WO2023227124A1 - Squelette pour la construction d'un gabarit de transcription in vitro d'arnm - Google Patents

Squelette pour la construction d'un gabarit de transcription in vitro d'arnm Download PDF

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WO2023227124A1
WO2023227124A1 PCT/CN2023/096643 CN2023096643W WO2023227124A1 WO 2023227124 A1 WO2023227124 A1 WO 2023227124A1 CN 2023096643 W CN2023096643 W CN 2023096643W WO 2023227124 A1 WO2023227124 A1 WO 2023227124A1
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mrna
utr
universal
sequence
vaccine
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张丹
邹军
王胜东
张建三
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深圳市三源生生物科技有限公司
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Publication of WO2023227124A1 publication Critical patent/WO2023227124A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the invention belongs to the field of biomedicine, and specifically relates to a skeleton for constructing an in vitro transcription template of mRNA and its application in the optimized design of mRNA.
  • mRNA vaccine is a new vaccine technology that combines molecular biology and immunology to transduce mRNA into somatic cells to express foreign antigens and activate the host's adaptive immunity.
  • scientists injected mRNA into mouse somatic cells, causing the mouse somatic cells to express fluorescent proteins, ⁇ -galactosidase and chloramphenicol acetyltransferase.
  • Jirikowski et al. injected mRNA encoding oxytocin and vasopressin into diabetic diabetes insipidus mice. As a result, the mice did not develop diabetes insipidus within hours after the injection. Since then, the development of mRNA vaccines has fallen into a trough.
  • mRNA vaccines are mainly to improve the stability and translation activity of mRNA and reduce the self-antigenicity of mRNA.
  • the new coronavirus vaccine which can be launched due to the urgency of research and development, the research and development of other mRNA vaccines have been hindered to some extent. The reason may be that when designing the mRNA vaccine, too much attention is paid to its ability to express antigens, while the risk of toxicity and the risk of apoptosis of surrounding host cells caused by the antigenicity and high expression ability of the mRNA vaccine itself are ignored.
  • the purpose of the present invention is to increase the versatility of the mRNA vaccine by using the UTR of immune-related proteins, while reducing potential side effects.
  • the universal framework can further accelerate the research and development of various infectious diseases.
  • a universal framework for constructing an mRNA transcript wherein the transcript includes an ORF to be expressed and a pair of genes located on both sides of the ORF. 5'-UTR region and 3'-UTR region, wherein one or two of the 5'-UTR region and 3'-UTR region are universal UTRs.
  • the universal UTR is selected from the following group: the UTR conserved sequence of a mutated or optimized antibody gene, the UTR conserved sequence of a mutated or optimized interferon gene, or a combination thereof.
  • the mRNA transcript includes the mRNA transcript in the mRNA vaccine.
  • the universal framework also includes additional UTR regions (ie, other UTR regions besides the universal UTR).
  • nucleotide sequence of the UTR conserved sequence of the antibody gene is shown in SEQ ID NO: 1 or 3.
  • the UTR conserved sequence of the antibody gene includes a 5' antibody conserved sequence and a 3' antibody conserved sequence.
  • nucleotide sequence of the 5' antibody conserved sequence is shown in SEQ ID NO: 1.
  • nucleotide sequence of the 3' antibody conserved sequence is shown in SEQ ID NO: 3.
  • sequence shown in SEQ ID NO:3 is conserved in different IGL genes.
  • nucleotide sequence of the UTR conserved sequence of the interferon gene is shown in SEQ ID NO: 7 or 9.
  • the UTR conserved sequence of the interferon gene includes a 5' conserved interferon sequence and a 3' conserved interferon sequence.
  • nucleotide sequence of the 5' interferon conserved sequence is shown in SEQ ID NO: 7.
  • nucleotide sequence of the 3' interferon conserved sequence is shown in SEQ ID NO: 9.
  • sequence shown in SEQ ID NO: 7 or 9 is conserved among different IFNA subtypes.
  • the universal UTR includes a universal 5'-UTR and a universal 3'-UTR.
  • the universal UTR includes: a 5'-UTR containing a Kozak sequence.
  • the AT-rich sequence in the 3'-UTR of the antibody gene and interferon gene is completely or partially deleted.
  • basically no AT-rich sequences means that in one UTR, the number of AT-rich sequences is ⁇ 2, and more preferably ⁇ 1.
  • the AT-rich sequence refers to a nucleic acid sequence rich in adenine and thymine bases.
  • the GC content in the universal UTR is 44%-64%.
  • nucleotide sequence of the universal 5'-UTR is shown in SEQ ID NO: 2 or 8.
