An optimized polyA sequence structure and its applications

Abstract

The present invention relates to an optimized polyA sequence and its applications. Specifically, the present invention provides a polyA tail element, and the 5'-end to 3'-end structure of the polyA tail element comprises the following elements: 2 to 7 successively connected X elements, at least 1 Y element, and 1 to 10 A bases located at the 3'-end. The polyA sequence of the present invention can improve the stability of mRNA, improve the translation efficiency of the protein coding region, thereby reducing the mRNA drug dosage and the side effects brought by the delivery system, and is used for the preparation of mRNA drugs.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and nucleic acid drugs, and specifically, to an optimized polyA sequence structure and its applications. Background Art

[0002] As an emerging vaccine, mRNA vaccine belongs to nucleic acid vaccines and is the third-generation vaccine developed on the basis of the first-generation live attenuated vaccines or inactivated vaccines and the second-generation subunit vaccines. Compared with DNA-based gene therapies, RNA does not need to enter the nucleus for expression, thus avoiding the risk of insertional mutations caused by integration into the host genome. In addition, mRNA has advantages such as greater simplicity in design and manufacturing, rapid mass production, and inherent immunogenicity. mRNA vaccines can not only be used for emerging infectious diseases, but also meet the prevention and treatment needs of major diseases such as genetic metabolic diseases and tumors. Therefore, establishing a rapid, accurate, and personalized new RNA drug design and vector R & D platform can better meet the market demand and provide more efficient and convenient preventive or therapeutic drugs for the population.

[0003] However, several factors limit the clinical applicability of synthetic mRNA, such as the short half-life of mRNA in cells and low protein production efficiency. Currently, some strategies have proven to be possible in increasing mRNA stability and translation efficiency. For example, the use of artificial cap analogs, optimization of UTRs, and the use of nucleotides including chemical modifications: such as 2-thiouridine and 5-methyl-cytidine.

[0004] Consistent with endogenous mRNA, in vitro transcribed mRNA also contains five parts: the 5' cap structure, the 5' untranslated region (5' UTR), the open reading frame (ORF) encoding the protein, the 3' untranslated region (3' UTR), and the polyadenylate tail (polyA), and these several parts will all have a certain impact on the stability and translation activity of mRNA vaccines.

[0005] In the cell nucleus, after mRNA is transcribed, it needs to undergo a series of processing to complete terminal modification. Except for the transcripts of individual mammalian histones, almost all eukaryotic mRNAs carry a polyA tail. The process of adding the polyA tail involves an endonuclease cleavage between the AAUAAA sequence upstream of the cleavage site and the downstream sequence rich in GU or U, generating an upstream sequence with a 3' OH at the end and a downstream sequence with a 5' phosphate group at the end. Polyadenylation occurs at the end of the upstream sequence, adding a polyA tail, and the downstream sequence is degraded. The polyA tail does not exist in the gene but is the end product of the polyadenylation reaction after internal cleavage at the 3' end of pre-mRNA. Its length varies, approximately 90 adenylate residues in yeast and about 250 adenylate residues in mammals.

[0006] In vitro mRNA synthesis, there are mainly two ways to add a polyA tail: enzymatic synthesis and co-transcription. Enzymatic synthesis is carried out by adding polyA polymerase derived from Escherichia coli, but it is difficult to ensure that the reaction end products contain tails of the same length. The resulting mRNA mixture carries tails of different lengths, and this enzyme requires an alkaline environment to function, which may cause a certain degree of mRNA degradation. Co-transcription is directly formed by transcribing the polyA sequence already present on the template plasmid DNA or PCR product. Its end products can largely maintain homogeneity, reduce process steps, and save costs.

[0007] However, there is still a major challenge in co-transcriptional tailing. Homopolymeric sequences (such as the sequence encoding the polyA tail) can recombine during the bacterial amplification of plasmid DNA, which may lead to the unpredictable loss of the tail over time. Therefore, the integrity of the plasmid template and polyA should be regarded as an important quality attribute in fermentation process development.

[0008] Therefore, there is an urgent need in the art to develop a polyA sequence that can be used as a template for large-scale in vitro transcription, which is definite and stable, and can improve the translation efficiency of the protein-coding region, providing technical support for the preparation of high-quality mRNA drugs. Summary of the Invention

[0009] The object of the present invention is to provide a polyA sequence that can be used as a template for large-scale in vitro transcription, which is definite and stable, and can improve the translation efficiency of the protein-coding region, and its application in the preparation of mRNA drugs.

[0010] In the first aspect of the present invention, a polyA tail element is provided. The 5'-to-3' end structure of the polyA tail element comprises the following elements:

[0011] (a) 2 to 7 successively connected X elements, each of which is independently composed of 10 to 70 consecutive A bases and 1 non-A base connected in sequence;

[0012] (b) at least 1 Y element, each of which is independently composed of 10 to 70 consecutive A bases and 10 to 30 consecutive C bases; and

[0013] (c) 1 to 10 A bases at the 3' end;

[0014] Wherein, the non-A base is preferably a T base or a G base.

[0015] In another preferred example, the 5'-end to 3'-end sequence of the polyA tail element has the following structure:

[0016] Xn-Y-Z;

[0017] Wherein, n is any positive integer selected from 2 to 7;

[0018] Each X is independently composed of 10 to 70 (preferably 20 to 50, more preferably 20 to 40) consecutive A bases and 1 non-A base (preferably a T, G or C base, more preferably a T or G base);

[0019] Y is composed of 10 to 70 consecutive A bases and 10 to 30 consecutive C bases;

[0020] Z is 1 to 10 A bases at the 3' end.

[0021] In another preferred example, the Z is 1 A base at the 3' end.

[0022] In another preferred example, the 5'-end to 3'-end sequence of the polyA tail element has the following structure:

[0023] X1-X2-X3-Y-Z;

[0024] Wherein,

[0025] X1 is composed of 10 to 19 (preferably 15 to 19, more preferably 16 to 19) consecutive A bases and 1 non-A base (preferably a T, G or C base, more preferably a T or G base);

[0026] X2 is composed of 20 to 40 (preferably 25 to 40, more preferably 25 to 30) consecutive A bases and 1 non-A base (preferably a T, G or C base, more preferably a T or G base);

[0027] X3 is either absent or consists of 20 to 50 (preferably 25 to 50, more preferably 30 to 49) consecutive A bases and 1 non-A base (preferably a T, G or C base, more preferably a G or C base) connected in sequence;

[0028] Y consists of 10 to 70 consecutive A bases and 10 to 30 consecutive C bases connected in sequence, independently of each other;

[0029] Z is 1 to 10 A bases located at the 3' end.

