24 hours after transfection, all RNAs including NF-KB and interferon pathways were most significantly upregulated in the wild-type saRNA group, followed by 191-saRNA, and the lowest in 190-saRNA.

[0478] In summary, it was detected that the transcription levels of genes in the innate immune pathway induced by different saRNAs were positively correlated with their replication ability. Since the ability of 190 / 191-saRNA to replicate and form double-stranded RNA was weakened, it induced a weaker innate immune response.

[0479] Example 14: Detection of protein translation activity of mutant saRNA in in vitro cell models

[0480] In order to detect the activity of different saRNAs expressing exogenous genes, different saRNAs encoding luciferase were transfected into Hela cells. The results showed that the expression of wild-type saRNA was higher than that of mutant saRNA at 2 hours and 6 hours, but at 24 hours, the expression of 190 / 191-saRNA was slightly higher, and at 48 hours, the expression of 190-saRNA was 6 times higher than that of wild-type saRNA (see Figure 34). It is speculated that the natural immune response induced by saRNA will limit the virus hijacking the cell translation mechanism for replication through host protein translation inhibition, so the cell translation activity after different saRNA transfection was detected. Before collecting the cells, puromycin was added to the culture medium to a final concentration of 5ug / mL and treated for 5 minutes to allow it to be incorporated into the polypeptide chain, and the intracellular labeled protein content was detected with anti-puromycin antibodies. The results showed that there was no significant difference in intracellular translation activity 2 hours after transfection, while at 6 hours and 24 hours, the translation activity of the wild-type saRNA group was weaker than that of the mutant saRNA group, while the 190-saRNA protein translation activity was slightly weaker than the control at 6 hours, but similar to the control group at 24 hours. In contrast, the translation activity of the 191-saRNA transfection group was slightly weaker at 24 hours (see Figure 35). This result proves that after saRNA transfection, protein translation in the cell is inhibited. Combined with the previous observation that PKR and OAS genes in dsRNA binding proteins are significantly upregulated after saRNA transfection, it is speculated that the activation of OAS / RNase L and PKR / eIF2α pathways may be involved in the inhibition of host protein translation, thereby limiting the expression of saRNA. Therefore, the integrity of ribosomal RNA and eIF2α phosphorylation at different time points after saRNA transfection were detected (see Figures 36 and 37). The results showed that the ribosomal RNA of cells transfected with wild-type saRNA remained relatively intact within 2 and 6 hours, but was severely degraded at 24 and 48 hours, while the integrity of the ribosomal RNA transfected with 190 / 191saRNA was not significantly reduced compared to the control group. This result suggests that ribosomal RNA is degraded after wild-type saRNA transfection, which is the result of activation of the OAS / RNase L pathway (see Figure 36). At the same time, the eIF2α phosphorylation test results showed that all groups of saRNA induced eIF2α phosphorylation 6 hours after transfection, and were positively correlated with the subgenomic level at 6h, with wild-type saRNA being the strongest and 191-saRNA being the weakest. Unexpectedly, 24 hours after transfection, the eIF2α phosphorylation level was significantly reduced, and the eIF2α phosphorylation induced by wild-type saRNA was even weaker than that of the control group (i.e., the mock group: only transfection reagent was added, and no RNA molecules were transfected), while the degree of eIF2α phosphorylation of the mutant saRNA was similar to that of the control group (see Figure 37).The above results show that host translation was not inhibited at 2 hours after transfection, but host translation mediated by PKR / eIF2α appeared at 6 hours. However, since the subgenomic RNA amplification level of wild-type saRNA was much higher than that of mutant saRNA, the expression level was still higher than that of the other two groups. At 24 hours, the phosphorylation level of wild-type eIF2α decreased. Therefore, host translation inhibition was largely promoted by rRNA degradation caused by OAS / RNase L activation. Therefore, the expression level of wild-type saRNA, which was most significantly inhibited, was the lowest.

