CN117165611A - Framework for constructing mRNA in-vitro transcription template - Google Patents

Framework for constructing mRNA in-vitro transcription template Download PDF

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CN117165611A
CN117165611A CN202210594377.9A CN202210594377A CN117165611A CN 117165611 A CN117165611 A CN 117165611A CN 202210594377 A CN202210594377 A CN 202210594377A CN 117165611 A CN117165611 A CN 117165611A
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mrna
utr
universal
sequence
vaccine
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张丹
邹军
陆杰
张建三
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Shenzhen Sanyuansheng Biotechnology Co ltd
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Shenzhen Sanyuansheng Biotechnology Co ltd
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Priority to CN202210594377.9A priority Critical patent/CN117165611A/en
Priority to PCT/CN2023/096643 priority patent/WO2023227124A1/en
Publication of CN117165611A publication Critical patent/CN117165611A/en
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    • A—HUMAN NECESSITIES
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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
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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
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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
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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
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    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Abstract

The application provides a framework for constructing mRNA transcripts. In particular, the application provides the use of a universal backbone for the construction of mRNA transcripts in mRNA preparation and/or optimization. The universal skeleton can effectively enhance the stability and translation activity of mRNA, thereby ensuring the stability of mRNA vaccine and optimizing the expression thereof. The universal skeleton of the application can accelerate the research and development of mRNA vaccine for various infectious diseases.

Description

Framework for constructing mRNA in-vitro transcription template
Technical Field
The application belongs to the field of biomedicine, and particularly relates to a framework for constructing an mRNA in-vitro transcription template and application of the framework in mRNA optimization design.
Background
mRNA vaccines are a novel vaccine technology that, through binding to molecular biology and immunology, transduce mRNA into somatic cells to express foreign antigens and thereby activate adaptive immunity of the host. As early as 1990, scientists expressed fluorescent protein, β -galactosidase, and chloramphenicol acetyl transferase by injecting mRNA into mouse somatic cells. In 1992, jirikowski et al injected diabetic diabetes insipidus mice with mRNA encoding oxytocin and vasopressin, and as a result mice did not develop insipidus within hours after injection. Thereafter, the development of mRNA vaccines falls into the valley phase.
In recent decades, researchers have studied and optimized mRNA vaccines at experimental levels for safety, expression efficiency, and industrial production. These advances have led to the preferential development of mRNA vaccines for the prevention of tumor and viral infections. Especially, the outbreak of new coronaviruses in recent two years has led to the development of mRNA vaccine technology and the clinical application of the technology.
mRNA vaccines have been developed primarily to improve mRNA stability and translational activity and to reduce mRNA autoantigens. At present, except that new coronavirus vaccines can be marketed due to the urgency of research and development, research and development of other mRNA vaccines are hindered. The reason for this is probably that the ability to express antigens is over-focused when designing mRNA vaccines, and the risk of toxicity and the risk of causing apoptosis in surrounding host cells, which may be caused by the antigenicity and high expression ability of mRNA vaccines themselves, is neglected.
There is a strong need in the art to develop an mRNA vaccine design method that has lower side effects and can accelerate development.
Disclosure of Invention
The application aims to increase the universality of an mRNA vaccine by adopting UTR of immune related proteins, reduce potential side effects and further accelerate the development of various infectious diseases by means of a universal skeleton.
In a first aspect of the application there is provided the use of a universal backbone for the construction of an mRNA transcript, wherein the transcript comprises an ORF to be expressed and a 5'-UTR region and a 3' -UTR region flanking the ORF, wherein 1 or 2 of the 5'-UTR region and the 3' -UTR region are universal UTRs.
In another preferred embodiment, the universal UTR is selected from the group consisting of: a UTR conserved sequence of a mutated or optimized antibody gene, a UTR conserved sequence of a mutated or optimized interferon gene, or a combination thereof.
In another preferred embodiment, the mRNA transcripts comprise mRNA transcripts in an mRNA vaccine.
In another preferred embodiment, the universal skeleton further comprises additional UTR regions (i.e., other UTR regions than the universal UTR).
In another preferred embodiment, the nucleotide sequence of the UTR conserved sequence of the antibody gene is shown in SEQ ID NO. 1 or 3.
In another preferred embodiment, the UTR conserved sequences of the antibody genes include 5 'antibody conserved sequences and 3' antibody conserved sequences.
In another preferred embodiment, the nucleotide sequence of the 5' antibody conserved sequence is shown as SEQ ID NO. 1.
In another preferred embodiment, the nucleotide sequence of the 3' antibody conserved sequence is shown in SEQ ID NO. 3.
In another preferred embodiment, the sequence shown as SEQ ID NO. 3 is conserved among the different IGL genes.
In another preferred embodiment, the nucleotide sequence of the UTR conserved sequence of the interferon gene is shown in SEQ ID NO. 7 or 9.
In another preferred embodiment, the UTR conserved sequences of the interferon genes include a 5 'interferon conserved sequence and a 3' interferon conserved sequence.
In another preferred embodiment, the nucleotide sequence of the 5' interferon conserved sequence is shown in SEQ ID NO. 7.
In another preferred embodiment, the nucleotide sequence of the 3' interferon conserved sequence is shown in SEQ ID NO. 9.
In another preferred embodiment, the sequence shown as SEQ ID NO 7 or 9 is conserved among the isoforms of different IFNA.
In another preferred embodiment, the universal UTR includes a universal 5'-UTR and a universal 3' -UTR.
In another preferred embodiment, the universal UTR includes: 5' -UTR containing Kozak sequence.
In another preferred embodiment, the AT-rich sequence in the 3' -UTR of the antibody gene and the interferon gene is deleted in whole or in part.
In another preferred embodiment, the universal UTR is free or substantially free of AT-rich sequences.