[0030] In another preferred example, the nucleotide sequence of X1 is positions 1 to 20 relative to the sequence shown in SEQ ID NO:1.

[0031] In another preferred example, the nucleotide sequence of X2 is positions 21 to 51 relative to the sequence shown in SEQ ID NO:1.

[0032] In another preferred example, the nucleotide sequence of Y is positions 52 to 120 relative to the sequence shown in SEQ ID NO:1.

[0033] In another preferred example, Z is 1 A base located at the 3' end.

[0034] In another preferred example, the nucleotide sequence length of the polyA tail element is 121 bases.

[0035] In another preferred example, the polyA tail element is used to construct an mRNA transcription template to improve the translation efficiency of the coding region in the mRNA transcribed from the mRNA transcription template and / or extend the half-life of the mRNA.

[0036] In another preferred example, the polyA tail element is used to construct plasmid DNA to improve the passage stability of the plasmid DNA, reduce the recombination occurring during the amplification of the plasmid DNA, and maintain the polyA tail length of the plasmid DNA.

[0037] In another preferred example, the nucleotide sequence of the polyA tail element is as shown in SEQ ID NO: 1 or SEQ ID NO:2, or has a homology of ≥70% with the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO:2.

[0038] In another preferred embodiment, the nucleotide sequence of the polyA tail element comprises: a nucleotide sequence having ≥70% (preferably ≥80%, more preferably ≥90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) homology with the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 and having the activity of enhancing the translation efficiency of the coding region in mRNA; or

[0039] a nucleotide sequence in which 1-50 (preferably 1-30, more preferably 1-10, still more preferably 1-5) nucleotides are added and / or deleted at the 5' end and / or 3' end of the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 and having the activity of enhancing the translation efficiency of the coding region in mRNA.

[0040] In another preferred embodiment, the nucleotide sequence of the polyA tail element is as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0041] In another preferred embodiment, the nucleotide sequence of the polyA tail element is as shown in SEQ ID NO: 1.

[0042] In the second aspect of the present invention, there is provided an mRNA transcription template construct having the structure of formula I:

[0043] Z1-Z2-Z3-Z4-Z5-Z6-Z7 (I)

[0044] In the formula,

[0045] Z1 and Z7 are none or restriction enzyme sites;

[0046] Z2 is none or a promoter element or an internal ribosome entry site sequence (IRES);

[0047] Z3 is a 5' UTR element;

[0048] Z4 is an alternative coding region;

[0049] Z5 is a 3' UTR element;

[0050] Z6 is the polyA tail element described in the first aspect of the present invention.

[0051] In another preferred embodiment, Z1 and Z7 are blunt-end restriction enzyme sites or sticky-end restriction enzyme sites.

[0052] In another preferred embodiment, Z2 is a promoter element, and the promoter is selected from the group consisting of: T7 promoter, T3 promoter, SP6 promoter, CAG promoter, UBC promoter, CMV promoter, U6 promoter, EF1a promoter, PGK1 promoter, TRE promoter, Ac5 promoter, UAS promoter, SV40 promoter, ADH1 promoter, CaMV35S promoter, Ubi promoter, Lac promoter, Ptac promoter, pL promoter, or a combination thereof.

[0053] In another preferred embodiment, the nucleotide sequence of Z2 is as shown in SEQ ID NO: 9.

[0054] In another preferred embodiment, Z4 is a luciferase-encoding gene or a protein-encoding gene for preventing and / or treating infectious diseases, rare genetic diseases, neurodegenerative diseases, retinopathies, cancers or tumors.

[0055] In another preferred embodiment, Z4 is selected from the group consisting of: luciferase-encoding gene, pathogen antigen gene, transposons in the genome (including silent and active transposons), cytokines, growth factors, protein hormones, polypeptide hormones, tumor-associated antigens (TAAs), tumor-specific antigens (TSAs), universal tumor mutation site antigens, protein adjuvants, polypeptide adjuvants, nucleic acid adjuvants, or a combination thereof.

[0056] In another preferred embodiment, the nucleotide sequence of Z3 is as shown in SEQ ID NO: 10.

[0057] In another preferred embodiment, the nucleotide sequence of Z4 is as shown in SEQ ID NO: 11.

[0058] In another preferred embodiment, the nucleotide sequence of Z5 is as shown in SEQ ID NO: 12.

[0059] In the third aspect of the present invention, a vector is provided, and the vector contains the mRNA transcription template construct as described in the second aspect of the present invention.

[0060] In another preferred embodiment, the vector is selected from the group consisting of: DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or a combination thereof; preferably, the vector is a plasmid.

[0061] In the fourth aspect of the present invention, a host cell is provided, and the host cell contains the vector as described in the third aspect of the present invention.

[0062] In another preferred embodiment, the host cell includes prokaryotic cells or eukaryotic cells.

[0063] In another preferred embodiment, the host cell is selected from the group consisting of: Escherichia coli, yeast cells, mammalian cells.

[0064] In a fifth aspect of the present invention, there is provided a method for producing optimized mRNA for preparing mRNA drugs, the method comprising the steps of:

[0065] (i) Culturing the host cell as described in the fourth aspect of the present invention under suitable conditions to obtain a culture containing a vector with an mRNA transcription template construct;

[0066] (ii) Isolating and / or recovering the vector described in (i) from the culture and linearizing it by enzymatic digestion into an mRNA transcription template; and

[0067] (iii) Transcribing the mRNA transcription template described in (ii) to obtain the optimized mRNA.

[0068] In another preferred embodiment, the method further comprises step (iv): purifying and / or modifying the optimized mRNA obtained in step (iii).

[0069] In a sixth aspect of the present invention, there is provided an optimized mRNA, which is prepared by the method as described in the fifth aspect of the present invention, and the optimized mRNA has the structure shown in Formula II:

[0070] M1-M2-M3-M4-M5-M6 (II)

[0071] In the formula,

[0072] M1 is a 5'-end cap element;

[0073] M2 is absent or an internal ribosome entry site sequence (IRES);

[0074] M3 is a 5' UTR element;

[0075] M4 is an alternative coding region;

[0076] M5 is a 3' UTR element;

[0077] M6 is the polyA tail element described in the first aspect of the present invention.

[0078] In another preferred embodiment, the M4 is a luciferase coding gene or a protein coding gene for preventing and / or treating infectious diseases, rare genetic diseases, neurodegenerative diseases, retinopathies, cancers or tumors.

