WO2024152996A1 - 自复制信使核糖核酸疫苗 - Google Patents

自复制信使核糖核酸疫苗 Download PDF

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WO2024152996A1
WO2024152996A1 PCT/CN2024/071926 CN2024071926W WO2024152996A1 WO 2024152996 A1 WO2024152996 A1 WO 2024152996A1 CN 2024071926 W CN2024071926 W CN 2024071926W WO 2024152996 A1 WO2024152996 A1 WO 2024152996A1
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mrna
mrna molecule
molecule according
virus
self
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French (fr)
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宫悦
刘晓虎
余志斌
徐江
丁隽
雍丹妮
刘根盛
贾为国
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上海复诺健生物科技有限公司
复诺健生物科技加拿大有限公司
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    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/127—RNA-directed RNA polymerase (2.7.7.48), i.e. RNA replicase
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K9/00—Medicinal preparations characterised by special physical form
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    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07048—RNA-directed RNA polymerase (2.7.7.48), i.e. RNA replicase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • C—CHEMISTRY; METALLURGY
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    • C12N2770/00011—Details
    • C12N2770/36011—Togaviridae
    • C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
    • C12N2770/36122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/36011—Togaviridae
    • C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
    • C12N2770/36134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to self-replicating messenger ribonucleic acid (mRNA) molecules, self-replicating mRNA vaccines, DNA molecules for transcribing into self-replicating mRNA molecules, use of self-replicating mRNA molecules in the manufacture of self-replicating mRNA vaccines that induce anti-HER2 immune responses, and methods for manufacturing self-replicating mRNA molecules.
  • mRNA messenger ribonucleic acid
  • HER2 Human epidermal growth factor receptor 2
  • HER2 Human epidermal growth factor receptor 2
  • the tumor will show stronger tumor invasiveness and higher recurrence frequency.
  • Current clinical data show that the patient group that has produced a cytotoxic immune response against HER2 in the body has significantly improved indicators such as survival or recurrence probability, suggesting that it is feasible to express HER2 by inducing the body to produce a specific immune response against HER2 to attack and kill tumor cells that highly express the antigen, while forming immune memory, so that the body can obtain long-term immunotherapy or prevention effects.
  • mRNA vaccines have shown unique advantages. mRNA vaccines can complete the translation and expression of antigens once they enter the cytoplasm, and are not integrated into the genome, avoiding the risk of antigen gene insertion. mRNA vaccines have proven to be able to induce both humoral immunity and cellular immunity, inducing strong immune responses in vivo. mRNA vaccines use a cell-free preparation method of in vitro transcription and microfluidic mixed encapsulation of nanolipid particles, which gets rid of the dependence on living cells for production, facilitates mass production, shortens the production cycle, and simplifies the production and purification process.
  • linear mRNA vaccines are divided into two types: non-replicating and self-replicating.
  • Self-replicating mRNA is derived from the modification of the RNA virus genome structure, and uses viral replicase to efficiently amplify the number of mRNA molecules that express antigens, so as to achieve the purpose of expressing a large number of antigen proteins with a small number of mRNA molecules, which can greatly reduce the amount of mRNA vaccine.
  • self-replicating mRNA can potentially activate the natural immune response, it can further enhance the body's immune response and form an adjuvant effect.
  • the mRNA tumor vaccines based on tumor-associated antigens that have entered clinical trials are divided into recombinant viral RNA replicon particle vaccines obtained through cell production or non-replicating mRNA vaccines based on lipid nanoparticles synthesized without cells.
  • the former is produced and packaged in cells to produce recombinant viral RNA replicon particles with only single infection ability, and the self-replicating mRNA molecules are efficiently delivered through the interaction between viral proteins and cell receptors. Due to the amplification effect of self-replicating mRNA molecules, high-level expression of tumor-associated antigens can be achieved to stimulate the body's immunity.
  • the recombinant viral vector technology has high requirements and the production process is complex, which will limit the rapid application of viral vector tumor vaccines to a certain extent.
  • Non-replicating mRNA vaccines based on lipid nanoparticle delivery use nanolipid particles to deliver non-replicating mRNA encoding tumor-associated antigens to cells for expression through potential lipid receptor interactions. Since the synthesis of mRNA molecules and their loading into nanolipid particles are both done through in vitro transcription and encapsulation of a cell-free matrix, the production and preparation process is simple, which is conducive to the rapid synthesis and use of vaccines. However, the number of antigens expressed by non-replicating mRNA is proportional to the number of mRNAs successfully delivered during the immunization process, and the half-life is short. Therefore, in order to obtain the antigen expression level required to induce sufficient immune protection in vivo, more doses of vaccine or repeated vaccinations are required.
  • a messenger RNA (mRNA) molecule comprising:
  • Expression cassette 1 which contains the coding sequence of RNA replicase
  • Expression cassette 2 which contains the coding sequence of the antigen.
  • RNA replicase is a viral RNA replicase
  • the virus of the genus Alphavirus is Venezuelan equine encephalitis virus (VEEV).
  • HER2 antigen variant has a modified intracellular domain (ICD) in which the tyrosine kinase (TK) active site is mutated by an inactivating mutation.
  • ICD intracellular domain
  • TK tyrosine kinase
  • HER2 antigen variant has a major histocompatibility complex 1 (MHC1) transport domain (MITD), wherein the MITD replaces the transmembrane domain (TMD) of the HER2 antigen.
  • MHC1 major histocompatibility complex 1
  • TMD transmembrane domain
  • the coding sequence of the HER2 antigen variant has the sequence shown in SEQ ID NO: 1 or 3.
  • the upstream of the expression cassette 1 also has a promoter site and 5'UTR for RNA polymerase;
  • the expression cassette 2 also has a 3'UTR and poly A downstream.
  • RNA polymerase is T7 RNA polymerase.
  • uridine triphosphate is natural uridine triphosphate (UTP) or N1-methylpseudouridine triphosphate (N1-Me-pUTP).
  • a messenger ribonucleic acid (mRNA) vaccine which is obtained by encapsulating the mRNA molecules according to the above embodiments into lipid nanoparticles (LNPs).
  • mRNA messenger RNA
  • a method for producing a messenger ribonucleic acid (mRNA) molecule comprising:
  • the virus of the genus Alphavirus is Venezuelan equine encephalitis virus (VEEV).
  • HER2 antigen variant has an engineered intracellular domain (ICD) in which the tyrosine kinase (TK) active site is mutated by an inactivating mutation.
  • ICD engineered intracellular domain
  • TK tyrosine kinase
  • the HER2 antigen variant has a major histocompatibility complex 1 (MHC1) transport domain (MITD), wherein the MITD replaces the transmembrane domain (TMD) of the HER2 antigen.
  • MHC1 major histocompatibility complex 1
  • TMD transmembrane domain
  • a nucleic acid vector for in vitro transcription into which the DNA molecule constructed by steps (1) to (3) is inserted,
  • adenosine triphosphate adenosine triphosphate
  • cytidine triphosphate adenosine triphosphate
  • guanosine triphosphate adenosine triphosphate
  • uridine triphosphate adenosine triphosphate
  • uridine triphosphate is natural uridine triphosphate (UTP) or N1-methylpseudouridine triphosphate (N1-Me-pUTP).
  • RNA polymerase is T7 RNA polymerase.
  • both the full-length self-replicating mRNA encoding the antigen molecule and the non-structural polyprotein and the full-length self-replicating mRNA encoding the antigen alone will undergo high-level replication, but the self-replicating mRNA encoding the antigen molecule alone will have a higher replication level, thereby achieving efficient expression of the antigen while minimizing the immune response and toxicity caused by the expression of viral components.
  • the present invention utilizes an in vitro transcription method to prepare self-replicating mRNA molecules and utilizes microfluidics technology to encapsulate mRNA into nanolipid particles, thereby realizing the cell-free preparation of self-replicating mRNA tumor vaccines.
  • this preparation method has a short production cycle, simple process and quality control, and is conducive to the rapid preparation of tumor vaccines.
  • FIG. 1A Schematic diagram of the structure of the HER2 antigen variant VG-HR1.
  • FIG. 1B Schematic diagram of the structure of the HER2 antigen variant VG-HR13.
  • FIG. 2A Schematic diagram of the structure of the expression cassette for expressing the self-replicating mRNA of VG-HR1.
  • FIG. 2B Schematic diagram of the structure of the expression cassette for expressing the self-replicating mRNA of VG-HR13.
  • FIG. 3A Schematic diagram of the structure of the expression cassette for expressing the non-self-replicating mRNA of VG-HR1.
  • FIG. 3B Schematic diagram of the structure of the expression cassette for expressing the non-self-replicating mRNA of VG-HR13.
  • FIG. 4 A map of plasmid psaRNA-VG-HR13 containing an expression cassette for self-replicating mRNA, obtained by inserting an expression cassette for expressing self-replicating mRNA of VG-HR13 into pUC57-Kan plasmid.
  • Figure 7 Relative changes in the copy number of the coding sequence portion of the non-structural polyprotein of the self-replicating mRNA expressing VG-HR1 (or the full-length self-replicating mRNA expressing VG-HR1) and the coding sequence portion of VG-HR1 (copy number 6 to 48 hours after transfection/copy number 2 hours after transfection) ( Figures 7A and 7B) and a schematic diagram of the self-replication principle ( Figure 7C).
  • Fig. 8 Changes in body weight of mice in the groups administered with LNPs encapsulating self-replicating mRNA and non-self-replicating mRNA expressing VG-HR13 (Groups 2 to 4 and 8 ( Fig. 8A ) and Groups 5 to 8 ( Fig. 8B )).
  • Granzyme B levels (CD8 + T cell immunity levels) at week 4 in mice in the groups (Groups 2 to 8) administered with LNPs encapsulating self-replicating mRNA and non-self-replicating mRNA expressing VG-HR13.
  • HER2 Human epidermal growth factor receptor-2
  • Wild-type HER2 is a 185kD transmembrane glycoprotein composed of 1255 amino acids and is an important member of the tumorigenesis pathway. HER2 can be roughly divided into a cysteine-rich extracellular domain (ECD), a lipophilic transmembrane domain (TMD), and an intracellular domain (ICD) with tyrosine kinase catalytic activity.
  • ECD cysteine-rich extracellular domain
  • TMD lipophilic transmembrane domain
  • ICD intracellular domain
  • HER2 variant antigens can be produced by transforming wild-type HER2.
  • HER2 variant antigens are produced by transforming the ICD of wild-type HER2.
  • the JAK-STAT signal transduction pathway is the main signal transduction mechanism of various cytokines and growth factors. JAK activation stimulates cell proliferation, differentiation, cell migration and apoptosis. These cell events are essential for hematopoiesis, immune development, mammary gland development and lactation, adipogenesis, bisexual growth and other processes.
  • TK tyrosine kinase active site in the coding sequence of the intracellular domain (ICD) of wild-type HER2 is inactivated by mutation to inhibit the unexpected activation of the JAK-STAT signal transduction pathway, thereby obtaining the DNA sequence of the antigen variant of HER2.
  • a HER2 variant antigen is produced by replacing the TMD of wild-type HER2 with another domain.
