Plasmid vectors used for in vitro mRNA transcription, construction methods, and fields of application technology

Abstract

The present invention provides a nucleic acid molecule comprising 1) a transcriptional nucleic acid fragment and / or a nucleic acid for introducing the transcriptional nucleic acid fragment; 2) a polyA nucleic acid fragment; and 3) a zyrase 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 loss / shortening, while simultaneously improving the stability of the nucleic acid molecule and reducing the probability of plasmid recombination during the amplification process, and is advantageous for the industrial production of mRNA products.

Description

Technology Field

[0001] The present invention belongs to the field of genetic engineering. Specifically, it relates to an in vitro mRNA transcription vector capable of improving plasmid stability, a method for constructing such a vector, and an application thereof. Background Technology

[0002] mRNA technology possesses numerous advantages: first, since mRNA is not integrated into the genome, there is no concern regarding insertion mutations; more importantly, unlike the relatively stable DNA, mRNA degrades after performing its function, thus avoiding other toxicities or side effects in the human body; furthermore, mRNA can be manufactured in a cell-free manner and produced on a large scale rapidly and economically through in vitro transcription technology; and due to its short research and development cycle, rapid, economical, and efficient production can be achieved. Additionally, a single mRNA can code for multiple antigens, proteins, or polypeptides, offers strong process applicability, and theoretically allows for the expression of any protein.

[0003] mRNA COVID-19 vaccines have demonstrated the applicability of mRNA technology platforms in the vaccine field. In addition to COVID-19 vaccines, mRNA technology can be widely utilized in fields such as infectious diseases, oncology, and protein replacement therapy.

[0004] Regarding mRNA technology, superior production processes begin with the design of safe, stable, and efficient plasmid vectors. Whether from the perspective of process production or therapeutic or regulatory applications, molecular weight must be minimized by removing most non-essential sequences from plasmid DNA to facilitate genetic manipulation; this is because if the plasmid is too large, it imposes a massive metabolic burden on host bacteria and reduces cellular resources used for plasmid replication, thereby decreasing plasmid DNA yield. Furthermore, in industrial production, plasmids must maintain high stability during continuous passage to ensure the widespread application and stability of mRNA technology.

[0005] One significant challenge in the production process of mRNA plasmid technology is that the PolyA sequence of the plasmid shortens as bacteria continue to amplify. This causes problems with the cloning and amplification of plasmids with PolyA tails, prevents the achievement of PolyA sequence uniformity in the produced plasmids, and fails to meet GMP-grade production process and pharmaceutical regulatory requirements. Furthermore, since the PolyA sequence is a critical factor for mRNA stability and translational expression efficiency, researchers are continuously striving to resolve the issue of PolyA sequence shortening during plasmid fermentation production.

[0006] To eliminate the instability of the PolyA tail in circular plasmids, Kyle Jacoby et al. developed a pEVL linear plasmid system derived from the PJazz plasmid. Compared to traditional circular plasmids, the pEVL plasmid has a significant advantage in maintaining the length of the PolyA tail even after continuous passage (Kyle Jacoby, 2016, Mol Ther Nucleic Acids). However, the pEVL plasmid is not suitable for large-scale production due to its very low copy number. In 2019, Zeljka Trepotec et al. discovered that segmenting the PolyA sequence reduces recombination during the plasmid DNA amplification process and maintains the PolyA tail length. Representative examples of using segmented polyA tails include the technical solution disclosed in BioNTech patent US10717982B2 and the technology disclosed in Ethris patent WO2020074642A1. Kristie Bloom et al. developed a plasmid capable of increasing PolyA length through enzymatic cleavage linkages and used it as a template for in vitro mRNA co-transcription, thereby improving the situation of PolyA shortening during plasmid passages. Additionally, other companies are using short PolyA sequences to ensure plasmid stability.

[0007] However, most of the aforementioned methods are improvements to polyA itself, and even if plasmid systems are developed to address the unstable state of polyA, they have proven unsuitable for large-scale production.

[0008] The present invention conducts additional research on plasmids suitable for RNA technology, breaks through most improved methods targeting only polyA, and solves the challenge of polyA loss from the perspective of the entire plasmid without affecting plasmid replication and yield, thereby improving the integrity and stability of the plasmid and making it suitable for large-scale production.

[0009] The present invention provides a nucleic acid molecule for improving the stability of a PolyA tail during plasmid passage in bacterial fermentation and its application.

[0010] The term "vector" of the present invention has the general meaning known to those skilled in the art herein and comprises any intermediate vector of nucleic acid, said nucleic acid may be introduced into a prokaryotic and / or eukaryotic host cell, for example, or incorporated into a single genome where appropriate, and the vector may preferably be replicated and / or expressed within the cell.