[0481] The above results demonstrate that saRNA transfection into cells activated PKR / eIF2α and OAS / RNase L-mediated host translation inhibition, but compared with wild-type saRNA, mutant saRNA had a lower degree of translation inhibition and therefore exhibited a higher expression level.

[0482] Example 15: Apoptosis induction detection of mutant saRNA in in vitro cell models

[0483] It was observed that 24 hours after transfection with wild-type saRNA, the cell nucleus underwent significant disintegration, which suggests that Hela cells may have undergone apoptosis (see Figure 38). Annexin-V and PI staining were performed on cells transfected for 24 hours and 48 hours. As shown in Figure 39, all cells transfected with saRNA underwent apoptosis, but the proportion of dead cells in the 190-saRNA group was significantly lower than that in other groups. Western blot detection was performed on the protein expression levels and activation states of the apoptosis initiator molecule caspase 8 and the effector molecule caspase 3. The results showed that all caspases were significantly activated in cells transfected with saRNA, but the caspase activation in the wild-type saRNA group was significantly stronger than that in other groups (see Figure 40). In addition, as shown in Figure 41, cells transfected with wild-type saRNA were treated with caspase inhibitors, and it was found that cell apoptosis was strongly inhibited after treatment, and the EGFP fluorescence intensity expressed by saRNA increased by 3 times. These results show that saRNA transfection of Hela cells leads to apoptosis, and inhibiting apoptosis can improve the expression efficiency of saRNA.

[0484] Example 16: Detection of in vivo expression levels of 190-saRNA

[0485] In order to verify whether mutant saRNA also has a high expression level in vivo, 190-saRNA, wild-type saRNA and non-replicating mRNA (nrmRNA) expressing the firefly luciferase gene were encapsulated with lipid nanoparticles (LNPs). As shown in Figure 42a, 4-6 week-old balb / c mice were intramuscularly immunized with 5 μg of different mRNA-LNPs expressing the firefly luciferase gene, and in vivo imaging was performed on days 1, 3, 7, 14, and 21. The results showed that the expression level of non-replicating mRNA was slightly higher than that of saRNA on the first day after immunization, but began to decay thereafter, and almost no expression was detected after day 7 (see Figures 42b and 43). The expression level of 190-saRNA continued to rise for at least 7 days and was higher than that of wild-type saRNA. Although it remained consistent with wild-type saRNA after 14 days, the total expression level was higher than that of wild-type saRNA and non-replicating mRNA (see Figure 44). Consistent with what was observed in vitro, 190-saRNA also showed a higher expression level in vivo.

[0486] Although specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details in light of all the teachings disclosed herein, and such modifications are within the scope of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. An mRNA molecule translation region encoding an alphavirus RNA replicase as shown in SEQ ID NO: 20, wherein: The 4500th base of the mRNA molecule is C.

2. The mRNA molecule translation region according to claim 1, wherein The 4509th base is T, C or G.

3. The mRNA molecule translation region according to any one of claims 1 to 2, wherein The 1672nd base is A, the 1673rd base is G, and the 1674th base is C.

4. The mRNA molecule translation region according to any one of claims 1 to 3, wherein The sequence of the translation region of the initial mRNA molecule encoding alphavirus RNA replicase is shown in SEQ ID NO:

10.

5. The mRNA molecule translation region according to any one of claims 1 to 4, wherein the sequence is shown in any one of SEQ ID NO: 11 to SEQ ID NO:

15.

6. An mRNA molecule comprising, in order: 5' untranslated region, RNA replicase coding region, RNA promoter, optional sequence encoding target protein, 3' untranslated region and polyadenylation sequence; Wherein, the RNA replicase coding region is the mRNA molecule translation region described in any one of claims 1 to 5.

7. The mRNA molecule according to claim 6, wherein in, The 5' untranslated region is derived from an alphavirus; preferably, derived from Venezuelan equine encephalomyelitis virus; Preferably, the sequence of the 5' untranslated region is as shown in SEQ ID NO:

16.