  • the major histocompatibility complex 1 (MHC1) trafficking domain (MITD) is a domain that guides the cytotoxic T lymphocyte (CTL) epitope of the antigen to the MHC1 molecule located on the endoplasmic reticulum to improve the efficiency of antigen presentation. Therefore, in a specific embodiment, a HER2 variant antigen is produced by replacing the TMD of wild-type HER2 with a MITD.
  • the HER2 variant antigen is produced by replacing the signal peptide (SP) of wild-type HER2 with another SP.
  • the HER2 variant antigen is produced by replacing the SP of wild-type HER2 with the SP of MITD.
  • amino acid sequence of the HER2 variant antigen is as shown in SEQ ID NO: 2 or 4.
  • the coding sequence of the HER2 variant antigen is human codon optimized.
  • the coding sequence of the HER2 variant antigen is as shown in SEQ ID NO: 1 or 3.
  • the 5' end of the coding sequence of the HER2 variant antigen is connected to a Kozak sequence.
  • the Kozak sequence is as shown in GCCACCATGG (DNA sequence) or GCCACCAUGG (RNA sequence).
  • RNA replicase is an RNA-dependent RNA polymerase (RdRp), which can synthesize RNA using RNA as a template, also known as RNA synthetase.
  • RdRp exists in most RNA viruses and plays a role in replicating viral RNA and synthesizing mRNA.
  • the mRNA vaccine of the present invention contains a coding sequence of an RNA replicase to achieve mRNA self-replication.
  • the mRNA vaccine of the present invention comprises a coding sequence of a non-structural polyprotein of a virus, wherein the coding sequence of the non-structural polyprotein of the virus contains a coding sequence of an RNA replicase to achieve mRNA self-replication.
  • the HER2 variant antigen may preferably be expressed in an expression cassette derived from a virus.
  • the virus is a virus of the Togaviridae family.
  • the virus is a virus of the genus Alphavirus, for example, it can be selected from: Aura virus, Barmah Forest virus, Bebaru virus, Cabassou virus, Chikungunya virus, Eastern equine encephalitis virus, Everglades virus, Fort Morgan virus, Getah virus, Highlands J virus, Mayaro virus, Middelburg virus, Mosso das Pedras virus, Mucambo virus, Ndumu virus.
  • the virus is Venezuelan equine encephalitis virus (VEEV).
  • the virus is VEEV TC-83 strain.
  • VEEV The genome of VEEV is roughly composed of the following parts: 5' untranslated sequence (UTR), coding sequence of nonstructural polyprotein, coding sequence of structural polyprotein and 3'UTR.
  • nonstructural polyprotein includes enzymes that maintain viral replication
  • structural polyprotein is mainly viral envelope protein.
  • the viral expression cassette is preferably prepared by replacing the coding sequence of the structural polyprotein in the viral genome (the part after the promoter of the structural polyprotein) with the coding sequence of the HER2 variant antigen.
  • the 5' end of the coding sequence of the HER2 variant antigen is connected to the Kozak sequence.
  • the Kozak sequence is shown as GCCACCATGG (DNA sequence) or GCCACCAUGG (RNA sequence).
  • an RNA polymerase promoter site is attached to the 5' side of the 5'UTR.
  • polyadenine (poly A) and a restriction endonuclease site are attached to the 3' side of the 3'UTR.
  • the viral expression cassette includes, from the 5' end to the 3' end: an RNA polymerase promoter site, a 5'UTR, a coding sequence of a non-structural polyprotein, a promoter sequence of a structural polyprotein, a coding sequence of a HER2 variant antigen, a 3'UTR, poly A and a restriction endonuclease site.
  • the RNA polymerase is preferably T7 RNA polymerase.
  • the promoter of the structural polyprotein is the 26S promoter of a virus of the genus Alphavirus.
  • the promoter of the structural polyprotein is the 26S promoter of VEEV.
  • the cleavage site of the restriction endonuclease can be the cleavage site of any restriction endonuclease, preferably the cleavage site of the BspQI restriction endonuclease.
  • the 5' end of eukaryotic mRNA usually has a bridged 7-methylguanosine (m7G) cap structure (Cap0).
  • m7G 7-methylguanosine
  • Cap1 Cap1 structure
  • the 5' cap structure can also protect mRNA from degradation by nuclease exonucleases, work in conjunction with translation initiation factor proteins, recruit ribosomes, and assist ribosomes in binding to mRNA, so that translation starts from AUG.
  • the Cap structure can recognize each other with the eukaryotic initiation factor 4E (eIF4E) at the initiation stage of translation and start the subsequent translation process.
  • eIF4E eukaryotic initiation factor 4E
  • the Cap1 structure can greatly reduce the immunogenicity of mRNA in vivo.
  • the 5' cap that can be used in the present invention is not particularly limited.
  • the 5' cap used for self-replication is m7G(5')ppp(5')(2'-OMeA)pU, whose molecular formula is C 31 H 42 N 12 O 25 P 4 and whose structural formula is as follows;
  • capping methods for preparing mRNA by in vitro transcription, including enzymatic capping, co-transcriptional capping, etc.
  • Enzymatic capping is a more traditional capping method. After the IVT reaction involving T7 polymerase is completed, the uncapped mRNA is purified first, and then Cap0 is produced by vaccinia virus capping enzyme (which has RNA triphosphatase activity, guanylyltransferase activity and guanine methyltransferase activity), which is then converted into Cap1 by 2'-O-methyltransferase and S-adenosylmethionine, and purified again to obtain the final mRNA.
  • vaccinia virus capping enzyme which has RNA triphosphatase activity, guanylyltransferase activity and guanine methyltransferase activity
  • One-step co-transcriptional capping is to directly add a cap analog to the IVT reaction system involving T7 polymerase to obtain mRNA containing the Cap1 structure in one step, and only one purification is required throughout the process.
  • This reaction method reduces the preparation steps, thereby effectively shortening the overall processing time, simplifying the purification steps, and reducing the number of enzymes required. Therefore, the chemical co-transcriptional capping process is relatively simple, introduces fewer impurities, and can rapidly increase the production capacity of mRNA vaccines and drugs.
  • one-step co-transcriptional capping is gradually becoming the mainstream technical route for mRNA preparation technology.
  • the template of self-replicating mRNA may start with 5'- TAATACGACTCACTATA AT...-3', wherein the underlined region is the promoter region of RNA polymerase, more preferably the promoter region of T7 RNA polymerase.
  • the uridine triphosphate that can be used in the present invention is not particularly limited, and can be natural uridine triphosphate or any modified uridine triphosphate commonly used in the art.
  • UTP is N1- methylpseudouridine triphosphate (N1-Me-pUTP, usually represented as " ⁇ "), whose molecular formula is C10H14N2Na3O15P3 , and whose structural formula is as follows :
  • the 5'UTR and 3'UTR are derived from the species from which the expression cassette containing the coding sequence of RNA replicase is derived.
  • the mRNA vaccine of the present invention is preferably encapsulated in a protective carrier.
  • the encapsulation carrier of mRNA that can be used in the present invention is not particularly limited.
  • nanoparticle-type carriers are used to encapsulate mRNA in the present invention.
  • nanoparticles containing lipids are used in the present invention to encapsulate mRNA.
  • LNP may include but is not limited to liposomes and micelles.
  • the lipid nanoparticles may include cationic and/or ionizable lipids, anionic lipids, neutral lipids, amphiphilic lipids, pegylated lipids and/or structural lipids.
  • the LNP may comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) cationic and/or ionizable lipids.
  • “Cationic lipid” generally refers to a lipid that carries any number of net positive charges at a certain pH (e.g., physiological pH).
  • the cationic lipids may include, but are not limited to, SM102, 3-(didodecylamino)-N1,N1,4-triadecyl-1-piperazineethylamine (KL10), N1-[2-(triadecylamino)ethyl]-N1,N4,N4-triadecyl-1,4-piperazinediethylamine (KL22), 14,25-tricosyl-15,18,21,24-tetraazaoctaporane (KL25), DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, octyl-CLinDMA, octyl-CLinDMA (2S), DODAC, DOTMA, DDAB, DOTAP, DOTAP.C1, DC-Choi, DOSPA, DOGS, DODAP, DODMA and DMRIE.
  • KL10 3-(didodecylamino)-N1,
  • the molar ratio of the cationic lipid in the lipid nanoparticle is about 40-70%, for example, about 40-65%, about 40-60%, about 45-55% or about 48-53%.
  • the LNP may include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) non-cationic lipids.
  • the non-cationic lipids may include anionic lipids.
  • Anionic lipids suitable for lipid nanoparticles of the present application may include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylphosphatidylethanolamine, and other neutral lipids having an anionic group connected thereto.
  • the non-cationic lipid may include a neutral lipid, which may include, for example, a phospholipid, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (PO ...phosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoylphosphatidyl
  • the molar ratio of the phospholipid in the lipid nanoparticles is about 5-20%.
  • the LNP may include lipid conjugates, for example, polyethylene glycol (PEG)-modified lipids and derived lipids.
  • PEG-modified lipids may include, but are not limited to, polyethylene glycol chains covalently linked to lipids with alkyl chains of lengths of C6 to C20, up to a length of 5 kDa. The addition of these components can prevent lipid aggregation, increase circulation duration, facilitate lipid-nucleic acid composition delivery to target cells, or quickly release nucleic acids.
  • the polyethylene glycol (PEG)-modified lipid molecule can be a PEG-ceramide with a shorter acyl chain (e.g., C14 or C18).
  • the polyethylene glycol (PEG)-modified lipid molecule has a molar ratio of about 0.5 to 2% in lipid nanoparticles, for example, about 1 to 2%, about 1.2 to 1.8%, or about 1.4 to 1.6%.
  • the polyethylene glycol (PEG)-modified lipid molecule can be PEG2000-DMG.
  • the LNP may further comprise cholesterol.
  • the molar ratio of cholesterol in the lipid nanoparticle is about 30-50%, for example, about 35-45%, or about 38-42%.
  • the LNP may include cationic lipids, cholesterol, phospholipids and lipid molecules modified with polyethylene glycol.
  • the molar ratio of the cationic lipids, cholesterol, phospholipids and lipid molecules modified with polyethylene glycol may be 45-55:35-45:5-15:0.5-2.
  • Example 1 Production of self-replicating mRNA and non-self-replicating mRNA expressing HER2 antigen variants
  • the DNA sequence of wild-type HER2 was obtained from NCBI (https://www.ncbi.nlm.nih.gov/), and the tyrosine kinase (TK) active site in the coding sequence of the intracellular domain (ICD) was inactivated to inhibit the unexpected activation of the JAK-STAT signaling pathway, thereby obtaining the DNA sequence of an antigen variant of HER2, VG-HR1 (SEQ ID NO: 1).
  • the amino acid sequence of VG-HR1 is shown in SEQ ID NO: 2, and its structural schematic diagram is shown in Figure 1A.
  • VG-HR13 SEQ ID NO: 3
  • the amino acid sequence of VG-HR13 is shown in SEQ ID NO: 4, and its structural schematic diagram is shown in Figure 1B.
  • the coding sequence of the structural polyprotein (the part after the promoter of the structural polyprotein) was replaced with "Kozak sequence + DNA sequence of VG-HR1 shown in SEQ ID NO: 1" or "Kozak sequence + DNA sequence of VG-HR13 shown in SEQ ID NO: 3".
  • a promoter site of T7 RNA polymerase was further added to the 5' side of the 5'UTR of the above construct.
