WO2024008192A2 - 经修饰的自复制mRNA - Google Patents

经修饰的自复制mRNA Download PDF

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WO2024008192A2
WO2024008192A2 PCT/CN2023/106610 CN2023106610W WO2024008192A2 WO 2024008192 A2 WO2024008192 A2 WO 2024008192A2 CN 2023106610 W CN2023106610 W CN 2023106610W WO 2024008192 A2 WO2024008192 A2 WO 2024008192A2
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mrna
replicating
modified
rbd
self
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PCT/CN2023/106610
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French (fr)
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WO2024008192A3 (zh
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王子豪
任晓光
唐喆伟
陈燕妮
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嘉晨西海(杭州)生物技术有限公司
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Publication of WO2024008192A2 publication Critical patent/WO2024008192A2/zh
Publication of WO2024008192A3 publication Critical patent/WO2024008192A3/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus

Definitions

  • the present invention belongs to the field of nucleotide mRNA, and specifically relates to a modified self-replicating mRNA.
  • mRNA is a rapid response vaccine development platform to respond to outbreaks.
  • modified nucleotide mRNA messenger ribonucleic acid
  • mRNA-1273 and Tozinameran were respectively launched for sale and used for vaccination to prevent COVID19, confirming the safety and effectiveness of the mRNA vaccine.
  • the mRNA vaccine is synthesized by using linearized plasmid DNA as a template and performing an enzyme transcription reaction in vitro. This synthesis strategy avoids the problems of live cell culture production, safety and complex production processes that need to be considered.
  • the mRNA vaccine platform is safe and effective, has a short production cycle, and has a simple process, making it particularly suitable for responding to outbreaks.
  • Self-replicating mRNA vaccines have strong immunogenicity in animal experiments and can use themselves as templates to copy their own sequences. Therefore, they require fewer doses than conventional mRNA vaccines and are And the adjuvant effect formed by the immune response induced during self-replication can induce stronger immune responses and further enhance humoral and cellular immune responses.
  • the first object of the present invention is to provide a modified self-replicating mRNA, the polynucleotide fragment of which contains one or more non-structural replicase domains from alphaviruses and an integrated target fragment, and the polynucleotide fragment contains a functional Nucleotide analogues;
  • the functional nucleotide analog includes: at least one of pseudouridine, N1-methylpseudouridine, 5-hydroxymethoxycytidine and N6 -methyladenosine.
  • the second object of the present invention is to provide a vaccine composition comprising modified self-replicating mRNA as described above.
  • a third object of the present invention is to provide a kit comprising a vaccine composition as described above, and optionally a container for administering said vaccine composition.
  • the method includes the step of replacing the corresponding unmodified form of the nucleoside in the mRNA with a functional nucleotide analog.
  • modified self-replicating mRNA has a better expression effect than self-replicating mRNA without nucleotide modifications, and has been found to be able to evade immune surveillance in cell experiments and induce weaker IFN-B1 and /or RIG-1 is transcribed, thereby maintaining long-term expression and inducing immune responses.
  • Figure 1 is a structural diagram of an mRNA-RBD plasmid according to an embodiment of the present invention, which contains a kanamycin resistance gene (Kan), a T7 promoter element, an nsp gene, an RBD gene and a polyA element.
  • Kan kanamycin resistance gene
  • Figure 2 is an electrophoresis result diagram of self-replicating mRNA-RBD (labeled SAM-CD5-RBD in the figure) after in vitro transcription according to an embodiment of the present invention; wherein ⁇ -UTP/1m ⁇ -UTP/5m-CTP respectively represent pseudouridine /N1-methylpseudouridine/5-hydroxymethoxycytidine, 0%, 10%, 20%, ..., 90%, 100% represents pseudouridine during in vitro transcription of self-replicating mRNA-RBD modified nucleotides /N1-methylpseudouridine/5-hydroxymethoxycytidine modification ratio, M represents RNA molecular weight standard (RNA ladder).
  • RNA ladder RNA molecular weight standard
  • Figure 3 is a comparison of modified nucleotides and conventional nucleotide self-replicating mRNA-RBD (labeled SAM-CD5-RBD in the figure) after transfection of BHK cells in vitro through western blot detection according to the embodiment, wherein A-C represent the expression results of RBD in cell lysates of self-replicating mRNA-RBD with different ratios of modified nucleotide pseudouridine/N1-methylpseudouridine/5-hydroxymethoxycytidine. D-F represent different self-replicating mRNA-RBDs. RBD expression results in the cell supernatant of the ratio-modified nucleotide pseudouridine/N1-methylpseudouridine/5-hydroxymethoxycytidine.
  • Figure 4 is a comparison of the RBD protein expression diagram of modified nucleotides and conventional nucleotide self-replicating mRNA-RBD (labeled as SAM-CD5-RBD) after transfection of BHK cells in vitro through western blot detection according to the embodiment.
  • the protein expression of RBD was quantitatively analyzed, using Actin as the internal control.
  • the AC diagram the upper part of the diagram represents the self-replicating mRNA-RBD modified nucleotide pseudouridine/N1-methylpseudouridine/5-hydroxymethoxy in different proportions.
  • RBD expression results in cell lysates based on cytidine, The lower half of the graph corresponds to the quantitative analysis of RBD expression.
  • Figure 5 is a qPCR detection according to the embodiment, comparing the natural immune regulator IFN- B1 gene expression map.
  • Figure 6 is a graph comparing IFN-B1 gene expression in mouse spleen cells using modified nucleotides and conventional nucleotide self-replicating mRNA-RBD (labeled SAM-CD5-RBD in the figure) detected by qPCR according to the embodiment.
  • Figure 7 is an animal experiment design plan according to the embodiment.
  • BALB/c female mice aged 6 to 8 weeks are divided into groups after adaptive feeding for 3 days. There are 6 mice in each group. One group is the control group and injected with preparation buffer; the remaining ten groups Conventional nucleotide self-replicating mRNA-RBD-LNP (marked as SAM-CD5-RBD (0% ⁇ -UTP) in the figure) and 10%, 20%, and 30% pseudouridine-modified self-replicating mRNA-RBD were injected respectively.
  • SAM-CD5-RBD 0% ⁇ -UTP
  • SAM-CD5-RBD 10%, 20%, 30% ⁇ -UTP
  • SAM-CD5-RBD 10%, 20%, 30% proportion of N1-methylpseudouridine-modified self-replicating mRNA-RBD- LNP
  • SAM-CD5-RBD 10%, 20%, 30% 1m ⁇ -UTP
  • 100% proportion of 5-methylcytidine-modified self-replicating mRNA-RBD-LNP marked in the figure is SAM-CD5 -RBD (100% 5m-CTP)
  • pseudouridine-modified non-self-replicating mRNA-RBD-LNP labeleled nSAM-CD5-RBD (100% ⁇ -UTP)
  • N1- Methyl pseudouridine-modified non-self-replicating mRNA-RBD-LNP marked as nSAM-CD5-RBD (100% 1m ⁇ -UTP) in the figure).
  • Each mouse was injected with 3ug of mRNA-RBD-LNP into the spine on day 0.
  • the spleens of three mice in each group were collected on day 2 to detect IFN-B1 expression.
  • the remaining three mice in each group were injected on day 7.
  • each mouse was injected with a second injection of 3ug mRNA-RBD-LNP into the spinal column, and then the mouse serum was collected on the 21st and 28th days respectively to detect the antibody titer against RBD.
  • Figure 8 shows the serum antibody titer detection in animal experiments according to the embodiment. Antibodies specific to RBD in the serum are detected by ELISA. The ordinate shows the logarithmic value of the antibody titer, and the abscissa shows the corresponding sample.
  • Figure 9 shows the effect of m 6 A modification on SAM-RNA expression in Examples.
  • Figure 10 is the WB detection result of RBD expression in the Example.
  • Figure 11 is an example of evaluating the impact of m 6 A modification on IFN-B1 expression gene expression; it can be seen from the figure that m 6 A modification can effectively reduce IFN-B1 expression.
  • the technical solution of "A, and/or, B, and/or, C, and/or, D” includes any one of A, B, C, and D (that is, they are all connected with "logical OR” technical solution), also includes any and all combinations of A, B, C, and D, that is, including combinations of any two or any three of A, B, C, and D, and also includes A, B, C , four combinations of D (that is, technical solutions that are all connected by "logical AND").
  • the present invention refers to concentration values, and their meaning includes fluctuations within a certain range. For example, it can fluctuate within the corresponding accuracy range. For example, 2% can allow fluctuation within the range of ⁇ 0.1%. For values that are large or do not require too fine control, the meaning is also allowed to include larger fluctuations. For example, 100mM can allow fluctuations within the range of ⁇ 1%, ⁇ 2%, ⁇ 5%, etc. Referring to molecular weight, fluctuations of ⁇ 10% are allowed.
  • the technical features described in open terms include closed technical solutions composed of the listed features, and also include open technical solutions including the listed features.
  • the term "functional nucleotide analog” refers to a modified version of the classic nucleotide A, G, C, U or T, said version (a) retaining the bases of the corresponding classic nucleotide pairing properties, and (b) contains at least one pair of (i) nucleobase, (ii) sugar group, (iii) phosphate group or (iv) any of (i) to (iii) of the corresponding natural nucleotide Combinatorial chemical modifications.
  • base pairing encompasses not only the classic Watson-Crick adenine-thymine, adenine-uracil, or guanine-cytosine base pairs, but also the classic nucleoside A base pair formed between an acid and a functional nucleotide analog, or between a pair of functional nucleotide analogs, in which the arrangement of the hydrogen bond donor and the hydrogen bond acceptor allows for the formation of a base pair between the modified nucleobase and between classical nucleobases or between two complementary modified nucleobases Hydrogen bonds are formed between base structures.
  • functional analogs of guanosine (G) retain the ability to base pair with cytosine (C) or functional analogs of cytosine.
  • nucleic acid molecules containing functional nucleotide analogs can have at least one modified nucleobase, sugar group, and/or internucleoside linkage.
  • nucleobase, sugar, or internucleoside linkages of nucleic acid molecules are exemplary chemical modifications of nucleobase, sugar, or internucleoside linkages of nucleic acid molecules.
  • Examples of functional nucleotide analogs include 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, dihydrouridine, 2'-O-methylpseudouridine, ⁇ ,D-galactose Q Nucleosides, 2'-O-methylguanosine, inosine, N 6 -prenyladenosine, 1-methyladenosine, 1-methylpseudouridine, 1-methylinosine, 2' 2-Dimethyladenosine, 2-methyladenosine, 2-methylguanosine, 5-methyluridine, 3-methylcytidine, 5-methylcytidine, N 6 -methyladenosine , 7-methylguanosine, 5-methylaminomethyluridine, 5-carboxymethylaminomethyluridine, 5-carboxymethylaminomethyl-2-thiouridine, ⁇ ,D-mannan Sugar Q nucleoside, 5-methoxycarbonylmethyl-2-thiouridine, 5-methoxy
  • the present invention relates to mRNA comprising functional nucleotide analogs.
  • the mRNA comprises self-replicating mRNA.
  • the mRNA comprises one or more polynucleotide fragments from the non-structural replicase domain of an alphavirus.
  • the mRNA comprises an integrated target polynucleotide fragment.
  • the present invention relates to modified self-replicating mRNAs, polynucleotide fragments thereof comprising one or more non-structural replicase domains from alphaviruses and an integrated target fragment, said polynucleotide fragments comprising functional nucleotide analogs .
