An mRNA molecule encoding erythropoietin and its application

Abstract

The present invention relates to the fields of biotechnology and medicine, and in particular to an mRNA molecule encoding erythropoietin and its application. The present invention provides an mRNA molecule encoding erythropoietin, which includes an ORF sequence encoding erythropoietin, and the ORF sequence is a nucleotide sequence shown in one of SEQ ID NO:7-9. The present invention provides a brand-new mRNA molecule encoding erythropoietin, which has a higher translation efficiency by rationally optimizing the nucleotide sequence of the functional region, and effectively improves the output of erythropoietin total protein, so the present invention has broad application prospects in the field of gene drug development.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and medicine, and in particular to an mRNA molecule encoding erythropoietin and an application thereof. Background Art

[0002] As an emerging biotechnology tool, mRNA has shown a wide range of application prospects in the medical field. In recent years, the development of mRNA vaccines has experienced significant breakthroughs and application expansion, especially in responding to public health emergencies and infectious disease outbreaks, showing great potential and influence. However, mRNA vaccines are susceptible to degradation by nucleases when delivered in vivo, which reduces the stability and effectiveness of mRNA. At present, the common solution is to use various types of lipid nanoparticles (LNPs) as delivery systems. LNP-mRNA technology is an emerging gene delivery method that protects the stability of mRNA in vivo and promotes its entry into target cells by encapsulating mRNA in lipid nanoparticles. This method is faster than traditional vaccine preparation, can be quickly designed, produced and distributed during an epidemic, and does not need to be integrated into the host genome and obtain efficient protein expression, which is safer and more efficient. The combination of mRNA technology and LNP system provides strong support for the development of mRNA vaccines and provides important guarantees for public health prevention and control. The successful application of COVID-19 vaccine is a typical example of this technological advantage.

[0003] Although mRNA vaccines have many advantages, their application still faces some important challenges. For example, the complex intracellular and extracellular environment, as well as the widespread existence of nucleases, can easily make mRNA inactive. To address these problems, researchers have developed a variety of strategies to enhance the stability and anti-degradation ability of mRNA. These include chemical modifications, such as the introduction of 2'-O-methylation (2'-O-methylation, Nm), 5-methylcytosine (5-methylcytosine, 5mC) or N1-methyl pseudouridine (N1-Methyl-Pseudouridine, m1Ψ), to reduce the risk of mRNA being recognized and degraded by nucleases and reduce its immunogenicity; and by optimizing the mRNA sequence and structure, reducing sequence elements and secondary structures that are easily degraded, and improving the stability and translation efficiency of mRNA. Therefore, mRNA optimization is a key step in improving the efficacy, stability and safety of mRNA vaccines and therapeutic mRNA.

[0004] At the current stage, the means of sequence optimization of mRNA mainly focus on adjusting the Codon Adaptation Index (CAI) and selecting the most suitable high-frequency codons for the target host cells to improve the translation efficiency of mRNA. At the same time, by introducing optimized ribosome binding sites (such as enhanced Kozak sequences) or adding specific regulatory elements in the 5'UTR to improve the efficiency of translation initiation; or introducing stable elements such as AU-rich elements or microRNA binding sites in the 3'UTR to extend the half-life of mRNA and regulate its translation. In addition, by adjusting the sequence, regulating the secondary structure of mRNA, avoiding the formation of a large number of structures such as hairpins or stem-loops, the translatability and stability of mRNA are improved. However, although mRNA sequence optimization can improve stability and translation efficiency in theory, it may be limited by immune response, production cost and structural complexity in practice. Therefore, in vitro and in vivo experimental testing of these sequence-optimized mRNAs can provide clearer and more intuitive guidance.

[0005] Erythropoietin (EPO), also known as erythrocyte stimulating factor or erythropoietin, is a glycoprotein hormone synthesized and secreted by the kidneys, which mainly regulates the production of red blood cells. EPO promotes the proliferation and differentiation of red blood cell precursor cells by binding to receptors on them, thereby improving the efficiency of red blood cell production. In biomedical research, EPO is often used as a model protein to study gene expression and protein secretion mechanisms due to its clear biological functions and easy detection characteristics. The secretion characteristics of EPO enable it to be directly detected in cell culture medium. By measuring the concentration of EPO, the expression level, translation efficiency and secretion process of the gene can be indirectly reflected. This method is similar to traditional reporter gene systems (such as fluorescent proteins or enzymes), but EPO itself has clear biological functions, and the detection is usually highly sensitive and specific. Therefore, in some experiments, EPO can be used as an effective reporter gene alternative. In the research and application of LNP-mRNA technology, using EPO as a reporter gene can effectively evaluate the delivery efficiency of mRNA and the level of protein expression. Therefore, sequence optimization of EPO can further improve its expression efficiency and stability, making it more suitable for use as a reporter gene. Summary of the invention

[0006] In order to solve the above problems, the present invention provides an mRNA molecule encoding erythropoietin with higher expression efficiency, which has higher protein production efficiency and can be used as a reporter gene, and has good potential in the fields of gene drug development.