[0079] In another preferred embodiment, M4 is selected from the group consisting of: luciferase-encoding genes, pathogen antigen genes, transposons in the genome (including silent and active transposons), cytokines, growth factors, protein hormones, polypeptide hormones, tumor-associated antigens (TAAs), tumor-specific antigens (TSAs), universal tumor mutation site antigens, protein adjuvants, polypeptide adjuvants, nucleic acid adjuvants, or combinations thereof.

[0080] In a seventh aspect of the present invention, there is provided a pharmaceutical composition comprising:

[0081] (c1) The mRNA transcription template construct as described in the second aspect of the present invention, or the optimized mRNA as described in the sixth aspect of the present invention, as an active ingredient; and

[0082] (c2) A pharmaceutically acceptable carrier.

[0083] In another preferred embodiment, the pharmaceutical composition is a vaccine composition.

[0084] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of: injections, lyophilized products, aerosol inhalation agents, and topical medications.

[0085] In another preferred embodiment, the pharmaceutical composition is administered by injection, i.e., intravenous, intramuscular, intradermal, subcutaneous, intrathecal, intraduodenal, or intraperitoneal injection.

[0086] In another preferred embodiment, the pharmaceutical composition is administered by inhalation, such as intranasal administration.

[0087] In another preferred embodiment, the pharmaceutical composition is administered transdermally, such as by transdermal application or iontophoresis.

[0088] In another preferred embodiment, the pharmaceutical composition is a lipid nanoparticle formed by encapsulating the mRNA with a cationic lipid, i.e., LNP-mRNA.

[0089] In another preferred embodiment, the vaccine composition comprises 0.01-99.99% of the optimized mRNA as described in the sixth aspect of the present invention and 0.01-99.99% of a pharmaceutically acceptable carrier, and the percentages are by mass of the vaccine composition.

[0090] In another preferred embodiment, the pharmaceutical composition is used for preparing a drug for preventing and / or treating (but not limited to) infectious diseases, rare genetic diseases, neurodegenerative diseases, retinopathies, cancers or tumors.

[0091] In the eighth aspect of the present invention, a method for preparing an mRNA pharmaceutical composition is provided, the method comprising: mixing the optimized mRNA as described in the sixth aspect of the present invention with a pharmaceutically acceptable carrier to obtain the mRNA pharmaceutical composition.

[0092] In another preferred embodiment, the mRNA pharmaceutical composition is an mRNA vaccine composition.

[0093] In the ninth aspect of the present invention, a use of the polyA tail element as described in the eighth aspect of the present invention is provided, for constructing an mRNA transcription template DNA plasmid, thereby improving the passage stability of the template DNA plasmid, and / or improving the translation efficiency of the coding region in the mRNA transcribed from the mRNA transcription template, and / or extending the half-life of the mRNA.

[0094] In the tenth aspect of the present invention, a use of the optimized mRNA as described in the sixth aspect of the present invention or the pharmaceutical composition as described in the seventh aspect of the present invention is provided, for preparing a drug for preventing and / or treating infectious diseases, rare genetic diseases, neurodegenerative diseases, retinopathies, cancers or tumors.

[0095] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Figure 1 The results of the passage stability of plasmids containing different polyA in Escherichia coli are shown. Among them, A is the passage of plasmids with different polyA in Escherichia coli, and at the corresponding passage times, the proportion of clones with no loss of polyA, that is, completely correct clones. The abscissa represents the passage times, such as P1 is the first generation and P30 is the 30th generation; B is the passage of plasmids with different polyA in Escherichia coli, and the result after summing up the proportions of clones with no loss of polyA, that is, completely correct clones, at different passage times (P1 - P30).

[0097] Figure 2 The results of the activity assay of luciferase mRNA transfected into 293T cells using different polyA are shown. Among them, A is the results of the activity assay of luciferase mRNA transfected into 293T cells at 24h, 48h, 72h, and 120h using different polyA; B is the results of the activity assay of luciferase mRNA transfected into 293T cells at 120h using different polyA. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0098] After extensive and in-depth research, the inventor of the present invention obtained an optimized polyA sequence structure through screening and optimization. Experiments have proved that the polyA sequence described in the present invention (such as the nucleotide sequence shown in SEQ ID NO: 1) can be used to construct an mRNA transcription template plasmid, and the template plasmid can be used for large-scale in vitro transcription; it can effectively reduce the recombination of plasmid DNA during amplification, and has a polyA sequence with definite and stable passaging; at the same time, this polyA can improve the translation efficiency of the coding region in the mRNA transcribed from the mRNA transcription template and extend the half-life. Furthermore, it reduces the mRNA drug dose and the potential side effects brought by the delivery system. On this basis, the present invention was completed.

[0099] Specifically, the nucleotide sequence (from the 5' end to the 3' end) structure of the polyA sequence provided by the present invention is a multi-segment type with greater than or equal to 3 segments and containing a continuous C base structure: except for the two segments near the 3' end, other segments are continuous A sequences, which are connected by non-A bases in the middle, the penultimate segment (i.e., the second segment near the 3' end) is a continuous C base, and there is no other base connection between it and the previous segment. In addition, the last segment (i.e., the first segment near the 3' end) is 1 to 10 non-C bases, preferably 1 A base.

[0100] Term

[0101] To make it easier to understand the present disclosure, certain terms are first defined. As used in this application, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below.

[0102] PolyA tail element

[0103] In one aspect of the present invention, a polyA tail element for constructing an mRNA transcription template is provided. As used herein, the terms "polyA tail element" and "polyA element" can be used interchangeably. Based on the original polyA tail sequence, the polyA tail element of the present invention has a multi-segment type with greater than or equal to 3 segments and containing a continuous C base structure: except for the last segment, other segments are continuous A sequences, which are connected by non-A bases in the middle, the last segment is a continuous C base, and there is no other base connection between the last segment and the previous segment. In addition, the last base is a non-C base. The nucleotide sequence of the polyA tail element of the present invention has a homology of ≥70% with the nucleotide sequence shown in SEQ ID NO: 1. Preferably, the nucleotide sequence of the polyA tail element of the present invention is as shown in SEQ ID NO: 1.

[0104] mRNA transcription template construct

[0105] As used herein, the terms "mRNA transcription template" and "mRNA transcript" are used interchangeably.

[0106] In another aspect of the present invention, there is provided an mRNA transcription template construct having a structure of Formula I:

[0107] Z1-Z2-Z3-Z4-Z5-Z6-Z7 (I)

[0108] Wherein,

[0109] Z1 and Z7 are either absent or restriction enzyme sites;

[0110] Z2 is either absent or a promoter element or an internal ribosome entry site sequence (IRES);

[0111] Z3 is a 5' UTR element;

[0112] Z4 is an alternative coding region;

[0113] Z5 is a 3' UTR element;

[0114] Z6 is the polyA tail element described in the first aspect of the present invention.