  • the content of any one type of functional nucleotide analogs in the mRNA/self-replicating mRNA is 0.01% to 100%, and 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30% , 35%, 40%, 50%, 60%, 70%, 80%, 90%.
  • the total amount of all types of functional nucleotide analogs in the mRNA/self-replicating mRNA is 0.01% to 100%, and 0.1%, 0.5%, and 1% can also be selected. ,2%,3%,4%,5%,6%,7%,8%,9%,10%,11%,12%,13%,14%,15%,20%,25%,30 %, 35%, 40%, 50%, 60%, 70%, 80%, 90%.
  • the functional nucleotide analog can be located at any position of the mRNA, for example, it can be located in the non-structural replicase domain of the alpha virus, or in the integrated target polynucleotide fragment, or both at the same time . In some embodiments, 0% to 100% of the functional nucleotide analogs are located in the non-structural replicase domain of the alphavirus, for example, at least 0.01%, 0.1%, 0.5%, 1%, 2 %, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%.
  • 0% to 100% of the functional nucleotide analogs are located in the target fragment, such as at least 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4% , 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%.
  • the "unmodified form" in the present invention refers to the nucleotide before modification of the functional nucleotide analogue, and they generally have the same base.
  • the unmodified form of pseudouridine and N1-methylpseudouridine can be uridine
  • the unmodified form of 5-hydroxymethoxycytidine can be cytidine.
  • the functional nucleotide analog includes: at least one of pseudouridine, N1-methylpseudouridine, 5-hydroxymethoxycytidine, and N6 -methyladenosine .
  • the functional nucleotide analog includes: at least one of pseudouridine, N1-methylpseudouridine, and 5-hydroxymethoxycytidine.
  • the uridine in the unmodified form of the polynucleotide fragment is 1% to 100%, or 1% to 50%, or 10% to 50%, or 20% to 40%, or 25% % ⁇ 35% modified to pseudouridine; modification ratio can also be selected from 5%, 10%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90 %.
  • the uridine in the unmodified form of the polynucleotide fragment is modified at a ratio of 1% to 100%, or 1% to 60%, or 10% to 60%, or 10% to 20%.
  • N1-methylpseudouridine; the modification ratio can also be selected from 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%.
  • the cytidine in the unmodified form of the polynucleotide fragment is modified into 5-hydroxymethoxycytidine at a ratio of 1% to 100%, or 80% to 100%, or 90% to 100%.
  • Glycoside the modification ratio can also be selected from 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%.
  • the adenosine in the unmodified form of the polynucleotide fragment is modified at a ratio of 1% to 100%, or 1% to 30%, or 1% to 10%, or 1% to 5% is N 6 -methyladenosine (m 6 A); the modification ratio can also be selected from 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12 %, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%.
  • the target fragment includes at least one mRNA encoding an antigen or a fragment or epitope thereof; preferably, the antigen is a pathogenic antigen, and more preferably, the antigen is a viral antigen, a bacterial antigen, or a parasite antigen. , fungal antigens, protozoal antigens, prion antigens or tumor antigens.
  • the viruses include: adenoviridae, arenaviridae, astroviridae, bunyaviridae, caliciviridae, flavivirus Flaviviridae, hepeviridae, mononegavirales, nidovirales, picornaviridae, orthocoronavirinae, orthomyxovirus Orthomyxoviridae, Papillomaviridae, Parvoviridae One or more of (parvoviridae), polyomaviridae, poxviridae, reoviridae, retroviridae, and togaviridae kind.
  • the bacteria include: Staphylococcus, Streptococcus, Listeria, Erysipelothrix, Nephrobacter, Bacillus, Clostridium, Mycobacterium, Actinomyces, Nocardia One or more of the genus Corynebacterium, Rhodococcus, and/or, Bacillus anthracis, Bacillus erysipelas, Bacillus tetanus, Listeria, Bacillus emphysema, Mycobacterium tuberculosis, Escherichia coli, Proteus, Shigella dysenteriae, pneumoniae, Brucella, Clostridium perfringens, Haemophilus influenzae, Haemophilus parainfluenzae, Moraxella catarrhalis, Acinetobacter, Yersinia, Legionella pneumophila, One or more of Bordetella pertussis, Bordetella parapertussis, Shigella, Past
  • the fungi include: Coccidioides immitis, Coccidioides coccidioides, Histoplasma capsulatum, Histoplasma donovani, Blastomyces loboi, Paracoccidioides brasiliensis, Blastomyces dermatitidis , Sporothrix schenckii, Penicillium marneffei, Candida albicans, Candida glabrata, Candida tropicalis, Candida portugueseus, Aspergillus, Exophyllum zhenii, Chromomyces pereii, Chromomyces compactum, warts Staphylococcus aureus, Staphylococcus aureus, Geotrichum candidum, Podosporium bodii, Cryptococcus neoformans, Rhizopus yeasts, Rhizopus oryzae, Mucor indica, Acanthus coccidioides, Acanthus racemosa, Frugophyllum faecalis, One or more of
  • the parasites include: parasites in the digestive tract, parasites in the liver, parasites in the lungs, parasites in brain tissue, parasites in blood vessels, parasites in lymphatic vessels, parasites in muscle tissue, and intracellular parasites.
  • parasites in the digestive tract include: parasites in the liver, parasites in the lungs, parasites in brain tissue, parasites in blood vessels, parasites in lymphatic vessels, parasites in muscle tissue, and intracellular parasites.
  • intracellular parasites include: parasites in the digestive tract, parasites in the liver, parasites in the lungs, parasites in brain tissue, parasites in blood vessels, parasites in lymphatic vessels, parasites in muscle tissue, and intracellular parasites.
  • bone tissue parasites and intraocular parasites include: bone tissue parasites in the digestive tract, parasites in the liver, parasites in the lungs, parasites in brain tissue, parasites in blood vessels, parasites in
  • the tumor includes: bone, bone connection, muscle, lung, trachea, heart, spleen, artery, vein, blood, capillary, lymph node, lymphatic vessel, lymph, oral cavity, pharynx, esophagus, stomach, ten Duodenum, small intestine, colon, rectum, anus, appendix, liver, gallbladder, pancreas, parotid gland, sublingual gland, urinary kidney, ureter, bladder, urethra, ovary, fallopian tube, uterus, vagina, vulva, scrotum, testis, Vas deferens, penis, eyes, ears, nose, tongue, skin, brain, brain stem, medulla oblongata, spinal cord, cerebrospinal fluid, nerves, thyroid, parathyroid gland, adrenal gland, pituitary gland, pineal gland, pancreatic islets, chest Tumors arising from lesions in any of the glands, gonads,
  • the target fragment is mRNA derived from SARS-CoV-2, preferably spike protein mRNA or a fragment thereof, more preferably it includes the RBD gene or a fragment thereof.
  • the alphavirus is selected from: Venezuelan Equine Encephalitis Virus (TC83Venezuelan Equine Encephalitis Virus, VEEV), Sin-dbis virus, Chikungunya virus, Eastern Equine Encephalomyelitis Eastern equine encephali-tis virus, Western equineencephalitis virus, Mayarovirus, Semliki forest virus and Venezuelan equine encephalomyelitis virus encephalitisvirus).
  • the virus in the alphavirus family is Venezuelan equine encephalomyelitis virus (TC83Venezuelan Equine Encephalitis Virus, VEEV).
  • TC83Venezuelan Equine Encephalitis Virus VEEV
  • the viruses of the alphavirus family listed herein are only illustrative and not limited thereto.
  • nucleotide sequence of the unmodified form of the modified self-replicating mRNA is as shown in SEQ ID NO: 3.
  • the invention also relates to a vaccine composition comprising modified self-replicating mRNA as described above.
  • the vaccine composition further includes at least one of a pharmaceutically acceptable carrier, diluent and excipient.
  • pharmaceutically acceptable means that the molecule itself, molecule fragments or compositions do not produce adverse, allergic or other adverse reactions when properly administered to animals or humans.
  • pharmaceutically acceptable carriers or components thereof include phosphoric acid, citric acid, and other organic acids; antioxidants (for example, ascorbic acid and methionine); antibacterial agents (for example, octadecane Dimethyl benzene chloride, hexahydrocarbon quaternary ammonium chloride, benzalkonium chloride, phenol, butanol or benzyl alcohol, alkylparaben, catechol, resorcinol, cyclohexanol, 3- amyl alcohol, or m-cresol); low molecular weight (less than about 10 kDa) polypeptides; proteins, e.g., serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino
  • the vaccine composition may be in solid, semi-solid or liquid form, preferably in liquid form.
  • the vaccine composition further includes a nucleic acid stabilizer.
  • stabilizing agents used to stabilize and retain nucleic acids include cationic compounds, detergents, chaotropic salts, ribonuclease inhibitors, chelating agents, and the like, and mixtures thereof.
  • Stabilizers may include, for example, cross-linking fixatives such as paraformaldehyde or precipitating agents such as ethanol. Stabilizers may act by forming covalent bonds between cellular molecules or by precipitating some intracellular molecules or by other methods.
  • the stabilizing agent includes cell lysis buffer.
  • Cell permeabilization buffers are also known in the art and may contain detergents that permeabilize the cell membrane allowing probes and dyes to pass through the membrane.
  • detergents used in cell lysis buffers include, but are not limited to, TweeruTriton X-100, saponins, NP-40, etc. Adjust the concentration of cell lysis and permeabilization agents for the given end use. When present at too low a concentration, cell lysis and permeabilization may not be optimal. At too high a concentration, undesirable cell destruction may occur. Routine empirical steps can be taken to determine the preferred route in each case.
  • stabilizers include chloroform, phenol, TRIZOL. However, in a more preferred embodiment, the stabilizer is an ingredient that is easily removable or less toxic to cells, most preferably a pharmaceutically acceptable ingredient.
  • the vaccine provided by the present invention preferably further includes an adjuvant.
  • adjuvants suitable for use in the vaccine of the present invention include those that enhance the immunogenicity against self-replicating mRNA, particularly target fragments thereof. For example, adjuvants that respond to antibody responses to B cell epitopes of an antigen, and adjuvants that enhance cell-mediated responses to T cell epitopes of the antigen, etc. These adjuvants are well known in the art.
  • the adjuvant is selected from alum, complete Freund's adjuvant, incomplete Freund's adjuvant, squalene, squalane, muramyl dipeptide, MF59, AS03, monophosphatidyl lipid A, Flagellum One or more of protein, CpG-ODN, Poly(I:C), and small molecules of aluminum or calcium salts. These adjuvants are well known in the art and are available from several commercial sources.
  • complete Freund's adjuvant complete Freund's adjuvant
  • incomplete Freund's adjuvant incomplete Freund's adjuvant
  • squalane squalane
  • alum alum
  • the vaccine is a water-in-oil emulsion having an aqueous phase and an oil phase.
  • the vaccine is an oil-in-water emulsion having an aqueous phase and an oil phase.
  • Vaccines are typically formulated for parenteral administration. Typical immunization is achieved by oral and subcutaneous (SC), nasal route, intramuscular (IM), intravenous (IV), intraperitoneal (IP) or intradermal (ID) injection.
  • SC subcutaneous
  • IM intramuscular
  • IV intravenous
  • IP intraperitoneal
  • ID intradermal
  • the vaccine is administered in a manner compatible with the dosage formulation and in an amount such as a therapeutically effective amount and an immunogenically effective amount.