[0007] The present invention provides an mRNA molecule encoding erythropoietin, which comprises an ORF sequence encoding erythropoietin, wherein the ORF sequence is a nucleotide sequence shown in one of SEQ ID NOs: 7-9.

[0008] Preferably, the mRNA molecule further comprises a 5'UTR and / or a 3'UTR.

[0009] More preferably, the 5'UTR is a nucleotide sequence shown in any one of SEQ ID NOs: 1-2, and the 3'UTR is a nucleotide sequence shown in any one of SEQ ID NOs: 4-5.

[0010] Specifically, the mRNA molecule comprises, in 5' to 3' order: a 5'UTR as shown in one of SEQ ID NOs: 1-2, an ORF region of an ORF sequence as shown in any one of SEQ ID NOs: 7-9, and a 3'UTR as shown in one of SEQ ID NOs: 4-5.

[0011] Further, the mRNA molecule comprises, in 5' to 3' order: the 5'UTR shown in SEQ ID NO: 1, the ORF sequence shown in SEQ ID NO: 7 and the 3'UTR shown in SEQ ID NO: 4; or,

[0012] The mRNA molecule comprises, in 5' to 3' order: a 5'UTR as shown in SEQ ID NO: 1, an ORF as shown in SEQ ID NO: 8, and a 3'UTR as shown in SEQ ID NO: 4; or,

[0013] The mRNA molecule comprises, in 5' to 3' order: a 5'UTR as shown in SEQ ID NO: 1, an ORF as shown in SEQ ID NO: 9, and a 3'UTR as shown in SEQ ID NO: 4; or,

[0014] The mRNA molecule comprises, in 5' to 3' order: a 5'UTR as shown in SEQ ID NO: 2, an ORF sequence as shown in SEQ ID NO: 7, and a 3'UTR as shown in SEQ ID NO: 5; or,

[0015] The mRNA molecule comprises, in 5' to 3' order: a 5'UTR as shown in SEQ ID NO: 2, an ORF as shown in SEQ ID NO: 8, and a 3'UTR as shown in SEQ ID NO: 5; or,

[0016] The mRNA molecule comprises, in 5' to 3' order: a 5'UTR as shown in SEQ ID NO: 2, an ORF as shown in SEQ ID NO: 9, and a 3'UTR as shown in SEQ ID NO: 5.

[0017] Furthermore, the mRNA molecule further comprises a 5' cap structure and / or a poly A sequence; the 5' cap structure is selected from a Cap0 cap structure, a Cap1 cap structure or a Cap2 cap structure; and the poly A sequence comprises 20-500 adenine nucleotides.

[0018] In some embodiments, the mRNA molecule comprises at least one chemical modification selected from at least one of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio T-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine or 5-methoxyuridine and 2'-O-methyluridine.

[0019] The present invention provides a vector containing the mRNA molecule encoding the gene.

[0020] The present invention also provides a cell, which comprises the mRNA molecule or the vector.

[0021] The present invention also provides a pharmaceutical composition comprising one of the following components (1) to (3), and a pharmaceutically acceptable carrier;

[0022] (1) the aforementioned mRNA molecule;

[0023] (2) the aforementioned vectors;

[0024] (3) The aforementioned cells.

[0025] The present invention also provides a method for preparing erythropoietin, which comprises culturing the aforementioned cells to obtain erythropoietin.

[0026] The present invention also provides the use of the aforementioned mRNA molecule or the aforementioned vector as a reporter gene.

[0027] The present invention also provides a lipid nanoparticle, which comprises the mRNA molecule or the polynucleotide or the vector.

[0028] The beneficial effects of the present invention are at least as follows:

[0029] The present invention provides a new erythropoietin mRNA molecule. By rationally optimizing the nucleotide sequence of the functional region, the erythropoietin mRNA molecule has a higher translation efficiency, effectively improves the output of erythropoietin total protein, and makes the mRNA encoding luciferase have broad application prospects in the field of gene drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the sequence information of an exemplary erythropoietin mRNA molecule.

[0031] Figure 2 It is an exemplary capillary electrophoresis detection result of erythropoietin mRNA molecule.

[0032] Figure 3 It is the relative light absorption ratio of candidate erythropoietin mRNA molecules in different cell lines, indicating its protein expression level. DETAILED DESCRIPTION

[0033] I. Definition and Explanation

[0034] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms and laboratory operation steps used herein are terms and routine steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definition and explanation of the relevant terms are provided below. It should be understood that the present invention is not limited to specific methods, reagents, compounds, compositions or biological systems, and of course the above can be changed. It should also be understood that the terms used in this application are only for describing specific embodiments and are not intended to be limited.

[0035] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0036] As used herein, the terms "include" and "have" and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, product or device comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps that are not listed, or may optionally include other steps that are inherent to these processes, methods, products or devices. The "plurality" mentioned in the present disclosure refers to two or more. "And / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B may represent: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the previously associated objects are in an "or" relationship.