[0115] As used herein, the terms "5'-UTR" and "5' UTR" are used interchangeably; "3'-UTR" and "3' UTR" are used interchangeably.

[0116] As used herein, the term "promoter" or "promoter region" refers to a nucleic acid sequence that accurately and effectively initiates gene transcription function, guiding the transcription of gene nucleic acid sequence into RNAs. It is usually present upstream (5' end) of the coding sequence of the target gene. Generally, the promoter or promoter region provides recognition sites for RNA polymerase and other factors necessary for correct initiation of transcription.

[0117] The promoter of the present invention can be operably linked to a foreign gene, and the foreign gene can be foreign (heterologous) relative to the promoter. The foreign gene (also referred to as the target gene) is not particularly limited, and representative examples include (but are not limited to): selection marker genes, resistance genes, antigen protein genes, and biological agent genes, etc.

[0118] In one embodiment of the present invention, the mRNA transcription template construct has a structure of Formula I, wherein Z1 and Z7 are restriction enzyme sites, Z2 is a promoter element or an internal ribosome entry site sequence (IRES), Z3 is a 5' UTR sequence, Z4 is an alternative coding region (such as the hEPO coding sequence or GFP coding sequence used in the screening in the examples), and Z5 is a 3' UTR sequence.

[0119] Optimized mRNA

[0120] Sequence optimization of mRNA is one of the methods to help improve the translation efficiency of mRNA. Sequence optimization of the 5'-UTR and 3'-UTR of mRNA can increase the half-life and translation activity of mRNA. Using different analogs for the Cap structure can increase the stability of mRNA, and adding a Cap structure to the 5' end of mRNA using an enzyme can have better efficacy than different forms of Cap analogs. The stabilizing effect of the polyA tail of mRNA is also very important. Studies have found that removing the polyA of mRNA makes mRNA extremely unstable, and at the same time reduces the number of polysome bindings, elongation speed, and translation rounds of mRNA. Therefore, polyA is crucial for the stability and efficient translation of mRNA. In addition, nucleotide modification and codon optimization 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 key regulatory sites affecting mRNA stability.

[0121] The optimized mRNA of the present invention has the structure shown in Formula II:

[0122] M1-M2-M3-M4-M5-M6 (II)

[0123] In the formula,

[0124] M1 is a 5' end cap element;

[0125] M2 is none or an internal ribosome entry site sequence (IRES);

[0126] M3 is a 5' UTR element;

[0127] M4 is an alternative coding region;

[0128] M5 is a 3' UTR element;

[0129] M6 is the polyA tail element described in the first aspect of the present invention.

[0130] In one embodiment of the present invention, the optimized mRNA has a structure of Formula II, wherein M1 is a 5'-end cap element, M2 is a non-existent or internal ribosome entry site sequence (IRES), M3 is a 5' UTR sequence of the nucleotide sequence shown in SEQ ID NO: 3, M4 is an alternative coding region (such as the hEPO coding sequence or GFP coding sequence used in the screening in the examples), M5 is a 3' UTR element, and its nucleotide sequence is shown in SEQ ID NO: 8; M6 is a polyA tail element, and its nucleotide sequence is shown in SEQ ID NO: 1.

[0131] The pharmaceutical composition and its application of the present invention

[0132] In one aspect of the present invention, a pharmaceutical composition is further provided. In one embodiment of the present invention, the pharmaceutical composition is an mRNA vaccine composition.

[0133] mRNA vaccines are divided into non-replicating RNA (nrRNA) and self-amplifying RNA (saRNA). Classical non-replicating RNA vaccines include a cap structure, 5'-UTR, open reading frame (ORF) (i.e., coding region), 3'-UTR, and polyA tail. The above five regions together determine the stability, translation activity, and immunogenicity of mRNA.

[0134] The structure of saRNA is derived from the α-virus genome. After saRNA enters the cell or body, it first translates into a replicon, and the replicon recognizes the viral element to initiate the transcription of the whole strand and subgenomic coding strands. The saRNA vaccine utilizes the property that the genome of the α-virus can self-replicate to achieve low-dose and long-lasting immune transformation. Based on saRNA, Beissert et al. also developed trans-amplifying RNA (taRNA), which splits saRNA into an mRNA encoding a replicon and an mRNA encoding an antigen gene. Through the mediation of viral elements, the replicon recognizes and amplifies the antigen-encoding strand. On the premise of maintaining the self-amplifying characteristics of saRNA, the modular structure makes the replacement of the antigen gene more convenient and economical.

[0135] One of the major challenges of mRNA vaccines is to reduce the immunogenicity of the exogenous mRNA itself. Under natural conditions, after entering the cell, exogenous mRNA can be recognized by retinoic acid-inducible gene I (RIG-I), activating the innate immune response and then being degraded. In vitro transcription (IVT) mRNA can activate immune cells and the inflammatory response mediated by Toll-like receptor (TLR). The U-rich sequence of mRNA is a key factor in activating Toll-like receptors. Reducing the immunogenicity of mRNA can be achieved through nucleotide chemical modification, adding a polyA tail, and optimizing the GC content of mRNA, etc.

[0136] Chemically modified nucleotides include 5-methylcytidine (m5C), 5-methyluridine (m5U), N1-methyladenosine (m1A), N6-methyladenosine (m6A), 2-thiouridine (s2U), 5-methoxyuridine (5moU), pseudouridine (ψ), and N1-methylpseudouridine (m1ψ), etc.

[0137] Adding a polyA tail can also reduce the U content and thus reduce the immunogenicity of mRNA. Some studies have attempted to transport erythropoietin-encoding mRNA into pigs through lipid nanoparticles. This mRNA has a high GC content and did not cause an obvious innate immune response, and successfully induced erythropoietin-related responses. However, too high a GC content will inhibit the translation activity of mRNA, which also needs to be noted during the vaccine development process.

[0138] The purification method of mRNA is also quite 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 method. The main purpose of purification is to remove truncated transcripts. A good example is that Pardi et al. designed to purify m1ψ-modified mRNA encoding HIV-1 antibodies by HPLC, which helped mice avoid HIV-1 infection through lipid nanoparticles (LNPs).

[0139] There are many current delivery methods for mRNA. Scientists have established methods such as liposome transportation, polymer transportation, peptide chain transportation, virus-like particle transportation, and cationic nanoemulsifier transportation. In addition, naked mRNA can also be directly injected into cells. The most commonly used delivery method for mRNA vaccines under research is lipid nanoparticle transportation. This method has advantages such as low toxicity and high delivery efficiency.