  • nucleotide sequence of the universal 3'-UTR is shown in SEQ ID NO: 4, 5, 6, 10, 11 or 12.
  • any one of the above nucleotide sequences also includes optionally adding, deleting, modifying and/or replacing at least one (such as 1-3) nucleotides and retaining Derived sequences for optimizing mRNA capacity.
  • a general-purpose skeleton is provided, and the general-purpose skeleton has the structure of formula I: Z1-Z2-Z3-Z4-Z5-Z6-Z7 (I)
  • Z1 and Z7 are no or enzyme cutting sites
  • Z2 is none or promoter element
  • Z3 is a 5'-UTR component
  • Z4 is a replaceable ORF region
  • Z5 is a 3'-UTR component
  • Z6 is a polyA tail component.
  • Z1 and Z7 are blunt end enzyme cleavage sites or sticky end enzyme cleavage sites.
  • the universal framework includes an enzyme cleavage site, a promoter, a 5'-UTR, an ORF, a 3'-UTR and polyA.
  • the blunt-end enzyme is selected from the following group: AelI, AatI, AluI, BavAI, BavBI, EcoRV, MlsI, or a combination thereof.
  • the blunt-end enzyme is AleI.
  • the sticky end enzyme is BspQI.
  • the Z2 is selected from the following group: T7 promoter, T3 promoter, SP6 promoter, or a combination thereof.
  • the Z2 is a T7 promoter.
  • one or two of Z3 and Z5 are universal UTRs.
  • the Z3 is selected from the following group: a mutated or optimized 5' antibody conserved sequence, a mutated or optimized 5' interferon conserved sequence, or a combination thereof.
  • the Z3 is selected from the following group: SEQ ID NO: 2, SEQ ID NO: 8, the 5'-UTR of human ⁇ -globin, or a combination thereof.
  • the Z4 can be replaced by a gene selected from the following group: hirudin, rabies virus G protein, dengue virus E protein, Mycobacterium tuberculosis ESAT-6 protein, Ag85A protein, or a combination thereof.
  • Z4 can be replaced by an antigen gene selected from the following group of pathogens: hirudin, cytomegalovirus (CMV), Zika virus (Zika), influenza virus (Influenza), respiratory syncytial virus ( RSV), Chikungunya, Rabies, HIV, Ebola virus, streptococci, malaria, Louping ill virus ), Toxoplasma gondii, dengue fever, plague, yellow fever, tuberculosis, herpes simplex virus, band virus, mycoplasma, chlamydia, foot-and-mouth disease virus, Pseudomonas aeruginosa, or combinations thereof
  • pathogens selected from the following group of pathogens: hirudin, cytomegalovirus (CMV), Zika virus (Zika), influenza virus (Influenza), respiratory syncytial virus ( RSV), Chikungunya, Rabies, HIV, Ebola virus, streptococci, malaria, Louping ill virus
  • Z4 is replaced with hirudin gene or rabies virus G protein gene.
  • the Z4 is codon optimized.
  • the Z4 replacement method includes homologous recombination and enzyme digestion.
  • the stop codon of Z4 is multiple stop codons.
  • the stop codon of Z4 is two stop codons.
  • the Z5 is selected from the following group: a mutated or optimized 3' antibody conserved sequence, a mutated or optimized 3' interferon conserved sequence, or a combination thereof.
  • the Z5 is selected from the following group: SEQ ID NO: 4-6, SEQ ID NO: 10-12, or a combination thereof.
  • the length of Z6 is preferably 100nt-150nt, more preferably 110nt-130nt, more preferably 120nt.
  • a universal UTR element in a third aspect of the present invention, includes:
  • a universal 5'-UTR wherein the sequence of the universal 5'-UTR is selected from the nucleotide sequence shown in SEQ ID NO: 2 or 8 or a derivative sequence thereof;
  • the derivative sequence refers to optionally adding, deleting, modifying and/or substituting at least one (such as 1-3) nuclei to any of the above nucleotide sequences. nucleotides and preserves the derived sequence used to optimize the ability of the mRNA.
  • (a) and (b) can be derived from the same transcript.
  • (a) and (b) can be derived from different transcripts.
  • a carrier which carrier contains the universal scaffold as described in the second aspect of the present invention.
  • the vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof.
  • the vector is a plasmid.
  • the vector is pUC57-Amp vector or pUC-Kana vector.
  • a host cell contains the vector as described in the fourth aspect of the present invention, or the universal framework as described in the second aspect of the present invention is integrated into its genome.
  • the host cells include prokaryotic cells or eukaryotic cells.
  • the host cell is selected from the following group: Escherichia coli, yeast cells, and mammalian cells.