[0037] As used herein, the term "nucleotide" refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and polymers thereof in single-stranded, double-stranded or multi-stranded form. The term includes, but is not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and / or pyrimidine bases or other natural, chemically modified, biochemically modified, non-natural, synthetic or derived nucleotide bases.

[0038] As used herein, the term "mRNA (messenger RNA)" refers to any polynucleotide that encodes a polypeptide or protein and can be translated to produce the encoded protein in vitro, in vivo, in situ or ex vivo.

[0039] As used herein, the term "open reading frame" (ORF) is a normal nucleotide sequence of a structural gene, which has the potential to encode a protein or polypeptide, starting from a start codon and ending at a stop codon, without a stop codon that interrupts translation. On an mRNA chain, the ribosome starts translating from the start codon, synthesizes a polypeptide chain along the RNA sequence and continues to extend, and when the stop codon is encountered, the extension reaction of the polypeptide chain is terminated.

[0040] As used herein, the term "identity" refers to the ratio of the total number of mismatched nucleotides to nucleotides in a homologous region expressed as a percentage. For example, a 20 base oligonucleotide hybridizing to a homologous region (site) in a target genome having two mismatches is referred to as having 90% identity to the region.

[0041] As used herein, the term "5'UTR" generally refers to the sequence between the 5' end of an mRNA molecule and the translation start codon, which is capable of recruiting ribosome complexes and initiating translation of mRNA. The 5'UTR includes the 5'UTR region structure on the mRNA or the structure corresponds to the coding sequence on the DNA template. The 5'UTR regulates post-transcriptional modification, the formation and stability of the translation initiation complex and other processes by interacting with transcription factors, ribosomes and other transcription regulatory proteins. Sequence design and optimization of this region are crucial for improving the efficiency of post-transcriptional modification and protein expression. The 5'UTR sequence involved in the present disclosure has a nucleotide sequence selected from the nucleotide sequence shown in one of SEQ ID NOs: 1-2 or its complementary sequence, or a nucleotide sequence having at least 80% identity with the nucleotide sequence shown in one of SEQ ID NOs: 1-2 or its complementary sequence.

[0042] As used herein, the term "3'UTR" refers to the sequence between the stop codon of the polypeptide coding sequence in the mRNA and the poly (A) sequence. The 3'UTR can regulate the translation of the mRNA by interacting with mRNA binding proteins, miRNA, etc. The 3'UTR includes the 3'UTR region structure on the mRNA or the structure corresponds to the coding sequence on the DNA template. It is closely related to post-transcriptional modification and mRNA stability. The sequence and structural characteristics of the 3'UTR can affect the stability of the mRNA, the scanning of the ribosome, the formation of the translation termination complex, etc., thereby affecting the expression level of the protein. The 3'UTR element involved in the present invention comprises a nucleotide sequence selected from one of SEQ ID NOs: 4-5 or its complementary sequence, or a nucleotide sequence having at least 80% identity with the nucleotide sequence shown in one of SEQ ID NOs: 4-5 or its complementary sequence.

[0043] As used herein, the term "complementary sequence" refers to a nucleic acid sequence that forms hydrogen bonds with another nucleic acid sequence through traditional Watson-Crick or other non-traditional types.

[0044] As used herein, the term "poly A sequence" includes a poly A sequence structure on mRNA or the structure corresponds to a coding sequence on a DNA template. The addition of a poly A sequence contributes to the stability and transport of mRNA, prevents its degradation, and plays an important role in the post-transcriptional modification process. The poly A sequence can be a continuous chain of pure adenine nucleotides, or it can contain non-adenine nucleotides. In any form, as long as its function is equivalent to that of a traditional poly A sequence, that is, it can provide biological functions similar to those of a traditional poly A sequence, such as affecting the stability of mRNA, translation efficiency, or ribosome binding, the sequence is identified as a poly A sequence. This includes but is not limited to known variants such as human growth hormone (hGH) poly A sequence and simian virus 40 (SV40) poly A sequence, which may differ in nucleotide composition but are functionally identified as equivalent to traditional poly A sequences. In the present invention, the poly A sequence comprises 20-500 adenine nucleotides, for example, 25, 50, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 175, 180, 190, 200, 210, 220, 230, 240, 250, 300, 350, 400, 450 or 500 adenine nucleotides.