[0140] The "active ingredient" in the pharmaceutical composition of the present invention refers to the mRNA transcription template construct or optimized mRNA described in the present invention. The "active ingredient", preparation and / or composition of the present invention can be used for preventing and / or treating diseases or disorders such as infectious diseases, rare genetic diseases, neurodegenerative diseases, retinopathies, cancers or tumors. "Safe and effective amount" means that the amount of the active ingredient is sufficient to significantly improve the condition or symptoms without causing serious side effects. "Pharmaceutically acceptable carrier" means one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the active ingredient of the present invention and with each other without significantly reducing the drug efficacy of the active ingredient.

[0141] The pharmaceutical composition can be liquid or solid, such as powder, gel or paste. Preferably, the composition is liquid, preferably an injectable liquid.

[0142] Some examples of pharmaceutically acceptable carriers are cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween®), wetting agents (such as sodium dodecyl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0143] The pharmaceutical composition can contain a physiologically acceptable sterile aqueous or non-aqueous solution, dispersion, suspension or emulsion, and a sterile powder for reconstituting into a sterile injectable solution or dispersion. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and their suitable mixtures.

[0144] The pharmaceutical composition of the present invention can be prepared into dosage forms such as injection, freeze-dried preparation, aerosol inhalation preparation, topical medicament, etc. The pharmaceutical composition of the present invention can be delivered (administered) by any suitable means, including oral, parenteral and topical methods. The pharmaceutical composition of the present invention can also be administered by injection, namely intravenous, intramuscular, intradermal, subcutaneous, intraduodenal, intrathecal, or intraperitoneal injection. In addition, the pharmaceutical composition described in the present invention can be administered by inhalation, such as intranasal administration. Additionally, the pharmaceutical composition of the present invention can be administered transdermally. The transdermal administration method through the topical route can be formulated into a medicated stick, solution, suspension, emulsion, gel, cream, ointment, paste, gel, paint, powder and aerosol. Furthermore, the pharmaceutical composition of the present invention can be actively administered to the intradermal, subcutaneous, muscle, tumor, tissue organ, central nerve and other sites through electrodes / electric fields / electric potential differences.

[0145] The pharmaceutical composition of the present invention can be co-administered with another active agent. Co-administration includes administering the compound and the active agent of the present invention within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20 or 24 hours of each other. Co-administration also includes simultaneously, substantially simultaneously (e.g., within about 1, 5, 10, 15, 20 or 30 minutes of each other) or sequentially in any order administering the compound and the active agent of the present invention. In some embodiments, co-administration can be accomplished by co-formulation, i.e., preparing a single pharmaceutical composition containing both the active ingredient (the mRNA transcription template construct or optimized mRNA described in the present invention) and the active agent of the present invention. In other embodiments, the active ingredient and the active agent of the present invention can be formulated separately.

[0146] The present invention also provides the use of the pharmaceutical composition for preparing a medicament for preventing and / or treating diseases, including (but not limited to) infectious diseases, rare genetic diseases, neurodegenerative diseases, retinopathies, cancers or tumors.

[0147] Among them, the infectious diseases include (but not limited to) the following diseases or diseases caused by the following pathogens: botulinum toxin, hirudin, cytomegalovirus (CMV), Zika virus, Influenzavirus, respiratory syncytial virus (RSV), Rabies, human immunodeficiency virus (HIV), Ebola virus, streptococci, malaria, Louping ill virus, Toxoplasma gondii Toxoplasma gondii) Dengue fever, plague, yellow fever, tuberculosis, herpes simplex virus, varicella-zoster virus, mycoplasma, chlamydia, foot-and-mouth disease virus, rotavirus, papillomavirus, poliovirus, coxsackievirus, rhinovirus, hand, foot and mouth disease virus, rubella virus, measles virus, Borna disease virus, retrovirus (T-lymphotropic virus), Nipah virus, polyomavirus, Pseudomonas aeruginosa, SARS-CoV-2, SARS, MERS, HBV, EBV, monkeypox, smallpox, Candida, Listeria, hepatitis C, hepatitis A, all viruses under the genus Alphavirus: Aura virus, Barmah Forest virus, Bebaru virus, Caaingua virus, Cabassou virus, Chikungunya virus, Eastern equine encephalitis virus, Eilat virus, Everglades virus, Fort Morgan virus, Getah virus, Highlands J virus, Madariaga virus, Mayaro virus, Middelburg virus, Mosso das Pedras virus, Mucambo virus, Ndumu virus, O'nyong-nyong virus, Pixuna virus, Rio Negro virus, Ross River virus, Salmon pancreas disease virus, Semliki Forest virus, Sindbis virus, Southern elephant seal virus, Tonate virus, Trocaravirus, Una virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus,Whataroa virus

[0148] The rare genetic diseases include (but are not limited to) rare genetic diseases selected from the following group: amyotrophic lateral sclerosis, Angelman syndrome, arginase deficiency, biotinidase deficiency, congenital myasthenic syndrome, Fabry disease, Gaucher disease, hemophilia, Huntington's disease, Leber hereditary optic neuropathy, multiple sclerosis, Parkinson's disease, pulmonary cystic fibrosis, sickle cell anemia, spinal muscular atrophy.

[0149] In another preferred embodiment, the pharmaceutical composition can also be used for the preparation of: all adoptive cell therapy drugs, all gene editing (TALEN, CRISPR, ZFN) and gene therapy mRNA alternative programs (replacing protein products, DNA, virus particles, or other nucleic acid protein products in the materials / raw materials), livestock vaccines, pet vaccines, etc.

[0150] The main innovation points of the present invention are as follows:

[0151] (1) The polyA sequence provided by the present invention can be stably passed on in DNA plasmids.

[0152] (2) The polyA sequence provided by the present invention can effectively reduce the recombination of plasmid DNA during amplification.

[0153] (3) The polyA sequence provided by the present invention can improve the translation efficiency of in vitro transcribed mRNA and prolong its half-life. Furthermore, it can reduce the mRNA drug dose and the potential side effects brought by the delivery system.

