An optimized polyA sequence and its application

Abstract

The present invention provides an optimized polyA sequence and its application. 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 dosage of mRNA drugs and the side effects caused 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 medicine, and in particular to an optimized polyA sequence and application thereof. Background Art

[0002] mRNA-based therapeutic methods are more accurate in practical applications and can be used for new vaccines and personalized treatments. They are a new direction for the development of modern medicine. Compared with recombinant proteins expressed in mammalian cell lines, mRNA production is faster and more flexible. In the past decade, technological developments in the field of mRNA modification and delivery systems have rapidly advanced the basic and clinical research of mRNA vaccines. However, the technical problems faced by mRNA therapy are also obvious, such as the low efficiency of mRNA expression in vivo, and the protective efficacy of mRNA vaccines is not optimistic. These are also key issues that need to be optimized in clinical applications.

[0003] mRNA optimized and developed based on stability and protein translation efficiency can maximize mRNA production and avoid loss problems during delivery. Among the structural characteristics of mRNA, several factors affect the expression and stability of mRNA vaccines, such as Cap, untranslated region (UTR), and polyA tail, which directly affect mRNA stability and translation efficiency. In the early days of mRNA research, scientists discovered that there is polyadenylic acid at its 3' tail. In fact, except for the transcripts of individual mammalian histones, almost all mRNAs in eukaryotes have a polyA tail. The polyA tail is added to the mRNA at the same time as transcription and is necessary for the export of mature mRNA from the nucleus to the cytoplasm. The polyA tail contributes to both the translation state and the stability of mRNA, and is therefore a key regulator of gene expression in the cytoplasm. In particular, the polyA tail can functionally synergize with the 7-methylguanine (m7G) at the 5' end of the mRNA to promote translation. When a transcript loses its polyA tail, its translation will be at a lower level, which also means that the cap structure at its 5' end is about to be removed, and decapping occurs. Some researchers have added a relatively short UGC sequence between polyadenylic acid sequences. For example, the mRNA vaccine BNT162b2 developed by Pfizer / BioNTech has a PolyA tail sequence consisting of 30A+10GCAUAUGACU+70A. The mRNA-1273 developed by Moderna has a PolyA sequence consisting of 100 A's, without other types of nucleotides.

[0004] There are two strategies for the in vitro synthesis of PolyA tails: the first is enzymatic capping, which uses recombinant PolyA polymerase to extend the mRNA synthesized by the IVT reaction. This enzymatic tailing method cannot produce a PolyA sequence of fixed length, which is not conducive to quality control during the process production process. The second is to add a long A sequence or a polyA tail of other nucleotides to the plasmid DNA template sequence to complete a one-step co-transcriptional tailing. One-step tailing can ensure that the length of the tail is highly controlled. The challenge of co-transcriptional tailing is that the plasmid amplification process may cause the tail to be lost. Therefore, the integrity of the plasmid template and PolyA should be regarded as an important quality attribute in the development of fermentation processes.

[0005] A classic RNA structure includes a 5' cap structure, a 5' untranslated region (UTR), a coding region (CDS), a 3' UTR, and a polyA tail. The 3' UTR and the polyA tail have a decisive influence on the stability of mRNA in cells and various tissues and organs. Its stability mechanism is mainly mediated by endogenous miRNA (microRNA), exogenous siRNA (small interference RNA), exogenous ASO (anti-sense oligonucleotides), and a more complex regulatory network composed of various RNA binding proteins (RBP).

[0006] In the field of mRNA drug development, most mRNA delivered by nanoliposome particles (Lipid Nano Particle, LNP) or other types of carriers are cleared, and only a small part of it escapes from the endosomal system to exert its efficacy. Even the escaped mRNA may be recognized as exogenous nucleic acid substances by the innate immune system and accelerated clearance. The poly A tail at the 3' end can protect the cap structure from degradation, and cooperate with the poly A binding protein, 5' Cap (cap structure) and translation initiation factor protein to initiate protein translation.