[0045] As used herein, the meaning of the term "5' cap structure" includes 5' cap structures and analogs thereof present on natural mRNA. The 5' cap structure on natural mRNA refers to that methylated guanylate is connected to the 5' terminal nucleotide of RNA via pyrophosphate to form a 5', 5'-triphosphate connection (5', 5'-triphosphate linkage). There are three types of 5' cap structures (m7G5'ppp5'Np, m7G5'ppp5'NmpNp, m7G5'ppp5'NmpNmpNp), which are respectively referred to as Cap0, Cap1 and Cap2. Cap0 refers to that the ribose of the terminal nucleotide is not methylated, Cap1 refers to that the ribose of one nucleotide at the end is methylated, and Cap2 refers to that the ribose of the two nucleotides at the end is methylated. Methods for capping mRNA molecules are known in the art. The 5' cap structure of the aforementioned mRNA molecule can be added by enzymatic reaction after obtaining the mRNA molecule by chemical synthesis or in vitro transcription (for example, by a commercial kit comprising vaccinia capping enzyme and mRNA cap structure 2'-O-methyltransferase). However, capped mRNA can also be produced by incorporating a capping nucleotide analog directly as the first nucleotide into the transcript during in vitro transcription.

[0046] As used herein, the term "chemical modification" or "chemically modified" refers to a modification of one or more of the position, pattern, percentage or population of adenosine (A), guanosine (G), uridine (U) or cytidine (C) ribonucleosides or deoxyribonucleosides. In this article, these terms are not intended to refer to ribonucleotide modifications in naturally occurring 5' cap structures.

[0047] As used herein, the term "vector" refers to a section of DNA extracted from a virus, plasmid or a cell of a higher organism, into which a foreign DNA fragment can be inserted or has been inserted for cloning and / or expression purposes. In certain embodiments, the vector can be stably maintained in an organism. The vector may include, for example, an origin of replication, a selective marker or a reporter gene, such as antibiotic resistance or GFP, and / or a multiple cloning site (MCS). The term includes linear DNA fragments (e.g., PCR products, linear plasmid fragments), plasmid vectors, viral vectors, cosmids, bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), etc.

[0048] As used herein, the terms "cell" and "host cell" are used interchangeably in the present invention and refer to cells that express or are capable of expressing a sequence to be expressed. The host cells of the present invention express polynucleotides encoding polypeptides or RNAs with a variety of uses, including biotechnology, molecular biology, and clinical applications. Host cells include prokaryotic cells or eukaryotic cells, and examples of suitable host cells in the present invention include, but are not limited to, bacteria, yeast cells, insect cells, animal cells, and mammalian cells.

[0049] As used herein, the term "pharmaceutically acceptable carrier" refers to one or more compatible solid, semisolid, liquid or gel fillers that are suitable for human or animal use and must have sufficient purity and sufficiently low toxicity. "Compatibility" refers to the components in the pharmaceutical composition and the active ingredients of the drug and their mutual blending without significantly reducing the efficacy. In the present invention, the aforementioned pharmaceutically acceptable carrier includes but is not limited to a buffer, an excipient, a stabilizer or a preservative. Examples of pharmaceutically acceptable carriers are physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, such as salts, buffers, sugars, antioxidants, aqueous or non-aqueous carriers, preservatives, wetting agents, surfactants or emulsifiers or combinations thereof. The amount of a pharmaceutically acceptable carrier in a pharmaceutical composition can be determined experimentally based on the activity of the carrier and the desired properties of the formulation, such as stability and / or minimal oxidation.

[0050] As used herein, the term "lipid nanoparticle" refers to a particle containing lipid components and having nanometer-scale dimensions.

[0051] As used herein, the term "ionizable cationic lipid" refers to a lipid molecule that can be positively charged under physiological pH conditions. For example, the ionizable cationic lipid is an amino lipid.

[0052] As used herein, the term "neutral lipid" refers to a lipid molecule that is uncharged under specific pH conditions, such as physiological pH conditions. Examples of neutral lipids include, but are not limited to, one or more of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), preferably DSPC and / or DOPE.

[0053] As used herein, the term "structured lipid" refers to a lipid that fills the gaps between lipids to enhance the stability of nanoparticles, such as steroids. Steroids are compounds with a cyclopentane pyrophenanthrene carbon skeleton. In a preferred embodiment, the steroid is selected from one or more of cholesterol, sitosterol, coprosterol, saposterol, brassicasterol, ergosterol, tomatine, ursolic acid, α-tocopherol, stigmasterol, avenasterol, ergocalciferol or campesterol, preferably cholesterol and / or β-sitosterol, more preferably cholesterol.

[0054] As used herein, the term "polymer lipid" refers to a molecule containing a polymer portion and a lipid portion. In some embodiments, the polymer lipid is a polyethylene glycol (PEG) lipid. Other lipids that can reduce aggregation, such as products of lipid coupling with compounds having uncharged, hydrophilic, steric barrier portions, can also be used. In a preferred embodiment, the PEGylated lipid is selected from: one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. Optionally, the PEGylated lipid contains a PEG portion of about 1000Da to about 20kDa, preferably a PEG portion of about 1000Da to about 5000Da. Optionally, the PEGylated lipid is selected from one or more of DMPE-PEG1000, DPPE-PEG1000, DSPE-PEG1000, DOPE-PEG1000, DMG-PEG2000, Ceramide-PEG2000, DMPE-PEG2000, DPPE-PEG2000, DSPE-PEG2000, Azido-PEG2000, DSPE-PEG2000-Mannose, Ceramide-PEG5000, and DSPE-PEG5000, preferably DMG-PEG2000.