[0154] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions or according to the conditions recommended by the manufacturers. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0155] Example 1 Design of candidate polyA sequences and preparation of mRNA

[0156] 1.1 Design of candidate polyA sequences

[0157] Four polyA sequences (named polyA1 to polyA4, and the sequences are shown in SEQ ID NO: 1 to 4 respectively) were rationally designed by the present invention and synthesized by a CRO company. Four positive control polyA sequences are divided into PC1 to PC4 (the sequences are shown in SEQ ID NO: 5 to 8 respectively). The relevant sequences are shown as follows:

[0158] >polyA1 (SEQ ID NO: 1)

[0159] AAAAAAAAAAAAAAAAAAATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACCCCCCCCCCCCCCCCCCCCA;

[0160] The polyA1 sequence successively contains 19 A's, 1 T, 30 A's, 1 G, 49 A's, 20 C's and 1 A (sequence abbreviation: 19A-T-30A-G-49A-20C-A), with a length of 121 nt, having 4 segments of A sequences, abbreviated as a 4-segment structure; the first 3 segments are consecutive A sequences, connected by non-A bases (such as T, G or C) in the middle, the 4th segment is 1 A base, and the 3rd and 4th segments are linked by consecutive C bases.

[0161] >polyA2 (SEQ ID NO: 2, with a length of 121 nt)

[0162] AAAAAAAAAAAAAAAAAAATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAACCCCCCCCCCA;

[0163] Similarly, the sequence of polyA2 is abbreviated as 19A-T-30A-G-30A-G-28A-10C-A, with a length of 121 nt, having 5 segments of A sequences, abbreviated as a 5-segment structure; the first 4 segments are consecutive A sequences, connected by non-A bases (such as T, G or C) in the middle, the 5th segment is 1 A base, and the 4th and 5th segments are linked by consecutive C bases.

[0164] >polyA3 (SEQ ID NO: 3, sequence abbreviation: 19A-G-30A-G-66A, with a length of 117 nt)

[0165] AAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA;

[0166] >polyA4 (SEQ ID NO: 4, the sequence abbreviation is 19A-T-30A-G-66A, length is 117 nt)

[0167] AAAAAAAAAAAAAAAAAAATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA;

[0168] Similarly, polyA3 and polyA4 are three-segment designs without consecutive C bases, and the consecutive A sequences are connected by non-A bases in the middle.

[0169] >PC1 (SEQ ID NO: 5, the sequence abbreviation is 30A - spacer sequence - 70A, length is 110 nt)

[0170] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA GCATATGACT AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0171] Among them, the underlined sequence represents the spacer sequence.

[0172] >PC2 (SEQ ID NO: 6, the sequence abbreviation is 100A, length is 100 nt)

[0173] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA;

[0174] >PC3 (SEQ ID NO: 7, the sequence abbreviation is 30A - spacer sequence - 90A, length 130 nt)

[0175] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA GCATATGACT AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA;

[0176] Among them, the underlined sequence represents the spacer sequence;

[0177] >PC4 (SEQ ID NO: 8, abbreviated as 120A in sequence, length 120 nt)

[0178] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA。

[0179] Similarly, the polyA sequence structures of PC1 and PC3 are both two-segment structures, with the continuous A sequences connected by spacer sequences (non-continuous C base sequences); the polyA sequence structures of PC2 and PC4 are both one-segment structures, both being continuous A sequences.

[0180] 1.2 Plasmid construction

[0181] The T7 promoter sequence, 5'UTR sequence, GOI sequence (this vector contains the coding region: firefly luciferase Luciferase), 3'UTR sequence, and PolyA sequence were combined by gene synthesis to construct a circular plasmid that can be used for in vitro transcription of mRNA. The naming of the plasmid is the same as that of polyA, that is, finally, plasmids polyA1, PC1, PC2, PC3, and PC4 with correct sequencing were obtained. Except for the different polyA, the T7 promoter sequence (sequence as shown in SEQ ID NO: 9), 5'UTR sequence (sequence as shown in SEQ ID NO: 10), Luciferase nucleic acid sequence (sequence as shown in SEQ ID NO: 11), and 3'UTR sequence (sequence as shown in SEQ ID NO: 12) are all the same. The DNA sequences are as follows.

[0182] >T7 promoter sequence (SEQ ID NO: 9)

[0183] TAATACGACTCACTATA

[0184] >5' UTR sequence (SEQ ID NO: 10)

[0185] GGGAGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCTCTAGAGCCGCCACC

[0186] >Luciferase nucleic acid sequence (SEQ ID NO: 11)

[0187]

[0188] >3' UTR sequence (SEQ ID NO: 12)

[0189] GACTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGC

[0190] The key DNA sequence structure on the vector is: T7 - 5' UTR - GOI - 3' UTR - PolyA, and the mRNA sequence structure obtained by in vitro transcription using this plasmid is: Cap - 5' UTR - GOI - 3' UTR - PolyA.

[0191] Example 2 Sub - culture stability of plasmids with different polyA in Escherichia coli

[0192] 2.1 Plasmid transformation

[0193] The correctly sequenced plasmids polyA1 - polyA4, PC1 - PC4 were chemically transformed into the selected stable Escherichia coli. After culturing, monoclonal colonies were screened on the resistance plate. The medium used was LB medium (peptone, yeast extract, sodium chloride), and the antibiotic was kanamycin (200 μg / mL).

[0194] 2.2 Selecting monoclonal colonies

[0195] The plate after transformation was cultured at 30 °C for 16 hours. Single colonies were randomly picked, and the colonies with completely correct sequencing were used as the P0 generation. The P0 generation was inoculated into LB liquid medium with a final concentration of 25 mg / L kanamycin and cultured at 30 °C, 220 rpm / min overnight for 16 hours to obtain the bacterial solution, which was the P1 - generation bacterial solution.

[0196] 2.3 Continuous sub - culture

[0197] Dilute the P1-generation bacterial solution with LB to an appropriate concentration and then streak it. Incubate the plate at 30 °C for 16 hours. After monoclonal colonies are formed, pick 10 monoclonal colonies to sequence the polyA tail and count the correct ratio of the polyA tail. At the same time, inoculate the P1-generation bacterial solution into an LB liquid medium containing 100 μg / mL kanamycin, incubate it at 30 °C and 220 rpm / min overnight for 16 hours to obtain the bacterial solution, which is the P2-generation bacterial solution. Continue to streak and inoculate it into a new medium to obtain bacterial solutions of different generations, and passage it until the 30th generation. Statistically analyze the correct ratio of the polyA tail every other generation from P1 to P10 and every 5 generations from P10 to P30.

[0198] The polyA of polyA1 to polyA4 and PC1 to PC4 is not lost, that is, the results of the completely correct clone ratio are shown in Table 1 or Figure 1 as shown.

