Plasmid vector for in vitro transcription of mRNA, construction method, and use thereof.

Abstract

The present invention provides a nucleic acid molecule comprising: 1) a transcribed nucleic acid fragment and / or a nucleic acid for introducing a transcribed nucleic acid fragment; 2) a polyA nucleic acid fragment; and 3) a gyrase-binding site nucleic acid fragment. The nucleic acid molecule of the present invention can improve the stability of PolyA and solve the problem of PolyA deletion / shortening, and at the same time improve the stability of the nucleic acid molecule and reduce the plasmid recombination probability during amplification, which is advantageous for the industrial production of mRNA products.

Description

TECHNICAL FIELD

[0001] The present invention belongs to the field of genetic engineering. Specifically, the present invention relates to an mRNA in vitro transcription vector capable of improving plasmid stability, a construction method thereof, and use thereof. BACKGROUND ART

[0002] mRNA technology has many advantages. First, since mRNA is not integrated into the genome, the concern of insertional mutagenesis can be avoided. More importantly, unlike relatively stable DNA, functional mRNA is subsequently degraded, so it does not cause other toxicities and side effects to the human body. mRNA can be produced by a cell-free method, and can be rapidly and economically produced on a large scale using in vitro transcription technology. Due to its short development cycle, rapid, economical and efficient production can be achieved. In addition, a single mRNA can encode various antigens, proteins or polypeptides, with strong process applicability, and in theory, any protein can be expressed.

[0003] mRNA-based COVID-19 vaccines have verified the applicability of the mRNA technology platform in the vaccine field. In addition to COVID-19 vaccines, mRNA technology can be widely applied in fields such as infectious diseases, tumors, and protein replacement therapy.

[0004] For mRNA technology, a good production process begins with the design of safe, stable, and efficient plasmid vectors. From both a process production perspective and a therapeutic or regulatory perspective, most non-essential sequences in plasmid DNA should be removed to reduce the molecular weight as much as possible and facilitate genetic manipulation. This is because if the plasmid is too large, it places a huge metabolic burden on the host organism, reducing cellular resources for plasmid replication and consequently lowering plasmid DNA yield. Furthermore, in industrial production, plasmids must maintain high stability even through successive passaging processes to ensure the widespread application and stability of mRNA technology.

[0005] One major challenge in mRNA plasmid technology production is the shortening of the plasmid's PolyA sequence during sustained bacterial amplification. This hinders the cloning and amplification of plasmids with PolyA tails, preventing uniformity in the produced plasmid PolyA sequence and failing to meet GMP-level production process and pharmaceutical regulatory requirements. Furthermore, since the PolyA sequence is a crucial element for mRNA stability and translational expression efficiency, resolving the issue of PolyA sequence shortening during plasmid fermentation production remains a constant focus for developers.

[0006] Kyle Jacoby et al. developed a pEVL linear plasmid system derived from the PJazz plasmid to overcome the instability of the PolyA tail in circular plasmids. Compared to conventional circular plasmids, pEVL plasmids exhibit a significant advantage in maintaining PolyA tail length through continuous subculturing (Kyle Jacoby, 2016, Mol Ther Nucleic Acids). However, the copy number of pEVL plasmids is very low, making them unsuitable for large-scale production. In 2019, Zeljka Trepotec et al. found that separating the PolyA sequence reduces recombination during plasmid DNA amplification, thereby maintaining the length of the PolyA tail. Representative techniques using fragmented polyA tails include the proposed technology disclosed in BioNTech patent US10717982B2 and the technology disclosed in Ethris patent WO2020074642A1. Kristie Bloom et al. developed a plasmid that can increase the length of the PolyA sequence through enzymatic cleavage and ligation, as a template for in vitro cotranscription of mRNA, in order to improve the situation of shortening of PolyA length during plasmid passage. Some companies also employ shorter polyA sequences to stabilize plasmids.

[0007] However, most of the methods described above are aimed at improving polyA itself, and developing plasmid systems to improve the instability of polyA has proven unsuitable for large-scale production and use. [Overview of the project]

[0008] This invention involves further research on plasmids applicable to RNA technology, overcoming the challenge of deleting only polyA from the entire plasmid, and addressing the difficult issue of polyA deletion starting from the whole plasmid, while ensuring that plasmid replication and yield are not affected. This improves plasmid integrity and stability, making it suitable for large-scale production.