[0055] II. Detailed description of specific implementation plan

[0056] On the one hand, the present invention provides an mRNA molecular sequence encoding erythropoietin, which comprises an ORF sequence encoding erythropoietin, wherein the ORF sequence is a nucleotide sequence shown in one of SEQ ID NOs: 7-9 or a nucleotide sequence having at least 80% identity thereto.

[0057] In some embodiments, the mRNA further comprises a 5'UTR and / or a 3'UTR; the 5'UTR is a nucleotide sequence as shown in one of SEQ ID NOs: 1-2 or a complementary sequence thereof, or a nucleotide sequence having at least 80% identity with a nucleotide sequence as shown in one of SEQ ID NOs: 1-2 or a complementary sequence thereof; the 3'UTR is a nucleotide sequence as shown in one of SEQ ID NOs: 4-5 or a complementary sequence thereof, or a nucleotide sequence having at least 80% identity with a nucleotide sequence as shown in one of SEQ ID NOs: 4-5 or a complementary sequence thereof.

[0058] In some embodiments, the above-mentioned 3'UTR further comprises a 3'UTR or its complementary sequence inserted with an additional sequence; preferably, the additional sequence is a miRNA binding site; more preferably, the additional sequence is selected from the full-length microRNA reverse complementary sequence or the reverse complementary sequence of its seed sequence; preferably, the full-length microRNA reverse complementary sequence is 19-25nt in length; preferably, the reverse complementary sequence of the seed sequence is 7-8nt in length; preferably, the aforementioned 3'UTR sequence further comprises a 3'UTR sequence or its complementary sequence inserted with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional sequences.

[0059] In some embodiments, the mRNA molecule comprises, in 5' to 3' order: a 5'UTR as shown in one of SEQ ID NOs: 1-2, an ORF sequence as shown in any one of SEQ ID NOs: 7-9, and a 3'UTR as shown in one of SEQ ID NOs: 4-5.

[0060] In some embodiments, the mRNA molecule comprises, in 5' to 3' order: a 5'UTR as shown in SEQ ID NO: 1, an ORF sequence as shown in SEQ ID NO: 7, and a 3'UTR as shown in SEQ ID NO: 4; or,

[0061] The mRNA molecule comprises, in 5' to 3' order: the 5'UTR shown in SEQ ID NO: 1, the ORF shown in SEQ ID NO: 8, and the 3'UTR shown in SEQ ID NO: 4; or,

[0062] The mRNA molecule comprises, in 5' to 3' order: the 5'UTR shown in SEQ ID NO: 1, the ORF shown in SEQ ID NO: 9, and the 3'UTR shown in SEQ ID NO: 4; or,

[0063] The mRNA molecule comprises, in 5' to 3' order: the 5'UTR shown in SEQ ID NO: 2, the ORF sequence shown in SEQ ID NO: 7, and the 3'UTR shown in SEQ ID NO: 5; or,

[0064] The mRNA molecule comprises, in 5' to 3' order: the 5'UTR shown in SEQ ID NO:2, the ORF shown in SEQ ID NO:8, and the 3'UTR shown in SEQ ID NO:5; or,

[0065] The mRNA molecule comprises, in 5' to 3' order: a 5'UTR as shown in SEQ ID NO:2, an ORF as shown in SEQ ID NO:9, and a 3'UTR as shown in SEQ ID NO:5.

[0066] In some optional embodiments, the mRNA further comprises a 5' cap structure and / or a poly A sequence; the 5' cap structure is selected from Cap0 cap structure, Cap1 cap structure or Cap2 cap structure, preferably Cap1 cap structure; the poly A sequence comprises 20-500 adenine nucleotides, preferably the poly-A sequence comprises 25, 50, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 175, 180, 190, 200, 210, 220, 230, 240, 250, 300, 350, 400, 450 or 500 adenine nucleotides, more preferably 120 adenine nucleotides.

[0067] In some embodiments, the above-mentioned mRNA molecule contains at least one chemical modification, and the chemical modification is selected from at least one of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio T-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine or 5-methoxyuridine and 2'-O-methyluridine.

[0068] In another aspect, the present invention provides a vector encoding the aforementioned RNA molecule.

[0069] In some embodiments, the aforementioned vectors include but are not limited to the following types: linear DNA fragments (such as PCR products, linear plasmid fragments), plasmid vectors, viral vectors, cosmids, bacterial artificial chromosomes (BAC) or yeast artificial chromosomes (YAC), etc. In some preferred embodiments, the aforementioned vector is a plasmid vector or a viral vector.

