WO2024055272A1 - 能高效表达目的基因的mRNA载体系统、其构建及应用 - Google Patents

能高效表达目的基因的mRNA载体系统、其构建及应用 Download PDF

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WO2024055272A1
WO2024055272A1 PCT/CN2022/119229 CN2022119229W WO2024055272A1 WO 2024055272 A1 WO2024055272 A1 WO 2024055272A1 CN 2022119229 W CN2022119229 W CN 2022119229W WO 2024055272 A1 WO2024055272 A1 WO 2024055272A1
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nucleic acid
expression vector
mrna
virus
utr
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PCT/CN2022/119229
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French (fr)
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徐建青
张晓燕
白诗梦
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复旦大学附属中山医院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression

Definitions

  • the present disclosure belongs to the field of biomedicine industry, especially involving the manufacturing of genetic engineering drugs and vaccines. Specifically, the present disclosure relates to an mRNA vector system that can efficiently express a target gene, its construction, and its application in the expression of the target gene. For example, it can be used as a nucleic acid vaccine vector.
  • mRNA vaccine technology has become one of the most popular forms of vaccine due to its many advantages. Unlike plasmid DNA and viral vector vaccines, mRNA is not integrated into the genome, thus avoiding the potential risks of insertional mutations.
  • the mRNA vaccine can be produced in a cell-free manner, with rapid production, simple process, and controllable cost.
  • a single mRNA vaccine can encode multiple antigens, allowing it to target tumor targets, different microorganisms, and enhance immune responses against hypermutated pathogens.
  • the in vitro transcribed mRNA molecule must simulate the structure of the endogenous mRNA molecule and must include the following elements in the 5' ⁇ 3' direction: 5' cap structure, 5'-UTR (untranslated region) sequence, coding sequence, 3'-UTR sequence, and poly(A) tail) sequence.
  • 5' cap structure 5'-UTR (untranslated region) sequence
  • coding sequence 3'-UTR sequence
  • poly(A) tail) sequence poly(A) tail
  • the 5' cap structure is mainly modified through co-transcriptional capping and enzymatic capping to protect the mRNA from degradation by exonucleases and works in conjunction with the poly(A) tail at the 3' end.
  • PABP poly(A) binding protein
  • binds to the poly(A) tail sequence it recruits the translation initiation factor proteins eIF4G and eIF4E, circularizes the mRNA and recruits ribosomes to initiate translation.
  • PABP poly(A) binding protein
  • eIF4G and eIF4E translation initiation factor proteins
  • the 5′-UTR and 3′-UTR of mRNA can significantly affect the translation speed and half-life of transcript products
  • optimizing UTR is one of the focuses of mRNA vaccine design. It is generally believed that the 3′-UTR is a region in mRNA where unstable factors are concentrated, and a sequence that stably expresses proteins or viral genomes needs to be selected.
  • poly-A tail protects the mRNA from degradation and facilitates subsequent binding of poly(A)-binding proteins. Therefore, adding a poly(A) tail to an antigen-encoding mRNA template results in higher levels of protein expression.
  • Long poly-A sequences are more conducive to the stability of mRNA and extend its half-life. According to earlier studies, in metazoans, poly-A tails are usually about 250 bp. In human monocyte-derived dendritic cells (DCs), the 120 bp poly-A sequence provides more stable IVT-mRNA (in vitro-transcribed mRNA) and more efficient translation than the short poly-A tail.
  • DCs human monocyte-derived dendritic cells
  • Poly-A sequences of more than 300 nucleotides are conducive to more efficient translation, in which IVT-mRNA with medium and long poly-A tails recruits PABP first and is sheared to 30A long, which is consistent with the size of naturally occurring mRNA poly(A) tails.
  • Adding poly(A) tails to DNA plasmids eliminates the in vitro tailing step, reduces the overall reaction time and loss of raw materials, and avoids enzymatic polyadenylation using poly(A) polymerase. Tail length changes.
  • poly(A) tails larger than 100 bp are optimal for therapeutic mRNA vaccines, the DNA sequences encoding these long poly(A)s can destabilize the DNA plasmids used for transcription. Moreover, studies have shown that when the length of the continuous Poly(A) tail is greater than 120 bp, the expression level of the corresponding protein does not increase.
  • the tail of the new coronavirus BNT162b2 mRNA vaccine developed by Pfizer-BioNTech, which has been successfully launched, contains 100A poly(A), with a 10bp UGC linker inserted in between, generating a sequence such as 30nt polA+GCATATGACT+70nt polA Poly( A) Tail.
  • This disclosure provides a new type of mRNA vector, its preparation method and application.
  • this article provides an expression vector containing an mRNA nucleic acid capable of expressing a protein of interest, which sequentially contains from the 5' end to the 3' end:
  • a polyadenylic acid tail element with a total length of more than 120 nt which includes: multiple adenylic acid strings, each adenylic acid string independently contains n consecutive adenylic acid residues, n is 10 to 80 an integer between, and the total number of adenylates in the plurality of adenylate strings is more than 100; and a linker located between the plurality of adenylate strings, each of the linkers independently does not Contains adenosine residues or only 1 or 2 adenosine residues.
  • the nucleic acid expression vector of the present disclosure can efficiently express the required target protein in vivo and in vitro, thereby achieving disease prevention and/or treatment effects, such as being used as therapeutic drugs, preventive drugs, protein replacement therapeutic molecules, gene editing therapeutic molecules, etc. wait.
  • the nucleic acid expression vector of the present disclosure can be used, for example, for the prevention and/or treatment of viral infections, cancer, genetic diseases (eg, single gene diseases).
  • composition comprising a nucleic acid expression vector herein, and packaging and/or a delivery system for the nucleic acid expression vector and/or a pharmaceutically or physiologically acceptable carrier.
  • this article also provides the use of the nucleic acid expression vector and/or composition of this article in the preparation of products for expressing the protein of interest, which products can be selected from: mRNA vaccines, therapeutic or preventive drugs, such as Protein replacement therapeutic drugs and gene editing therapeutic drugs.
  • methods for disease prevention and/or treatment include administering to a subject in need thereof a prophylactically and/or therapeutically effective amount of the nucleic acid expression vector and/or composition herein.
  • nucleic acid expression vectors and/or compositions herein for expressing a protein of interest.
  • nucleic acid expression vectors and/or compositions herein are further provided for use in disease prevention and/or treatment.
  • this article also provides a method for preparing a nucleic acid expression vector or composition as described herein, which method includes: providing individual or connected elements; assembling each element into a nucleic acid expression vector.
  • Figure 1 Schematic diagram of the linearized pCDNA3.1+ plasmid transformed from the empty vector of mRNA transcription template in Figure 1.
  • Figure 2 Positive rate (A) and expression level (B) of HEK293T cells transfected with 3′-UTR from different sources and eGFP mRNA encoded by the 30poly(A) vector.
  • Figure 3 Positive rate (A) and expression level (B) of HEK293T cells transfected with eGFP mRNA encoded by 120poly(A) and 250poly(A) vectors with 3′-UTR from different sources.
  • Figure 4 Comparison of the positive rate (A) and expression level (B) of HEK293T cells transfected with eGFP mRNA encoded by the 250poly(A) vector in different tandem 3′-UTRs.
  • Figure 5 Comparison of the positive rate (A) and expression level (B) of HEK293T cells transfected with eGFP mRNA encoded by the same tandem 3'-UTR with 120poly(A) and 250poly(A) vectors.
  • Figure 6 Comparison of humoral response levels after the same tandem 3'-UTR carrying 120poly(A) and 250poly(A) mRNA vectors were used in mice vaccinated with rabies mRNA (the ordinate is the logarithm of log base 10).
  • Figure 7 Comparison of humoral response levels after the same tandem 3′-UTR carrying 120poly(A) and 250poly(A) mRNA vectors were used in mice vaccinated with influenza virus mRNA (the ordinate is the logarithm of log base 10) .
  • the present disclosure provides an mRNA vector that can efficiently express a target protein.
  • the mRNA vector can be a non-replicating mRNA vector that can efficiently express different target genes, whether at the in vitro cell expression level or by delivering the mRNA via a delivery tool.
  • the target gene can achieve efficient translation and stable and sustained expression.
  • the eGFP green fluorescent protein gene is used as a reference to conduct a series of optimizations on the components of the mRNA vector.
  • a non-replicating mRNA vector that can efficiently express the target gene is selected.
  • This mRNA vector can both efficiently translate , and can also be stably and continuously expressed at the cellular level or in vivo.
  • the nucleic acid expression vector herein can efficiently express various target molecules, such as various antigen molecules; through the selection, modification, transformation and combination of each element in the expression vector, the target gene can be efficiently expressed It achieves efficient and stable translation at the cellular level or in vivo, and can effectively regulate the expression level and half-life of the target gene in vivo, making it achieve high immunogenicity and long-term sustainability. Therefore, the expression vector and related products of the present application have broad application prospects in the application of preventive and therapeutic vaccines, expression of specific antibodies, expression of therapeutic or targeted drugs, protein replacement therapy, etc.
  • eukaryote may include humans, primates, rodents (eg, rats, mice, guinea pigs, hamsters), domestic animals, or livestock mammals.
  • a high degree of sequence identity as described herein includes a sequence identity of more than 70%, more than 75%, more than 80%, more preferably more than 85%, such as 85%, 90%, 95%, 98% or even 99% or more. , these high-identity sequences are also within the scope of equivalence that is preferably considered in the present invention. Methods and tools for comparing sequence identities are also well known in the art, such as BLAST.
  • This article provides an mRNA nucleic acid expression vector capable of expressing the target protein, which contains in sequence from the 5' end to the 3' end:
  • Poly(A) tail elements with a total length of more than 120 nt which include:
  • each adenylate string independently contains n consecutive adenylates, n is an integer between 10 and 80, and the total number of adenylates in the multiple adenylate strings The number is more than 100;
  • a linker is located between the plurality of adenylate strings, each of the linkers independently contains no adenylate or only contains 1 or 2 adenylate.
  • the length of the 5'-UTR element used in the present disclosure ranges from 10 to 200 nt, such as from 15 to 100 nt.
  • the 5'-UTR element used in the present disclosure is derived from one or more 5'-UTRs from the following group: human alpha-globulin, beta-globulin, ribosomal protein (RP), micron Tubulin ⁇ -2B, complement factor 3 (C3), cytochrome P4502E1 (CYP2E1), apolipoprotein A-II (APOA2), human hemoglobin subunit ⁇ (hHBB), hemoglobin A1 (HBAl), hemoglobin A2 (HBA2) , Dengue virus (DENV).
  • human alpha-globulin beta-globulin
  • RP ribosomal protein
  • C3 complement factor 3
  • CYP2E1 cytochrome P4502E1
  • APOA2E1 apolipoprotein A-II
  • hHBB human hemoglobin subunit ⁇
  • HBAl hemoglobin A1
  • HBA2 hemoglobin A2
  • DEV Dengue virus
  • the 5'-UTR element used in the present disclosure has the sequence set forth in SEQ ID NO: 1 or has at least 80% sequence identity thereto.
  • the 3'-UTR element used in the present disclosure is a 3'-UTR derived from a mammal or a virus, for example, a 3'-UTR derived from a source selected from the group consisting of, or a combination thereof (e.g., a tandem sequence): human alpha Sequences of globulin, human beta globin, human albumin, human actin, human hemoglobin subunit alpha 1 (HBAl), cytochrome B-245 alpha chain (CYBA), eukaryotic mitochondria (Mit), SARAS-Cov- 2. Dengue virus (DENV), turnip wrinkle virus (TCV), tobacco mosaic virus (TMV) and tobacco etch virus (TEV).
  • a source selected from the group consisting of, or a combination thereof (e.g., a tandem sequence): human alpha Sequences of globulin, human beta globin, human albumin, human actin, human hemoglobin subunit alpha 1 (HBAl), cytochrome B-245
  • the 3'-UTR element used in the present disclosure comprises one or more 3'-UTR molecules selected from the group consisting of alpha-globulin 3'-UTR, eukaryotic mitochondrial 3'-UTR, albumin 3 '-UTR, ⁇ -globin 3'-UTR or any tandem sequence thereof, preferably ⁇ -globin 3'-UTR, eukaryotic mitochondria 3'-UTR, or the 3'-UTR formed by their tandem connection.
  • the 3'-UTR used in the present disclosure has one or more sequences as shown in SEQ ID NO: 2-8, preferably a sequence as shown in SEQ ID NO: 2, 5 or 8, or Sequences with at least 80% sequence identity to any one of them.