  • Poly A and BspQI restriction endonuclease cleavage sites were further added to the 3' side of the 3'UTR of the above construct.
  • the expression cassette for expressing the self-replicating mRNA of VG-HR1 derived from VEEV TC-83 strain ( Figure 2A) and the expression cassette for expressing the self-replicating mRNA of VG-HR13 ( Figure 2B) constructed in this way include from the 5' end to the 3' end:
  • FIG. 2A An expression cassette for expressing the self-replicating mRNA of VG-HR1 ( FIG. 2A ) (SEQ ID NO: 5): a promoter site of T7 RNA polymerase, 5′UTR, a coding sequence of a nonstructural polyprotein, a 26S promoter, “Kozak sequence + the DNA sequence of VG-HR1 shown in SEQ ID NO: 1”, a 3′UTR, a poly A and a cleavage site of a BspQI restriction endonuclease;
  • Expression cassette for expressing self-replicating mRNA of VG-HR13 ( Figure 2B) (SEQ ID NO: 6): promoter site of T7 RNA polymerase, 5'UTR, coding sequence of non-structural polyprotein, 26S promoter, "Kozak sequence + DNA sequence of VG-HR13 shown in SEQ ID NO: 3", 3'UTR, poly A and cleavage site of BspQI restriction endonuclease.
  • an expression cassette for expressing non-self-replicating mRNA of VG-HR1 ( FIG. 3A ) and an expression cassette for expressing non-self-replicating mRNA of VG-HR13 ( FIG. 3B ) were constructed in a similar manner, and they included from the 5′ end to the 3′ end:
  • FIG. 3A An expression cassette for expressing non-self-replicating mRNA of VG-HR1 ( FIG. 3A ): a promoter site of T7 RNA polymerase, a 5′UTR, a “Kozak sequence + a DNA sequence of VG-HR1 shown in SEQ ID NO: 1”, a 3′UTR, a poly A and a cleavage site of a BspQI restriction endonuclease;
  • Expression cassette for expressing non-self-replicating mRNA of VG-HR13 ( Figure 3B): promoter site of T7 RNA polymerase, 5'UTR, "Kozak sequence + DNA sequence of VG-HR13 shown in SEQ ID NO: 3", 3'UTR, poly A and cleavage site of BspQI restriction endonuclease.
  • the expression cassettes for expressing the self-replicating mRNA of VG-HR1 and the self-replicating mRNA of VG-HR13 constructed in Example 1_2, as well as the expression cassettes for expressing the non-self-replicating mRNA of VG-HR1 and the non-self-replicating mRNA of VG-HR13 constructed in Example 1_2 were respectively inserted into the pUC57-Kan plasmid to obtain the plasmids psaRNA-VG-HR1 and psaRNA-VG-HR13 containing the expression cassette of the self-replicating mRNA ( Figure 4 ) and the plasmids pnsaRNA-VG-HR1 and pnsaRNA-VG-HR13 containing the expression cassette of the non-self-replicating mRNA, respectively.
  • the plasmid containing the expression cassette of the self-replicating mRNA and the expression cassette of the non-self-replicating mRNA obtained in Example 1_3 was linearized by incubation at 50°C for 2 hours. After the linearization was completed, ddH 2 O was further added to the reaction system to a total volume of 100 ⁇ l.
  • the linearized plasmid containing the expression cassette of the self-replicating mRNA and the expression cassette of the non-self-replicating mRNA was purified and recovered using an agarose gel DNA recovery kit and used as an in vitro transcription (IVT) template.
  • Self-replicating mRNA was obtained by IVT at 37°C for 2 hours. After the reaction, 4U DNase I (Novozyme, EN401) was added to the reaction system and reacted at 37°C for 15 minutes. RNase-free ddH 2 O was added to the reaction system to a total volume of 50 ⁇ L, and 25 ⁇ L of 5M LiCl solution was further added and mixed. After standing at -20°C for 30 minutes, centrifuged at 13000 ⁇ g at 4°C for 10 minutes and the supernatant was discarded. The precipitate was washed with 70% ethanol, centrifuged at 13000 ⁇ g at 4°C for 2 minutes and the supernatant was discarded, and the precipitate was dissolved in 30 ⁇ L of water.
  • 4U DNase I Novozyme, EN401
  • the concentration was determined using a spectrophotometer. 500 ng of self-replicating mRNA obtained by in vitro transcription was mixed with 2 ⁇ l 2 ⁇ RNA Loading Dye, incubated at 70°C for 10 minutes, and then detected by denaturing agarose gel electrophoresis. The electrophoresis spectrum when linearized psaRNA-VG-HR1 was used as an IVT template is shown in Figure 5A. At the same time, the integrity of the self-replicating mRNA obtained by in vitro transcription was detected by capillary electrophoresis. The capillary electrophoresis integrity detection data when linearized psaRNA-VG-HR1 was used as an IVT template are shown in Figure 5B. These data indicate that psaRNA-VG-HR1 obtained by in vitro transcription has higher integrity, which is conducive to the effective replication and expression of mRNA.
  • the self-replicating mRNA and non-self-replicating mRNA obtained as IVT products in Example 1-5 were dissolved in citrate buffer, and then mixed with a lipid mixture dissolved in ethanol (ionizable lipid ALC-0315: distearoylphosphatidylcholine: cholesterol: PEG lipid ALC-0159) using a microfluidic device NanoAssembler to obtain lipid nanoparticles (lipid nanoparticals, LNPs) encapsulating self-replicating mRNA and non-self-replicating mRNA. These lipid nanoparticles were mixed with phosphate buffered saline, and residual ethanol was removed by ultrafiltration. The LNPs thus obtained were characterized, and the results are shown in Table 3 below.
  • LNPs were mixed with cryoprotectants and stored at -80°C.
  • HEK-293T cells derived from human embryonic kidney cells were inoculated in a 75 cm2 cell culture flask containing DMEM high glucose medium (+10% fetal bovine serum +1% double antibody) to a confluence of more than 80% in the culture flask. The cells were digested and counted with trypsin. An appropriate number of suspended HEK-293T cells were plated in a 24-well cell culture plate containing Opti-MEM medium and cultured overnight at 37°C in a CO2 incubator.
  • DMEM high glucose medium +10% fetal bovine serum +1% double antibody
  • Example 2_3 After 24 hours of transfection of the HEK-293T cells in the above-mentioned 24-well cell culture plate with 0.5 ⁇ g of LNPs encapsulating the self-replicating mRNA and non-self-replicating mRNA expressing the HER2 antigen variants VG-HR1 and VG-HR13 obtained in Example 1, the transfected cell culture was used for the protein blot (Western Blot) assay in the following Example 2_2 and the detection of the amplification efficiency of the self-replicating mRNA in Example 2_3.
  • the transfected cell culture was used for the protein blot (Western Blot) assay in the following Example 2_2 and the detection of the amplification efficiency of the self-replicating mRNA in Example 2_3.
  • Example 2_1 After adding 90 ⁇ l of cell lysate containing 1% protease inhibitor to the cell culture obtained by transfection in Example 2_1, place on ice for 5 minutes. Centrifuge at 14000 ⁇ g for 10 minutes, transfer the supernatant to a new centrifuge tube, add NuPAGE LDS sample buffer, and place in a metal bath at 95°C for 10 minutes. The cell lysate obtained in this way is stored at -20°C.
  • the cell lysate was subjected to polyacrylamide gel electrophoresis (PAGE) at 200V for 30 minutes. After the electrophoresis, the gel was transferred to the membrane at 25V for 7 minutes using a transfer kit (Trans-Blot Turbo 0.2 ⁇ m PVDF Transfer Kit, Bio-rad 1704272). After the transfer, the membrane was blocked with TBSTw blocking solution (Biyuntian, ST673) for 15 minutes, and then incubated with anti-HER2 monoclonal antibody (Abcam, ab221438) diluted 1:1000 as the first antibody at room temperature for 1 hour. Wash with 1 ⁇ TBST 5 times, 5 minutes each time.
  • PAGE polyacrylamide gel electrophoresis
  • Example 2_1 Aspirate the supernatant from the cell culture obtained by transfection in Example 2_1, rinse the cells once with 500 ⁇ l PBS and then aspirate the supernatant. After further adding 500 ⁇ l PBS, blow the cells until the cells fall off. Collect the detached cells into an Eppendorf centrifuge tube, centrifuge at 1000rpm for 5 minutes and then discard the supernatant. Store the obtained cells at -80°C.
  • Upstream primer F of the coding sequence of the nonstructural polyprotein of VEEV TC-83 strain 5'-AGCAGAGATA GTATTGAAC-3' (SEQ ID NO: 7);
  • the downstream primer R of the coding sequence of the non-structural polyprotein of VEEV TC-83 strain is 5'-TAATGGATAA CGGAACAG-3' (SEQ ID NO: 8);
  • Upstream primer F of the coding sequence of VG-HR1 5'-ACTACCTCAG CCTCCTAT-3' (SEQ ID NO: 9);
  • Upstream primer F of the coding sequence of GAPDH 5'-GGTATCGTGG AAGGACTC-3' (SEQ ID NO: 11);
  • ⁇ Downstream primer R of the coding sequence of GAPDH 5'-GTAGAGGCAG GGATGATG-3' (SEQ ID NO: 12).
  • the relative change in the copy number of the self-replicating mRNA expressing VG-HR1 (copy number 6 to 48 hours after transfection/copy number 2 hours after transfection) is shown in Figure 7.
  • Figure 7 The copy number of the coding sequence part of the non-structural polyprotein (or the full length of the self-replicating mRNA expressing VG-HR1) 24 hours after transfection is 98.85 times the copy number 2 hours after transfection ( Figure 7A), while the copy number of the coding sequence part of VG-HR1 24 hours after transfection is 4666.18 times the copy number 2 hours after transfection ( Figure 7B).
  • mice Female BALB/c mice aged 6 to 8 weeks were divided into 8 groups of 5 mice each. After 3 days of adaptive feeding observation, the LNPs encapsulating the self-replicating mRNA and non-self-replicating mRNA of the antigenic variants VG-HR1 and VG-HR13 expressing HER2 prepared in Example 1 were initially administered by intramuscular injection on day 0, and the corresponding LNPs were administered by intramuscular injection on day 14. Physiological saline was administered to Group 1 (control group). The properties of the LNPs administered in each group are shown in Table 4 below.
  • mice in groups (groups 2 to 8) administered with LNPs encapsulating self-replicating mRNA and non-self-replicating mRNA expressing VG-HR13 showed a weight loss of less than 10%, but their weight returned to normal levels within 4 days. This shows that whether the mRNA in the LNP is self-replicating mRNA or non-self-replicating mRNA, whether the uridine triphosphate in the mRNA is UTP or N1-Me-pUTP, there is no obvious side effect on the vaccinated object, and it is safe enough.
  • Dilute ELISA Plate Coating buffer (Elabscience, E-ELIR-001) with deionized water to the working concentration (1 ⁇ ), and then prepare a coating solution containing 1.5 ⁇ g/ml HER2 (Yeasen, 93098ES20). Then, add 100 ⁇ l HER2 coating solution to the high binding EIA/RIA plate (Corning costar, 42592), cover with sealing film, and incubate overnight at 4°C.