[0007] Therefore, there is an urgent need in this field to develop polyA sequences that can improve the translation efficiency of protein coding regions to provide technical support for the preparation of high-quality mRNA drugs. Summary of the invention

[0008] The purpose of the present invention is to provide a polyA sequence capable of improving the translation efficiency of a protein coding region and its application in preparing mRNA drugs.

[0009] In a first aspect of the present invention, a polyA tail element is provided, which is used to construct an mRNA transcription template to improve the translation efficiency of the coding region in the mRNA obtained by transcription of the mRNA transcription template, characterized in that the nucleotide sequence of the polyA element is as shown in SEQ ID NO: 1, or has ≥70% homology with the nucleotide sequence shown in SEQ ID NO: 1.

[0010] 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, and having the activity of improving the translation efficiency of the coding region in mRNA; or

[0011] The 5' end and / or 3' end of the nucleotide sequence as shown in SEQ ID NO: 1 increases and / or decreases 1-50 (preferably 1-30, more preferably 1-10, more preferably 1-5) nucleotides, and has a nucleotide sequence that increases the translation efficiency of the coding region in the mRNA.

[0012] The second aspect of the present invention provides an mRNA transcription template construct, wherein the construct has a structure of Formula I:

[0013] Z1-Z2-Z3-Z4-Z5-Z6-Z7 (I)

[0014] In the formula,

[0015] Z1 and Z7 are no or restriction sites;

[0016] Z2 is no or promoter element or internal ribosome entry site sequence (IRES);

[0017] Z3 is the 5`UTR element;

[0018] Z4 is a replaceable coding region;

[0019] Z5 is the 3`UTR element;

[0020] Z6 is the polyA tail element described in the first aspect of the present invention.

[0021] In another preferred embodiment, Z1 and Z7 are blunt-end restriction sites or sticky-end restriction sites.

[0022] In another preferred embodiment, the Z2 is a promoter element, and the promoter is selected from the following group: 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.

[0023] In another preferred embodiment, the Z4 is a protein encoding gene for preventing and / or treating infectious diseases, rare genetic diseases, neurodegenerative diseases, retinopathy, cancer or tumors.

[0024] In another preferred embodiment, the Z4 is selected from the following group: pathogen antigen genes, transposons in the genome (including silent and active transposons), cytokines, growth factors, protein hormones, polypeptide hormones, tumor-associated antigens (TAA), tumor-specific antigens (TSA), universal tumor mutation site antigens, protein adjuvants, polypeptide adjuvants, nucleic acid adjuvants, or a combination thereof.

[0025] The third aspect of the present invention provides a vector, wherein the vector contains the mRNA transcription template construct as described in the second aspect of the present invention.

[0026] In another preferred embodiment, the vector is selected from the following group: DNA, RNA, viral vector, plasmid, transposon, other gene transfer system, or a combination thereof; preferably, the vector is a plasmid.

[0027] The fourth aspect of the present invention provides a host cell, wherein the host cell contains the vector as described in the third aspect of the present invention.

[0028] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0029] In another preferred embodiment, the host cell is selected from the following group: Escherichia coli, yeast cells, and mammalian cells.

[0030] A fifth aspect of the present invention provides a method for producing optimized mRNA for preparing mRNA drugs, the method comprising the steps of:

[0031] (i) culturing the host cell according to the fourth aspect of the present invention under suitable conditions to obtain a culture containing a vector of an mRNA transcription template construct;

[0032] (ii) isolating and / or recovering the vector in (i) from the culture, and linearizing it with enzymes to form an mRNA transcription template; and

[0033] (iii) transcribing the mRNA transcription template described in (ii) to obtain the optimized mRNA.

[0034] In another preferred embodiment, the method further comprises step (iv): purifying and / or modifying the optimized mRNA obtained in step (iii).

[0035] The sixth aspect of the present invention provides an optimized mRNA, wherein the optimized mRNA is prepared by the method described in the fifth aspect of the present invention, and the optimized mRNA has a structure shown in Formula II:

[0036] M1-M2-M3-M4-M5-M6 (II)

[0037] In the formula,

[0038] M1 is a 5' end cap component;

[0039] M2 is none or internal ribosome entry site sequence (IRES);

[0040] M3 is the 5`UTR element;

[0041] M4 is a replaceable coding region;

[0042] M5 is the 3`UTR element;

[0043] M6 is the polyA tail element described in the first aspect of the present invention.