  • an engineered cell contains: the vector as described in the fourth aspect of the present invention, or its genome is integrated with a universal vector as described in the second aspect of the present invention. sexual skeleton and contains the target gene fragment.
  • the engineered cells are stable3 Escherichia coli competent cells.
  • the target gene fragment contains homologous arm sequences.
  • the vector or universal framework contains homologous arm sequences.
  • homologous recombination occurs between the target gene fragment and the vector or universal scaffold.
  • the target gene fragment is connected to the vector or universal scaffold and circularized.
  • the target gene is selected from the following group: hirudin, cytomegalovirus (CMV), Zika virus (Zika), influenza virus (Influenza), respiratory syncytial virus (RSV), chikungunya Chikungunya, Rabies, HIV, Ebola virus, streptococci, malaria, Louping ill virus, Toxoplasma gondii Toxoplasma gondii), dengue fever, plague, yellow fever, tuberculosis vaccine disease, herpes simplex virus, band viruses, mycoplasma, chlamydia, foot-and-mouth disease virus, Pseudomonas aeruginosa, or combinations thereof.
  • a method for producing optimized mRNA for preparing a vaccine comprising the steps:
  • step (d) Optionally, purify and/or modify the optimized mRNA obtained in step (c).
  • a method for preparing an mRNA vaccine includes the steps:
  • kit in a ninth aspect of the present invention, includes:
  • the description also describes a method of using the first plasmid as a template to amplify the first fragment with the homology arm sequence.
  • the description also describes a method of using the second plasmid as a template to amplify a second fragment with a homology arm sequence.
  • the description also describes a method of circularizing the first fragment and the second fragment through homologous recombination and transferring them into a suitable host cell.
  • the description also describes a method for obtaining optimized mRNA from the host cell.
  • the present invention also provides a kit, which includes:
  • an mRNA vaccine composition which vaccine composition contains:
  • the immunogen is selected from the following group: hirudin, cytomegalovirus (CMV), Zika virus (Zika), influenza virus (Influenza), respiratory syncytial virus (RSV), chikungunya Chikungunya, Rabies, HIV, Ebola virus, streptococci, malaria, Louping ill virus, Toxoplasma gondii Toxoplasma gondii), dengue fever, plague, yellow fever, tuberculosis, herpes simplex virus, band viruses, mycoplasma, chlamydia, foot-and-mouth disease virus, Pseudomonas aeruginosa, or combinations thereof.
  • the mRNA itself in the vaccine composition can also serve as an adjuvant.
  • the dosage form of the vaccine composition is selected from the following group: injection and lyophilized agent.
  • the vaccine composition includes 0.01 to 99.99% of the universal framework as described in the second aspect of the present invention and 0.01 to 99.99% of a pharmaceutically acceptable carrier, and the percentage is The mass percentage of the vaccine composition.
  • an mRNA vaccine composition as described in the tenth aspect of the present invention, or the use of engineered cells as described in the sixth aspect of the present invention, which is used to prepare a drug said medicament is used to prevent pathogens selected from the following group: hirudin, cytomegalovirus (CMV), Zika virus (Zika), influenza virus (Influenza), respiratory syncytial virus (RSV), chikungunya disease (Chikungunya), Rabies, HIV, Ebola virus, streptococci, malaria, jumping disease Louping ill virus, Toxoplasma gondii, dengue fever, plague, yellow fever, tuberculosis, herpes simplex virus, band virus, mycoplasma, chlamydia, foot-and-mouth disease virus, Pseudomonas aeruginosa, or combinations thereof.
  • pathogens selected from the following group: hirudin, cytomegalovirus (CMV), Zika virus (Zika), influenza virus
  • Figure 1 is a schematic structural diagram of a carrier in an embodiment of the present invention.
  • Figure 2 shows the sequence IGL-5-O before 5'-UTR optimization, the sequence IGL-5'UTR-F after optimization and their corresponding GC contents.
  • Figure 3 shows the sequence IFN-5-O before 5'-UTR optimization, the sequence IFN-5'UTR-F after optimization and their corresponding GC contents.
  • Figure 4 shows the sequence IGL-3-O before 3'-UTR optimization, the sequence IGL-3'UTR-F after optimization and their corresponding GC contents.
  • Figure 5 shows the sequence INF-3-O before 3'-UTR optimization, the sequence IFN-3'UTR-F after optimization and their corresponding GC contents.
  • Figure 6 is a schematic diagram of inserting two stop codons after the ORF sequence.
  • Figure 7 is a schematic diagram of the hirudin gene synthesis fragment.
  • Figure 8 is a schematic diagram of the backbone amplified fragment.
