CN117721129A - mRNA vector system capable of efficiently expressing target gene, construction and application thereof - Google Patents

Abstract

The present invention relates to mRNA vector system capable of expressing target gene with high efficiency, its construction and application. Specifically, provided herein is an mRNA expression vector capable of efficiently expressing a protein of interest, comprising, in order from the 5 'end to the 3' end: (a) a 5' -UTR element; (b) an open reading frame element encoding a protein of interest; (c) a 3' -UTR element; (d) a polyadenylation tail element with a total length of 120nt or more. Also provided herein are construction of the mRNA expression vectors, related products and uses thereof, e.g., as mRNA vaccines.

Description

mRNA vector system capable of efficiently expressing target gene, construction and application thereof

Technical Field

The present disclosure is in the field of biomedical industry, and in particular relates to genetically engineered drug and vaccine manufacturing. In particular, the present disclosure relates to an mRNA vector system capable of efficiently expressing a gene of interest, its construction, and its use in expression of a gene of interest, for example, as a nucleic acid vaccine vector.

Background

In recent years, mRNA vaccine technology has become one of the most interesting vaccine forms due to its numerous advantages. Unlike plasmid DNA and viral vector vaccines, mRNA is not integrated into the genome, thereby avoiding potential hazards associated with insertional mutagenesis. mRNA vaccine can be produced in a cell-free mode, and has the advantages of rapid production, simple process and controllable cost. In addition, a single mRNA vaccine can encode multiple antigens, enabling it to target tumor targets, different microorganisms, and enhance immune responses to highly mutated pathogens.

With the maturation of mRNA preparation technology, in vitro transcription to produce mRNA molecules is becoming simpler. The in vitro transcribed mRNA molecules must mimic the structure of the endogenous mRNA molecule and, in the 5 '. Fwdarw.3' direction, include the following elements in order: a 5' cap structure, a 5' -UTR (untranslated region) sequence, a coding sequence, a 3' -UTR sequence, and a poly (A) tail sequence. Methods for improving intracellular mRNA stability and translation include: modification of the 5' cap structure, selection of 5' -UTR and 3' -UTR, structural modification of poly (A) tail and optimization of coding sequence.

Currently, the 5 'cap structure is modified primarily by co-transcriptional capping and enzymatic capping to protect the mRNA from exonuclease degradation and work in concert with the poly (A) tail at the 3' end. After binding of poly (A) binding protein (PABP) to poly (A) tail sequence, the translation initiation factor proteins eIF4G and eIF4E are recruited, the mRNA is cyclized and ribosome-initiated translation is recruited. Whereas the 5'-UTR and 3' -UTR of mRNA can significantly affect the translation rate and half-life of the transcription product, optimizing UTR is one of the key points of mRNA vaccine design. The 3' -UTR is generally considered to be a region of mRNA where unstable factors are concentrated, and it is necessary to select sequences for stably expressing proteins or viral genomes.

The last step in mRNA transcription is the addition of a poly-A tail, which protects the mRNA from degradation and promotes subsequent binding of the poly (A) binding protein. Thus, the addition of poly (A) tails to mRNA templates encoding antigens results in higher levels of protein expression. The long poly-A sequence is more beneficial to the stability of mRNA and prolongs the half life of mRNA. According to earlier studies, in metazoans the poly-A tail is typically about 250bp. In human monocyte-derived Dendritic Cells (DCs), the 120bp poly-A sequence provides for more stable IVT-mRNA (in vitro-transcribed mRNA) and more efficient translation than the short poly-A tail. In human primary T cells, poly-A sequences of over 300 nucleotides favor more efficient translation, where IVT-mRNAs with medium and long Poly-A tails recruit PABP first and are sheared to 30A long, consistent with naturally occurring mRNApoly (A) tail sizes. The addition of poly (A) tails to DNA plasmids eliminates the step of in vitro tailing, reduces overall reaction time and raw material loss, and avoids tail length variation due to enzymatic polyadenylation using poly (A) polymerase.

However, while Poly (A) tails greater than 100bp are the best choice for therapeutic mRNA vaccines, the DNA sequences encoding these long Poly (A) s would disrupt the stability of the DNA plasmid used for transcription. Furthermore, studies have shown that when the length of the continuous Poly (A) tail is greater than 120bp, the expression level of the corresponding protein is not increased.

The tail in the novel coronavirus BNT162b2mRNA vaccine developed by Pfizer-Biontech, which has been successfully marketed at present, contains 100A Poly (A) with a 10bp UGC linker inserted between them, generating a Poly (A) tail with a sequence such as 30nt polA+GCATATGACT+70nt polA.

Most of the template plasmids currently used for preparing mRNA are loaded with 100-120 poly-A sequences of unequal length, and few reports on mRNA vector construction and optimization are made.

Improving intracellular mRNA stability, translational efficiency, and long lasting persistence has been one of the difficulties that mRNA vaccines need to overcome, which is closely related to the expression vectors used to make mRNA. Thus, there is an urgent need in the art to provide novel mRNA vectors capable of overcoming the technical problems as described above.

Disclosure of Invention

It is in this disclosure to provide a novel mRNA vector, its preparation method and use.

In some aspects, provided herein is an mRNA nucleic acid expression vector comprising a nucleic acid capable of expressing a protein of interest, comprising, in order from the 5 'end to the 3' end:

(a) A 5' -UTR element;

(b) An open reading frame element encoding a protein of interest;

(c) 3' -UTR elements;

(d) A polyadenylation tail element having a total length of 120nt or more comprising: a plurality of adenylate strings, each adenylate string independently comprising n consecutive adenylates, n being an integer between 10 and 80, and the total number of adenylates of the plurality of adenylate strings being 100 or more; and a linker located between the plurality of adenylate strings, each independently comprising no adenylate or only 1 or 2 adenylates.

The nucleic acid expression vectors of the present disclosure are capable of efficiently expressing a desired protein of interest in vivo and in vitro, thereby achieving, for example, disease prevention and/or therapeutic effects, for example, use as therapeutic drugs, prophylactic drugs, protein replacement therapeutic molecules, gene editing therapeutic molecules, and the like. The nucleic acid expression vectors of the present disclosure can be used, for example, for the prevention and/or treatment of viral infections, cancers, genetic diseases (e.g., monogenic diseases).

In some aspects, provided herein is a composition comprising a nucleic acid expression vector herein, and a package and/or a delivery system for the nucleic acid expression vector and/or a pharmaceutically or physiologically acceptable carrier.

In some aspects, also provided herein is the use of a nucleic acid expression vector and/or composition herein in the preparation of a product for expressing a protein of interest, which product may be selected, for example, from: mRNA vaccines, therapeutic or prophylactic agents, such as protein replacement therapy agents, gene editing therapy agents.

In some aspects, provided herein are also methods of preventing and/or treating a disease comprising administering to a subject in need thereof a prophylactically and/or therapeutically effective amount of a nucleic acid expression vector and/or composition herein.

In some aspects, provided herein are also nucleic acid expression vectors and/or compositions herein for expressing a protein of interest. In some aspects, further provided are nucleic acid expression vectors and/or compositions herein for use in the prevention and/or treatment of a disease.