In another preferred embodiment, the substantial absence of AT-rich sequences means that in one UTR the number of AT-rich sequences is.ltoreq.2, more preferably.ltoreq.1.
In another preferred embodiment, the AT-rich sequence refers to a nucleic acid sequence enriched in adenine and thymine bases.
In another preferred embodiment, the GC content in the universal UTR is from 44% to 64%.
In another preferred embodiment, the nucleotide sequence of the universal 5' -UTR is as shown in SEQ ID NO. 2 or 8.
In another preferred embodiment, the nucleotide sequence of the universal 3' -UTR is as shown in SEQ ID NO. 4, 5, 6, 10, 11 or 12.
In another preferred embodiment, any of the above nucleotide sequences further comprises a derivative sequence optionally having at least one (e.g., 1-3) nucleotide added, deleted, modified and/or substituted and capable of retaining the ability to optimize mRNA.
In a second aspect of the present application, there is provided a universal skeleton having the structure of formula I:
Z1-Z2-Z3-Z4-Z5-Z6-Z7 (I)
in the method, in the process of the application,
z1 and Z7 are non-or enzyme cutting sites;
z2 is a no or promoter element;
z3 is a 5' -UTR element;
z4 is an alternative ORF region;
z5 is a 3' -UTR element;
z6 is a polyA tail element.
In another preferred embodiment, Z1 and Z7 are blunt-end cleavage sites or cohesive-end cleavage sites.
In another preferred embodiment, the universal backbone comprises a cleavage site, a promoter, a 5'-UTR, an ORF, a 3' -UTR, and a polyA.
In another preferred embodiment, the blunt-ended enzyme is selected from the group consisting of: aleI, aatI, aluI, bavAI, bavBI, ecoRV, mlsI, or a combination thereof.
In another preferred embodiment, the blunt-ended enzyme is AleI.
In another preferred embodiment, the Z2 is selected from the group consisting of: a T7 promoter, a T3 promoter, an SP6 promoter, or a combination thereof.
In another preferred embodiment, the Z2 is a T7 promoter.
In another preferred embodiment, 1 or 2 of the Z3 and Z5 are universal UTRs.
In another preferred embodiment, Z3 is selected from the group consisting of: mutated or optimized 5 'antibody-conserved sequences, mutated or optimized 5' interferon-conserved sequences, or combinations thereof.
In another preferred embodiment, Z3 is selected from the group consisting of: SEQ ID NO. 2, SEQ ID NO. 8, the 5' -UTR of human alpha globin, or a combination thereof.
In another preferred embodiment, the Z4 is replaced with a gene selected from the group consisting of: hirudin, rabies G protein, dengue virus E protein, mycobacterium tuberculosis ESAT-6 protein, ag85A protein, or combinations thereof.
In another preferred embodiment, said Z4 is replaced by an antigenic gene of a pathogen selected from the group consisting of: hirudin, cytomegalovirus (CMV), zika virus (Zika), influenza virus (Influnza), respiratory Syncytial Virus (RSV), chikungunya, rabies (Rabies), HIV, ebola virus (Ebola virus), streptococcus, malaria (Malaria), jumping virus (Louping ill virus), toxoplasma gondii (Toxoplasma gondii), dengue fever, plague, yellow fever, tuberculosis, herpes simplex virus, ribbonvirus, mycoplasma, chlamydia, foot and mouth disease virus, pseudomonas aeruginosa, or combinations thereof
In another preferred embodiment, said Z4 is replaced by a hirudin gene.
In another preferred embodiment, the Z4 is codon optimized.
In another preferred embodiment, the replacement method of Z4 comprises homologous recombination and cleavage.
In another preferred embodiment, the stop codon of Z4 is a plurality of stop codons.
In another preferred embodiment, the stop codon for Z4 is 2 stop codons.
In another preferred embodiment, Z5 is selected from the group consisting of: mutated or optimized 3 'antibody-conserved sequences, mutated or optimized 3' interferon-conserved sequences, or combinations thereof.
In another preferred embodiment, Z5 is selected from the group consisting of: SEQ ID NO. 4-6, SEQ ID NO. 10-12, or combinations thereof.
In another preferred embodiment, the length of Z6 is preferably 100nt to 150nt, more preferably 110nt to 130nt, and even more preferably 120nt.
In a third aspect of the present application, there is provided a universal UTR element comprising:
(a) A universal 5'-UTR, wherein the sequence of the universal 5' -UTR is selected from the nucleotide sequence shown as SEQ ID NO. 2 or 8 or a derivative sequence thereof; and/or
(b) Universal 3'-UTR, wherein the sequence of said universal 3' -UTR is selected from the nucleotide sequences shown as SEQ ID NOs 4, 5, 6, 10, 11 or 12 or derived sequences thereof.
In another preferred embodiment, the derivative sequence is a derivative sequence which is obtained by optionally adding, deleting, modifying and/or substituting at least one (e.g., 1 to 3) nucleotide to any one of the above nucleotide sequences and which can retain the ability to optimize mRNA.
In another preferred embodiment, the (a) and (b) may be derived from the same transcript.
In another preferred embodiment, the (a) and (b) may be derived from different transcripts.
In a fourth aspect of the application there is provided a vector comprising a versatile framework according to the second aspect of the application.
In another preferred embodiment, the carrier is selected from the group consisting of: DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof. Preferably, the vector is a plasmid.
In another preferred embodiment, the vector is a pUC57-Amp vector.
In a fifth aspect of the application there is provided a host cell comprising a vector according to the fourth aspect of the application, or having integrated into its genome a universal backbone according to the second aspect of the application.
In another preferred embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell.
In another preferred embodiment, the host cell is selected from the group consisting of: coli, yeast cells, mammalian cells.
