WO2025016031A1 - Self-amplifying nucleic acid molecule and use thereof - Google Patents

Abstract

The present invention relates to an alphavirus RNA replicase coding sequence, an optimized self-amplifying nucleic acid molecule, a pharmaceutical composition, and a use. 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 an improved self-amplifying mRNA comprising a mutated replicase; and the mutated replicase comprises a mutated macro domain. The present invention further relates to a preparation method for the self-amplifying nucleic acid molecule.

Description

Self-amplifying nucleic acid molecules and their applications

CROSS-REFERENCE TO RELATED APPLICATIONS

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

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

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 stimulate a stronger immune response due to the immunostimulatory effect brought about by replication.

Generally, the in vitro synthesis of mRNA is mainly obtained 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, the plasmid needs to be linearized using a specific DNA endonuclease (such as BspQI or XbaI). However, due to or in part due to the non-specific digestion of the DNA endonuclease, unexpected linearization products are produced, resulting in non-target in vitro transcription products, which ultimately reduce 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.

mRNA vaccine is a new vaccine technology, which uses synthetic mRNA molecules to encode target non-endogenous antigenic proteins and stimulates immune responses against targets such as pathogens or tumors by injecting them into the human body. Currently, mRNA vaccines have been successfully used to prevent the new coronavirus and have broad application prospects in research fields such as infectious disease prevention vaccines, tumor treatment, and protein replacement therapy.

Common mRNA forms include linear mRNA and circular mRNA, among which linear mRNA is divided into non-replicating type and self-amplifying mRNA with self-replication ability. Self-amplifying RNA is regarded as the next generation of mRNA molecules. Due to its long expression cycle and self-adjuvant effect, it has shown its advantages in effectively inducing specific immunity in infectious diseases and tumor vaccines. In addition, self-amplifying RNA also has unique potential in protein replacement therapies that expect a long half-life. In 2023, Japan approved a new crown vaccine based on self-amplifying RNA, further proving the safety of self-amplifying RNA as a vaccine. Compared with non-replicating mRNA, an important advantage of self-amplifying RNA is that the expression level of exogenous genes is high and the duration is long, which can significantly reduce the administration dose and reduce side effects. Therefore, further improving the expression efficiency has always been one of the directions of self-amplifying RNA sequence optimization.

For the optimization of non-replicating mRNA, the use of nucleotide modification to circumvent the recognition of mRNA by pattern recognition receptors has been proven to be a very successful strategy aimed at improving translation efficiency by reducing the innate immune response. However, since self-amplifying RNA essentially mimics the replication and expression mechanism of viruses, it is more complicated than non-replicating mRNA. In addition to the presence of unmodified nucleotides, self-amplifying RNA replication products including double-stranded RNA, daughter RNA with Cap0 structure and uncapped 5'-triphosphate daughter RNA can be recognized by pattern recognition receptors (PRR) and initiate antiviral immune responses including but not limited to interferon or NF-KB signaling pathways. The interferon-stimulated factors induced by this, such as protein kinase R (PKR), 2-5-oligoadenylate synthetase (OAS) and interferon induced proteins with tetratricopeptide repeats (IFIT), will perform host translation inhibition and induce cell apoptosis to limit the replication and expression of self-amplifying RNA. 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.

The sequence structure of self-amplifying mRNA is relatively complex compared to mRNA, especially the presence of viral replicase and replication-related elements such as RNA promoters. Self-amplifying mRNA uses the RNA replicase from alphavirus contained in it to enable mRNA to replicate itself. Therefore, the injected mRNA can continuously produce the target protein in the body, thereby enhancing the durability and intensity of the vaccine immune response, or enhancing the half-life of protein replacement therapy.

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

In addition, after the 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 the lipid nanoparticles may mediate the self-amplifying mRNA to enter the liver and other parenchymal tissues, where it may express a large amount of the target gene encoded by the self-amplifying mRNA, causing potential liver toxicity.

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 level.

Summary of the invention

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

The present invention obtains an optimized alphavirus RNA replicase coding sequence (translation region), and further obtains an optimized self-amplifying mRNA backbone sequence, which can significantly reduce the production of non-specific enzyme cleavage products of DNA endonuclease BspQI compared with the original sequence, 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:

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

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

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

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.

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.

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.

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.

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 The 1673rd base is A, the 1674th base is G, and the 1674th base is C.

In some embodiments of the present invention, the translation region of the mRNA molecule encodes the alpha virus 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.

In some embodiments of the present invention, the translation region of the mRNA molecule encodes the alpha virus 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.

In some embodiments of the present invention, the translation region of the mRNA molecule encodes the alpha virus 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.

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

In some embodiments of the present invention, the translation region of the mRNA molecule has a sequence as shown in any one of SEQ ID NO:11 to SEQ ID NO:15.

Another aspect of the present invention relates to an mRNA molecule comprising, in sequence:

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 the present invention.

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.

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

In some embodiments of the present invention, the mRNA molecule, wherein

Wherein, the 5' untranslated region is derived from an alpha virus; preferably, it is derived from Venezuelan equine encephalomyelitis virus;

Preferably, the sequence of the 5’ non-translated region is as shown in SEQ ID NO:16.

In some embodiments of the present invention, the mRNA molecule, wherein

Wherein, the 3' untranslated region is derived from an alpha virus; preferably, derived from Venezuelan equine encephalomyelitis virus;

Preferably, the sequence of the 3’ non-translated region is as shown in SEQ ID NO:17.

In some embodiments of the present invention, the mRNA molecule, 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 as shown in SEQ ID NO:18.

In some embodiments of the present invention, the mRNA molecule, wherein the sequence of the polyadenylic acid sequence is as shown in SEQ ID NO:19.

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.

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

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

Another aspect of the present invention relates to a DNA molecule encoding the translation region of the mRNA molecule described in any one of the present invention or encoding the mRNA molecule described in any one of the present invention.

Another aspect of the present invention relates to a recombinant vector, which contains the DNA molecule of the present invention; preferably, the recombinant vector is a recombinant prokaryotic expression vector or a recombinant eukaryotic expression vector.

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

Another aspect of the present invention relates to a kit, which contains 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 the present invention, and a liposome delivery system.

Another aspect of the present invention relates to a pharmaceutical composition, which contains 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 the present invention, and one or more pharmaceutically acceptable excipients; preferably, the excipient is a liposome delivery system.

Another aspect of the present invention relates to a vaccine preparation, which contains 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 the present invention;

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.

Lipid delivery systems include liposomes, lipid nanoparticles (LNP), and lipid polymer nanocarriers (LPP).

LPP among lipids is a double-layer structure with polymer-encapsulated mRNA as the core and phospholipids as the shell. The double-layer liposome membrane of LPP has a better effect of encapsulating and protecting mRNA, and the core of LPP can gradually release mRNA molecules as the polymer degrades. LPP has excellent targeting effect on dendritic cells and can better activate the immune response of T cells through antigen presentation to achieve ideal therapeutic effects.

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 a variety of 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 is required and the effectiveness is high; it does not enter the cell nucleus and has good safety. The mRNA COVID-19 vaccine verifies the applicability of the mRNA technology platform in the vaccine field.

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.

The microRNA specifically expressed by a specific tissue binds to and regulates the replication of self-amplifying mRNA molecules. Amplification of mRNA backbone

The present invention also obtains a self-amplifying mRNA skeleton that can be bound by microRNA specifically expressed in a specific tissue and then regulate the replication of self-amplifying mRNA molecules, thereby achieving the degradation of self-amplifying mRNA molecules in tissues expressing specific microRNAs, while existing normally in tissues that low-express or do not express specific microRNAs, so as to achieve tissue-specific expression of target gene molecules in the form of self-amplifying mRNA. The following invention is provided:

Another aspect of the present invention relates to an mRNA molecule, comprising:

5' non-translated sequence, RNA replicase sequence, RNA promoter, and optional sequence encoding target protein column, 3' untranslated 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.

In some embodiments of the present invention, the mRNA molecule, 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 as shown in SEQ ID NO:41; preferably, the coding sequence of the RNA replicase is as shown in SEQ ID NO:42.

In some embodiments of the present invention, the mRNA molecule, 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.

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 alpha virus; optimally, it is derived from Venezuelan equine encephalitis virus.

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;

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.

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.

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.

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 needs to be greater than or equal to 20 nt. For example, the length of MicroRNA-122-5p is 22 nt. The length of the binding sequence can be designed to be 20-24 nt, such as 20 nt, 21 nt, 22 nt, 23 nt or 24 nt.

Without being limited by theory, miRNA binding sites are usually located in the 3' untranslated region (3'UTR) of the target mRNA, and can also be located in the 5'UTR or coding region. MicroRNA binds to the target mRNA, thereby inhibiting the translation of the target mRNA or degrading the target mRNA, thereby affecting the expression of the target gene.