[0011] The term "pharmaceuticalally acceptable carrier" in the present invention refers to an inactive substance combined with a pharmaceutically active substance to meet or promote requirements regarding the dosage, absorption, solubility, or pharmacokinetic aspects of the active substance.

[0012] The term "plasmid" in the present invention typically relates to a composition of extrachromosomal genetic material, typically a cyclic DNA duplex, which can replicate independently of chromosomal DNA.

[0013] The term “host cell” in the present invention 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). Particularly preferably, it is a mammalian cell, such as a cell derived from a human, mouse, hamster, pig, goat, or primate. Cells may be derived from various tissue types and include primary cells and cell lines.

[0014] The terms "peptide" and "protein" of the present invention include substances containing not only amino acid components but also non-amino acid components such as sugars and phosphate structures, and also substances containing bonds such as ester bonds, thioether bonds, or disulfide bonds.

[0015] The term "nucleotide fragment encoding a peptide or protein" of the present invention refers to a substance that, when present in a suitable environment, preferably within a cell, can induce the assembly of amino acids in the corresponding process to produce the translation of a peptide or protein.

[0016] The term “immune response” of the present invention relates to the response of the immune system to an immunogenic organism, such as, for example, bacteria or viruses, cells or substances. The term “immune response” includes innate immune responses and adaptive immune responses. Preferably, the immune response involves the activation of immune cells, the induction of cytokine biosynthesis, and / or antibody production. Preferably, the immune response comprises the steps of activating antigen-presenting cells, such as dendritic cells and / or macrophages, presenting an antigen or a fragment thereof by said antigen-presenting cells, and activating cytotoxic T cells as a result of said presentation.

[0017] In a first aspect of the present invention, a nucleic acid molecule is provided comprising: 1) a transcribable nucleic acid fragment or a nucleic acid for introducing a transcribable nucleic acid fragment; 2) a nucleic acid fragment comprising polydeoxyadenylic acid polyA; and 3) a nucleic acid fragment comprising a gyrase binding site (SGS).

[0018] The above-mentioned transcriptional nucleic acid fragment includes a nucleic acid fragment encoding a peptide or protein.

[0019] The nucleic acid for introducing the above-mentioned transcriptional nucleic acid fragment includes a polyclonal site or a homologous recombination site.

[0020] The above polyA contains at least 50 consecutive deoxyadenilic acid groups.

[0021] The nucleic acid fragment containing the polyA comprises at least two elements having a length of at least 20 consecutive deoxyadenylic acid sequences, and at least one non-T deoxynucleotide is spaced between the two elements.

[0022] In some specific embodiments, the nucleic acid fragment containing the zyrase binding site is derived from a virus or bacterium; preferably, the nucleic acid fragment containing the zyrase binding site is derived from a virus; more preferably, the nucleic acid fragment containing the zyrase binding site is derived from a phage.

[0023] In some specific embodiments, the nucleic acid fragment containing the zyrase binding site is derived from a plasmid; preferably, the nucleic acid fragment containing the zyrase binding site is derived from a ColE1 type plasmid.

[0024] In some specific embodiments, the SGS nucleic acid fragment is constructed upstream of the T7 promoter in the plasmid vector; and in some specific embodiments, the SGS nucleic acid fragment is constructed downstream of the PolyA in the plasmid vector. Those skilled in the art may select a suitable location for inserting the SGS as needed, in accordance with the contents disclosed in this application.

[0025] In another aspect, the present invention provides a DNA expression vector or a recombinant plasmid, wherein the sequence of the DNA expression vector or recombinant plasmid 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.

[0026] The present invention also provides a method for preparing an expression vector or a recombinant plasmid, wherein the above-described expression vector or recombinant plasmid is transformed into an E. coli competent cell, an appropriate amount is taken and coated onto a resistant LB medium plate and cultured, monoclonal colonies are selected and sequenced to obtain a positive bacterial suspension with accurate sequencing results, and then cultured on a large scale.

[0027] The present invention provides a method for amplifying a nucleic acid molecule comprising the steps of replicating the above-described nucleic acid molecule in a host cell and proliferating the host cell.

[0028] In some specific embodiments, the host cell is a eukaryotic cell or a prokaryotic cell; preferably, the host cell is E. coli.

[0029] The present invention provides a method for obtaining RNA comprising the steps of amplifying a nucleic acid molecule according to the method described above and transcribing the nucleic acid molecule into RNA in vitro using the nucleic acid molecule as a template.