  • Figure 9 shows the electrophoretic identification results of the insert fragment and the plasmid backbone fragment.
  • Figure 10 shows the results of Hirudin plasmid gel electrophoresis.
  • Figure 11 shows the results of AleI digestion of Hirudin plasmid.
  • Figure 12 shows the changes in plasmid yield during fermentation.
  • Figure 13 shows the expression results of rabies virus G protein after mRNA transfection of cells.
  • Figure 14 shows the agarose gel electrophoresis and enzyme digestion pattern verification results of the universal in vitro transcription template plasmid containing the universal backbone.
  • Figure 15 shows the sequence alignment results of a universal in vitro transcription template plasmid containing a universal scaffold.
  • the inventor developed for the first time a universal UTR and a universal skeleton for constructing mRNA transcripts, thereby obtaining optimized mRNA with improved stability and translation activity. This enables applications in the preparation and/or optimization of mRNA vaccines. Universal use of the present invention The sexual skeleton could speed up the development of mRNA vaccines for a variety of infectious diseases. On this basis, the present invention was completed.
  • general-purpose skeleton of the present invention As used herein, "general-purpose skeleton of the present invention”, “general-purpose skeleton”, “skeleton of the present invention”, “skeleton”, “general-purpose component”, “a series of universal components”, “general-purpose component” “Universal element series” are used interchangeably, and both refer to the framework for constructing mRNA transcripts by replacing the ORF region of the universal UTR element, which is composed of a series of universal element arrangements, typically with the aforementioned formula I The structure shown.
  • UTR conserved sequence of antibody gene and “antibody conserved sequence” can be used interchangeably, both refer to the conserved sequence of the UTR region in the antibody gene, preferably its nucleotide sequence is as shown in SEQ ID NO: 1 or 3 Show.
  • UTR conserved sequence of interferon gene and “interferon conserved sequence” can be used interchangeably, both referring to the conserved sequence of the UTR region in the interferon gene, preferably its nucleotide sequence such as SEQ ID NO:7 Or as shown in 9.
  • the terms "universal skeleton of the invention”, “skeleton of the invention”, “general component”, “general framework”, “a series of universal components”, “general component” “Series” are used interchangeably, and both refer to the universal skeleton composed of a series of universal component arrangements described in the second aspect of the present invention.
  • the general-purpose skeleton of the present invention has the structure of formula I: Z1-Z2-Z3-Z4-Z5-Z6-Z7 (I)
  • Z1 to Z7 are as described above.
  • FIG. 1 A schematic structural diagram of a representative carrier containing the universal scaffold of the present invention is shown in Figure 1.
  • the proteins or polypeptides suitable for expression using the universal framework of the present invention are not particularly limited, including antigenic proteins or antigenic peptides, or other useful proteins.
  • the ORF of the foreign protein can be placed in the universal framework of the present invention, thereby achieving efficient expression.
  • the ORF carries a stop codon.
  • one or more additional stop codons can also be introduced, as shown in Figure 6.
  • mRNA vaccines are divided into self-amplifying RNA (saRNA) and non-amplifying RNA (non-replicating mRNA).
  • Classic non-amplified RNA vaccines include cap, 5'-untranslated regions (5'-UTR), open reading frame (open reading frame, ORF), 3'-untranslated regions (3'- untranslated regions, 3'-UTR) and polyA tail (polyA tail).
  • the ORF region is responsible for encoding antigen expression, but the above five regions Together they determine the stability, expression activity and immunogenicity of mRNA.
  • saRNA The structure of saRNA is derived from the alphavirus genome.
  • the saRNA vaccine utilizes the self-replicating properties of the alphavirus genome to enable the DNA or RNA that enters the body cells to first self-amplify and then transcribe the antigen-encoding mRNA.
  • saRNA vaccines There are currently two types of saRNA vaccines: saRNA based on DNA plasmids and saRNA delivered by virus-like particles.
  • Beissert et al. also developed transgenic amplifying RNA (taRNA), which places the gene encoding the antigen in the alphavirus genome, increasing the safety of the vaccine.
  • taRNA transgenic amplifying RNA
  • non-amplified RNA is smaller, expresses antigens more specifically and does not cause non-specific immunity.
  • a major challenge for mRNA vaccines is reducing the immunogenicity of the exogenous mRNA itself.
  • exogenous mRNA can be recognized by retinoic acid-inducible gene I (RIG-I), activate the innate immune response, and then be degraded.
  • IIG-I retinoic acid-inducible gene I
  • ITT In vitro transcription
  • mRNA can activate immune cells and Toll-like receptor (Toll-like receptor)-mediated inflammatory responses.