8. The mRNA molecule according to any one of claims 6 to 7, wherein in, The 3' untranslated region is derived from an alphavirus; preferably, from Venezuelan equine encephalomyelitis poison; Preferably, the sequence of the 3' untranslated region is as shown in SEQ ID NO:

17.

9. The mRNA molecule according to any one of claims 6 to 8, wherein The RNA promoter is a subgenomic promoter; Preferably, the RNA promoter is a subgenomic promoter derived from an alphavirus; Preferably, the RNA promoter is a subgenomic promoter derived from Venezuelan equine encephalitis virus; Preferably, the RNA promoter is a 26S promoter; Preferably, the sequence of the RNA promoter is shown in SEQ ID NO:

18.

10. The mRNA molecule according to any one of claims 6 to 9, wherein The sequence of the polyadenylation sequence is shown in SEQ ID NO:

19.

11. The mRNA molecule according to any one of claims 6 to 10, wherein The sequence encoding the target protein is a sequence encoding a vaccine antigen, a therapeutic protein, or an antibody targeting an immune checkpoint.

12. The mRNA molecule according to any one of claims 6 to 11, wherein the sequence is shown in SEQ ID NO: 3 and any one of SEQ ID NO: 6 to SEQ ID NO:

9.

13. A DNA molecule encoding the translation region of the mRNA molecule according to any one of claims 1 to 5 or encoding the mRNA molecule according to any one of claims 6 to 12.

14. A recombinant vector comprising the DNA molecule according to claim 13; preferably, the recombinant vector is a recombinant prokaryotic expression vector or a recombinant eukaryotic expression vector.

15. A recombinant host cell, comprising the translation region of the mRNA molecule according to any one of claims 1 to 5, the mRNA molecule according to any one of claims 6 to 12, the DNA molecule according to claim 13 or the recombinant vector according to claim 14.

16. A kit comprising the translation region of the mRNA molecule according to any one of claims 1 to 5, the mRNA molecule according to any one of claims 6 to 12 or the DNA molecule according to claim 13, and a liposome delivery system.

17. A pharmaceutical composition comprising the translation region of the mRNA molecule according to any one of claims 1 to 5, the mRNA molecule according to any one of claims 6 to 12 or the DNA molecule according to claim 13, and one or more pharmaceutically acceptable excipients; preferably, the excipient is a liposome delivery system.

18. A vaccine preparation comprising the translation region of the mRNA molecule according to any one of claims 1 to 5, the mRNA molecule according to any one of claims 6 to 12, or the DNA molecule according to claim 13; Preferably, the mRNA molecule or DNA molecule is encapsulated by a liposome-based delivery system; Optionally, the vaccine formulation further comprises one or more vaccine adjuvants; Preferably, the vaccine preparation is a vaccine preparation for preventing viral infection such as novel coronavirus infection or preventing severe illness caused by novel coronavirus infection.

19. Use of the translation region of the mRNA molecule according to any one of claims 1 to 5, the mRNA molecule according to any one of claims 6 to 12, or the DNA molecule according to claim 13 in the preparation of a drug for treating or preventing viral infection, a drug for treating or preventing tumors, or a drug for protein replacement therapy.

20. An mRNA molecule comprising, in order: 5' non-translated sequence, RNA replicase sequence, RNA promoter, optional sequence encoding target protein, 3' non-translated sequence and 3' PolyA tail; in, A microRNA binding site sequence is contained between nsp1 and nsp2, between nsp2 and nsp3, between nsp3 and nsp4, and / or between the sequence encoding the target protein and the 3'-end non-translated sequence of the RNA replicase.

21. The mRNA molecule according to claim 20, wherein The RNA replicase is an RNA replicase derived from an alphavirus; preferably, it is an RNA replicase derived from Venezuelan equine encephalomyelitis virus; Preferably, the amino acid sequence of the RNA replicase is shown as SEQ ID NO:41; preferably, the coding sequence of the RNA replicase is shown as SEQ ID NO:

42.