[0009] This invention provides nucleic acid molecules and their use for improving the stability of PolyA tails during plasmid passage in bacterial fermentation.

[0010] In this specification, the term “vector” in the present invention has the general meaning known to those skilled in the art and includes any intermediate vector of nucleic acid that enables, for example, the introduction of such nucleic acid into prokaryotic and / or eukaryotic host cells, or, where appropriate, integration into a single genome, the vector is preferably replicated and / or expressed within the cell.

[0011] In this invention, the term "pharmaceutically acceptable carrier" refers to an inactive substance prepared together with a pharmaceutically active substance to satisfy or enhance the requirements of the active substance in terms of dose, adsorption, solubility, or pharmacokinetics. In this invention, the term "plasmid" usually refers to a construct consisting of extrachromosomal genetic material, and is generally a circular double-stranded DNA that can replicate independently of chromosomal DNA.

[0012] In this invention, the term "host cell" refers to any cell that can be transformed or transfected with exogenous nucleic acids, including prokaryotic cells (e.g., Escherichia coli) or eukaryotic cells (e.g., yeast cells and insect cells). Mammalian cells, such as those derived from humans, mice, hamsters, pigs, goats, and primates, are particularly preferred. The cells may be derived from various tissue types and include primary cells and cell lines.

[0013] The terms "peptide" and "protein" in this invention include substances containing not only amino acid components but also non-amino acid components such as sugars and phosphate structures, and also include substances containing bonds, such as ester bonds, thioether bonds, or disulfide bonds.

[0014] In this invention, the term "nucleotide fragment encoding a peptide or protein" refers to a nucleotide fragment that, when present in a suitable environment, preferably in a cell, can guide translation to assemble amino acids and produce a peptide or protein.

[0015] The term "immune response" in this invention relates to the immune system's response to an immunogenic organism, cell, or substance, such as bacteria or viruses. The term "immune response" includes innate and adaptive immune responses. Preferably, the immune response involves the activation of immune cells, induction of cytokine biosynthesis, and / or antibody production. Preferably, the immune response includes the steps of activating antigen-presenting cells such as dendritic cells and / or macrophages, the antigen-presenting cells presenting an antigen or a fragment thereof, and the activation of cytotoxic T cells by such presentation.

[0016] A first aspect of the present invention provides a nucleic acid molecule comprising: 1) a transcribed nucleic acid fragment or a nucleic acid for introducing a transcribed nucleic acid fragment; 2) a nucleic acid fragment containing polydeoxyadenylate (polyA); and 3) a nucleic acid fragment containing a gyrase binding site (SGS).

[0017] The above-mentioned transcribed nucleic acid fragments include nucleic acid fragments that encode peptides or proteins.

[0018] The nucleic acid used to introduce the above-mentioned transcribable nucleic acid fragment includes a polyclonal site or a homologous recombination site.

[0019] The above polyA contains at least 50 consecutive deoxyadenylic acid molecules.

[0020] The nucleic acid fragment containing the above polyA comprises at least two elements, each having a continuous sequence of at least 20 deoxyadenylic acids, with at least one non-T deoxynucleotide interposed between the two elements.

[0021] In some specific embodiments, the nucleic acid fragment containing the gyrase binding site is derived from a virus or bacteria, preferably from a virus, and more preferably from a phage.

[0022] In some specific embodiments, the nucleic acid fragment comprising the gyrase binding site is derived from a plasmid, preferably, the nucleic acid fragment comprising the gyrase binding site is derived from a ColE1-type plasmid.

[0023] In some specific embodiments, the above SGS nucleic acid fragment is constructed upstream of the T7 promoter in a plasmid vector, and in some specific embodiments, the above SGS nucleic acid fragment is constructed downstream of PolyA in a plasmid vector. A person skilled in the art can select a suitable position to insert SGS as needed based on the disclosure content of the present application.