  • the amount administered depends on the subject being treated, the ability of the subject's immune system to synthesize antibodies, and the degree of protection expected.
  • the exact amount of active ingredient to be administered depends on the physician's judgment and will vary from individual to individual.
  • the appropriate schedule for initial administration and booster vaccination may vary, but typically a further injection or other administration will be given at some interval (weeks or months) after the initial administration.
  • the vaccine composition is packaged and delivered in the form of plasmids, viral vectors, liposomes, dendrimers, inorganic nanoparticles or cell-penetrating peptides.
  • the mRNA molecule can be packaged directly, or in the form of its precursor, and the plasmids and viral vectors can contain a selectable marker (e.g., a tag that facilitates enrichment, such as his tag; or a tag that facilitates detection, such as GFP), and An origin of replication matches the cell type specified by the cloning vector, and the expression vector contains the regulatory elements necessary to affect expression in the specified target cell.
  • the viral vector may be a bacteriophage, lentivirus, retrovirus, adenovirus or adeno-associated virus.
  • Liposomes can be cationic liposomes or neutral liposomes, which can be prepared or modified by known methods. For example, adding polyethylene glycol (PEG)-modified liposomes can effectively prevent the aggregation of liposome carriers. and increase its stability.
  • PEG polyethylene glycol
  • Dendrimers are molecules with a well-defined, precisely controllable chemical structure and Special polymer families with unique multivalent properties have gradually become non-viral vectors for gene delivery.
  • Typical dendrimers include poly(amidoamine) (PAMAM) dendrimers, which can be further modified, such as modifying the nucleobase analogue 2-amino-6-chloropurine on the surface of PAMAM to construct the derivative AP- PAMAM, or CS-PAMAM prepared by coupling chondroitin sulfate (CS) with PAMAM, etc.
  • PAMAM poly(amidoamine)
  • CS-PAMAM prepared by coupling chondroitin sulfate
  • Inorganic nanoparticles can include gold nanoparticles (AuNPs), magnetic nanoparticles, mesoporous silica nanoparticles (MSNs), etc.
  • AuNPs gold nanoparticles
  • MSNs mesoporous silica nanoparticles
  • CPPs Cell-penetrating peptides
  • CPPs such as TAT, Penetratin, Polyarginine, P22N, DPV3 and DPV6, etc.
  • amphipathic CPPs which can be covalently linked to hydrophobic peptide sequences and NLSs, or isolated from natural proteins, such as pVEC, ARF(1-22) and BPrPr(1-28)
  • hydrophobic CPPs generally containing only non-polar amino acid residues, the net charge is about less than 20% of the total charge of the amino acid sequence).
  • Another embodiment of the invention relates to a kit comprising a vaccine as described above, and a container for administering said vaccine composition.
  • the inoculation container is preferably a medical syringe.
  • the present invention also relates to methods for changing the following properties of mRNA after inoculation into animals:
  • the method includes the step of replacing the corresponding unmodified form of the nucleoside in the mRNA with a functional nucleotide analog.
  • the mRNA is a self-replicating mRNA, and preferably the modified self-replicating mRNA as described above is obtained after replacement.
  • the animal is a chicken, duck, goose, cat, dog, cow, sheep, horse, donkey, pig, giant panda, monkey, rabbit, rat, or human.
  • the measurement parameters of raw material components are involved. Unless otherwise specified, there may be slight deviations within the range of weighing accuracy. Temperature and time parameters are involved, allowing for acceptable deviations due to instrument testing accuracy or operating accuracy.
  • SARS-CoV-2 spike protein
  • RBD receptor binding domain
  • Self-replicating mRNA was designed based on the genome of Venezuelan equine encephalomyelitis virus (TC83, VEEV) in the alphavirus family. It contains genes encoding the self-replicating components of alphaviruses, but lacks the coding for making infectious alphaviruses.
  • the structural protein of the particle is directly obtained through synthesis of the designed sequence.
  • the nucleotide sequence is shown in SEQ ID NO: 2.
  • the preparation method of recombinant plasmid JCXH-107 is as follows (the prepared recombinant plasmid JCXH-107 is shown in Figure 1)
  • pUC57-RBD and TC83 self-replicating vectors were digested with ApaI and NotI, and the enzyme digestion system was 20 ⁇ L: pUC57-RBD or TC83 self-replicating vector ⁇ 1 ⁇ g, ApaI 1 ⁇ L, NotI 1 ⁇ L, 10 ⁇ CutSmart buffer 2 ⁇ L, ddH 2 O.
  • Digest the system Make up 20 ⁇ L. Digest the plasmid in a water bath at 25°C for 1 hour, then digest it in a water bath at 37°C for 1 hour. The vector fragment needs to be added with 0.5 ⁇ L CIP, and dephosphorylated in a water bath at 37°C for 30 minutes.
  • the enzyme digestion mixture was mixed with 6 ⁇ loading buffer, electrophoresed (1% agarose, 94V) and gelled to recover fragments of corresponding lengths (pUC57-RBD recovery fragment RBD gene fragment, length 2574bp; TC83 self-replicating vector recovery fragment, length Approximately 9.5Kb), elute with 30 ⁇ L elution buffer.
  • PCR screening Use the bacterial plasmid extracted by the boiling method as a template to amplify the spike protein of the target band.
  • the upstream primer F 5'-TATGGCCATGACTACTCTAGCTA-3'
  • the downstream primer R 5'-GGGAAAC GCCTGGTATCTTT-3'
  • the reaction cycle conditions are: : 94°C 3min ⁇ (94°C 1min, 47°C 30s, 72°C 3min) ⁇ 30 cycles ⁇ 72°C 10min ⁇ 4°C; observe the PCR results by electrophoresis (electrophoresis conditions: 1% agarose gel; 90V, loading volume : 5 ⁇ l PCR product), expected result: RBD fragment approximately 2977bp.
  • Enzyme digestion verification Extract plasmids from positive bacteria verified by PCR screening, perform enzyme digestion with ApaI and NotI according to the above enzyme digestion system, mix the enzyme digestion mixture with 6 ⁇ loading buffer and perform electrophoresis (1% agarose, 94V).
  • Sequencing verification Extract the plasmids that meet the expectations by PCR and enzyme digestion and send them to a sequencing company for sequencing.
  • the E. coli carrying the positive plasmid that was completely correct after sequencing verification was stored at -80°C.
  • the linearized JCXH-107 plasmid uses an in vitro transcription reaction (IVT) initiated by T7 RNA polymerase, Turbo DNase enzyme to degrade the template DNA, and capping enzyme (Vaccinia capping enzyme) to cap the 7-methylated guanylate structure. (7-methylguanylate cap structure, called Cap 0) is added to the 5' end of the transcribed mRNA.
  • IVTT in vitro transcription reaction
  • Turbo DNase enzyme to degrade the template DNA
  • capping enzyme Vaccinia capping enzyme
  • 10 ⁇ reaction buffer 2 ⁇ L, NTP each 0.5mM, uridine (U) can be according to the proportion of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% Replaced with pseudouridine, or uridine (U) can be replaced with N1-methyl in the ratio of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% Pseudouridine, or cytidine (C) can be replaced with 5-hydroxymethoxy in the proportions 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% Cytidine; Linearized JCXH-107 1 ⁇ g, T7RNA Polymerase 2 ⁇ L, add water to 20 ⁇ L, react at 30°C for 1 hour, add 1 ⁇ L TURBO TM DNase, 4 ⁇ L 10 ⁇ capping buffer, 2 ⁇ L GTP (10mM), 2 ⁇ L SAM (2mM), Add 2 ⁇ L of capping enzyme, add water to the total volume to 40 ⁇ L, and react at 30°
  • Cell culture and plating Inoculate the recovered BHK cells into a 75cm2 culture bottle.
  • the medium is DMEM high sugar medium + 5% double antibody + 10% fetal calf serum. Wait until the density of the cells at the bottom of the bottle reaches When reaching more than 80%, the cells were digested with trypsin and counted. Plate an appropriate number of cells in a 6-well cell culture plate and incubate overnight in a 37°C CO2 incubator.
  • Electrophoresis The polyacrylamide separation gel concentration is 6%, the protein loading volume is 20 ⁇ L, the stacking gel electrophoresis conditions are 150V, 10min, and the separation gel electrophoresis conditions are 200V, 30min.
  • Electroporation Use nitrocellulose membrane and transfer the membrane at a voltage of 100V for 1 hour.
  • Blocking Use 1 ⁇ PBST to prepare 5% BSA as blocking solution, and block overnight at 4°C.
  • Primary antibody incubation Use blocking solution to dilute the SARA-Cov-2 (2019-nCov) spike protein antibody at 1:2000 and incubate at room temperature for 1 hour. Wash 3 times with 1 ⁇ PBST for 10 minutes each time.
  • Secondary antibody incubation Dilute the secondary antibody with blocking solution to a dilution ratio of 1:5000 and incubate at room temperature for 1 hour. Wash 3 times with 1 ⁇ PBST for 10 minutes each time.
  • Figure 3-1 and Figure 3-2 compare the self-replicating mRNA-RBD of modified nucleotides and conventional nucleotides (labeled SAM-CD5-RBD in the figure) in vitro.
  • RBD protein expression chart after transfection of BHK cells including detection of RBD protein expression in cell lysate and cell supernatant, in which the cell lysate uses Actin protein as an internal reference, and the cell supernatant uses protein Coomassie staining as an internal reference;
  • Figure 4 This is a quantitative statistical diagram of RBD protein in cell lysate in Figure 3-1 and Figure 3-2. From Figure 3-1, Figure 3-2, and Figure 4, it can be seen that self-replicating mRNA-RBD transfected BHK cells in vitro The expression of RBD protein is better in the rear.
  • the present invention uses real-time fluorescence quantitative PCR to detect and compare the use of modified nucleotides and conventional nucleotides to self-replicate mRNA-RBD (marked as SAM-CD5-RBD in the figure) in vitro transfection of the mRNA-RBD obtained by the in vitro transcription reaction. After HeLa cells, the transcription level of the natural immune regulator IFN-B1 was detected.
  • the specific method is as follows:
  • Cell RNA extraction Aspirate off the cell culture medium, wash the cells once with PBS, add 1mL RNAiso, pipette the cells, and collect in an EP tube. Add 0.2 mL of chloroform, shake and mix, let stand for 5 minutes, centrifuge at 12000g for 15 minutes, and absorb the supernatant. Add the same volume of isopropyl alcohol as the supernatant, shake to mix, and let stand at room temperature for 10 minutes. Centrifuge at 12000g for 10 minutes and discard the supernatant. Add 1 mL of Nuclease-Free water containing 75% ethanol and wash the pellet. Centrifuge at 12000g for 5 minutes and discard the supernatant. Dry the precipitate, add 20 ⁇ L of Nuclease-Free water, and pipet gently to dissolve the precipitate. Use UV spectrophotometer to measure OD260/OD280 value and RNA concentration.
  • the upstream primer sequence used to detect the expression of the internal reference GAPDH gene is GAAGGCTGGG GCTCATTT, and the downstream primer sequence is CAGGAGGCATTGCTGATGAT.
  • the upstream primer sequence used to detect RI GI gene expression is GTTGTCCCCATGCTGTTCTT, and the downstream primer sequence is GCAAGTCTTACATGGCAGCA. Detection was performed using the Thermo Fisher Quant Studio 1 real-time fluorescence quantitative PCR system.
