WO2023227124A1 - Skeleton for constructing mrna in-vitro transcription template - Google Patents

Abstract

Provided is a skeleton for constructing an mRNA transcript. Specifically, provided is an application of a universal skeleton for constructing an mRNA transcript in mRNA preparation and/or optimization. The universal skeleton can effectively enhance the stability and translation activity of mRNA, so that the stability of an mRNA vaccine is ensured, and the expression of the mRNA vaccine is optimized.

Description

A scaffold for constructing mRNA in vitro transcription templates Technical field

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.

Background technique

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. As early as the 1990s, scientists injected mRNA into mouse somatic cells, causing the mouse somatic cells to express fluorescent proteins, β-galactosidase and chloramphenicol acetyltransferase. In 1992, 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.

In recent decades, researchers have studied and optimized the safety, expression efficiency and industrial production of mRNA vaccines at the experimental level. These advances have prioritized the development of mRNA vaccines for the prevention of tumors and viral infections. In particular, the outbreak of the new coronavirus in the past two years has made great progress in the development and clinical application of mRNA vaccine technology.

The development of mRNA vaccines is mainly to improve the stability and translation activity of mRNA and reduce the self-antigenicity of mRNA. At present, except for 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.

There is an urgent need in this field to develop an mRNA vaccine design method with lower side effects and faster development.

Contents of the invention

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.

In a first aspect of the present invention, there is provided the use of 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.

In another preferred embodiment, 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.

In another preferred embodiment, the mRNA transcript includes the mRNA transcript in the mRNA vaccine.

In another preferred embodiment, the universal framework also includes additional UTR regions (ie, other UTR regions besides 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 sequence of the antibody gene includes a 5' antibody conserved sequence and a 3' antibody conserved sequence.

In another preferred embodiment, the nucleotide sequence of the 5' antibody conserved sequence is shown in 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 in SEQ ID NO:3 is conserved in 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 example, the UTR conserved sequence of the interferon gene includes a 5' conserved interferon sequence and a 3' conserved interferon sequence.

In another preferred embodiment, the nucleotide sequence of the 5' interferon conserved sequence is shown in SEQ ID NO: 7.

In another preferred example, the nucleotide sequence of the 3' interferon conserved sequence is shown in SEQ ID NO: 9.

In another preferred embodiment, the sequence shown in SEQ ID NO: 7 or 9 is conserved among different IFNA subtypes.

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: a 5'-UTR containing a Kozak sequence.

In another preferred embodiment, the AT-rich sequence in the 3'-UTR of the antibody gene and interferon gene is completely or partially deleted.

In another preferred embodiment, there is no or substantially no AT-rich sequence in the universal UTR.

In another preferred embodiment, basically no AT-rich sequences means that in one UTR, the number of AT-rich sequences is ≤ 2, and more preferably ≤ 1.

In another preferred embodiment, the AT-rich sequence refers to a nucleic acid sequence rich in adenine and thymine bases.

In another preferred embodiment, the GC content in the universal UTR is 44%-64%.

In another preferred embodiment, the nucleotide sequence of the universal 5'-UTR is shown in SEQ ID NO: 2 or 8.

In another preferred embodiment, the nucleotide sequence of the universal 3'-UTR is shown in SEQ ID NO: 4, 5, 6, 10, 11 or 12.

In another preferred embodiment, 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.

In a second aspect of the present invention, 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)

In the formula,

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.

In another preferred embodiment, Z1 and Z7 are blunt end enzyme cleavage sites or sticky end enzyme cleavage sites.

In another preferred embodiment, the universal framework includes an enzyme cleavage site, a promoter, a 5'-UTR, an ORF, a 3'-UTR and polyA.

In another preferred embodiment, the blunt-end enzyme is selected from the following group: AelI, AatI, AluI, BavAI, BavBI, EcoRV, MlsI, or a combination thereof.

In another preferred embodiment, the blunt-end enzyme is AleI.

In another preferred embodiment, the sticky end enzyme is BspQI.

In another preferred embodiment, the Z2 is selected from the following group: T7 promoter, T3 promoter, SP6 promoter, or a combination thereof.

In another preferred example, the Z2 is a T7 promoter.

In another preferred example, one or two of Z3 and Z5 are universal UTRs.

In another preferred embodiment, 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.

In another preferred embodiment, the Z3 is selected from the following group: SEQ ID NO: 2, SEQ ID NO: 8, the 5'-UTR of human α-globin, or a combination thereof.

In another preferred example, 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.

In another preferred embodiment, 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

In another preferred example, Z4 is replaced with hirudin gene or rabies virus G protein gene.