22. The mRNA molecule according to any one of claims 20 to 21, wherein The RNA promoter is a subgenomic promoter; Preferably, the RNA promoter is a subgenomic promoter derived from an alphavirus; Preferably, the RNA promoter is a subgenomic promoter derived from Venezuelan equine encephalitis virus; Preferably, the RNA promoter is the 26S promoter.

23. The mRNA molecule according to any one of claims 20 to 22, wherein The 5' non-translated sequence and / or the 3' non-translated sequence is derived from an alphavirus; optimally, derived from Venezuelan equine encephalomyelitis virus.

24. The mRNA molecule according to any one of claims 20 to 23, wherein The microRNA binding site sequence is a binding site sequence corresponding to the microRNA expressed specifically in the tissue; Preferably, the tissue-specifically expressed microRNA is a microRNA that is lowly expressed in tumor tissue; Preferably, the microRNA lowly expressed in tumor tissue is selected from miRNA-122, miRNA-143, miRNA-1, miRNA-124, miRNA-217 and miRNA-126.

25. The mRNA molecule according to any one of claims 20 to 24, wherein The microRNA binding site sequence comprises one or more microRNA mature sequences; preferably, the microRNA binding site sequence comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 microRNA mature sequences that are identical or different in sequence.

26. The mRNA molecule according to any one of claims 20 to 25, wherein The microRNA mature sequence is shown in any one of SEQ ID NO:30 to SEQ ID NO:

32.

27. The mRNA molecule according to any one of claims 20 to 26, wherein Each microRNA mature sequence contains one or more identical or different internal spacer sequences; Preferably, the internal spacer sequence is shown in any one of SEQ ID NO:33 to SEQ ID NO:

34.

28. The mRNA molecule according to any one of claims 20 to 27, wherein The 5' end and / or 3' end of the microRNA binding site sequence contains one or more identical or different external spacer sequences; Preferably, the external spacer sequence at the 5' end is as shown in any one of SEQ ID NO:35 to SEQ ID NO:36; Preferably, the external spacer sequence at the 3' end is as shown in any one of SEQ ID NO:37 to SEQ ID NO:

38.

29. The mRNA molecule according to any one of claims 20 to 28, wherein The 5' end and / or 3' end of the microRNA binding site sequence contains one or more restriction site sequences that can be recognized by alphavirus RNA replicase or nsp2; Preferably, it is a restriction site sequence between nsp1 and nsp2 that can be recognized by the alphavirus replicase or nsp2; Preferably, the restriction site sequence is as shown in SEQ ID NO:

53.

30. The mRNA molecule according to any one of claims 20 to 29, wherein The microRNA binding site sequence is shown in SEQ ID NO:

22.

31. The mRNA molecule according to any one of claims 20 to 30, further comprising a 5' end cap structure.

32. The mRNA molecule according to any one of claims 20 to 31, whose sequence is shown in SEQ ID NO:24 or SEQ ID NO:

28.

33. An mRNA molecule combination, comprising a first mRNA molecule and a second mRNA molecule, wherein: The first mRNA molecule comprises, in order: The first 5' untranslated sequence, the sequence of RNA replicase, the first 3' untranslated sequence and the 3' PolyA tail; The second mRNA molecule comprises, in order: The second 5' non-translated sequence, the sequence essential for alphavirus replication, the RNA promoter, the sequence encoding the target protein, the second 3' non-translated sequence and the 3' PolyA tail; Wherein, a microRNA binding site sequence is contained between nsp1 and nsp2, between nsp2 and nsp3, between nsp3 and nsp4, and / or between the sequence encoding the target protein and the second 3'-end non-translated sequence of the RNA replicase.

34. The mRNA molecule combination according to claim 33, wherein: The first 5' non-translated sequence is the same as or different from the second 5' non-translated sequence; and / or The first 3' non-translated sequence is the same as or different from the second 3' non-translated sequence.