  • the detection program is: Stage 1: 95°C, 3min, repeated once; Stage 2: 95°C, 10s, 60°C, 30s, repeated 40 times; Stage 3 dissolution curve: 95°C, 15s; 60°C, 60s; 95°C , 15s, repeat once.
  • mRNA and lipids including 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), cholesterol, distearoylphosphatidylcholine (DSPC) ) and cationic lipids, dissolved in alcohol
  • DMG-PEG2000 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000
  • DSPC distearoylphosphatidylcholine
  • cationic lipids dissolved in alcohol
  • BALB/c female mice aged 6 to 8 weeks were adaptively fed for 3 days and then divided into groups, with 6 mice in each group.
  • One group was the control group and injected with preparation buffer; the remaining ten groups were injected with conventional nucleotide self-replicating mRNA-RBD- LNP, 10%, 20%, and 30% pseudouridine-modified self-replicating mRNA-RBD-LNP, 10%, 20%, and 30% proportions of N1-methylpseudouridine-modified self-replicating mRNA-RBD-LNP, 100% proportion of 5-methylcytidine modified self-replicating mRNA-LNP, 100% proportion of pseudouridine-modified non-self-replicating mRNA-RBD-LNP, 100% proportion of N1-methylpseudouridine-modified non-self-replicating mRNA -RBD-LNP.
  • Each mouse was injected with 3ug of mRNA-RBD-LNP into the spine on day 0.
  • the spleens of three mice in each group were taken to detect IFN-B1 expression on day 2.
  • the results are shown in Figure 6; the remaining three mice in each group were After the serum was collected from the mice on the 7th and 14th days, each mouse was injected with a second spinal injection of 3ug of mRNA-RBD-LNP, and then the mouse serum was collected on the 21st and 28th days respectively, and ELISA was used to detect anti-RBD antibody drops.
  • the experimental process and design are shown in Figure 7.
  • the antibody titer detection method is ELISA.
  • the specific operation is as follows. Each well of a 96-well enzyme plate is coated with 50ng of spike protein RBD domain protein at room temperature overnight. The next day, it is washed 3 times with PBST, blocked with 5% milk, and kept at 37°C for 1 After 1 hour, wash with PBST 3 times, add mouse serum corresponding to the dilution ratio, keep at 37°C for 1 hour, wash with PBST 3 times, add anti-mouse IgG heavy chain-light chain HRP, keep at 37°C for 1 hour, wash with PBST 3 times, and finally add color developer to develop color.
  • the experimental results are shown in Figure 8.
  • mice injected with 100% pseudouridine-modified non-self-replicating mRNA-RBD-LNP and 100% N1-methylpseudouridine-modified non-self-replicating mRNA-RBD-LNP middle Have higher RBD-specific antibodies;
  • mice injected with 30% pseudouridine-modified self-replicating mRNA-RBD-LNP have higher RBD-specific antibodies in their serum.
  • the self-replicating mRNA obtained by nucleotide modification using the method of the present invention can be well expressed in cells and can induce better antibody responses in animals.
  • This experiment uses BHK and Hela cell lines to transfect, using Lipofectamine lipofectamine transfection reagent, the transfection dose is 0.5ug, and the samples are collected in 24 hours;
  • WB detects changes in RBD protein expression levels in BHK and Hela cells, and detects changes in IFN-B1 and/or RIG-I gene expression in Hela cells.
  • LNP-hEPO-mRNA animal experiment adopts intravenous injection.
  • LNP-Luciferase- mRNA animal experiments use intramuscular injection;
  • 2.4 hEPO detects protein expression in vivo through WB
  • Luciferase detects protein expression in vivo through fluorescence imaging.
  • RBD protein is basically unaffected or significantly increased under m6A modification at a specific ratio (for example, below 20%, or below 10%, or below 5%).
  • m6A modification at a specific ratio (for example, below 20%, or below 10%, or below 5%).
  • Figures 9 and 10 The results are shown in Figures 9 and 10; where Figure 9, where A and C are protein expressions in cell lysates, and B and D are protein expressions in supernatants. It can be seen that m 6 A modification below 10% does not significantly reduce the expression of RBD.
  • the results of RBD expression detected by WB are shown in Figure 10. It can be seen from the figure that 1% and 5% m 6 A modification does not destroy the expression of RBD.
  • the Luciferase fluorescence signal is enhanced when modified with a specific ratio of m 6 A;
  • m 6 A-modified self-replicating mRNA has basically no effect on protein expression, or may have a promoting effect; m 6 A-modified self-replicating mRNA can effectively reduce inflammatory gene expression.

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Abstract

本发明属于核苷酸mRNA领域，具体地，涉及一种经修饰的自复制mRNA。其多核苷酸片段包含一个或多个来自α病毒的非结构性复制酶域以及整合的目标片段，所述多核苷酸片段包含功能性核苷酸类似物；所述功能性核苷酸类似物包括：假尿苷、N1-甲基假尿苷、5-羟甲氧基胞苷以及N6-甲基腺苷中的至少一种。

Description

经修饰的自复制mRNA
优先权声明
本申请要求于2022年7月8日提交的CN 2022108058522的优先权和利益，并通过参考以全文引入。
技术领域
本发明属于核苷酸mRNA领域，具体地，涉及一种经修饰的自复制mRNA。
背景技术