[0044] In another preferred embodiment, the M4 is a protein encoding gene for preventing and / or treating infectious diseases, rare genetic diseases, neurodegenerative diseases, retinopathy, cancer or tumors.

[0045] In another preferred embodiment, the M4 is selected from the following group: pathogen antigen genes, transposons in the genome (including silent and active transposons), cytokines, growth factors, protein hormones, polypeptide hormones, tumor-associated antigens (TAA), tumor-specific antigens (TSA), universal tumor mutation site antigens, protein adjuvants, polypeptide adjuvants, nucleic acid adjuvants, or a combination thereof.

[0046] The seventh aspect of the present invention provides a pharmaceutical composition, comprising:

[0047] (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

[0048] (c2) a pharmaceutically acceptable carrier.

[0049] In another preferred embodiment, the pharmaceutical composition is a vaccine composition.

[0050] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: injection, lyophilized agent, nebulized inhalation agent, and smearable agent.

[0051] In another preferred embodiment, the pharmaceutical composition is administered by injection, i.e., intravenous, intramuscular, intradermal, subcutaneous, intrathecal, intraduodenal or intraperitoneal injection.

[0052] In another preferred embodiment, the pharmaceutical composition is administered by inhalation, such as intranasal administration.

[0053] In another preferred embodiment, the pharmaceutical composition is administered transdermally, such as by transdermal application or electrode introduction.

[0054] In another preferred embodiment, the pharmaceutical composition is lipid nanoparticles formed by encapsulating the mRNA with cationic lipids, namely LNP-mRNA.

[0055] In another preferred embodiment, the vaccine composition comprises 0.01 to 99.99% of the optimized mRNA as described in the sixth aspect of the present invention and 0.01 to 99.99% of a pharmaceutically acceptable carrier, and the percentages are the mass percentages of the vaccine composition.

[0056] In another preferred embodiment, the pharmaceutical composition is used to prepare drugs for preventing and / or treating diseases including (but not limited to) infectious diseases, rare genetic diseases, neurodegenerative diseases, retinopathy, cancer or tumors.

[0057] The eighth aspect of the present invention provides a method for preparing an mRNA pharmaceutical composition, 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.

[0058] In another preferred embodiment, the mRNA pharmaceutical composition is an mRNA vaccine composition.

[0059] The ninth aspect of the present invention provides a use of the polyA tail element as described in the first aspect of the present invention for constructing an mRNA transcription template, thereby improving the translation efficiency of the coding region in the mRNA transcribed from the mRNA transcription template.

[0060] The tenth aspect of the present invention provides 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 for preparing a drug for preventing and / or treating infectious diseases, rare genetic diseases, neurodegenerative diseases, retinopathy, cancer or tumors.

[0061] 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 a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 The experimental procedure for in vivo expression of polyA-hEPO is shown.

[0063] Figure 2 The results of the in vivo expression experiment of polyA-hEPO are shown.

[0064] Figure 3 The sequence alignment results of polyA-GFP cell passage stability are shown. DETAILED DESCRIPTION

[0065] After extensive and in-depth research, the inventors obtained an optimized polyA sequence through screening and optimization. Experiments have shown that the polyA sequence of the present invention (such as the nucleotide sequence shown in SEQ ID NO: 1) can improve the translation efficiency of the mRNA protein coding region, thereby reducing the mRNA drug dosage and reducing the potential side effects caused by the delivery system. On this basis, the present invention was completed.

[0066] the term

[0067] In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms shall have the meaning given below.

[0068] PolyA tail element

[0069] 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. The polyA tail element of the present invention inserts a newly designed spacer sequence (GGGTACCACT, SEQ ID NO: 9) on the basis of the original polyA tail sequence, and replaces a segment of adenine nucleotides near the 3' end with cytosine nucleotides. 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.