In another preferred example, the Z4 is codon optimized.

In another preferred embodiment, the Z4 replacement method includes homologous recombination and enzyme digestion.

In another preferred example, the stop codon of Z4 is multiple stop codons.

In another preferred example, the stop codon of Z4 is two stop codons.

In another preferred embodiment, 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.

In another preferred example, the Z5 is selected from the following group: SEQ ID NO: 4-6, SEQ ID NO: 10-12, or a combination thereof.

In another preferred example, the length of Z6 is preferably 100nt-150nt, more preferably 110nt-130nt, more preferably 120nt.

In a third aspect of the present invention, a universal UTR element is provided, and the universal UTR element includes:

(a) 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; and/or

(b) Universal 3'-UTR, wherein the sequence of the universal 3'-UTR is selected from the nucleotide sequence shown in SEQ ID NO: 4, 5, 6, 10, 11 or 12 or derivatives thereof sequence.

In another preferred embodiment, 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.

In another preferred embodiment, (a) and (b) can be derived from the same transcript.

In another preferred embodiment, (a) and (b) can be derived from different transcripts.

In the fourth aspect of the present invention, a carrier is provided, which carrier contains the universal scaffold as described in the second aspect of the present invention.

In another preferred embodiment, the vector 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 pUC57-Amp vector or pUC-Kana vector.

In the fifth aspect of the present invention, a host cell is provided, the 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.

In another preferred embodiment, the host cells include prokaryotic cells or eukaryotic cells.

In another preferred embodiment, the host cell is selected from the following group: Escherichia coli, yeast cells, and mammalian cells.

In the sixth aspect of the present invention, an engineered cell is provided. The 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.

In another preferred embodiment, the engineered cells are stable3 Escherichia coli competent cells.

In another preferred embodiment, the target gene fragment contains homologous arm sequences.

In another preferred embodiment, the vector or universal framework contains homologous arm sequences.

In another preferred embodiment, homologous recombination occurs between the target gene fragment and the vector or universal scaffold.

In another preferred embodiment, the target gene fragment is connected to the vector or universal scaffold and circularized.

In another preferred embodiment, 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.

In a seventh aspect of the present invention, a method for producing optimized mRNA for preparing a vaccine is provided, comprising the steps:

(a) Under appropriate conditions, culture the engineered cells as described in the sixth aspect of the present invention, thereby obtaining a culture containing a vector for transcribing DNA templates;

(b) Isolating and/or recovering the vector described in (a) from the culture, and enzymatically digesting and linearizing it into a DNA template;

(c) transcribing the DNA template described in (b) to obtain the optimized mRNA; and

(d) Optionally, purify and/or modify the optimized mRNA obtained in step (c).

In an eighth aspect of the present invention, a method for preparing an mRNA vaccine is provided, which method includes the steps:

(i) Obtain optimized mRNA by the method described in the seventh aspect of the present invention;

(ii) Mix the optimized mRNA obtained in (i) with a pharmaceutically acceptable carrier to obtain the mRNA vaccine.

In a ninth aspect of the present invention, a kit is provided, which kit includes:

(a) a first plasmid or a linearized DNA fragment thereof, which contains the gene of interest; and

(b) a second plasmid containing a universal scaffold according to the second aspect of the invention; and

(c) Instructions describing a method of using said first plasmid or a linearized DNA fragment thereof and a second plasmid to produce optimized mRNA useful in the preparation of a vaccine.

In another preferred embodiment, the description also describes a method of using the first plasmid as a template to amplify the first fragment with the homology arm sequence.

In another preferred embodiment, the description also describes a method of using the second plasmid as a template to amplify a second fragment with a homology arm sequence.

In another preferred embodiment, 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.

In another preferred embodiment, the description also describes a method for obtaining optimized mRNA from the host cell.

In another preferred embodiment, the present invention also provides a kit, which includes:

(Z1) a plasmid containing a universal scaffold as described in the second aspect of the present invention; and

(Z2) Instructions describing a method of using said plasmid to produce optimized mRNA useful in the preparation of a vaccine.

In a tenth aspect of the present invention, an mRNA vaccine composition is provided, which vaccine composition contains:

(a) mRNA for expressing an immunogen, said mRNA comprising a general scaffold as described in the second aspect of the invention; and

(b) Pharmaceutically acceptable carrier.

In another preferred embodiment, 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.

In another preferred embodiment, the mRNA itself in the vaccine composition can also serve as an adjuvant.

In another preferred embodiment, the dosage form of the vaccine composition is selected from the following group: injection and lyophilized agent.

In another preferred embodiment, 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.