[0030] The present invention provides a method for obtaining a peptide or protein, comprising the step of obtaining mRNA encoding a peptide or protein according to the RNA acquisition method described above, and translating the mRNA into an amino acid fragment of the peptide or protein.

[0031] The present invention provides a system for generating translatable mRNA in a eukaryote comprising 1) a vector or plasmid containing the nucleic acid molecule described above; and 2) a host cell.

[0032] In another aspect, the present invention provides an application of a nucleic acid nucleotide fragment encoding a gyrase binding site in the construction of a plasmid or vector, said plasmid or vector used in in vitro RNA transcription.

[0033] In another aspect, the present invention provides a composition comprising RNA, peptide, or protein obtained according to the method described above.

[0034] In some specific embodiments, the composition comprises one or more of pharmaceutically acceptable excipients, carriers, buffers, protective agents, stabilizers, surfactants, osmotic pressure regulators, adjuvants, preservatives, and inactivators.

[0035] The buffer of 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 ethylenediaminetetraacetic acid (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 glycerol; the osmotic pressure regulator may include or omit sodium chloride; and the excipient component comprises one or more of mannitol, sucrose, sodium chloride, magnesium chloride, HEPES, polysorbate 80, and glycerol.

[0036] In some specific embodiments, the composition of the present invention may be prepared as a mucosal immune agent, a humoral immune agent, a cellular immune agent, or a skin immune agent.

[0037] In some specific embodiments, the composition of the present invention may be prepared as a liquid formulation, a solid formulation, a semi-solid formulation, a gaseous formulation, or an inhalation formulation.

[0038] 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, respiratory administration, and transdermal administration, wherein respiratory administration refers to oral inhalation, nasal inhalation, or inhalation after being atomized through a nebulizing device.

[0039] The nucleic acid molecules, plasmids, or vectors containing nucleic acid molecules of the present invention can be used to transcribe and express recombinant proteins in cells. More specifically, when producing recombinant proteins, the expression vector of the present invention is 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.

[0040] The nucleic acid molecule, plasmid, or vector containing the nucleic acid molecule of the present invention can be used, for example, for transient gene expression, and possible applications include RNA-based vaccines that are transfected into cells in vitro or administered to directly and transiently express functional proteins in the body.

[0041] The present invention provides uses for the composition described above, including medicinal use, transgenic delivery to host cells, induction of a primary immune response to treat or prevent at least one disease in animals, enhancement of an immune response in animals, and induction of an immune response to break resistance to autoantigens in animals. The immune response includes vaccines for cancer prevention, therapeutic tumor vaccines, prevention of viral infection, prevention of bacterial infection, and prevention of fungal infection.

[0042] The present invention provides the use of any of the above-described nucleic acid molecules, expression vectors, or compositions in the manufacture of drugs or vaccines for treating or preventing diseases.

[0043] In some specific embodiments, the disease is: novel coronavirus, dengue virus, malaria parasite, herpes zoster virus, feline parvovirus, feline calicivirus, feline herpesvirus, feline leukemia virus, feline coronavirus, porcine genital 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 herpes simplex virus type 8, human papillomavirus, BK virus, JC virus, smallpox, poliovirus, hepatitis B virus, human bocavirus, parvovirus B19, human astrovirus, Norwalk virus, Coxsackie virus, hepatitis A virus, rhinovirus, severe acute respiratory syndrome virus, hepatitis C virus, yellow fever virus, dengue virus, western Nile virus, rubella virus, hepatitis E virus, human immunodeficiency virus (HIV), influenza virus type A or B, guanarito virus, zunin It is caused by one or more of viruses, bacteria, or protozoa such as the 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, E. coli virus, human enterovirus, Japanese encephalitis virus, herpes vesicular virus, Eastern equine encephalitis virus, hantavirus, Middle East respiratory coronavirus, chikungunya virus, or banna virus.

[0044] The beneficial effects of the present invention include the following:

[0045] In the first aspect, the stability of PolyA is improved through SGS sequencing, and the problem of PolyA loss / shortening is resolved.

[0046] In the second aspect, the stability of nucleic acid molecules is enhanced through SGS sequences.

[0047] In a third aspect, the SGS sequence reduces the probability of plasmid recombination during the amplification process and further enhances the safety of plasmid applications.