[0070] mRNA transcription template construct

[0071] As used herein, the terms "mRNA transcription template" and "mRNA transcript" are used interchangeably.

[0072] In another aspect of the present invention, an mRNA transcription template construct is provided, wherein the construct has a structure of Formula I:

[0073] Z1-Z2-Z3-Z4-Z5-Z6-Z7 (I)

[0074] In the formula,

[0075] Z1 and Z7 are no or restriction sites;

[0076] Z2 is no or promoter element or internal ribosome entry site sequence (IRES);

[0077] Z3 is the 5`UTR element;

[0078] Z4 is a replaceable coding region;

[0079] Z5 is the 3`UTR element;

[0080] Z6 is the polyA tail element described in the first aspect of the present invention.

[0081] As used herein, the terms "5'-UTR" and "5'UTR" can be used interchangeably; "3'-UTR" and "3'UTR" can be used interchangeably.

[0082] As used herein, the term "promoter" or "promoter region (domain)" refers to a nucleic acid sequence that accurately and effectively initiates gene transcription function, guides the transcription of gene nucleic acid sequences into RNAs, and is usually present upstream (5' end) of the target gene coding sequence. Generally, the promoter or promoter region provides recognition sites for RNA polymerase and other factors necessary for the correct initiation of transcription.

[0083] The promoter of the present invention can be operably linked to an exogenous gene, which can be foreign (heterologous) relative to the promoter. The exogenous gene (also called target gene) is not particularly limited, and representative examples include (but are not limited to): screening marker genes, resistance genes, antigen protein genes, and biological agent genes.

[0084] In one embodiment of the present invention, the mRNA transcription template construct has a structure of Formula I, wherein Z1 and Z7 are restriction sites, Z2 is a promoter element or an internal ribosome entry site sequence (IRES), Z3 is a 5'UTR sequence, Z4 is a replaceable coding region (e.g., the hEPO coding sequence or GFP coding sequence used for screening in the embodiment), and Z5 is a 3'UTR sequence.

[0085] Optimizing mRNA

[0086] Sequence optimization of mRNA is one of the methods to help mRNA improve translation efficiency. Sequence optimization of 5`-UTR and 3`-UTR of mRNA can increase the half-life and translation activity of mRNA. Using different analogs of Cap structure can increase the stability of mRNA. Using enzymes to add Cap structure to the 5` end of mRNA can have better performance than different forms of Cap analogs. The effect of polyA tail of mRNA on stabilizing mRNA is also very important. Some studies have shown that removing polyA of mRNA makes mRNA extremely unstable, and also reduces the number of polyribosomes, extension rate and translation rounds of mRNA. Therefore, polyA is crucial for the stability and effective translation of mRNA. In addition, nucleotide modification and synonymous substitution 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 adjustable sites that affect mRNA stability.

[0087] The optimized mRNA of the present invention has the structure shown in Formula II:

[0088] M1-M2-M3-M4-M5-M6 (II)

[0089] In the formula,

[0090] M1 is a 5' end cap component;

[0091] M2 is none or internal ribosome entry site sequence (IRES);

[0092] M3 is the 5`UTR element;

[0093] M4 is a replaceable coding region;

[0094] M5 is the 3`UTR element;

[0095] M6 is the polyA tail element described in the first aspect of the present invention.

[0096] In one embodiment of the present invention, the optimized mRNA has a structure of formula II, wherein M1 is a 5' cap element, M2 is a non- or internal ribosome entry site sequence (IRES), M3 is a 5'UTR sequence of a nucleotide sequence as shown in SEQ ID NO: 3, M4 is a replaceable coding region (for example, the hEPO coding sequence or GFP coding sequence used for screening in the embodiment), M5 is a 3'UTR element, whose nucleotide sequence is shown in SEQ ID NO: 8; M6 is a polyA tail element, whose nucleotide sequence is shown in SEQ ID NO: 1.

[0097] The pharmaceutical composition of the present invention and its application

[0098] In one aspect of the present invention, a pharmaceutical composition is also provided. In one embodiment of the present invention, the pharmaceutical composition is an mRNA vaccine composition.