The present invention introduces the binding sites corresponding to the 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 the differential expression of mRNA in different tissues, and further achieving the tissue-specific expression of mRNA.

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;

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

In some embodiments of the present invention, the mRNA molecule, wherein the 5' end and/or the 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 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.

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;

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.

Without being bound by theory, nsp2 in the alphavirus replicase is a part that exerts protease activity, which can recognize a specific amino acid sequence between the four components of the alphavirus replicase, thereby cutting the replicase polyprotein into a single component to exert replication function. Therefore, the restriction site here should be a protease restriction site sequence that can be recognized by the alphavirus replicase or nsp2.

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

In some embodiments of the present invention, the mRNA molecule, wherein the microRNA binding site The sequence is shown in SEQ ID NO:22.

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

In some embodiments of the present invention, the mRNA molecule has a sequence as shown in SEQ ID NO:24 or SEQ ID NO:28.

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

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

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

Another aspect of the present invention relates to 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' non-translated sequence, the RNA replicase sequence, the first 3' non-translated 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.

In some embodiments of the present invention, the mRNA molecule combination, 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.

In some embodiments of the present invention, the mRNA molecule combination, wherein the first 5' non-translated sequence and the first 3' non-translated sequence are not derived from alpha virus.

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 an alpha virus; preferably, from Venezuelan equine encephalitis virus.

In some embodiments of the present invention, the mRNA molecule combination, 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 as shown in SEQ ID NO:41; preferably, the coding sequence of the RNA replicase is as shown in SEQ ID NO:42.

In some embodiments of the present invention, the mRNA molecule combination, 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.

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;

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.

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.

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.

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;

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

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;

Preferably, the external spacer sequence at the 5' end is 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.

In some embodiments of the present invention, the mRNA molecule combination, wherein the microRNA The 5' end and/or 3' end of the cleavage site sequence contains one or more restriction site sequences that can be recognized by the 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.

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

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

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

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

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

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

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, and the first mRNA molecule and the second mRNA molecule are on the same DNA molecule.

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.

In some embodiments of the invention, the DNA molecule is an isolated DNA molecule.

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

The first DNA molecule encodes the first mRNA molecule described in any one of the present invention; and

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

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;

Preferably, the viral infection refers to novel coronavirus infection;

Preferably, the tumor is selected from liver cancer, rhabdomyosarcoma, glioma, bladder cancer, colorectal cancer, pancreatic cancer, adenocarcinoma 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.

MicroRNA is a type of non-coding single-stranded RNA molecule with a length of about 22nt encoded by endogenous genes. They participate in post-transcriptional gene expression regulation in animals and plants. MicroRNA recognizes the mRNA sequence of the target gene and binds to it, resulting in the inhibition of mRNA translation or the degradation of mRNA, thereby regulating the expression of the mRNA encoding gene. This gene expression regulation mechanism is called microRNA-mediated post-transcriptional regulation. MicroRNA has tissue specificity and its expression levels vary in different tissues. For example:

miRNA-122 is highly expressed in the liver, but is less expressed in non-hepatic cells (e.g., muscle cells) and liver cancer tissues;

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

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

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

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

miRNA-126 and miRNA-143 were highly expressed in lung tissues, but lowly expressed in lung cancer tissues.

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

Without being limited by theory, any pathogen infection or any tumor type is applicable to this requirement. More specifically, it may also correspond to a tumor type with low expression of a certain type of 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.

Another aspect of the present invention relates to a recombinant vector, which contains 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.

Another aspect of the present invention relates to a recombinant host cell, which contains 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.

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 The DNA molecule combination described above, and the liposome delivery system.

Another aspect of the present invention relates to a pharmaceutical composition, which contains 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.

Another aspect of the present invention relates to a vaccine preparation, which contains 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;

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.

Lipid delivery systems include liposomes, lipid nanoparticles (LNP), and lipid polymer nanocarriers (LPP).

LPP among lipids is a double-layer structure with polymer-encapsulated mRNA as the core and phospholipids as the shell. The double-layer liposome membrane of LPP has a better effect of encapsulating and protecting mRNA, and the core of LPP can gradually release mRNA molecules as the polymer degrades. LPP has excellent targeting effect on dendritic cells and can better activate the immune response of T cells through antigen presentation to achieve ideal therapeutic effects.

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 a variety of 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 is required and the effectiveness is high; it does not enter the cell nucleus and has good safety. The mRNA COVID-19 vaccine verifies the applicability of the mRNA technology platform in the vaccine field.

Another aspect of the present invention relates to the use of 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 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.

Another aspect of the present invention relates to a method for treating or preventing viral infection or tumor or a protein replacement therapy, comprising the step of administering to a subject in need thereof an effective amount of 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;

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.

Self-amplifying mRNA nucleic acid sequences with enhanced expression levels of foreign genes

The present invention also provides an improved saRNA construct, and a method for preparing and using the construct. The present invention further provides the use of the saRNA to carry a target gene for treatment, prevention and/or diagnosis. Specifically, after in-depth research and creative work, the present invention has obtained an optimized self-amplified 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:

One aspect of the present invention provides an mRNA molecule encoding an RNA replicase composed of non-structural proteins 1, 2, 3 and 4 derived from an alphavirus, wherein the non-structural 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 non-structural proteins 1, 2, 3 and 4 are derived from Venezuelan equine encephalitis virus.

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: 73. 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 the N-terminus.

In some embodiments, the mRNA molecule comprises a nucleotide sequence encoding the macro domain as shown in SEQ ID NO:68 or 70.

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

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.

In some embodiments, the mRNA molecule further comprises:

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

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 can be connected to the 3' end of the RNA promoter.

In some embodiments, the mRNA molecule comprises the following operably linked nucleotide sequences in order 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,

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

In some embodiments, the coding sequences of non-structural proteins 1, 2, 3 and 4 comprise 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.

In some embodiments, the coding sequences of non-structural proteins 1, 2, 3 and 4 include 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.

In some embodiments, the 5' untranslated region is derived from an alpha virus; 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.

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

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.

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.

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

In some embodiments, 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 (eg, a non-coding gene).

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.

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

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.

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.

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 delivery system.

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 the DNA molecule is encapsulated by a liposome delivery system. The vaccine formulation may also contain 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 novel coronavirus infection or preventing severe illness caused by novel coronavirus infection. Accordingly, the target gene encoded by the mRNA may encode an immunogenic peptide of the novel coronavirus.

In one aspect, the present invention provides the use of the mRNA molecule or DNA molecule disclosed herein 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. Accordingly, the target gene encoded by the mRNA can be a vaccine antigen gene, a tumor killing gene, a therapeutic protein, or a Protein genes, antibody genes, etc.

In one aspect, the invention provides a kit comprising an mRNA molecule or a DNA molecule as disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

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

Figure 2: Overlay of RNA capillary electrophoresis results of in vitro transcription of different saRNA templates modified using psaRNA-152 as a template. saRNA-062 is the original saRNA, and saRNA-183 to 185 are the modified saRNAs.

Figure 3: Capillary electrophoresis results of in vitro transcribed RNA after modification of positions 1680-1676 in psaRNA-183 as template. The black box indicates the electrophoresis detection peak shape of the short transcript before and after backbone optimization.

Figure 4: Expression level results of optimized luciferase-encoding saRNA-152 and original saRNA-062 after transfection of BHK-21 cells for 24 and 72 hours.

Figure 5: Expression level results of saRNA-243 encoding luciferase and original saRNA-062 obtained after detection optimization after transfection of BHK-21 cells for 24 hours.

Figure 6: Replication results of optimized luciferase-encoding saRNA-152 and original saRNA-062 after transfection of BHK-21 cells for 24 and 72 hours. The data were normalized to the results 2 hours after transfection.

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

Figure 8: E7-ELISA test results for detecting the secreted HPV16 antigen expression of S23H02, M22H04 and negative control after transfection into tissue culture containing Lipofectamine 3000.

Figure 9: Tumor volume data of C57bl/6 mice challenged with TC1 tumor cells and injected with 3 doses of therapeutic vaccine. Tumor volume and mouse body weight were recorded. The dose per mouse per dose was calculated based on the active ingredient.

Figure 10: Body weight data of C57bl/6 mice after being challenged with TC1 tumor cells and injected with 3 doses of therapeutic vaccine. The dose per mouse per dose was calculated based on the active ingredient.

Figure 11: Flow cytometry was used to detect the number of E7-specific CD8 T cells in the spleen cells of C57bl/6 mice injected with one dose of therapeutic vaccine. The dose per mouse per dose was calculated based on the active ingredient.

Figure 12: Schematic diagram of the miRNA122 binding site sequence box. After the inner spacer sequence is separated, an outer spacer sequence is added at both ends, and the outermost portion is a protease cleavage site that can be recognized by the replication enzyme.