  • the mouse serum obtained on the specified date after immunization was diluted 100 times for detection, and the diluted sample was added to the blocked ELISA plate, 100 ⁇ l per well, and incubated at room temperature for 2h. After completion, the supernatant was discarded and the washing buffer was added to wash 3 times.
  • HRP-conjugated Affinipure goat anti-mouse IgG H+L was diluted 1:10000 times with sample diluent, and then added to the ELISA plate and incubated at room temperature for 1h. After incubation, the supernatant was discarded and the washing buffer was added to wash 3 times.
  • TMB substrate (Solarbio, PR1200) solution was added to each reaction well, and the color was developed at room temperature for about 10 minutes, and then 0.1ml of stop solution was added to each reaction well.
  • concentration of specific anti-HER2 antibodies was detected by measuring the absorbance (OD450) at 450nm using an ELISA reader.
  • the concentration of specific anti-HER2 antibodies in the serum of mice in the groups (groups 2 to 8) that were administered with LNPs encapsulating self-replicating mRNA and non-self-replicating mRNA expressing VG-HR13 at week 4 was significantly higher than that in the serum of mice in week 2, compared with group 1 (control group) administered with normal saline.
  • group 1 control group
  • the serum antibody level equivalent to or even higher than that when 44 ⁇ g of non-self-replicating mRNA/mouse was administered could be achieved. This indicates that self-replicating mRNA has a significantly better antibody induction effect than non-self-replicating mRNA.
  • mice were killed on the 28th day, and the spleens were collected to obtain spleen cells.
  • spleen cells of each group were resuspended in 1640 culture medium, they were evenly inoculated into U-bottom 96-well plates, and HER2 protein with a final concentration of 1 ⁇ g/ml was added for 24 hours. During the period, after 18 hours of culture, the cells were centrifuged, the culture medium was removed, and 100 ⁇ l of fresh culture medium containing Golgiplug (BD, 555029) was added. The positive control group was added with culture medium containing T cell stimulators and monnesin, and culture was continued for 4 to 6 hours.
  • Golgiplug Golgiplug
  • the cells were centrifuged and washed once with PBS, and 50 ⁇ l of cell surface antibodies prepared with PBS were added, and incubated at 4°C in the dark for 30 minutes. After that, the cells were centrifuged and washed once with 1640 culture medium containing 2% FBS, and 50 ⁇ l of fixative was added, and fixed at 4°C for 1 hour. After that, the cells were centrifuged, rinsed once with washing buffer, and 50 ⁇ l of cytokine antibodies were added, and incubated at 4°C for 1 hour. Finally, the cells were centrifuged, rinsed once with washing buffer, and the cells were resuspended with PBS and detected on a flow cytometer.

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Abstract

一种信使核糖核酸（mRNA）分子，其包含：表达盒1，其含有RNA复制酶的编码序列、和表达盒2，其含有抗原的编码序列。一种mRNA疫苗，其通过将上述mRNA分子包封为脂质纳米颗粒（LNP）得到。一种DNA分子，其被转录成上述mRNA分子。一种mRNA分子在制造引发抗HER2免疫应答的mRNA疫苗中的用途。一种mRNA分子的制造方法，其包括：（1）基于病毒的基因组的DNA序列，将其中结构性多蛋白的编码序列中的结构性多蛋白的启动子之后的部分替换为抗原的编码序列；（2）向5'UTR的5'侧附加RNA聚合酶的启动子位点；（3）向3'UTR的3'侧附加多聚A；及（4）将通过步骤（1）～（3）构建的DNA分子转录为mRNA分子。

Description

自复制信使核糖核酸疫苗 【技术领域】
本发明涉及自复制信使核糖核酸(mRNA)分子、自复制mRNA疫苗、用于转录成自复制mRNA分子的DNA分子、自复制mRNA分子在制造引发抗HER2免疫应答的自复制mRNA疫苗中的用途、及自复制mRNA分子的制造方法。
 【背景技术】
人类表皮生长因子受体2(HER2)是一种表达于正常细胞表面的功能受体，但在30％的乳腺癌患者的瘤组织会中出现异常高表达，是一类肿瘤相关抗原。在HER2高表达的患者中，肿瘤会表现出更强的肿瘤侵袭性及更高的复发频率。目前临床数据显示，机体内产生了针对HER2的细胞毒性免疫反应的患者群体，其生存期或复发概率等指标都有明显提升，提示通过表达HER2通过诱导机体产生针对HER2的特异性免疫反应来攻击和杀死高表达该抗原的肿瘤细胞，同时形成免疫记忆，使机体获得长期的免疫治疗或预防效果的可行性。
在基于免疫细胞、多肽、病毒载体和核酸分子在内的多种肿瘤疫苗形式中，mRNA疫苗表现出了独特的优势。mRNA疫苗进入细胞质中即可完成抗原的翻译表达，且不整合至基因组中，避免了抗原基因插入风险。mRNA疫苗证明了能够同时诱导体液免疫和细胞免疫，在体内诱导强免疫应答。mRNA疫苗使用体外转录及微流控混合包封纳米脂质颗粒的无细胞制备方法，摆脱活细胞依赖生产，便于大量生产，缩短生产周期，简化了生产纯化工艺。
目前，线性mRNA疫苗又分为非复制和自复制两种，自复制mRNA通过改造RNA病毒基因组结构而来，利用病毒复制酶来高效扩增表达抗原的mRNA分子数量，以达到以少量mRNA分子表达大量抗原蛋白的目的，可以大大降低mRNA疫苗接种量。另外，由于自复制mRNA可以潜在激活天然免疫反应，因此可以进一步提高机体的免疫反应，形成佐剂效应。
已经进入临床试验的基于肿瘤相关抗原的mRNA肿瘤疫苗分为通过细胞生产得到的重组病毒RNA复制子颗粒疫苗或者无细胞合成的基于脂质纳米颗粒的非复制mRNA疫苗。前者通过在细胞中生产包装出仅有单次感染能力的重组病毒RNA复制子颗粒，通过病毒蛋白与细胞受体的相互作用将自复制mRNA分子高效递送，由于自复制mRNA分子的放大作用，可以实现肿瘤相关抗原高水平表达以刺激机体免疫，但由于病毒载体本身的免疫原性，有可能导致非预期的针对载体本身的免疫反应，进而可能阻止未来继续使用相同的病毒载体进行加强接种的应用。此外，重组病毒载体技术要求较高且生产工艺复杂，一定程度上会限制病毒载体肿瘤疫苗的快速应用。
基于脂质纳米颗粒递送的非复制mRNA疫苗，通过纳米脂质颗粒以潜在的脂质受体相互作用将编码肿瘤相关抗原的非复制mRNA递送至细胞进行表达。由于mRNA分子的合成及装载至纳米脂质颗粒都通过无细胞基质的体外转录及包封，因此生产制备工艺简单，有利于疫苗的快速合成使用。但非复制mRNA表达抗原的数量与在免疫过程中递送成功的mRNA数量成正比，而且半衰期较短，因此，为了获得在体内引起足够免疫保护效果所需的抗原表达水平，需要接种较多剂量的疫苗或者反复接种。
【发明内容】
本发明涉及下列实施方式：
1.信使核糖核酸(mRNA)分子，其包含：
表达盒1，其含有RNA复制酶的编码序列、和
表达盒2，其含有抗原的编码序列。
2.根据前述实施方式所述的mRNA分子，其中所述RNA复制酶是病毒的RNA复制酶。
3.根据前述实施方式所述的mRNA分子，其中所述表达盒1源于病毒的非结构性多蛋白的编码序列。
4.根据前述实施方式所述的mRNA分子，其中所述病毒是披膜病毒科(Togaviridae)的病毒。
5.根据前述实施方式所述的mRNA分子，其中所述披膜病毒科(Togaviridae)的病毒是甲病毒属(Alphavirus)的病毒。