[0099] mRNA vaccines are divided into self-amplifying RNA (saRNA) and non-replicating RNA (non-replicating mRNA). Classic non-amplifying RNA vaccines include cap, 5`-UTR, open reading frame (ORF) (i.e. coding region), 3`-UTR and poly A tail. The ORF region is responsible for encoding antigen expression, but the above five regions jointly determine the stability, expression activity and immunogenicity of mRNA.

[0100] The structure of saRNA is derived from the alphavirus genome. The saRNA vaccine uses the self-replication property of the alphavirus genome to allow the DNA or RNA that enters the somatic cells to self-amplify first, 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 amplified 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 nonspecific immunity.

[0101] A major challenge for mRNA vaccines is to reduce the immunogenicity of the exogenous mRNA itself. Under natural conditions, after exogenous mRNA enters the cell, 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-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 by chemical modification of nucleotides, adding a polyA tail, and optimizing the GC content of mRNA.

[0102] 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).

[0103] In addition, adding a polyA tail can also reduce the U content and thus reduce the immunogenicity of mRNA. CureVac and Acuita Therapeutics have tried 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, too high a GC content will inhibit the translation activity of mRNA, which is also something that needs to be paid attention to during vaccine development.

[0104] The method of mRNA purification is also very important in reducing the immunogenicity of the 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 HPLC purification of m1ψ-modified mRNA encoding anti-HIV-1 antibodies to help mice avoid HIV-1 infection through lipid nanoparticles (LNP).

[0105] There are many methods for delivering mRNA. Scientists have established methods such as liposome delivery, polymer delivery, peptide chain delivery, virus-like replicon particle delivery, and cationic nanoemulsifier delivery. In addition, naked mRNA can also be directly injected into cells. The most commonly used delivery method for mRNA vaccines under development is lipid nanoparticle delivery. This method has the advantages of low toxicity and high delivery efficiency.

[0106] The "active ingredient" in the pharmaceutical composition of the present invention refers to the mRNA transcription template construct or optimized mRNA of the present invention. The "active ingredient", preparation and / or composition of the present invention can be used to prevent and / or treat diseases or conditions such as infectious diseases, rare genetic diseases, neurodegenerative diseases, retinopathy, cancer or tumors. "Safe and effective amount" means: the amount of active ingredient is sufficient to significantly improve the condition or symptoms without causing serious side effects. "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which 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 ingredients of the present invention and with each other without significantly reducing the efficacy of the active ingredients.

[0107] The pharmaceutical composition may be a liquid or a solid, such as a powder, a gel or a paste. Preferably, the composition is a liquid, preferably an injectable liquid.

[0108] Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, 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 lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0109] Pharmaceutical compositions may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0110] The pharmaceutical composition of the present invention can be prepared into dosage forms such as injection, lyophilized agent, aerosol inhalation, smear-type medicament. 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, i.e. intravenous, intramuscular, intradermal, subcutaneous, intraduodenal, intrathecal, or intraperitoneal injection. In addition, the pharmaceutical composition of the present invention can be administered by inhalation, such as intranasal administration. In addition, the pharmaceutical composition of the present invention can be administered transdermally. The transdermal administration method through the local route can be formulated into a coating stick, solution, suspension, emulsion, gel, cream, ointment, paste, gel, paint, powder and aerosol. In addition, the pharmaceutical composition of the present invention can be actively administered to the intradermal, subcutaneous, muscle, tumor, tissue organ, central nervous system and other parts through electrodes / electric field / potential difference.

[0111] The pharmaceutical composition of the present invention can be co-administered with another active agent. Co-administration includes administering the compound of the present invention and the active agent within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20 or 24 hours of each other. Co-administration also includes administering the compound of the present invention and the active agent simultaneously, approximately simultaneously (e.g., within about 1, 5, 10, 15, 20 or 30 minutes of each other) or sequentially in any order. In some embodiments, co-administration can be accomplished by co-formulation, i.e., preparing a single pharmaceutical composition comprising both the active ingredient of the present invention (the mRNA transcription template construct of the present invention or the optimized mRNA) and the active agent. In other embodiments, the active ingredient of the present invention and the active agent can be separately formulated.