In a sixth aspect of the application, there is provided an engineered cell comprising: the vector according to the fourth aspect of the present application, or a genome thereof, has incorporated therein the universal backbone according to the second aspect of the present application, and contains a gene fragment of interest.
In another preferred embodiment, the engineered cell is a stable3 E.coli competent cell.
In another preferred embodiment, the gene segment of interest contains a homology arm sequence.
In another preferred embodiment, the vector or universal backbone contains a homology arm sequence.
In another preferred embodiment, the gene fragment of interest is homologous recombined with the vector or the universal backbone.
In another preferred embodiment, the gene fragment of interest is circularized with the vector or universal backbone ligation.
In another preferred embodiment, the gene of interest is selected from the group consisting of: hirudin, cytomegalovirus (CMV), zika virus (Zika), influenza virus (Influenza), respiratory Syncytial Virus (RSV), chikungunya, rabies (Rabies), aids virus (HIV), ebola virus (Ebola virus), streptococcus (streptococci), malaria (malaria), jumping virus (Louping ill virus), toxoplasma gondii (Toxoplasma gondii), dengue fever, plague, yellow fever, tuberculosis vaccine disease, herpes simplex virus, banded virus, mycoplasma, chlamydia, foot and mouth disease virus, pseudomonas aeruginosa, or combinations thereof.
In a seventh aspect of the application, there is provided a method of producing an optimised mRNA for use in the preparation of a vaccine, comprising the steps of:
(a) Culturing the engineered cell according to the sixth aspect of the application under suitable conditions, thereby obtaining a culture containing the vector transcribing the DNA template;
(b) Isolating and/or recovering the vector of (a) from the culture and enzymatically tangentially forming a DNA template;
(c) Transcribing the DNA template of (b) to obtain the optimized mRNA; and
(d) Optionally, purifying and/or modifying the optimized mRNA obtained in step (c).
In an eighth aspect of the present application, there is provided a method for preparing an mRNA vaccine, the method comprising the steps of:
(i) Obtaining an optimized mRNA by a method according to the seventh aspect of the application;
(ii) Mixing the optimized mRNA obtained in (i) with a pharmaceutically acceptable carrier, thereby obtaining the mRNA vaccine.
In a ninth aspect of the application, there is provided a kit comprising:
(a) A first plasmid containing a gene of interest; and
(b) A second plasmid comprising a universal backbone according to the second aspect of the application; and
(c) The specification describes a method for producing optimized mRNA useful for preparing a vaccine using the first and second plasmids.
In another preferred embodiment, the specification also describes a method for amplifying a first fragment with a homology arm sequence using a first plasmid as a template.
In another preferred embodiment, the specification also describes a method for amplifying a second fragment with a homology arm sequence using a second plasmid as a template.
In another preferred embodiment, the specification also describes methods for circularizing the first fragment and the second fragment by homologous recombination ligation and transferring the fragments into a suitable host cell.
In another preferred embodiment, the specification also describes a method for obtaining optimized mRNA from the host cell.
In a tenth aspect of the application, there is provided an mRNA vaccine composition comprising:
(a) An mRNA for expressing an immunogen comprising a universal scaffold according to the second aspect of the application; and
(b) A pharmaceutically acceptable carrier.
In another preferred embodiment, the immunogen is selected from the group consisting of: hirudin, cytomegalovirus (CMV), zika virus (Zika), influenza virus (Influenza), respiratory Syncytial Virus (RSV), chikungunya, rabies (Rabies), aids virus (HIV), ebola virus (Ebola virus), streptococcus (streptococci), malaria (malaria), jumping virus (Louping ill virus), toxoplasma gondii (Toxoplasma gondii), dengue fever, plague, yellow fever, tuberculosis, herpes simplex virus, banded virus, mycoplasma, chlamydia, foot and mouth disease virus, pseudomonas aeruginosa, or combinations thereof.
In another preferred embodiment, the mRNA itself in the vaccine composition may also act as an adjuvant.
In another preferred embodiment, the vaccine composition is in a dosage form selected from the group consisting of: injection and freeze-dried preparation.
In another preferred embodiment, the vaccine composition comprises 0.01-99.99% of the universal backbone according to the second aspect of the application and 0.01-99.99% of a pharmaceutically acceptable carrier, said percentages being mass percentages of the vaccine composition.
In an eleventh aspect of the application there is provided the use of an mRNA vaccine composition according to the tenth aspect of the application, or an engineered cell according to the sixth aspect of the application, for the preparation of a medicament for the prevention of a pathogen selected from the group consisting of: hirudin, cytomegalovirus (CMV), zika virus (Zika), influenza virus (Influenza), respiratory Syncytial Virus (RSV), chikungunya, rabies (Rabies), aids virus (HIV), ebola virus (Ebola virus), streptococcus (streptococci), malaria (malaria), jumping virus (Louping ill virus), toxoplasma gondii (Toxoplasma gondii), dengue fever, plague, yellow fever, tuberculosis, herpes simplex virus, banded virus, mycoplasma, chlamydia, foot and mouth disease virus, pseudomonas aeruginosa, or combinations thereof.
It is understood that within the scope of the present application, the above-described technical features of the present application and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
Fig. 1 is a schematic structural view of a carrier according to an embodiment of the present application.
FIG. 2 shows the pre-optimized sequence IGL-5-O of the 5'-UTR, the optimized sequence IGL-5' UTR-F and their corresponding GC contents.
FIG. 3 shows the sequence IFN-5-O before 5'-UTR optimization, the sequence IFN-5' UTR-F after optimization and their corresponding GC contents.
FIG. 4 shows the pre-3-O sequence of the 3'-UTR, the optimized sequence IGL-3' UTR-F and their corresponding GC contents.