[0070] In some embodiments, the aforementioned vector comprises an RNA polymerase promoter sequence, an RNA coding sequence, and / or a restriction endonuclease site and a selective marker. The RNA polymerase promoter sequence is operably connected to the RNA coding sequence, which allows RNA molecules to be transcribed in vivo and / or in vitro. The aforementioned RNA polymerase promoter can be various types, including but not limited to T7 RNA polymerase promoter, T6 viral RNA polymerase promoter, SP6 viral RNA polymerase promoter, T3 viral RNA polymerase promoter or T4 viral RNA polymerase promoter. In some preferred embodiments, the aforementioned vector comprises a restriction endonuclease site at the 3' flank of the coding sequence of the aforementioned mRNA molecule, such as an IIS type restriction endonuclease site. Suitable restriction endonucleases include but are not limited to BsmBI, BsaI or SapI etc. The aforementioned restriction endonuclease site can be used for linearizing the vector to carry out in vitro transcription.

[0071] In another aspect, the present invention provides a cell comprising the aforementioned RNA molecule and / or the aforementioned vector.

[0072] In some embodiments, the aforementioned cells include prokaryotic cells or eukaryotic cells. In some preferred embodiments, the aforementioned cells are selected from the group consisting of Escherichia coli, yeast cells, or mammalian cells. In some more preferred embodiments, the cells are mammalian cells, including but not limited to rodent cells such as mouse cells or rat cells, etc.; primate cells such as monkey cells or human cells, etc. In some more preferred embodiments, the aforementioned cells are human cells. Furthermore, the aforementioned cells are Hela cells and 293T cells.

[0073] In another aspect, the present invention provides a pharmaceutical composition comprising one of the following components (1) to (3), and a pharmaceutically acceptable carrier;

[0074] (1) The aforementioned mRNA;

[0075] (2) the aforementioned vectors;

[0076] (3) The aforementioned cells.

[0077] In some embodiments, the mRNA itself in the aforementioned pharmaceutical composition can also serve as an adjuvant.

[0078] In some embodiments, the dosage form of the aforementioned pharmaceutical composition is selected from an injection or a lyophilized preparation.

[0079] On the other hand, the present invention provides a method for promoting the expression of erythropoietin, which comprises introducing the aforementioned RNA molecule and / or the aforementioned vector into a host cell. By the method of the present invention, the aforementioned host cell can contain the aforementioned RNA molecule or the aforementioned vector, or the aforementioned sequence or RNA molecule can be integrated into its genome, so that the cell expresses erythropoietin.

[0080] In some embodiments, the aforementioned sequences or RNA molecules can be introduced into the aforementioned cells by microinjection or liposome-mediated transfection or electroporation.

[0081] In some embodiments, the aforementioned mRNA is sequentially coupled and cloned into the multiple cloning site of the vector in a "head-to-tail relationship" order, and an IIS type restriction endonuclease site is added after the poly A tail to guide the IIS type restriction endonuclease to cut the DNA template. The multiple cloning site of the vector refers to a nucleic acid region containing a restriction endonuclease, any of which can be used to cut the vector and insert a sequence. The restriction endonuclease can recognize a specific sequence (binding site) on a double-stranded DNA molecule and cut the phosphodiester bond. The vector will be cut under the action of the IIS type restriction endonuclease, and the cutting site is located at a determined distance from the DNA binding site. The product is recovered to obtain a linearized plasmid. The IIS type restriction endonuclease involved in the present invention includes, but is not limited to, BsmBI, BsaI or SapI.

[0082] The linearized plasmid template is transcribed in vitro using T7 DNA polymerase to produce mRNA molecules, and a 5' cap structure is added at the same time. The 5' cap structure can be generated by co-transcriptional capping or post-transcriptional capping. The difference is that co-transcriptional capping is the incorporation of a cap structure analog as the first nucleotide into the transcript during in vitro transcription to directly produce mRNA with a cap1 cap structure; while post-transcriptional capping is the use of vaccinia capping enzyme to first produce a cap0 cap structure after in vitro transcription interpretation, and then the mRNA cap structure 2'-O-methyltransferase is used to add a cap1 structure. The mRNA produced is purified and resuspended in water for subsequent transfection and detection.

[0083] The above mRNA molecules encoding erythropoietin were transfected into mammalian cells Hela and 293T cells. At 24 hours after transfection, cell samples were collected and subjected to enzyme-linked immunosorbent assay to compare the protein expression levels produced by different optimized EPO mRNAs, thereby proving that the erythropoietin mRNA sequence designed in the present invention has high protein output.

[0084] In another aspect, the present invention provides a method for preparing erythropoietin, which comprises culturing the aforementioned cells to obtain erythropoietin.

[0085] On the other hand, the present invention provides a use of the aforementioned RNA molecule, the aforementioned vector, the aforementioned cell, the aforementioned pharmaceutical composition and / or the aforementioned method in promoting the expression of erythropoietin.

[0086] In another aspect, the present invention provides use of the aforementioned mRNA molecule or vector as a reporter gene.

[0087] The present invention finally provides a lipid nanoparticle comprising the above mRNA.

[0088] In some preferred embodiments, the lipid nanoparticles further comprise one or more lipid moieties, wherein the one or more lipid moieties are selected from the group consisting of ionizable cationic lipids, structural lipids, neutral lipids or polymer lipids.