In some aspects, also provided herein are methods of making a nucleic acid expression vector or composition as described herein, the method comprising: providing separate or connected elements; the elements are assembled into a nucleic acid expression vector.

Any combination of the technical solutions and features described above can be made by a person skilled in the art without departing from the inventive concept and the scope of protection of the present invention. Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

The present invention will be further described with reference to the accompanying drawings, wherein these drawings are provided only for illustrating embodiments of the present invention and are not intended to limit the scope of the present invention.

Fig. 1: FIG. 1 schematic representation of linearized pCDNA3.1+ plasmid following transformation of mRNA transcription template empty vector.

Fig. 2: 3' -UTR from different sources and with 30poly (A) vector encoded eGFP mRNA transfected HEK293T cells positive rate (A) and expression level (B).

Fig. 3: 3' -UTR from different sources and with 120poly (A) and 250poly (A) vector encoded eGFP mRNA transfected HEK293T cells positive rate (A) and expression level (B).

Fig. 4: the positive rate (A) and expression level (B) of HEK293T cells transfected with eGFP mRNA encoded by the 250poly (A) vector were compared for the different tandem 3' -UTRs.

Fig. 5: 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 were compared.

Fig. 6: the same tandem 3' -UTR with 120poly (A) and 250poly (A) mRNA vectors was used for comparison of the humoral response levels (log on ordinate with 10 as bottom) after rabies mRNA vaccination of mice.

Fig. 7: the same tandem 3' -UTR with 120poly (A) and 250poly (A) mRNA vectors was used for comparison of humoral response levels (log 10 base log on ordinate) after influenza mRNA vaccination of mice.

Detailed Description

The present disclosure provides an mRNA vector capable of efficiently expressing a target protein, which may be a non-replicative mRNA vector capable of efficiently expressing different target genes, and the target genes can be efficiently translated and stably and continuously expressed, both at the level of in vitro cellular expression and by delivering the mRNA into an organism through a delivery means.

In the application, the eGFP green fluorescent protein gene is taken as a reference, a series of optimization is carried out on the elements of the mRNA vector, and the non-replicative mRNA vector capable of efficiently expressing the target gene is screened out by comparing the expression level of the eGFP, so that the mRNA vector can be efficiently translated and can be stably and continuously expressed in a cell level or an organism.

Compared with the prior art, the nucleic acid expression vector can efficiently express various target molecules, such as various antigen molecules; through selection, modification, transformation and combination of elements in the expression vector, the target gene can be translated efficiently and stably at the cellular level or in organisms, and the expression level and in vivo half-life of the target gene can be effectively regulated and controlled, so that the target gene has high immunogenicity and long-acting persistence. Therefore, the expression vector and the related products thereof have wide application prospects in the aspects of preventive and therapeutic vaccines, specific antibody expression, therapeutic or targeting drug expression, protein replacement therapy and the like.

All numerical ranges provided herein are intended to expressly include all values and ranges of values between the endpoints of the range. The features mentioned in the description or the features mentioned in the examples can be combined. All of the features disclosed in this specification may be combined with any combination of the features disclosed in this specification, and the various features disclosed in this specification may be substituted for any alternative feature serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the disclosed features are merely general examples of equivalent or similar features.

As used herein, "comprising," having, "or" including "includes" including, "" consisting essentially of … …, "" consisting essentially of … …, "and" consisting of … …; "consisting essentially of … …", "consisting essentially of … …" and "consisting of … …" are under the notion of "containing", "having" or "including".

As used herein, "eukaryotic" may include humans, primates, rodents (e.g., rats, mice, guinea pigs, hamsters), domestic animals, or farm mammals.

The high degree of sequence identity described herein, including sequence identity of 70% or more, 75% or more, 80% or more, more preferably 85% or more, such as 85%, 90%, 95%, 98% or even 99% or more, is also within the equivalent scope of the preferred embodiments of the present invention. Methods and tools for aligning sequence identity are also well known in the art, such as BLAST.

mRNA nucleic acid expression vector and composition comprising same

Provided herein is an mRNA nucleic acid expression vector comprising a nucleic acid capable of expressing a protein of interest comprising, in order from the 5 'end to the 3' end:

(a) A 5' -UTR element;

(b) An open reading frame element encoding a protein of interest;

(c) 3' -UTR elements;

(d) A polyadenylation tail element having a total length of 120nt or more comprising:

a plurality of adenylate strings, each adenylate string independently comprising n consecutive adenylates, n being an integer between 10 and 80, and the total number of adenylates of the plurality of adenylate strings being 100 or more; and

a linker located between the plurality of adenylate strings, each independently comprising no adenylate or only 1 or 2 adenylates.

In some embodiments, the 5' -UTR elements used in the present disclosure have a length of 10 to 200nt, such as 15 to 100nt.

In some embodiments, the 5'-UTR elements used in the present disclosure are derived from one or more 5' -UTRs from the group consisting of: human α -globulin, β -globulin, ribosomal Protein (RP), tubulin β -2B, complement factor 3 (C3), cytochrome P450 2E1 (CYP 2E 1), apolipoprotein a-II (APOA 2), human hemoglobin subunit β (hbb), hemoglobin A1 (HBA 1), hemoglobin A2 (HBA 2), dengue virus (DENV).

In some embodiments, the 5' -UTR elements used in the present disclosure have the sequence as shown in SEQ ID NO. 1 or have at least 80% sequence identity thereto.

In some embodiments, the 3' -UTR element used in the present disclosure is a 3' -UTR derived from a mammal or virus, e.g., a 3' -UTR derived from a source selected from the group consisting of: human alpha globulin, human beta globulin, human albumin, human actin, human hemoglobin subunit alpha 1 (HBA 1), cytochrome B-245 alpha Chain (CYBA), sequences of eukaryotic mitochondria (Mit), SARAS-Cov-2, dengue virus (DENV), radish wrinkle virus (TCV), tobacco Mosaic Virus (TMV), and Tobacco Etch Virus (TEV).

In some embodiments, the 3'-UTR element used in the present disclosure comprises one or more 3' -UTR molecules selected from the group consisting of: the α -globulin 3' -UTR, eukaryotic mitochondrial 3' -UTR, albumin 3' -UTR, β -globulin 3' -UTR or any of the tandem sequences thereof is preferably α -globulin 3' -UTR, eukaryotic mitochondrial 3' -UTR or the 3' -UTR formed by the tandem of these.

In some embodiments, the 3' -UTR used in the present disclosure has one or more of the sequences shown in SEQ ID NOS.2-8, preferably the sequence shown in SEQ ID NOS.2, 5 or 8, or a sequence having at least 80% sequence identity to any one thereof.

In some embodiments, the total length of the polyadenylation tail element used in the present disclosure is 120-400 nt, e.g., 120-350 nt, 120-320 nt, or any integer therein, e.g., 120, 304nt.

In some embodiments, each adenylate cluster independently comprises 10 to 80, 20 to 70, 25 to 60, 30 to 50, or any integer number thereof, consecutive adenylates, such as 20, 30, 33, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70 consecutive adenylates.

In some embodiments, the polyadenylation tail element further comprises a linker at one or both ends of the element.

In some embodiments, the linkers are each independently 3 to 15nt in length, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15nt. In some embodiments, the linkers each independently do not comprise an adenylate, or comprise only 1 adenylate or 2 adenylates.