传统灭活疫苗、重组蛋白疫苗生产周期长，工艺复杂无法应对突发性大规模流行病的大规模接种需要。mRNA则是一种应对爆发性疫情的快速反应疫苗开发平台。2020年Moderna和BioNTech公司开发的修饰核苷酸的mRNA(信使核糖核酸)疫苗针对COVID19大流行的mRNA-1273和Tozinameran分别上市销售，用于接种预防COVID19，证实了mRNA疫苗的安全性和有效性。
mRNA疫苗是利用线性化的质粒DNA为模板，在体外进行酶转录反应合成的，该合成策略避免了活细胞培养生产方式，需要考虑的安全性以及复杂的生产工艺等问题。mRNA疫苗平台具有安全有效、生产周期短、工艺简洁的特点，因而特别适合应对爆发性疫情。
自复制mRNA疫苗在动物实验中有很强的免疫原性，能够以自身为模板复制出自身的序列，因此相比常规mRNA疫苗需要的接种剂量更少，并 且自复制时所诱导的免疫反应形成的佐剂效应，能够诱导出更强的免疫应答，进一步增强体液和细胞免疫反应。
然而，关于经核苷酸修饰的自复制mRNA还需要进一步研究。
发明内容
本发明第一目的在于提供一种经修饰的自复制mRNA，其多核苷酸片段包含一个或多个来自α病毒的非结构性复制酶域以及整合的目标片段，所述多核苷酸片段包含功能性核苷酸类似物；
所述功能性核苷酸类似物包括：假尿苷、N1-甲基假尿苷、5-羟甲氧基胞苷以及N6-甲基腺苷中的至少一种。
本发明的第二目的在于提供一种疫苗组合物，其包含如上所述的经修饰的自复制mRNA。
本发明的第三目的在于提供成套试剂盒，其包含如上所述的疫苗组合物，以及任选地用于接种所述疫苗组合物的容器。
本发明的第四目的在于提供一种方法，其用于改变mRNA在接种于动物体内后的如下特性：
a)提高所述mRNA的免疫原性；和/或
b)提高所述mRNA逃避免疫监视的能力；
所述方法包括用功能性核苷酸类似物替换所述mRNA中相应的非修饰形式核苷的步骤。
经研究发现，经修饰的自复制mRNA相比于未进行核苷酸修饰的自复制mRNA具有更好的表达效果，并且发现其在细胞实验中能逃避免疫监视，诱导较弱的IFN-B1和/或RIG-1转录，进而能够维续长时间表达，可诱导免疫反应。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1是根据本发明的实施例的mRNA-RBD质粒结构图谱，其含有卡那霉素抗性基因(Kan)、T7启动子元件、nsp基因、RBD基因和polyA元件。
图2是根据本发明的实施例的自复制mRNA-RBD(图中标为SAM-CD5-RBD)体外转录后的电泳结果图；其中Ψ-UTP/1mΨ-UTP/5m-CTP分别表示假尿苷/N1-甲基假尿苷/5-羟甲氧基胞苷，0％、10％、20％、…、90％、100％表示自复制mRNA-RBD修饰核苷酸体外转录时假尿苷/N1-甲基假尿苷/5-羟甲氧基胞苷修饰的比例，M表示RNA分子量标准(RNA ladder)。
图3是根据实施例的通过western blot检测，比较修饰核苷酸和常规核苷酸自复制mRNA-RBD(图中标为SAM-CD5-RBD)在体外转染BHK细胞后RBD蛋白表达图，其中A-C表示自复制mRNA-RBD不同比例修饰核苷酸假尿苷/N1-甲基假尿苷/5-羟甲氧基胞苷的细胞裂解液中RBD表达结果，D-F表示自复制mRNA-RBD不同比例修饰核苷酸假尿苷/N1-甲基假尿苷/5-羟甲氧基胞苷的细胞上清中RBD表达结果。
图4是根据实施例的通过western blot检测，比较修饰核苷酸和常规核苷酸自复制mRNA-RBD(图中标为SAM-CD5-RBD)在体外转染BHK细胞后RBD蛋白表达图，对RBD的蛋白表达进行定量分析，以Actin作为内参，其中A-C图，上半部分图表示自复制mRNA-RBD不同比例修饰核苷酸假尿苷/N1-甲基假尿苷/5-羟甲氧基胞苷的细胞裂解液中RBD表达结果， 下半部分图对应RBD表达的定量分析。
图5是根据实施例的通过qPCR检测，比较修饰核苷酸和常规核苷酸自复制mRNA-RBD(图中标为SAM-CD5-RBD)在体外转染Hela细胞后，天然免疫调节因子IFN-B1基因表达图。
图6是根据实施例的通过qPCR检测，比较修饰核苷酸和常规核苷酸自复制mRNA-RBD(图中标为SAM-CD5-RBD)在小鼠脾脏细胞中IFN-B1的基因表达图。
图7是根据实施例的动物实验设计方案，6～8周龄的BALB/c雌鼠适应性喂养3天后分组，每组6小鼠，一组为对照组，注射制剂缓冲液；其余十组分别注射常规核苷酸自复制mRNA-RBD-LNP(图中标为SAM-CD5-RBD(0％ψ-UTP))，10％、20％、30％比例假尿苷修饰的自复制mRNA-RBD-LNP(图中标为SAM-CD5-RBD(10％,20％,30％ψ-UTP)),10％、20％、30％比例N1-甲基假尿苷修饰的自复制mRNA-RBD-LNP(图中标为SAM-CD5-RBD(10％,20％,30％1mψ-UTP)),100％比例5-甲基胞苷修饰的自复制mRNA-RBD-LNP(图中标为SAM-CD5-RBD(100％5m-CTP))，100％比例假尿苷修饰的非自复制mRNA-RBD-LNP(图中标为nSAM-CD5-RBD(100％ψ-UTP))，100％比例N1-甲基假尿苷修饰的非自复制mRNA-RBD-LNP(图中标为nSAM-CD5-RBD(100％1mψ-UTP))，。每只小鼠在第0天脊柱注射3ug mRNA-RBD-LNP，每组的三只小鼠分别在第2天取脾脏检测IFN-B1表达；每组剩余的三只小鼠在第七天，第14天取血清后，每只小鼠脊柱注射第二针3ug mRNA-RBD-LNP，然后分别在第21、28天取小鼠血清，检测针对RBD抗体滴度。
图8是根据实施例的动物实验血清抗体效价检测，通过ELISA检测血清中特异针对RBD的抗体，纵坐标显示是对抗体效价取对数的数值，横坐标表示了对应的样本。
图9是实施例的m6A修饰对SAM-RNA表达的影响。
图10是实施例的WB检检测RBD表达结果。
图11是实施例的评估m6A修饰对IFN-B1表达基因表达的影响；从图中可见m6A修饰能够有效降低IFN-B1表达。
具体实施方式
现将详细地提供本发明实施方式的参考，其一个或多个实例描述于下文。提供每一实例作为解释而非限制本发明。实际上，对本领域技术人员而言，显而易见的是，可以对本发明进行多种修改和变化而不背离本发明的范围或精神。例如，作为一个实施方式的部分而说明或描述的特征可以用于另一实施方式中，来产生更进一步的实施方式。
除非另有说明，用于披露本发明的所有术语(包括技术和科学术语)的意义与本发明所属领域普通技术人员所通常理解的相同。通过进一步的指导，随后的定义用于更好地理解本发明的教导。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。
本文所使用的术语“和/或”、“或/和”、“及/或”的选择范围包括两个或两个以上相关所列项目中任一个项目，也包括相关所列项目的任意的和所有的组合，所述任意的和所有的组合包括任意的两个相关所列项目、任意的更多个相关所列项目、或者全部相关所列项目的组合。需要说明的是，当用至少两个选自“和/或”、“或/和”、“及/或”的连词组合连接至少三个项目时，应当理解，在本申请中，该技术方案毫无疑问地包括均用“逻辑与”连接的技术方案，还毫无疑问地包括均用“逻辑或”连接的技术方案。比如，“A及/或B”包括A、B和A+B三种并列方案。又比如，“A，及/或，B，及/或，C，及/或，D”的技术方案，包括A、B、C、D中任一项(也即均用“逻辑或”连接的技术方案)，也包括A、B、C、D的任意的和所有的组合，也即包括A、B、C、D中任两项或任三项的组合，还包括A、B、C、D的四项组合(也即均用“逻辑与”连接的技术方案)。
本发明中所使用的术语“含有”、“包含”和“包括”是同义词，其是包容性或开放式的，不排除额外的、未被引述的成员、元素或方法步骤。
本发明中用端点表示的数值范围包括该范围内所包含的所有数值及分数，以及所引述的端点。
本发明中涉及浓度数值，其含义包括在一定范围内的波动。比如，可以在相应的精度范围内波动。比如2％，可以允许±0.1％范围内波动。对于数值较大或无需过于精细控制的数值，还允许其含义包括更大波动。比如100mM，可以允许±1％、±2％、±5％等范围内的波动。涉及分子量，允许其含义包括±10％的波动。
本发明中，涉及“多个”、“多种”等描述，如无特别限定，指在数量上指大于等于2。
本发明中，以开放式描述的技术特征中，包括所列举特征组成的封闭式技术方案，也包括包含所列举特征的开放式技术方案。
本发明中，“优选”、“更好”、“更佳”、“为宜”仅为描述效果更好的实施方式或实施例，应当理解，并不构成对本发明保护范围的限制。本发明中，“可选地”、“可选的”、“可选”，指可有可无，也即指选自“有”或“无”两种并列方案中的任一种。如果一个技术方案中出现多处“可选”，如无特别说明，且无矛盾之处或相互制约关系，则每项“可选”各自独立。
如本文所使用，术语“功能性核苷酸类似物”是指经典核苷酸A、G、C、U或T的经修饰型式，所述型式(a)保留相应经典核苷酸的碱基配对特性，并且(b)含有至少一种对相应天然核苷酸的(i)核碱基、(ii)糖基、(iii)磷酸酯基或(iv)(i)至(iii)的任何组合的化学修饰。如本文所使用，碱基配对不仅涵盖经典沃森-克里克(Watson-Crick)腺嘌呤-胸腺嘧啶、腺嘌呤-尿嘧啶或鸟嘌呤-胞嘧啶碱基对，而且还涵盖在经典核苷酸与功能性核苷酸类似物之间或在一对功能性核苷酸类似物之间形成的碱基对，其中氢键供体与氢键受体的布置允许在经修饰的核碱基与经典核碱基之间或在两个互补的经修饰核碱 基结构之间形成氢键。举例来说，鸟苷(G)的功能性类似物保留与胞嘧啶(C)或胞嘧啶的功能性类似物碱基配对的能力。此类非经典碱基配对的一个实例是经修饰核苷酸肌苷与腺嘌呤、胞嘧啶或尿嘧啶之间的碱基配对。如本文所述，功能性核苷酸类似物可为天然存在或非天然存在的。因此，含有功能性核苷酸类似物的核酸分子可具有至少一个经修饰的核碱基、糖基和/或核苷间键联。本文提供对核酸分子的核碱基、糖基或核苷间键联的示例性化学修饰。功能性核苷酸类似物的实例包括4-乙酰胞苷、5-(羧羟甲基)尿苷、二氢尿苷、2'-O-甲基假尿苷、β,D-半乳糖Q核苷、2'-O-甲基鸟苷、肌苷、N6-异戊烯基腺苷、1-甲基腺苷、1-甲基假尿苷、1-甲基肌苷、2'2-二甲基腺苷、2-甲基腺苷、2-甲基鸟苷、5-甲基尿苷、3-甲基胞苷、5-甲基胞苷、N6-甲基腺苷、7-甲基鸟苷、5-甲基氨基甲基尿苷、5-羧甲基氨甲基尿苷、5-羧甲基氨甲基-2-硫代尿苷、β,D-甘露糖Q核苷、5-甲氧基羰基甲基-2-硫代尿苷、5-甲氧基羰基甲基尿苷、5-甲氧基尿苷、2-硫代甲基-N6-异戊烯基腺苷、N-((9-β-D-呋喃核糖基-2-硫代甲基嘌呤-6-Yl)氨基甲酰)苏氨酸、N-((9-β-D-呋喃核糖嘌呤-6-yl)N-甲基氨基甲酰)苏氨酸、尿苷-5-氧化乙酸-甲基酯、尿苷-5-氧化乙酸、wybutoxosine、假尿苷、N1-甲基假尿苷、5-羟甲氧基胞苷、Q核苷、2-硫代胞苷、5-甲基-2硫代尿苷、2-硫代尿苷、4-硫代尿苷、5-硫代尿苷、N-((9-β-D-呋喃核糖基-6-基)-氨基甲酰)苏氨酸、2'-O-甲基腺苷-5甲基尿苷、2'-O-甲基腺苷、2'-O-甲基胞苷、Wybutosine、3-(3-氨基-3-羧基-丙基)尿苷、N6-乙酰基腺苷以及2-甲硫基-N6-甲基腺苷中的一种、两种或更多种；优选的功能性核苷酸类似物包括：假尿苷、N1-甲基假尿苷以及5-羟甲氧基胞苷中的至少一种、两种或三种。
本发明涉及mRNA，其包含功能性核苷酸类似物。
在一些实施方式中，所述mRNA包含自复制mRNA。
在一些实施方式中，所述mRNA包含一个或多个来自α病毒的非结构性复制酶域的多核苷酸片段。
在一些实施方式中，所述mRNA包含整合的目标多核苷酸片段。