35. The mRNA molecule combination according to any one of claims 33 to 34, wherein: The first 5' non-translated sequence and the first 3' non-translated sequence are not derived from alphavirus.

36. The mRNA molecule combination according to any one of claims 33 to 35, wherein: The second 5' non-translated sequence and the second 3' non-translated sequence are derived from an alphavirus; preferably, derived from Venezuelan equine encephalitis virus.

37. The mRNA molecule combination according to any one of claims 33 to 36, wherein: The RNA replicase is an RNA replicase derived from an alphavirus; preferably, it is an RNA replicase derived from Venezuelan equine encephalomyelitis virus; Preferably, the amino acid sequence of the RNA replicase is shown as SEQ ID NO:41; preferably, the coding sequence of the RNA replicase is shown as SEQ ID NO:

42.

38. The mRNA molecule combination according to any one of claims 33 to 37, wherein: The RNA promoter is a subgenomic promoter; Preferably, the RNA promoter is a subgenomic promoter derived from an alphavirus; Preferably, the RNA promoter is a subgenomic promoter derived from Venezuelan equine encephalitis virus; Preferably, the RNA promoter is the 26S promoter.

39. The mRNA molecule combination according to any one of claims 33 to 38, wherein: The microRNA binding site sequence is a binding site sequence corresponding to the microRNA expressed specifically in the tissue; Preferably, the tissue-specifically expressed microRNA is a microRNA that is lowly expressed in tumor tissue; Preferably, the microRNA lowly expressed in tumor tissue is selected from miRNA-122, miRNA-143, miRNA-1, miRNA-124, miRNA-217 and miRNA-126.

40. The mRNA molecule combination according to any one of claims 33 to 39, wherein: The microRNA binding site sequence comprises one or more microRNA mature sequences; preferably, the microRNA binding site sequence comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 microRNA mature sequences that are identical or different in sequence.

41. The mRNA molecule combination according to any one of claims 33 to 40, wherein: The microRNA mature sequence is shown in any one of SEQ ID NO:30 to SEQ ID NO:

32.

42. The mRNA molecule combination according to any one of claims 33 to 41, wherein: Each microRNA mature sequence contains one or more identical or different internal spacer sequences; Preferably, the internal spacer sequence is shown in any one of SEQ ID NO:33 to SEQ ID NO:

34.

43. The mRNA molecule combination according to any one of claims 33 to 42, wherein: The 5' end and / or 3' end of the microRNA binding site sequence contains one or more identical or different external spacer sequences; Preferably, the external spacer sequence at the 5' end is as shown in any one of SEQ ID NO:35 to SEQ ID NO:36; Preferably, the external spacer sequence at the 3' end is as shown in any one of SEQ ID NO:37 to SEQ ID NO:

38.

44. The mRNA molecule combination according to any one of claims 33 to 43, wherein: The 5' end and / or 3' end of the microRNA binding site sequence contains one or more restriction site sequences that can be recognized by alphavirus RNA replicase or nsp2; Preferably, it is a restriction site sequence between nsp1 and nsp2 that can be recognized by the alphavirus replicase or nsp2; Preferably, the restriction site sequence is as shown in SEQ ID NO:

53.

45. The mRNA molecule combination according to any one of claims 33 to 44, wherein: The microRNA binding site sequence is shown in SEQ ID NO:

22.

46. ​​The mRNA molecule combination according to any one of claims 33 to 45, wherein: The first mRNA molecule and / or the second mRNA molecule further comprises a 5' end cap structure.

47. A DNA molecule encoding the mRNA molecule of any one of claims 20 to 32, or encoding the first mRNA molecule and the second mRNA molecule of any one of claims 33 to 46, wherein the first mRNA molecule and the second mRNA molecule are on the same DNA molecule.

48. The DNA molecule according to claim 47, comprising a T7 promoter, an SP6 promoter or a T3 promoter upstream of the sequence encoding the alphavirus RNA replicase.