[0089] For purposes of clarity and concise description, features are described herein as part of the same or separate embodiments, however, it will be understood that the scope of the invention may include embodiments having a combination of all or some of the described features.

[0090] III. Example

[0091] Example 1: Construction of a vector containing erythropoietin mRNA sequence

[0092] The present invention selects a UTR combination that can effectively enhance translation efficiency (5'UTR shown in one of SEQ ID NO:1-2, 3'UTR shown in one of SEQ ID NO:4-5), and further performs sequence design and screening to increase the yield of erythropoietin protein. For the erythropoietin protein determined by the amino acid sequence (SEQ ID NO:11), the conservative codon sequence is screened out by comparison, and the technology in the field of natural language processing and the RNA secondary structure prediction model (such as CDSfold, Ribotree) are further used to assign weights and identify stable mRNA sequences. The two parameters of CAI and MFE are balancedly combined to screen out a batch of mRNA sequences that may achieve higher erythropoietin protein yields. These mRNA sequences are subjected to gene synthesis, in vitro transcription template preparation, plasmid linearization, IVT mRNA synthesis, purification quality inspection and other steps to obtain mRNA molecules for in vitro cell experiments. Finally, candidate mRNA sequences are obtained (see hEPO-1, hEPO-2, hEPO-3, hEPO-4, hEPO-5 and hEPO-6 in Table 2). The erythropoietin mRNA sequence used as a control (see control in Table 2) was derived from the erythropoietin gene sequence CleanCap® M6 EPO mRNA (N1MePsU)-(L-8109) (SEQ ID NO: 10) publicly provided by Trilink, combined with the 5'UTR (SEQ ID NO: 3) and 3'UTR sequences (SEQ ID NO: 6) from Modern mRNA1273 expression vector, complete sequence (GenBank: OR134578.1).

[0093] Table 1. UTR sequences

[0094]

[0095] Table 2. Sequences of mRNA molecules encoding erythropoietin

[0096]

[0097] To test candidate mRNA sequences, a nucleic acid fragment containing a T7 promoter, a 5'UTR, an ORF sequence encoding erythropoietin, a 3'UTR, a poly(A) sequence containing 120 A nucleotide residues, and a type IIS restriction endonuclease cleavage site was synthesized in vitro and cloned into an in vitro transcription vector (pIVTRup, Addgene plasmid #101362).

[0098] Example 2: mRNA molecule length and integrity detection

[0099] The vector obtained in Example 1 was linearized and transcribed in vitro using T7-RNA polymerase to produce mRNA molecules, and a 5' cap structure was added at the same time. The 5' cap structure is capped by co-transcription, and the cap analog is incorporated into the transcript as the first nucleotide during in vitro transcription to directly produce mRNA molecules with Cap1 structure. Figure 1 The sequence of an exemplary mRNA comprising the erythropoietin mRNA molecule of the present invention is shown. In order to produce the plasmid of other candidate mRNAs or control mRNAs in Table 2, Figure 1 The 5'UTR sequence (underlined) - ORF sequence encoding erythropoietin (bold) - 3'UTR sequence (underlined) can be replaced with other candidate mRNA sequences or control mRNA sequences, which are also synthesized and cloned into the vector, and other sequence elements remain unchanged.

[0100] The mRNA molecules thus obtained were purified and resuspended in water. The mRNA molecules were quality checked using a 5200 fragment analyzer (Agilent) to detect whether the length and integrity of the mRNA molecules (values ​​derived from the area under the curve of the expected length fragments) met the requirements (see Figure 2 ), which can be used for subsequent testing of different candidate sequences.

[0101] Example 3: Verification of in vitro expression level of erythropoietin mRNA sequence

[0102] The candidate erythropoietin mRNA sequences generated by the algorithm were tested in vitro to see whether they had higher protein output, as follows:

[0103] The same amount of the candidate mRNA molecules and Renilla luciferase mRNA molecules were co-transfected into mammalian cells Hela and 293T. The absorbance value of erythropoietin was detected by enzyme linked immunosorbent assay (ELISA) 24 hours after transfection, representing its protein expression; the chemiluminescent light absorption value of Renilla luciferase was used to equalize the cell amount and mRNA transfection efficiency of the group. The light absorption value reading obtained by ELISA detection of erythropoietin was divided by the light absorption reading of Renilla luciferase to obtain the relative light unit of the group. Each candidate mRNA molecule was processed to the corresponding relative light unit after detection in this experiment, which can refer to the protein expression level of the candidate erythropoietin mRNA molecule.

[0104] The specific experimental procedure for erythropoietin protein expression used to determine translation efficiency is as follows:

[0105] Hela human cervical cancer cells and 293T human embryonic kidney cells were cultured at 5×10 4 Cells were seeded in 48-well plates at a density of 10 cells / well. The next day, the cells were washed in Opti-MEM, and then 2 μl of Lipofectamine2000, 200 ng / well of candidate mRNA molecules encoding erythropoietin, and 100 ng / well of mRNA molecules encoding Renilla luciferase were added to Opti-MEM for co-transfection. Cells without any RNA molecules were used as background groups. After 6 hours of transfection, the mixed culture medium was aspirated and replaced with complete culture medium.