In some embodiments, the sequences of the linkers are each independently selected from, for example: GCTATGACT, GTATGT, GCAAGT, GATTGC, GGCTGC, TACTGC, GGCTTC, GCATATGACT. In some embodiments, the polyadenylation tail element has the sequence of SEQ ID NO. 10 or SEQ ID NO. 11, or at least 80% sequence identity thereto.

In some embodiments herein, the 250 polyadenylation sequences contained in the polyadenylation tail element are discontinuous, with 5-10 bp of linker sequences containing no A, or only 1 or 2A bases, per 20-40 (e.g., 30, 40) A intervals, to facilitate more stable, efficient and durable translation of the coding sequence, and to extend its half-life.

In some embodiments, the nucleic acid expression vector of the present disclosure is a non-replicating mRNA vector or as a nucleic acid vaccine. In some embodiments, the element encoding the protein of interest in the present disclosure is a monocistronic, bicistronic, or polycistronic mRNA. In some embodiments, the bicistronic or polycistronic mRNA is an mRNA comprising two or more coding regions.

In some embodiments, the element encoding the protein of interest is codon optimized, with or without base modification and/or nucleoside analogs, e.g., employing one or more modified bases or nucleoside analogs in the encoding element selected from the group consisting of: at least one of pseudouridine, N1-methyl-pseudouridine, N1-ethyl-pseudouridine, 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-dihydro-pseudouridine, 2-thio-dihydro-uridine, 2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydro-pseudouridine or 5-methoxy-uridine and 2' -O-methyluridine, preferably pseudouridine or N1-methyl-pseudouridine or N1-ethyl-pseudouridine, more preferably N1-methyl-pseudouridine.

In some embodiments, the expression vector sequence may be codon optimized and/or comprise modified bases and/or nucleoside analogs. For example, in some embodiments, 50% to 100% of uracil in an expression vector sequence is replaced. The stability of mRNA in vivo can be improved by substitution.

In some embodiments, the nucleic acid expression vectors of the present disclosure further comprise a 5 '-cap element, which is optionally modified, e.g., the 5' -cap element is selected from the group consisting of: m7GpppXpYp, m7GpppXmpYp, m7GpppXmpYmp, or methylation modified sequences, reverse binding isomers, anti-reverse cap analogues (ARCA), N7-benzyl dinucleotide tetraphosphate analogues.

In some embodiments, the nucleic acid expression vectors of the present disclosure further comprise a promoter element, such as a T7 promoter, sp6 promoter, or T3 promoter.

In some embodiments, the nucleic acid expression vectors of the present disclosure further include a signal peptide coding element, e.g., a signal peptide (e.g., transmembrane signal peptide, secretory signal peptide, nuclear localization signal peptide) coding element that directs subcellular localization of the protein of interest.

In some embodiments, the nucleic acid expression vectors of the present disclosure further comprise a cleavage site, e.g., xbaI, ecoRV, bamHI, xhoI.

In some embodiments, the nucleic acid expression vectors of the present disclosure further include a tag, e.g., a molecular tag, such as a Flag tag, HA tag, for use in the identification, isolation, or purification of a molecule of interest.

In some embodiments, the nucleic acid expression vectors of the present disclosure comprise mRNA capable of expressing one or more proteins of interest selected from the group consisting of: an immunogenic molecule, an antibody molecule, a therapeutic drug, a prophylactic drug, a protein replacement therapy molecule, and a gene editing therapy molecule.

In some embodiments, the nucleic acid expression vectors of the present disclosure may be used to express a variety of different exogenous genes, including, but not limited to, expression of specific antibodies, expression of therapeutic or targeted drugs, protein replacement therapies, and the like.

In some embodiments, the nucleic acid expression vector may be used to prepare a nucleic acid vaccine that may be used to prepare a cancer vaccine, a viral vaccine, wherein the virus may be a variety of infectious disease viruses including ebola virus, rabies virus, zika virus, yellow fever virus, dengue virus, cytomegalovirus, blue ear 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, human metapneumovirus, and the like. The cancer comprises squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, hepatocellular carcinoma, renal cell carcinoma, bladder carcinoma, intestinal cancer, cervical cancer, colon cancer, esophagus cancer, head cancer, kidney cancer, liver cancer, lung cancer, neck cancer, ovarian cancer, pancreatic cancer, prostatic cancer, gastric cancer, leukemia, lymphoma, burkitt's lymphoma and non-Hodgkin's lymphoma; melanoma; myeloproliferative diseases; sarcomas, hemangiosarcomas, kaposi's sarcoma, liposarcoma, myosarcomas, peripheral neuroepithelial tumors, synovial sarcomas, gliomas, astrocytomas, oligodendrogliomas, ependymomas, glioblastomas, neuroblastomas, gangliocytomas, gangliogliomas, medulloblastomas, pineal tumors, meningiomas, neurofibromas, and schwannomas; breast cancer, uterine cancer, testicular cancer, thyroid cancer, astrocytoma, esophageal cancer, carcinoma sarcoma, hodgkin's disease, wilms' tumor, teratocarcinoma, and the like.

In some embodiments, the polypeptide of interest in the nucleic acid expression vector is an immunogen, the site of expression of which includes intracellular, cell membrane or secretory expression. In some embodiments, the immunogen is a viral immunogen, for example, from a viral Envelope protein, including but not limited to, hemagglutinin protein (HA), neuraminidase (NA), matrix protein (M), envelope protein (Envelope), spike protein (Spike), membrane protein (Membrane, M), hemolysin (Haemolysin, HL), fusion protein (Fusion, F), glycoprotein (G).

In some embodiments, the nucleic acid expression vectors of the present disclosure comprise, from 5 'to 3': a 5' -UTR element comprising the sequence shown in SEQ ID NO. 1; an open reading frame element encoding a protein of interest; a 3' -UTR element comprising the sequence as shown in SEQ ID NO. 2, 5 or 8; a polyadenylation tail element comprising a sequence as set forth in SEQ ID NO 10 or 11; or a sequence having at least 80% sequence identity to the preceding sequence, respectively.

In some embodiments, the nucleic acid expression vectors of the present disclosure comprise a sequence as set forth in any one of SEQ ID NOS.15-22, 24-25 and 27-28, a sequence having at least 80% sequence identity thereto, or a sequence in which an open reading frame element encoding a protein of interest in any one of the foregoing sequences is replaced with an open reading frame element encoding a desired protein of interest.

In some embodiments herein, a non-replicating mRNA expression vector is provided that is a plasmid template required to encode mRNA for different genes. The mRNA vector plasmid template includes the essential elements required for in vitro transcription of mRNA molecules.

In some embodiments, the expression vector comprises the following elements: t7 promoter, 5'-UTR sequence, coding sequence, 3' -UTR sequence, polyadenylation tail sequence, linearization site. These elements may be as described herein.

In specific embodiments of the invention, the non-replicating mRNA vector comprises a 5'-UTR sequence, a 3' -UTR sequence, a poly A tail sequence selected from the group consisting of 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 non-replicating mRNA vector comprises a 5'-UTR sequence, a 3' -UTR sequence, a poly A tail sequence that is at least 75% homologous to any of the above sequences (e.g., has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology to SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO: 8), and the non-replicating mRNA vector can express a variety of exogenous genes.