6.根据前述实施方式所述的mRNA分子，其中所述甲病毒属(Alphavirus)的病毒是委内瑞拉马脑炎病毒(Venezuelan equine encephalitis virus，VEEV)。
7.根据前述实施方式所述的mRNA分子，其中所述抗原是HER2抗原或其变体。
8.根据前述实施方式所述的mRNA分子，其中所述HER2抗原变体具有经改造的细胞内结构域(ICD)，其中酪氨酸激酶(TK)活性位点被失活突变。
9.根据前述实施方式所述的mRNA分子，其中所述HER2抗原变体具有主要组织相容性复合体1(MHC1)转运结构域(MITD)，所述MITD置换了HER2抗原的跨膜结构域(TMD)。
10.根据前述实施方式所述的mRNA分子，其中所述HER2抗原变体具有MITD的信号肽(SP)，所述MITD的SP置换了HER2抗原的SP。
11.根据前述实施方式所述的mRNA分子，其中所述HER2抗原变体具有SEQ ID NO：2或4所示的序列。
12.根据前述实施方式所述的mRNA分子，其中所述HER2抗原变体的编码序列具有SEQ ID NO：1或3所示的序列。
13.根据前述实施方式所述的mRNA分子，其中所述HER2抗原或其变体的编码序列的5'侧具有Kozak序列。
14.根据前述实施方式所述的mRNA分子，其中所述Kozak序列是GCCACCAUGG。
15.根据前述实施方式所述的mRNA分子，其中所述表达盒2具有启动子，所述抗原的编码序列位于所述启动子下游。
16.根据前述实施方式所述的mRNA分子，其中所述启动子源于所述表达盒1所来源的物种。
17.根据前述实施方式所述的mRNA分子，其中所述启动子是26S启动子。
18.根据前述实施方式所述的mRNA分子，其中
所述表达盒1的上游还具有RNA聚合酶的启动子位点和5'UTR；且
所述表达盒2的下游还具有3'UTR和多聚A。
19.根据前述实施方式所述的mRNA分子，其中所述RNA聚合酶是T7 RNA聚合酶。
20.根据前述实施方式所述的mRNA分子，其中所述5'UTR和3'UTR源于所述表达盒1所来源的物种。
21.根据前述实施方式所述的mRNA分子，其对应的DNA序列具有SEQ ID NO：5或6所示的序列。
22.根据前述实施方式所述的mRNA分子，其中的尿苷三磷酸是天然尿苷三磷酸(UTP)或N1-甲基假尿苷三磷酸(N1-Me-pUTP)。
23.信使核糖核酸(mRNA)疫苗，其通过将根据前述实施方式所述的mRNA分子包封为脂质纳米颗粒(LNP)得到。
24.DNA分子，其被转录成根据前述实施方式所述的mRNA分子。
25.根据前述实施方式所述的mRNA分子在制造引发抗HER2免疫应答的信使核糖核酸(mRNA)疫苗中的用途。
26.信使核糖核酸(mRNA)分子的制造方法，其包括：
(1)基于病毒的基因组的DNA序列，将其中结构性多蛋白的编码序列中的结构性多蛋白的启动子之后的部分替换为抗原的编码序列；
(2)向5'UTR的5'侧附加RNA聚合酶的启动子位点；
(3)向3'UTR的3'侧附加多聚A；及
(4)将通过步骤(1)～(3)构建的DNA分子转录为mRNA分子。
27.根据前述实施方式所述的方法，其中所述病毒是披膜病毒科(Togaviridae)的病毒。
28.根据前述实施方式所述的方法，其中所述披膜病毒科(Togaviridae)的病毒是甲病毒属(Alphavirus)的病毒。
29.根据前述实施方式所述的方法，其中所述甲病毒属(Alphavirus)的病毒是委内瑞拉马脑炎病毒(Venezuelan equine encephalitis virus，VEEV)。
30.根据前述实施方式所述的方法，其中所述抗原是HER2抗原或其变体。
31.根据前述实施方式所述的方法，其中所述HER2抗原变体具有经改造的细胞内结构域(ICD)，其中酪氨酸激酶(TK)活性位点被失活突变。
32.根据前述实施方式所述的方法，其中所述HER2抗原变体具有主要组织相容性复合体1(MHC1)转运结构域(MITD)，所述MITD置换了HER2抗原的跨膜结构域(TMD)。
33.根据前述实施方式所述的方法，其中所述HER2抗原变体具有MITD的信号肽(SP)，所述MITD的SP置换了HER2抗原的SP。
34.根据前述实施方式所述的方法，其中所述HER2抗原变体具有SEQ ID NO：2或4所示的序列。
35.根据前述实施方式所述的方法，其中所述HER2抗原变体的编码序列具有SEQ ID NO：1或3所示的序列。
36.根据前述实施方式所述的方法，其中所述HER2抗原或其变体的编码序列的5'侧具有Kozak序列。
37.根据前述实施方式所述的方法，其中所述Kozak序列是GCCACCATGG。
38.根据前述实施方式所述的方法，其中通过步骤(1)～(3)构建的DNA分子具有SEQ ID NO：5或6所示的序列。
39.根据前述实施方式所述的方法，其中所述转录是体外转录。
40.根据前述实施方式所述的方法，其中所述体外转录使用：
体外转录用核酸载体，其中插入通过步骤(1)～(3)构建的DNA分子，
腺苷三磷酸、胞苷三磷酸、鸟苷三磷酸和尿苷三磷酸，及
RNA聚合酶。
41.根据前述实施方式所述的方法，其中所述尿苷三磷酸是天然尿苷三磷酸(UTP)或N1-甲基假尿苷三磷酸(N1-Me-pUTP)。
42.根据前述实施方式所述的方法，其中所述RNA聚合酶是T7 RNA聚合酶。
【技术效果】
通过上述实施方式，本发明至少达到了如下技术效果：
(1)自复制mRNA在进入细胞进行表达复制时，共同编码抗原分子及非结构多蛋白及单独编码抗原的全长自复制mRNA都会发生高水平复制，但单独编码抗原分子的自复制mRNA会有更高的复制水平，从而在尽量避免病毒组分表达带来的免疫反应及毒性的情况下实现抗原的高效表达。
(2)自复制mRNA疫苗相比非复制mRNA疫苗激活了更强的针对抗原的免疫反应及肿瘤特异性杀伤的效果。
(3)与非复制mRNA相比，仅有1/10剂量的自复制mRNA肿瘤疫苗可实现相同水平免疫刺激效果，从而可降低mRNA免疫剂量，并降低由于mRNA剂量引起的不良反应，提高安全性。
(4)本发明利用体外转录方法制备自复制mRNA分子并利用微流控技术将mRNA包封至纳米脂质颗粒，实现自复制mRNA肿瘤疫苗的无细胞制备，相比传统疫苗制备路线，该制备方法生产周期短，工艺及质控简单，有利于肿瘤疫苗的快速制备。
【附图简述】
【图1A】HER2的抗原变体VG-HR1的结构示意图。
【图1B】HER2的抗原变体VG-HR13的结构示意图。
【图2A】表达VG-HR1的自复制mRNA的表达盒的结构示意图。
【图2B】表达VG-HR13的自复制mRNA的表达盒的结构示意图。
【图3A】表达VG-HR1的非自复制mRNA的表达盒的结构示意图。
【图3B】表达VG-HR13的非自复制mRNA的表达盒的结构示意图。
【图4】向pUC57-Kan质粒中插入表达VG-HR13的自复制mRNA的表达盒而得到的含自复制mRNA的表达盒的质粒psaRNA-VG-HR13的图谱。
【图5】作为IVT模板使用线性化的psaRNA-VG-HR1时的变性琼脂糖凝胶电泳图谱(图5A)和毛细管电泳完整性检测结果(图5B)。
【图6】用包封了表达VG-HR1的自复制mRNA和非自复制mRNA的LNP的转染HEK-293T细胞后的蛋白印迹(Western Blot)测定结果。
【图7】表达VG-HR1的自复制mRNA的非结构性多蛋白的编码序列部分(或表达VG-HR1的自复制mRNA全长)和VG-HR1的编码序列部分的拷贝数相对变化(转染后6～48小时的拷贝数/转染后2小时的拷贝数)(图7A和图7B)及自复制原理示意图(图7C)。
[根据细则91更正 31.01.2024]
【图8】施用了包封了表达VG-HR13的自复制mRNA和非自复制mRNA的LNP的组(组2～4和8(图8A)和组5～8(图8B))的小鼠的体重变化。
【图9】施用了包封了表达VG-HR13的自复制mRNA和非自复制mRNA的LNP的组(组2～8)的小鼠的第2周和第4周血清中的特异性抗HER2抗体的浓度。
【图10】施用了包封了表达VG-HR13的自复制mRNA和非自复制mRNA的LNP的组(组2～8)的小鼠的第4周的颗粒酶B水平(CD8+T细胞免疫水平)。
 【具体实施方式】
【人表皮生长因子受体-2(HER2)及其变体抗原】
野生型HER2是由1255个氨基酸组成的185kD的跨膜糖蛋白，是肿瘤发生通路中的重要一员。HER2可大致划分为富含半胱氨酸的细胞外结构域(extracellular domain，ECD)、亲脂性的跨膜结构域(transmembrane domain，TMD)和具有酪氨酸激酶催化活性的细胞内结构域(Intracellular domain，ICD)。
在具体的实施方式中，可通过改造野生型HER2而制造出HER2变体抗原。在一个具体实施方式中，通过对野生型HER2的ICD进行改造而制造出HER2变体抗原。JAK-STAT信号传导通路是多种细胞因子和生长因子的主要信号传导机制。JAK激活刺激细胞增殖，分化，细胞迁移和凋亡。这些细胞事件对于造血，免疫发育，乳腺发育和泌乳，脂肪形成，两性性生长和其他过程至关重要。因此，在一个进一步的实施方式中，通过将野生型HER2的细胞内结构域(ICD)的编码序列中的酪氨酸激酶(TK)活性位点进行失活突变，以抑制JAK-STAT信号传导通路的非预期激活，由此得到HER2的抗原变体的DNA序列。
在一个进一步的实施方式中，通过将野生型HER2的TMD替换为别的结构域而制造出HER2变体抗原。主要组织相容性复合体1(major histocompatibility complex 1，MHC1)转运结构域(MHC1 trafficking domain，MITD)是将抗原的细胞毒性T淋巴细胞(CTL)表位引导到位于内质网上的MHC1分子上来提高抗原呈递效率的结构域。因此，在一个具体实施方式中，通过将野生型HER2的TMD替换为MITD而制造出HER2变体抗原。
在一个进一步的实施方式中，通过将野生型HER2的信号肽(Signal Peptide，SP)替换为别的SP而制造出HER2变体抗原。在一个具体实施方式中，通过将野生型HER2的SP替换为MITD的SP而制造出HER2变体抗原。
在优选的实施方式中，HER2变体抗原的氨基酸序列如SEQ ID NO：2或4所示。
在优选的实施方式中，HER2变体抗原的编码序列经人密码子优化。在优选的实施方式中，HER2变体抗原的编码序列如SEQ ID NO：1或3所示。在优选的实施方式中，HER2变体抗原的编码序列的5'端连接了Kozak序列。在一个具体的实施方式中，Kozak序列如GCCACCATGG(DNA序列)或GCCACCAUGG(RNA序列)所示。
【RNA复制酶】
RNA复制酶是依赖于RNA的RNA聚合酶(RNA dependent RNA polymerase，RdRp)，能以RNA为模板合成RNA，亦称RNA合成酶。RdRp存在于大部分RNA病毒中，起到复制病毒RNA以及合成mRNA的作用。
在一个实施方式中，本发明的mRNA疫苗含有RNA复制酶的编码序列，以实现mRNA自复制。在一个实施方式中，本发明的mRNA疫苗包含病毒的非结构性多蛋白的编码序列，所述病毒的非结构性多蛋白的编码序列中含有RNA复制酶的编码序列，以实现mRNA自复制。
【源于病毒的表达盒】