Figure 13: Self-amplifying mRNA structure, the structural components from left to right are cap structure-5-terminal alphavirus non-translated sequence (5'UTR)-alphavirus replicase sequence-26S promoter sequence-arbitrary target protein sequence X-3-terminal alphavirus non-translated sequence (3'UTR)-polyadenine sequence (polyA sequence).

Figure 14: Schematic diagram of the self-amplified mRNA structure with microRNA122 binding sites inserted in different positions. Arrows represent different insertion positions of microRNA122 binding sites. The microRNA122 binding site is obtained by combining 6 microRNA122 binding sites in series.

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

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

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

Figure 18: Expression of Nluc luciferase 24 hours after transfection of self-amplified mRNA expressing Nluc-EGFP with microRNA122 binding site inserted into Huh7.5.1 cells and C2C12 cells. The control is self-amplified mRNA without microRNA122 binding site inserted. The expression value is normalized with firefly luciferase.

Figure 19: Relative RNA levels of self-amplified mRNA in C2C12 cells 24 hours after transfection of self-amplified mRNA expressing Nluc-EGFP with microRNA122 binding site inserted into cells. Control is self-amplified mRNA without microRNA122 binding site inserted. The relative RNA level value after 24 hours of transfection is normalized with that after 2 hours of transfection. nsp1 represents the relative level of RNA encoding replicase sequence, and EGFP represents the relative level of RNA encoding EGFP sequence.

Figure 20: Relative RNA level of self-amplified mRNA in Huh7.5.1 cells 24 hours after transfection of self-amplified mRNA expressing Nluc-EGFP with microRNA122 binding site inserted into the cells. Control is self-amplified mRNA without microRNA122 binding site inserted. The relative RNA level value after 24 hours of transfection is normalized with that 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.

Figure 21: Schematic diagram of self-amplified mRNA with different copy numbers of liver-specific microRNA-122 binding sequences inserted intention.

FIG. 22 : Expression of Nluc luciferase 24 hours after transfection of self-amplified mRNA expressing Nluc-EGFP and inserted with different copies of microRNA122 binding sites into Huh7.5.1 or C2C12 cells.

Figure 23: Schematic diagram of mRNA molecule assembly.

Figure 24: The position of the mutated amino acid in the macrodomain of saRNA and the comparison results of the amino acid sequences of different virus macrodomains. Macro is the macrodomain, AUD is the alphavirus-unique domain, HVD is the hypervariable domain, different shapes represent different mutations, and the amino acids with more than 50% homology in the amino acid comparison results are marked with shadows. 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.

Figure 25: Schematic diagram of the protein structure of the saRNA macrodomain. The positions marked by arrows are ADP ribose, glutamine at position 48 (abbreviated Q), and isoleucine at position 113 (abbreviated I) bound to the macrodomain.

Figure 26: Western blot test results of the effect of macrodomain amino acid mutations on total cellular protein ADP-ribosylation. Mock is a simulated transfection group, in which only transfection reagent was added without transfection of any RNA molecules. GAPDH protein was used as a cellular protein internal reference. The bottom value is the ratio of total protein ADP-ribosylation level of each group relative to the control group.

Figure 27: Comparison of mutant saRNA subgenomic RNA replication capacity in hela cells. The significant differences between the groups were statistically analyzed using two-way ANOVA. **** and ^^^^ represent that the P values of WT-saRNA and 190-saRNA or 191-saRNA groups are less than 0.0001, respectively.

Figure 28: Comparison of the ability of mutant saRNA to form double-stranded RNA during hela cell replication. Figure 28a is a schematic diagram of the flow cytometry results 24 hours after transfection, and Figure 28b is a statistical bar graph of the proportion of double-stranded RNA.

Figure 29: Principal component analysis of transcriptome levels 24 hours after Hela cells were transfected with different saRNAs.

FIG. 30 : Venn diagram of the number of genes upregulated or downregulated in different saRNAs relative to the control group.

Figure 31: Ridge plot of differential gene cluster analysis in wild-type saRNA relative to the control group. Figure 31a is a biological function cluster analysis of differential genes; Figure 31b is a molecular function cluster analysis of differential genes; the horizontal axis is the log2 value of the change fold, green represents the adjusted P value, blue dots represent each down-regulated gene, and red dots represent each up-regulated gene.

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

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.

Figure 34: Luciferase expression levels after HeLa cells were transfected with different saRNAs for 2, 6, 24 and 48 hours.

Figure 35: Western blot detection results of cell translation activity after 2, 6, and 24 hours of transfection with different saRNAs. GAPDH was used as an internal reference.

Figure 36: Detection of ribosomal RNA integrity after 2, 6, 24 and 48 hours of transfection of Hela cells with different saRNAs. Figure 36a is a schematic diagram of the detection of ribosomal RNA integrity 24 hours after transfection of saRNA using capillary electrophoresis, with arrows marking degraded ribosomal RNA. Figure 36b is the statistical results of the detection of ribosomal RNA integrity at different times.

Figure 37: Detection of eIF2α phosphorylation level after 6 and 24 hours of transfection with different saRNAs. peIF2α is phosphorylated eIF2α, and total eIF2α protein and GAPDH are used as internal references.

Figure 38: Fluorescence microscopy images 24 hours after transfection with different saRNAs, red arrows mark cells with nuclear shrinkage and their EGFP expression. DAPI represents stained cell nuclei, and EGFP represents cells expressing EGFP.

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

Figure 40: Detection results of activated caspase 8 and caspase 3 proteins that were cleaved 24 hours after transfection with different saRNAs, with the uncleaved full-length caspase as a control. GAPDH was used as an internal reference.

Figure 41: Detection of cell apoptosis and EGFP expression intensity in Hela cells transfected with wild-type saRNA treated or not with Caspase inhibitors.

Figure 42: Schematic diagram of in vivo expression level detection of different mRNA-LNPs. Figure 42a is a schematic diagram of the detection process, D0 is the immunization time. Figure 42b is a fluorescent image of in vivo imaging of mice on days 1, 3, and 7 after immunization.

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

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

Detailed description of the invention

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 a cell. saRNA is similar to mRNA and generally contains a 5'UTR, a 3'UTR, and a poly(A) tail, but additionally contains a non-structural protein sequence derived from a virus (e.g., an alphavirus) and a subgenomic promoter (sgPr) located upstream of the target protein coding sequence. saRNA is a positive-strand RNA molecule that is first translated by the ribosome into several non-structural protein components and assembled into RNA replicase after entering the cell. RNA replicase first uses the saRNA that first enters the cell to synthesize the saRNA negative strand, and then uses the negative strand 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, used 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 without a target protein coding sequence and saRNA that operably connects the saRNA backbone sequence to the target protein coding sequence.

As used herein, the term "macrodomain" refers to a conserved protein domain encoded by viruses such as alphaviruses, coronaviruses, 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 reduction of ADP ribose hydrolysis activity in the viral macrodomain will lead to a reduction in the ability of the virus to replicate, so the macrodomain is one of the potential hotspots for the development of antiviral drugs in the near future. 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%.

As used herein, the term "RNA replicase" is also called RNA-dependent RNA replicase (RdRp), which 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 required for the replication of other RNA viruses and viroids except retroviruses. The RNA replicase used for saRNA is usually derived from alphaviruses and contains nonstructural proteins 1, 2, 3, and 4 (nsP1, nsP2, nsP3, and nsP4).

The term "subgenomic promoter" (SGP) refers to a nucleic acid sequence upstream (5' end) of a nucleic acid sequence (e.g., a coding sequence) that controls the expression of a nucleic acid sequence by providing recognition and structural sites for RNA polymerase, usually RNA-dependent RNA polymerase, and in particular functional alphavirus nonstructural proteins. The subgenomic promoter is a genetic element of a positive-strand RNA virus (e.g., an alphavirus). The subgenomic promoter of an alphavirus is a nucleic acid sequence contained 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., a functional alphavirus nonstructural protein). The RNA (-) strand (i.e., the complementary strand of the alphavirus genomic RNA) serves as a template for the synthesis of a (+) strand subgenomic transcript, and the synthesis of a (+) strand subgenomic transcript typically begins at or near the subgenomic promoter.

As used herein, the term "subgenomic" refers to a nucleotide sequence (such as 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 be a region encoding a VEEV structural protein, and a subgenomic RNA may be transcribed from a subgenomic region using an internal subgenomic promoter, the sequence of which is located in the genomic viral RNA or its complement. Transcription of the subgenomic region may be mediated by a virally encoded polymerase associated with a host cell-encoded protein (e.g., nsP1-4).

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 non-structural proteins. In particular, the alphavirus replicase uses the (-) strand complementary strand of the genomic RNA as a template to synthesize the (+) strand subgenomic transcript. The (+) strand subgenomic transcript encodes 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 as a polyprotein, and the polyprotein is cleaved to produce the structural protein. At the late stage 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 packaged by structural proteins (White et al., 1998, J. Virol., vol. 72, pp. 4320-4326).

