Mrna encoding luciferase and its use

Abstract

The application belongs to the technical field of biotechnology and medicine, and discloses an mRNA coding luciferase and application thereof. The mRNA coding luciferase comprises an ORF sequence coding luciferase, and the ORF sequence coding luciferase is a nucleotide sequence shown in one of SEQ ID NO: 10-13. The disclosure provides a brand-new mRNA coding luciferase. By reasonably optimizing the nucleic acid sequence of a functional region, the mRNA is stable in structure, not easy to degrade, and has high translation efficiency, high protein yield, high biological availability and safety, and can be used as a reporter gene, and has good potential.

Description

[0001] This application is a divisional application of the original application No. 2024109783015, entitled "mRNA encoding luciferase and its application", filed on July 22, 2024. TECHNICAL FIELD

[0002] The present application belongs to the field of biotechnology and medicine, and more specifically relates to mRNA encoding luciferase and its application. BACKGROUND

[0003] In recent years, mRNA technology has shown unprecedented potential in the field of biomedicine, especially in vaccine development and genetic disease treatment. During the global response to the Sars-CoV-2 epidemic, mRNA technology played a key role in vaccine development. By rapidly designing and producing mRNA vaccines against specific viral antigens, mRNA technology has shown great value and potential in public health emergencies. It is particularly worth mentioning that by chemically modifying mRNA molecules, such as replacing uracil with N1-methyl-pseudouridine (m1Ψ) or 5-methoxy-uridine (5moU), the immunogenicity of mRNA is significantly reduced, making mRNA-based vaccines and enzyme replacement therapy applications possible. This modification not only increases the stability of mRNA molecules in the body, but also optimizes their translation efficiency in cells, providing a new way to treat various genetic diseases.

[0004] However, the application of mRNA still faces many challenges, and efficient delivery is one of them. Because the extracellular and intracellular environments are filled with various nucleases, unprotected mRNA molecules are difficult to maintain stability in the body. In order to effectively deliver mRNA to target cells, it is often necessary to rely on the packaging of carrier materials such as lipid nanoparticles, and these additional components sometimes trigger an immune response in the human body. Therefore, the key is how to deliver the minimum amount of mRNA while ensuring that it can be translated into the target protein for a long time and in large quantities in the body to maximize the therapeutic effect. Therefore, through the design and optimization of mRNA, to achieve high-level and stable expression of the corresponding protein is the top priority of the entire technology.

[0005] Currently, the optimization of mRNA sequences mainly focuses on adjusting the Codon Adaptation Index (CAI) to match the antisense codon-tRNA abundance in host (human or other model organisms) cells, thereby improving the translation rate. In addition, by changing the base pairing within the mRNA, i.e., Watson-Crick base pairing, the secondary structure of the mRNA can be adjusted, thereby affecting the stability of the mRNA molecule. However, although this method is theoretically feasible, the existing technology still faces limitations in exhaustively designing mRNA sequences, exploring the boundaries of their expression levels and stability, and finding the optimal balance point.

[0006] On the other hand, in the process of developing and exploring the functions of carrier materials such as lipid nanoparticles, in vivo tracing experiments are often widely conducted to indicate the delivery efficiency, distribution, and metabolism of lipid nanoparticles. The usual practice includes encapsulating mRNA containing a firefly luciferase reporter gene into lipid nanoparticles (LNP) with luciferin substrate, injecting it into the animal body, and then detecting the luminescence intensity through imaging technology to evaluate its delivery efficiency, distribution, and metabolic status. In experimental design, in order to ensure experimental results while reducing material costs and meeting animal ethics requirements, it is particularly important to choose the lowest possible LNP-mRNA dose. At the same time, ensuring that the mRNA sequence encapsulated by the lipid nanoparticle (LNP) carrier injected into the body has high safety is crucial for ensuring treatment effectiveness and patient safety. This is related to reducing potential immune responses and side effects, ensuring the long-term stability and biocompatibility of the drug delivery system in the body. Therefore, accurately evaluating and designing the safety of mRNA sequences is not only a necessary step in drug development, but also a key to improving efficacy and reducing treatment risks. On this basis, developing a firefly luciferase mRNA sequence that produces higher protein yield will greatly enhance the economic benefits and scientific value of the experiment. SUMMARY

[0007] To solve the above problems, the present application provides an mRNA encoding luciferase and its application. The mRNA has stable structure, is not easily degraded, has high translation efficiency, has high protein yield, has high biological availability and safety, can be used as a reporter gene, and has good potential.

[0008] The present application provides an mRNA encoding luciferase, which comprises an ORF sequence encoding luciferase, and the ORF sequence is a nucleotide sequence shown in one of SEQ ID NO: 7-13.

[0009] Further, 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 NO: 1-2, and the 3'UTR is a nucleotide sequence as shown in one of SEQ ID NO: 4-5.