HER2变体抗原可优选在源于病毒的表达盒中表达。在优选的实施方式中，病毒是披膜病毒科(Togaviridae)的病毒。在进一步优选的实施方式中，病毒是甲病毒属(Alphavirus)的病毒，例如，可选自：奥拉病毒(Aura virus)、巴马森林病毒(Barmah Forest virus)、比巴鲁病毒(Bebaru virus)、卡巴斯欧病毒(Cabassou virus)、基孔肯雅病毒(Chikungunya virus)、东方马脑炎病毒(Eastern equine encephalitis virus)、沼泽地病毒(Everglades virus)、摩根堡病毒(Fort Morgan virus)、盖塔病毒(Getah virus)、高地J病毒(Highlands J virus)、马亚罗病毒(Mayaro virus)、米德尔堡病毒(Middelburg virus)、莫斯达斯佩德拉斯病毒(Mosso das Pedras virus)、穆坎布病毒(Mucambo virus)、恩杜穆病毒(Ndumu virus)、欧尼恩病毒(O’nyong-nyong virus)、那皮舒纳病毒(Pixuna virus)、里奥内格罗病毒(Rio Negro virus)、罗斯河病毒(Ross River virus)、鲑鱼胰腺病病毒(Salmon pancreas disease virus)、西门利启森林病毒(Semliki Forest virus)、辛德比斯病毒(Sindbis virus)、南方象海豹病毒(Southern elephant seal virus)、图那特病毒(Tonate virus)、特罗卡拉病毒(Trocara virus)、乌纳病毒(Una virus)、委内瑞拉马脑炎病毒(Venezuelan equine encephalitis virus)、西方马脑炎病毒(Western equine encephalitis virus)及瓦塔罗阿病毒(Whataroa virus)。在进一步优选的实施方式中，病毒是委内瑞拉马脑炎病毒(Venezuelan equine encephalitis virus，VEEV)。在进一步优选的实施方式中，病毒是VEEV TC-83株。
VEEV的基因组大致由如下部分构成：5'非翻译序列(UTR)、非结构性多蛋白(nonstructural polyprotein)的编码序列、结构性多蛋白(structural polyprotein)的编码序列和3'UTR。其中，非结构性多蛋白包括维持病毒复制的酶等，结构性多蛋白主要是病毒包膜蛋白。
在优选的实施方式中，优选通过将病毒基因组中的结构性多蛋白的编码序列(结构性多蛋白的启动子之后的部分)替换为HER2变体抗原的编码序列来制造源于病毒的表达盒。在优选的实施方式中，HER2变体抗原的编码序列的5'端连接了Kozak序列。在一个具体的实施方式中，Kozak序列如GCCACCATGG(DNA序列)或GCCACCAUGG(RNA序列)所示。在进一步的实施方式中，在5'UTR的5'侧附加RNA聚合酶启动子位点。在进一步的实施方式中，在3'UTR的3'侧附加多聚腺嘌呤(多聚A)和限制性内切酶位点。优选的实施方式中，源于病毒的表达盒从5'端至3'端依此包括：RNA聚合酶启动子位点、5'UTR、非结构性多蛋白的编码序列、结构性多蛋白的启动子序列、HER2变体抗原的编码序列、3'UTR、多聚A和限制性内切酶位点。在进一步优选的实施方式中，RNA聚合酶优选为T7 RNA聚合酶。在进一步优选的实施方式中，结构性多蛋白的启动子是甲病毒属(Alphavirus)的病毒的26S启动子。在进一步优选的实施方式中，结构性多蛋白的启动子是VEEV的26S启动子。在进一步优选的实施方式中，限制性内切酶的切割位点可为任何限制性内切酶的的切割位点，优选为BspQI限制性内切酶的切割位点。
【5'帽】
真核生物mRNA的5'端通常具有桥接的7-甲基鸟苷(m7G)帽子结构(Cap0)，Cap0结构中m7G后面第一个核苷的2'羟基甲基化后形成Cap1结构(m7GpppmN)。现有研究发现，5'端帽子结构可以调节mRNA的剪切成熟，并帮助RNA转录产物穿过核膜的选择性孔道而进入细胞质。此外，5'帽子结构还可以保护mRNA不被核酸外切酶降解，与翻译起始因子蛋白协同工作，招募核糖体，并协助核糖体与mRNA结合，使翻译从AUG开始。通常情况下，Cap结构可以与真核起始因子4E(eIF4E)在翻译起始阶段相互识别，开启后续翻译过程，同时Cap1结构能够极大降低mRNA在体内的免疫原性。
只要不妨碍本发明的技术效果的实现，可在本发明中使用的5'帽无特别限制。在优选的实施方式中，用于自复制的5'帽是m7G(5')ppp(5')(2'-OMeA)pU，其分子式为C31H42N12O25P4，结构式如下；
在体外转录制备mRNA有不同的“加帽”方法，包括酶加帽、共转录加帽等。
酶法加帽是较为传统的加帽方式，该方法需在T7聚合酶参与的IVT反应结束后，先纯化获得未加帽的mRNA，再通过牛痘病毒加帽酶(兼具RNA三磷酸酯酶活性、鸟苷酰基转移酶活性和鸟嘌呤甲基转移酶活性)产生Cap0，再通过2'-O-甲基转移酶和S-腺苷甲硫氨酸转化为Cap1，再次纯化获得最终的mRNA。
一步法共转录加帽，就是在T7聚合酶参与的IVT反应体系中直接加入帽类似物，实现一步法获得含Cap1结构的mRNA，全程只需一次纯化。此法反应减少了制备步骤，进而有效缩短整体处理时间、简化纯化步骤，减少所需酶的数量。因此，化学法共转录加帽在工艺上相对简单，引入杂质少，能够迅速提升mRNA疫苗和药物的产能。目前，一步法共转录加帽正在逐步成为了mRNA制备工艺的主流技术路线。
【自复制mRNA的模板】
只要是可用于自复制mRNA，其模板无特别限制。在优选的实施方式中，自复制mRNA的模板可始于5'-TAATACGACTCACTATAAT…-3'，其中加下划线区域是RNA聚合酶的启动子区域，更优选为T7 RNA聚合酶的启动子区域。
由于自复制mRNA模板序列通常较长，IVT产量及产物完整性较常规模板低。
【尿苷三磷酸(UTP)】
只要不妨碍本发明的技术效果的实现，可在本发明中使用的尿苷三磷酸无特别限制，可为天然的尿苷三磷酸或任何本领域常用的经修饰的尿苷三磷酸。在优选的实施方式中，UTP是N1-甲基假尿苷三磷酸(N1-Me-pUTP，通常表示为“Ψ”)，其分子式为C10H14N2Na3O15P3，结构式如下：
在mRNA疫苗和药物生产过程中掺入N1-甲基假尿苷三磷酸可提高mRNA的翻译效率并降低mRNA在体内的免疫原性。
【5'UTR和3'UTR】
只要不妨碍本发明的技术效果的实现，可在本发明中使用的5'UTR和3'UTR无特别限制。在优选的实施方式中，5'UTR和3'UTR源于含有RNA复制酶的编码序列的表达盒所来源的物种。
【mRNA的包封载体及利用该载体的递送方式】
由于裸mRNA无法有效进入机体的细胞内进行蛋白表达，且mRNA的稳定性较差，易降解，所以本发明的mRNA疫苗优选包封在保护性载体中。只要足以保持本发明的mRNA疫苗在足够长的时间不降解，且不妨碍本发明的技术效果的实现，可在本发明中使用的mRNA的包封载体无特别限制。在优选的实施方式中，本发明中使用纳米颗粒型载体包封mRNA。在进一步优选的实施方式中，本发明中使用包含脂质的纳米颗粒(也称为“脂质纳米颗粒(lipid nanopartical,LNP)”)包封mRNA。在进一步优选的实施方式中，LNP可以包括但不限于脂质体和胶束。在具体的实施方式中，所述脂质纳米颗粒可以包括阳离子和/或可离子化的脂质、阴离子脂质、中性脂质、两亲性脂质、聚乙二醇化的脂质和/或结构性脂质。
在一个具体实施方式中，所述LNP可包含一种或多种(例如1、2、3、4、5、6、7或8)阳离子和/或可离子化的脂质。“阳离子脂质”通常指在一定pH(例如，生理pH)下携带任意数目的净正电荷的脂质。所述阳离子脂质可包括但不限于SM102、3-(双十二烷基氨基)-N1,N1,4-三十二烷基-1-哌嗪乙胺(KL10)、N1-[2-(二十二烷基氨基)乙基]-N1,N4,N4-三十二烷基-1,4-哌嗪二乙胺(KL22)、14,25-二十三烷基-15,18,21,24-四氮杂八孔并烷(KL25)、DLin-DMA、DLin-K-DMA、DLin-KC2-DMA、辛基-CLinDMA、辛基-CLinDMA(2S)、DODAC、DOTMA、DDAB、DOTAP、DOTAP.C1、DC-Choi、DOSPA、DOGS、DODAP、DODMA和DMRIE。
在某些实施方式中，所述阳离子脂质在脂质纳米颗粒中的摩尔比例为约40～70％，例如，约40～65％、约40～60％、约45～55％或约48～53％。
在一个具体实施方式中，所述LNP可包含一种或多种(例如1、2、3、4、5、6、7或8)非阳离子脂质。所述非阳离子脂质可以包括阴离子脂质。适用于本申请的脂质纳米颗粒的阴离子脂质可包括磷脂酰甘油、心磷脂、二酰基磷脂酰丝氨酸、二酰基磷脂酸、N-十二烷酰基磷脂酰乙醇胺、N-琥珀酰基磷脂酰乙醇胺、N-戊二酰基磷脂酰磷酸乙醇基，以及其他连接了阴离子基团的中性脂质。
在更具体的实施方式中，所述非阳离子脂质可以包括中性脂质，所述中性脂质可例如包括磷脂，例如二硬脂酰基磷脂酰胆碱(DSPC)、二油酰基磷脂酰胆碱(DOPC)、二棕榈酰基磷脂酰胆碱(DPPC)、二油酰基磷脂酰甘油(DOPG)、二棕榈酰基磷脂酰甘油(DPPG)、二油酰基磷脂酰乙醇胺(DOPE)、棕榈酰基油酰基磷脂酰胆碱(POPC)、棕榈酰基油酰基-磷脂酰乙醇胺(POPE)、二油酰基-磷脂酰乙醇胺4-(N-马来酰亚胺基甲基)-环己烷-1-羧酸酯(DOPE-mal)、二棕榈酰基磷脂酰基乙醇胺(DPPE)、二肉豆蔻酰基磷酸乙醇胺(DMPE)、二硬脂酰基-磷脂酰基-乙醇胺(DSPE)、16-O-单甲基PE、16-O-二甲基PE、18-1-反式PE、1-硬脂酰基-2-油酰基-磷脂酰乙醇胺(SOPE)，或其混合物。另外，可以使用具有饱和和不饱和脂肪酸链的混合物的脂质。例如，本申请所述的中性脂质可以选自DOPE、DSPC、DPPC、POPC或任何相关的磷脂酰胆碱。
在某些实施方式中，所述磷脂在脂质纳米颗粒中的摩尔比例为约5～20％。
在某些实施方式中，所述LNP可包含脂质缀合物，例如，聚乙二醇(PEG)修饰的脂质和衍生的脂质。PEG修饰的脂质可包括但不限于与具有C6～C20长度的烷基链的脂质共价连接的长度至多为5kDa的聚乙二醇链。这些组分的加入可防止脂质聚集，也可增加循环持续时间，易于脂质-核酸组合物递送至靶细胞，或快速释放出核酸。例如，所述聚乙二醇(PEG)修饰的脂分子可以是具有较短的酰基链(例如，C14或C18)的PEG-神经酰胺。在某些实施方式中，所述聚乙二醇(PEG)修饰的脂分子在脂质纳米颗粒中的摩尔比例为约0.5～2％，例如，约1～2％、约1.2～1.8％或约1.4～1.6％。在某些实施方式中，所述聚乙二醇(PEG)修饰的脂分子可以为PEG2000-DMG。
在某些实施方式中，所述LNP还可包含胆固醇。在某些实施方式中，所述胆固醇在脂质纳米颗粒中的摩尔比例为约30～50％，例如，约35～45％、或约38～42％。
在某些实施方式中，所述LNP可包括阳离子脂质、胆固醇、磷脂以及聚乙二醇修饰的脂分子。在某些实施方式中，所述阳离子脂质、胆固醇、磷脂以及聚乙二醇修饰的脂分子的摩尔比可以为45～55：35～45：5～15：0.5～2。
利用上述包封载体的递送方式无特别限制，可采用任何本领域常规使用的递送方式，例如可采用US20160376224A1或WO2015199952A1中提到的递送方式。
【实施例】
【实施例1：表达HER2抗原变体的自复制mRNA和非自复制mRNA的制造】