FIG. 5 shows the sequence INF-3-O before 3'-UTR optimization, the optimized sequence IFN-3' UTR-F and their corresponding GC contents.
FIG. 6 is a schematic representation of the insertion of 2 stop codons after ORF sequence.
FIG. 7 is a schematic diagram of a synthetic fragment of the hirudin gene.
FIG. 8 is a schematic diagram of a backbone amplified fragment.
FIG. 9 shows the results of electrophoretic identification of inserts and plasmid backbone fragments.
FIG. 10 shows the results of Hirudin plasmid gel electrophoresis.
FIG. 11 shows the cleavage results of the Hirudin plasmid AleI.
FIG. 12 shows the plasmid yield change during fermentation.
Detailed Description
Through extensive and intensive research and a large number of screening, the inventor develops a universal UTR and a universal skeleton for constructing mRNA transcripts for the first time, so that optimized mRNA with improved stability and translation activity is obtained, and the application in aspects of mRNA vaccine preparation and/or optimization and the like is realized. The universal skeleton of the application can accelerate the research and development of mRNA vaccine for various infectious diseases. The present application has been completed on the basis of this finding.
Terminology
In order that the present disclosure may be more readily understood, certain terms are first defined. As used in the present application, each of the following terms shall have the meanings given below, unless explicitly specified otherwise herein.
As used herein, "universal scaffold of the application", "universal scaffold", "scaffold of the application", "scaffold" are used interchangeably and refer to a scaffold comprising the ORF region of a universal UTR element that is used interchangeably to construct an mRNA transcript.
As used herein, "UTR conserved sequence of an antibody gene", "antibody conserved sequence" are used interchangeably and refer to a conserved sequence of a UTR region in an antibody gene, preferably the nucleotide sequence of which is shown in SEQ ID No. 1 or 3.
As used herein, "UTR conserved sequence of an interferon gene", "interferon conserved sequence" are used interchangeably and refer to a conserved sequence of the UTR region of an interferon gene, preferably the nucleotide sequence of which is shown in SEQ ID No. 7 or 9.
Universal skeleton and expression vector
As used herein, the terms "generic skeleton of the application", "skeleton of the application" are used interchangeably to refer to the generic skeleton described in the second aspect of the application.
Typically, the universal frameworks of the present application have the structure of formula I:
Z1-Z2-Z3-Z4-Z5-Z6-Z7 (I)
wherein Z1 to Z7 are as described above.
A schematic structural diagram of a representative vector containing the universal backbone of the present application is shown in FIG. 1.
It will be appreciated that proteins or polypeptides suitable for expression with the universal frameworks of the present application are not particularly limited and include antigenic proteins or peptides, or other useful proteins. In the present application, the ORF of the foreign protein can be placed in the universal skeleton of the present application, thereby realizing efficient expression. Typically, the ORF carries a stop codon. However, if desired, one or more additional stop codons may also be introduced, as shown in FIG. 6.
mRNA vaccine types
mRNA vaccines are classified into self-amplifying RNA (saRNA) and non-amplifying mRNA. Classical non-amplified RNA vaccines include cap caps, 5 'non-coding regions (5' -untranslated regions,5 '-UTR), open reading frames (open reading frame, ORF), 3' non-coding regions (3 '-untranslated regions,3' -UTR) and poly A tails (polyA tails). The ORF region is responsible for encoding antigen expression, but the above 5 regions together determine mRNA stability, expression activity and immunogenicity.
Whereas the structure of saRNA is derived from the alphavirus genome. The saRNA vaccine utilizes the characteristic that the genome of the alphavirus can self-replicate to self-amplify DNA or RNA entering a somatic cell and then transcribe antigen-encoding mRNA. There are currently two types of saRNA vaccines, DNA plasmid-based and virus-like particle delivered saRNA. Based on saRNA, beisset et al have also developed a transgenic amplified RNA (taRNA) that places the gene encoding the antigen in the alphavirus genome, increasing the safety of the vaccine. Compared with self-amplified RNA, the non-amplified RNA has the characteristics of smaller size, more specific expression antigen and no non-specific immunity.
mRNA vaccine immunogenicity
One challenge of mRNA vaccines is to reduce the immunogenicity of the exogenous mRNA itself. Naturally, exogenous mRNA enters cells and can be identified by retinoic acid-induced gene I (retinoic acid-iinducible gene I, RIG-I), so as to activate an innate immune response and then be degraded. In vitro transcribed (in vitro transcription, IVT) mRNA is capable of activating immune cells and Toll-like receptor (Toll-like receptor) -mediated inflammatory responses. The U-rich (U-rich) sequence of mRNA is a key factor in activating Toll-like receptors. The immunogenicity of mRNA can be reduced by nucleotide chemical modification, addition of polyA tail, optimization of mRNA GC content, and the like.
Chemically modified nucleotides include 5-methylcytidine (5-methylcytidine, m 5C), 5-methyluridine (5-methyluridine, m 5U), N1-methyladenosine (m 1A), N6-methyladenosine (N6-methyluridine, m 6A), 2-thiouridine (2-thiouridine, s 2U), 5-oxomethyluridine (5-methoxyuridine, 5 moU), pseudouridine (pseudouridine, m1 psi) and N1-methylparaben (N1-methylparaben, m1 psi).
Furthermore, the addition of polyA tails can also reduce U content and thus mRNA immunogenicity. CureVac and Acuita Therapeutics have attempted to transport erythropoietin-encoding mRNA, which has a relatively high GC content and is consequently capable of causing an erythropoietin-associated response without immunogenicity, into pigs via lipid nanoparticles. However, too high a GC content can inhibit the translation activity of the mRNA, which is also a concern during vaccine development.