In the present invention, the term "sequence essential for alphavirus replication" is a sequence that can be recognized by alphavirus replicase and initiate subsequent RNA transcription and replication, which is intercepted from the alphavirus replicase sequence. The sequence essential for alphavirus replication must contain a minimum sequence (shown in SEQ ID NO:54), and the length can be increased or adjusted based on the shortest sequence.

In the present invention, the term "microRNA mature sequence" refers to a microRNA molecule formed after the microRNA precursor (pre-miRNA) that forms a hairpin structure is processed by the Dicer enzyme. The two arms of the microRNA precursor each produce a functional mature microRNA, which targets different sites. They are generally named "-5p" (or 5p) and "-3p" (or 3p), such as hsa-miR-21-5p and hsa-miR-21-3p, respectively, indicating that the mature microRNA sequence is derived from hsa- The 5' and 3' arms of the mir-21 precursor are processed. From the sequence point of view, most of the sequences of 5p and 3p are complementary.

In the present invention, if not otherwise specified, the term "integrity" refers to the percentage of the amount of the target RNA product produced after the RNA in vitro transcription reaction is completed (including the target RNA product and the unexpected RNA product) produced; the amount of the target RNA product and the amount of the total RNA product can be determined by electrophoresis. For example, if the amount of the target saRNA product determined by the electrophoresis method accounts for 80% of the amount of all RNA products, the saRNA integrity is 80%.

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.

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 described in the present invention, or reduce and/or alleviate the indications or symptoms in a subject.

mRNA Therapeutics

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 a vaccine or drug, mRNA can be used to prevent infectious diseases, treat tumors, and for protein replacement therapy. mRNA drugs have rich targets and are not restricted by the drugability of the target protein or intracellular/extracellular restrictions. The mRNA sequence is easy to design, and the patient's own cells are used to produce molecules, bypassing the challenges of chemical synthesis, and the self-induced molecules have stronger efficacy. The mRNA production platform has strong scalability and replicability, and is easy to scale up.

(1) Preventive vaccines for infectious diseases

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

(2) Tumor therapeutic vaccines/drugs

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

(3) Protein replacement therapy

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 plant is theoretically an economically feasible and efficient way. 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 required.

Self-amplifying RNA

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

In another aspect, the present application reduces the ADP ribose hydrolysis activity by amino acid mutation of the macro domain in the replicase protein domain encoded by saRNA, thereby reducing the replication efficiency of saRNA, thereby reducing the 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 a nucleic acid sequence encoding the replicase with a sequence encoding a target gene.

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 compared to the wild-type macrodomain from VEEV (as shown in SEQ ID NO: 55) (numbering according to SEQ ID NO: 56). 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.

In some embodiments, the saRNA disclosed herein comprises nucleotide sequences encoding replicase constituent proteins nsP1, nsP2, nsP3 and nsP4 derived from alphavirus, wherein the four replicase constituent proteins are capable of assembling into an RNA replicase complex, wherein the mutant macrodomain is located at the N-terminus of nsP3. The four non-structural protein components nsP1, nsP2, nsP3 and nsP4 are assembled into an RNA replicase complex in the form of a polyprotein. nsP1-4 all have their unique functions, wherein nsP4 plays the role of an RNA polymerase with RNA as a template. The sequence of the replicase constituent protein encoded by the saRNA may be derived from, for example, Venezuelan equine encephalitis virus (VEEV) and forest encephalitis virus (SFV), etc. In some embodiments, the domain sequence of at least one non-structural 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), etc. In some embodiments, the plurality of non-structural protein sequences forming the replicase are derived from an alphavirus such as VEEV. Specifically, the saRNA may comprise a nucleotide sequence encoding a replicase constituent protein as shown in SEQ ID NO: 67 or SEQ ID NO: 69.

Although the native alphavirus genome encodes structural proteins in addition to the non-structural replicase, in some embodiments, the alphavirus-based saRNA of the present invention does not encode alphavirus structural proteins.

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 may be derived from a variety of viruses, such as alphaviruses and coronaviruses. In some embodiments, the 5' untranslated region is from Venezuelan equine encephalitis virus. Specifically, the 5' untranslated region may comprise a nucleotide sequence as shown in SEQ ID NO: 63. The 3' untranslated region may be derived from a variety of viruses, such as alphaviruses and coronaviruses. In some embodiments, the 3' untranslated region is from Venezuelan equine encephalitis virus. Specifically, the 3' untranslated region may include a nucleotide sequence as shown in SEQ ID NO: 64. In some embodiments, the poly(A) sequence includes 20-200 adenylic acids. The poly(A) sequence may include a restriction enzyme cleavage site inside or at the 3' end. Location.

In some embodiments, the saRNA further comprises a nucleotide sequence encoding a target protein or a target gene (GOI) coding sequence, wherein the target protein is or the target gene encodes any one selected from the following: a therapeutic polypeptide, a preventive polypeptide, a diagnostic polypeptide, a reporter gene, an antigen, or a gene encoding a regulatory structure. For example, the target protein 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 siRNA, microRNA, gRNA, etc.

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.

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.

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, 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 at the 5' end of the second expression unit. In some embodiments, the subgenomic promoter is a 26S promoter, such as shown in SEQ ID NO: 65.

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

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,

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

Wherein, 5'UTR is 5' untranslated region, nsP (including nsP1, nsP2, nsP3, nsP4) is a plurality of 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' polyadenylic acid tail, and wherein nsP comprises a mutated macrodomain. When multiple GOIs are present, each GOI can be operably linked to its respective SGP. In some embodiments, the SGP is a 26S promoter, for example as shown in SEQ ID NO:65.

In some embodiments, the saRNA disclosed herein comprises a 5' untranslated sequence that is at least 85% identical to SEQ ID NO: 63, at least 85% identical to SEQ ID NO: 68 or 70 and comprises a sequence encoding a sequence such as SEQ ID NO: 57. or a nonstructural protein coding sequence of the nucleotide sequence of the macrodomain shown in 59, and/or a 3' non-translated sequence that is at least 85% identical to SEQ ID NO: 64. The at least 85% identical can 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.

In some embodiments, 5'UTR, nsP, SGP and 3'UTR sequences are all derived from alphavirus. 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 viral particles) 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 (-) strand complementary strand of the genomic RNA as a template to synthesize the (+) strand 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 as a polyprotein, and the polyprotein is cleaved to produce structural proteins.

The saRNA disclosed herein can linearize the DNA plasmid template before being prepared by in vitro transcription. To this end, a restriction enzyme site can be added to the 3' end of the saRNA to facilitate cutting. 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, for example, for encoding other desired gene products.

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 adding a target protein coding sequence or a 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.

Methods for preparing saRNA

saRNA is generally obtained by in vitro transcription (IVT) by 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 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, wherein the DNA Examples of the RINA-dependent polymerases include 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.

The transcription reaction will contain nucleotides as well as 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.

Applications of saRNA

mRNA-based therapy is the delivery of in vitro synthesized mRNA to specific cells in the human body, where it is translated into the desired protein in the cytoplasm. As a vaccine or drug, mRNA can be used to prevent infectious diseases, treat tumors, and for protein replacement therapy. mRNA drugs have rich targets and are not restricted by the drugability of the target protein or intracellular/extracellular restrictions. The mRNA sequence is easy to design, and the patient's own cells are used to produce molecules, bypassing the challenges of chemical synthesis, and the self-induced molecules have stronger efficacy. The mRNA production platform has strong scalability and replicability, and is easy to scale up.

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.

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 of a pathogen related to an mRNA vaccine encoding an infectious disease. After injection, specific antigens 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 pathogens. The vaccine can be a tumor therapeutic vaccine. For example, mRNA encoding tumor-specific antigen targets is delivered to 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.

In one aspect, the saRNA disclosed herein can be used to treat diseases, for example, by providing therapeutic proteins or immune regulatory proteins for diseases. Typically, protein replacement therapy is used to treat rare monogenic diseases, aiming to restore enzyme function. Protein synthesis is difficult, administration is difficult and expensive, while the use of mRNA Turning the human body into a processing plant for its own proteins is theoretically an economically feasible and efficient way. The saRNA disclosed in the present invention can theoretically synthesize any target protein and can be used as a protein supplement or alternative therapy to treat a variety of diseases.

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.

Advantageous Effects of the Invention

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

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

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

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

In addition, the current self-amplifying mRNA is an mRNA vector modified from the alphavirus genome, and has no specific microRNA binding site. After being encapsulated and delivered into the body by the currently commonly used nanolipid particles (lipid nanoparticles, LNP), due to the inherent physical and chemical properties of the nanolipid particles, the self-amplifying mRNA may be mediated to enter the liver and other parenchymal tissues for expression in large quantities, which may cause potential liver toxicity in some application examples. Therefore, in another aspect, the technical effect achieved by the present invention also includes that the present invention utilizes the microRNA highly expressed in liver tissue to degrade the self-amplifying mRNA, thereby reducing liver toxicity.