  • the U-rich sequence of mRNA is a key factor in activating Toll-like receptors.
  • the immunogenicity of mRNA can be reduced through chemical modification of nucleotides, adding polyA tails, and optimizing the GC content of mRNA.
  • Chemically modified nucleotides include 5-methylcytidine (m5C), 5-methyluridine (m5U), N1-methyladenosine (m1A), N6 -Methyladenosine (N6-methyladenosine, m6A), 2-thiouridine (s2U), 5-oxymethyluridine (5-methoxyuridine, 5moU), pseudouridine (psi) and N1-methylpseudouridine (N1-methylpseudouridine, m1 ⁇ ).
  • adding polyA tail can also reduce the U content and thereby reduce the immunogenicity of mRNA.
  • CureVac and Acuita Therapeutics are trying to transport erythropoietin-encoding mRNA into pigs through lipid nanoparticles.
  • the mRNA has a high GC content and can cause erythropoietin-related reactions without immunogenicity.
  • excessive GC content will inhibit the translation activity of mRNA, which is something that needs to be paid attention to during vaccine development.
  • the purification method of mRNA is also very important in reducing the immunogenicity of mRNA itself.
  • Currently commonly used purification methods include high performance liquid chromatography (HPLC), anion exchange chromatography, affinity chromatography and particle size separation.
  • HPLC high performance liquid chromatography
  • anion exchange chromatography anion exchange chromatography
  • affinity chromatography particle size separation.
  • the purpose of purification is mainly to remove truncated transcripts.
  • Pardi et al. designed to purify m1 ⁇ -modified mRNA encoding anti-HIV-1 antibodies through HPLC through lipid nanoparticles (LNP) to help mice avoid HIV-1 infection.
  • LNP lipid nanoparticles
  • Sequence optimization of mRNA is one of the methods to help stabilize mRNA. Sequence optimization of the 5'-UTR and 3'-UTR of the mRNA can increase the half-life and translation activity of the mRNA. Cap structure using different analogs can To increase the stability of mRNA, the use of enzymes to add a Cap structure to the 5' end of mRNA can have better performance than different forms of Cap analogs. The stabilizing effect of the polyA tail of mRNA is also very important. Some studies have shown that removing the polyA of mRNA makes the mRNA extremely unstable. It also reduces the number of polyribosomes, elongation speed and number of translation rounds of the mRNA. Therefore polyA is crucial for the stable and efficient translation of mRNA.
  • nucleotide modifications and synonymous substitutions of codons can also affect the stability and translation activity of mRNA.
  • sequence optimization may affect the secondary structure and post-translational modification of mRNA.
  • increasing the GC content of mRNA can also increase mRNA stability.
  • 5'-UTR, 3'-UTR, 5'Cap, polyA tail, codon optimization and GC content are all modifiable sites that enhance mRNA stability.
  • LNP lipid nanoparticles
  • mRNA vaccines activate innate and adaptive immunity
  • mRNA vaccines can activate the innate and adaptive immune systems. Direct recognition of mRNA by pattern recognition receptors such as TLRs in somatic cells will lead to the degradation of mRNA and at the same time enhance the IFN pathway.
  • mRNA vaccines play a major role by stimulating adaptive immune responses.
  • the antigen translated from the mRNA is directly presented through MHC-I to activate CD4+ T cells (Helper T cells) or after the antigen is secreted, other cells phagocytose it and present the antigen through the MHC-II pathway to activate CD8+ T cells (cytotoxic T cells).
  • MHC-I CD4+ T cells
  • cytotoxic T cells CD8+ T cells
  • mRNA-containing particles are absorbed by local cells at the injection site, the mRNA is recognized by pattern recognition receptors and also begins to translate the antigen, causing local inflammation at the injection site and promoting the infiltration of immune cells, including neutrophils and monocytes. cells, myeloid dendritic cells (MDCs) and plasmacytoid dendritic cells (PDCs). Neutrophils can efficiently take up LNPs, but monocytes and MDCs translate mRNA more efficiently. The secretion of type I interferon (IFN) is stimulated.
  • IFN type I interferon
  • the mechanism of the mRNA vaccine is to inoculate the mRNA encoding the antigenic protein into the host, and then use the host's genetic material to express and synthesize the antigenic protein in cells in the body.
  • the antigenic protein induces and activates the body's immune system to produce an immune response, thereby achieving prevention. and the purpose of treating disease. Its unique advantages are: 1) Monitoring and quality control of all production processes can be easily realized; 2) The research and development and production cycle of mRNA is short, it is easy to achieve mass production, and the vaccine production capacity is high, which is very important for quickly responding to new emerging diseases worldwide.