  • the total length of the poly(A) tail element used in the present disclosure is 120-400 nt, such as 120-350 nt, 120-320 nt, or any integer therein, such as 120, 304 nt.
  • each adenylate string independently contains 10 to 80, 20 to 70, 25 to 60, 30 to 50, or any integer number of consecutive adenylates therein, such as 20, 30 1, 33, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70 consecutive adenosine nucleotides.
  • the poly(A) tail element further comprises a linker at one or both ends of the element.
  • the length of the linker is each independently 3 to 15 nt, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 nt.
  • the linkers each independently comprise no adenylate, or only 1 adenylate or 2 adenylate.
  • the sequence of the linker is each independently selected from, for example: GCTATGACT, GTATGT, GCAAGT, GATTGC, GGCTGC, TACTGC, GGCTTC, GCATATGACT.
  • the poly(A) tail element has the sequence of SEQ ID NO: 10 or SEQ ID NO: 11, or has at least 80% sequence identity with either one.
  • the 250 poly(A) sequences included in the poly(A) tail element are discontinuous, and every 20 to 40 (eg, 30, 40) A's are separated by a 5 ⁇
  • the 10 bp linker sequence does not contain A, or contains only 1 or 2 A bases, to facilitate more stable, efficient and sustained translation of the coding sequence and extend its half-life.
  • the nucleic acid expression vector of the present disclosure is a non-replicating mRNA vector or serves as a nucleic acid vaccine.
  • the element encoding a protein of interest in the present disclosure is a monocistronic, bicistronic, or polycistronic mRNA.
  • the bicistronic or polycistronic mRNA is an mRNA containing two or more coding regions.
  • the element encoding the protein of interest is codon-optimized or not, includes or does not include base modifications and/or nucleoside analogs, for example, one or more elements selected from the group consisting of: Modified bases or nucleoside analogues: pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio T-methyl-pseudouridine, 2-thio-5-aza- Uridine, 2-thio-dihydropseudine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4- Methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine,
  • expression vector sequences may be codon optimized and/or contain modified bases and/or nucleoside analogs. For example, in some embodiments, 50% to 100% of the uracils in the expression vector sequence are substituted. Substitution can improve the stability of mRNA in vivo.
  • the nucleic acid expression vector of the present disclosure also includes a 5'-cap element, which can optionally be modified.
  • the 5'-cap element is selected from: m7GpppXpYp, m7GpppXmpYp, m7GpppXmpYmp, or its methylation modification sequence, Reverse binding isomer, anti-reverse cap analog (ARCA), N7-benzyldinucleoside tetraphosphate cap analog.
  • the nucleic acid expression vector of the present disclosure further includes a promoter element, such as a T7 promoter, sp6 promoter or T3 promoter.
  • a promoter element such as a T7 promoter, sp6 promoter or T3 promoter.
  • the nucleic acid expression vector of the present disclosure also includes a signal peptide coding element, such as a signal peptide coding element that guides the subcellular localization of the target protein (such as a transmembrane signal peptide, a secretion signal peptide, a nuclear localization signal peptide).
  • a signal peptide coding element such as a signal peptide coding element that guides the subcellular localization of the target protein (such as a transmembrane signal peptide, a secretion signal peptide, a nuclear localization signal peptide).
  • the nucleic acid expression vector of the present disclosure also includes restriction sites, such as XbaI, EcoRV, BamHI, and XhoI.
  • the nucleic acid expression vector of the present disclosure also includes tags, such as molecular tags used for identification, isolation or purification of target molecules, such as Flag tags and HA tags.
  • the nucleic acid expression vector of the present disclosure contains mRNA capable of expressing one or more proteins of interest selected from the following group: immunogenic molecules, antibody molecules, therapeutic drugs, preventive drugs, protein replacement therapeutic molecules, Gene editing therapeutic molecules.
  • the nucleic acid expression vector of the present disclosure can be used to express various foreign genes, including but not limited to expression of specific antibodies, expression of therapeutic or targeted drugs, protein replacement therapy, etc.
  • the nucleic acid expression vector can be used to prepare nucleic acid vaccines.
  • the nucleic acid vaccines can be used to prepare cancer vaccines and virus vaccines, wherein the viruses can be various infectious disease viruses, including Ebola. Viruses, rabies virus, Zika virus, yellow fever virus, dengue virus, cytomegalovirus, PRRS virus, swine fever virus, enterovirus, hepatitis B virus, respiratory syncytial virus, herpes simplex virus, human papillomavirus tumor virus, human immunodeficiency virus, influenza virus, coronavirus, parainfluenza virus, measles virus, mumps virus, Nipah virus, human metapneumovirus, etc.
  • Ebola Viruses, rabies virus, Zika virus, yellow fever virus, dengue virus, cytomegalovirus, PRRS virus, swine fever virus, enterovirus, hepatitis B virus, respiratory syncytial virus, herpes simple
  • the cancers include squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, hepatocellular carcinoma, renal cell carcinoma, bladder cancer, intestinal cancer, cervical cancer, colon cancer, esophageal cancer, head cancer, kidney cancer, liver cancer, and lung cancer.
  • neck cancer neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, leukemia, lymphoma, Burkitt's lymphoma, non-Hodgkin's lymphoma; melanoma; myeloproliferative diseases; sarcoma, angiosarcoma, cardia Bowel's sarcoma, liposarcoma, sarcoma, peripheral neuroepithelialoma, synovial sarcoma, glioma, astrocytoma, oligodendroglioma, ependymoma, glioblastoma, neuroblastoma tumors, ganglioneuromas, gangliogliomas, medulloblastomas, pineal cell tumors, meningiomas, meningiosarcomas, neurofibromas, and schwannomas; breast, uterine, testicular, thyroid carcinoma, astrocytoma, es
  • the target polypeptide in the nucleic acid expression vector is an immunogen, and its expression site includes intracellular, cell membrane or secreted expression.
  • the immunogen is a viral immunogen, which is, for example, derived from viral envelope proteins, including but not limited to hemagglutinin (HA), neuraminidase (NA), matrix protein (Matrix protein, M), envelope protein (Envelope), spike protein (Spike), membrane protein (Membrane, M), hemolysin (Haemolysin, HL), fusion protein (Fusion, F), glycoprotein (Glycoprotein, G).
  • the nucleic acid expression vector of the present disclosure includes from the 5′ end to the 3′ end: a 5′-UTR element including the sequence shown in SEQ ID NO: 1; an open reading frame element encoding the target protein; including SEQ ID NO.
  • the nucleic acid expression vector of the present disclosure comprises the sequence described in any one of SEQ ID NO: 15-22, 24-25 and 27-28, a sequence having at least 80% sequence identity therewith, or the aforementioned A sequence obtained by replacing the open reading frame element encoding the protein of interest in any sequence with an open reading frame element encoding the desired protein of interest.
  • a non-replicating mRNA expression vector which is a plasmid template required to encode the mRNA of different genes.
  • the mRNA vector plasmid template includes the basic elements required for in vitro transcription of mRNA molecules.
  • the expression vector includes the following elements: T7 promoter, 5'-UTR sequence, coding sequence, 3'-UTR sequence, poly(A) tail sequence, and linearized restriction site. These elements may be as described herein.
  • the 5'-UTR sequence, 3'-UTR sequence, and poly(A) tail sequence contained in the non-replicating mRNA vector are selected from SEQ ID NO: 1, SEQ ID NO: 2 , SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or the 5′-UTR sequence, 3′-UTR sequence, and poly(A) tail sequence contained in the non-replicating mRNA vector are sequences that are at least 75% homologous to any of the above sequences ( For example, with SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11 have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99% homology), and the non-replicating mRNA vector can express various foreign genes
  • the present disclosure also provides expression of the non-replicating mRNA vector at the cellular level in vitro.
  • eGFP fluorescent protein gene as a reference, a series of optimizations were performed on its components including 5′-UTR sequence, 3′-UTR sequence, and poly(A) tail sequence.
  • a type of gene that can be expressed efficiently was selected.
  • Non-replicating mRNA vector IV-eGFP ( ⁇ -globin+Mit) 250A).
  • eGFP can be replaced through molecular biology or genetic engineering techniques so that the exemplary mRNA vectors herein can load different genes.
  • the nucleic acid expression vector of the present disclosure is included in a package alone, or is combined with a carrier in a delivery system, for example, the delivery system is selected from: lipid delivery system, lipid delivery system, polymer delivery The system or its combination delivery system, for example, loaded with lipid nanoparticles, polyurethane (PAA), poly ⁇ -aminoester (PBAE), polyethylenimine (PEI), lipid-wrapped polymer micelles.
  • PAA polyurethane
  • PBAE poly ⁇ -aminoester
  • PEI polyethylenimine
  • composition comprising a nucleic acid expression vector of the present disclosure, and a packaging and/or a delivery system for the nucleic acid expression vector and/or a pharmaceutically or physiologically acceptable carrier.
  • the composition of the present disclosure is in a form suitable for one or more administration or delivery methods selected from the group consisting of: respiratory aerosol inhalation, nasal instillation, oral administration, direct injection (e.g., intravenous injection, subcutaneous injection , intradermal injection, intramuscular injection), mucosal administration.
  • respiratory aerosol inhalation e.g., nasal instillation
  • oral administration e.g., direct injection (e.g., intravenous injection, subcutaneous injection , intradermal injection, intramuscular injection), mucosal administration.
  • compositions of the present disclosure further comprise or are used in combination with an adjuvant
  • the adjuvant is selected from: aluminum adjuvant, cholera toxin and its subunits, oligodeoxynucleotides, manganese ion adjuvant agent, colloidal manganese adjuvant, Freund's adjuvant, MF59 adjuvant, QS-21 adjuvant, Poly I: C and other TLR ligands, GM-CSF, IL-2, IL-3, IL-7, IL- 11. IL-12, IL-18, IL-21.
  • the form of the composition of the present disclosure is suitable for combined administration of two or more drugs or vaccines, such as combined vaccination or sequential vaccination.
  • the composition of the present disclosure is selected from: mRNA vaccines, therapeutic or preventive drugs, such as protein replacement therapeutic drugs, gene editing therapeutic drugs.
  • a method of preparing a nucleic acid expression vector or composition described herein includes: providing individual or connected elements; and assembling each element into a nucleic acid expression vector.
  • the method includes employing one or more materials selected from the group consisting of: DNA template (eg, PCR product or linearized plasmid DNA), nuclease, polymerase, capping enzyme, polyadenylation Ulate synthase, DNase, one or more component molecules, linker molecules, natural or modified nucleic acid molecules, buffers, solvents.
  • the method further includes one or more steps selected from the group consisting of: designing, optimizing, transforming and/or modifying each component; and isolating, purifying, and identifying intermediate products and/or final products. , quantification, packaging and/or activity testing; combining the nucleic acid expression vector with a delivery system for the nucleic acid expression vector and/or a pharmaceutically or physiologically acceptable carrier.
  • nucleic acid expression vectors and/or compositions herein in the preparation of products for expressing a protein of interest.
  • the product is selected from: mRNA vaccines, therapeutic or preventive drugs, such as protein replacement therapeutic drugs, gene editing therapeutic drugs.
  • the nucleic acid expression vectors of the present disclosure are used for disease prevention and/or treatment.
  • diseases that can be prevented and/or treated with the nucleic acid expression vectors of the present disclosure or related products thereof include, but are not limited to, diseases selected from the group consisting of viral infections, cancers, genetic diseases (eg, single gene diseases).
  • diseases that can be prevented and/or treated with the nucleic acid expression vectors of the present disclosure or related products thereof include but are not limited to one or more viral infections selected from the following: rabies virus, influenza virus, coronavirus, Ebola virus Latina virus, Zika virus, yellow fever virus, dengue virus, cytomegalovirus, PRRS virus, swine fever virus, enterovirus, hepatitis B virus, respiratory syncytial virus, herpes simplex virus, human papilloma virus , human immunodeficiency virus, influenza virus, coronavirus, parainfluenza virus, measles virus, mumps virus, Nipah virus and human metapneumovirus.
  • viral infections selected from the following: rabies virus, influenza virus, coronavirus, Ebola virus Latina virus, Zika virus, yellow fever virus, dengue virus, cytomegalovirus, PRRS virus, swine fever virus, enterovirus, hepatitis B virus, respiratory sy
  • the target protein expressed by the nucleic acid expression vector of the present disclosure is a viral immunogen, for example, derived from viral envelope proteins, including hemagglutinin. Protein (Hemagglutinin, HA), neuraminidase (NA), matrix protein (Matrix protein, M), envelope protein (Envelope), spike protein (Spike), membrane protein (Membrane, M), hemolysin (Haemolysin, HL), fusion protein (Fusion, F), glycoprotein (Glycoprotein, G).