The disclosure also provides for expression of the non-replicating mRNA vector at the in vitro cellular level. The non-replicating mRNA vector IV-eGFP (alpha-globin+Mit) (250A) which can be expressed efficiently is screened by taking eGFP fluorescent protein genes as references and carrying out a series of optimization on elements comprising 5'-UTR sequences, 3' -UTR sequences and polyadenylation tail sequences and comparing the expression level of eGFP. Those skilled in the art will recognize that eGFP can be replaced by molecular biology or genetic engineering techniques to load the exemplary mRNA vectors herein with different genes.

In some embodiments, a nucleic acid expression vector of the disclosure is contained in a package alone, or in combination with a carrier in a delivery system, e.g., selected from the group consisting of: lipid delivery systems, polymer delivery systems, or combinations thereof, such as delivery systems loaded on lipid nanoparticles, polyurethanes (PAA), poly-beta-amino esters (PBAE), polyethylenimines (PEI), lipid-encapsulated polymer micelles.

Preparation method, related product and application thereof

Also provided in some aspects of the present disclosure is a composition comprising a nucleic acid expression vector of the present disclosure, and a package and/or a delivery system for the nucleic acid expression vector and/or a pharmaceutically or physiologically acceptable carrier.

In some embodiments, the compositions of the present disclosure are in a form suitable for administration or delivery by one or more modes selected from the group consisting of: respiratory tract aerosol inhalation, nasal drops, oral administration, direct injection (e.g., intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection), mucosal administration.

In some embodiments, the compositions of the present disclosure further comprise or are used in combination with an adjuvant, e.g., selected from the group consisting of: aluminum adjuvants, cholera toxin and subunits thereof, oligodeoxynucleotides, manganese ion adjuvants, colloidal manganese adjuvants, freund's adjuvant, MF59 adjuvants, QS-21 adjuvants, poly I: C and other TLR ligands, GM-CSF, IL-2, IL-3, IL-7, IL-11, IL-12, IL-18, IL-21.

In some embodiments, the compositions of the present disclosure are in a form suitable for combined administration of 2 or more drugs or vaccines, e.g., co-vaccination or sequential vaccination. In some embodiments, the compositions of the present disclosure are selected from: mRNA vaccines, therapeutic or prophylactic agents, such as protein replacement therapy agents, gene editing therapy agents.

In some aspects herein, there is provided a method of making a nucleic acid expression vector or composition described herein, the method comprising: providing separate or connected elements; the elements are assembled into a nucleic acid expression vector.

In some embodiments, the method comprises using one or more materials selected from the group consisting of: DNA templates (e.g., PCR products or linearized plasmid DNA), nucleases, polymerases, capping enzymes, poly-a-synthases, dnases, one or more element molecules, linker molecules, natural or modified nucleic acid molecules, buffers, solvents.

In some embodiments, the method further comprises one or more steps selected from the group consisting of: designing, optimizing, reforming and/or modifying each element; isolating, purifying, identifying, quantifying, packaging and/or testing the activity of the intermediate product and/or the final product; the nucleic acid expression vector is combined with a delivery system and/or a pharmaceutically or physiologically acceptable carrier for the nucleic acid expression vector.

In some aspects of the disclosure, there is provided the use of a nucleic acid expression vector and/or composition herein in the preparation of a product for expressing a protein of interest. In some embodiments, the product is selected from: mRNA vaccines, therapeutic or prophylactic agents, such as protein replacement therapy agents, gene editing therapy agents.

In some embodiments, the nucleic acid expression vectors of the present disclosure are used for disease prevention and/or treatment. In some embodiments, 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 infection, cancer, genetic disease (e.g., monogenic disease).

In some embodiments, diseases that may 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, viral infections selected from one or more of the following: rabies virus, influenza virus, coronavirus, ebola virus, zika virus, yellow fever virus, dengue virus, cytomegalovirus, blue ear 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.

In some embodiments, in the context of use in the prevention and/or treatment of viral infectious diseases, the proteins of interest expressed by the nucleic acid expression vectors of the present disclosure are viral immunogens, e.g., from viral Envelope proteins, including Hemagglutinin (HA), neuraminidase (NA), matrix protein (M), envelope protein (Envelope), spike protein (Spike), membrane protein (M), hemolysin (HL), fusion protein (Fusion, F), glycoprotein (G).

In some embodiments, diseases that may 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 group consisting of: squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, hepatocellular carcinoma, renal cell carcinoma, bladder carcinoma, intestinal cancer, cervical cancer, colon cancer, esophageal carcinoma, head cancer, renal cancer, liver cancer, lung cancer, neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, leukemia, lymphoma, burkitt's lymphoma, non-hodgkin's lymphoma; melanoma; myeloproliferative diseases; sarcomas, hemangiosarcomas, kaposi's sarcoma, liposarcoma, myosarcomas, peripheral nerve epithelial tumors, synovial sarcomas, gliomas, astrocytomas, oligodendrogliomas, ependymomas, glioblastomas, neuroblastomas, gangliocytomas, gangliogliomas, medulloblastomas, pineal tumors, meningiomas, neurofibromas and schwannomas, breast cancer, uterine cancer, testicular cancer, thyroid cancer, astrocytomas, esophageal cancer, carcinoma sarcomas, hodgkin's disease, wilms ' tumors, and teratocarcinomas.

In some embodiments, diseases that may 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 group consisting of: methylmalonic acid, acute intermittent porphyrin, fabry disease, albinism, hemophilia, phenylketonuria, galactosylation, mucopolysaccharidosis and congenital adrenocortical hyperplasia.

As used herein, the term "pharmaceutically or physiologically acceptable" ingredients are substances that are suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., commensurate with a reasonable benefit/risk ratio. As used herein, the term "effective amount" refers to an amount that is functional or active in and acceptable to a human and/or animal.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents. The term refers to such agent carriers: they are not per se essential active ingredients and are not overly toxic after administration. Suitable vectors are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington pharmaceutical sciences (Remington's Pharmaceutical Sciences, mack Pub.Co., N.J.1991).

Pharmaceutically acceptable carriers in the compositions can contain liquids such as water, saline, glycerol, and ethanol. In addition, auxiliary substances such as fillers, disintegrants, lubricants, glidants, effervescent agents, wetting or emulsifying agents, flavoring agents, pH buffering substances, etc. may also be present in these carriers. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8.

As used herein, the term "unit dosage form" refers to a dosage form in which the active substances herein are formulated for ease of administration as required for a single administration, including, but not limited to, various solid (e.g., tablets), liquid, capsules, sustained release formulations.

It will be appreciated that the effective dose of the active agent used may vary with the severity of the subject to be administered or treated. The specific conditions are determined according to the individual condition of the subject (e.g., the subject's weight, age, physical condition, effect to be achieved), which is within the scope of judgment of a skilled physician.