本发明涉及经修饰的自复制mRNA，其多核苷酸片段包含一个或多个来自α病毒的非结构性复制酶域以及整合的目标片段，所述多核苷酸片段包含功能性核苷酸类似物。
在一些实施方式中，所述功能性核苷酸类似物中的任意一种类型在所述mRNA/自复制mRNA中含量为0.01％～100％，还可以选择0.1％、0.5％、1％、2％、3％、4％、5％、6％、7％、8％、9％、10％、11％、12％、13％、14％、15％、20％、25％、30％、35％、40％、50％、60％、70％、80％、90％。
在一些实施方式中，所述功能性核苷酸类似物中的所有类型的总量在所述mRNA/自复制mRNA中含量为0.01％～100％，还可以选择0.1％、0.5％、1％、2％、3％、4％、5％、6％、7％、8％、9％、10％、11％、12％、13％、14％、15％、20％、25％、30％、35％、40％、50％、60％、70％、80％、90％。
所述功能性核苷酸类似物可位于所述mRNA的任意位置，例如可以位于所述α病毒的非结构性复制酶域，或位于所述整合的目标多核苷酸片段，或二者同时出现。在一些实施方式中，所述功能性核苷酸类似物中的0％～100％位于所述α病毒的非结构性复制酶域，例如至少0.01％、0.1％、0.5％、1％、2％、3％、4％、5％、10％、20％、30％、40％、50％、60％、70％、80％、90％。在一些实施方式中，所述功能性核苷酸类似物中的0％～100％位于所述目标片段，例如至少0.01％、0.1％、0.5％、1％、2％、3％、4％、5％、10％、20％、30％、40％、50％、60％、70％、80％、90％。
容易理解，在本发明中属于“非修饰形式”是指所述功能性核苷酸类似物修饰前的核苷酸，它们一般具有相同的碱基。例如假尿苷和N1-甲基假尿苷的非修饰形式可以为尿苷，5-羟甲氧基胞苷的非修饰形式则可以为胞苷。
在一些实施方式中，所述功能性核苷酸类似物包括：假尿苷、N1-甲基假尿苷、5-羟甲氧基胞苷以及N6-甲基腺苷中的至少一种。
在一些实施方式中，所述功能性核苷酸类似物包括：假尿苷、N1-甲基假尿苷以及5-羟甲氧基胞苷中的至少一种。
在一些实施方式中，其中所述多核苷酸片段非修饰形式中的尿苷按照1％～100％，或1％～50％，或10％～50％，或20％～40％，或25％～35％比例修饰为假尿苷；修饰比例还可以选择5％、10％、20％、25％、30％、35％、40％、50％、60％、70％、80％、90％。
在一些实施方式中，其中所述多核苷酸片段非修饰形式中的尿苷按照1％～100％，或1％～60％，或10％～60％，或10％～20％比例修饰为N1-甲基假尿苷；修饰比例还可以选择5％、10％、15％、20％、30％、40％、50％、60％、70％、80％、90％。
在一些实施方式中，其中所述多核苷酸片段非修饰形式中的胞苷按照1％～100％，或80％～100％，或90％～100％比例修饰为5-羟甲氧基胞苷；修饰比例还可以选择5％、10％、20％、30％、40％、50％、60％、70％、80％、90％。
在一些实施方式中，其中所述多核苷酸片段非修饰形式中的腺苷按照1％～100％，或1％～30％，或1％～10％，或1％～5％的比例修饰为N6-甲基腺苷(m6A)；修饰比例还可以选择2％、3％、4％、5％、6％、7％、8％、9％、10％、11％、12％、13％、14％、15％、20％、25％、30％、35％、40％、50％、60％、70％、80％、90％。
在一些实施方式中，所述目标片段包含至少一种编码抗原或其片段或表位的mRNA；优选所述抗原是致病性抗原，更优选所述抗原为病毒抗原、细菌抗原、寄生虫抗原、真菌抗原、原生动物抗原、朊病毒抗原或肿瘤抗原。
可选的，所述病毒包括：腺病毒科(adenoviridae)、沙粒病毒科(arenaviridae)、星状病毒科(astroviridae)、本雅病毒科(bunyaviridae)、杯状病毒科(caliciviridae)、黄病毒科(flaviviridae)、肝炎病毒科(hepeviridae)、单分子负链RNA病毒目(mononegavirales)、网巢病毒目(nidovirales)、小RNA病毒科(picornaviridae)、正冠状病毒亚科(orthocoronavirinae)、正黏液病毒科(orthomyxoviridae)、乳头瘤病毒科(papillomaviridae)、细小病毒科 (parvoviridae)、多瘤病毒科(polyomaviridae)、痘病毒科(poxviridae)、呼肠孤病毒科(reoviridae)、反转录病毒科(retroviridae)以及披膜病毒科(togaviridae)中的一种或多种。
可选的，所述细菌包括：葡萄球菌属、链球菌属、李式杆菌属、丹毒丝菌属、肾杆菌属、芽孢杆菌属、梭菌属、分支杆菌属、放线菌属、奴卡菌属、棒状杆菌属、红球菌属中的一种或多种，和/或，炭疽杆菌、丹毒杆菌、破伤风杆菌、李氏杆菌、气肿疽杆菌结核杆菌、大肠杆菌外、变形杆菌、痢疾杆菌、肺炎杆菌、布氏杆菌、产气夹膜杆菌、流感嗜血杆菌、副流感嗜血杆菌、卡他摩拉克氏菌、不动杆菌属、耶尔森菌属、嗜肺军团菌、百日咳杆菌、副百日咳杆菌、志贺菌属、巴斯德菌属、霍乱弧菌以及副溶血性杆菌中的一种或多种。
可选的，所述真菌包括：粗球孢子菌、普赛德斯球抱子菌、荚膜组织胞浆菌、杜氏组织胞浆菌、洛博芽生菌、巴西副球孢子菌、皮炎芽生菌、申克氏孢子丝菌、马尔尼菲青霉菌、白色念珠菌、光滑念珠菌、热带念珠菌、葡萄牙假丝酵母、曲霉菌、甄氏外瓶霉、裴氏着色霉、紧密着色霉、疣状着色霉、皮炎着色霉、白地霉、波氏足肿菌、新型隐球菌、丝孢酵母菌、米根霉、印度毛霉、伞枝犁头霉、总状共头霉、蛙粪霉、冠状耳霉、异孢耳霉、西伯鼻孢子菌、透明丝孢霉以及暗色丝孢霉中的一种或多种。
可选的，所述寄生虫包括：消化道内寄生虫、肝内寄生虫、肺内寄生虫、脑组织寄生虫、血管内寄生虫、淋巴管内寄生虫、肌肉组织寄生虫、细胞内寄生虫、骨组织寄生虫以及眼内寄生虫中的一种或多种。
可选的，所述肿瘤包括：骨、骨连接、肌肉、肺、气管、心脏、脾脏、动脉、静脉、血液、毛细血管、淋巴结、淋巴管、淋巴液、口腔、咽、食管、胃、十二指肠、小肠、结肠、直肠、肛门、阑尾、肝、胆、胰腺、腮腺、舌下腺、泌尿肾、输尿管、膀胱、尿道、卵巢、输卵管、子宫、阴道、外阴部、阴囊、睾丸、输精管、阴茎、眼、耳、鼻、舌、皮肤、脑、脑干、延髓、脊髓、脑脊液、神经、甲状腺、甲状旁腺、肾上腺、垂体、松果体、胰岛、胸 腺、性腺、舌下腺以及腮腺中任一处病变生成的肿瘤。
在一些实施方式中，所述目标片段为源自SARS-CoV-2的mRNA，优选为刺突蛋白mRNA或其片段，更优选其包含RBD基因或其片段。
在一些实施方式中，所述α病毒选自：委内瑞拉马脑脊髓炎病毒(TC83Venezuelan Equine Encephalitis Virus，VEEV)，辛德毕斯病毒(Sin-dbis virus)、屈曲病毒(Chikungunya virus)、东方马脑脊髓炎病毒(Eastern equine encephali-tis virus)、西方马脑脊髓炎病毒(Western equineencephalitis virus)、马雅鲁病毒(Mayarovirus)、生里基森林病毒(Semliki forest virus)以及委内瑞拉马脑脊髓炎病毒(Venezuelan equine encephalitisvirus)中的至少一种。优选地，所述α病毒家族中的病毒为委内瑞拉马脑脊髓炎病毒(TC83Venezuelan Equine Encephalitis Virus，VEEV)。然而，应当理解的是，本文中列举的α病毒家族的病毒仅是例示性的，但并不限于此。
在一些实施方式中，所述的经修饰的自复制mRNA的非修饰形式的核苷酸序列如SEQ ID NO：3所示。
本发明还涉及疫苗组合物，其包含如上所述的经修饰的自复制mRNA。
在一些实施方式中，所述的疫苗组合物还包含药学上可接受的载体、稀释剂和赋形剂中的至少一种。
术语“药学上可接受的”指当分子本体、分子片段或组合物适当地给予动物或人时，它们不会产生不利的、过敏的或其他不良反应。可作为药学上可接受的载体或其组分的一些物质的具体示例包括磷酸，柠檬酸，和其它有机酸；抗氧化剂(例如，抗坏血酸和甲硫氨酸)；抗菌剂(例如，十八烷基二甲基苯氯化铵，氯化六烃季铵，苯扎氯铵，酚，丁醇或苯甲醇，烷基尼泊金，邻苯二酚，间苯二酚，环己醇，3-戊醇，或间甲酚)；低分子量(不到约10kDa)多肽；蛋白，例如，血清白蛋白，明胶，或免疫球蛋白；亲水性聚合物，例如，聚乙烯吡咯烷酮；氨基酸(例如，甘氨酸，谷氨酰胺，天冬酰胺，组氨酸，精氨酸，或赖氨酸)；单糖，二糖和其它碳水化合物(包括例如，葡萄糖， 甘露糖，或葡聚糖)；螯合剂(例如，EDTA)；糖(例如，蔗糖，甘露醇，海藻糖，或山梨醇)；成盐反离子；金属复合物；和/或非离子型表面活性剂(例如，包括TWEENTM，PLURONICSTM，或聚乙二醇)。此外，根据配制方法，可以由本领域普通技术人员适当选择常用的填充剂，稀释剂，结合剂，增湿剂，崩解剂，和/或表面活性剂。疫苗组合物可以以固体、半固体或液体形式存在，优选以液体形式存在。
在一些实施方式中，所述疫苗组合物还包含核酸稳定剂。
用于稳定化和保持核酸的稳定化药剂的例子包括阳离子化合物、去污剂、离液盐、核糖核酸酶抑制剂、螯合剂等及其混合物。稳定剂可以包括例如诸如多聚甲醛的交联固定剂或诸如乙醇的沉淀剂。稳定剂可以通过在细胞分子之间形成共价键或通过将一些细胞内分子沉淀或通过其它方法来起作用。在一些实施方式中，稳定剂包括细胞裂解缓冲液。细胞透化缓冲液也是本领域已知的，并且可以包含使细胞膜透化从而允许探针和染料穿过膜的去污剂。在细胞裂解缓冲液中使用的去污剂的例子包括但不限于TweeruTriton X-100、皂草苷、NP-40等。针对给定的最终用途调整细胞裂解和透化剂的浓度。当以过低的浓度存在时，细胞裂解和透化可能不能达到最佳。在过高的浓度下，可能出现不希望的细胞破坏。可以进行常规的根据经验的步骤来确定在每种情况下优选的路线。在一些实施方式中，稳定剂包括氯仿、苯酚、TRIZOL。但在更优选的实施方式中，稳定剂是易于被除去的或者对细胞毒性较低的成分，最优选是药学上可接受的成分。
本发明所提供的疫苗优选地还包括佐剂。适用于本发明疫苗的佐剂包括可增强针对自复制mRNA，特别是其中的目标片段的免疫原性。例如，对于抗原的B细胞表位的抗体反应的佐剂，以及可增强细胞介导的针对所述抗原中T细胞表位的反应的佐剂等。这些佐剂是本领域所熟知的。
在一些实施方式中，所述佐剂选自明矾、完全弗氏佐剂、不完全弗氏佐剂、角鲨烯、角鲨烷、胞壁酰二肽、MF59、AS03、单磷脂酰脂质A，鞭毛 蛋白、CpG-ODN、Poly(I：C)，以及铝或钙盐的小分子中的一种或多种。这些佐剂均是本领域所熟知并可通过若干商业渠道获得的。
其中完全弗氏佐剂、不完全弗氏佐剂、角鲨烷和明矾一般不用于人。
在一些实施方式中，所述疫苗是具有水相和油相的油包水乳液。
在一些实施方式中，所述疫苗是具有水相和油相的水包油乳液。
疫苗典型地被配制用于肠胃外施用。典型的免疫接种是通过口腔和皮下(SC)、鼻腔途径、肌内(IM)、静脉内(IV)、腹膜内(IP)或真皮内(ID)注射实现。
上述疫苗是以与剂量配方相容的方式，以及诸如治疗有效量和免疫原性有效量的用量被施用的。施用量取决于接受治疗的对象、该对象的免疫系统合成抗体的能力，以及预期的保护程度。需施用的活性成分的准确数量取决于医师的判断，个体不同，用量也不同。最初施用和加强接种的合适方案也可变化，但典型地在首次施用后的一定间隔时间(数周或数月)后再进行1次注射或以其它方式施用。
在一些实施方式中，所述的疫苗组合物以质粒、病毒载体、脂质体、树状大分子、无机纳米粒子或细胞穿膜肽的形式进行包装递送。