  • viral envelope proteins including hemagglutinin. Protein (Hemagglutinin, HA), neuraminidase (NA), matrix protein (Matrix protein, M), envelope protein (Envelope), spike protein (Spike), membrane protein (Membrane, M), hemolysin (Haemolysin, HL), fusion protein (Fusion, F), glycoprotein (Glycoprotein, G).
  • diseases that can be prevented and/or treated with the nucleic acid expression vectors of the present disclosure or related products thereof include, but are not limited to, one or more cancers selected from the following: squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, Hepatocellular carcinoma, renal cell carcinoma, bladder cancer, intestinal cancer, cervical cancer, colon cancer, esophageal cancer, head cancer, kidney cancer, liver cancer, lung cancer, neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer, leukemia, lymphoma Neoplasm, Burkitt's lymphoma, non-Hodgkin's lymphoma; melanoma; myeloproliferative disorders; sarcoma, angiosarcoma, Kaposi's sarcoma, liposarcoma, sarcoma, peripheral neuroepithelialoma, synovial sarcoma , glioma, astrocytoma, oligodendro
  • diseases that can be prevented and/or treated with the nucleic acid expression vectors of the present disclosure or related products thereof include, but are not limited to, one or more genetic diseases selected from the following: methylmalonic acidemia, acute intermittent Porphyria, Fabry disease, albinism, hemophilia, phenylketonuria, galactosemia, mucopolysaccharidoses, and congenital adrenocortical hyperplasia.
  • the term "pharmaceutically or physiologically acceptable” ingredients are suitable for use in humans and/or animals without undue adverse side effects (e.g., toxicity, irritation, and allergic reactions), i.e., with a reasonable benefit/risk ratio substance.
  • the term “effective amount” refers to an amount that produces a function or activity in humans and/or animals and is acceptable to humans and/or animals.
  • the term "pharmaceutically acceptable carrier” refers to a vehicle for administration of a therapeutic agent, including various excipients and diluents. This term refers to pharmaceutical carriers that do not themselves require the active ingredient and are not unduly toxic upon administration. Suitable carriers are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences, Mack Pub. Co., N.J. 1991.
  • compositions may contain liquids such as water, saline, glycerin and ethanol.
  • these carriers may also contain auxiliary substances, such as fillers, disintegrants, lubricants, glidants, effervescent agents, wetting agents or emulsifiers, flavoring agents, pH buffering substances, etc.
  • these materials may be formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, typically at a pH of about 5-8, preferably at a pH of about 6-8.
  • unit dosage form refers to the preparation of the active substance herein into a dosage form required for a single administration for the convenience of administration, including but not limited to various solid dosage forms (such as tablets), liquid dosage forms, and capsules. agent, sustained-release agent.
  • the effective dose of active substance employed may vary depending on the severity of the subject to be administered or treated. The specific situation is determined based on the individual situation of the subject (such as the subject's weight, age, physical condition, and desired effects), which is within the scope of a skilled physician's judgment.
  • the products herein may be in solid state (such as granules, tablets, freeze-dried powder, suppositories, capsules, sublingual tablets) or liquid state (such as oral liquid) or other suitable shapes.
  • the route of administration can be: (1) direct naked nucleic acid injection; (2) connecting the mRNA expression vector to the transferrin/poly-L-lysine complex to enhance its biological effect; (3) expressing the mRNA The carrier forms a complex with positively charged lipids to overcome the difficulty in crossing the cell membrane caused by the negative charge of the phosphate backbone; (4) wrapping the mRNA expression vector with liposomes and mediating its entry into cells is beneficial to the smooth movement of macromolecules Enter and avoid hydrolysis by various extracellular enzymes; (5) Combine the mRNA expression vector with cholesterol to increase its retention time; (6) Use immunoliposomes to transport the mRNA to specifically transport it to the target tissue and target cells; (7) transfecting the mRNA expression vector into the transfected cells in vitro; (8) electropor
  • the plasmid was linearized by digestion with XbaI at the single enzyme site and used as a template for in vitro transcription.
  • the purified eGFP mRNA is added with a 7-methylguanosine cap structure.
  • the capped mRNA is purified with lithium chloride and transfected.
  • the reagent was Lipofectamine 3000, and the HEK293T cell transfection experiment was performed, and the expression level of eGFP green fluorescent protein was analyzed by BD LSRFortessa flow cytometer.
  • rabies mRNA vaccine In order to construct a rabies mRNA vaccine, the DNA sequence containing the Pitman-Moore (PM) vaccine strain glycoprotein (RABV-G) was used as the basis for construction, and was optimized according to the preference of eukaryotic codons for subsequent in vitro transcription experiments. .
  • the corresponding DNA template sequence is as described in SEQ ID NO: 23.
  • the DNA sequence of the extracellular end of the HA2 protein in the stem region of the influenza virus hemagglutinin protein was used as the basis for construction, and was optimized according to the preference of eukaryotic codons for subsequent in vitro transcription experiments.
  • the corresponding DNA template sequence is as described in SEQ ID NO: 26.
  • sequence optimization and modification were carried out using similar methods as described above, and the obtained mRNA (SEQ ID NO: 27, 28) was used for the preparation of lipid nanoparticles.
  • rabies mRNA liposome nanoparticles (Maiana (Shanghai) Instrument Technology Co., Ltd.): Combine the cationic lipid Dlin-MC3-DMA, structural lipid cholesterol, auxiliary lipid DSPC and stable lipid DMG-PEG2000, according to Dissolve in ethanol at a molar ratio of 50:38:10:2, and the ethanol concentration is 30% (v/v) to obtain an oil phase mixture. Then, the oil phase mixture was added to 50mM pH 4.0 citrate buffer at room temperature to obtain a lipid mixture.
  • lipid mixture to a liposome extruder, first squeeze and filter it with a 200nm filter membrane, and then filter it with a 100nm filter membrane to change the solution from milky white to clear, and obtain cationic liposome nanoparticles.
  • RABV-G mRNA cationic lipid nanoparticles Dissolve the optimized nucleoside-modified RABV-G mRNA in citrate buffer (pH 4.0), and add it dropwise to the cationic lipid nanoparticles at a ratio of cationic lipid nanoparticles to mRNA mass ratio of 20:1. , use a Vortex shaker to mix to obtain a mixed solution. After thorough mixing, heat and incubate in a metal bath at 42°C for 1 hour. The mixture is then dialyzed into sterile PBS and filtered with a 0.22 ⁇ m sterile filter to obtain RABV-G mRNA cationic lipid nanoparticles, which is the rabies virus nucleic acid vaccine.
  • Rabies virus envelope protein G protein was used as the immunogen to immunize BALB/c mice, 5 in each group.
  • the initial immunization dose is 10 ⁇ g
  • the boosting dose is 1 ⁇ g
  • the intramuscular inoculation route is 100 ⁇ l
  • immunization twice is set, 2 weeks and 2 weeks after the second immunization.
  • Blood was collected at 1 month, 5 months, and 7 months, and the levels of rabies virus G protein-specific antibodies were evaluated by ELISA.
  • influenza virus mRNA liposome nanoparticles As in II, the company was sent to prepare influenza virus mRNA liposome nanoparticles, and the HA2 protein at the extracellular end of the stem region of the influenza virus hemagglutinin protein was used as the immunogen to immunize BALB/c mice, 5 in each group. Both the primary and booster doses were 5 ⁇ g, administered intramuscularly, with an interval of 3 weeks, and a liposome empty vector control group was also set up. Blood was collected 2 weeks after the second immunization to analyze specific antibody levels, and ELISA was used to evaluate and analyze the extracellular activity in the HA stem region. Segment HA2 protein-specific antibody levels.
  • the mRNA vectors are named in sequence: I-eGFP actin (30A), as shown in SEQ ID NO: 12; I-eGFP albumin (30A), as shown in SEQ ID NO: 13; I-eGFP- ⁇ -globin (30A ), as shown in SEQ ID NO: 14;
  • eGFP-mRNA vectors derived from the 3′-UTR of human albumin and human ⁇ -globulin and each with a poly(A) tail of 120A, named in turn: II-eGFP-albumin (120A), such as SEQ ID NO: 15; II-eGFP- ⁇ -globin (120A), as SEQ ID NO: 16;
  • III-eGFP-albumin such as SEQ ID NO: 17
  • III-eGFP- ⁇ -globin as SEQ ID NO: 18
  • SEQ ID NO: 18 eGFP-mRNA vectors derived from the 3′-UTR of human albumin and human ⁇ -globulin and each with a 250A poly(A) tail
  • the UTR sequence ⁇ -globin+albumin
  • the tandem sequence of human albumin and eukaryotic mitochondria albumin+Mit
  • the tandem sequence of human ⁇ -globin and eukaryotic mitochondria ⁇ -globin+Mit
  • IV-eGFP( ⁇ -globin+albumin)(250A) as shown in SEQ ID NO: 19
  • IV-eGFP(albumin+Mit)(250A) as shown in SEQ ID NO: 20
  • IV-eGFP( ⁇ -globin+Mit)(250A) as shown in SEQ ID NO: 21;
  • the eGFP-mRNA vector contains a tandem 3′-UTR with a 120A poly(A) tail, where the tandem 3′-UTR is a 3′-UTR derived from human ⁇ -globin and eukaryotic mitochondria.
  • the tandem sequence is named: IV-eGFP( ⁇ -globin+Mit)(120A), as shown in SEQ ID NO: 22;
  • rabies mRNA vaccines The screened mRNA vectors each with a 120A poly(A) tail and a 250A poly(A) mRNA vector (3′-UTR are both ⁇ -globin+Mit) are used for rabies mRNA vaccines, which are named in turn. :RABV-G mRNA(120A)(SEQ ID NO:25) and RABV-G mRNA(250A)(SEQ ID NO:24);
  • the screened mRNA vectors each with a 120A poly(A) tail and a 250A poly(A) tail (3′-UTR are both ⁇ -globin+Mit) are used for influenza virus mRNA vaccines, They are named in sequence: HA2 mRNA (120A) (SEQ ID NO: 28) and HA2 mRNA (250A) (SEQ ID NO: 27).
  • Example 1 Expression verification of HEK293T cells transfected with eGFP-mRNA vectors having 3′-UTRs from different sources and carrying a 30A poly(A) tail
  • HEK293T cells were transfected with eGFP-mRNA prepared according to the above experimental method I, and expression verification was performed.
  • HEK293 cells were seeded 24 hours before transfection, and the cells were seeded into a 12-well plate at a density of 200,000 cells per well.
  • the medium was DMEM complete medium (containing 10% FBS and 1% P.S.).
  • the transfection reagent is Lipofectamine 3000.
  • the transfection ratio of mRNA to Lipofectamine 3000 is 1:2.
  • Each well plate is transfected with 2 ⁇ g eGFP-mRNA and cultured in a 37°C incubator.
  • the expression level of eGFP was detected by flow cytometry, and the detection time points were 12h, Day1, Day2, Day3, Day4, Day5, Day6, and Day7.
  • I-eGFP actin (30A), I-eGFP albumin (30A), and I-eGFP- ⁇ -globin (30A) can all be expressed in large amounts after transfection into HEK293T cells, and the transfection positive rates are all within More than 60%, reaching the highest within 24h to 48h after transfection (Figure 2A).
  • the average fluorescence intensity of eGFP is 2 to 3 times higher than that of I-eGFP actin (30A), and can last for many days. Relatively high expression (Figure 2B).
  • I-eGFP albumin (30A) and I-eGFP- ⁇ -globin (30A) can efficiently transfect cells, and the 3'-UTR they contain contributes to a significant increase in expression levels.
  • Example 2 Expression verification of HEK293T cells transfected with eGFP-mRNA vectors with 3′-UTR from different sources and carrying a 120A poly(A) tail or a 250A poly(A) tail.
  • Example 2 The same method as in Example 1 was used to transfect HEK293T cells for expression verification, except that the expression vectors used were poly(A) tails with 3'-UTRs from different sources and with 120 A's or 250 A's.
  • II-eGFP-albumin (120A) and II-eGFP- ⁇ -globin (120A) can be expressed in large amounts after transfection into HEK293T cells, and the transfection positive rates reached more than 80% ( Figure 3A).