The products herein may be in solid form (e.g., granules, tablets, lyophilized powders, suppositories, capsules, sublingual tablets) or liquid form (e.g., oral liquid) or other suitable form. The administration route can be as follows: (1) direct naked nucleic acid injection; (2) Ligating the mRNA expression vector with a transferrin/poly L-lysine complex to enhance its biological effect; (3) Forming complexes of mRNA expression vectors with positively charged lipids to overcome the difficulties of crossing cell membranes due to negative charge of the phosphate backbone; (4) After the mRNA expression vector is wrapped by the liposome, the liposome mediates to enter cells, thereby being beneficial to the smooth entry of macromolecules and avoiding the hydrolysis of various extracellular enzymes; (5) Binding of the mRNA expression vector to cholesterol increases its retention time; (6) Using immunoliposomes to transport mRNA for specific transport to target tissue and target cells; (7) transfecting the mRNA expression vector in vitro into a tranfusion cell; (8) Electroporation (electric corporation), i.e., the introduction of mRNA vectors into target cells by means of electric current.

Examples

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Appropriate modifications and variations of the invention may be made by those skilled in the art, and are within the scope of the invention.

The experimental procedures described in the following examples, which are not explicitly described in the specification, may be carried out by methods conventional in the art, for example, by reference to the molecular cloning laboratory Manual (third edition, new York, cold spring harbor laboratory Press, new York: cold Spring Harbor Laboratory Press, 1989) or according to the conditions suggested by the suppliers. Methods for sequencing DNA are routine in the art and can also be provided for testing by commercial companies.

Percentages and parts are by weight unless otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.

Materials, methods and animals

The mRNA preparation, mRNA transfection HEK293T cells, mouse immunization and detection methods involved in the examples were as follows:

Preparation of mRNA

Preparing a proper DNA template, taking pCDNA3.1+ as a template, modifying, and sequentially carrying out the following steps from the N end to the C end after the T7 promoter: the coding sequence is inserted into the coding sequence by taking eGFP fluorescent protein genes as references, and EcoRV and Flag tags of enzyme cutting sites are inserted into the C end of the coding sequence, and the Flag tags are added for subsequent identification of different gene expression, as shown in figure 1.

The plasmid was linearized by single cleavage site XbaI and used as template for in vitro transcription. Adding NTP required by transcription according to a proper proportion by using an in vitro transcription kit and a capping kit purchased by a company according to a specification operation, using T7 polymerase to obtain corresponding eGFP mRNA by transcription, then capping by an enzyme capping method, namely a one-step method of simultaneously using vaccinia virus capping enzyme and 2' -O-methyltransferase, adding a 7-methylguanosine cap structure to the purified eGFP mRNA, purifying the capped mRNA by lithium chloride, using a transfection reagent of Lipofectamine 3000, carrying out HEK293T cell transfection experiments, and analyzing the expression level of eGFP green fluorescent protein by a BD LSRFortessa flow cytometry.

For construction of rabies mRNA vaccine, the DNA sequence containing the glycoprotein of Pitman-Moore (PM) vaccine strain (RABV-G) was used as the basis for construction, and optimized according to the preference of eukaryotic codons for subsequent in vitro transcription experiments. The corresponding DNA template sequence is shown in SEQ ID NO. 23. Taking plasmid of SEQ ID NO. 23 as a template after single enzyme digestion, adding NTP required by transcription according to a proper proportion, using 1-methyl uracil nucleoside to replace uracil nucleoside, using T7 polymerase to obtain corresponding RABV-G mRNA (SEQ ID NO. 24 and 25), then capping by an enzyme capping method by using vaccinia virus capping enzyme and 2' -O-methyltransferase simultaneously, adding 7-methyl guanosine cap structure to the purified RABV-G mRNA, and purifying the capped mRNA by lithium chloride for preparing lipid nanoparticles.

In order to construct an influenza virus mRNA vaccine, the DNA sequence of the HA2 protein at the extracellular end of the stem region of the influenza virus hemagglutinin protein is used as a construction basis, and is optimized according to the preference of eukaryotic codons for subsequent in vitro transcription experiments. The corresponding DNA template sequence is shown in SEQ ID NO. 26. In vitro transcription, sequence optimization and modification were performed using similar methods as described previously, and the resulting mRNA (SEQ ID NOS: 27, 28) was used for the preparation of lipid nanoparticles.

Screening non-replicating mRNA vector for rabies mRNA vaccinated mice

Preparation of rabies mRNA liposome nanoparticles (micana (Shanghai) instruments technologies limited): dissolving cationic lipid Dlin-MC3-DMA, structural lipid cholesterol, auxiliary lipid DSPC and stable lipid DMG-PEG2000 in ethanol according to the molar ratio of 50:38:10:2, wherein the ethanol concentration is 30% (v/v), so as to obtain an oil phase mixed solution. The oil phase mixture was then added to 50mM citrate buffer pH 4.0 at room temperature to give a lipid mixture. Adding the lipid mixture into a liposome extruder, extruding and filtering with a 200nm filter membrane, and filtering with a 100nm filter membrane to change the solution from milky white to clear, thereby obtaining the cationic liposome nano-particles.

Dissolving the optimized nucleoside modified RABV-G mRNA in a citrate buffer solution (pH 4.0), dropwise adding the citrate buffer solution into the cationic lipid nanoparticle according to the mass ratio of the cationic lipid nanoparticle to the mRNA of 20:1, and uniformly mixing by using an oscillator Vortex to obtain a mixed solution. After thorough mixing, heating and incubating for 1 hour in a metal bath at 42 ℃, then dialyzing the mixed solution to sterile PBS, and filtering by a 0.22 mu m sterile filter to obtain RABV-G mRNA cationic lipid nano particles, namely the rabies virus nucleic acid vaccine.

The rabies virus envelope protein G protein is used as immunogen to immunize BALB/c mice, and 5 mice are immunized in each group. The priming dose was 10 μg, the boosting dose was 1 μg, the muscle vaccination route, the injection volume was 100 μl, the immunization was twice, 3 weeks apart, and a liposome empty-load group control was set, blood was collected 2 weeks, 2 months, 5 months, 7 months after the second immunization, respectively, and rabies virus G protein specific antibody levels were evaluated by ELISA.

Screened non-replicating mRNA vectors for influenza mRNA vaccinated mice

In the same II, the company is sent to prepare influenza virus mRNA liposome nano particles, and the HA2 protein at the extracellular end of the stem region of the influenza virus hemagglutinin protein is used as an immunogen to immunize BALB/c mice, wherein 5 mice are used in each group. The initial dose and the booster dose are 5 mug, the muscle is inoculated at intervals of 3 weeks, a liposome empty vector group control is arranged at the same time, the specific antibody level is analyzed by blood sampling 2 weeks after the secondary immunization, and the specific antibody level to the HA2 protein at the extracellular section of the HA stem region is evaluated and analyzed by ELISA.