49. A DNA molecule combination, comprising a first DNA molecule and a second DNA molecule, wherein: The first DNA molecule encodes the first mRNA molecule of any one of claims 33 to 46; and The second DNA molecule encodes the second mRNA molecule according to any one of claims 33 to 46.

50. A recombinant vector comprising the DNA molecule according to any one of claims 47 to 48; preferably, the recombinant vector is a recombinant prokaryotic expression vector or a recombinant eukaryotic expression vector.

51. A recombinant host cell comprising the mRNA molecule of any one of claims 20 to 32, the mRNA molecule combination of any one of claims 33 to 46, the DNA molecule of any one of claims 47 to 48, or the DNA molecule combination of claim 49.

52. A kit comprising an mRNA molecule according to any one of claims 20 to 32, an mRNA molecule combination according to any one of claims 33 to 46, a DNA molecule according to any one of claims 47 to 48 or a DNA molecule combination according to claim 49, and a liposome-based delivery system.

53. A pharmaceutical composition comprising the mRNA molecule described in any one of claims 20 to 32, the mRNA molecule combination described in any one of claims 33 to 46, the DNA molecule described in any one of claims 47 to 48 or the DNA molecule combination described in claim 49, and one or more pharmaceutically acceptable excipients; preferably, the excipient is a liposome delivery system.

54. A vaccine formulation comprising the mRNA molecule of any one of claims 20 to 32, the mRNA molecule combination of any one of claims 33 to 46, the DNA molecule of any one of claims 47 to 48, or the DNA molecule combination of claim 49; Preferably, the mRNA molecule, the combination of mRNA molecules, the DNA molecule or the combination of DNA molecules is encapsulated by a liposome-based delivery system; Optionally, the vaccine formulation further comprises one or more vaccine adjuvants; Preferably, the vaccine preparation is a vaccine preparation for preventing viral infection such as novel coronavirus infection or preventing severe illness caused by novel coronavirus infection.

55. Use of the mRNA molecule of any one of claims 20 to 32, the mRNA molecule combination of any one of claims 33 to 46, the DNA molecule of any one of claims 47 to 48, or the DNA molecule combination of claim 49 in the preparation of a drug for treating or preventing viral infection, a drug for treating or preventing tumors, or a drug for protein replacement therapy; Preferably, the viral infection refers to novel coronavirus infection; Preferably, the tumor is one or more selected from liver cancer, rhabdomyosarcoma, glioma, bladder cancer, colorectal cancer, pancreatic cancer and lung cancer; Preferably, the tumor is a tumor with low expression of one microRNA or multiple microRNAs; preferably, the microRNA is one or more selected from miRNA-122, miRNA-143, miRNA-1, miRNA-124, miRNA-217 and miRNA-126.

56. An mRNA molecule encoding non-structural proteins 1, 2, 3 and 4 derived from an alphavirus, wherein the non-structural protein 3 comprises a macro domain as shown in the amino acid sequence of SEQ ID NO: 57 or 59 at its N-terminus.

57. The mRNA molecule according to claim 56, comprising a nucleotide sequence encoding the macro domain as shown in SEQ ID NO:68 or 70.

58. The mRNA molecule according to any one of claims 56 to 57, wherein the nonstructural proteins 1, 2, 3 and 4 are derived from Venezuelan equine encephalitis virus, Optionally, the mRNA molecule encodes nonstructural protein 1 as shown in SEQ ID NO:71, nonstructural protein 2 as shown in SEQ ID NO:72, nonstructural protein 3 as shown in SEQ ID NO:73 or 74, and nonstructural protein 4 as shown in SEQ ID NO:

75.

59. The mRNA molecule according to any one of claims 56 to 58, comprising in the 5' to 3' direction an operably linked nucleotide sequence encoding nonstructural protein 1, a nucleotide sequence encoding nonstructural protein 2, a nucleotide sequence encoding nonstructural protein 3, and a nucleotide sequence encoding nonstructural protein 4, Optionally, the mRNA molecule comprises or consists of the nucleotide sequence of SEQ ID NO: 67 or 69.