[0112] The present invention also provides the use of the pharmaceutical composition for preparing a drug for preventing and / or treating diseases, including (but not limited to) infectious diseases, rare genetic diseases, neurodegenerative diseases, retinopathy, cancer or tumors.

[0113] The infectious diseases include (but are not limited to) the following diseases or diseases caused by the following pathogens: botulinum toxin, hirudin, cytomegalovirus (CMV), Zika virus, influenza virus, respiratory syncytial virus (RSV), Chikungunya, rabies, HIV, Ebola virus, streptococci, malaria, Louping illvirus, Toxoplasma gondii gondii), dengue fever, plague, yellow fever, tuberculosis, herpes simplex virus, herpes zoster virus, mycoplasma, chlamydia, foot-and-mouth disease virus, rotavirus, varicella virus, 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, all viruses of the genus Alphavirus (Aura virus, Barmah Forest virus, Bebaru virus, Caaingua virus, Cabassou virus, Chikungunya virus, Eastern equine encephalitis virus, Eilat virus, virus, Evergladesvirus, 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, Hepatitis C, Hepatitis A.

[0114] 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, cystic fibrosis, sickle cell anemia, and spinal muscular atrophy.

[0115] In another preferred embodiment, the pharmaceutical composition can also be used to prepare: all adoptive cell therapy drugs, all gene editing (TALEN, CRISPR, ZFN) and gene therapy mRNA alternatives (protein products, DNA, viral particles, or other nucleic acid protein products in alternative materials / raw materials), livestock vaccines, pet vaccines, etc.

[0116] The main innovative features of the present invention are:

[0117] After screening and optimization, the inventors designed a new spacer sequence to be inserted into the polyA sequence. Using the spacer sequence to separate the polyA sequence can effectively reduce the recombination of plasmid DNA during amplification, maintain the length of the polyA tail in the DNA template, and do not affect the translation efficiency and half-life of the mRNA generated by in vitro transcription. In addition, a section of adenine nucleotides in the PolyA tail sequence was replaced with cytosine nucleotides to enhance the stability of the mRNA generated by in vitro transcription, while maintaining the length of the polyA tail of the mRNA and significantly improving the translation efficiency of the mRNA in mice.

[0118] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for the unrecorded specific conditions in the following examples are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0119] Example 1 Candidate polyA sequence design and mRNA preparation

[0120] 1.1 Design of candidate polyA sequences

[0121] The present invention rationally designed a polyA sequence (named Da5) and handed it over to CRO for synthesis. The positive control (PC) used the 120A sequence. The candidate polyA and positive control sequences are as follows:

[0122] >polyA-Da5 (SEQ ID NO: 1)

[0123] AAAAAAAAAAAAAAAAAAAAAAAAAAA GGGTACCACT AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACCCCCCCCCCCCCCCCCCCCCCCAAAAAA

[0124] Among them, the underlined part is the spacer sequence;

[0125] >PC (SEQ ID NO: 2)

[0126] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0127] 1.2 mRNA preparation

[0128] (1) The candidate polyA sequence (shown in SEQ ID NO: 1) was ligated to the 3′ end of the hEPO coding region (amino acid sequence shown in SEQ ID NO: 4; nucleic acid sequence shown in SEQ ID NO: 5) using T4 ligase to construct the hEPO open reading frame.

[0129] (2) Transform competent E. coli, sequence and select bacterial strains containing the correct sequence

[0130] (3) The bacteria are cultured and the target gene is extracted, and the target gene is cut from a double-stranded closed circular structure into a double-stranded linear structure to obtain an mRNA transcription template.

[0131] (4) In the presence of a buffer system and nucleotide raw materials, the mRNA is catalyzed by T7 RNA transcriptase and then capped by a capping enzyme to obtain a complete mRNA crude product with expression capacity, wherein the mRNA has a polyA tail (transcribed from the sequence shown in SEQ ID NO: 1).