mRNA分子可直接包装，或以其前体的方式进行包装，所述质粒和病毒载体可以包含选择标记(例如便于富集的标签，例如his tag；或便于被检测的标签，例如GFP)，以及与所述克隆载体所指定的细胞类型相匹配的复制起点，而表达载体则包含对于影响指定靶细胞中的表达必要的调节元件。病毒载体可以为噬菌体、慢病毒、逆转录病毒、腺病毒或腺相关病毒。
脂质体可以为阳离子脂质体或中性脂质体，其可通过公知的方法进行制备或修饰，例如加入聚乙二醇(PEG)修饰的脂质体可以有效防止脂质体载体的聚集并增加其稳定性。
树枝状大分子是一种具有明确的分子结构、可精确控制的化学结构和 独特的多价性质的特殊聚合物家族，逐渐成为基因传递的非病毒载体。典型的树枝状大分子例如聚(酰氨基胺)(PAMAM)树枝状聚合物，其可做进一步修饰，例如在PAMAM表面修饰核碱基类似物2-氨基-6-氯嘌呤构建衍生物AP-PAMAM，或者通过硫酸软骨素(CS)与PAMAM偶联制备CS-PAMAM等等。
无机纳米粒子可选择金纳米粒子(AuNPs)、磁性纳米粒子、介孔二氧化硅纳米粒子(MSNs)等。
细胞穿膜肽(cell-penetrating peptides，CPPs)是一类具有较强跨膜转运能力的小分子肽，可携带多肽、蛋白质和核酸等多种大分子物质进入细胞。其可以为阳离子型CPPs(如TAT，Penetratin，Polyarginine，P22N，DPV3和DPV6等)、两亲型CPPs(可以由疏水性肽序列和NLSs共价连接而成，或者从天然蛋白质中分离获得，如pVEC，ARF(1-22)和BPrPr(1-28))、疏水型CPPs(一般只含有非极性氨基酸残基，净电荷量约低于氨基酸序列总电荷量的20％)。
本发明的另一个实施方式涉及成套试剂盒，所述试剂盒包含如上所述的疫苗，以及用于接种所述疫苗组合物的容器。
接种容器优选为医用注射器。
本发明还涉及方法，其用于改变mRNA在接种于动物体内后的如下特性：
a)提高所述mRNA的免疫原性；和/或
b)提高所述mRNA逃避免疫监视的能力；
所述方法包括用功能性核苷酸类似物替换所述mRNA中相应的非修饰形式核苷的步骤。
在一些实施方式中，所述mRNA为自复制mRNA，优选替换后得到如上所述的经修饰的自复制mRNA。
在一些实施方式中，所述动物为鸡、鸭、鹅、猫、犬、牛、羊、马、驴、猪、大熊猫、猴子、兔、鼠或人。
下面将结合实施例对本发明的实施方案进行详细描述。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，优先参考本发明中给出的指引，还可以按照本领域的实验手册或常规条件，还可以参考本领域已知的其它实验方法，或者按照制造厂商所建议的条件。
下述的具体实施例中，涉及原料组分的量度参数，如无特别说明，可能存在称量精度范围内的细微偏差。涉及温度和时间参数，允许仪器测试精度或操作精度导致的可接受的偏差。
实施例1
1、RBD基因片段合成
从NCBI上查询刺突蛋白(SARS-CoV-2)再设计受体结合区(RBD)基因，然后根据人密码子进行相应优化，核苷酸序列如SEQ ID NO：1所示。并在上游加入ApaI酶切位点和启动子，下游加入NotI酶切位点，最后通过合成直接获得DNA序列(以公司提供克隆质粒pUC57-RBD的形式获得)。
2、构建TC83自复制载体
自复制mRNA是根据α病毒家族中委内瑞拉马脑脊髓炎病毒(TC83，VEEV)的基因组进行设计的，其包含可编码α病毒自复制组件的基因，但是缺乏编码用于制造具有传染性的α病毒颗粒的结构蛋白，将设计好的序列通过合成直接获得，核苷酸序列如SEQ ID NO：2所示。
3、重组质粒JCXH-107的制备方法如下(所制备的重组质粒JCXH-107如图1所示)
pUC57-RBD和TC83自复制载体用ApaⅠ和NotⅠ双酶切，酶切体系20μL：pUC57-RBD或TC83自复制载体<1μg，ApaⅠ 1μL，NotⅠ 1μL，10×CutSmart缓冲液2μL，ddH2O将体系补足20μL。25℃水浴1h酶切质粒，然后37℃水浴1h酶切质粒，载体片段需加0.5μL CIP，37℃水浴30min去磷酸化。酶切混合物与6×上样缓冲液混合，电泳(1％琼脂糖，94V)并胶回收相应长度的片段(pUC57-RBD回收片段RBD基因片段，长度为2574bp；TC83自复制载体回收片段，长度约9.5Kb)，以30μL洗脱缓冲液洗脱。
分别取RBD基因片段与TC83自复制载体片段按照连接体系：载体50ng，插入片段摩尔数：载体片段摩尔数＝5:1，T4连接酶1μL，10×连接酶缓冲液5μL，ddH2O将体系补足10μl，22℃连接1h。将连接产物按体积1:10与大肠杆菌DH5α的感受态细胞轻柔混匀，冰浴30min，42℃热激45s，冰浴3min，加入无抗LB培养基500μL，混匀后于37℃，180rpm培养1h，涂布于LK平板(LB-Kan平板：含50μg/mL Kan的LB平板)，37℃培养16-20h。
PCR筛选：以煮沸法提取的细菌质粒为模板，扩增目的条带刺突蛋白，上游引物F：5’-TATGGCCATGACTACTCTAGCTA-3’，下游引物R：5’-GGGAAAC GCCTGGTATCTTT-3’，反应循环条件为：94℃3min→(94℃1min，47℃30s，72℃3min)×30个循环→72℃10min→4℃；电泳观察PCR结果(电泳条件：1％琼脂糖凝胶；90V，上样量：5μl PCR产物)，预期结果：RBD片段约2977bp。
酶切验证：PCR筛选验证的阳性菌提取质粒，按照上述酶切体系以ApaⅠ和NotⅠ进行酶切，将酶切混合物与6×上样缓冲液混合进行电泳(1％琼脂糖，94V)。
测序验证：提取PCR及酶切验证符合预期的质粒送测序公司测序。将测序验证后完全正确的携带阳性质粒的大肠杆菌保存于-80℃。
4、JCXH-107质粒用BspQI酶切线性化及回收方法如下
JCXH-107质粒10μg，BspQI 1μL，10×NE缓冲液3.1 5μL，ddH2O将体系补足50μL，50℃水浴1h酶切质粒。将酶切混合物与6×上样缓冲液混合，电泳(1％琼脂糖，94V)并胶回收相应长度的片段(JCXH-107质粒用，长度约12Kb)，以30μL洗脱缓冲液洗脱。
5、线性化JCXH-107质粒使用T7RNA聚合酶开始的体外转录反应(IVT)，Turbo DNase酶对模板DNA进行降解，和加帽酶(Vaccinia capping enzyme)将7-甲基化鸟苷酸帽结构(7-methylguanylate cap structure，称为Cap 0)加至转录的mRNA的5’端，具体方法如下：
10×反应缓冲液2μL，NTP每种0.5mM，尿苷(U)可按照比例10％，20％，30％，40％，50％，60％，70％，80％，90％，100％替换为假尿苷，或者尿苷(U)可按照比例10％，20％，30％，40％，50％，60％，70％，80％，90％，100％替换为N1-甲基假尿苷，或胞苷(C)可按照比例10％，20％，30％，40％，50％，60％，70％，80％，90％，100％替换为5-羟甲氧基胞苷；线性化JCXH-107 1μg，T7RNA Polymerase 2μL，补水至20μL，30℃反应1小时后加入1μL TURBOTMDNase，10×加帽缓冲液4μL，GTP(10mM)2μL，SAM(2mM)2μL，加帽酶2μL，总体积补水至总体积40μL，30℃反应1小时。后补水至200μL再加入7.5M氯化锂120μL混匀后，-20℃静置30分钟后，14000g 4℃离心30分钟，弃上清后用70％酒精清洗沉淀，14000g4℃离心5分钟，弃上清后风干5分钟，溶解在40μL水内。分光光度计定量后，取400ng和10μL Northern Max-Gly Sample Loading Dye混合，然而50℃孵育30分钟后，用Northern Max-Gly Gel Prep/Running缓冲液电泳(1％琼脂糖，70V)。自复制mRNA-RBD(图中标为SAM-CD5-RBD)体外转录后的电泳结果图片参见图2，其非修饰形式核苷酸序列如SEQ ID NO：3所示。
6、Western blot检测自复制mRNA-RBD在细胞中的表达情况。将购于上海细胞中心的BHK细胞传代培养，待细胞数量足够，胰酶消化至细胞培养6孔板每孔，铺板37℃CO2培养箱过夜。第二天将脂质体包裹的 mRNA-RBD转染到铺好BHK细胞中，培养24小时后，将细胞裂解收集蛋白样品。具体方法：
1)细胞培养及铺板：将复苏好的BHK细胞接种在75cm2的培养瓶中，培养基为DMEM高糖培养基+5％的双抗+10％胎牛血清，待细胞在瓶底的密度达到80％以上，用胰酶将细胞消化下来，并计数。在6孔细胞培养板中铺入适宜数量的细胞，37℃CO2培养箱过夜。
2)脂质体mRNA转染BHK或Hela细胞：吸干铺好的6孔板中的培养基，用PBS缓冲液清洗一遍，将脂质体包裹的mRNA 0.5μg与200μL Opti-MEM培养基混合，加至清洗好的6孔板中，37℃CO2培养箱6小时后补充200μL含有20％胎牛血清的DMEM高糖培养基。37℃CO2培养箱，继续培养18小时。
3)蛋白样品的处理：在24小时后的BHK细胞中加入200μL细胞裂解液及1％的PMSF，冰上放置5分钟，离心机14000g离心5分钟，将上清转移至新的离心管，加入5×SDS 95℃金属浴12分钟。放在-20℃备用。
4)Western blot检测：
电泳：聚丙稀酰胺分离胶浓度为6％，蛋白上样量20μL，积层胶电泳条件150V，10min，分离胶电泳条件为200V，30min。
电转：使用硝酸纤维素膜，在100V电压下转膜1小时。
封闭：使用1×PBST配制5％BSA作为封闭液，4℃封闭过夜。
一抗孵育：使用封闭液将SARA-Cov-2(2019-nCov)刺突蛋白抗体以1：2000进行稀释，在室温下孵育1小时。用1×PBST洗3次，每次10分钟。
二抗孵育：使用封闭液稀释二抗，稀释比为1：5000，在室温下孵育1小时。用1×PBST洗3次，每次10分钟。
显影：显影液A液：B液＝1：1，在成像系统中进行显影。
实验结果参见图3-1和图3-2，图3-1和图3-2是比较修饰核苷酸和常规核苷酸自复制mRNA-RBD(图中标为SAM-CD5-RBD)在体外转染BHK细胞后RBD蛋白表达图，包括RBD蛋白在细胞裂解液及细胞上清中的表达检测，其中细胞裂解液以Actin蛋白作为内参，细胞上清以蛋白考马斯染色作为内参；图4是针对图3-1和图3-2里RBD蛋白在细胞裂解液中的量化统计图，由图3-1、图3-2、图4可知，自复制mRNA-RBD在体外转染BHK细胞后RBD蛋白表达情况较好。
7、本发明通过实时荧光定量PCR检测比较使用修饰核苷酸和常规核苷酸自复制mRNA-RBD(图中标为SAM-CD5-RBD)在体外转录反应所得到的mRNA-RBD在体外转染Hela细胞后，检测天然免疫调节因子IFN-B1转录水平。具体方法如下：
1)细胞RNA抽提：吸去细胞培养基，用PBS洗细胞一次，加入1mL RNAiso吹打细胞，收集于EP管中。加入0.2mL氯仿，震荡混合，静置5分钟后，12000g离心15分钟，吸取上清。加入与上清同体积异丙醇，震荡混合，室温静置10分钟。12000g离心10分钟，弃上清。加入1mL含75％乙醇的Nuclease-Free water，洗涤沉淀。12000g离心5分钟，弃上清。晾干沉淀，加入Nuclease-Free水20μL，轻轻吹打，溶解沉淀。紫外分光光度计测定OD260/OD280值、RNA浓度。