【1_1.HER2抗原变体的制造】
从NCBI(https://www.ncbi.nlm.nih.gov/)得到野生型HER2的DNA序列，将其中细胞内结构域(ICD)的编码序列中的酪氨酸激酶(TK)活性位点进行失活突变，以抑制JAK-STAT信号传导通路的非预期激活，由此得到HER2的一个抗原变体VG-HR1的DNA序列(SEQ ID NO：1)。该VG-HR1的氨基酸序列如SEQ ID NO：2所示，其结构示意图如图1A所示。
在如上VG-HR1的DNA序列的基础上，将中HER2的TMD的编码序列替换为MITD的编码序列，进一步将HER2的SP的编码序列替换为MITD的SP的编码序列而得到HER2的另一个抗原变体VG-HR13的DNA序列(SEQ ID NO：3)。该VG-HR13的氨基酸序列如SEQ ID NO：4所示，其结构示意图如图1B所示。
【1_2.源于VEEV TC-83株的自复制mRNA的表达盒和非自复制mRNA的表达盒的构建】
基于VEEV TC-83株的基因组DNA序列，将其中结构性多蛋白的编码序列(结构性多蛋白的启动子之后的部分)替换为“Kozak序列+SEQ ID NO：1所示的VG-HR1的DNA序列”或“Kozak序列+SEQ ID NO：3所示的VG-HR13的DNA序列”。进一步向上述构建体的5'UTR的5'侧附加T7 RNA聚合酶的启动子位点。进一步向上述构建体的3'UTR的3'侧附加多聚A和BspQI限制性内切酶的切割位点。如此构建的源于VEEV TC-83株的表达VG-HR1的自复制mRNA的表达盒(图2A)和表达VG-HR13的自复制mRNA的表达盒(图2B)从5'端至3'端依此包括：
·表达VG-HR1的自复制mRNA的表达盒(图2A)(SEQ ID NO：5)：T7 RNA聚合酶的启动子位点、5'UTR、非结构性多蛋白的编码序列、26S启动子、“Kozak序列+SEQ ID NO：1所示的VG-HR1的DNA序列”、3'UTR、多聚A和BspQI限制性内切酶的切割位点；
·表达VG-HR13的自复制mRNA的表达盒(图2B)(SEQ ID NO：6)：T7 RNA聚合酶的启动子位点、5'UTR、非结构性多蛋白的编码序列、26S启动子、“Kozak序列+SEQ ID NO：3所示的VG-HR13的DNA序列”、3'UTR、多聚A和BspQI限制性内切酶的切割位点。
同时，作为对照，以类似方式构建表达VG-HR1的非自复制mRNA的表达盒(图3A)和表达VG-HR13的非自复制mRNA的表达盒(图3B)，它们从5'端至3'端依此包括：
·表达VG-HR1的非自复制mRNA的表达盒(图3A)：T7 RNA聚合酶的启动子位点、5'UTR、“Kozak序列+SEQ ID NO：1所示的VG-HR1的DNA序列”、3'UTR、多聚A和BspQI限制性内切酶的切割位点；
·表达VG-HR13的非自复制mRNA的表达盒(图3B)：T7 RNA聚合酶的启动子位点、5'UTR、“Kozak序列+SEQ ID NO：3所示的VG-HR13的DNA序列”、3'UTR、多聚A和BspQI限制性内切酶的切割位点。
【1_3.含自复制mRNA的表达盒和非自复制mRNA的表达盒的质粒的构建】
向pUC57-Kan质粒中分别插入实施例1_2中构建的表达VG-HR1的自复制mRNA的表达盒和表达VG-HR13的自复制mRNA的表达盒及表达VG-HR1的非自复制mRNA的表达盒和表达VG-HR13的非自复制mRNA的表达盒而分别得到含自复制mRNA的表达盒的质粒psaRNA-VG-HR1和psaRNA-VG-HR13(图4)及含非自复制mRNA的表达盒的质粒pnsaRNA-VG-HR1和pnsaRNA-VG-HR13。
【1_4.含自复制mRNA的表达盒和非自复制mRNA的表达盒的质粒的线性化】
配制下表1所示的50μl的质粒线性化反应体系。
【表1：50μL的含自复制mRNA的表达盒和非自复制mRNA的表达盒的质粒的线性化体系】
通过于50℃温育2小时，将实施例1_3中得到的含自复制mRNA的表达盒和非自复制mRNA的表达盒的质粒线性化。在线性化完成后，向反应体系进一步添加ddH2O至总体积100μl。使用琼脂糖凝胶DNA回收试剂盒纯化并回收线性化的含自复制mRNA的表达盒和非自复制mRNA的表达盒的质粒而作为作为体外转录(in vitro transcription，IVT)模板使用。
【1_5.自复制mRNA和非自复制mRNA的IVT】
配制下表2所示的20μL的IVT反应体系。
【表2：20μL的IVT反应体系】
通过于37℃反应2小时来进行IVT而得到自复制mRNA。在反应结束后，向反应体系中加入4U DNA酶I(诺唯赞，EN401)，于37℃反应15分钟。向反应体系中添加无RNA酶的ddH2O至总体积50μL，进一步添加25μL 5M的LiCl溶液后混匀。于-20℃静置30分钟后，于4℃以13000×g离心10分钟后弃上清。用70％乙醇清洗沉淀，于4℃以13000×g离心2分钟后弃上清，将沉淀溶解在30μL水中。
用分光光度计测定浓度。取500ng体外转录得到的自复制mRNA和2μl 2×RNA Loading Dye混合，于70℃温育10分钟后，进行变性琼脂糖凝胶电泳检测。作为IVT模板使用线性化的psaRNA-VG-HR1时的电泳图谱如图5A所示。同时，对体外转录得到的自复制mRNA进行毛细管电泳检测完整性。作为IVT模板使用线性化的psaRNA-VG-HR1时的毛细管电泳完整性检测数据如图5B所示。这些数据表明，通过体外转录得到的psaRNA-VG-HR1有较高的完整性，有利于mRNA的有效复制和表达。
【1_6.包封自复制mRNA和非自复制mRNA的脂质纳米颗粒的制造】
将实施例1_5中得到的作为IVT产物的自复制mRNA和非自复制mRNA溶解于柠檬酸盐缓冲液中，再将其使用微流体装置NanoAssembler与溶解于乙醇中的脂质混合物(可电离脂质ALC-0315:二硬脂酰磷脂酰胆碱:胆固醇:PEG脂质ALC-0159)混合而得到包裹着自复制mRNA和非自复制mRNA的脂质纳米颗粒(lipid nanopartical，LNP)。将这些脂质纳米颗粒与磷酸盐缓冲盐混合，通过超滤除去残余乙醇。对如此得到的LNP进行表征，结果如下表3所示。
【表3：LNP的表征结果】
这表明，成功获得了良好包封了表达HER2的抗原变体VG-HR1和VG-HR13的自复制mRNA和非自复制mRNA的良好纯化的LNP。轻微负的ζ电位适用于静电稳定性和细胞内摄取。
将这些LNP与冷冻保护剂混合后储存在-80℃下。
【实施例2：自复制mRNA的自复制效率】
【2_1.细胞转染】
将衍生自人胚胎肾细胞的HEK-293T细胞接种在含有DMEM高糖培养基(+10％胎牛血清+1％双抗)的75cm2细胞培养瓶中，使细胞在培养瓶中汇合度达到80％以上。用胰酶将该细胞消化、计数。将适宜数量的悬浮的HEK-293T细胞铺入含有Opti-MEM培养基的24孔细胞培养板中，在CO2培养箱中于37℃过夜培养。
将上述24孔细胞培养板中的HEK-293T细胞用0.5μg实施例1中得到的包封了表达HER2的抗原变体VG-HR1和VG-HR13的自复制mRNA和非自复制mRNA的LNP转染24小时后，将经转染得到的细胞培养物用于如下实施例2_2中的蛋白印迹(Western Blot)测定及实施例2_3中的自复制mRNA的扩增效率的检测。
【2_2.蛋白印迹(Western Blot)测定】
向实施例2_1中经转染得到的细胞培养物中加入含有1％的蛋白酶抑制剂的90μl细胞裂解液后，在冰上放置5分钟。以14000×g离心10分钟，将上清转移至新的离心管中，加入NuPAGE LDS样品缓冲液，于95℃金属浴10分钟。将如此得到的细胞裂解物保存于-20℃。
取上述细胞裂解物在200V电压下进行聚丙烯酰胺凝胶电泳(PAGE)30分钟。电泳结束后，利用转膜试剂盒(Trans-Blot Turbo 0.2μm PVDF Transfer Kit，Bio-rad 1704272)将凝胶在25V电压下转膜7分钟。转膜完成后，用TBSTw封闭液(碧云天，ST673)封闭15分钟后，与以1:1000稀释的作为第一抗体的抗HER2单克隆抗体(Abcam，ab221438)在室温下温育1小时。用1×TBST洗涤5次，每次5分钟。之后与经TBST以1:20000稀释的辣根过氧化物标记的作为第二抗体的羊抗兔IgG(Abcam，ab205718)在室温下温育40分钟。用1×TBST洗涤5次，每次5分钟。将以1:1比例混合的显影液A液和B液的混合液滴加在膜上，在成像系统中进行显影。
用包封了表达VG-HR1的自复制mRNA和非自复制mRNA的LNP的转染HEK-293T细胞后的蛋白印迹(Western Blot)测定结果显示于图6中。结果表明，在相同的转染时间内，自复制mRNA的表达量(图6中的泳道1)远大于非自复制mRNA的表达量(图6中的泳道2)。
【2_3.自复制mRNA的自复制效率的检测】
将实施例2_1中经转染得到的细胞培养物中的上清吸弃，使用500μl PBS润洗细胞一次后吸弃上清。进一步加入500μl PBS后，吹打细胞至细胞脱落。将脱落的细胞收集至Eppendorf离心管中，以1000rpm离心5分钟后弃去上清。将得到的细胞保存于-80℃。
使用RNeasy Mini试剂盒(Qiagen，74106)从上述细胞提取总RNA，用紫外分光光度计对提取的RNA进行定量后，使用HiScript III 1st Strand cDNA Synthesis Kit(诺唯赞，R312)将RNA逆转录为DNA。利用2×ChamQ Universal SYBR qPCR Master Mix(诺唯赞，Q711)，用如下引物(针对表达VG-HR1的自复制mRNA)，以95℃ 30s→(95℃ 10s→60℃ 30s)40的条件进行荧光定量PCR：
·VEEV TC-83株的非结构性多蛋白的编码序列的上游引物F：5'-AGCAGAGATA GTATTGAAC-3'(SEQ ID NO：7)；
·VEEV TC-83株的非结构性多蛋白的编码序列的下游引物R：5'-TAATGGATAA CGGAACAG-3'(SEQ ID NO：8)；
·VG-HR1的编码序列的上游引物F：5'-ACTACCTCAG CCTCCTAT-3'(SEQ ID NO：9)；
·VG-HR1的编码序列的下游引物R：5'-CACTCGCTGT CAATCATC-3'(SEQ ID NO：10)；
·GAPDH的编码序列的上游引物F：5'-GGTATCGTGG AAGGACTC-3'(SEQ ID NO：11)；
·GAPDH的编码序列的下游引物R：5'-GTAGAGGCAG GGATGATG-3'(SEQ ID NO：12)。
表达VG-HR1的自复制mRNA的拷贝数相对变化(转染后6～48小时的拷贝数/转染后2小时的拷贝数)如图7所示。从图7可以看出，表达VG-HR1的自复制mRNA中不同部分的拷贝数相对变化不同。非结构性多蛋白的编码序列部分(或表达VG-HR1的自复制mRNA全长)的转染后24小时的拷贝数是转染后2小时的拷贝数的98.85倍(图7A)，而VG-HR1的编码序列部分的转染后24小时的拷贝数是转染后2小时的拷贝数的4666.18倍(图7B)。这表明，尤其是在转染后24小时，自复制mRNA在经其转染的细胞内高效自复制，而且，自复制主要发生在VG-HR1的编码序列部分(其示意图如图7C所示)。