[0024] In another aspect, the present invention provides a DNA expression vector or a recombinant plasmid, and the DNA expression vector or recombinant plasmid sequence comprises 1) a first nucleic acid fragment encoding an mRNA molecule, 2) a second nucleic acid fragment comprising polydeoxyadenylic acid polyA, and 3) a third nucleic acid fragment comprising a gyrase binding site.

[0025] The present invention further provides a method for producing the expression vector or the recombinant plasmid, which comprises transforming the above expression vector or recombinant plasmid into E. coli competent cells, taking an appropriate amount, spreading it on a resistant LB medium plate for culture, picking a monoclonal colony for sequencing, obtaining a positive bacterial solution with correct sequencing, and carrying out large-scale culture.

[0026] The present invention provides a method for amplifying a nucleic acid molecule, and the method comprises replicating the above nucleic acid molecule in a host cell and propagating the above host cell.

[0027] In some specific embodiments, the above host cell is a eukaryotic cell or a prokaryotic cell, preferably, the above host cell is Escherichia coli.

[0028] The present invention provides a method for obtaining RNA, which comprises amplifying a nucleic acid molecule based on the above method, and transcribing the nucleic acid molecule into RNA in vitro using the nucleic acid molecule as a template.

[0029] The present invention provides a method for obtaining a peptide or protein, comprising obtaining an mRNA encoding the peptide or protein based on the above method for obtaining RNA, and translating the mRNA into an amino acid fragment of the peptide or protein.

[0030] The present invention provides a system for producing eukaryotically translatable mRNA, comprising 1) the vector or plasmid containing the above nucleic acid molecule, and 2) a host cell.

[0031] In another aspect, the present invention provides use of a nucleic acid nucleotide fragment encoding a gyrase binding site in the construction of a plasmid or vector, wherein the plasmid or vector is used for being transcribed into RNA in vitro.

[0032] In another aspect, the present invention provides a composition comprising the RNA, peptide, or protein obtained by the method described above.

[0033] In some specific embodiments, the above composition comprises one or more of pharmaceutically acceptable excipients, carriers, buffers, protective agents, stabilizers, surfactants, osmotic pressure regulators, adjuvants, preservatives, and inactivating agents.

[0034] The buffer according to the present invention comprises one or more of HEPES, HIS, TRIS, PB, succinic acid, and citric acid, the protective agent comprises one or more of gelatin, ethanol, ethylenediaminetetraacetic acid (EDTA), disodium ethylenediaminetetraacetate (EDTA-2Na), and magnesium chloride, the stabilizer comprises one or more of sucrose, mannitol, fucose, and maltose, the surfactant comprises one or more of Tween, Span, and glycerin, the osmotic pressure regulator comprises sodium chloride or is omitted, and the excipient component comprises one or more of mannitol, sucrose, sodium chloride, magnesium chloride, HEPES, polysorbate 80, and glycerin.

[0035] In several specific embodiments, the compositions described in the present invention can be manufactured as mucosal immunotherapy preparations, humoral immunotherapy preparations, cellular immunotherapy preparations, and skin immunotherapy preparations.

[0036] In some specific embodiments, the compositions described in the present invention can be manufactured as liquid dosage forms, solid dosage forms, semi-solid dosage forms, gaseous dosage forms, and inhalation dosage forms.

[0037] In some specific embodiments, the method of administration of the composition is intravenous injection, intramuscular injection, subcutaneous injection, oral administration, oral administration, sublingual administration, rectal administration, intra-airway administration, or transdermal administration, and intra-airway administration is by oral inhalation, nasal inhalation, or inhalation after spraying with a spray administration device.

[0038] The nucleic acid molecules, plasmids, or vectors containing nucleic acid molecules according to the present invention can be used for the expression of recombinant proteins in cell transcription and expression. More specifically, when producing recombinant proteins, the expression vector according to the present invention can be used to transcribe recombinant nucleic acids and express recombinant proteins in a cell-based system. Examples include the production of recombinant antibodies, hormones, cytokines, enzymes, etc.

[0039] The nucleic acid molecules, plasmids, or vectors containing nucleic acid molecules according to the present invention can be used, for example, for transient gene expression. Possible application areas include RNA-based vaccines, which are transfected into cells in vitro or applied directly in vivo or in vitro to transiently express functional proteins.