In the eleventh aspect of the present invention, there is provided 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.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described one by one here.

Description of the drawings

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.

Detailed ways

After extensive and in-depth research and extensive screening, 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.

the term

In order that this disclosure may be more easily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning given below unless expressly stated otherwise herein.

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.

As used herein, "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.

As used herein, "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.

Universal scaffolds and expression vectors

As used herein, 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.

Typically, the general-purpose skeleton of the present invention has the structure of formula I:
Z1-Z2-Z3-Z4-Z5-Z6-Z7 (I)

Among them, Z1 to Z7 are as described above.

A schematic structural diagram of a representative carrier containing the universal scaffold of the present invention is shown in Figure 1.

It should be understood that 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. In the present invention, the ORF of the foreign protein can be placed in the universal framework of the present invention, thereby achieving efficient expression. Typically, the ORF carries a stop codon. However, if desired, one or more additional stop codons can also be introduced, as shown in Figure 6.

mRNA vaccine types

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.

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. There are currently two types of saRNA vaccines: saRNA based on DNA plasmids and saRNA delivered by virus-like particles. Based on saRNA, 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. Compared with self-amplifying RNA, non-amplified RNA is smaller, expresses antigens more specifically and does not cause non-specific immunity.

mRNA vaccine immunogenicity

A major challenge for mRNA vaccines is reducing the immunogenicity of the exogenous mRNA itself. Under natural circumstances, after exogenous mRNA enters cells, it can be recognized by retinoic acid-inducible gene I (RIG-I), activate the innate immune response, and then be degraded. In vitro transcription (IVT) 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, m1ψ).

In addition, 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. However, 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. The purpose of purification is mainly to remove truncated transcripts. A good example is that Pardi et al. designed to purify m1ψ-modified mRNA encoding anti-HIV-1 antibodies through HPLC through lipid nanoparticles (LNP) to help mice avoid HIV-1 infection.

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 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. In addition, nucleotide modifications and synonymous substitutions of codons can also affect the stability and translation activity of mRNA. At the same time, sequence optimization may affect the secondary structure and post-translational modification of mRNA. In addition, increasing the GC content of mRNA can also increase mRNA stability. In summary, 5'-UTR, 3'-UTR, 5'Cap, polyA tail, codon optimization and GC content are all modifiable sites that enhance mRNA stability.

mRNA delivery

There are currently many delivery methods for mRNA. Scientists have established methods such as liposome transport, polymer transport, peptide chain transport, virus-like replicon particle transport, and cationic nanoemulsifier transport. In addition, naked mRNA can also be injected directly into cells. . The most commonly used delivery method for mRNA vaccines under development is lipid nanoparticles (LNP) delivery. This method has the advantages of low toxicity and high delivery efficiency.

mRNA vaccines activate innate and adaptive immunity

By expressing antigens in somatic cells and presenting them through the antigen presentation system, 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). After the antigen is expressed on the cell surface, it can also be recognized by B cell receptors and activate the antibody expression of B cells and the formation of memory B cells, thereby causing the apoptosis of infected cells and the neutralization of pathogens.

Events that generate adaptive immune responses after mRNA vaccination

a) After the vaccine’s 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.

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 leading to the formation of germinal centers where memory B cells and antibody-producing plasma cells are produced. These cells reside in the bone marrow.

Advantages of mRNA vaccines against infectious diseases

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.

Optimization strategy for mRNA vaccines

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. For the design of 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. For the 3'-UTR, 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→Ψ changes, followed by the 139nt mtRNR1 sequence. At present, the truly effective UTR design method is to learn from natural genes and optimize based on experience (PMID: 34358150).

In addition, the method of adding 1-2 stop codons after the stop codon of the antigen protein has also been adopted to more effectively depolymerize the translation elongation complex and facilitate the translation and stability of mRNA. 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.

Infectious disease mRNA vaccines

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.

In addition, many 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. These 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.

Antibody gene UTR

In order to improve the versatility of UTR and reduce immunogenicity, 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 μ chain, δ chain, γ chain, α chain and ε chain respectively. Compared with these heavy chains There are two types of coordinated light chains, namely kappa (κ) chain and lambda (λ) chain. According to the differences in individual amino acids in the constant region of the λ chain, the λ chain can be divided into four subtypes: λl, λ2, λ3 and λ4. . 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.

In the present invention, 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.

In the present invention, 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 preferred 3'-UTR based on the sequence optimization of the antibody gene UTR, its nucleoside The acid sequence is shown in SEQ ID NO: 4, 5 or 6.

interferon gene UTR

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.

In the present invention, 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.