【实施例3：包封了表达HER2的抗原变体VG-HR1和VG-HR13的自复制mRNA和非自复制mRNA的LNP的安全性及功效】
【3_1.用LNP免疫接种】
将6～8周龄的雌性BALB/c小鼠以每组5只分为8组，在适应性喂养观察3天后，通过在第0天通过肌肉注射初始(Prime)施用实施例1中制备的包封了表达HER2的抗原变体VG-HR1和VG-HR13的自复制mRNA和非自复制mRNA的LNP，并在第14天通过肌肉注射追加(Boost)施用相应LNP来实现疫苗接种。组1(对照组)施用生理盐水。各组中施用的LNP的属性如下表4所示。
【表4：各组中施用的LNP的属性】
【3_2.用LNP免疫接种后的体重变化】
对于实施例3_1中处理的各组小鼠，在第1、4、6、8、10、16、19、20、22和25天测量体重。
结果如图8A和8B所示，相比施用了生理盐水的组1(对照组)，施用了包封了表达VG-HR13的自复制mRNA和非自复制mRNA的LNP的组(组2～8)的小鼠呈现10％以内的体重下降，但在4天内体重又会恢复到正常水平。这表明，无论LNP中的mRNA是自复制mRNA还是非自复制mRNA，无论mRNA中的尿苷三磷酸是UTP还是N1-Me-pUTP，均对被接种对象无明显的副作用，足够安全。
【3_3.用LNP免疫接种后产生的特异性抗HER2抗体】
对于实施例3_1中处理的各组小鼠，在第13(2周)和28天(4周)采集血清，检测其中特异性抗HER2抗体的水平。
将ELISA Plate Coating缓冲液(Elabscience，E-ELIR-001)用去离子水稀释成工作浓度(1×)，然后配置成含有1.5μg/ml HER2的包被液(Yeasen，93098ES20)，之后在high binding EIA/RIA plate(Corning costar，42592)中加入100μl HER2的包被液，盖上封膜，4℃过夜温育。
取出包被的检测板，用移液器吸弃去包被液，在滤纸上拍干。加入300μl洗涤缓冲液，静置30s以上，甩去液体后在吸水纸上拍干，重复洗涤三次。完毕后每孔加入100μl ELISA Plate封闭缓冲液，室温温育2h。完成后弃去孔内液体，在滤纸上拍干。加入300μl洗涤缓冲液，静置30s以上，甩去液体后在滤纸上拍干，重复洗涤三次后待用。
将免疫后指定日期获得的小鼠血清稀释100倍进行检测，将稀释过的样本加入到封闭完成的酶标板中，每孔100μl，室温温育2h。完成后弃去上清，加入洗涤缓冲液洗涤3次。同时使用sample diluent稀释HRP-conjugated Affinipure山羊抗小鼠IgG(H+L)1:10000倍，然后加入到酶标板中，室温温育1h。温育完毕弃去上清，加入洗涤缓冲液洗涤3次。清洗完成后于各反应孔中加入TMB底物(Solarbio，PR1200)溶液0.1ml，室温显色约10分钟，之后于各反应孔中加入终止液0.1ml。通过使用酶标仪测定450nm处的吸光度(OD450)来检测其中特异性抗HER2抗体的浓度。
结果如图9所示，相比施用了生理盐水的组1(对照组)，施用了包封了表达VG-HR13的自复制mRNA和非自复制mRNA的LNP的组(组2～8)的小鼠的第4周血清中的特异性抗HER2抗体的浓度均显著高于第2周血清中的特异性抗HER2抗体的浓度。而且，只要施用10μg自复制mRNA/只，就能达到相当于甚至高于施用44μg非自复制mRNA/只时的血清抗体水平。这表明，自复制mRNA有比非自复制mRNA显著更优的抗体诱导效果。
【3_4.用LNP免疫接种后产生的针对HER2的特异性细胞免疫反应】
对于实施例3_1中处理的各组小鼠，在第28天处死小鼠，采集脾脏，获得脾细胞。
将各组的脾细胞重悬于1640培养基后，均分接种至U型底96孔板，分别加入终浓度1μg/ml的HER2蛋白培养24小时，期间在培养18小时后，将细胞离心，并去除培养基，加入含有Golgiplug(BD，555029)的新鲜培养基100μl，阳性对照组则加入含有T细胞刺激物和monnesin的培养基，继续培养4～6小时。刺激完毕后，细胞离心并用PBS洗一遍，加入用PBS配制的细胞表面抗体50μl，于4℃避光温育30分钟。之后，将细胞离心，并用含2％FBS的1640培养基洗一遍，加入固定液50μl，于4℃固定1小时。之后，将细胞离心，并用洗涤缓冲液漂洗一次，并加入细胞因子抗体50μl，于4℃温育1小时。最后，将细胞离心，并用洗涤缓冲液漂洗一次后，用PBS重悬细胞，在流式细胞仪上进行检测。
结果如图10所示，在免疫4周后，除组5(自复制mRNA，N1-Me-pUTP，1μg/只)外，其余各自复制mRNA施用组(组2～4，6～7)均比非自复制mRNA施用组(组8，44μg/只)产生了显著更高水平的颗粒酶B，即引发了显著更高水平的CD8+T细胞免疫。这表明，自复制mRNA有比非自复制mRNA显著更优的细胞免疫诱导效果。

Claims (42)

  1. 信使核糖核酸(mRNA)分子，其包含：
    表达盒1，其含有RNA复制酶的编码序列、和
    表达盒2，其含有抗原的编码序列。
  2. 根据权利要求1所述的mRNA分子，其中所述RNA复制酶是病毒的RNA复制酶。
  3. 根据权利要求1所述的mRNA分子，其中所述表达盒1源于病毒的非结构性多蛋白的编码序列。
  4. 根据权利要求2或3所述的mRNA分子，其中所述病毒是披膜病毒科(Togaviridae)的病毒。
  5. 根据权利要求4所述的mRNA分子，其中所述披膜病毒科(Togaviridae)的病毒是甲病毒属(Alphavirus)的病毒。
  6. 根据权利要求4所述的mRNA分子，其中所述甲病毒属(Alphavirus)的病毒是委内瑞拉马脑炎病毒(Venezuelan equine encephalitis virus，VEEV)。
  7. 根据权利要求1所述的mRNA分子，其中所述抗原是HER2抗原或其变体。
  8. 根据权利要求7所述的mRNA分子，其中所述HER2抗原变体具有经改造的细胞内结构域(ICD)，其中酪氨酸激酶(TK)活性位点被失活突变。
  9. 根据权利要求7或8所述的mRNA分子，其中所述HER2抗原变体具有主要组织相容性复合体1(MHC1)转运结构域(MITD)，所述MITD置换了HER2抗原的跨膜结构域(TMD)。
  10. 根据权利要求9所述的mRNA分子，其中所述HER2抗原变体具有MITD的信号肽(SP)，所述MITD的SP置换了HER2抗原的SP。
  11. 根据权利要求8或10所述的mRNA分子，其中所述HER2抗原变体具有SEQ ID NO：2或4所示的序列。
  12. 根据权利要求11所述的mRNA分子，其中所述HER2抗原变体的编码序列具有SEQ ID NO：1或3所示的序列。
  13. 根据权利要求7所述的mRNA分子，其中所述HER2抗原或其变体的编码序列的5'侧具有Kozak序列。
  14. 根据权利要求13所述的mRNA分子，其中所述Kozak序列是GCCACCAUGG。
  15. 根据权利要求1所述的mRNA分子，其中所述表达盒2具有启动子，所述抗原的编码序列位于所述启动子下游。
  16. 根据权利要求15所述的mRNA分子，其中所述启动子源于所述表达盒1所来源的物种。
  17. 根据权利要求15或16所述的mRNA分子，其中所述启动子是26S启动子。
  18. 根据权利要求1所述的mRNA分子，其中
    所述表达盒1的上游还具有RNA聚合酶的启动子位点和5'UTR；且
    所述表达盒2的下游还具有3'UTR和多聚A。
  19. 根据权利要求18所述的mRNA分子，其中所述RNA聚合酶是T7 RNA聚合酶。
  20. 根据权利要求18所述的mRNA分子，其中所述5'UTR和3'UTR源于所述表达盒1所来源的物种。
  21. 根据权利要求19或20所述的mRNA分子，其对应的DNA序列具有SEQ ID NO：5或6所示的序列。
  22. 根据权利要求1所述的mRNA分子，其中的尿苷三磷酸是天然尿苷三磷酸(UTP)或N1-甲基假尿苷三磷酸(N1-Me-pUTP)。
  23. 信使核糖核酸(mRNA)疫苗，其通过将根据权利要求1～22之任一项所述的mRNA分子包封为脂质纳米颗粒(LNP)得到。
  24. DNA分子，其被转录成根据权利要求1～22之任一项所述的mRNA分子。
  25. 根据权利要求1～22之任一项所述的mRNA分子在制造引发抗HER2免疫应答的信使核糖核酸(mRNA)疫苗中的用途。
  26. 信使核糖核酸(mRNA)分子的制造方法，其包括：
    (1)基于病毒的基因组的DNA序列，将其中结构性多蛋白的编码序列中的结构性多蛋白的启动子之后的部分替换为抗原的编码序列；
    (2)向5'UTR的5'侧附加RNA聚合酶的启动子位点；
    (3)向3'UTR的3'侧附加多聚A；及
    (4)将通过步骤(1)～(3)构建的DNA分子转录为mRNA分子。
  27. 根据权利要求26所述的方法，其中所述病毒是披膜病毒科(Togaviridae)的病毒。
  28. 根据权利要求27所述的方法，其中所述披膜病毒科(Togaviridae)的病毒是甲病毒属(Alphavirus)的病毒。
  29. 根据权利要求28所述的方法，其中所述甲病毒属(Alphavirus)的病毒是委内瑞拉马脑炎病毒(Venezuelan equine encephalitis virus，VEEV)。
  30. 根据权利要求26所述的方法，其中所述抗原是HER2抗原或其变体。
  31. 根据权利要求30所述的方法，其中所述HER2抗原变体具有经改造的细胞内结构域(ICD)，其中酪氨酸激酶(TK)活性位点被失活突变。
  32. 根据权利要求30或31所述的方法，其中所述HER2抗原变体具有主要组织相容性复合体1(MHC1)转运结构域(MITD)，所述MITD置换了HER2抗原的跨膜结构域(TMD)。
  33. 根据权利要求32所述的方法，其中所述HER2抗原变体具有MITD的信号肽(SP)，所述MITD的SP置换了HER2抗原的SP。
  34. 根据权利要求31或33所述的方法，其中所述HER2抗原变体具有SEQ ID NO：2或4所示的序列。
  35. 根据权利要求34所述的方法，其中所述HER2抗原变体的编码序列具有SEQ ID NO：1或3所示的序列。
  36. 根据权利要求30所述的方法，其中所述HER2抗原或其变体的编码序列的5'侧具有Kozak序列。
  37. 根据权利要求36所述的方法，其中所述Kozak序列是GCCACCATGG。
  38. 根据权利要求26所述的方法，其中通过步骤(1)～(3)构建的DNA分子具有SEQ ID NO：5或6所示的序列。
  39. 根据权利要求26所述的方法，其中所述转录是体外转录。
  40. 根据权利要求39所述的方法，其中所述体外转录使用：
    体外转录用核酸载体，其中插入通过步骤(1)～(3)构建的DNA分子，
    腺苷三磷酸、胞苷三磷酸、鸟苷三磷酸和尿苷三磷酸，及
    RNA聚合酶。
  41. 根据权利要求40所述的方法，其中所述尿苷三磷酸是天然尿苷三磷酸(UTP)或N1-甲基假尿苷三磷酸(N1-Me-pUTP)。
  42. 根据权利要求40所述的方法，其中所述RNA聚合酶是T7 RNA聚合酶。
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