[0040] The present invention provides the use of the above composition, which includes being used for pharmaceuticals, for delivering recombinant genes to host cells, for inducing a primary immune response in animals to treat or prevent at least one disease, for enhancing an immune response in animals, and for inducing an immune response that overcomes resistance to autoantigens in animals. The above immune response includes vaccines for the prevention of cancer, therapeutic tumor vaccines, prevention of viral infections, prevention of bacterial infections, and prevention of fungal infections.

[0041] The present invention provides the use of any one of the above-mentioned nucleic acid molecules, expression vectors, or compositions in the manufacture of a drug or vaccine for treating or preventing a disease.

[0042] In some specific embodiments, the above-mentioned diseases include novel coronavirus, dengue virus, malaria parasite, herpes zoster virus, feline parvovirus, feline calicivirus, feline herpesvirus, feline leukemia virus, feline coronavirus, porcine reproductive and respiratory syndrome virus, herpes simplex virus type 1, herpes simplex virus type 2, encephalitis virus, papillomavirus, varicella-zoster virus, Epstein-Barr virus, human cytomegalovirus, human herpesvirus type 8, human papillomavirus, BK virus, JC virus, smallpox, poliovirus, hepatitis B virus, human bocavirus, parvovirus B19, human astrovirus, Norwalk virus, coxsackievirus, hepatitis A virus, rhinovirus, severe acute respiratory syndrome virus, hepatitis C virus, yellow fever virus, dengue virus, It is caused by one or more viruses, bacteria, or protozoa, including West Nile virus, rubella virus, hepatitis E virus, human immunodeficiency virus (HIV), influenza A or B virus, guanalitovirus, Junin virus, lassa virus, Machupo virus, Sabia virus, Crimean-Congo hemorrhagic fever virus, Ebola virus, Marburg virus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, human metapneumovirus, Hendra virus, Nipah virus, rabies virus, hepatitis D, rotavirus, orbivirus, Escherichia coli virus, human enterovirus, Japanese encephalitis virus, vesicular herpesvirus, eastern equine encephalitis virus, hantavirus, Middle East respiratory coronavirus, chikungunya virus, or vannavirus.

[0043] The beneficial effects of the present invention include the following: In the first embodiment, the problem of PolyA deletion / shortening is solved by improving the stability of PolyA with the SGS sequence. In a second embodiment, the stability of nucleic acid molecules is improved by the SGS sequence. In a third aspect, the SGS sequence reduces the recombination probability of the plasmid during the amplification period, further improving the safety of plasmid application. In a fourth aspect, SGS sequencing allows for the acquisition of RNA molecules, peptides, or proteins with higher purity that meet GMP-level production process and pharmaceutical regulatory requirements, making them suitable for large-scale production. [Brief explanation of the drawing]

[0044] [Figure 1] This study examines the influence of SGS sequences of different origins on the stability of the polyA sequence under non-resistance conditions. [Figure 2] This study examines the influence of SGS sequences of different origins on the stability of polyA sequences under kanamycin-resistant conditions. [Figure 3] This study describes the effect of the SGS Mu398 sequence on the stability of different length types of PolyA under non-resistance conditions. [Figure 4] This study examines the effect of the SGS Mu398 sequence on the stability of different length types of PolyA under kanamycin-resistant conditions. [Figure 5] This describes the applicability of the SGS Mu398 sequence to different target antigen sequences under resistance-free conditions. [Figure 6] This study examines the applicability of the Mu398 SGS sequence to different target antigen sequences under kanamycin resistance conditions. [Figure 7] This is the result of integrity detection of the 10th generation transcription mRNA of the F001 recombinant plasmid. [Figure 8] This is the result of integrity detection of the 10th generation transcription mRNA of the F002 recombinant plasmid. [Figure 9] This is the result of integrity detection of the 10th generation transcription mRNA of the F003 recombinant plasmid. [Figure 10] This is the result of integrity detection of the 10th generation transcription mRNA of the F004 recombinant plasmid. [Figure 11] This is the result of integrity detection of the 10th generation transcription mRNA of the F005 recombinant plasmid. [Modes for carrying out the invention]

[0045] Example 1: Effect of different SGS sequences on the stability of plasmid polyA To investigate the effect of SGS sequences on plasmid stability, SGS sequences from different sources were inserted downstream of the vector polyA or upstream of the T7 promoter. polyA was a continuous 100A sequence, and target gene sequences were simultaneously inserted into the plasmid vector. The effect of SGS sequences on plasmid stability was observed, and the design method is shown in Table 1. The results showed that insertion of SGS sequences significantly improved the passage stability of polyA under both non-resistance and kanamycin-resistant conditions (Figures 1 and 2).