[0199] The structure shows that the polyA1 and polyA2 sequences designed in this application are significantly superior to the control groups PC2 to PC4 in terms of the passage stability in Escherichia coli cells; compared with the control group PC1, the passage stability of polyA1 is higher, and the polyA2 sequence is comparable to it; while the passage stability of the polyA3 and polyA4 sequences in Escherichia coli cells is significantly lower than that of the polyA1 and polyA2 sequences.

[0200] Table 1

[0201]

[0202] Preparation of mRNA Samples Corresponding to Candidate polyA Sequences in Example 3

[0203] 3.1 Plasmid Extraction

[0204] After the plasmid construction is completed and the sequence is confirmed to be correct, preserve the strain. Inoculate the strain into a 2YT medium, add kanamycin to the medium, inoculate according to the ratio of 100 μL of bacterial solution per 200 mL of volume medium, and incubate it on a shaker at 200 rpm at 37 °C overnight for 14 - 16 hours until the OD 600 is between 2.0 and 3.0, and collect the bacterial cells by centrifugation.

[0205] Extract plasmid DNA by the alkaline lysis method. The required buffer preparation system is shown in Table 2 below:

[0206] Table 2

[0207]

[0208] Resuspend the collected bacterial cells in P1 buffer, then add P2 buffer for lysis, and finally add P3 buffer for neutralization. Centrifuge and take the supernatant for Bestarose 6FF gel filtration. Finally, transfer the sample to a 100 kD ultrafiltration tube for concentration. After concentration, pipette the sample into a new collection tube, measure the concentration and store it.

[0209] 3.2 Preparation of Linearized Plasmid Template

[0210] Since circular plasmids have no effective termination, RNAs of different lengths will be transcribed. To obtain RNAs of a specific length, the plasmid must be completely linearized, and the linearized plasmid should ensure that the double-stranded ends are blunt or have a protruding structure at the 5' end. When designing the sequence, type IIS restriction enzymes Bsa I is selected as the plasmid linearization digestion site. Digest the supercoiled plasmid. The 50 µL plasmid linearization system is shown in Table 3 below:

[0211] Table 3

[0212]

[0213] Add each component to a centrifuge tube, mix well, and incubate in an incubator at 37°C ± 0.5°C for 16 h. After the reaction, take a sample for agarose gel electrophoresis to confirm the linearization effect. The electrophoresis result shows a single band with the correct size. Use isopropanol precipitation to recover the completely linearized plasmid, and detect the residual RNA enzyme in the recovered linearized plasmid. The linearized plasmid without obvious degradation is used for the next step.

[0214] 3.3 In Vitro Transcription

[0215] Preparation before in vitro transcription: First, surface disinfect the prepared centrifuge tubes, pipettes, sterile gun tips, etc. with 75% ethanol and then put them into the laminar flow hood. Turn on the power and ultraviolet lamp of the laminar flow hood, disinfect for 30 min, and let it self-purify for 10 min.

[0216] Before transcription, place the T7 RNA transcriptase mixture on an ice box, and melt NTPs, 10× transcription buffer, cap structure analog, and DNA template at room temperature. Each material should be fully melted and mixed well before use. Wear gloves, disinfect hands with 75% ethanol, and perform operations in the laminar flow hood. After mixing the melted materials, prepare to formulate the in vitro transcription system. The co-transcription capping in vitro transcription system is shown in Table 4 below. All components except nuclease-free water are purchased from Shanghai Novoprotein Biotechnology Co., Ltd.

[0217] Table 4

[0218]

[0219] After the in vitro transcription system with cotranscriptional capping was mixed evenly, it was placed in an incubator and left to stand at 37°C ± 0.5°C for 3 h. After the transcription reaction ended, the template was digested with DNase I, and then it was left to stand in the incubator at 37°C ± 0.5°C for 30 min. After the digestion of DNase I ended, precipitation centrifugation recovery was carried out using an RNA precipitation solution (lithium chloride precipitation solution), washed twice with 75% ethanol, air-dried, and the precipitate was dissolved in nuclease-free water to obtain a clear and transparent mRNA solution.

[0220] Example 4 In vitro cell transfection experiment of mRNA

[0221] In this example, the luciferase activity was detected to reflect the expression levels of mRNAs with different polyAs in 293T cells, and then the effects on translation efficiency and half-life were investigated. If the luciferase activity is higher, it indicates that the expression levels of mRNAs with different polyAs in 293T cells are higher and the translation efficiency is higher; if the luciferase can still maintain a high activity under long-term transfection conditions, it indicates that the mRNA half-life is longer. The specific process is as follows:

[0222] 4.1 Cell culture and in vitro transfection

[0223] 293T cells were cultured in a 75 cm 2 vented-cap right-angle culture flask with DMEM medium supplemented with 10% fetal bovine serum (FBS). When the cells grew to 70–90% confluence, the cells were digested with trypsin to make a cell suspension, and the density of the cell suspension was 2.5×10 5 . The cells were seeded in a 24-well plate, 500 μL per well, and cultured overnight. The next day, before transfection, the original medium was discarded and replaced with Opti-MEM™ I reduced-serum medium. 450 μL of Opti-MEM™ I medium was slowly added along the side wall to each well of the cells. Prepare 2 Tube tubes, and dilute the transfection reagent and mRNA samples with Opti-MEM™ I medium respectively. 1.5 μL of the transfection reagent Lipofectamin mRNA Max (Thermo Fisher Scientific) was added to 25 μL of Opti-MEM™ I medium, and 2 μg of the mRNA stock solution was added to 25 μL of Opti-MEM™ I medium. The mixtures in the two tubes were pipetted and mixed evenly, and left to stand for 10 min. After the waiting time ended, the above transfection reagent and mRNA stock solution were mixed evenly and left to stand for 5 min. 50 μL of the mRNA-transfection reagent mixture was slowly added dropwise into the culture plate, mixed evenly, and cultured at 37°C for 24 h, 48 h, 72 h, and 120 h, with each sample repeated 2 times.

[0224] 4.2 Firefly luciferase assay

[0225] The luciferase activity was detected using the luciferase assay system of Promega (Beijing) Biotechnology Co., Ltd. The culture medium was discarded, and 500 μL of lysis buffer was added to each well of a 24-well culture plate and lysed at room temperature for 5 min. Reaction and reading: 100 μL of the cell lysate supernatant was pipetted into a black 96-well plate, 100 μL of luciferase substrate was added, the luminescence detection mode of the instrument was selected, and the processed samples were quickly read using a microplate reader. The results are shown in Table 5 and Figure 2 as follows:

[0226] Table 5

[0227]

[0228] Note: Each underscore in the polyA sequence represents 1 segment; for example, polyA1 has a 4-segment structure, and the others are similar and will not be elaborated further.