In the present invention, 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 main advantages of the present invention include:

(1) By constructing a plasmid with a replaceable ORF template backbone, the development of mRNA vaccines can be accelerated.

(2) By selecting 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 present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the invention and are not intended to limit the scope of the invention. Experimental methods without specifying specific conditions in the following examples usually follow conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to manufacturing Conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

Example 1. Screening and optimization method of UTR sequences

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.

Typically, this vector includes a vector backbone, AleI restriction site or BspQI restriction site, 5'-UTR, ORF, 3'-UTR, and polyA. In addition, 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.

Based on the expression characteristics of antibodies and interferons, 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.

Table 1 Participating comparison sequences and their numbers

Example 2. Selection and optimization of 5'-UTR and 3'-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.

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 above sequence information is shown in Table 2:

Table 2 Wild-type sequence and optimized sequence

Since 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. In addition, 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.

Example 3. Construction of hirudin mRNA vaccine engineering strain

(1) Use the plasmid containing the hirudin gene as a template, and PCR amplify the hirudin gene (Hirudin) fragment from it, as shown in Figure 7, introduce the homology arm sequence, T7 promoter, and AleI digestion through primers locus, GFP and 6×His tags.

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.

As shown in Figure 9, gene synthesis fragments and PCR products were electrophoresed in agarose gels for identification.

(2) Obtain the two fragments obtained in (1) through gel recovery, then use homologous recombination to connect, circularize and transfer them into stable3 Escherichia coli competent cells to construct engineering bacteria, select single clones and culture them into bacterial liquid. Sanger sequencing was performed to identify the engineering bacteria.

As shown in Figure 10, the plasmid extracted from the successfully constructed engineering bacteria was subjected to agarose gel electrophoresis.

As shown in Figure 11, the plasmid extracted from the successfully constructed engineering bacteria was digested with AleI enzyme.

Example 4. Identification of leech plasmid engineering bacteria

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.

Table 3 Bacterial plasmid copy number of hirudin engineering bacteria

As shown in Table 3, bacterial plasmid copy number detection shows that the plasmid copy number of the engineering bacteria is approximately 53.17-240.28 copies/cell.

Table 4 Test results of bacterial plasmid loss rate of leech plasmid engineered bacteria

As shown in Table 4, the bacterial plasmid loss rate test showed that no plasmid loss occurred in the engineering bacteria during the passage process.

Example 5. Fermentation plasmid yield of hirudin engineering bacteria

In the 5L fermentation tank, add 3L LB culture medium, and perform bacterial liquid fermentation under the conditions of tank pressure 0.05±0.02MPa, ventilation 2-10L/min, stirring speed 100-250rpm, temperature 37±0.5℃, and pH 7.0±0.1. During the 10-hour fermentation process, take 2 tubes of 5mL bacterial liquid every hour for plasmid extraction. The plasmid concentration results are shown in Table 5 and Figure 12.

Table 5 Plasmid yield during fermentation process of hirudin engineering bacteria

The experimental results shown in Figure 12 show that the highest plasmid yield can be obtained by fermenting the seed bacteria for 4 hours.

Example 6. Verification of in vitro transcription efficiency

The transcription template plasmid constructed using the universal UTR element of the present invention has a high-level in vitro transcription effect. Referring to the construction method of Example 3, 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. In vitro transcription (IVT) experiments were performed based on this plasmid.

After linearizing the plasmid using BspQI, the linearized DNA fragment was used as the template for IVT. T7 Polymerase, NTPs, RNase inhibitor, inorganic pyrophosphatase PPasse and Corresponding buffer system. After reacting at a constant temperature of 37°C for 3 hours, and purifying using the LiCl precipitation method, 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.

A total of 8 batches of IVT reactions and purification were carried out. The input amount of template DNA in the reaction was 2.20 μg, the RNA yield was 0.27-0.40 mg, and the ratio of RNA yield/DNA template amount (transcription efficiency) was about 122-182 times. The transcription template plasmid constructed by the skeleton in the present invention has high in vitro transcription activity in in vitro transcription.

Table 6 IVT mRNA yield using universal scaffolds to construct transcription templates (DNA input amount is 2.20 μg)

Example 7. Verification of expression ability of mRNA obtained by construction

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. Two transfection reagents, transfection reagent A (Lipofectamine ^TM MessengerMAX ^TM ) and transfection reagent B (YEASEN-Hieff Trans mRNA), were used at the same time to conduct the mRNA transfection experiment on 293T cells. The amount of mRNA used was 2 μg/well, and the number of cells in the 6-well plate was 10 ⁶ cells/well.