In another aspect, the saRNA of the present invention has the advantages of known saRNA over traditional mRNA, that is, it can achieve the same protein expression level as traditional mRNA with a very low dose, and prolong the existence time of antigen protein in the body, thereby possibly enhancing the immune response. In terms of production, a lower effective dose may reduce the production cost of saRNA. As a therapy, the dose and number of injections used in mRNA therapy can be reduced, thereby prolonging the efficacy while reducing the possible toxic side effects of mRNA and delivery vectors.

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

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

(2) Reduced the intensity of the natural immune response induced by saRNA;

(3) Reduced translational inhibition of saRNA;

(4) Reduced saRNA-induced cell apoptosis;

(5) Improved the expression efficiency of saRNA.

The saRNA disclosed herein contains a mutated macrodomain, which weakens the ADP ribose hydrolysis activity, thereby causing The ability of the virus to replicate is weakened and the ability to stimulate the innate immune system is reduced. 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.

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 positions are replaced by GOI, so that the saRNA encodes the required gene products, rather than the structural proteins of the alphavirus viral particles.

More specifically, the saRNA containing a mutant macrodomain of the present invention leads to a significant reduction in the dsRNA formed during replication. There are sensors in human cells that recognize foreign viral invasions, which are called pattern recognition receptors. One of the signals they recognize is double-stranded RNA that appears in the cytoplasm, because this may represent the replication of viral RNA in the cell. During the replication process, saRNA forms double-stranded RNA, which is very similar to the viral RNA in replication, and therefore may stimulate the innate immune response of the cell. This may further enhance the effect of the vaccine. However, this stimulated immune response may promote the immune response as a vaccine, but on the other hand, as a therapy, the stimulated immune response may cause side effects. If the stimulated innate immune response is too strong, the expression of mRNA may be inhibited, which in turn affects the efficacy of therapy and vaccines. In addition, the double-stranded RNA produced during saRNA replication is the key to activating the interferon or NFKB signaling pathway, thereby inducing a large number of downstream antiviral genes to inhibit saRNA replication. Therefore, the immunogenicity of saRNA requires precise design and adjustment, so as to moderately reduce the replication ability of saRNA and the production of double-stranded RNA, and the sgRNA disclosed in the present invention achieves this purpose.

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 (TLR), which lead to the production of type I interferon and inhibition of translation. However, the translation inhibition of the saRNA of the present invention is reduced due to reduced immunogenicity, so although its replication ability is reduced, the final expression efficiency in the cell is improved compared to the control saRNA encoding the wild-type macro domain.

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

Sequence 2: saRNA-062 sequence

Sequence 3: saRNA-152 sequence

Sequence 4: saRNA-153 sequence

Sequence 5: saRNA-154 sequence

Sequence 6: saRNA-183 sequence

Sequence 7: saRNA-184 sequence

Sequence 8: saRNA-185 sequence

Sequence 9: saRNA-243 sequence

In the above sequence 2-9:

“NNNNNNNNNNNNNNNN” indicates exogenous genes, including but not limited to: vaccine antigen genes, tumor killing genes, therapeutic protein genes, antibody genes; exogenous genes can be adjusted according to different purposes;

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.

Sequence 10:062 Replicase coding sequence (SEQ ID NO:10)

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

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

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

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

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

Sequence 16: 5' non-translated sequence (SEQ ID NO: 16)

Sequence 17: 3’ non-translated sequence (SEQ ID NO: 17)

Sequence 18: Subgenomic promoter sequence (SEQ ID NO: 18)

Sequence 19: Polyadenylation sequence (SEQ ID NO: 19)

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

Sequence 21: S23H02 sequence

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

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

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

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

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

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

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

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

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

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

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

Sequence 33: Inner spacer sequence 1

AGCGCGA (SEQ ID NO:33)

Sequence 34: Inner spacer sequence 2

ACGCGAA (SEQ ID NO:34)

Sequence 35: 5’outer spacer sequence 1 (SEQ ID NO: 35)

Sequence 36: 5’outer spacer sequence 2 (SEQ ID NO: 36)

Sequence 37: 3’outer spacer sequence 1 (SEQ ID NO: 37)

Sequence 38: 3’outer spacer sequence 2 (SEQ ID NO: 38)

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

Sequence 40: 3’ replicase recognition protease cleavage site 1 (SEQ ID NO: 40)

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

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

Sequence 54: The shortest sequence contained in the essential sequence for virus replication: (SEQ ID NO: 54)

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

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

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)

Sequence 56: Wild-type saRNA sequence

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

Sequence 58: saRNA-190 sequence (different from sequence 57 only by the introduction of the Q48P mutation in the macrodomain) (SEQ ID NO: 58)

Sequence 60: saRNA-191 sequence (different from sequence 57 only by the introduction of the I113F mutation in the macrodomain) (SEQ ID NO: 60)

Sequence 61: saRNA-190 backbone sequence

Sequence 62: saRNA-191 backbone sequence

In the sequence 61-62 above:

“NNNNNNNNNNNNNNNN” indicates exogenous genes, including but not limited to: vaccine antigen genes, tumor killing genes, therapeutic protein genes, antibody genes; exogenous genes can be adjusted according to different purposes;

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.

Sequence 63: 5' non-translated sequence (SEQ ID NO: 63)

Sequence 64: 3' non-translated sequence (SEQ ID NO: 64)

Sequence 65: Subgenomic promoter sequence (SEQ ID NO: 65)

Sequence 66: Polyadenylation sequence (SEQ ID NO: 66)

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

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

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

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

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

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

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

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

Sequence 75: Nonstructural protein 4 amino acid sequence (SEQ ID NO: 75)

DETAILED DESCRIPTION

The embodiments of the present invention will be described in detail below with reference to the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. If the specific conditions are not specified, the experiments were carried out under conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased from the market.

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

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

Lipofectamine transfection reagents -mRNA Transfection Kit was purchased from Mirus, catalog number MIR 2225.

Example 1: Modification and completion of positions 4497-4503 in the saRNA-EGFP template plasmid psaRNA-062 Sexual testing

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

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

3. Preparation of modified saRNA by in vitro transcription:

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), BspQ I 4 μL, 10×Digestion Buffer 4 μL in sequence, and finally add DEPC water to the system to 40 μL, and incubate at 50°C for 2 h to linearize the plasmid.

b. In vitro transcription to obtain modified saRNA: Specifically, add 10×T7 polymerase buffer, 7.5mM ATP, 7.5mM GTP, 7.5mM UTP, 7.5mM CTP, 1μg linearized in vitro transcription template, 400U T7 RNA polymerase, 20U RNase I inhibitor, 6mM cap analog, add water to a total volume of 20μL, and react at 37°C for 2 hours. After the reaction, add 2U DNase I enzyme and react at 37°C for 15 minutes. The reaction system is replenished to 50μL, 25μL 7.5M lithium chloride solution is added and mixed, and after standing at -20°C for 30 minutes, centrifuged at 13000g 4°C for 10 minutes, the supernatant is discarded and the precipitate is washed with 70% ethanol, centrifuged at 13000g 4°C for 2 minutes, the supernatant is discarded, and dissolved in 30μL water. After measuring the concentration with a spectrophotometer, an appropriate volume of RNA was taken and the RNA integrity was tested using the 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%.

Example 2: Integrity test of different saRNAs constructed with the optimized psaRNA-152 template plasmid as the backbone Test

Using the psaRNA-152 plasmid as a template, the A base at position 4509 was mutated to T, C, and G, respectively, and the template plasmids thus obtained 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 mutation and detection methods were the same as those in Example 1. From the capillary electrophoresis detection results in Figure 2, it can be seen that the integrity of different saRNAs constructed with psaRNA-152 as the backbone is significantly improved compared to the original backbone psaRNA-062.

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

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

Example 4: Verification of the replication and expression efficiency of luciferase gene saRNA after skeleton optimization

1. Luciferase Assay

a. Cell inoculation: An appropriate number of Huh7.5.1 cells were inoculated into a 24-well cell culture plate and cultured overnight at 37°C with 5% CO ₂ .

b. Self-amplified mRNA transfection of Huh7.5.1 cells: 0.5 μg of saRNA-152, saRNA-243 or unoptimized saRNA-062 expressing luciferase gene after backbone optimization was transfected into Huh7.5.1 cells using liposome transfection reagent, and cultured overnight at 5% CO _{2 and} 37° C. for 72 hours.

c. Luciferase detection: 24 and 72 hours after transfection, use Nano-Glo Luciferase Assay System detection reagent, transfer 25μl Nano-Glo Luciferase Assay Reagent to a 96-well white opaque bottom plate, take 25μl supernatant sample to the well and mix evenly, wait for 3 minutes and use a microplate reader to detect the signal. The test results are shown in Figures 4 and 5. There is no significant difference in the luciferase expression levels of saRNA-152 and saRNA-243 after backbone optimization and at different time points compared with the unoptimized backbone, indicating that backbone optimization does not affect saRNA expression.