[0132] (5) Purified hEPO mRNA containing the candidate polyA sequence was obtained by LiCl precipitation and 75% ethanol washing.

[0133] (6) Liposome encapsulates hEPO mRNA. Dissolve hEPO mRNA in aqueous buffer and mix well as the aqueous phase. Dissolve ionizable lipids (SM-102), DSPC, cholesterol and DMG-PEG2000 in anhydrous ethanol and mix well in a certain proportion as the organic phase. Transfer the aqueous phase and organic phase to syringes respectively, set the parameters using the PNI microfluidic nanopreparation instrument, and prepare LNP-hEPO (named LNP-hEPO-Da5). LNP-hEPO is concentrated, purified, sterilized and filtered, and injected into mice after passing quality control.

[0134] The hEPO mRNA containing the positive control sequence was prepared according to the same steps as above and encapsulated with LNP, named LNP-hEPO-120A.

[0135] Example 2 LNP-mRNA expression verification experiment in mice

[0136] according to Figure 1 The process shown was used to conduct the in vivo expression verification experiment of the LNP-mRNA prepared in Example 1 in mice.

[0137] 2.1 Tail vein injection of mice

[0138] The mice were fixed and the tail vein was selected for injection. The LNP-hEPO injection volume was 10 µg. LNP-hEPO-Da5 was set as the sample group, LNP-hEPO-120A was set as the positive control group, and only PBS and LNP empty bags were injected as the negative control group. Three mice were injected with each LNP-hEPO sample.

[0139] 2.2 Submandibular blood collection of mice

[0140] 6 h and 24 h after injection, submandibular blood was collected and gently mixed in EDTA anticoagulant tubes, and numbered for later use.

[0141] 2.3 Serum extraction

[0142] After blood was collected, it was centrifuged at 2000 g for 10 min, and the supernatant was plasma, which was stored at -80°C.

[0143] 2.4 Detection of hEPO expression using human Erythropoietin ELISA kit

[0144] The operation was performed according to the instructions, and two replicate wells were set for each serum sample. The OD value was detected by an enzyme-labeled instrument, and the expression level was calculated according to the standard curve and the dilution ratio. The expression levels of LNP-hEPO-Da5 and LNP-hEPO-120A are shown in Tables 1 and Figure 2 As shown:

[0145] Table 1: hEPO expression in mouse peripheral blood

[0146]

[0147] The negative control group injected with only PBS and empty LNP did not express hEPO. The expression of LNP-hEPO-Da5 at 6h was 37% higher than that of LNP-hEPO-120A ((434780.1-319314.2) / 319314.2×100%=36.2%); the expression at 24h was 26.5% higher than that of LNP-hEPO-120A ((98739-78054) / 78054×100%=26.5%). The above results show that the expression of Da5 in mice significantly improves the translation efficiency of mRNA. It can be seen that the insertion of the spacer sequence in the PolyA tail sequence and the replacement of the 105-129 position of the PolyA tail sequence with cytosine nucleotides improve the translation efficiency of mRNA delivered into mice.

[0148] Example 3 Stability of transcription template containing Da5 in Escherichia coli

[0149] 3.1 Plasmid transformation

[0150] The candidate Da5 sequence was connected to the 3' end of the GFP coding region (amino acid sequence as shown in SEQ ID NO:6, nucleotide sequence as shown in SEQ ID NO:7) by seamless cloning to construct the GFP open reading frame. The selected stable Escherichia coli was introduced by chemical transformation, and after cultivation, single clones were screened on resistance plates. The culture medium used was LB medium (peptone, yeast powder, sodium chloride), and the antibiotic was ampicillin (200ug / ml).

[0151] 3.2 Single clone selection

[0152] After transformation, the plate was cultured at 30°C for 16 hours (called the first generation), and 10 single colonies were randomly picked and inoculated into LB liquid culture medium containing 100ug / ml ampicillin, and cultured at 30°C, 220rpm / min overnight.