Two days after transfection, collect the cell pellet in a 1.5 mL centrifuge tube, add lysis buffer (RIPA:PMSF=99:1), lyse on ice for 5 minutes, and collect the protein lysate. After using the BCA method to detect the protein concentration, add 6×Protein Loading buffer (full gold, DL101-02), and heat and denature in boiling water for 10 minutes.

Denatured protein samples were subjected to WB experiments, and 7.5% SDS-PAGE gel was electrophoresed. A total of approximately 20 μg of protein was added to each well. Anti-His mouse monoclonal antibody (Beijing Quanshijin, HL102-01) and HRP-labeled Goat Anti-Mouse IgG (H+L) were used as secondary antibodies (1:5000, diluted in PBS) to label rabies virus G protein. After the incubation is completed, use ultra-sensitive ECL luminescent solution (Yase, SQ201) for development.

As shown in Figure 13, 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.

Referring to the construction method of Example 3, 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 A recombinant engineered bacterium was constructed on the vector skeleton), and a rabies virus mRNA experimental vaccine was produced based on this recombinant engineered bacterium.

As shown in Figure 14, the results of agarose gel electrophoresis and enzyme digestion map verification of the plasmid extracted from the successfully constructed engineering bacteria are shown. Among them, lane P1 is the plasmid band, and 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.

As shown in Figure 15, the comparison results between the plasmid sequence extracted from the successfully constructed engineering bacteria and the designed sequence are shown. The comparison results showed that the sequencing results were completely consistent with the designed sequence.

The above results show that the rabies virus mRNA vaccine engineering strain has been successfully constructed.

Next, 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 μg/mouse) intramuscularly; the high-dose group, 16 mice (8 males and 8 females), received a higher dose intramuscularly. mRNA vaccine (13 μg/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".

As shown in Table 7, 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.

Table 7 Mouse serum anti-rabies virus neutralizing antibody levels

All documents mentioned in this application are incorporated by reference in this application to the same extent as if each individual document was individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims (10)

1. The use of a universal framework, characterized in that the universal framework is used to construct mRNA transcripts, wherein the transcripts include the ORF to be expressed and the 5'-UTR regions located on both sides of the ORF and a 3'-UTR region, wherein one or both of the 5'-UTR region and the 3'-UTR region are universal UTRs.
2. A general-purpose skeleton, characterized in that the general-purpose skeleton has the structure of formula I:
   Z1-Z2-Z3-Z4-Z5-Z6-Z7 (I)

   In the formula,

   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.
3. A universal UTR element, characterized in that the universal UTR element includes:

   (a) 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; and/or

   (b) Universal 3'-UTR, wherein the sequence of the universal 3'-UTR is selected from the nucleotide sequence shown in SEQ ID NO: 4, 5, 6, 10, 11 or 12 or derivatives thereof sequence.
4. A carrier, characterized in that the carrier contains the universal skeleton as claimed in claim 2.
5. A host cell, characterized in that the host cell contains the vector according to claim 4, or the universal framework according to claim 2 is integrated into its genome.
6. An engineered cell, characterized in that the engineered cell contains: the vector according to claim 4, or the universal scaffold according to claim 2 integrated into its genome, and contains a target gene fragment.
7. A method of producing optimized mRNA for use in preparing a vaccine, comprising the steps of:

   (a) Under suitable conditions, culture the engineered cells as claimed in claim 6 to obtain a culture containing a vector that transcribes the DNA template;

   (b) Isolating and/or recovering the vector described in (a) from the culture, and enzymatically digesting and linearizing it into a DNA template;

   (c) transcribing the DNA template described in (b) to obtain the optimized mRNA; and

   (d) Optionally, purify and/or modify the optimized mRNA obtained in step (c).
8. A method for preparing an mRNA vaccine, characterized in that the method includes the steps:

   (i) Obtain optimized mRNA by the method as claimed in claim 7;

   (ii) Mix the optimized mRNA obtained in (i) with a pharmaceutically acceptable carrier to obtain the mRNA vaccine.
9. A test kit, characterized in that the test kit includes:

   (a) a first plasmid or a linearized DNA fragment thereof, which contains the gene of interest; and

   (b) a second plasmid containing the universal scaffold of claim 2; and

   (c) Instructions describing a method of using said first plasmid or a linearized DNA fragment thereof and a second plasmid to produce optimized mRNA useful in the preparation of a vaccine.
10. An mRNA vaccine composition, characterized in that the vaccine composition contains:

    (a) mRNA for expressing an immunogen, said mRNA comprising a universal scaffold as claimed in claim 2; and

    (b) Pharmaceutically acceptable carrier.