The manner in which mRNA is purified is also important in reducing the immunogenicity of mRNA itself. Purification methods commonly used at present include high performance liquid chromatography (high performance liquid chromatography, HPLC), anion exchange chromatography, affinity chromatography and particle size separation. The purpose of purification is mainly to remove truncated transcripts. A good example is the purification by HPLC of m1ψmodified mRNA encoding anti-HIV-1 antibodies designed by Pardi et al, which helps mice avoid HIV-1 infection by lipid nanoparticles (lipid nanoparticles, LNP).
Sequence optimization of mRNA
Sequence optimization of mRNA is one of the methods to help stabilize mRNA. Sequence optimization of the 5'-UTR and 3' -UTR of mRNA can increase half-life and translational activity of mRNA. The Cap structure adopts different analogues to increase the stability of mRNA, and enzyme is utilized to enable the 5' end of mRNA to be added with the Cap structure, so that the Cap structure has better efficiency than different forms of Cap analogues. The stabilizing effect of the polyA tail of mRNA is also very important, and studies have been made to remove polyA from mRNA to make mRNA extremely unstable, while also reducing the number of polysomes, the rate of extension and the number of translation rounds of mRNA. polyA is thus critical for stable and efficient translation of mRNA. In addition, nucleotide modifications and synonymous substitutions of codons can also affect mRNA stability and translational activity. While optimization of the sequence may affect the secondary structure and post-translational modification of the mRNA. In addition, increasing the GC content of the mRNA can also increase the mRNA stability. In summary, 5' -UTR, 3' -UTR, 5' Cap, polyA tail, codon optimization and GC content are all regulatory sites that enhance mRNA stability.
mRNA delivery
There are many current methods of mRNA delivery, scientists have established liposome transport, polymer transport, peptide chain transport, viral-like replicon particle transport, and cationic nanoemulsion transport, and in addition, naked mRNA can be injected directly into cells. The most common delivery method in the research of mRNA vaccines is lipid nanoparticle (lipid nanoparticles, LNP) transport. The method has the advantages of low toxicity, high delivery efficiency and the like.
mRNA vaccine activates innate and adaptive immunity
mRNA vaccines are capable of activating the innate and adaptive immune systems by expressing antigens in somatic cells and presenting them via an antigen presentation system. Direct recognition of mRNA by pattern recognition receptors such as TLRs in somatic cells can lead to degradation of mRNA, while at the same time being able to enhance IFN pathways.
mRNA vaccines play a major role by eliciting an adaptive immune response. The antigen translated from mRNA is presented by MHC-I to activate CD4+ T cells (Helper T cells) or by other cells to activate CD8+ T cells (cytotoxic T cells) by presenting antigen via MHC-II pathway after phagocytosis. The antigen, after being expressed on the cell surface, is also recognized by B cell receptors to activate antibody expression and memory B cell formation by B cells, thereby causing apoptosis of the infected cells and neutralization of pathogens.
Events that produce adaptive immune responses following mRNA vaccination
a) After the mRNA-containing particles of the vaccine are taken up by local cells at the injection site, the mRNA is recognized by the pattern recognition receptor and translation of the antigen is also initiated, resulting in local inflammation at the injection site, promoting infiltration of immune cells, including neutrophils, monocytes, myeloid dendritic cells (myeloid dendritic cells, MDCs) and plasmacytoid dendritic cells (plasmacytoid dendritic cells, PDCs). Neutrophils can efficiently take up LNPs, but monocytes and MDCs translate mRNA more efficiently. Secretion of type I Interferon (IFN) is stimulated.
b) The mRNA/LNP and protein antigens will spread and the cells will migrate to the vaccinated lymph nodes.
c) Antigen presentation to T cells and interaction of antigen with B cells occurs resulting in the formation of germinal centers where memory B cells and antibody-producing plasma cells are produced, these cells residing in the bone marrow.
Advantages of mRNA vaccine against infectious diseases
The mechanism of mRNA vaccine is to inoculate mRNA encoding antigen protein into host, express and synthesize antigen protein in vivo cell with host genetic material, induce and activate immune system of organism to produce immune response, so as to prevent and treat diseases. The unique advantages are as follows: 1) The monitoring and quality control of all production processes can be easily realized; 2) The research and development and production cycle of mRNA are short, mass production is easy to realize, and the vaccine productivity is high, which is important for rapidly coping with new infectious diseases on the global scale; 3) mRNA can be quickly degraded after immunization due to the characteristics of the mRNA, so that the safety risk is low; 4) The vaccine has good immune efficacy, can induce humoral immunity and cellular immunity at the same time, and has potential to develop more effective vaccines for infectious diseases without better vaccines at present.
mRNA vaccine optimization strategy
Challenges that mRNA vaccines need to face to function include: 1) The half life of the self-body is prolonged, and the stability is enhanced; 2) Enhancing translational activity; 3) Reduces the immunogenicity of mRNA and avoids rapid clearance.
The method for achieving these effects is to design specific 5'-UTR, 3' -UTR, stop codon and polyA amount, etc. There are three effective approaches to the design of 5'-UTR and 3' -UTR: 1) UTR of human gene with high expression is adopted; 2) Use of the UTR of the antigenic protein itself; 3) Exponential enrichment ligand systematic evolution techniques (systematic evolution of ligands by exponential enrichment, SELEX). Both the first two methods are relatively simple and the third method is relatively complex, requiring continuous attempts and optimization of the sequence by in vitro experiments, thus requiring a long time, but the third method is still the best choice when time is plentiful.