  • the average fluorescence intensity of eGFP is higher than that of eGFP-mRNA with the same 3'-UTR of 30A ( Figure 3B); the same 3'-UTR of eGFP-mRNA with 250 poly(A) is III-eGFP-albumin (250A) , III-eGFP- ⁇ -globin (250A), the positive rate reached more than 85% ( Figure 3A), which is 1.5 to 2 times higher than the average fluorescence intensity of eGFP with 120poly(A), although the fluorescence intensity decreased from the 2nd day onwards. There was a decrease at first, but the decrease of eGFP-mRNA with 250 poly(A) was less, and it could be maintained for at least 7 days (Figure 3B).
  • Example 3 Expression verification of HEK293T cells transfected with eGFP-mRNA vectors with different tandem 3′-UTRs and a 250A poly(A) tail.
  • HEK293T cells were transfected and detected at different time points for expression verification.
  • IV-eGFP ( ⁇ -globin+Mit) (250A) mRNA has the highest average eGFP fluorescence intensity, which can reach 3E4 and above, compared with The other two tandem combinations were 2 to 4 times higher ( Figure 4B); and although the fluorescence intensity began to decrease after the second day, the eGFP-mRNA of IV-eGFP ( ⁇ -globin+Mit) (250A) decreased more than less, and the average fluorescence intensity remained above 1E4 for at least 7 days (Figure 4C).
  • Example 4 Expression verification of HEK293T cells transfected with eGFP-mRNA vectors with the same tandem 3′-UTR and a poly(A) tail of 120A or 250A
  • Example 2 The same method as in Example 1 was used to transfect HEK293T cells and detect expression at different time points, except that the expression vector used was an eGFP-mRNA vector with the same tandem 3′-UTR and a poly(A) tail of 120A or 250A.
  • IV-eGFP( ⁇ -globin+Mit)(120A) and IV-eGFP( ⁇ -globin+Mit)(250A) can be expressed in large amounts after transfection into HEK293T cells, and the transfection positive rates are all within More than 85%, up to more than 95% (Figure 5A), and the average fluorescence intensity of eGFP-mRNA with the same tandem 3'-UTR but with a 250A poly(A) tail after transfection is higher than that of the eGFP-mRNA with 120A poly( A) The average fluorescence intensity of eGFP-mRNA in the tail was about 2 times higher (Figure 5B); and the fluorescence intensity also decreased after the second day (Figure 5B).
  • Example 5 The humoral response level after vaccinating mice with rabies mRNA vaccine prepared using an mRNA vector with the same tandem 3′-UTR and a 120A poly(A) tail or a 250A poly(A) tail.
  • this example applies it to the preparation of mRNA rabies vaccine.
  • the mRNA selected in this example is: RABV-G mRNA (120A) and RABV-G mRNA (250A) were made into cationic lipid nanoparticles encapsulating RABV-G mRNA, and BALB/c mice were immunized to evaluate their immunogenicity. . There were 5 mice in each group. The initial dose was 10 ⁇ g and the boosting dose was 1 ⁇ g. The injection volume was 100 ⁇ l per intramuscular route. The mice were immunized twice with an interval of 3 weeks. Blood was collected from the orbit 2 weeks, 2 months, 5 months, and 7 months after the secondary immunization. The negative control was an equal volume of cationic lipid nanoparticles without nucleic acid encapsulation (i.e., no-load control).
  • Example 6 The humoral response level after vaccinating mice with influenza virus mRNA prepared using an mRNA vector with the same tandem 3′-UTR and a 120A poly(A) tail or a 250A poly(A) tail.
  • the selected non-replicating mRNA vector is used for the preparation and vaccination of influenza virus mRNA vaccine.
  • the influenza virus mRNA used in this example is HA2 mRNA (120A) and HA2 mRNA (250A).
  • HA2 mRNA liposome nanoparticles were prepared and BALB/c mice were immunized to evaluate their immunogenicity. Immunogenicity was evaluated 2 weeks and 4 weeks after boosting as described in Materials and Methods III.
  • the non-replicating mRNA vector selected in this disclosure (especially IV-eGFP-( ⁇ -globin+Mit) (250A)) can be loaded with different target genes (eGFP Gene replacement with various target genes), and can achieve efficient translation and stable and sustained expression.

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Abstract

提供了一种能够高效表达目的蛋白的mRNA 表达载体，其从5'端至3'端依次包含:（a）5'-UTR元件;（b）编码目的蛋白的开放阅读框元件；（c）3'-UTR元件;（d）总长度为120 nt以上的多聚腺苷酸尾元件。还提供了该mRNA 表达载体的构建、相关产品及其应用，例如用作mRNA疫苗。

Description

能高效表达目的基因的mRNA载体系统、其构建及应用 技术领域
本公开属于生物医药产业领域，尤其涉及基因工程药物和疫苗制造。具体而言，本公开涉及一种能高效表达目的基因的mRNA载体系统、其构建以及在目的基因表达中的应用，例如其可用作核酸疫苗载体。
背景技术
近年来，mRNA疫苗技术因其众多的优点成为了最受关注的疫苗形式之一。与质粒DNA和病毒载体疫苗不同，mRNA不会整合到基因组中，从而避免了插入突变可能带来的隐患。mRNA疫苗可以以无细胞的方式生产，生产迅速，工艺简单、成本可控。此外，一种单一的mRNA疫苗可以编码多种抗原，使其能够靶向肿瘤靶点、不同的微生物、并增强对高突变病原体的免疫应答。
随着mRNA制备技术的成熟化，体外转录生产mRNA分子愈来愈简单。体外转录的mRNA分子必须模拟内源mRNA分子的结构，按照5′→3′方向依次需包括以下元件：5′帽结构，5′-UTR(非翻译区域)序列、编码序列、3′-UTR序列、和聚腺苷酸(poly(A)尾)序列。提高细胞内mRNA稳定性和翻译的方法包括：5′帽结构的修饰、5′-UTR和3′-UTR的选择、poly(A)尾的结构修饰和编码序列的优化。
目前，5′帽结构主要通过共转录加帽和酶加帽法进行修饰，以保护mRNA不被外切核酸酶降解，并与3′端的poly(A)尾协同工作。poly(A)结合蛋白(PABP)与poly(A)尾序列结合后，招募翻译起始因子蛋白eIF4G和eIF4E，使mRNA环状化并招募核糖体启动翻译。鉴于mRNA的5′-UTR和3′-UTR可显著影响转录产物的翻译速度和半衰期，优化UTR是mRNA疫苗设计的重点之一。通常认为3′-UTR是mRNA中不稳定因子较集中的区域，需选用稳定表达蛋白质或病毒基因组的序列。
mRNA转录的最后一个步骤是添加poly-A尾，poly(A)尾部保护mRNA免受降解，并促进poly(A)结合蛋白的后续结合。因此，将poly(A)尾加入到编码 抗原的mRNA模板中会导致更高水平的蛋白表达。长poly-A序列更有利于mRNA的稳定性，延长其半衰期。根据早期的研究，在后生动物中，poly-A尾通常约为250bp。在人单核细胞来源的树突状细胞(DCs)中，120bp的poly-A序列比短的poly-A尾提供更稳定的IVT-mRNA(in vitro-transcribed mRNA)和更高效的翻译。在人原代T细胞中，超过300个核苷酸的Poly-A序列有利于更高效的翻译，其中具有中、长poly-A尾的IVT-mRNA最先招募PABP，并被剪切到30A长，这与自然产生的mRNA poly(A)尾大小一致。在DNA质粒中加入poly(A)尾省去了体外加尾的步骤，可减少整体反应时间和原材料的损失，也避免了因使用poly(A)聚合酶进行的酶多聚腺苷化造成的尾部长度变化。