2)制备cDNA样品：各取1.5μg RNA样品加入5×gDNA Digester Mix和Nuclease-Free水定容至15μL，42℃孵育2min。加入III Super Buffer 2μL、III RT Enzyme Mix 1μL、Random Primers N6 1μL、Nuclease-Free water1μL，混合均匀，进行反转录反应。反转录程序为：25℃，5min；60℃，15min；85℃，5min。
3)实时荧光定量PCR检测：将cDNA样品稀释5倍，配置反应体系：2×ChamQ Universal SYBR qPCR Master Mix 5μL、10μM上游引物0.2μ L、10μM下游引物0.2μL、稀释cDNA样品1μL、Nuclease-Free water 3.6μL，混合均匀并检测。用于检测IFN-B1基因表达的上游引物序列为CA TTACCTGAAGGCCAAGGA，下游引物序列为CAGCATCTGCTGGTTGA AGA。用于检测内参GAPDH基因表达的上游引物序列GAAGGCTGGG GCTCATTT，下游引物序列CAGGAGGCATTGCTGATGAT。用于检测RI G-I基因表达的上游引物序列为GTTGTCCCCATGCTGTTCTT，下游引物序列GCAAGTCTTACATGGCAGCA。使用Thermo Fisher Quant Studio 1实时荧光定量PCR系统进行检测。检测程序为，阶段1：95℃，3min，重复1次；阶段2：95℃，10s，60℃，30s，重复40次；阶段3溶解曲线：95℃，15s；60℃，60s；95℃，15s，重复1次。采用相对定量法分析目的基因表达情况：倍数变化＝2-ΔΔCT，结果如图5所示，作为阳性对照，100％比例假尿苷修饰的非自复制mRNA-RBD，100％比例N1-甲基假尿苷修饰的非自复制mRNA-RBD转染细胞后，几乎不会刺激天然免疫调节因子IFN-B1基因的高表达；而常规核苷酸修饰的自复制mRNA-RBD转染细胞后，刺激IFN-B1高表达；相比于常规核苷酸修饰的自复制mRNA-RBD，10％、20％、30％比例假尿苷修饰的自复制mRNA-RBD转染细胞后，可以降低对IFN-B1的刺激，且随着修饰核苷酸比例的增加，IFN-B1降低越明显，且修饰核苷酸对RBD蛋白的翻译影响不大；10％、20％、30％比例N1-甲基假尿苷修饰的自复制mRNA-RBD转染细胞后，同样具有降低对IFN-B1高表达的效果，同时对RBD蛋白翻译影响不大；而100％比例假尿苷修饰的自复制mRNA-RBD，100％比例N1-甲基假尿苷修饰的自复制mRNA-RBD转染细胞后，几乎不会刺激IFN-B1基因的高表达，但是对RBD蛋白翻译具有明显的抑制效果；100％比例5-甲基胞苷修饰的自复制mRNA-RBD转染细胞后，在一定程度上降低IFN-B1的高表达，但是其降低效果不如30％假尿苷修饰的自复制mRNA-RBD，其对RBD蛋白翻译影响不大；综合比较不同种类和不同程度核苷酸修饰对IFN-B1基因表达及RBD蛋白翻译的影响，30％假尿苷修饰的自复制mRNA-RBD比常规核苷酸修饰的自复制mRNA-RBD具有较好的炎症基因抑制效果并保留了较好的RBD蛋白翻译 表达。
8、mRNA-RBD与脂类包裹步骤如下。
mRNA与脂类(包括1,2-二肉豆蔻酰基-外消旋-甘油-3-甲氧基聚乙二醇-2000(DMG-PEG2000)、胆固醇、二硬脂酰基磷脂酰胆碱(DSPC)以及阳离子脂类，溶解在酒精内)通过Nanoassemblr混合器进行快速的混合，从而引起脂类的沉淀及在电荷作用mRNA包裹进入LNP内。mRNA-RBD-LNP复合物随后通过浓缩及换液至制剂溶液中。
9、mRNA-RBD-LNP疫苗免疫小鼠诱导的抗刺突蛋白抗体滴度检测
6～8周龄的BALB/c雌鼠适应性喂养3天后分组，每组6小鼠，一组为对照组，注射制剂缓冲液；其余十组分别注射常规核苷酸自复制mRNA-RBD-LNP，10％、20％、30％比例假尿苷修饰的自复制mRNA-RBD-LNP,10％、20％、30％比例N1-甲基假尿苷修饰的自复制mRNA-RBD-LNP,100％比例5-甲基胞苷修饰的自复制mRNA-LNP，100％比例假尿苷修饰的非自复制mRNA-RBD-LNP，100％比例N1-甲基假尿苷修饰的非自复制mRNA-RBD-LNP。每只小鼠在第0天脊柱注射3ug mRNA-RBD-LNP，每组的三只小鼠分别在第2天取脾脏检测IFN-B1表达，结果如图6所示；每组剩余的三只小鼠在第7天，第14天取血清后，每只小鼠脊柱注射第二针3ug mRNA-RBD-LNP，然后分别在第21、28天取小鼠血清，并用ELISA检测针对RBD抗体滴度，实验流程及设计见图7。
抗体滴度检测方法为ELISA具体操作如下，96孔酶标板每孔包被50ng刺突蛋白RBD结构域蛋白室温过夜，第二天用PBST清洗3遍，用5％牛奶封闭，37℃下1小时后，PBST清洗3遍，加入对应稀释比例的小鼠血清，37℃下1小时后，PBST清洗3遍，加入抗小鼠IgG重链-轻链HRP，37℃下1小时后，PBST清洗3遍，最后加入显色液显色。实验结果见图8，注射100％比例假尿苷修饰的非自复制mRNA-RBD-LNP和100％比例N1-甲基假尿苷修饰的非自复制mRNA-RBD-LNP的小鼠，其血清中 具有较高的RBD特异性抗体；在自复制mRNA-RBD-LNP中，注射30％假尿苷修饰的自复制mRNA-RBD-LNP的小鼠，其血清中具有较高RBD特异性抗体。
从上述实施例的结果可知，运用本发明的方法进行核苷酸修饰得到的自复制mRNA能够很好的在细胞中表达，并且能够在动物中诱导产生较好的抗体反应。
实施例2
在实施例1的基础之上改变修饰类型为m6A，简述之：
一、实验方法
1.细胞水平检测
1.1本实验使用SrRNA-TC83自复制mRNA系统，插入片段为RBD；
1.2体外合成m6A修饰自复制mRNA：设计合成不同m6A修饰比例的自复制mRNA：0％、1％、5％、10％、20％、30％、40％、50％、60％、70％、80％、90％、100％；
1.3细胞转染：本实验使用BHK和Hela细胞系转染，使用Lipofectamine脂质体转染试剂，转染剂量为0.5ug，24小时收样；
1.4结果检测：WB检测BHK、Hela细胞RBD蛋白表达水平变化，检测Hela细胞IFN-B1和/或RIG-I基因表达变化。
2.动物实验：
2.1使用LNP包裹不同比例m6A修饰的自复制mRNA做动物实验；
2.2使用SrRNA-TC83作为载体，在动物实验上检测hEPO(human Erythropoietin)及Luciferase基因蛋白的表达；
2.3 LNP-hEPO-mRNA动物实验采用静脉注射的方式，LNP-Luciferase- mRNA动物实验采用肌肉注射的方式；
2.4 hEPO通过WB检测蛋白在体内表达效果，Luciferase通过荧光成像检测蛋白在体内表达效果。
二、实验结果：
1.细胞水平实验
1.1 RBD蛋白表达在特定比例(例如20％以下，或10％以下，或5％以下)m6A修饰下表达基本不受影响，或显著性增加，结果如图9和10所示；其中图9中，其中A和C为细胞裂解液中的蛋白表达，B和D为上清液中的蛋白表达，可以看出，10％以下的m6A修饰基本不显著较少RBD的表达。WB检检测RBD表达结果如图10所示，从图中可以看出1％、5％的m6A修饰不破坏RBD的表达。
1.2 IFN-B1基因的表达结果图11所示，可见m6A修饰能够有效降低IFN-B1表达。
2.动物水平实验
2.1 Luciferase荧光信号在特定比例m6A修饰下，信号增强；
2.2 hEPO蛋白表达在特定比例m6A修饰下，蛋白表达增加，小鼠炎症基因表达明显下降。
由上可知，m6A修饰的自复制mRNA，对蛋白表达基本不影响，或有促进作用；m6A修饰的自复制mRNA能够有效降低炎症基因表达。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准，说明书及附图可以用于解释权利要求的内容。

Claims (17)

  1. 经修饰的自复制mRNA，其多核苷酸片段包含一个或多个来自α病毒的非结构性复制酶域以及整合的目标片段，所述多核苷酸片段包含功能性核苷酸类似物；
    所述功能性核苷酸类似物包括：假尿苷、N1-甲基假尿苷、5-羟甲氧基胞苷以及N6-甲基腺苷中的至少一种。
  2. 根据权利要求1所述的经修饰的自复制mRNA，其中所述多核苷酸片段非修饰形式中的尿苷按照1％～100％，或1％～50％，或10％～50％，或20％～40％，或25％～35％比例修饰为假尿苷。
  3. 根据权利要求1所述的经修饰的自复制mRNA，其中所述多核苷酸片段非修饰形式中的尿苷按照1％～100％，或1％～60％，或10％～60％，或10％～20％比例修饰为N1-甲基假尿苷。
  4. 根据权利要求1所述的经修饰的自复制mRNA，其中所述多核苷酸片段非修饰形式中的胞苷按照1％～100％，或80％～100％，或90％～100％比例修饰为5-羟甲氧基胞苷。
  5. 根据权利要求1所述的经修饰的自复制mRNA，其中所述多核苷酸片段非修饰形式中的腺苷按照1％～100％，或1％～10％比例修饰为N6-甲基腺苷。
  6. 根据权利要求1～5任一项所述的经修饰的自复制mRNA，所述目标片段包含至少一种编码抗原或其片段或表位的mRNA，优选所述抗原是致病性抗原，更优选所述抗原为病毒抗原、细菌抗原、寄生虫抗原、真菌抗原、原生动物抗原、朊病毒抗原或肿瘤抗原。
  7. 根据权利要求6所述经修饰的自复制mRNA，所述目标片段为源自SARS-CoV-2的mRNA，优选为刺突蛋白mRNA或其片段，更优选其包含RBD基因或其片段。
  8. 根据权利要求1～5、7任一项所述的经修饰的自复制mRNA，所述α病毒选自：委内瑞拉马脑脊髓炎病毒(TC83 Venezuelan Equine Encephalitis Virus，VEEV)，辛德毕斯病毒(Sin-dbis virus)、屈曲病毒(Chikungunya virus)、东方马脑脊髓炎病毒(Eastern equine encephali-tis virus)、西方马脑脊髓炎病毒(Western equineencephalitis virus)、马雅鲁病毒(Mayarovirus)、生里基森林病毒(Semliki forest virus)以及委内瑞拉马脑脊髓炎病毒(Venezuelan equine encephalitisvirus)中的至少一种。
  9. 根据权利要求8所述的经修饰的自复制mRNA，其非修饰形式的核苷酸序列如SEQ ID NO：3所示。
  10. 疫苗组合物，其包含权利要求1～9任一项所述的经修饰的自复制mRNA。
  11. 根据权利要求10所述的疫苗组合物，其还包含药学上可接受的载体、稀释剂和赋形剂中的至少一种。
  12. 根据权利要求10所述的疫苗组合物，其还包含核酸稳定剂和/或免疫佐剂。
  13. 根据权利要求10～12任一项所述的疫苗组合物，其以质粒、病毒载体、脂质体、树状大分子、无机纳米粒子或细胞穿膜肽的形式进行包装递送。
  14. 成套试剂盒，其包含权利要求10～12任一项所述的疫苗组合物，以及任选地用于接种所述疫苗组合物的容器。
  15. 方法，其用于改变mRNA在接种于动物体内后的如下特性：
    a)提高所述mRNA的免疫原性；和/或
    b)提高所述mRNA逃避免疫监视的能力；
    所述方法包括用功能性核苷酸类似物替换所述mRNA中相应的非修饰形式核苷的步骤。
  16. 根据权利要求15所述的方法，所述mRNA为自复制mRNA，优选 替换后得到权利要求1～9任一项所述的经修饰的自复制mRNA。
  17. 根据权利要求15或16所述的方法，所述动物为鸡、鸭、鹅、猫、犬、牛、羊、马、驴、猪、大熊猫、猴子、兔、鼠或人。
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