[0048] In a fourth aspect, SGS sequencing allows for the acquisition of RNA molecules, peptides, or proteins with higher purity that meet GMP-grade production processes and pharmaceutical regulatory requirements, making them suitable for large-scale production. Brief explanation of the drawing

[0049] Fig. 1, Effect of SGS sequences of different origins on the stability of a polyA sequence under non-tolerance conditions. Fig. 2, Effect of SGS sequences of different origins on the stability of polyA sequences under Kana resistance conditions. Fig. 3, Effect of SGS Mu398 sequence on the stability of PolyA of different length types under non-tolerance conditions. Fig. 4, Effect of SGS Mu398 sequence on the stability of PolyA of different length types under Kana resistance conditions. Fig. 5, Applicability of the SGS Mu398 sequence to different target antigen sequences under resistance-free conditions. Fig. 6, Applicability of the Mu398 SGS sequence to different target antigen sequences under Kana resistance conditions. Fig. 7, 10th generation transcription mRNA integrity detection results of F001 recombinant plasmid. Fig. 8, 10th generation transcription mRNA integrity detection results of F002 recombinant plasmid. Fig. 9, 10th generation transcription mRNA integrity detection results of F003 recombinant plasmid. Fig. 10, 10th generation transcription mRNA integrity detection results of F004 recombinant plasmid. Fig. 11, 10th generation transcription mRNA integrity detection results of F005 recombinant plasmid. Specific details for implementing the invention

[0050] Example 1: Effect of SGS sequences of different origins on the stability of plasmid polyA

[0051] To investigate the effect of SGS sequences on plasmid stability, SGS sequences of different origins were inserted downstream of the vector polyA or upstream of the T7 promoter, where polyA is a continuous sequence of 100A. Simultaneously, target gene sequences were inserted into the plasmid vectors, and the effect of SGS sequences on plasmid stability was observed. The design method is shown in Table 1. According to the results, the insertion of SGS sequences was found to significantly improve the passage stability of polyA under non-resistant and Kana resistant conditions (Figs. 1 and 2).

[0052] The transcription vector of 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, wherein each of the sequences was synthesized into a pUC57-kana vector by Nanjing GenScript Biotechnology Co., Ltd. and then sequentially linked to a universal transcription vector via a homologous recombination method. The constructed plasmid was introduced into Trans1-Blue E. coli competent cells, an appropriate amount was taken and coated onto a Kana-resistant LB medium plate, and cultured at 37°C for a certain period of time. Monoclonal colonies were selected and sequenced, and positive bacterial suspensions with accurate sequencing results were stored. Subsequently, the bacterial suspension containing the positive plasmid was shaken at 37°C, the plasmid was extracted in large quantities to obtain an in vitro transcription template, and mRNA was produced through a co-transcription capping method.

[0053] Table 1. Design method for the effect of SGS sequence on plasmid stability

[0054]

[0055] Example 2: Effect of SGS Sequence on the Stability of PolyA of Different Length Types

[0056] To further verify whether the SGS sequence could improve the stability of PolyA sequences of different lengths, the Mu398 SGS sequence and different types of polyA sequences were inserted into the aforementioned plasmid vector, and the passage stability of the polyA sequences was observed. The specific design is shown in Table 2.

[0057] Table 2. Design method of the effect of SGS sequences on the stability of PolyA of different length types.

[0058]

[0059] According to the results, the addition of the Mu398 SGS sequence was found to increase the passage stability of various types of polyA sequences and maintain their integrity within 10 generations (Figs. 3 and 4).

[0060] Example 3: Determination of a universal high-stability plasmid vector

[0061] To further verify the universality of the aforementioned plasmid vectors, the present invention constructed expression vectors F001, F002, F003, F004, and F005 by inserting target gene sequences derived from five different pathogens—novel coronavirus (COVID-19), dengue virus (DENV), malaria, herpes zoster virus (VZV), and feline parvovirus (FPV)—into the aforementioned vectors containing SGS sequences, respectively. 100-A consecutive passage stability was observed during the plasmid passage process. According to the results, the SGS sequences can be universally used for different target gene sequences and can guarantee passage stability of each recombinant plasmid polyA within 10 generations (as shown in FIGS. 5 and 6).

[0062] To further observe the transcriptional effect of the transcription vector, a large amount of the 10th generation plasmid of the aforementioned constructed recombinant vector plasmid was extracted and transcription was performed in vitro, and the results indicated that each plasmid was capable of transcribing high-quality mRNA (as illustrated in Figures 7 to 11, and the data are referenced in Table 3).

[0063] Table 3. Detection result data of Figures 7–11

[0064]

[0065] In summary, the present invention provides a universal, highly stable plasmid vector that has general applicability and can solve the problem of plasmid instability caused by polyA sequences in the mRNA field.