Sequence naming

The following is a nomenclature for the names of sequences referred to herein, which have the same 5' -UTR:

-an eGFP-mRNA vector comprising 3' -UTRs derived from human actin (actin), human albumin (albumin), human α -globulin), respectively, each with a poly (a) tail of 30A, named in order: I-eGFP actin (30A), shown as SEQ ID NO. 12; I-eGFP album (30A) as shown in SEQ ID NO. 13; I-eGFP-alpha-globin (30A) as shown in SEQ ID NO. 14;

-an eGFP-mRNA vector comprising poly (a) tails derived from human albumin, human α -globulin 3' -UTR, respectively, each with 120A, named in turn: II-eGFP-album (120A) as shown in SEQ ID NO. 15; II-eGFP-alpha-globin (120A) as shown in SEQ ID NO. 16;

-an eGFP-mRNA vector comprising poly (a) tails derived from human albumin, human α -globulin 3' -UTR, respectively, each with 250A, named in order: III-eGFP-album (250A) as shown in SEQ ID NO. 17; III-eGFP-alpha-globin (250A), shown as SEQ ID NO. 18;

eGFP-mRNA vector comprising different tandem 3' -UTRs with a 250A poly (A) tail, wherein the tandem 3' -UTRs comprise tandem sequences derived from human α -globulin and human albumin 3' -UTR sequences (α -globin+album), human albumin and eukaryotic mitochondria (album+mit), and human α -globulin and eukaryotic mitochondria sequences (α -globin+mit), named in turn: IV-eGFP (alpha-globin+album) (250A) as shown in SEQ ID NO 19; IV-eGFP (album+Mit) (250A) as shown in SEQ ID NO. 20; IV-eGFP (alpha-globin+mit) (250A) as shown in SEQ ID NO. 21;

-eGFP-mRNA vector comprising tandem 3' -UTR with 120A poly (a) tail, wherein the tandem 3' -UTR is a 3' -UTR tandem sequence derived from human α -globulin and eukaryotic mitochondria, designated: IV-eGFP (alpha-globin+mit) (120A) as shown in SEQ ID NO. 22;

-the selected mRNA vector each carrying a poly (a) tail of 120A and poly (a) mRNA vector carrying 250A (3' -UTR is α -globin+mit) were used for rabies mRNA vaccine, named in sequence: RABV-G mRNA (120A) (SEQ ID NO: 25) and RABV-G mRNA (250A) (SEQ ID NO: 24);

-the selected mRNA vector each carrying a poly (a) tail of 120A and mRNA vector carrying a poly (a) tail of 250A (3' -UTR is α -globin+mit) were used for influenza virus mRNA vaccine, 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 with different sources of 3' -UTR and with a 30A poly (A) tail

The eGFP-mRNA prepared in the above experimental method I was transfected into HEK293T cells for expression verification. HEK293 cells were plated 24h before transfection and cells were seeded at a density of 200000 per well in 12-well plates in DMEM complete medium (10% fbs and 1% p.s.). The transfection reagent is Lipofectamine 3000, the transfection ratio of mRNA to Lipofectamine 3000 is 1:2, 2 mug of eGFP-mRNA is transfected per well plate, and the culture is carried out in incubator at 37 ℃. The expression level of eGFP was detected in a flow assay at 12h, day1, day2, day3, day4, day5, day6, day7.

The results are shown in FIG. 2: I-eGFP actin (30A), I-eGFP album (30A) and I-eGFP-alpha-globin (30A) can be expressed in large quantities after HEK293T cells are transfected, the transfection positive rate is above 60%, and the highest transfection positive rate is achieved within 24-48 h after transfection (figure 2A). Furthermore, after transfection of I-eGFP albumin (30A) and I-eGFP-alpha-globin (30A) eGFP-mRNA, the average fluorescence intensity of eGFP was 2-3 times higher than that of I-eGFP actin (30A), and the expression was relatively high for many days (FIG. 2B).

The above results demonstrate that I-eGFP album (30A) and I-eGFP-alpha-globin (30A) can transfect cells with high efficiency, and that the 3' -UTR contained therein contributes to a significant increase in expression level.

Example 2 expression verification of HEK293T cells transfected with eGFP-mRNA vectors having different sources of 3' -UTR and either 120A poly (A) tail or 250A poly (A) tail

HEK293T cells were transfected for expression verification in the same manner as in example 1, except that the expression vector used was an eGFP-mRNA vector having a different source 3' -UTR and carrying either 120A poly (A) tails or 250A poly (A) tails.

The results are shown in FIG. 3: II-eGFP-album (120A) and II-eGFP-alpha-globin (120A) can be expressed in large quantity after HEK293T cells are transfected, the transfection positive rate is more than 80% (figure 3A), and the eGFP average fluorescence intensity is higher than that of eGFP-mRNA with the same 3' -UTR of 30A (figure 3B); the same 3' -UTR with 250poly (A) eGFP-mRNA, i.e., III-eGFP-album (250A), III-eGFP- α -globin (250A) had a positive rate of 85% or more (FIG. 3A), which was 1.5-2 times higher than the average fluorescence intensity of eGFP with 120poly (A), although the fluorescence intensity was reduced from day 2 onwards, the decrease in eGFP-mRNA with 250poly (A) was less, and was maintained for at least 7 days (FIG. 3B).

The above results demonstrate that vectors III-eGFP-album (250A), III-eGFP- α -globin (250A) with 250A poly (A) are able to transfect cells more efficiently and further increase expression levels, with the same 3' -UTR.

Example 3 expression verification of HEK293T cells transfected with eGFP-mRNA vectors with different tandem 3' -UTRs and with a 250A poly (A) tail

HEK293T cells were transfected and tested for expression verification at different time points using the same method as in example 1, except that the expression vector used was an eGFP-mRNA vector with a different tandem 3' -UTR and with a 250A poly (A) tail.

The results are shown in FIG. 4: IV-eGFP (alpha-globin+album) (250A), IV-eGFP (album+mit) (250A) and IV-eGFP (alpha-globin+mit) (250A) can be expressed in large quantity after being transfected into HEK293T cells, the positive rate is above 85% (figure 4A), and the average fluorescence intensity of eGFP of the transfected IV-eGFP (alpha-globin+mit) (250A) mRNA is highest, can reach 3E4 and above, and is 2-4 times higher than the combination of the other two serial connection types (figure 4B); and although the fluorescence intensity was reduced from day 2 onwards, the decrease in eGFP-mRNA of IV-eGFP (. Alpha. -globin+Mit) (250A) was small, and the average fluorescence intensity was maintained at a level of 1E4 or more for at least 7 days (FIG. 4B).

The above results demonstrate that mRNA vectors with 3' -UTRs in tandem from different sources and with a 250A poly (A) tail are able to transfect cells efficiently and to boost expression levels, with IV-eGFP (α -globin+mit) (250A) having the highest expression level.

Example 4 expression verification of HEK293T cells transfected with eGFP-mRNA vectors having the same tandem 3' -UTR and with a poly (A) tail of 120A or 250A

HEK293T cells were transfected and tested for expression verification at different time points using the same method as in example 1, except that the expression vector used was an eGFP-mRNA vector with the same tandem 3' -UTR and with a poly (A) tail of 120A or 250A.

The results are shown in FIG. 5: IV-eGFP (alpha-globin+Mit) (120A), IV-eGFP (alpha-globin+Mit) (250A) were all abundantly expressed after transfection of HEK293T cells, with transfection positivity rates above 85% and up to 95% (FIG. 5A), and the average fluorescence intensity of eGFP-mRNA having the same tandem 3' -UTR but with a poly (A) tail of 250A after transfection was about 2-fold higher than that of eGFP-mRNA with a poly (A) tail of 120A (FIG. 5B); and also the fluorescence intensity was reduced from day 2 (fig. 5B).

The above results demonstrate at the cellular level that expression vectors with the same tandem 3'-UTR with 250poly (A) perform better than expression vectors with 120A, with 3' -UTR in tandem from different sources and with 250poly (A) expression vectors performing best.