[0106] 24 hours after transfection, the cell culture supernatant was collected for ELISA detection.

[0107] The following steps were performed: After collecting the cell culture supernatant, centrifuge (500 g, 5 minutes) and take the supernatant. Next, prepare 7 concentration gradient standard solutions for subsequent standard curve drawing. Add 100 μL of sample or standard of different concentrations to the pre-coated well plate, and set up blank wells as background correction blanks. Seal the reaction wells with a sealing film and incubate the well plate at room temperature (25°C) for 120 minutes. Next, add 300 μL of washing solution to each well, wash the plate 5 times with an interval of 30 seconds each time, and pat the well plate dry on thick absorbent paper for the last time. Subsequently, add 100 μL of biotinylated antibody to each well, seal the reaction well with a sealing film, and incubate at room temperature for 60 minutes. Wash the plate five times as before. Then, add 100 μL of horseradish peroxidase-labeled streptavidin to each well, seal the reaction well with a sealing film again, and incubate at room temperature in the dark for 20 minutes. Wash the plate five times as before. Subsequently, 100 μL of TMB was added to each well, the reaction wells were sealed with a sealing film, and incubated at room temperature in the dark for 20 minutes. Finally, 50 μL of stop solution (such as sulfuric acid or other appropriate stop reagent) was added to each well, and after shaking and mixing, the A450 value was immediately measured and the A450 value of each experimental group was recorded as the absorbance value of erythropoietin.

[0108] Add 100 μL of lysis buffer (Promega) to the culture wells from which the cell supernatant was removed, lyse at room temperature for 5 minutes, and measure the Renilla luciferase activity in relative light units (RLU) in a multiwell plate reader (Agilent). Pipette 20 μL of lysis buffer, add 50 μL of buffer containing sea cucumber luciferase substrate, shake the plate to mix, and detect the sea cucumber luciferin light absorption value.

[0109] Subtract the absorbance value of the background correction well from the absorbance value of each standard and sample. Furthermore, use the standard concentration as the horizontal coordinate, the A450 value after subtracting the background correction well as the vertical coordinate, and connect the coordinate points of the standard in each well with a smooth line to draw a standard curve. Substitute the absorbance value of the sample into the drawn standard curve to calculate the corresponding concentration of erythropoietin protein. After subtracting the absorption value of the respective background group from the Renilla fluorescein absorbance value, the concentration value of the erythropoietin protein is divided by the Renilla fluorescein absorption value, and then the relative value of the erythropoietin protein level that has been normalized for transfection efficiency and cell number can be obtained. Make a bar graph of different candidate mRNA molecules for relative value analysis, such as Figure 3 .from Figure 3 It can be seen that the candidate mRNA molecular sequence encoding erythropoietin has a higher protein output in the host cells Hela and 293T cells, indicating that the mRNA molecular sequence encoding erythropoietin can be efficiently expressed in the host cells Hela and 293T cells and has a high protein expression efficiency.

Claims

1. An mRNA molecule encoding erythropoietin, characterized in that It consists of, in order from 5' to 3', a 5'UTR of the nucleotide sequence shown in SEQ ID NO: 2, an ORF sequence encoding erythropoietin, and a 3'UTR of the nucleotide sequence shown in SEQ ID NO: 5, wherein the ORF sequence is the nucleotide sequence shown in SEQ ID NO: 8 or 9.

2. The mRNA molecule according to claim 1, characterized in that The mRNA molecule further comprises a 5' cap structure and / or a poly A sequence; the 5' cap structure is selected from a Cap0 cap structure, a Cap1 cap structure or a Cap2 cap structure; and the poly A sequence comprises 20-500 adenine nucleotides.

3. The mRNA molecule according to claim 1 or 2, characterized in that The mRNA molecule includes at least one chemical modification selected from at least one of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-T-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine or 5-methoxyuridine and 2'-O-methyluridine.

4. A vector containing an mRNA molecule encoding any one of claims 1-3.

5. A cell comprising the mRNA molecule according to any one of claims 1 to 3 or the vector according to claim 4.

6. A pharmaceutical composition comprising one of the following components (1) to (3), and a pharmaceutically acceptable carrier; (1) The mRNA molecule according to any one of claims 1 to 3; (2) The vector according to claim 4; (3) The cell according to claim 5.

7. A method for preparing erythropoietin, characterized in that: The method comprises culturing the cell according to claim 5 to obtain erythropoietin.

8. Use of the mRNA molecule according to any one of claims 1 to 3 or the vector according to claim 4 as a reporter gene.

9. A lipid nanoparticle comprising the mRNA molecule according to any one of claims 1 to 3 or the vector according to claim 4.

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