  • Infectious diseases are crucial; 3) Due to its own characteristics, mRNA can be degraded quickly after immunization, and the safety risk is low; 4) The immune effect is good, and it can induce both humoral immunity and cellular immunity at the same time, and is effective against infections for which there is currently no better vaccine. There may be potential for more effective vaccines against diseases.
  • the challenges that mRNA vaccines need to face to work include: 1) extending its half-life and enhancing stability; 2) enhancing translation activity; 3) reducing the immunogenicity of mRNA to avoid rapid clearance.
  • the way to achieve these effects is to design special 5'-UTR, 3'-UTR, stop codon, polyA number, etc.
  • 5'-UTR and 3'-UTR there are three effective methods: 1) using the UTR of highly expressed human genes; 2) using the UTR of the antigen protein itself; 3) exponential enrichment ligand system evolution technology ( systematic evolution of ligands by exponential enrichment, SELEX).
  • the first two methods are relatively simple, while the third method is relatively complex and requires continuous trying and optimizing the sequence through in vitro experiments, so it takes a long time. However, the third method is still the best choice when time is sufficient.
  • the 5'-UTR of Pfizer/BioNTech's BNT162b2 vaccine which is currently approved by the FDA, uses the 5'-UTR of human alpha globin and optimizes the Kozak sequence and 5' end sequence, adjusting the second half of the 5'-UTR. hierarchical structure, while the 5'-UTR of Moderna's mRNA-1273 vaccine uses a sequence designed and optimized by its computer.
  • Moderna's mRNA-1273 vaccine uses the 110nt base in the 3'-UTR of human alpha globin (HBA1), while Pfizer's BNT162b2 vaccine uses a SELEX method based on natural genes to select The 3'-UTR of human 12S rRNA (mtRNR1) and AES/TLE5 genes were identified. On this basis, the Pfizer vaccine selected the 136nt sequence of AES 3'-UTR and made two C ⁇ changes, followed by the 139nt mtRNR1 sequence.
  • the truly effective UTR design method is to learn from natural genes and optimize based on experience (PMID: 34358150).
  • Pfizer's BNT162b2 vaccine uses the termination signal UGAUGA, while Moderna's mRNA-1273 vaccine uses UGAUAAUAG.
  • the number of polyA is an important factor affecting the stability of mRNA. 80nt-150n or 100nt-150nt is suitable, and 120nt is better. An appropriate amount of polyA can have higher protein production than an inappropriate amount of mRNA. expression ability and mRNA stability.
  • the framework for constructing mRNA transcripts of the present invention can be used to construct mRNA vaccines against infectious diseases.
  • the vaccine using the skeleton of the mRNA transcript of the present invention can be applied to a variety of different pathogens.
  • Representative pathogens include (but are not limited to): coronavirus (such as new coronavirus), cytomegalovirus (CMV), Zika virus ( Zika), Influenza, Respiratory Syncytial Virus (RSV), Chikungunya, Rabies, HIV, Ebola virus, Streptococci ), malaria, jumping disease virus, Toxoplasma gondii, etc.
  • infectious diseases lack effective inactivated vaccines and recombinant protein vaccines, so it may be hoped that mRNA vaccines can stimulate the body's preventive immunity against these pathogens and then develop effective vaccines.
  • diseases include dengue fever (existing vaccines can only protect one serotype), plague, yellow fever, tuberculosis vaccine, herpes simplex virus, band virus, mycoplasma, chlamydia, foot and mouth disease virus, etc., and can also be used in some anti-tumor drugs of treatment.
  • the inventor used the UTR of the antibody gene as the original UTR.
  • Antibodies are divided into 5 categories (classes), namely IgM, IgD, IgG, IgA and IgE. Their corresponding heavy chains are ⁇ chain, ⁇ chain, ⁇ chain, ⁇ chain and ⁇ chain respectively. Compared with these heavy chains There are two types of coordinated light chains, namely kappa ( ⁇ ) chain and lambda ( ⁇ ) chain. According to the differences in individual amino acids in the constant region of the ⁇ chain, the ⁇ chain can be divided into four subtypes: ⁇ l, ⁇ 2, ⁇ 3 and ⁇ 4. .
  • the recombination rates of the above-mentioned peptide chain genes are very high, suggesting that their 5'-UTR and 3'-UTR sequences may have strong compatibility with changed ORF regions and changed peptide chain expression. Therefore, the 5'-UTR and 3'-UTR of antibody genes may have versatility to support efficient translation of different ORFs.
  • preferred 5'-UTR and 3'-UTR are sequence-optimized UTRs.