The 5' -UTR of the BNT162b2 vaccine, which has been currently marketed by FDA approved (Pfizer/Biontech), uses the 5' -UTR of human alpha globin and optimizes the Kozak sequence and the 5' -terminal sequence, the secondary structure of the 5' -UTR is adjusted, and the 5' -UTR of the mRNA-1273 vaccine of Muldena (Moderna) uses its sequence designed and optimized by computer. For 3' -UTR, the mRNA-1273 vaccine of Modena uses 110nt base in 3' -UTR of human alpha globin (HBA 1), while the BNT162b2 vaccine of pyroxene uses a method of SELEX based on natural gene, and the 3' -UTR of human 12S rRNA (mtRNR 1) and AES/TLE5 genes are selected. The psilosis vaccine uses 136nt sequence of AES 3' -UTR on the basis, and makes two C- & gt- ψ changes, and then continues 139nt mtRNR1 sequence. The current truly effective UTR design approach is also based on natural genes and experience-based optimization (PMID: 34358150).
In addition, a method of adding 1-2 stop codons after the stop codons of the antigen protein is also adopted to more effectively depolymerize the translation extension complex, thereby facilitating translation and stabilization of mRNA. The BNT162b2 vaccine of the pyroxene uses the termination signal UGAUGAUGA, while the mRNA-1273 vaccine of the Mudana uses UGAAAUAG.
The amount of polyA is an important factor affecting the stability of mRNA, and is preferably 80nt-150n or 100nt-150nt, more preferably 120nt. The appropriate amount of polyA can have higher protein expression ability and mRNA stability than the unsuitable amount of mRNA.
mRNA vaccine for infectious diseases
The framework of the present application for constructing mRNA transcripts can be used to construct mRNA vaccines against infectious diseases.
Vaccines employing the backbone of the mRNA transcripts of the present application may be suitable for use with a variety of different pathogens, representative pathogens including (but not limited to): coronavirus (e.g., new coronavirus), cytomegalovirus (CMV), zika virus (Zika), influenza virus (Influenza), respiratory Syncytial Virus (RSV), chikungunya, rabies (Rabies), aids virus (HIV), ebola virus (Ebola virus), streptococcus (streptococci), malaria (malaria), jumping disease virus (Louping ill virus), toxoplasma gondii (Toxoplasma gondii), and the like.
In addition, many infectious diseases lack effective inactivated vaccines and recombinant protein vaccines, and thus it may be desirable to develop effective vaccines if mRNA vaccines can elicit preventive immunity against these pathogens in humans. Such diseases include dengue fever (only one serotype is protected by the existing vaccine), plague, yellow fever, tuberculosis vaccines, herpes simplex virus, banded virus, mycoplasma, chlamydia, foot and mouth disease virus, etc., and may also be used in the treatment of some antitumor drugs.
Antibody Gene UTR
In order to improve the universal use of UTRs and reduce immunogenicity, the inventors used UTRs of antibody genes as original UTRs. Antibodies (anti) are classified into 5 classes (class), namely IgM, igD, igG, igA and IgE, the corresponding heavy chains are mu, delta, gamma, alpha and epsilon, respectively, and the light chains associated with these heavy chains are two, kappa (kappa) and lambda (lambda) chains, respectively, and lambda chains can be further classified into four subtypes of lambda l, lambda 2, lambda 3 and lambda 4 based on the differences in the individual amino acids of the lambda constant regions. The recombination rates of these peptide chain genes are high, suggesting that their 5'-UTR and 3' -UTR sequences may have strong compatibility with the altered ORF region and altered peptide chain expression. Thus, the 5'-UTR and the 3' -UTR of the antibody gene may have versatility to support efficient translation of different ORFs.
In the present application, preferred 5'-UTR and 3' -UTR are sequence-optimized UTR. The pre-optimized sequence IGL-5-O of the 5'-UTR and the optimized sequence IGL-5' UTR-F are shown in FIG. 2, with the GC content being increased from 54% to 64% after optimization. The pre-3 '-UTR optimized sequence IGL-3-O and the optimized sequence IGL-3' UTR-F are shown in FIG. 4, with the GC content being increased from 54% to 56% after optimization.
In the application, the nucleotide sequence of the optimized 5' -UTR based on the antibody gene UTR is shown as SEQ ID NO. 2; the nucleotide sequence of the optimized 3' -UTR based on the antibody gene UTR is shown as SEQ ID NO. 4, 5 or 6.
Interferon gene UTR
In order to improve the universal use of UTRs and reduce immunogenicity, the inventors have also adopted UTRs of genes of immune-related protein interferons widely expressed in humans as original UTRs.
The interferon is a glycoprotein which is generated by viruses or other interferon inducers and stimulates cells such as cells in the net, megasaliva cells, lymphocytes and the like, can be dissociated outside the cells and has broad-spectrum antiviral effect. It is stably expressed in a variety of cells and is optimized using its 5'-UTR and 3' -UTR, potentially reducing its immunogenicity and thus reducing the side effects of mRNA vaccines.
In the present application, preferred 5'-UTR and 3' -UTR are sequence-optimized UTR. The pre-optimized sequence IFN-5-O and the optimized sequence IFN-5' UTR-F with GC content increased from 49% to 52% after optimization are shown in FIG. 3. In FIG. 5, the sequence INF-3-O before 3'-UTR optimization and the sequence IFN-3' UTR-F after optimization are shown, the GC content of which is increased from 31% to 44% after optimization.
In the application, the nucleotide sequence of the optimized preferred 5' -UTR based on the interferon gene UTR is shown as SEQ ID NO. 8; the nucleotide sequence of the optimized 3' -UTR based on the interferon gene UTR is shown as SEQ ID NO. 10, 11 or 12.
The main advantages of the application include:
(1) By constructing a plasmid with a framework of a replaceable ORF template, the development speed of mRNA vaccines can be increased.
(2) The UTR of antibody and interferon gene is selected as UTR part of general plasmid skeleton, which is the UTR part for producing mRNA later, so that the stability and translation activity of mRNA are raised and the general property of plasmid skeleton and the stability of mRNA vaccine are ensured.