然而，虽然大于100bp的Poly(A)尾是治疗性mRNA疫苗的最佳选择，编码这些长的poly(A)DNA序列却会破坏用于转录的DNA质粒的稳定性。并且，有研究表明当连续的Poly(A)尾长度大于120bp时，相应蛋白质的表达水平并不提高。
在目前已成功上市的Pfizer-BioNTech开发的新型冠状病毒BNT162b2 mRNA疫苗中的尾部包含100A的poly(A)，其间插入一个10bp的UGC链接子，生成序列如30nt polA+GCATATGACT+70nt polA的Poly(A)尾。
当前用于制备mRNA的载体大多数模板质粒装载100-120个长度不等的poly-A序列，而关于mRNA载体构建及优化的报道甚少。
提高细胞内mRNA稳定性、翻译效率和长效持续性一直是mRNA疫苗需要攻克的难点之一，这与用于制备mRNA的表达载体密切相关。由此，本领域中迫切需要提供能够克服如上技术问题的新型mRNA载体。
发明内容
本公开中正是提供了一种新型的mRNA载体、其制备方法和应用。
在一些方面中，本文提供了一种包含能够表达目的蛋白的mRNA核酸表达载体，其从5′端至3′端依次包含：
(a)5′-UTR元件；
(b)编码目的蛋白的开放阅读框元件；
(c)3′-UTR元件；
(d)总长度为120nt以上的多聚腺苷酸尾元件，其包含：多个腺苷酸串，每个腺苷酸串各自独立地包含n个连续的腺苷酸，n为10～80之间的整数，且所述多个腺苷酸串的腺苷酸总个数为100个以上；以及位于所述多个腺苷酸串之间的连接子，所述连接子各自独立地不包含腺苷酸或仅包含1个或2个腺苷酸。
本公开的核酸表达载体能够在体内外高效表达所需的目的蛋白，从而实现诸如疾病预防和/或治疗效果，例如用作治疗性药物、预防性药物、蛋白质替代治疗分子、基因编辑治疗分子等等。本公开的核酸表达载体能够用于例如病毒感染、癌症、遗传疾病(例如单基因疾病)的预防和/或治疗。
在一些方面中，本文提供了一种组合物，其包含本文的核酸表达载体，以及包装物和/或用于该核酸表达载体的递送系统和/或药学上或生理学上可接受的运载体。
在一些方面中，本文还提供了本文核酸表达载体和/或组合物在制备用于表达目的蛋白的产品中的应用，所述产品可例如选自：mRNA疫苗、治疗性或预防性药物，如蛋白质替代治疗药物、基因编辑治疗药物。
在一些方面中，本文还提供了疾病预防和/或治疗的方法，所述方法包括给予有需要的对象预防和/或治疗有效量的本文核酸表达载体和/或组合物。
在一些方面中，本文还提供了用于表达目的蛋白的本文核酸表达载体和/或组合物。在一些方面中，进一步提供了用于疾病预防和/或治疗的本文核酸表达载体和/或组合物。
在一些方面中，本文还提供了制备如本文所述核酸表达载体或组合物的方法，所述方法包括：提供独立或连接的各元件；将各元件组装成核酸表达载体。
本领域的技术人员可对前述的技术方案和技术特征进行任意组合而不脱离本发明的发明构思和保护范围。本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
下面结合附图对本发明作进一步说明，其中这些显示仅为了图示说明本发明的实施方案，而不是为了局限本发明的范围。
图1：图1mRNA转录模板空载体改造后的线性化pCDNA3.1+质粒示意图。
图2：不同来源的3′-UTR并带有30poly(A)载体编码的eGFP mRNA转染HEK293T细胞的阳性率(A)和表达水平(B)。
图3：不同来源的3′-UTR并带有120poly(A)和250poly(A)载体编码的eGFP mRNA转染HEK293T细胞的阳性率(A)和表达水平(B)。
图4：不同串联的3′-UTR带有250poly(A)载体编码的eGFP mRNA转染HEK293T细胞的阳性率(A)和表达水平(B)比较。
图5：相同串联3′-UTR带有120poly(A)和250poly(A)载体编码的eGFP mRNA转染HEK293T细胞的阳性率(A)和表达水平(B)比较。
图6：相同串联3′-UTR带有120poly(A)和250poly(A)mRNA载体用于狂犬病mRNA疫苗接种小鼠后的体液应答水平比较(纵坐标为log以10为底的对数)。
图7：相同串联3′-UTR带有120poly(A)和250poly(A)mRNA载体用于流感病毒mRNA疫苗接种小鼠后的体液应答水平比较(纵坐标为log以10为底的对数)。
具体实施方式
本公开中提供一种可高效表达目的蛋白的mRNA载体，该mRNA载体可为非复制型mRNA载体，其能够高效的表达不同目的基因，无论是在体外细胞表达水平上，还是通过递送工具将mRNA递送至生物体内，目的基因均可实现高效翻译、稳定持续性表达。
本申请中以eGFP绿色荧光蛋白基因为参考，针对mRNA载体的元件进行一系列优化，通过比较eGFP的表达水平，筛选出可高效表达目的基因的非复制型mRNA载体，该mRNA载体既可高效翻译，还可在细胞水平或生物体内稳定持续性表达。
与现有技术相比，本文的核酸表达载体可高效的表达各种目的分子，例如高效表达各种不同抗原分子；通过对表达载体中各元件的选择、修饰、改造和组合，可使目的基因在细胞水平或生物体内实现高效稳定的翻译，且可有效调控目的基因的表达水平和体内半衰期，使其达到高效免疫原性的同时具有长效持续性。由此，本申请的表达载体及其相关产品在预防性和治疗性疫苗的应用、特异性抗体表达、治疗性或靶向性药物的表达、蛋白替换疗法等方面均有广阔应用前景。
本文中提供的所有数值范围旨在清楚地包括落在范围端点之间的所有数值及它们之间的数值范围。可对本发明提到的特征或实施例提到的特征进行组合。本说明书所揭示的所有特征可与任何组合物形式并用，说明书中所揭示的各个特征，可以任何可提供相同、均等或相似目的的替代性特征取代。因此除有特别说明，所揭示的特征仅为均等或相似特征的一般性例子。
如本文所用，“含有”、“具有”或“包括”包括了“包含”、“主要由……构成”、“基本上由……构成”、和“由……构成”；“主要由……构成”、“基本上由……构成”和“由……构成”属于“含有”、“具有”或“包括”的下位概念。
如本文所用，“真核生物”可包括人、灵长类动物、啮齿动物(例如大鼠、小鼠、豚鼠、仓鼠)、驯养动物或畜牧哺乳动物。
本文所述的高度序列同一性，包括具有70％以上、75％以上、80％以上，更优选85％以上，如85％、90％、95％、98％甚至99％或以上的序列同一性，这些高同一性序列也在本发明优选考虑的等同范围之内。比对序列同一性的方法和工具也是本领域周知的，如BLAST。
mRNA核酸表达载体及包含该载体的组合物
本文中提供了一种包含能够表达目的蛋白的mRNA核酸表达载体，其从5′端至3′端依次包含：
(a)5′-UTR元件；
(b)编码目的蛋白的开放阅读框元件；
(c)3′-UTR元件；
(d)总长度为120nt以上的多聚腺苷酸尾元件，其包含：
多个腺苷酸串，每个腺苷酸串各自独立地包含n个连续的腺苷酸，n为10～80之间的整数，且所述多个腺苷酸串的腺苷酸总个数为100个以上；以及
位于所述多个腺苷酸串之间的连接子，所述连接子各自独立地不包含腺苷酸或仅包含1个或2个腺苷酸。
在一些实施方式中，本公开中所用5′-UTR元件的长度为10～200nt，例如15～100nt。
在一些实施方式中，本公开中所用5′-UTR元件源自下组中的一种或多种5′-UTR：人α-球蛋白、β-球蛋白、核糖体蛋白(RP)、微管蛋白β-2B、补体因子3(C3)、细胞色素P4502E1(CYP2E1)、载脂蛋白A-II(APOA2)、人类血红蛋白亚基β(hHBB)、血红蛋白A1(HBAl)、血红蛋白A2(HBA2)、登革热病毒(DENV)。
在一些实施方式中，本公开中所用5′-UTR元件具有如SEQ ID NO：1所示的序列或与其具有至少80％的序列同一性。
在一些实施方式中，本公开中所用3′-UTR元件为来源于哺乳动物或病毒的3′-UTR，例如来源选自下组的3′-UTR或其组合(如串联序列)：人α球蛋白、人β球蛋白、人白蛋白、人肌动蛋白、人血红蛋白亚基α1(HBAl)、细胞色素B-245α链(CYBA)、真核生物线粒体(Mit)的序列、SARAS-Cov-2、登革热病毒(DENV)、萝卜皱病毒(TCV)、烟草花叶病毒(TMV)和烟草蚀刻病毒(TEV)。
在一些实施方式中，本公开中所用3′-UTR元件包含选自下组一个或多个3′-UTR分子：α球蛋白3′-UTR、真核生物线粒体3′-UTR、白蛋白3′-UTR、β球蛋白3′-UTR或其任意串联序列，优选α球蛋白3′-UTR、真核生物线粒体3′-UTR、或它们串联形成的3′-UTR。
在一些实施方式中，本公开中所用3′-UTR具有如SEQ ID NO：2～8中所示的一个或多个序列，优选如SEQ ID NO：2、5或8所示的序列，或与其中任何一者具有至少80％的序列同一性的序列。
在一些实施方式中，本公开中所用多聚腺苷酸尾元件的总长度为120～400nt，例如120～350nt，120～320nt，或其中的任意整数，例如120、304nt。
在一些实施方式中，各腺苷酸串各自独立地包含10～80个、20～70个、25～60个、30～50个或其中任意整数个连续的腺苷酸，如20个、30个、33个、35个、36个、37个、38个、39个、40个、45个、50个、55个、60个、65个、70个连续腺苷酸。
在一些实施方式中，所述多聚腺苷酸尾元件还进一步包含位于所述元件一个或两个端部的连接子。
在一些实施方式中，所述连接子的长度各自独立地为3～15nt，例如3、4、5、6、7、8、9、10、11、12、13、14或15nt。在一些实施方式中，所述连接子各自独立地不包含腺苷酸，或仅包含1个腺苷酸或2个腺苷酸。
在一些实施方式中，所述连接子的序列各自独立地选自例如：GCTATGACT、GTATGT、GCAAGT、GATTGC、GGCTGC、TACTGC、GGCTTC、GCATATGACT。在一些实施方式中，所述多聚腺苷酸尾元件具有SEQ ID NO：10或SEQ ID NO：11的序列，或与其中任何一者具有至少80％的序列同一性。
在本文的一些实施方式中，所述多聚腺苷酸尾元件包含的250个多聚腺苷酸序列是不连续的，每20～40个(例如30个、40个)A间隔一个5～10bp的不含A、或仅含1个或2个A碱基的链接子序列，以利于编码序列更稳定、并可高效持久的翻译，延长其半衰期。
在一些实施方式中，本公开的核酸表达载体为非复制型mRNA载体或作为核酸疫苗。在一些实施方式中，本公开中编码目的蛋白的所述元件为单顺反子、双顺反子或多顺反子mRNA。在一些实施方式中，所述的双顺反子或多顺反子mRNA即含有两个及以上编码区的mRNA。
在一些实施方式中，编码目的蛋白的所述元件经过或未经密码子优化、包含或不包含碱基修饰和/或核苷类似物，例如在编码元件中采用选自下组的一个或多个修饰碱基或核苷类似物：假尿苷、N1-甲基假尿苷、N1-乙基假尿苷、2-硫尿苷、4′-硫尿苷、5-甲基胞嘧啶、5-甲基尿苷、2-硫基-1-甲基-1-去氮杂-假尿苷、2-硫基T-甲基-假尿苷、2-硫基-5-氮杂-尿苷、2-硫基-二氢假尿苷、2-硫基-二氢尿苷、2-硫基-假尿苷、4-甲氧基-2-硫基-假尿苷、4-甲氧基-假尿苷、4-硫基-1-甲基-假尿苷、4-硫基-假尿苷、5-氮杂-尿苷、二氢假尿苷或5-甲氧基尿苷 和2′-0-甲基尿苷中的至少一种，优选假尿苷或N1-甲基假尿苷或N1-乙基假尿苷，进一步优选为N1-甲基假尿苷。
在一些实施方式中，表达载体序列可经密码子优化和/或包含修饰碱基和/或核苷类似物。例如，在一些实施方式中，表达载体序列中的50％～100％尿嘧啶被置换。通过置换能够提高mRNA在生物体内的稳定性。
在一些实施方式中，本公开核酸表达载体还包括5′-帽元件，其可选被修饰，例如所述5′-帽元件选自：m7GpppXpYp、m7GpppXmpYp、m7GpppXmpYmp、或其甲基化修饰序列、反向结合异构体、抗-反转帽类似物(ARCA)、N7-苄基二核苷四磷酸帽类似物。
在一些实施方式中，本公开核酸表达载体还包括启动子元件，例如T7启动子、sp6启动子或T3启动子。
在一些实施方式中，本公开核酸表达载体还包括信号肽编码元件，例如指导目的蛋白亚细胞定位的信号肽(如跨膜信号肽、分泌信号肽、核定位信号肽)编码元件。
在一些实施方式中，本公开核酸表达载体还包括酶切位点，例如XbaI、EcoRV、BamHI、XhoI。
在一些实施方式中，本公开核酸表达载体还包括标签，例如用于目的分子鉴定、分离或纯化的分子标签，如Flag标签、HA标签。