EXAMPLE 5 humoral response levels after Vaccination of mice with rabies mRNA prepared with mRNA vectors having the same tandem 3' -UTR and either a poly (A) tail of 120A or a poly (A) tail of 250A

To further verify the expression effect of non-replicating mRNA vectors screened according to the techniques herein in vivo, this example was applied to the preparation of mRNA rabies vaccine.

The mRNA selected in this example was: RABV-G mRNA (120A) and RABV-G mRNA (250A) were prepared into cationic lipid nanoparticles encapsulating RABV-G mRNA, and BALB/c mice were immunized to evaluate their immunogenicity. Each group of 5 mice was vaccinated at a priming dose of 10. Mu.g, a boosting dose of 1. Mu.g, a muscle inoculation route, 100. Mu.l per injection volume, two immunizations, 3 weeks apart. The orbits were collected 2 weeks, 2 months, 5 months, 7 months after the secondary immunization, respectively, and the negative control was an equal volume of non-nucleic acid coated cationic lipid nanoparticles (i.e., no-load control).

The results are shown in fig. 6, where the specific antibody titer of RABV-G mRNA (120A) and RABV-G mRNA (250A) groups was 281231 and the RABV-G mRNA (250A) group was 902035, i.e., the antibody titer of the RABV-G mRNA (250A) group was about 3.2 times that of the RABV-G mRNA (120A) group, for both the RABV-GmRNA (120A) group and the RABV-G mRNA (250A) group showing significantly increased antibody response levels compared to the empty control 14 days after the 1 μg dose boost injection. Also, following booster immunization, the RABV-GmRNA (250A) group was able to maintain higher titers of antibody concentrations up to 7 months.

The experimental results prove that the rabies virus G protein antigen loaded by the vector disclosed herein has stronger immunogenicity and can continuously induce high-level antibody response in vivo.

Example 6 humoral response levels after Vaccination of mice with influenza Virus mRNA prepared with mRNA vectors having the same tandem 3' -UTR and either a poly (A) tail of 120A or a poly (A) tail of 250A

The present example uses the screened non-replicating mRNA vector for the preparation and vaccination of influenza mRNA vaccines. The influenza virus mRNAs used in this example were HA2mRNA (120A) and HA2mRNA (250A), HA2mRNA liposome nanoparticles were prepared, and BALB/c mice were immunized to evaluate their immunogenicity. Immunogenicity was assessed 2 weeks and 4 weeks after boosting, respectively, as described in material method III.

The results are shown in FIG. 7: the HA2mRNA (120A) boost increased the antibody titer levels to some extent compared to the no-load control, whereas the HA2mRNA (250A) boost significantly increased the specific antibodies to HA2 to an average titer of about 8400 even further than the HA2mRNA (120A) group, with about a 12-21 fold difference in antibody titers between the two groups.

The above experimental results demonstrate that vectors with specific 3' -UTRs and long poly (a) tails herein can effectively carry influenza antigens and induce high titer antibody levels. Particularly, when influenza hemagglutinin antigen (stem region antigen) is carried on a vector having a poly (A) tail of 250A, an extremely strong immune response can be induced.

In summary, whether it is at the cellular level or in vivo animal efficacy validation, the non-replicating mRNA vector (especially IV-eGFP- (α -globin+mit) (250A) screened by the present disclosure can be loaded with different genes of interest (replace eGFP gene with various genes of interest), and can achieve efficient translation, stable and sustained expression.

The above specific embodiments are used for further detailed description of the objects, technical solutions and advantageous effects of the present invention. It is to be understood that the above description is only of specific embodiments of the invention and is not intended to limit the invention.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto. In other words, any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

The method comprises the following steps: sequence list correspondence information

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Claims (14)

1. An mRNA nucleic acid expression vector capable of expressing a protein of interest comprising, in order from the 5 'end to the 3' end:

(a) A 5' -UTR element;

(b) An open reading frame element encoding a protein of interest;

(c) 3' -UTR elements;

(d) A polyadenylation tail element having a total length of 120nt or more comprising:

a plurality of adenylate strings, each adenylate string independently comprising n consecutive adenylates, n being an integer between 10 and 80, and the total number of adenylates of the plurality of adenylate strings being 100 or more; and

a linker located between the plurality of adenylate strings, each independently comprising no adenylate or only 1 or 2 adenylates.

2. A nucleic acid expression vector according to claim 1, wherein the 5' -UTR element is 10 to 200nt, such as 15 to 100nt, in length; and/or

The 5'-UTR elements are derived from one or more 5' -UTRs from the group consisting of: human α -globulin, β -globulin, ribosomal Protein (RP), tubulin β -2B, complement factor 3 (C3), cytochrome P450 2E1 (CYP 2E 1), apolipoprotein a-II (APOA 2), human hemoglobin subunit β (hbb), hemoglobin A1 (HBA 1), hemoglobin A2 (HBA 2), dengue virus (DENV); and/or

The 5' -UTR element has the sequence as shown in SEQ ID No. 1 or has at least 80% sequence identity thereto.

3. A nucleic acid expression vector according to claim 1, wherein the 3' -UTR element is a 3' -UTR of mammalian or viral origin, e.g. derived from a 3' -UTR selected from the group consisting of: human alpha globulin, human beta globulin, human albumin, human actin, human hemoglobin subunit alpha 1 (HBA 1), cytochrome B-245 alpha Chain (CYBA), sequences of eukaryotic mitochondria (Mit), sars-Cov-2, dengue virus (DENV), turnip Crinkle Virus (TCV), tobacco Mosaic Virus (TMV), and Tobacco Etch Virus (TEV); and/or

The 3'-UTR element comprises one or more 3' -UTR molecules selected from the group consisting of: alpha globulin 3' -UTR, eukaryotic mitochondrial 3' -UTR, albumin 3' -UTR, beta globulin 3' -UTR or any of their tandem sequences, preferably alpha globulin 3' -UTR, eukaryotic mitochondrial 3' -UTR, or 3' -UTR formed by tandem connection thereof; and/or

The 3' -UTR has one or more of the sequences shown in SEQ ID NOS.2 to 8, preferably the sequence shown in SEQ ID NOS.2, 5 or 8, or a sequence having at least 80% sequence identity to any one thereof.

4. The nucleic acid expression vector of claim 1, wherein the total length of the polyadenylation tail element is 120-400 nt, such as 120-350 nt, 120-320 nt, or any integer therein, such as 120, 304nt; and/or

Each adenylate cluster independently comprises 10 to 80, 20 to 70, 25 to 60, 30 to 50 or any integer number of consecutive adenylates therein, such as 20, 30, 33, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70 consecutive adenylates; and/or

The polyadenylation tail element further comprises a linker at one or both ends of the element; and/or

The linkers are each independently 3 to 15nt in length, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15nt; and/or

For example, the sequences of the linkers are each independently selected from: GCTATGACT, GTATGT, GCAAGT, GATTGC, GGCTGC, TACTGC, GGCTTC, GCATATGACT; and/or

The polyadenylation tail element has the sequence of SEQ ID NO. 10 or SEQ ID NO. 11, or at least 80% sequence identity thereto.