  • Figure 2 shows the sequence IGL-5-O before 5'-UTR optimization and the sequence IGL-5'UTR-F after optimization. The GC content increased from 54% to 64% after optimization.
  • Figure 4 shows the sequence IGL-3-O before 3'-UTR optimization and the sequence IGL-3'UTR-F after optimization. The GC content increased from 54% to 56% after optimization.
  • the preferred 5'-UTR based on the sequence optimization of the antibody gene UTR, its nucleotide sequence is shown in SEQ ID NO: 2;
  • the acid sequence is shown in SEQ ID NO: 4, 5 or 6.
  • the inventor In order to improve the versatility of UTR and reduce immunogenicity, the inventor also used the UTR of the gene of immune-related protein interferon, which is widely expressed in the human body, as the original UTR.
  • Interferon is a glycoprotein produced by viruses or other interferon-inducing agents that can be released outside the cells and has broad-spectrum antiviral effects by stimulating intraretinal cells, macrosialocytes, lymphocytes and other cells. It can be stably expressed in a variety of cells, and optimization using its 5'-UTR and 3'-UTR may reduce its immunogenicity and thereby reduce the side effects of mRNA vaccines.
  • preferred 5'-UTR and 3'-UTR are sequence-optimized UTRs.
  • Figure 3 shows the sequence IFN-5-O before 5'-UTR optimization and the sequence IFN-5'UTR-F after optimization. Its GC content increased from 49% to 52% after optimization.
  • Figure 5 shows the sequence INF-3-O before 3'-UTR optimization and the sequence IFN-3'UTR-F after optimization. The GC content increased from 31% to 44% after optimization.
  • the preferred 5'-UTR based on the sequence optimization of the interferon gene UTR its nucleotide sequence is shown in SEQ ID NO: 8; the preferred 3'-UTR based on the sequence optimization of the interferon gene UTR, whose The nucleotide sequence is shown in SEQ ID NO: 10, 11 or 12.
  • the UTR of the antibody and interferon genes as the UTR part of the universal plasmid skeleton, that is, the UTR part of the subsequent generated mRNA, the stability and translation activity of the mRNA are enhanced, mainly to ensure the universality of the plasmid skeleton. and the stability of mRNA vaccines.
  • the inventors designed a plasmid that can replace the ORF, and uses blunt-end or sticky-end restriction sites to facilitate subsequent linearized fragment testing of in vitro transcription.
  • this vector includes a vector backbone, AleI restriction site or BspQI restriction site, 5'-UTR, ORF, 3'-UTR, and polyA.
  • a special competent state was selected and ORF codon optimization was performed.
  • the inventors used high-copy plasmid vector pUC57-Amp or pUC-Kana to amplify the fragments.
  • the high-efficiency blunt-end restriction enzyme AleI or the sticky-end restriction enzyme BspQI is used as an enzyme to cut the inserted fragment from the plasmid.
  • the blunt-end or sticky end it can produce can facilitate subsequent experiments.
  • the promoter selected was T7 promoter.
  • the inventor downloaded the sequences of 10 different antibody peptide chains and different types of interferons from the NCBI database (as shown in Table 1). After comparison, they found out that they are relatively conserved. and a UTR sequence of appropriate length. And based on the natural UTR sequence, the optimized UTR sequence was obtained by optimizing the Kozak sequence and reducing the AT-rich sequence.
  • 5'-UTR Two types of 5'-UTR were selected: antibody conserved sequences and interferon conserved sequences, and Kozak sequence optimization was performed. And the GC content of the 5'-UTR of the antibody and interferon was optimized before vector construction.
  • 3'-UTR Two types of 3'-UTR were selected: antibody conserved sequences and interferon conserved sequences, and AT-rich sequences were eliminated. And the GC content of the 3'-UTR of the antibody and interferon was optimized before vector construction.
  • the translation elongation complex recognizes multiple stop codons at the stop codon, which is beneficial to the depolymerization of the complex and thereby enhances the translation activity of the mRNA, the inventors added two stop codons.
  • the selected polyA length is 120 ⁇ 10nt.
  • the ORF sequence in the plasmid backbone can be easily replaced through homologous recombination or enzyme digestion, which can then be used to construct mRNA vaccines for different antigens.
  • the plasmid backbone fragment was amplified by PCR using the pUC57-Amp vector as a template, as shown in Figure 8. A fragment of the vector backbone of the source arm.
  • the plasmid extracted from the successfully constructed engineering bacteria was subjected to agarose gel electrophoresis.
  • the plasmid extracted from the successfully constructed engineering bacteria was digested with AleI enzyme.