[0046] The transcription vector according to the present invention comprises a 5'UTR sequence, a Kozak sequence, an antigen-coding sequence, a 3'UTR sequence, a polyA tail sequence, and an SGS sequence. Of these, the above sequences were synthesized into the pUC57-kana vector by Nanjing Jinsirui Biotechnology Co., Ltd., and then sequentially ligated into a general-purpose transcription vector by homologous recombination. The constructed plasmid was transferred to Trans1-Blue competent E. coli cells, an appropriate amount was taken and spread onto a kanamycin-resistant LB medium plate, and cultured at 37°C for a certain period of time. Monoclonal colonies were selected and sequenced, and a positive bacterial suspension with accurate sequencing was stored. Subsequently, the bacterial suspension containing the positive plasmid was shaken at 37°C, the plasmid was maxiprepped, an in vitro transcription template was obtained, and mRNA was produced by co-transcription capping.

[0047] [Table 1]

[0048] Example 2: Effect of SGS sequence on the stability of PolyA of different length types To further investigate whether SGS sequences can improve the stability of PolyA sequences of different lengths, Mu398 SGS sequences and different types of polyA sequences were inserted into the plasmid vector described above, and the passage stability of the polyA sequences was observed. The specific design is shown in Table 2.

[0049] [Table 2]

[0050] The results showed that the addition of the Mu398 SGS sequence could increase the passage stability of various types of polyA sequences and maintain their integrity for up to 10 generations (Figures 3 and 4).

[0051] Example 3: Determination of a general-purpose, highly stable plasmid vector. To further verify the versatility of the plasmid vector described above, the present invention inserted target gene sequences from five different pathogens—novel coronavirus (COVID-19), dengue virus (DENV), malaria, herpes zoster virus (VZV), and feline parvovirus (FPV)—into the vector containing the SGS sequence, respectively, and constructed them as expression vectors F001, F002, F003, F004, and F005. The continuous 100A passage stability during plasmid passage was observed. As a result, it was shown that the SGS sequence is universally applicable to different target gene sequences and can guarantee passage stability of each recombinant plasmid polyA within 10 generations (as shown in Figures 5 and 6).

[0052] To further observe the transcription effect of the transcription vector, the 10th generation of the recombinant vector plasmid constructed above was transcribed in vitro after maxiprep of the plasmid. As a result, it was confirmed that each plasmid was able to transcribe high-quality mRNA (as shown in Figures 7 to 11; see Table 3 for data).

[0053] [Table 3]

[0054] In summary, the present invention provides a versatile, highly stable plasmid vector, which has universal applicability and can solve the problem of plasmid instability caused by polyA sequences in the mRNA field.

Claims

1. A nucleic acid molecule characterized by comprising: 1) a transcribable nucleic acid fragment and / or a nucleic acid for introducing a transcribable nucleic acid fragment; 2) a nucleic acid fragment containing polydeoxyadenylate polyA; and 3) a nucleic acid fragment containing a gyrase binding site.

2. The nucleic acid molecule according to claim 1, characterized in that the transcribable nucleic acid fragment encodes a peptide or a protein.

3. The nucleic acid molecule according to claim 1 or 2, characterized in that the nucleic acid fragment for introducing the transcribable nucleic acid fragment includes a polyclonal site or a homologous recombination site.

4. The nucleic acid molecule according to any one of claims 1 to 3, characterized in that the polyA contains at least 50 consecutive deoxyadenylic acid molecules.

5. The nucleic acid molecule according to any one of claims 1 to 4, characterized in that the nucleic acid fragment containing polyA comprises at least two elements which are consecutive deoxyadenylic acid sequences of at least 20 in length, and at least one non-T deoxynucleotide is interposed between the two elements.