The application will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.
Example 1 screening and optimization method of UTR sequences
The present inventors devised a plasmid that replaced the ORF and made it possible to facilitate subsequent in vitro transcription of linearized fragment experiments using blunt-ended cleavage sites.
The vector comprises a vector skeleton, aleI enzyme cutting sites, 5'-UTR, ORF, 3' -UTR and polyA. In addition, special competence is selected and ORF codon optimization is performed.
The present inventors used the high copy plasmid vector pUC57-Amp for amplifying fragments. The efficient blunt end restriction enzyme AleI was used as an enzyme for cleaving the insert from the plasmid, which was able to produce blunt ends for later experiments. The promoter selected is T7 promoter.
Based on the characteristics of antibody and interferon expression, the inventor downloads 10 sequences of different antibody peptide chains and different types of interferons from NCBI database, and finds out the UTR sequences which are relatively conservative and have proper length after alignment. And based on the natural UTR sequence, the optimized UTR sequence is obtained by optimizing the Kozak sequence, reducing the AT-rich sequence and the like.
Table 1 contains the alignment and numbering thereof
Example 2.5'-UTR and selection and optimization of 3' -UTR
The 5' -UTR was selected for both antibody-and interferon-conserved sequences, respectively, and was subjected to Kozak sequence optimization. And the 5' -UTR of the antibody and the interferon were optimized for GC content, respectively, prior to vector construction.
The 3' -UTR selects two kinds of antibody conserved sequences and interferon conserved sequences, and eliminates AT-rich sequences. And the 3' -UTR of the antibody and the interferon, respectively, was optimized for GC content prior to vector construction.
The above sequence information is shown in table 2:
TABLE 2 wild-type and optimized sequences
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The inventors increased 2 stop codons since the translation extension complex recognizes multiple stop codons at the stop codons favors the depolymerization of the complex, thereby enhancing the translation activity of the mRNA. In addition, the length of the selected polyA is 120+/-10 nt.
The ORF sequence in the plasmid skeleton can be conveniently replaced by a homologous recombination or enzyme digestion method, so that the method can be used for constructing mRNA vaccines of different antigens.
EXAMPLE 3 construction of hirudin mRNA vaccine engineering Strain
(1) A Hirudin gene (Hirudin) fragment was PCR-amplified therefrom using a plasmid carrying the Hirudin gene as a template, and as shown in FIG. 7, a homology arm sequence, a T7 promoter, an AleI cleavage site, GFP and a 6 XHis tag were introduced by primers.
The plasmid backbone fragment was amplified by PCR using pUC57-Amp vector as a template, as shown in FIG. 8.
As shown in FIG. 9, the gene synthesis fragment and the PCR product were separately identified by electrophoresis in agarose gel.
(2) And (3) obtaining the two fragments obtained in the step (1) through glue recovery, then adopting a homologous recombination method to connect and cyclize, transferring the fragments into stable3 escherichia coli competent cells to construct engineering bacteria, selecting monoclonal culture bacterial liquid, and carrying out sanger sequencing to identify the engineering bacteria.
As shown in FIG. 10, the plasmids extracted from the engineering bacteria successfully constructed were subjected to agarose gel electrophoresis.
As shown in FIG. 11, the plasmid extracted from the engineering bacteria successfully constructed was subjected to AleI enzyme single cleavage.
EXAMPLE 4 identification of hirudin plasmid engineering bacteria
And carrying out long-term passage on the hirudin plasmid engineering bacteria, and carrying out strain identification and bacterial plasmid copy number and bacterial plasmid loss rate detection on part of algebraic preservation bacterial liquid. The results of the detection are shown in tables 1-2.
3. IMVC biochemical experiment results of the 5, 10, 15 and 20 generation bacterial solutions show that the bacterial solutions are typical Escherichia coli; gram staining results showed short bar gram negative bacteria, no contamination with infectious microbe.
TABLE3 hirudin engineering bacteria bacterial plasmid copy number
As shown in Table3, bacterial plasmid copy number detection showed that the plasmid copy number of the engineering bacteria was about 53.17-240.28copies/cell.
TABLE 4 hirudin plasmid engineering bacteria bacterial plasmid loss Rate assay results
As shown in Table 4, the bacterial plasmid loss rate detection showed that the engineering bacteria did not exhibit plasmid loss during passage.
EXAMPLE 5 hirudin engineering bacteria fermentation plasmid yield
3L LB culture medium is added into a 5L fermentation tank, and bacterial liquid fermentation is carried out under the conditions of tank pressure of 0.05+/-0.02 MPa, ventilation of 2-10L/min, stirring speed of 100-250rpm, temperature of 37+/-0.5 ℃ and pH of 7.0+/-0.1. 2 bacterial solutions of 5mL are taken per hour in the fermentation process for 10 hours and used for extracting plasmids. The plasmid concentration results are shown in Table 5 and FIG. 12.
TABLE 5 plasmid yield during fermentation of hirudin engineering bacteria
Time Plasmid concentration 1 Plasmid concentration 2 Mean value of
1 15.2 5.5 10.35
2 25.6 14.4 20
3 56 38.8 47.4
4 115 95 105
5 99 93.3 96.15
6 76.9 60.5 68.7
7 63.4 75.4 69.4
8 36.6 48.6 42.6
9 35.2 53.4 44.3
10 31.1 41.2 36.15
As shown in the experimental results of FIG. 12, the highest plasmid yield was obtained by fermentation of the seed bacteria for 4 hours.
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.