在一些实施方式中，本公开核酸表达载体包含能够表达选自下组中的一种或多种目的蛋白的mRNA：免疫原分子、抗体分子、治疗性药物、预防性药物、蛋白质替代治疗分子、基因编辑治疗分子。
在一些实施方式中，本公开的核酸表达载体可以用来表达各种不同的外源基因，包括但不限于特异性抗体的表达，治疗性或靶向性药物的表达，蛋白替换疗法等。
在一些实施方式中，所述核酸表达载体可以制备核酸疫苗，所述核酸疫苗可以用于制备癌症类疫苗、病毒类疫苗，其中所述的病毒可以是各类传染性疾病病毒，包括埃博拉病毒、狂犬病毒、寨卡病毒、黄热病病毒、登革热病毒、巨细胞病毒、蓝耳病病毒、猪瘟病毒、肠道病毒、乙型肝炎病毒、呼吸道合胞 病毒、单纯疱疹病毒、人乳头瘤病毒、人免疫缺陷病毒、流感病毒、冠状病毒、副流感病毒、麻疹病毒、腮腺炎病毒、尼帕病毒、人偏肺病毒等。所述的癌症，包括鳞状细胞癌、基底细胞癌、腺癌、肝细胞癌、肾细胞癌、膀胱癌、肠癌、宫颈癌、结肠癌、食道癌、头癌、肾癌、肝癌、肺癌、颈癌、卵巢癌、胰腺癌、前列腺癌、胃癌、白血病、淋巴瘤、伯基特氏淋巴瘤、非霍奇金氏淋巴瘤；黑素瘤；骨髓增生性疾病；肉瘤、血管肉瘤、卡波氏肉瘤、脂肪肉瘤、肌肉瘤、外周神经上皮瘤、滑膜肉瘤、神经胶质瘤、星形细胞瘤、少突神经胶质瘤、室管膜瘤、成胶质细胞瘤、成神经细胞瘤、神经节细胞瘤、神经节神经胶质瘤、成神经管细胞瘤、松果体细胞肿瘤、脑膜瘤、脑膜肉瘤、神经纤维瘤和神经鞘瘤；乳腺癌、子宫癌、睾丸癌、甲状腺癌、星形细胞瘤、食道癌、癌肉瘤、霍奇金氏病、维尔姆斯瘤和畸胎癌等。
在一些实施方式中，所述核酸表达载体中的目的多肽为免疫原，其表达部位包括细胞内、细胞膜上或分泌性表达。在一些实施方式中，所述免疫原为病毒免疫原，其例如来自于病毒囊膜蛋白，包括但不限于血凝素蛋白(Hemagglutinin，HA)、神经氨酸酶(Neuraminidase，NA)、基质蛋白(Matrix protein，M)、包膜蛋白(Envelope)、刺突蛋白(Spike)、膜蛋白(Membrane，M)、溶血素(Haemolysin，HL)、融合蛋白(Fusion，F)、糖蛋白(Glycoprotein，G)。
在一些实施方式中，本公开核酸表达载体从5′端到3′端包含：包含SEQ ID NO：1所示序列的5′-UTR元件；编码目的蛋白的开放阅读框元件；包含如SEQ ID NO：2、5或8所示序列的3′-UTR元件；包含如SEQ ID NO：10或11所示序列的多聚腺苷酸尾元件；或与前述序列分别具有至少80％序列同一性的序列的元件。
在一些实施方式中，本公开核酸表达载体包含如SEQ ID NO：15～22、24～25和27～28中任一项所述的序列、与其具有至少80％序列同一性的序列、或前述任一序列中编码其目的蛋白的开放阅读框元件被替换为编码所需目的蛋白的开放阅读框元件所得的序列。
在本文的一些实施方式中，提供了一种非复制型mRNA表达载体，所述非复制型mRNA载体为可编码不同基因mRNA所需的质粒模板。所述的mRNA 载体质粒模板包括体外转录mRNA分子所需的基本元件。
在一些实施方式中，所述表达载体包含以下元件：T7启动子、5′-UTR序列、编码序列、3′-UTR序列、聚腺苷酸尾序列、线性化酶切位点。这些元件可如本文中所描述。
在本发明的具体实施方式中，所述的非复制型mRNA载体包含的5′-UTR序列、3′-UTR序列、聚腺苷酸尾序列选自SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11，或者，所述的非复制型mRNA载体包含的5′-UTR序列、3′-UTR序列、聚腺苷酸尾序列为与上述任意序列至少75％的同源性的序列(例如，与SEQ ID NO：1、SEQ ID NO：5、SEQ ID NO：8、SEQ ID NO：11具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的同源性)，并且所述的非复制型mRNA载体可表达各种不同的外源基因。
本公开还提供了所述的非复制型mRNA载体在体外细胞水平上的表达。以eGFP荧光蛋白基因为参考，针对其元件包括5′-UTR序列、3′-UTR序列、聚腺苷酸尾序列进行一系列优化，通过比较eGFP的表达水平，筛选出一种可高效表达的非复制型mRNA载体IV-eGFP(α-globin+Mit)(250A)。本领域技术人员知晓可通过分子生物学或遗传工程学技术对eGFP进行替换，以使本文的示例性mRNA载体装载不同的基因。
在一些实施方式中，本公开核酸表达载体单独包含于包装物中，或与递送系统中的运载体组合，例如，所述递送系统选自：脂质递送系统、类脂递送系统、聚合物递送系统或其组合递送系统，例如加载于脂质纳米颗粒、聚氨酯(PAA)、聚β氨基酯(PBAE)、聚乙烯亚胺(PEI)、脂质包裹的聚合物胶束。
制备方法、相关产品及其应用
在本公开的一些方面中还提供了一种组合物，其包含本公开的核酸表达载体，以及包装物和/或用于该核酸表达载体的递送系统和/或药学上或生理学上可接受的运载体。
在一些实施方式中，本公开的组合物，其形式适于选自下组的一种或多种给予或递送方式：呼吸道雾化吸入、滴鼻、口服、直接注射(例如静脉注射、皮下注射、皮内注射、肌肉注射)、黏膜给药。
在一些实施方式中，本公开的组合物还包含佐剂或与佐剂联合使用，例如所述佐剂选自：铝佐剂、霍乱毒素及其亚单位、寡脱氧核苷酸、锰离子佐剂、胶体锰佐剂、弗氏佐剂、MF59佐剂、QS-21佐剂、Poly I：C及其他TLR配体、GM-CSF、IL-2、IL-3、IL-7、IL-11、IL-12、IL-18、IL-21。
在一些实施方式中，本公开的组合物的形式适于进行2种或以上药物或疫苗的组合给予，例如联合接种或序贯接种。在一些实施方式中，本公开的组合物选自：mRNA疫苗、治疗性或预防性药物，如蛋白质替代治疗药物、基因编辑治疗药物。
在本文的一些方面中，提供了制备本文所述的核酸表达载体或组合物的方法，所述方法包括：提供独立或连接的各元件；将各元件组装成核酸表达载体。
在一些实施方式中，所述方法包括采用选自下组的一种或多种材料：DNA模板(例如PCR产物或已线性化的质粒DNA)、核酸酶、聚合酶、加帽酶、聚腺苷酸合酶、DNA酶、一个或多个元件分子、接头分子、天然或修饰的核酸分子、缓冲液、溶剂。
在一些实施方式中，所述方法还包括选自下组的一个或多个步骤：对各元件进行设计、优化、改造和/或修饰；对中间产物和/或最终产物进行分离、纯化、鉴定、定量、包装和/或活性测试；将核酸表达载体与用于该核酸表达载体的递送系统和/或药学上或生理学上可接受的运载体组合。
在本公开的一些方面中，提供了本文的核酸表达载体和/或组合物在制备用于表达目的蛋白的产品中的应用。在一些实施方式中，所述产品选自：mRNA疫苗、治疗性或预防性药物，如蛋白质替代治疗药物、基因编辑治疗药物。
在一些实施方式中，本公开的核酸表达载体用于疾病预防和/或治疗。在一些实施方式中，可用本公开的核酸表达载体或其相关产品预防和/或治疗的疾病包括但不限于选自下组的疾病：病毒感染、癌症、遗传疾病(例如单基因疾病)。
在一些实施方式中，可用本公开的核酸表达载体或其相关产品预防和/或治 疗的疾病包括但不限于选自如下一种或多种病毒感染：狂犬病病毒、流感病毒、冠状病毒、埃博拉病毒、寨卡病毒、黄热病病毒、登革热病毒、巨细胞病毒、蓝耳病病毒、猪瘟病毒、肠道病毒、乙型肝炎病毒、呼吸道合胞病毒、单纯疱疹病毒、人乳头瘤病毒、人免疫缺陷病毒、流感病毒、冠状病毒、副流感病毒、麻疹病毒、腮腺炎病毒、尼帕病毒和人偏肺病毒。
在一些实施方式中，在用于病毒感染性疾病的预防和/或治疗的情况下，本公开的核酸表达载体表达的目的蛋白为病毒免疫原，例如来自于病毒囊膜蛋白，包括血凝素蛋白(Hemagglutinin，HA)、神经氨酸酶(Neuraminidase，NA)、基质蛋白(Matrix protein，M)、包膜蛋白(Envelope)、刺突蛋白(Spike)、膜蛋白(Membrane，M)、溶血素(Haemolysin，HL)、融合蛋白(Fusion，F)、糖蛋白(Glycoprotein，G)。
在一些实施方式中，可用本公开的核酸表达载体或其相关产品预防和/或治疗的疾病包括但不限于选自如下一种或多种癌症：鳞状细胞癌、基底细胞癌、腺癌、肝细胞癌、肾细胞癌、膀胱癌、肠癌、宫颈癌、结肠癌、食道癌、头癌、肾癌、肝癌、肺癌、颈癌、卵巢癌、胰腺癌、前列腺癌、胃癌、白血病、淋巴瘤、伯基特氏淋巴瘤、非霍奇金氏淋巴瘤；黑素瘤；骨髓增生性疾病；肉瘤、血管肉瘤、卡波氏肉瘤、脂肪肉瘤、肌肉瘤、外周神经上皮瘤、滑膜肉瘤、神经胶质瘤、星形细胞瘤、少突神经胶质瘤、室管膜瘤、成胶质细胞瘤、成神经细胞瘤、神经节细胞瘤、神经节神经胶质瘤、成神经管细胞瘤、松果体细胞肿瘤、脑膜瘤、脑膜肉瘤、神经纤维瘤和神经鞘瘤、乳腺癌、子宫癌、睾丸癌、甲状腺癌、星形细胞瘤、食道癌、癌肉瘤、霍奇金氏病、维尔姆斯瘤和畸胎癌。
在一些实施方式中，可用本公开的核酸表达载体或其相关产品预防和/或治疗的疾病包括但不限于选自如下一种或多种遗传疾病：甲基丙二酸血症、急性间歇性卟啉症、法布里病、白化病、血友病、苯丙酮尿症、半乳糖血症、粘多糖病和先天性肾上腺皮质增生症。
如本文所用，术语“药学上或生理上可接受的”成分是适用于人和/或动物而无过度不良副反应(如毒性、刺激和变态反应)的，即有合理的效益/风险比的物质。如本文所用，术语“有效量”是指可对人和/或动物产生功能或活性的且可 被人和/或动物所接受的量。
如本文所用，术语“药学上可接受的运载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。该术语指这样一些药剂载体：它们本身并不是必要的活性成分，且施用后没有过分的毒性。合适的载体是本领域普通技术人员所熟知的。在《雷明顿药物科学》(Remington′s Pharmaceutical Sciences，Mack Pub.Co.，N.J.1991)中可找到关于药学上可接受的赋形剂的充分讨论。
在组合物中药学上可接受的运载体可含有液体，如水、盐水、甘油和乙醇。另外，这些载体中还可能存在辅助性的物质，如填充剂、崩解剂、润滑剂、助流剂、泡腾剂、润湿剂或乳化剂、矫味剂、pH缓冲物质等。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地，pH约为6-8。
如本文所用，术语“单位剂型”是指为了给药方便，将本文的活性物质制备成单次给药所需的剂型，包括但不限于各种固体剂(如片剂)、液体剂、胶囊剂、缓释剂。
应理解，所用活性物质的有效剂量可随待施用或治疗的对象的严重程度而变化。具体情况根据对象的个体情况(例如对象体重、年龄、身体状况、所需达到的效果)来决定，这在熟练医师可以判断的范围内。
本文的产品可以为固态(如颗粒剂、片剂、冻干粉、栓剂、胶囊、舌下含片)或液态(如口服液)或其它合适的形状。给药途径可采用：(1)直接裸核酸注射法；(2)将mRNA表达载体与转铁蛋白/多聚L-赖氨酸复合物连接，以增强其生物效应；(3)使mRNA表达载体与带正电荷的脂类形成复合物，以克服磷酸骨架负电荷所致的穿越细胞膜的困难；(4)用脂质体包裹mRNA表达载体后介导进入细胞，既有利于大分子的顺利进入又免受细胞外各种酶的水解作用；(5)使mRNA表达载体与胆固醇结合使其保持时间增加；(6)用免疫脂质体转运mRNA以使其特异性转运至靶组织和靶细胞；(7)将mRNA表达载体体外转染给转载细胞；(8)电打孔(electroporation)，即借助于电流将mRNA载体导入靶细胞。
实施例
下面结合具体实施例，进一步阐述本发明。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。本领域技术人员可对本发明做出适当的修改、变动，这些修改和变动都在本发明的范围之内。
下列实施例中未注明具体条件的实验方法，可采用本领域中的常规方法，例如参考《分子克隆实验指南》(第三版，纽约，冷泉港实验室出版社，New York：Cold Spring Harbor Laboratory Press，1989)或按照供应商所建议的条件。DNA的测序方法为本领域常规的方法，也可由商业公司提供测试。
除非另外说明，否则百分比和份数按重量计算。除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。
材料、方法与动物
实施例中涉及到的mRNA制备、mRNA转染HEK293T细胞、小鼠免疫及检测方法如下：
I.mRNA的制备
制备合适的DNA模板，以pCDNA3.1+为模板，进行改造，在T7启动子后从N端-C端依次为：5′-UTR序列、编码序列、3′-UTR序列、不同长度的聚腺苷酸尾序列、线性化酶切位点XbaI，并以eGFP荧光蛋白基因为参考，插入到编码序列，并在编码序列C端插入酶切位点EcoRV和Flag标签，加入Flag标签以便后续不同基因表达鉴定，如图1所示。