  • the leech plasmid engineered bacteria were passaged for a long time, and part of the bacterial liquid was preserved for several generations to conduct strain identification and detection of bacterial plasmid copy number and bacterial plasmid loss rate.
  • the test results are shown in Table 1-2.
  • the IMVC biochemical test results of the 3rd, 5th, 10th, 15th and 20th generations of bacterial fluids showed that they were typical Escherichia coli; the Gram staining results showed that they were short rod-shaped Gram-negative bacteria without miscellaneous bacterial contamination.
  • bacterial plasmid copy number detection shows that the plasmid copy number of the engineering bacteria is approximately 53.17-240.28 copies/cell.
  • the bacterial plasmid loss rate test showed that no plasmid loss occurred in the engineering bacteria during the passage process.
  • the transcription template plasmid constructed using the universal UTR element of the present invention has a high-level in vitro transcription effect.
  • the gene encoding the rabies virus G protein (GeneBank: GQ918139.1) was inserted into the replaceable ORF plasmid as an ORF to construct recombinant engineering bacteria and plasmids.
  • IVT In vitro transcription
  • the linearized DNA fragment was used as the template for IVT.
  • the amount of synthesized RNA was detected, and the IVT yield was calculated as the multiple of the amount of RNA relative to the amount of DNA template added.
  • the transcription template plasmid constructed by the skeleton in the present invention has high in vitro transcription activity in in vitro transcription.
  • the mRNA constructed using the universal UTR element of the present invention has the ability to express the corresponding protein.
  • Cell transfection experiments were performed using the mRNA prepared in Example 6, which contained a 6 ⁇ His tag at the C terminus of the rabies virus G protein.
  • the amount of mRNA used was 2 ⁇ g/well, and the number of cells in the 6-well plate was 10 6 cells/well.
  • the results show that rabies virus G protein expression exists in the transfected cells, indicating that the mRNA produced by the universal UTR element and skeleton of the present invention has the ability to express the corresponding protein in cells.
  • Example 8 The rabies virus mRNA vaccine constructed using the universal UTR element of the present invention can induce mice to produce extremely high levels of antibodies.
  • the rabies virus G protein encoding gene is used as an ORF to be inserted into the replaceable ORF plasmid (including the universal UTR element of the present invention and the BspQI restriction site), and the high-copy plasmid vector pUC-Kana
  • the replaceable ORF plasmid including the universal UTR element of the present invention and the BspQI restriction site
  • the high-copy plasmid vector pUC-Kana was constructed on the vector skeleton
  • a rabies virus mRNA experimental vaccine was produced based on this recombinant engineered bacterium.
  • lane P1 is the plasmid band
  • lane 1 is the result of ApaLI and PvuII double enzyme digestion.
  • the assay results showed that they were consistent with the expected results of the plasmid design.
  • mice 6-8 weeks old BALB/C mice were used for experiments.
  • the experiment was divided into 4 groups: positive control group, 16 mice (8 males and 8 females), and intramuscular injection of chicken embryo inactivated vaccine (0.6IU/mouse). ); the low-dose group, 16 mice (8 males and 8 females), received a lower dose of mRNA vaccine (5 ⁇ g/mouse) intramuscularly; the high-dose group, 16 mice (8 males and 8 females), received a higher dose intramuscularly. mRNA vaccine (13 ⁇ g/animal); negative control group, no intramuscular injection. Except for the negative control group, two injections were performed on days 0 and 14 to enhance the immune effect, and blood was taken from half of the mice on days 14 and 28 respectively. Serum samples were tested for rabies virus G protein antibody levels by fluorescence focus inhibition method according to the method of "Chinese Pharmacopoeia".
  • the results show that the chicken embryo inactivated vaccine can induce mice to produce antibody levels that are theoretically sufficient to resist rabies virus infection.
  • the rabies virus mRNA vaccine produced more antibodies than the inactivated vaccine under both low-dose and high-dose conditions. High antibody levels.
  • the results indicate that an mRNA vaccine constructed and produced using universal elements can induce a high level of immune response.

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Abstract

L'invention concerne un squelette pour construire un transcrit d'ARNm. En particulier, l'invention concerne une application d'un squelette universel pour construire un transcrit d'ARNm dans la préparation et/ou l'optimisation d'ARNm. Le squelette universel peut améliorer efficacement la stabilité et l'activité de traduction de l'ARNm, de telle sorte que la stabilité d'un vaccin à ARNm est assurée, et l'expression du vaccin à ARNm est optimisée.
PCT/CN2023/096643 2022-05-27 2023-05-26 Squelette pour la construction d'un gabarit de transcription in vitro d'arnm WO2023227124A1 (fr)

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