Claims

Claim 1 A nucleic acid molecule, wherein the nucleic acid molecule comprises 1) a transcriptional nucleic acid fragment and / or a nucleic acid for introducing the transcriptional nucleic acid fragment; 2) a nucleic acid fragment comprising polydeoxyadenylic acid polyA; and 3) a nucleic acid fragment comprising a gyrase binding site. Claim 2 A nucleic acid molecule according to claim 1, wherein the transcriptional nucleic acid fragment codes for a peptide or a protein. Claim 3 A nucleic acid molecule according to claim 1 or 2, wherein the nucleic acid fragment for introducing the transcriptional nucleic acid fragment comprises a polyclonal site or a homologous recombination site. Claim 4 A nucleic acid molecule according to any one of claims 1 to 3, wherein the polyA comprises at least 50 consecutive deoxyadenilic acid groups. Claim 5 A nucleic acid molecule according to any one of claims 1 to 4, wherein the nucleic acid fragment containing polyA comprises at least two elements having a length of at least 20 consecutive deoxyadenylic acid sequences, and at least one non-T deoxynucleotide is spaced apart between the two elements. Claim 6 A nucleic acid molecule according to any one of claims 1 to 5, wherein the nucleic acid fragment containing the zyrase binding site is derived from a virus or bacterium; preferably, the nucleic acid fragment containing the zyrase binding site is derived from a virus; and more preferably, the nucleic acid fragment containing the zyrase binding site is derived from a phage. Claim 7 A nucleic acid molecule according to any one of claims 1 to 5, wherein the nucleic acid fragment containing the zyrase binding site is derived from a plasmid; preferably, the nucleic acid fragment containing the zyrase binding site is derived from a ColE1 type plasmid. Claim 8 A plasmid vector, wherein the plasmid vector comprises: 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. Claim 9 A method for preparing a plasmid vector, characterized by transforming the plasmid vector according to claim 8 into E. coli competent cells, taking an appropriate amount and coating it onto a resistant LB medium plate for culture, selecting monoclonal colonies, sequencing, obtaining a positive bacterial suspension with accurate sequencing results, and then performing large-scale culture. Claim 10 A method for amplifying a nucleic acid molecule, comprising the step of multiplying a nucleic acid molecule according to claims 1 to 7 in a host cell and multiplying the host cell, wherein the host cell comprises bacteria or fungi; and wherein the host cell is preferably Escherichia coli. Claim 11 A method for obtaining RNA, characterized by comprising the step of amplifying a nucleic acid molecule by the method according to claim 10 and transcribing the nucleic acid molecule into RNA in vitro using the nucleic acid molecule as a template. Claim 12 A method for obtaining a peptide or protein, characterized by comprising the step of obtaining mRNA encoding the peptide or protein by the method according to claim 11 and translating the mRNA into an amino acid fragment of the peptide or protein. Claim 13 A system for generating translatable mRNA in eukaryotes, characterized by comprising: 1) a plasmid vector containing a nucleic acid molecule according to claims 1 to 7; and 2) a host cell. Claim 14 Application of a nucleic acid fragment encoding a zyrase binding site in the construction of a plasmid vector, characterized in that the plasmid vector is used for in vitro RNA transcription. Claim 15 A composition characterized by comprising RNA, peptide, or protein obtained by the method according to claim 11 or 12. Claim 16 A composition according to claim 15, characterized in that the composition comprises one or more of pharmaceutically acceptable excipients, carriers, buffers, protective agents, stabilizers, surfactants, osmotic pressure regulators, adjuvants, preservatives, and inactivators. Claim 17 The 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. Claim 18 In paragraph 17, the above diseases are: novel coronavirus, dengue virus, malaria parasite, herpes zoster virus, feline parvovirus, feline calicivirus, feline herpesvirus, feline leukemia virus, feline coronavirus, porcine genital 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 herpes virus type 8, human papillomavirus, BK virus, JC virus, smallpox, poliovirus, hepatitis B virus, human bocavirus, parvovirus B19, human astrovirus, Norwalk virus, Coxsackie virus, hepatitis A virus, rhinovirus, severe acute respiratory syndrome virus, hepatitis C virus, yellow fever virus, western Nile virus, rubella virus, hepatitis E virus, human immunodeficiency virus (HIV), influenza virus type A or B, guanarito virus, zunin virus, Lassa Use characterized by being caused by one or more of viruses, bacteria, or protozoa, such as viruses, Machupovirus, 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, E. coli virus, human enterovirus, Japanese encephalitis virus, herpes vesicular virus, Eastern equine encephalitis virus, hantavirus, Middle East respiratory coronavirus, chikungunya virus, or banna virus.

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