60. according to the mRNA molecule described in any one in claim 56 to 59, it also comprises the target gene coding sequence and optionally, the RNA promoter upstream of the target gene coding sequence.

61. The mRNA molecule according to any one of claims 56 to 60, further comprising: 5' untranslated region, 3' untranslated region and polyadenylation sequence, Optionally, the mRNA molecule further comprises a signal peptide coding sequence, a 5' cap sequence, a Kozak sequence and / or a restriction enzyme cleavage site.

62. according to the mRNA molecule described in claim 61, wherein said mRNA molecule comprises the following operably linked nucleotide sequences in the 5' to 3' direction: 5' untranslated region, nucleotide sequences encoding nonstructural proteins 1, 2, 3 and 4, RNA promoter, target gene coding sequence, 3' untranslated region and polyadenylation sequence, Optionally, the nucleotide sequences are connected via a linker sequence.

63. The mRNA molecule according to claim 61 or 62, wherein The 5' untranslated region is derived from an alphavirus; preferably, derived from Venezuelan equine encephalomyelitis virus; For example, the 5' untranslated region comprises the sequence shown in SEQ ID NO:

63.

64. The mRNA molecule according to any one of claims 61 to 63, wherein in, The 3' untranslated region is derived from an alphavirus; preferably, derived from Venezuelan equine encephalomyelitis virus; For example, the 3' untranslated region comprises the sequence shown in SEQ ID NO:

64.

65. The mRNA molecule according to any one of claims 61 to 64, wherein The RNA promoter is a subgenomic promoter; Preferably, the RNA promoter is a subgenomic promoter derived from an alphavirus; Preferably, the RNA promoter is a subgenomic promoter derived from Venezuelan equine encephalitis virus; Preferably, the RNA promoter is a 26S promoter; Preferably, the RNA promoter comprises the sequence shown in SEQ ID NO:

65.

66. The mRNA molecule according to any one of claims 61 to 65, wherein The polyadenylic acid sequence comprises a restriction enzyme cleavage site, for example, the polyadenylic acid sequence comprises a sequence as shown in SEQ ID NO: 66, Optionally, the 3' end of the poly(A) sequence comprises a restriction enzyme site.

67. The mRNA molecule according to any one of claims 60 to 66, wherein The target gene coding sequence is a sequence encoding a therapeutic polypeptide, a preventive polypeptide, a diagnostic polypeptide, a reporter gene, an antigen or a sequence encoding a regulatory structure.

68. The mRNA molecule according to any one of claims 60 to 67, wherein the sequence is as shown in any one of SEQ ID NOs: 58 and 60-62.

69. A DNA molecule encoding the mRNA molecule of any one of claims 56 to 68.

70. A recombinant vector comprising the DNA molecule of claim 69; preferably, the recombinant vector is a prokaryotic expression vector or a eukaryotic expression vector.

71. A recombinant host cell comprising the mRNA molecule of any one of claims 56 to 68, the DNA molecule of claim 69, or the recombinant vector of claim 70.

72. A pharmaceutical composition comprising the mRNA molecule of any one of claims 56 to 68 or the DNA molecule of claim 69, and one or more pharmaceutically acceptable carriers; optionally, the carrier is a liposome delivery system or a nanoparticle composition.

73. A vaccine formulation comprising the mRNA molecule of any one of claims 56 to 68 or the DNA molecule of claim 69; Optionally, the mRNA molecule or DNA molecule is encapsulated by a liposome-based delivery system; Optionally, the vaccine formulation further comprises one or more vaccine adjuvants; Optionally, the vaccine formulation is a vaccine formulation for preventing viral infection.

74. Use of the mRNA molecule described in any one of claims 56 to 68 or the DNA molecule described in claim 69 in the preparation of a drug for treating or preventing viral infection, a drug for treating or preventing tumors, or a drug for protein replacement therapy.

75. A kit comprising the mRNA molecule of any one of claims 56 to 68 or the DNA molecule of claim 69.

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