2. Self-amplification mRNA replication efficiency detection

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

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 by pipetting into EP tubes, and store in a -80°C refrigerator.

Reverse transcription fluorescence quantitative PCR was used to detect the replication of saRNA: total RNA was extracted from the cell sample using RNeasy Mini Kit, and RNA was quantified using a UV spectrophotometer. After reverse transcription was completed using 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 30s-(95°C 10s-60°C 30s)×40 cycles, and the test results are shown in Figure 6. The results show that there is no significant difference between the saRNA-152 after skeleton optimization and the unoptimized skeleton at different time points, indicating that skeleton optimization also does not affect the replication ability of saRNA.

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

1. Construction of in vitro transcription template plasmid

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 to obtain a self-amplified mRNA in vitro transcription template plasmid expressing Ag9.1, named L23H02.

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

a. Linearization of in vitro transcription template plasmid

Add 8 μg of in vitro transcription template plasmid L23H04, 4 μL of BspQI, and 4 μL of 10×Digestion Buffer in sequence, and finally make up the system to 40 μL with double distilled water. Incubate at 50°C for 2 hours to linearize the plasmid. After agarose gel electrophoresis to detect that the plasmid is completely linearized, it can be directly used as an in vitro transcription template without purification.

b. Obtaining L23H02 self-amplified mRNA by in vitro transcription

Add 10×T7 polymerase buffer, 7.5mM ATP, 7.5mM GTP, 7.5mM UTP, 7.5mM CTP, 1 μg linearized in vitro transcription template, 400U T7 RNA polymerase, 20U RNase I inhibitor, 6mM cap analog, add water to a total volume of 20 μL, and react at 37°C for 2 hours. After the reaction, add 2U DNase I enzyme and react at 37°C for 15 minutes. The reaction system was added with water to 50 μL, 25 μL 7.5M lithium chloride solution was added and mixed, and after standing at -20°C for 30 minutes, centrifuged at 13000g 4°C for 10 minutes, the supernatant was discarded and the precipitate was washed with 70% ethanol, centrifuged at 13000g4°C for 2 minutes, the supernatant was discarded, and the washing was repeated once, the supernatant was removed and dissolved in 30 μL water to obtain saRNA expressing Ag9.1, named S23H02 (sequence as shown in SEQ ID NO: 21). After the concentration was determined by a spectrophotometer, the integrity was detected using Qsep400. The capillary electrophoresis detection 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 was no obvious short transcription product.

Example 6: Preparation of saRNA lipid nanoparticle formulations expressing Ag9.1

The S23H02 prepared in Example 5 was encapsulated in four component lipids to form mRNA lipid nanoparticles, specifically: a lipid mixture (ionizable lipid ALC-0315: distearoylphosphatidylcholine: cholesterol: PEG lipid ALC-0159) dissolved in ethanol and S23H02 dissolved in citrate buffer were mixed 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 a lipid nanoparticle preparation S23H02-LNP for the following experiments.

Example 7: Tumors expressing optimized backbone saRNA of HPV antigens and mRNA expressing HPV antigens Comparison of treatment effects

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

Table 1

S23H02, M22H04 and negative control were transfected into tissue culture containing lipofectamine 3000. Supernatants were collected at the time points marked in Figure 8, and the expression of secreted HPV16 antigens was detected using an ELISA test for detecting the E7 domain, and the results are shown in Figure 8. S23H02 showed stronger humoral immunity than M22H04 against specific antigens.

C57bl/6 mice were challenged with TC1 tumor cells, set as day 0; then three doses of therapeutic vaccine were injected on days 5, 8, and 11, respectively; injected into the hind leg muscle.

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

The spleen cells of C57bl/6 mice injected with one dose of therapeutic vaccine were collected. After labeling, the number of E7-specific CD8 T cells in the spleen was detected by flow cytometry. The results are shown in Figure 11. S23H02 showed significantly stronger cellular immunity than M22H04 against specific antigens.

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.

Preparation Example 1: Self-amplification of liver-specific microRNA-122 binding sequences inserted at six different locations Preparation of mRNA

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 to both ends. The outermost site is a protease cleavage site that can be recognized by the replicase, as shown in Figure 12.

2. Design of self-amplifying mRNA structure: It is modified from the genome of Venezuelan equine encephalitis virus TC-83 strain in the alphavirus family. Specifically, the non-structural protein, namely the viral replicase sequence and the non-translated sequence, including the non-translated sequence at the 5-end and 3-end of the viral genome and the 26S promoter after the non-structural protein, is retained, and the alphavirus structural protein coding sequence is replaced with any target protein sequence X. At the same time, a T7 polymerase promoter site is added before the alphavirus non-translated sequence at the 5-end, and a polyadenine sequence (polyA sequence) is added after the alphavirus non-translated sequence at the 3-end. The specific structure is shown in Figure 13.

3. Preparation of self-amplified mRNA in vitro transcription templates with microRNA122 sequences inserted in six different locations Preparation: The T7 polymerase promoter site, the 5-terminal alphavirus non-translated sequence, the alphavirus replicase sequence, the 26S promoter sequence, the Nluc-EGFP reporter gene sequence, the 3-terminal alphavirus non-translated sequence, the polyadenylation sequence and the restriction endonuclease BspQI restriction site sequence were constructed into the pUC57-Kan plasmid by gene synthesis to obtain the self-amplifying mRNA plasmid psaRNA-Nluc (fluorescein)-EGFP (green fluorescent protein), named saRNA-EGFP (SEQ ID NO: 29); then the 6 microRNA-122 binding sequence tandem 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, which were named N21091, N21092, N21093, N21094, N21095 and N21096, respectively. The corresponding self-amplified mRNA structures are shown in Figure 14, and the corresponding sequences are shown in SEQ ID NO:23 to SEQ ID NO:28.

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

a. Linearization of in vitro transcription template plasmid: Specifically, add 8 μg of in vitro transcription template plasmid psaRNA-EGFP-microRNA, 4 μL of BspQI, and 4 μL of 10×Digestion Buffer in sequence, and finally add ddH ₂ O to the system to 40 μL, and incubate at 50°C for 2 hours to linearize the plasmid. After completion, add ddH ₂ O to the enzyme digestion mixture system to 100 μl, and use a large amount of agarose gel DNA recovery kit to purify and recover the linearized in vitro transcription template.

b. In vitro transcription to obtain self-amplified mRNA: Specifically, add 10×T7 polymerase buffer, 7.5mM ATP, 7.5mM GTP, 7.5mM UTP, 7.5mM CTP, 1μg linearized in vitro transcription template, 400U T7RNA polymerase, 20U RNase I inhibitor, 6mM cap analog, add water to a total volume of 20μL, and react at 37℃ for 2 hours. After the reaction, add 2U DNase I enzyme and react at 37℃ for 15 minutes. Add water to the reaction system to 50μL, add 25μL 7.5M lithium chloride solution and mix well, stand at -20℃ for 30 minutes, centrifuge at 13000g at 4℃ for 10 minutes, discard the supernatant and wash the precipitate with 70% ethanol, centrifuge at 13000g at 4℃ for 2 minutes, discard the supernatant, repeat the washing once, remove the supernatant and dissolve in 30μL water. After measuring the concentration with 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, and 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, and the results show that the self-amplified mRNA was successfully prepared.

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

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

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

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

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

U6 promoter downstream primer R: 5’-TTTGCGTGTCATCCTTGCG-3’ (SEQ ID NO:46).

After normalizing the microRNA-122 CT values of C2C12 and huh7.5.1 cells with the CT values 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 about 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.

Example 9: Self-amplification of liver-specific microRNA-122 binding sequences inserted at six different locations In vitro mRNA expression and replication assays

1. Fluorescent protein and luciferase detection

a. Cell inoculation: Huh7.5.1 cells with high expression of microRNA-122 and C2C12 cells with low expression of microRNA-122 were inoculated in ^75cm2 cell culture flasks. The culture medium was DMEM high glucose medium + 10% fetal bovine serum + 1% double antibody. When the confluence of cells in the culture flask reached more than 80%, the cells were digested and counted with trypsin. An appropriate number of cells were plated in a 24-well cell culture plate and cultured overnight at 37°C in a _CO2 incubator.

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 cultured in a _CO2 incubator at 37°C 24 hours.

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.