通过单酶切位点XbaI酶切质粒线性化，并以其为模板，进行体外转录。利用公司购买的体外转录套盒及加帽套盒，按照说明书操作按合适的比例加入转录所需的NTP，使用T7聚合酶，转录得到相应的eGFP mRNA，然后通过酶加帽法即同时使用牛痘病毒加帽酶和2′-O-甲基转移酶一步法进行加帽，将纯化后的eGFP mRNA加上7-甲基鸟苷帽结构，用氯化锂纯化加帽后的mRNA，转染试剂为Lipofectamine 3000，进行HEK293T细胞转染实验，并通过BD  LSRFortessa流式细胞分析仪分析eGFP绿色荧光蛋白的表达水平。
为构建狂犬病mRNA疫苗，以含Pitman-Moore(PM)疫苗株糖蛋白(RABV-G)的DNA序列为构建基础，并且根据真核生物密码子的偏好性进行优化，用于后续的体外转录实验。相对应的DNA模板序列如SEQ ID NO：23所述。以SEQ ID NO：23的质粒单酶切后为模板，按照合适的比例加入转录所需的NTP，并用1-甲基尿嘧啶核苷替代尿嘧啶核苷，使用T7聚合酶，转录得到相应的RABV-G mRNA(SEQ ID NO：24、25)，然后通过酶加帽法同时使用牛痘病毒加帽酶和2’-O-甲基转移酶一步法进行加帽，将纯化后的RABV-G mRNA加上7-甲基鸟苷帽结构，用氯化锂纯化加帽后的mRNA，用于脂质纳米颗粒的制备。
为构建流感病毒mRNA疫苗，以流感病毒血凝素蛋白茎干区胞外端HA2蛋白的DNA序列为构建基础，并且根据真核生物密码子的偏好性进行优化，用于后续的体外转录实验。相对应的DNA模板序列如SEQ ID NO：26所述。采用如前所述的类似方法对其进行体外转录、序列优化与修饰，将得到的mRNA(SEQ ID NO：27、28)用于脂质纳米颗粒的制备。
II.筛选的非复制型mRNA载体用于狂犬病mRNA疫苗接种小鼠
狂犬病mRNA脂质体纳米颗粒的制备(迈安纳(上海)仪器科技有限公司)：将阳离子脂质Dlin-MC3-DMA、结构脂质胆固醇、辅助脂质DSPC和稳定脂质DMG-PEG2000，按50∶38∶10∶2的摩尔比例溶解于乙醇中，乙醇浓度为30％(v/v)，得到油相混合液。然后，将该油相混合液于室温加入到50mM pH 4.0的柠檬酸盐缓冲液中得到脂质混合物。将该脂质混合物加入脂质体挤出器，先用200nm滤膜挤出过滤，然后再用100nm滤膜过滤，使溶液从乳白变为澄清，得到阳离子脂质体纳米颗粒。
将优化后核苷修饰的RABV-G mRNA溶于柠檬酸盐缓冲液(pH 4.0)中，按照阳离子脂质纳米颗粒与mRNA质量比为20∶1的比例，逐滴加入至阳离子脂质纳米颗粒中，使用振荡仪Vortex混匀得到混合液。彻底混匀后42℃金属浴加热孵育1小时，随后将混合液透析至无菌PBS，0.22μm无菌过滤器过滤后得到RABV-G mRNA阳离子脂质纳米颗粒，即狂犬病毒核酸疫苗。
以狂犬病毒囊膜蛋白G蛋白为免疫原，免疫BALB/c小鼠，每组5只。初免剂量为10μg，加强剂量为1μg，肌肉接种途径，每只注射体积为100μl，免疫两次，间隔3周，并设置脂质体空载组对照，分别在二次免疫后2周、2个月、5个月、7个月采血，通过ELISA评价狂犬病毒G蛋白特异性抗体水平。
III.筛选的非复制型mRNA载体用于流感病毒mRNA疫苗接种小鼠
同II中，送公司制备流感病毒mRNA脂质体纳米颗粒，以流感病毒血凝素蛋白茎干区胞外端HA2蛋白为免疫原，免疫BALB/c小鼠，每组5只。初免和加强剂量均为5μg，肌肉接种，间隔3周，同时设置脂质体空载体组对照，二次免疫后2周采血分析特异性抗体水平，通过ELISA评价分析对HA茎干区胞外段HA2蛋白特异性抗体水平。
IV.序列命名
以下为本文中涉及的序列名称的命名，这些序列具有相同的5′-UTR：
——包含分别来源于人肌动蛋白(actin)、人白蛋白(albumin)、人α-球蛋白(α-globin)的3′-UTR并各自带有30A的poly(A)尾的eGFP-mRNA载体，依次命名为：I-eGFP actin(30A)，如SEQ ID NO：12所示；I-eGFP albumin(30A)，如SEQ ID NO：13所示；I-eGFP-α-globin(30A)，如SEQ ID NO：14所示；
——包含分别来源于人白蛋白、人α-球蛋白3′-UTR并各自带有120A的poly(A)尾的eGFP-mRNA载体，依次命名为：II-eGFP-albumin(120A)，如SEQ ID NO：15所示；II-eGFP-α-globin(120A)，如SEQ ID NO：16所示；
——包含分别来源于人白蛋白、人α-球蛋白3′-UTR并各自带有250A的poly(A)尾的eGFP-mRNA载体，依次命名为：III-eGFP-albumin(250A)，如SEQ ID NO：17所示；III-eGFP-α-globin(250A)，如SEQ ID NO：18所示；
——包含不同串联的3′-UTR并带有250A的poly(A)尾的eGFP-mRNA载体，其中串联的3′-UTR包括来源于人α-球蛋白和人白蛋白串连3′-UTR序列(α-globin+albumin)、人白蛋白和真核生物线粒体的串联序列(albumin+Mit)、人α-球蛋白和真核生物线粒体的串联序列(α-globin+Mit)，依次命名为：IV-eGFP(α-globin+albumin)(250A)，如SEQ ID NO：19所示；IV-eGFP(albumin+Mit)(250A)，如SEQ ID NO：20所示；IV-eGFP(α-globin+Mit)(250A)，如SEQ  ID NO：21所示；
——包含串联的3′-UTR并带有120A的poly(A)尾的eGFP-mRNA载体，其中串联的3′-UTR为来源于人α-球蛋白和真核生物线粒体的3′-UTR串联序列，命名为：IV-eGFP(α-globin+Mit)(120A)，如SEQ ID NO：22所示；
——筛选的各自带有120A的poly(A)尾的mRNA载体和带有250A的poly(A)mRNA载体(3′-UTR均为α-globin+Mit)用于狂犬病mRNA疫苗，依次命名为：RABV-G mRNA(120A)(SEQ ID NO：25)和RABV-G mRNA(250A)(SEQ ID NO：24)；
——筛选的各自带有120A的poly(A)尾的mRNA载体和带有250A的poly(A)尾的mRNA载体(3′-UTR均为α-globin+Mit)用于流感病毒mRNA疫苗，依次命名为：HA2 mRNA(120A)(SEQ ID NO：28)和HA2 mRNA(250A)(SEQ ID NO：27)。
实施例1.用具有不同来源3′-UTR并带有30A的poly(A)尾的eGFP-mRNA载体转染HEK293T细胞的表达验证
将按上述实验方法I中制备的eGFP-mRNA转染HEK293T细胞，进行表达验证。转染前24h铺HEK293细胞，将细胞以每孔200000个的密度接种到12孔板，培养基为DMEM完全培养基(含10％FBS和1％P.S.)。转染试剂为Lipofectamine 3000，mRNA与Lipofectamine 3000转染比例为1∶2，每孔板转染2μg eGFP-mRNA，37℃孵箱培养。流式检测eGFP的表达水平，检测时间点为12h、Day1、Day2、Day3、Day4、Day5、Day6、Day7。
结果如图2所示：I-eGFP actin(30A)、I-eGFP albumin(30A)、I-eGFP-α-globin(30A)，转染HEK293T细胞后均可大量表达，转染阳性率均在60％以上，转染后24h～48h内达到最高(图2A)。并且，I-eGFP albumin(30A)、I-eGFP-α-globin(30A)eGFP-mRNA转染后，eGFP平均荧光强度比I-eGFP actin(30A)高2～3倍，且可持续多日相对高表达(图2B)。
以上结果证明了I-eGFP albumin(30A)、I-eGFP-α-globin(30A)可高效转染细胞，且其所含的3′-UTR有助于表达水平的显著提高。
实施例2.用具有不同来源3′-UTR并带有120A的poly(A)尾或250A的poly(A)尾的eGFP-mRNA载体转染HEK293T细胞的表达验证
采用与实施例1相同方法，转染HEK293T细胞进行表达验证，不同之处在于所用表达载体为具有不同来源3′-UTR并带有120个A的poly(A)尾或250个A的poly(A)尾的eGFP-mRNA载体。
结果如图3所示：II-eGFP-albumin(120A)、II-eGFP-α-globin(120A)转染HEK293T细胞后均可大量表达，转染阳性率均达80％以上(图3A)，并且其eGFP平均荧光强度高于带有30A的相同3′-UTR的eGFP-mRNA(图3B)；相同3′-UTR带有250poly(A)的eGFP-mRNA即III-eGFP-albumin(250A)、III-eGFP-α-globin(250A)阳性率均达85％以上(图3A)，比带有120poly(A)的eGFP平均荧光强度高1.5～2倍，虽然荧光强度在从第2天后均开始有所降低，但带有250poly(A)的eGFP-mRNA下降较少，且可维持至少7天(图3B)。
以上结果证明了在包含相同3′-UTR的情况下，带有250A poly(A)的载体III-eGFP-albumin(250A)、III-eGFP-α-globin(250A)能够更高效地转染细胞，并进一步提升表达水平。
实施例3.用具有不同串联3′-UTR并带有250A的poly(A)尾的eGFP-mRNA载体转染HEK293T细胞的表达验证
采用与实施例1相同的方法，转染HEK293T细胞并在不同时间点检测进行表达验证，不同之处在于所用表达载体为具有不同串联3′-UTR并带有250A的poly(A)尾的eGFP-mRNA载体。
结果如图4所示：IV-eGFP(α-globin+albumin)(250A)、IV-eGFP(albumin+Mit)(250A)、IV-eGFP(α-globin+Mit)(250A)转染HEK293T细胞后均可大量表达，阳性率均在85％以上(图4A)，并且转染后IV-eGFP(α-globin+Mit)(250A)mRNA的eGFP平均荧光强度最高，可达到3E4及以上，比其他两种串联的组合高2～4倍(图4B)；并且虽然从第2天后荧光强度均开始有所降低，但IV-eGFP(α-globin+Mit)(250A)的eGFP-mRNA下降较少，平均荧光强度维持至少7天在 1E4以上的水平(图4C)。
以上结果证明了具有不同来源串联的3′-UTR并带有250A的poly(A)尾的mRNA载体能够高效转染细胞，并提升表达水平，其中IV-eGFP(α-globin+Mit)(250A)具有最高的表达水平。
实施例4.用具有相同串联的3′-UTR并带有120A或250A的poly(A)尾的eGFP-mRNA载体转染HEK293T细胞的表达验证
采用与实施例1相同的方法，转染HEK293T细胞并在不同时间点检测进行表达验证，不同之处在于所用表达载体为具有相同串联的3′-UTR并带有120A或250A的poly(A)尾的eGFP-mRNA载体。
结果如图5所示：IV-eGFP(α-globin+Mit)(120A)、IV-eGFP(α-globin+Mit)(250A)转染HEK293T细胞后均可大量表达，转染阳性率均在85％以上，最高可达95％以上(图5A)，并且转染后具有相同串联3′-UTR但带有250A的poly(A)尾的eGFP-mRNA平均荧光强度比带有120A的poly(A)尾的eGFP-mRNA平均荧光强度高2倍左右(图5B)；并且也都从第2天后荧光强度有所降低(图5B)。
以上结果从细胞水平上证明了具有相同串联3′-UTR带有250poly(A)的表达载体效果优于带有120A的表达载体，其中不同来源串联的3′-UTR并带有250个poly(A)表达载体效果最好。
实施例5.用具有相同串联的3′-UTR并带有120A的poly(A)尾或250A的poly(A)尾的mRNA载体制备的狂犬病mRNA疫苗接种小鼠后的体液应答水平
为进一步在体内验证根据本文的技术所筛选出的非复制型mRNA载体的表达效果，本实施例将其应用于mRNA狂犬病疫苗的制备。
本实施例选用的mRNA为：将RABV-G mRNA(120A)、RABV-G mRNA(250A)，制成包裹RABV-G mRNA的阳离子脂质纳米颗粒，免疫BALB/c小鼠评价其免疫原性。每组5只小鼠，初免剂量均为10μg，加强剂量为1μg，肌肉接种途径，每只注射体积为100μl，免疫两次，间隔为3周。分别在二次免疫 后2周、2个月、5个月、7个月眼眶采血，阴性对照为等体积的未包裹核酸的阳离子脂质纳米颗粒(即空载对照)。