5. The nucleic acid expression vector of claim 1, wherein the nucleic acid expression vector is a non-replicating mRNA vector or is a nucleic acid vaccine; and/or

The nucleic acid expression vector is codon optimized or comprises modified bases and/or nucleoside analogues;

the element encoding the protein of interest is a single, double or polycistronic mRNA; and/or

The elements encoding the protein of interest are optionally codon optimized, with or without base modification and/or nucleoside analogs.

6. The nucleic acid expression vector of claim 1, wherein the nucleic acid expression vector further comprises one or more elements selected from the group consisting of:

a 5 '-cap element, optionally modified, for example the 5' -cap element is selected from: m7GpppXpYp, m7 gpppxpmpyp, or methylation modified sequences, reverse binding isomers, anti-reverse cap analogues (ARCA), N7-benzyl dinucleotide tetraphosphate cap analogues;

promoter elements, such as the T7 promoter, sp6 promoter or T3 promoter;

signal peptide coding elements, e.g., signal peptide (e.g., transmembrane signal peptide, secretory signal peptide, nuclear localization signal peptide) coding elements that direct subcellular localization of the protein of interest;

cleavage sites, e.g., xbaI, ecoRV, bamHI, xhoI;

tags, e.g. molecular tags for identification, isolation or purification of a molecule of interest, such as Flag tags, HA tags.

7. The nucleic acid expression vector of any one of claims 1-6, wherein the nucleic acid expression vector comprises mRNA capable of expressing one or more proteins of interest selected from the group consisting of: an immunogenic molecule, an antibody molecule, a therapeutic drug, a prophylactic drug, a protein replacement therapy molecule, a gene editing therapy molecule; and/or

The nucleic acid expression vector is used for preventing and/or treating a disease selected from the group consisting of: viral infection, cancer, genetic disease (e.g., monogenic disease), such as:

the disease is selected from one or more of the following viral infections: rabies virus, influenza virus, coronavirus, ebola virus, zika virus, yellow fever virus, dengue virus, cytomegalovirus, blue ear 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; and/or

In the case of use in the prevention and/or treatment of viral infectious diseases, the protein of interest is a viral immunogen, for example from viral Envelope proteins, including Hemagglutinin protein (HA), neuraminidase (NA), matrix protein (M), envelope protein (Envelope), spike protein (Spike), membrane protein (Membrane, M), hemolysin (Haemolysin, HL), fusion protein (Fusion, F), glycoprotein (G); and/or, the protein of interest is expressed in a cell, on a cell membrane, or expressed secreted; and/or

The disease is selected from one or more of the following cancers: squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, hepatocellular carcinoma, renal cell carcinoma, bladder carcinoma, intestinal cancer, cervical cancer, colon cancer, esophageal carcinoma, head cancer, renal cancer, liver cancer, lung cancer, neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, leukemia, lymphoma, burkitt's lymphoma, non-hodgkin's lymphoma; melanoma; myeloproliferative diseases; sarcomas, hemangiosarcomas, kaposi's sarcoma, liposarcoma, myosarcomas, peripheral nerve epithelial tumors, synovial sarcomas, gliomas, astrocytomas, oligodendrogliomas, ependymomas, glioblastomas, neuroblastomas, gangliocytomas, gangliogliomas, medulloblastomas, pineal tumors, meningiomas, neurofibromas and schwannomas, breast cancer, uterine cancer, testicular cancer, thyroid cancer, astrocytomas, esophageal cancer, carcinoma sarcomas, hodgkin's disease, wilms ' tumors, and teratocarcinomas;

the disease is selected from one or more of the following genetic diseases: methylmalonic acid, acute intermittent porphyrin, fabry disease, albinism, hemophilia, phenylketonuria, galactosylation, mucopolysaccharidosis and congenital adrenocortical hyperplasia.

8. The nucleic acid expression vector of claim 1, wherein the nucleic acid expression vector comprises, from 5 'to 3':

a 5' -UTR element comprising the sequence shown in SEQ ID NO. 1; an open reading frame element encoding a protein of interest; a 3' -UTR element comprising the sequence as shown in SEQ ID NO. 2, 5 or 8; a polyadenylation tail element comprising a sequence as set forth in SEQ ID NO 10 or 11; or a sequence having at least 80% sequence identity to said sequence; and/or

The nucleic acid expression vector comprises a sequence as set forth in any one of SEQ ID NOS.15-22, 24-25 and 27-28, a sequence having at least 80% sequence identity thereto, or a sequence in which an open reading frame element encoding a protein of interest in any one of the foregoing sequences is replaced with an open reading frame element encoding a desired protein of interest.

9. The nucleic acid expression vector of claim 1, which is contained in a package alone or in combination with a carrier in a delivery system, e.g., selected from the group consisting of: lipid delivery systems, polymer delivery systems, or combinations thereof, such as delivery systems loaded on lipid nanoparticles, polyurethanes (PAA), poly-beta-amino esters (PBAE), polyethylenimines (PEI), lipid-encapsulated polymer micelles.

10. A composition comprising the nucleic acid expression vector of any one of claims 1-9, and a package and/or a delivery system for the nucleic acid expression vector and/or a pharmaceutically or physiologically acceptable carrier.

11. The composition of claim 10 in a form suitable for administration or delivery by one or more modes selected from the group consisting of: respiratory tract aerosol inhalation, nasal drip, oral administration, direct injection (e.g., intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection), mucosal administration; and/or

The composition further comprises or is used in combination with an adjuvant, for example selected from the group consisting of: aluminum adjuvants, cholera toxin and subunits thereof, oligodeoxynucleotides, manganese ion adjuvants, colloidal manganese adjuvants, freund's adjuvant, MF59 adjuvants, QS-21 adjuvants, poly I: C and other TLR ligands, GM-CSF, IL-2, IL-3, IL-7, IL-11, IL-12, IL-18, IL-21; and/or

The composition is in a form suitable for combined administration of 2 or more drugs or vaccines, such as co-vaccination or sequential vaccination; and/or

The composition is selected from: mRNA vaccines, therapeutic or prophylactic agents, such as protein replacement therapy agents, gene editing therapy agents.

12. Use of a nucleic acid expression vector according to any one of claims 1 to 9 and/or a composition according to any one of claims 10 to 11 for the preparation of a product for expression of a protein of interest,

for example, the product is selected from: mRNA vaccines, therapeutic or prophylactic agents, such as protein replacement therapy agents, gene editing therapy agents.

13. A method of preparing the nucleic acid expression vector of any one of claims 1-9 or the composition of any one of claims 10-11, the method comprising: providing separate or connected elements; the elements are assembled into a nucleic acid expression vector.

14. The method of claim 13, wherein the method comprises using one or more materials selected from the group consisting of: DNA templates (e.g., PCR products or linearized plasmid DNA), nucleases, polymerases, capping enzymes, poly-a synthases, dnases, one or more element molecules, linker molecules, natural or modified nucleic acid molecules, buffers, solvents; and/or

The method further comprises one or more steps selected from the group consisting of: designing, optimizing, reforming and/or modifying each element; isolating, purifying, identifying, quantifying, packaging and/or testing the activity of the intermediate product and/or the final product; the nucleic acid expression vector is combined with a delivery system and/or a pharmaceutically or physiologically acceptable carrier for the nucleic acid expression vector.