6. The nucleic acid molecule according to any one of claims 1 to 5, characterized in that the nucleic acid fragment containing the gyrase binding site is derived from a virus or bacteria, preferably from a virus, and more preferably from a phage.

7. The nucleic acid molecule according to any one of claims 1 to 5, characterized in that the nucleic acid fragment containing the gyrase binding site is derived from a plasmid, and preferably the nucleic acid fragment containing the gyrase binding site is derived from a ColE1 type plasmid.

8. A plasmid vector characterized by comprising: 1) a first nucleic acid fragment encoding an mRNA molecule; 2) a second nucleic acid fragment containing polyA; and 3) a third nucleic acid fragment containing a gyrase binding site.

9. A method for producing a plasmid vector, characterized by transforming competent Escherichia coli cells with the plasmid vector described in claim 8, taking an appropriate amount and spreading it on a resistant LB medium plate for cultivation, selecting monoclonal colonies and sequencing them, obtaining a positive bacterial suspension with accurate sequencing, and subsequently performing large-scale culture.

10. A method for amplifying nucleic acid molecules, comprising growing the nucleic acid molecules described in claims 1 to 7 in host cells, and growing the host cells, wherein the host cells include bacteria or fungi, and the host cells are preferably Escherichia coli.

11. A method for obtaining RNA, comprising amplifying a nucleic acid molecule based on the method of claim 10 and transcribing the nucleic acid molecule into RNA in vitro using the nucleic acid molecule as a template.

12. A method for obtaining a peptide or protein, characterized by comprising obtaining mRNA encoding a peptide or protein based on the method of claim 11, and translating the mRNA into amino acid fragments of a peptide or protein.

13. A system for generating eukaryotically translatable mRNA, comprising: 1) a plasmid vector containing the nucleic acid molecules described in claims 1 to 7; and 2) a host cell.

14. A use of a nucleic acid fragment encoding a gyrase binding site in the construction of a plasmid vector, wherein the plasmid vector is used for transcription into RNA in vitro.

15. A composition comprising RNA, peptide, or protein obtained by the method described in claim 11 or 12.

16. The composition according to claim 15, characterized in that the composition comprises one or more pharmaceutically acceptable excipients, carriers, buffers, protective agents, stabilizers, surfactants, osmotic pressure regulators, adjuvants, preservatives, and inactivators.

17. Use of a nucleic acid molecule according to any one of claims 1 to 7, a plasmid vector according to claim 8, or a composition according to any one of claims 15 to 16 in the manufacture of a drug or vaccine for treating or preventing a disease.

18. The aforementioned diseases include: novel coronavirus, dengue virus, malaria parasite, herpes zoster virus, feline parvovirus, feline calicivirus, feline herpesvirus, feline leukemia virus, feline coronavirus, porcine reproductive and respiratory syndrome virus, herpes simplex virus type 1, herpes simplex virus type 2, encephalitis virus, papillomavirus, varicella-zoster virus, Epstein-Barr virus, human cytomegalovirus, human herpesvirus type 8, human papillomavirus, BK virus, JC virus, smallpox, poliovirus, hepatitis B virus, human bocavirus, parvovirus B19, human astrovirus, Norwalk virus, coxsackievirus, hepatitis A virus, rhinovirus, severe acute respiratory syndrome virus, hepatitis C virus, yellow fever virus, West Nile virus, rubella virus, and hepatitis E. The use according to claim 17, characterized in that it is caused by one or more of the following viruses, bacteria, or protozoa: viruses, human immunodeficiency virus (HIV), influenza A or B virus, guanalitovirus, Junin virus, lassa virus, Machupo virus, Sabia virus, Crimean-Congo hemorrhagic fever virus, Ebola virus, Marburg virus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, human metapneumovirus, Hendra virus, Nipah virus, rabies virus, hepatitis D, rotavirus, orbivirus, Escherichia coli virus, human enterovirus, Japanese encephalitis virus, vesicular herpesvirus, eastern equine encephalitis virus, hantavirus, Middle East respiratory coronavirus, chikungunya virus, or vannavirus.

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