Sequence listing
<110> Shenzhen City Sanyuan Biotechnology Co., ltd
<120> a framework for constructing an in vitro mRNA transcription template
<130> P2022-0776
<160> 12
<170> PatentIn version 3.5
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<220>
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<213> Homo sapiens (Homo sapiens)
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gttctcatcc ctcacccccc accacgggag actagagctg caggatccca ggggaggggt 60
ctctcctccc accccaaggc atcaagccct tctccctgca ctcaataaac cctcaataaa 120
tattctcatt gtcaatt 137
<210> 4
<211> 115
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<213> Artificial sequence (Artificial Sequence)
<220>
<223> optimization of sequence IGL-3' UTR-F1
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<220>
<223> optimization of sequence IGL-3' UTR-F2
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gttctcatcc ctcacccccc accacgggag actagagctg caggatccca ggggaggggt 60
ctctcctccc accccaaggc atcaagccct tctccctgca ctcaaaaccc tcaactctgt 120
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gttctcatcc ctcacccccc accacgggag actagagctg caggatccca ggggaggggt 60
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ttatctattt aaatatttat ttaactattc ataagattta aattattttt gttcatataa 180
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ttgttcaacg tcgtgcacct ttacactgtg gttagtgtaa aaacgttcct tttactc 177

Claims (10)

1. Use of a universal backbone for constructing an mRNA transcript, wherein the transcript comprises an ORF to be expressed and a 5'-UTR region and a 3' -UTR region flanking the ORF, wherein 1 or 2 of the 5'-UTR region and the 3' -UTR region are universal UTRs.
2. A universal skeleton, wherein the universal skeleton has a structure of formula I:
Z1-Z2-Z3-Z4-Z5-Z6-Z7 (I)
in the method, in the process of the application,
z1 and Z7 are non-or enzyme cutting sites;
z2 is a no or promoter element;
z3 is a 5' -UTR element;
z4 is an alternative ORF region;
z5 is a 3' -UTR element;
z6 is a polyA tail element.
3. A universal UTR element, wherein the universal UTR element comprises:
(a) A universal 5'-UTR, wherein the sequence of the universal 5' -UTR is selected from the nucleotide sequence shown as SEQ ID NO. 2 or 8 or a derivative sequence thereof; and/or
(b) Universal 3'-UTR, wherein the sequence of said universal 3' -UTR is selected from the nucleotide sequences shown as SEQ ID NOs 4, 5, 6, 10, 11 or 12 or derived sequences thereof.
4. A vector comprising the universal skeleton of claim 2.
5. A host cell comprising the vector of claim 4, or having integrated into its genome the universal backbone of claim 2.
6. An engineered cell, the engineered cell comprising: the vector according to claim 4, or a genome thereof, which has the universal backbone according to claim 2 integrated therein, and which contains a gene fragment of interest.
7. A method of producing optimized mRNA for use in preparing a vaccine, comprising the steps of:
(a) Culturing the engineered cell of claim 6 under suitable conditions to obtain a culture containing a vector of transcribed DNA template;
(b) Isolating and/or recovering the vector of (a) from the culture and enzymatically tangentially forming a DNA template;
(c) Transcribing the DNA template of (b) to obtain the optimized mRNA; and
(d) Optionally, purifying and/or modifying the optimized mRNA obtained in step (c).
8. A method of preparing an mRNA vaccine, the method comprising the steps of:
(i) Obtaining an optimized mRNA by the method of claim 7;
(ii) Mixing the optimized mRNA obtained in (i) with a pharmaceutically acceptable carrier, thereby obtaining the mRNA vaccine.
9. A kit, comprising:
(a) A first plasmid containing a gene of interest; and
(b) A second plasmid comprising the universal backbone of claim 2; and
(c) The specification describes a method for producing optimized mRNA useful for preparing a vaccine using the first and second plasmids.
10. An mRNA vaccine composition, characterized in that the vaccine composition comprises:
(a) An mRNA for expressing an immunogen comprising the universal scaffold of claim 2; and
(b) A pharmaceutically acceptable carrier.
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CN117487809A (en) * 2023-12-29 2024-02-02 艾斯拓康医药科技(北京)有限公司 Optimized 5' -UTR sequence and application thereof
CN117535295A (en) * 2024-01-09 2024-02-09 艾斯拓康医药科技(北京)有限公司 Optimized 3' -UTR sequence and application thereof
CN117568338A (en) * 2024-01-17 2024-02-20 艾斯拓康医药科技(北京)有限公司 Optimized polyA sequence and application thereof
CN117535295B (en) * 2024-01-09 2024-04-26 艾斯拓康医药科技(北京)有限公司 Optimized 3' -UTR sequence and application thereof

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US5824497A (en) * 1995-02-10 1998-10-20 Mcmaster University High efficiency translation of mRNA molecules
CN111607613A (en) * 2020-05-19 2020-09-01 深圳市新合生物医疗科技有限公司 Plasmid vector for expressing mRNA of cellular immune vaccine and construction method and application thereof
CN111607612A (en) * 2020-05-19 2020-09-01 深圳市新合生物医疗科技有限公司 Plasmid vector for expressing humoral immunity vaccine mRNA and construction method and application thereof

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CN117487809A (en) * 2023-12-29 2024-02-02 艾斯拓康医药科技(北京)有限公司 Optimized 5' -UTR sequence and application thereof
CN117487809B (en) * 2023-12-29 2024-03-19 艾斯拓康医药科技(北京)有限公司 Optimized 5' -UTR sequence and application thereof
CN117535295A (en) * 2024-01-09 2024-02-09 艾斯拓康医药科技(北京)有限公司 Optimized 3' -UTR sequence and application thereof
CN117535295B (en) * 2024-01-09 2024-04-26 艾斯拓康医药科技(北京)有限公司 Optimized 3' -UTR sequence and application thereof
CN117568338A (en) * 2024-01-17 2024-02-20 艾斯拓康医药科技(北京)有限公司 Optimized polyA sequence and application thereof

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