The results showed that: in the same transfection time, N21091, N21093, and N21094 self-amplified mRNAs were not significantly expressed in C2C12 cells with low expression of microRNA-122, while N21092 and N21096 were expressed normally, with the same level as the control self-amplified mRNA. In Huh7.5.1 cells with high expression of microRNA-122, the expression of N21092 and N21096 was significantly reduced, indicating that N21092 and N21096 self-amplified mRNAs were regulated by intracellular microRNAs as expected and had good cell expression specificity. N21095 self-amplified mRNA showed similar expression to the control self-amplified mRNA, inferring that placing the microRNA-122 binding site at the corresponding site did not affect the normal expression of the self-amplified mRNA, but was not effectively regulated by microRNA.

d. Luciferase detection: 24 hours after transfection, use Nano-Glo Luciferase Assay System detection reagent, transfer 25μl Nano-Glo Luciferase Assay Reagent to a 96-well white opaque bottom plate, take 25μl supernatant sample to the well and mix evenly, wait for 3 minutes and use an ELISA reader to detect the signal. The detection results are shown in Figure 18, which also show that the expression levels of N21092 and N21096 self-amplified mRNAs in Huh7.5.1 cells with high expression of microRNA-122 and C2C12 cells with low expression of microRNA-122 are significantly different, indicating their good cell expression specificity.

2. Self-amplification mRNA replication efficiency detection

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

b. Processing of cell samples: 2 hours and 24 hours after transfection, discard the supernatant, rinse the cells once with 500μl PBS, discard the supernatant, add 500μl cell lysis buffer and collect by pipetting into EP tubes, and store in a -80℃ refrigerator.

c. Reverse transcription fluorescence quantitative PCR detection of saRNA replication: After the total RNA of the cell sample is extracted using RNeasy Mini Kit, RNA is quantified using a UV spectrophotometer. After reverse transcription is completed using HiScript III 1st Strand cDNA Synthesis Kit, a fluorescence quantitative PCR experiment is performed using 2×ChamQ Universal SYBR qPCR Master Mix. The detection primers are as follows:

Primer F upstream of alphavirus replicase sequence (nsp1): 5'-GACGGACCGACAAGTCTCTA-3' (SEQ ID NO: 47),

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

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

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

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

GAPDH downstream primer R: 5’-GTAGAGGCAGGGATGATG-3’ (SEQ ID NO:52).

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

The detection results are shown in Figures 19 and 20.

The results showed that the relative expression of N21092 and N21096 self-amplified mRNAs in Huh7.5.1 cells with high expression of microRNA-122 and C2C12 cells with low expression of microRNA-122 were significantly different. In Huh7.5.1 cells, the relative expression of N21092 and N21096 self-amplified RNAs was significantly downregulated compared with C2C12 cells, indicating that microRNA-122 can regulate gene expression by effectively degrading the level of self-amplified RNA.

Example 10: Self-amplifying mRNA with different copy numbers of liver-specific microRNA-122 binding sequences inserted In vitro expression and replication assay

1. The structure of self-amplifying 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-amplifying mRNA with different copy numbers of liver-specific microRNA-122 binding sequences inserted. The preparation method refers to Preparation Example 1.

2. The fluorescent protein and luciferase detection method is the same as in Example 8. The relative expression results 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 FIG. 22 .

The results showed that in Huh7.5.1 cells with high expression of microRNA-122, the relative expression level of self-amplifying mRNA bound 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 was reduced 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%.

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

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 microRNA binding sequence placement site.

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

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 conservative between different viruses, with a homology of more than 50%. Therefore, we selected the 113th amino acid close to the ADP ribose hydrolysis active center and the 48th amino acid away from the ADP ribose hydrolysis active center for amino acid mutation, and the specific positions are shown in Figure 25. In coronaviruses and alphaviruses, the 113th position encodes isoleucine (I) or valine (V), which is highly conservative. We infer that it involves the ADP ribose hydrolysis function. The 48th position 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 with only an amino group on the side chain or isoleucine with a methyl group and an ethyl group. Therefore, we constructed the following three saRNAs through homologous recombination and in vitro transcription:

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 protein in the cells was detected with an anti-mono-ADP-ribosylation antibody. As shown in Figure 26, 24 hours after transfection, the ADP-ribosylation of 190/191-saRNA cell proteins was significantly stronger than that of 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. The data show that the design of amino acid mutations at positions 113 and 48 of the macrodomain successfully reduced the ADP ribosyl hydrolase activity.

Example 12: Detection of replication levels of mutant saRNAs with impaired ADP ribose hydrolase activity

After verifying that the ADP ribose hydrolase activity of the two mutant saRNAs was reduced, we tested whether the replication ability of the mutant saRNAs would change. After transfecting saRNA-190, saRNA-191 and wild-type saRNA into Hela cells for 2, 6, 24 and 48 hours, RNA was extracted and the EGFP gene encoded by saRNA was subjected to fluorescence quantitative PCR.

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 of 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. More than half of the cells transfected with wild-type saRNA were detected with dsRNA 24 hours after transfection, 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 the replication ability of 190/191-saRNA with macrodomain amino acid mutations in in vitro cells is weakened.

Example 13: Detection of differences in natural immunity induced by mutant saRNA

Considering that double-stranded RNA can significantly trigger the innate immune response, it is necessary to examine the changes in the innate immune level induced by mutant saRNA and wild-type saRNA. After transfecting Hela cells with 190/191-saRNA and wild-type saRNA for 24 hours, we extracted total cell RNA for RNA sequencing and analyzed the differences in transcriptome levels.

According to the results of principal component analysis, compared with the control group, the transcriptome difference of the transfected 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 downregulated genes, and most of the upregulated genes in the 190 and 191 groups were almost the same as the wild-type saRNA group. However, we found that the number of genes that were downregulated in common between different groups was 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, the differential genes of the wild-type saRNA group relative to the control group were clustered. 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 them 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 function of the up-regulated genes It can be related to cytokine/chemokine/growth factor/receptor binding and dsRNA/ssRNA binding (see Figure 31b). It is known that the proportion of cells that form double-stranded RNA after 190/191-saRNA transfection is significantly lower than that of wild-type RNA. Therefore, it is not difficult to associate the difference in the proportion of double-stranded RNA formed during the replication of different saRNAs may affect the induction of natural immune response.

Next, we checked the upregulated genes in the double-stranded RNA/single-stranded RNA binding protein. We saw that the intracellular RNA sensor RIG-I, MDA-5 and the endosomal RNA sensor TLR3 as well as the interferon-stimulated genes downstream of the IFN pathway, such as the OAS family and EIF2AK2, were highly upregulated (see Figure 32), indicating that the double-stranded RNA produced in saRNA replication is the key to activating the interferon or NFKB signaling pathway, thereby inducing a large number of downstream antiviral genes to inhibit saRNA replication. From the gene heat map of the interferon pathway between different groups, it can be seen that compared with the control group, the transcription levels of interferon pathway genes in all cells transfected with saRNA were significantly upregulated. In addition to the RNA binding proteins mentioned, there were interferon beta, transcription factors IRF7/9, STAT1 and many downstream interferon-stimulated genes. In addition, the upregulation of cytokines was also seen, suggesting that the NF-kb pathway was also effectively activated. As expected, the transcription levels of interferon signaling pathway genes induced by saRNA in different groups were significantly different. Compared with the control group, 190 induced the weakest IFN immune response, 191 was slightly stronger, and the wild type was the strongest. As shown in Figure 33, the mRNA levels of interferon pathway and cytokines were verified by fluorescence 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, while at 6 hours, the IFN-beta, CXCL10, CCL5 and IFIT2 RNA levels in the wild-type saRNA group were significantly upregulated compared with the control group, and 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.

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.

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

In order to detect the activity of exogenous genes expressed by different saRNAs, different saRNAs encoding luciferase were transfected into HeLa cells. The results showed that the expression level of wild-type saRNA was higher than that of mutant saRNA at 2 hours and 6 hours, but the expression level of 190/191-saRNA was slightly higher at 24 hours, and the expression level of 190-saRNA was higher at 48 hours. 6 times higher than 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 transfections was detected. Before collecting cells, puromycin was added to the culture medium to a final concentration of 5ug/mL and treated for 5 minutes to incorporate it 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, and the 190-saRNA protein translation activity was slightly weaker than the control at 6 hours, but similar to the control group at 24h. In contrast, the translation activity of cells in the transfected 191-saRNA group was slightly weaker at 24h (see Figure 35). The results prove that after saRNA transfection, protein translation in cells 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 was still relatively intact within 2 and 6 hours, but severely degraded at 24 and 48 hours, while the integrity of ribosomal RNA transfected with 190/191saRNA was not significantly reduced relative to the control group. The results suggest 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 results of eIF2α phosphorylation detection showed that after 6 hours of transfection, each group of saRNA induced eIF2α phosphorylation, and was positively correlated with the subgenomic level of 6h, with wild-type saRNA being the strongest and 191-saRNA being the weakest. Unexpectedly, 24 hours after transfection, the phosphorylation level of eIF2α was significantly reduced, and the phosphorylation of eIF2α induced by wild-type saRNA was even weaker than that of the control group (i.e., the mock group: only the transfection reagent was added, and no RNA molecules were transfected), while the phosphorylation degree of eIF2α of the mutant saRNA was similar to that of the control group (see Figure 37). Based on the above results, it was found that host translation was not inhibited at 2 hours of transfection, and host translation mediated by PKR/eIF2α appeared at 6 hours, but at this time, because 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, wild-type eIF2α phosphorylation levels were reduced, and thus host translational repression was largely promoted by rRNA degradation caused by OAS/RNase L activation, so the wild-type saRNA that was most significantly inhibited had the lowest expression level.