[0153] 3.3 Serial passaging

[0154] The bacterial solution was diluted with LB to an appropriate concentration and then streaked. The plate was cultured at 30°C for 16 hours (called the second generation). After a single clone was formed, the single clone was picked, shaken, and streaked in sequence. DNA sequencing and sampling were performed after each single clone was picked and shaken. The bacterial solutions of different generations were obtained and stored at -80°C and passed to the 10th generation.

[0155] The transcription template sequences in the first and tenth generations of E. coli were determined by Sanger sequencing technology. The results are as follows Figure 3 As shown. The first and tenth generation sequences of the transcription template (Da5) in E. coli were compared. Since the existing Sanger sequencing technology cannot ensure the fidelity of the enzyme when sequencing repeated sequences, except for the linker region in the middle, the other sequences are the same. It can be seen that the transcription template containing the polyA tail sequence (Da5) of the present invention can be stably propagated to the tenth generation in E. coli.

[0156] discuss:

[0157] It has been more than 50 years since the discovery of polyA at the 3' end of mRNA. During this period, studies have confirmed that the polyA tail should be the core factor regulating mRNA translation and stability. Early studies have shown that if the polyA sequence on the transcription template exceeds 100 nt, deletion mutations will occur, and if it exceeds 150 nt, it is difficult to obtain correct cloning. Therefore, the composition of template polyA is very important for the stability and translation of mRNA molecules and the stability of the template plasmid itself. Rational design of polyA sequence is of great significance for production practice.

[0158] The insertion of non-A ribonucleotides into polyA can slow down deadenylation in a transcript-specific manner. The polyA tail function regulates the degradation of mRNA, and deadenylation (enzymatic trimming of the tail from the 3' end) is directly related to the degradation of mRNA in cells. From the perspective of synthetic mRNA drug development, the present invention provides a new idea for improving the efficacy and reducing the dosage of synthetic mRNA therapeutic drugs. This study proved that the use of cytidine (C) sequences as the tail of synthetic mRNA can improve the expression intensity of synthetic mRNA through experiments on the replacement of non-A nucleotides at the tail.

[0159] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings 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 claims attached to this application.

Claims

1. A polyA tail element, which 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, characterized in that: The polyA tail element is based on the original polyA tail sequence shown in SEQ ID NO: 2, with a spacer sequence shown in SEQ ID NO: 9 inserted, and a segment of adenine nucleotides near the 3' end are replaced with cytosine nucleotides; the nucleotide sequence of the polyA tail element is shown in SEQ ID NO:

1.

2. 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 no or restriction sites; Z2 is no or promoter element or internal ribosome entry site sequence (IRES); Z3 is the 5`UTR element; Z4 is a replaceable coding region; Z5 is the 3`UTR element; Z6 is the polyA tail element as described in claim 1.

3. A vector comprising the mRNA transcription template construct according to claim 2.

4. A host cell comprising the vector according to claim 3 or the mRNA transcription template construct according to claim 2.

5. A method for producing mRNA for preparing an mRNA drug, the method comprising the steps of: (i) culturing the host cell according to claim 4 under suitable conditions to obtain a culture containing the vector of the mRNA transcription template construct; (ii) isolating and / or recovering the vector in (i) from the culture, and linearizing it with enzymes to form an mRNA transcription template; and (iii) transcribing the mRNA transcription template described in (ii) to obtain the mRNA.

6. An mRNA, wherein the mRNA is prepared by the method according to claim 5, and the mRNA has a structure as shown in Formula II: M1-M2-M3-M4-M5-M6 (II) In the formula, M1 is a 5' end cap component; M2 is none or internal ribosome entry site sequence (IRES); M3 is the 5`UTR element; M4 is a replaceable coding region; M5 is the 3`UTR element; M6 is the polyA tail element as described in claim 1.

7. A pharmaceutical composition, comprising: (c1) the mRNA transcription template construct according to claim 2, or the mRNA according to claim 6 as an active ingredient; and (c2) a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition is a lipid nanoparticle formed by encapsulating the mRNA with cationic lipids.

9. Use of the polyA tail element as claimed in claim 1 in constructing an mRNA transcription template, which can improve the translation efficiency of the coding region in the mRNA transcribed from the mRNA transcription template.

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