[0229] The results showed that compared with the control group, the expression activities of polyA1 and polyA2 were slightly higher than those of the control group (PC1 - PC4 groups) at the time points of 48 h, 72 h, and 120 h, indicating that the mRNAs of polyA sequences (such as polyA1 - polyA4) containing at least a 3-segment structure (such as 3-segment, 4-segment, or more than 5-segment structures) had higher translation ability than the mRNAs of polyA sequences (such as PC1 - PC4) containing 1 - 2-segment structures; among them, compared with the control group, the mRNAs of the polyA1 sequence with a 4-segment structure maintained a high expression activity from 24 h to 120 h, indicating that the mRNAs of the polyA1 sequence with a 4-segment structure had higher translation ability and a longer half-life ( Figure 2 A and B in

[0230] Although polyA3 with a 3-segment structure had higher or similar translation ability and half-life compared with the control group, compared with the plasmids of polyA with a structure greater than 3 segments and containing consecutive C bases (such as polyA1 or polyA2), the plasmids of 3-segment structure polyA without consecutive C bases (polyA3 or polyA4) had poor passage stability in Escherichia coli ( Figure 1 A and B in

[0231] In summary, polyA1 and polyA2 have a multi-segment structure with more than 3 segments and contain a consecutive C base structure. Their plasmids have good passage stability in Escherichia coli, can effectively reduce the recombination of plasmid DNA during amplification, and maintain the length of the polyA tail in the DNA template; at the same time, they improve the translation efficiency of in vitro transcribed mRNA and extend the half-life of mRNA.

[0232] All documents mentioned in this invention are cited herein by reference as if each individual document was cited by reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A polyA tail element, characterized in that, The 5'-to-3' structure of the polyA tail element consists of the following elements: (a) 2 to 7 sequentially connected X elements, each of which independently consists of 10 to 70 consecutive A bases and 1 non-A base connected in sequence; (b) at least 1 Y element, each of which independently consists of 10 to 70 consecutive A bases and 10 to 30 consecutive C bases; and (c) 1 to 10 A bases located at the 3' end; wherein, the non-A base is selected from the group consisting of: T base or G base; The nucleotide sequence of the polyA tail element is as shown in SEQ ID NO: 1 or SEQ ID NO:

2.

2. The polyA tail element according to claim 1, characterized in that, The 5'-to-3' sequence of the polyA tail element has the following structure: X1-X2-X3-Y-Z; wherein, X1 consists of 10 to 19 consecutive A bases and 1 non-A base connected in sequence; X2 consists of 20 to 40 consecutive A bases and 1 non-A base connected in sequence; X3 is absent or consists of 20 to 50 consecutive A bases and 1 non-A base connected in sequence; Y independently consists of 10 to 70 consecutive A bases and 10 to 30 consecutive C bases; Z is 1 to 10 A bases located at the 3' end; The nucleotide sequence of X1 is positions 1 to 20 relative to the sequence shown in SEQ ID NO:1; The nucleotide sequence of X2 is positions 21 to 51 relative to the sequence shown in SEQ ID NO:1; The nucleotide sequence of Y is positions 52 to 120 relative to the sequence shown in SEQ ID NO:1; Z is 1 A base located at the 3' end.

3. The polyA tail element according to claim 1, characterized in that, The nucleotide sequence length of the polyA tail element is 121 bases.

4. The polyA tail element according to claim 1, characterized in that, The nucleotide sequence of the polyA tail element is as shown in SEQ ID NO:

2.

5. The polyA tail element according to claim 1, characterized in that, The nucleotide sequence of the polyA tail element is as shown in SEQ ID NO:

1.

6. An mRNA transcription template construct, characterized in that, The construct has the structure of formula I: Z1-Z2-Z3-Z4-Z5-Z6-Z7 (I) In the formula, Z1 and Z7 are absent or restriction enzyme sites; Z2 is absent or a promoter element or an internal ribosome entry site sequence IRES; Z3 is a 5' UTR element; Z4 is an alternative coding region; Z5 is a 3' UTR element; Z6 is the polyA tail element as claimed in claim 1; Z4 is a luciferase coding gene or a protein coding gene for preventing and / or treating infectious diseases, rare genetic diseases, neurodegenerative diseases, retinopathies, cancers or tumors; Z4 is selected from the group consisting of: luciferase coding gene, pathogen antigen gene, cytokine, growth factor, protein hormone, polypeptide hormone, tumor-associated antigen, tumor-specific antigen, universal tumor mutation site antigen, protein adjuvant, polypeptide adjuvant, nucleic acid adjuvant, or a combination thereof.

7. A carrier, characterized in that, The vector contains the mRNA transcription template construct as claimed in claim 6.

8. A host cell, characterized in that, The host cell contains the vector as claimed in claim 7.

9. A method for generating mRNA for preparing mRNA drugs, characterized in that, The method comprises the steps of: (i) culturing the host cell as claimed in claim 8 under suitable conditions to obtain a culture containing the vector with the mRNA transcription template construct; (ii) isolating and / or recovering the vector as described in (i) from the culture and linearizing it by enzymatic digestion to obtain the mRNA transcription template; and (iii) transcribing the mRNA transcription template as described in (ii) to obtain the mRNA.

10. An mRNA, characterized in that, The mRNA is prepared by the method as claimed in claim 9 and has the structure shown in formula II: M1-M2-M3-M4-M5-M6 (II) wherein, M1 is a 5'-end capping element; M2 is an optional or internal ribosome entry site sequence IRES; M3 is a 5' UTR element; M4 is an alternative coding region; M5 is a 3' UTR element; M6 is the polyA tail element as claimed in claim 1; the M4 is a luciferase coding gene or a protein coding gene for preventing and / or treating infectious diseases, rare genetic diseases, neurodegenerative diseases, retinopathies, cancers or tumors; the M4 is selected from the group consisting of: a luciferase coding gene, a pathogen antigen gene, a cytokine, a growth factor, a protein hormone, a polypeptide hormone, a tumor-associated antigen, a tumor-specific antigen, a common tumor mutation site antigen, a protein adjuvant, a polypeptide adjuvant, a nucleic acid adjuvant, or a combination thereof.

11. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (c1) the mRNA transcription template construct as claimed in claim 6, or the mRNA as claimed in claim 10 as an active ingredient; and (c2) a pharmaceutically acceptable carrier.

12. A method for preparing an mRNA pharmaceutical composition, characterized in that, The method comprises: mixing the mRNA as claimed in claim 10 with a pharmaceutically acceptable carrier to obtain the mRNA pharmaceutical composition.

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