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

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

It was observed that 24 hours after the wild-type saRNA was transfected, the cell nucleus collapsed significantly, suggesting 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 transfected group was significantly lower than that in other groups. Western blot was performed to detect the protein expression level and activation state 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 fluorescence intensity of EGFP 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.

Example 16: Detection of in vivo expression level of 190-saRNA

In order to verify whether the 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 nanolipid particles (LNP). As shown in Figure 42a, 4-6 week-old balb/c mice were intramuscularly immunized with 5 μg of different mRNA-LNP expressing the firefly luciferase gene, and live 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 it began to decay afterwards, and almost no expression was detected after the 7th day (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 higher expression levels in vivo.

Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art. According to all the teachings disclosed, various modifications and replacements can be made to those details, and these changes are all within the protection scope of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims (75)

A translation 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. The mRNA molecule translation region according to claim 1, wherein the 4509th base is T, C or G. 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. The mRNA molecular translation region according to any one of claims 1 to 3, wherein the sequence of the initial mRNA molecular translation region encoding the alphavirus RNA replicase is as shown in SEQ ID NO:10. The translation region of the mRNA molecule according to any one of claims 1 to 4, whose sequence is shown in any one of SEQ ID NO:11 to SEQ ID NO:15. 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. The mRNA molecule according to claim 6, wherein Wherein, the 5' untranslated region is derived from an alpha virus; preferably, it is derived from Venezuelan equine encephalomyelitis virus; Preferably, the sequence of the 5’ non-translated region is as shown in SEQ ID NO:16. The mRNA molecule according to any one of claims 6 to 7, wherein Wherein, the 3' untranslated region is derived from an alpha virus; preferably, from a Venezuelan equine encephalomyelitis virus. poison; Preferably, the sequence of the 3’ non-translated region is as shown in SEQ ID NO:17. 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 as shown in SEQ ID NO:18. The mRNA molecule according to any one of claims 6 to 9, wherein the sequence of the polyadenylic acid sequence is as shown in SEQ ID NO:19. 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. The mRNA molecule according to any one of claims 6 to 11, whose sequence is shown in SEQ ID NO:3 and any one of SEQ ID NO:6 to SEQ ID NO:9. 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. 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. 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. 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. 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. 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. Use of the mRNA molecule translation region 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. 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. 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 as shown in SEQ ID NO:41; preferably, the coding sequence of the RNA replicase is as shown in SEQ ID NO:42. 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. 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, from Venezuelan equine encephalitis virus. 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 a tissue-specifically expressed microRNA; 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. 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. 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. 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. The mRNA molecule according to any one of claims 20 to 27, wherein the 5' end and/or the 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 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. 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. The mRNA molecule according to any one of claims 20 to 29, wherein the microRNA binding site sequence is as shown in SEQ ID NO:22. The mRNA molecule according to any one of claims 20 to 30, further comprising a 5' end cap structure. The mRNA molecule according to any one of claims 20 to 31, wherein the sequence is shown in SEQ ID NO: 24 or SEQ ID NO: 28. 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' non-translated sequence, the RNA replicase sequence, the first 3' non-translated 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. 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. 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. 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, from a Venezuelan equine encephalitis virus. 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 as shown in SEQ ID NO:41; preferably, the coding sequence of the RNA replicase is as shown in SEQ ID NO:42. 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. 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 a tissue-specifically expressed microRNA; 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. 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. The mRNA molecule combination according to any one of claims 33 to 40, wherein the microRNA mature sequence is shown as any one of SEQ ID NO:30 to SEQ ID NO:32. 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. The mRNA molecule combination according to any one of claims 33 to 42, wherein the 5' end and/or the 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. 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. The mRNA molecule combination according to any one of claims 33 to 44, wherein the microRNA binding site sequence is as shown in SEQ ID NO:22. 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 contains a 5' end cap structure. A DNA molecule encoding the mRNA molecule according to any one of claims 20 to 32, or encoding the first mRNA molecule and the second mRNA molecule according to any one of claims 33 to 46, wherein the first mRNA molecule and the second mRNA molecule are on the same DNA molecule. 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. 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. 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. 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. A kit comprising the mRNA molecule according to any one of claims 20 to 32, the mRNA molecule combination according to any one of claims 33 to 46, the DNA molecule according to any one of claims 47 to 48 or the DNA molecule combination according to claim 49, and a liposome delivery system. A pharmaceutical composition 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, and one or more pharmaceutically acceptable excipients; preferably, the excipient is a liposome delivery system. A vaccine preparation 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. 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. A mRNA molecule that encodes non-structural proteins 1, 2, 3 and 4 derived from an alphavirus, wherein the non-structural protein 3 comprises a macrodomain at its N-terminus as shown in the amino acid sequence of SEQ ID NO:57 or 59. The mRNA molecule according to claim 56 comprises a nucleotide sequence encoding the macro domain as shown in SEQ ID NO:68 or 70. The mRNA molecule according to any one of claims 56 to 57, wherein the non-structural proteins 1, 2, 3 and 4 are derived from Venezuelan equine encephalomyelitis virus, Optionally, the mRNA molecule encodes non-structural protein 1 as shown in SEQ ID NO:71, non-structural protein 2 as shown in SEQ ID NO:72, non-structural protein 3 as shown in SEQ ID NO:73 or 74, and non-structural protein 4 as shown in SEQ ID NO:75. The mRNA molecule according to any one of claims 56 to 58, which comprises in the 5' to 3' direction an operably linked 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, Optionally, the mRNA molecule contains or consists of the nucleotide sequence of SEQ ID NO:67 or 69. The mRNA molecule according to any one of claims 56 to 59, further comprising a target gene coding sequence and, optionally, an RNA promoter upstream of the target gene coding sequence. 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. The mRNA molecule according to claim 61, wherein the mRNA molecule comprises the following operably linked nucleotide sequences in the 5' to 3' direction: a 5' untranslated region, a nucleotide sequence encoding nonstructural proteins 1, 2, 3 and 4, an RNA promoter, a target gene coding sequence, a 3' untranslated region and a polyadenylation sequence, Optionally, the nucleotide sequences are connected via a linker sequence. The mRNA molecule according to claim 61 or 62, wherein The 5' untranslated region is derived from an alpha virus; preferably, derived from Venezuelan equine encephalomyelitis virus; For example, the 5’ non-translated region contains a sequence as shown in SEQ ID NO:63. The mRNA molecule according to any one of claims 61 to 63, wherein Wherein, the 3' untranslated region is derived from an alpha virus; preferably, it is derived from Venezuelan equine encephalomyelitis virus; For example, the 3’ non-translated region contains a sequence as shown in SEQ ID NO:64. 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 a sequence as shown in SEQ ID NO:65. The mRNA molecule according to any one of claims 61 to 65, wherein the poly(A) sequence comprises a restriction enzyme cleavage site, for example, the poly(A) 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. 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. The mRNA molecule according to any one of claims 60 to 67, whose sequence is as shown in SEQ ID NO: 58 and Any sequence from 60-62 is shown. A DNA molecule encoding the mRNA molecule of any one of claims 56 to 68. A recombinant vector comprising the DNA molecule of claim 69; preferably, the recombinant vector is a prokaryotic expression vector or a eukaryotic expression vector. A recombinant host cell comprising the mRNA molecule according to any one of claims 56 to 68, the DNA molecule according to claim 69, or the recombinant vector according to claim 70. A pharmaceutical composition comprising the mRNA molecule described in any one of claims 56 to 68 or the DNA molecule described in claim 69, and one or more pharmaceutically acceptable carriers; optionally, the carrier is a liposome delivery system or a nanoparticle composition. A vaccine preparation comprising the mRNA molecule according to any one of claims 56 to 68 or the DNA molecule according to 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. Use of the mRNA molecule according to any one of claims 56 to 68 or the DNA molecule according to 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. A kit comprising the mRNA molecule according to any one of claims 56 to 68 or the DNA molecule according to claim 69.