结果如图6所示，1μg剂量加强注射后的14天，与空载对照相比，RABV-G mRNA(120A)组和RABV-G mRNA(250A)组均显示显著提高的抗体应答水平，其中RABV-G mRNA(120A)组免疫小鼠RABV-G的特异性抗体滴度为281231，RABV-G mRNA(250A)组为902035，即RABV-G mRNA(250A)组的抗体滴度达到RABV-G mRNA(120A)组的3.2倍左右。并且，加强免疫后，RABV-G mRNA(250A)组能够维持较高滴度的抗体浓度直至7个月。
上述实验结果证明了采用本文所公开载体搭载狂犬病毒G蛋白抗原后，不仅有较强的免疫原性，而且能够在体内持续诱导高水平的抗体应答。
实施例6.用具有相同串联的3′-UTR并带有120A的poly(A)尾或250A的poly(A)尾的mRNA载体制备的流感病毒mRNA疫苗接种小鼠后的体液应答水平
本实施例将筛选出的非复制型mRNA载体用于流感病毒mRNA疫苗的制备和接种。本实施例中所用流感病毒mRNA为HA2 mRNA(120A)和HA2 mRNA(250A)，制备HA2 mRNA脂质体纳米颗粒，免疫BALB/c小鼠评价其免疫原性。按材料方法III中所述，加强后2周、4周分别评价其免疫原性。
结果如图7所示：HA2 mRNA(120A)加强接种后可较空载对照在一定程度上提高抗体滴度水平，而HA2 mRNA(250A)接种后，较HA2 mRNA(120A)组可更进一步显著提升针对HA2的特异性抗体至约8400的平均滴度，两组的抗体滴度具有约12～21倍的差异。
以上实验结果证明了，本文中带有特定3′-UTR和长poly(A)尾的载体可有效搭载流感抗原，并诱导高滴度抗体水平。尤其是采用具有250A的poly(A)尾的载体搭载流感血凝素抗原(茎干区抗原)后，可诱导出极强的免疫应答。
综上可知，无论是细胞水平还是动物体内效果验证，本公开筛选出的非复制型mRNA载体(尤其是IV-eGFP-(α-globin+Mit)(250A)，可装载不同目的基因 (将eGFP基因替换为各种目的基因)，且可实现高效翻译、稳定持续性表达。
以上所述的具体实施例，对本发明的目的、技术方案和有益效果进行了进一步详细说明。应理解的是，以上所述仅为本发明的具体实施例而已，并不用于限制本发明。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。换言之，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
附：序列表对应信息
Figure PCTCN2022119229-appb-000001

Claims (14)

  1. 一种能够表达目的蛋白的mRNA核酸表达载体，其从5′端至3′端依次包含：
    (a)5′-UTR元件；
    (b)编码目的蛋白的开放阅读框元件；
    (c)3′-UTR元件；
    (d)总长度为120nt以上的多聚腺苷酸尾元件，其包含：
    多个腺苷酸串，每个腺苷酸串各自独立地包含n个连续的腺苷酸，n为10～80之间的整数，且所述多个腺苷酸串的腺苷酸总个数为100个以上；以及
    位于所述多个腺苷酸串之间的连接子，所述连接子各自独立地不包含腺苷酸或仅包含1个或2个腺苷酸。
  2. 如权利要求1所述的核酸表达载体，其中，所述5′-UTR元件的长度为10～200nt，例如15～100nt；和/或
    所述5′-UTR元件源自下组中的一种或多种5′-UTR：人α-球蛋白、β-球蛋白、核糖体蛋白(RP)、微管蛋白β-2B、补体因子3(C3)、细胞色素P450 2E1(CYP2E1)、载脂蛋白A-II(APOA2)、人类血红蛋白亚基β(hHBB)、血红蛋白A1(HBA1)、血红蛋白A2(HBA2)、登革热病毒(DENV)；和/或
    所述5′-UTR元件具有如SEQ ID NO：1所示的序列或与其具有至少80％的序列同一性。
  3. 如权利要求1所述的核酸表达载体，其中，所述3′-UTR元件为来源于哺乳动物或病毒的3′-UTR，例如来源选自下组的3′-UTR或其组合(如串联序列)：人α球蛋白、人β球蛋白、人白蛋白、人肌动蛋白、人血红蛋白亚基α1(HBA1)、细胞色素B-245α链(CYBA)、真核生物线粒体(Mit)的序列、SARAS-Cov-2、登革热病毒(DENV)、萝卜皱病毒(TCV)、烟草花叶病毒(TMV)和烟草蚀刻病毒(TEV)；和/或
    所述3′-UTR元件包含选自下组一个或多个3′-UTR分子：α球蛋白3′-UTR、 真核生物线粒体3′-UTR、白蛋白3′-UTR、β球蛋白3′-UTR或其任意串联序列，优选α球蛋白3′-UTR、真核生物线粒体3′-UTR、或它们串联形成的3′-UTR；和/或
    所述3′-UTR具有如SEQ ID NO：2～8中所示的一个或多个序列，优选如SEQ ID NO：2、5或8所示的序列，或与其中任何一者具有至少80％的序列同一性的序列。
  4. 如权利要求1所述的核酸表达载体，其中，所述多聚腺苷酸尾元件的总长度为120～400nt，例如120～350nt，120～320nt，或其中的任意整数，例如120、304nt；和/或
    各腺苷酸串各自独立地包含10～80个、20～70个、25～60个、30～50个或其中任意整数个连续的腺苷酸，如20个、30个、33个、35个、36个、37个、38个、39个、40个、45个、50个、55个、60个、65个、70个连续腺苷酸；和/或
    所述多聚腺苷酸尾元件还进一步包含位于所述元件一个或两个端部的连接子；和/或
    所述连接子的长度各自独立地为3～15nt，例如3、4、5、6、7、8、9、10、11、12、13、14或15nt；和/或
    例如，所述连接子的序列各自独立地选自：GCTATGACT、GTATGT、GCAAGT、GATTGC、GGCTGC、TACTGC、GGCTTC、GCATATGACT；和/或
    所述多聚腺苷酸尾元件具有SEQ ID NO：10或SEQ ID NO：11的序列，或与其中任何一者具有至少80％的序列同一性。
  5. 如权利要求1所述的核酸表达载体，其中，所述核酸表达载体为非复制型mRNA载体或作为核酸疫苗；和/或
    所述核酸表达载体经密码子优化或包含修饰碱基和/或核苷类似物；
    编码目的蛋白的所述元件为单、双或多顺反子mRNA；和/或
    编码目的蛋白的所述元件经过或未经密码子优化、包含或不包含碱基修饰和/或核苷类似物。
  6. 如权利要求1所述的核酸表达载体，其中，所述核酸表达载体还包括选自下组的一种或多种元件：
    5′-帽元件，其可选被修饰，例如所述5′-帽元件选自：m7GpppXpYp、m7GpppXmpYp、m7GpppXmpYmp、或其甲基化修饰序列、反向结合异构体、抗-反转帽类似物(ARCA)、N7-苄基二核苷四磷酸帽类似物；
    启动子元件，例如T7启动子、sp6启动子或T3启动子；
    信号肽编码元件，例如指导目的蛋白亚细胞定位的信号肽(如跨膜信号肽、分泌信号肽、核定位信号肽)编码元件；
    酶切位点，例如XbaI、EcoRV、BamHI、XhoI；
    标签，例如用于目的分子鉴定、分离或纯化的分子标签，如Flag标签、HA标签。
  7. 如权利要求1～6中任一项所述的核酸表达载体，其中，所述核酸表达载体包含能够表达选自下组中的一种或多种目的蛋白的mRNA：免疫原分子、抗体分子、治疗性药物、预防性药物、蛋白质替代治疗分子、基因编辑治疗分子；和/或
    所述核酸表达载体用于疾病预防和/或治疗，所述疾病选自：病毒感染、癌症、遗传疾病(例如单基因疾病)，例如：
    所述疾病选自如下一种或多种病毒感染：狂犬病病毒、流感病毒、冠状病毒、埃博拉病毒、寨卡病毒、黄热病病毒、登革热病毒、巨细胞病毒、蓝耳病病毒、猪瘟病毒、肠道病毒、乙型肝炎病毒、呼吸道合胞病毒、单纯疱疹病毒、人乳头瘤病毒、人免疫缺陷病毒、流感病毒、冠状病毒、副流感病毒、麻疹病毒、腮腺炎病毒、尼帕病毒和人偏肺病毒；和/或
    在用于病毒感染性疾病的预防和/或治疗的情况下，所述目的蛋白为病毒免疫原，例如来自于病毒囊膜蛋白，包括血凝素蛋白(Hemagglutinin，HA)、神经氨酸酶(Neuraminidase，NA)、基质蛋白(Matrix protein，M)、包膜蛋白(Envelope)、刺突蛋白(Spike)、膜蛋白(Membrane，M)、溶血素(Haemolysin，HL)、融合蛋白(Fusion，F)、糖蛋白(Glycoprotein，G)；和/或，所述目的蛋白表达于细胞内、细胞膜上或分泌性表达；和/或
    所述疾病选自如下一种或多种癌症：鳞状细胞癌、基底细胞癌、腺癌、肝细胞癌、肾细胞癌、膀胱癌、肠癌、宫颈癌、结肠癌、食道癌、头癌、肾癌、肝癌、肺癌、颈癌、卵巢癌、胰腺癌、前列腺癌、胃癌、白血病、淋巴瘤、伯基特氏淋巴瘤、非霍奇金氏淋巴瘤；黑素瘤；骨髓增生性疾病；肉瘤、血管肉瘤、卡波氏肉瘤、脂肪肉瘤、肌肉瘤、外周神经上皮瘤、滑膜肉瘤、神经胶质瘤、星形细胞瘤、少突神经胶质瘤、室管膜瘤、成胶质细胞瘤、成神经细胞瘤、神经节细胞瘤、神经节神经胶质瘤、成神经管细胞瘤、松果体细胞肿瘤、脑膜瘤、脑膜肉瘤、神经纤维瘤和神经鞘瘤、乳腺癌、子宫癌、睾丸癌、甲状腺癌、星形细胞瘤、食道癌、癌肉瘤、霍奇金氏病、维尔姆斯瘤和畸胎癌；
    所述疾病选自如下一种或多种遗传疾病：甲基丙二酸血症、急性间歇性卟啉症、法布里病、白化病、血友病、苯丙酮尿症、半乳糖血症、粘多糖病和先天性肾上腺皮质增生症。
  8. 如权利要求1所述的核酸表达载体，其中，所述核酸表达载体从5′端到3′端包含：
    包含SEQ ID NO：1所示序列的5′-UTR元件；编码目的蛋白的开放阅读框元件；包含如SEQ ID NO：2、5或8所示序列的3′-UTR元件；包含如SEQ ID NO：10或11所示序列的多聚腺苷酸尾元件；或与所述序列具有至少80％序列同一性的序列的元件；和/或
    所述核酸表达载体包含如SEQ ID NO：15～22、24～25和27～28中任一项所述的序列、与其具有至少80％序列同一性的序列、或前述任一序列中编码其目的蛋白的开放阅读框元件被替换为编码所需目的蛋白的开放阅读框元件所得的序列。
  9. 如权利要求1所述的核酸表达载体，其单独包含于包装物中，或与递送系统中的运载体组合，例如，所述递送系统选自：脂质递送系统、类脂递送系统、聚合物递送系统或其组合递送系统，例如加载于脂质纳米颗粒、聚氨酯(PAA)、聚β氨基酯(PBAE)、聚乙烯亚胺(PEI)、脂质包裹的聚合物胶束。
  10. 一种组合物，其包含如权利要求1～9中任一项所述的核酸表达载体， 以及包装物和/或用于该核酸表达载体的递送系统和/或药学上或生理学上可接受的运载体。
  11. 如权利要求10所述的组合物，其形式适于选自下组的一种或多种给予或递送方式：呼吸道雾化吸入、滴鼻、口服、直接注射(例如静脉注射、皮下注射、皮内注射、肌肉注射)、黏膜给药；和/或
    所述组合物还包含佐剂或与佐剂联合使用，例如所述佐剂选自：铝佐剂、霍乱毒素及其亚单位、寡脱氧核苷酸、锰离子佐剂、胶体锰佐剂、弗氏佐剂、MF59佐剂、QS-21佐剂、Poly I：C及其他TLR配体、GM-CSF、IL-2、IL-3、IL-7、IL-11、IL-12、IL-18、IL-21；和/或
    所述组合物的形式适于进行2种或以上药物或疫苗的组合给予，例如联合接种或序贯接种；和/或
    所述组合物选自：mRNA疫苗、治疗性或预防性药物，如蛋白质替代治疗药物、基因编辑治疗药物。
  12. 如权利要求1～9中任一项所述的核酸表达载体和/或如权利要求10～11中任一项所述的组合物在制备用于表达目的蛋白的产品中的应用，
    例如，所述产品选自：mRNA疫苗、治疗性或预防性药物，如蛋白质替代治疗药物、基因编辑治疗药物。
  13. 制备如权利要求1～9中任一项所述的核酸表达载体或如权利要求10～11中任一项所述的组合物的方法，所述方法包括：提供独立或连接的各元件；将各元件组装成核酸表达载体。
  14. 如权利要求13所述的方法，其中，所述方法包括采用选自下组的一种或多种材料：DNA模板(例如PCR产物或已线性化的质粒DNA)、核酸酶、聚合酶、加帽酶、聚腺苷酸合酶、DNA酶、一个或多个元件分子、接头分子、天然或修饰的核酸分子、缓冲液、溶剂；和/或
    所述方法还包括选自下组的一个或多个步骤：对各元件进行设计、优化、改造和/或修饰；对中间产物和/或最终产物进行分离、纯化、鉴定、定量、包装和/或活性测试；将核酸表达载体与用于该核酸表达载体的递送系统和/或药学上或生理学上可接受的运载体组合。
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