[0010] 4-5 in the specific embodiments, the mRNA comprises, in the order from 5' to 3': 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,

[0011] the mRNA comprises, in the order from 5' to 3': a 5'UTR as shown in SEQ ID NO: 1, a CDS as shown in SEQ ID NO: 8, and a 3'UTR as shown in SEQ ID NO: 4; or,

[0012] the mRNA comprises, in the order from 5' to 3': a 5'UTR as shown in SEQ ID NO: 1, an ORF sequence as shown in SEQ ID NO: 9, and a 3'UTR as shown in SEQ ID NO: 4; or,

[0013] the mRNA comprises, in the order from 5' to 3': a 5'UTR as shown in SEQ ID NO: 1, an ORF sequence as shown in SEQ ID NO: 10, and a 3'UTR as shown in SEQ ID NO: 4; or,

[0014] the mRNA comprises, in the order from 5' to 3': a 5'UTR as shown in SEQ ID NO: 1, an ORF sequence as shown in SEQ ID NO: 11, and a 3'UTR as shown in SEQ ID NO: 4; or,

[0015] the mRNA comprises, in the order from 5' to 3': a 5'UTR as shown in SEQ ID NO: 2, an ORF sequence as shown in SEQ ID NO: 12, and a 3'UTR as shown in SEQ ID NO: 5; or,

[0016] the mRNA comprises, in the order from 5' to 3': a 5'UTR as shown in SEQ ID NO: 2, an ORF sequence as shown in SEQ ID NO: 13, and a 3'UTR as shown in SEQ ID NO: 5; or,

[0017] the mRNA comprises, in the order from 5' to 3': a 5'UTR as shown in SEQ ID NO: 2, an ORF sequence as shown in SEQ ID NO: 11, and a 3'UTR as shown in SEQ ID NO: 5; or,

[0018] The mRNA comprises, in 5' to 3' order: a 5' UTR as set forth in SEQ ID NO: 2, an ORF sequence as set forth in SEQ ID NO: 10, and a 3' UTR as set forth in SEQ ID NO: 5.

[0019] In further embodiments, the mRNA 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; the poly-A sequence comprises 20-500 adenine nucleotides.

[0020] In further embodiments, the mRNA 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.

[0021] The present application provides a nucleotide molecule encoding the above mRNA.

[0022] The present application also provides a vector comprising the above nucleotide molecule.

[0023] The present application also provides a cell comprising the above mRNA, the above nucleotide molecule, or the above vector.

[0024] The present application further provides a method of producing luciferase by culturing the above cell to obtain luciferase.

[0025] The present application also provides the use of the above mRNA or the above vector as a reporter gene.

[0026] The present application finally provides a lipid nanoparticle comprising the above mRNA.

[0027] The present application has at least the following advantages:

[0028] The present disclosure provides a brand new luciferase-encoding mRNA, by rationally optimizing the nucleic acid sequence of the functional region, so that the luciferase-encoding mRNA has a high translation efficiency, thereby increasing the output of luciferase protein, and the luciferase-encoding mRNA of the present disclosure has a comparable or even better effect on aggregate control than the industry standard, stability, bioavailability and safety, so that the luciferase-encoding mRNA has a wide application prospect in the field of gene drug development. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The sequence information of an exemplary luciferase mRNA molecule mRNA is shown below.

[0030] Figure 2 The capillary electrophoresis detection results of an exemplary luciferase mRNA molecule are shown below.

[0031] Figure 3 The relative chemiluminescence intensity of luciferase expressed by candidate mRNA molecules at different times is shown below.

[0032] Figure 4 The size-exclusion chromatography analysis of candidate mRNA molecules is shown below. DETAILED DESCRIPTION

[0033] I. Definitions and Explanation

[0034] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by a person skilled in the art. Also, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms and laboratory operation steps used herein are the terms and conventional steps widely used in the corresponding field. At the same time, in order to better understand the present disclosure, the definitions and explanations of related terms are provided below. It should be understood that the present disclosure is not limited to specific methods, reagents, compounds, compositions or biological systems, and of course changes can be made to the above. It should also be understood that the terms used in the present application are only for the purpose of describing specific embodiments and are not intended to be limiting.

[0035] Unless otherwise expressly specified, the singular forms "a", "an", and "the" as used in this specification and the appended claims include plural referents.

[0036] As used herein, the terms “comprising” and “having” and any variations thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, article, or apparatus that comprises a list of steps or modules are not necessarily limited to those listed steps or modules, but can optionally include additional steps or modules not listed, or optionally include steps or modules inherent to such process, method, article, or apparatus. “A plurality” as referred to herein means two or more. “And / or” as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items. The term “ / ” generally indicates a relationship between the preceding and following associated objects.

[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-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and / or pyrimidine bases or other natural, chemically modified, biochemically modified, non-natural, synthetic, or derivatized nucleotide bases.

[0038] As used herein, the term “mRNA (messenger RNA)” refers to any polynucleotide that encodes a polypeptide or protein and is capable of being translated in vitro, in vivo, in situ, or ex vivo to produce the encoded polypeptide.

[0039] As used herein, the term “open reading frame” (ORF) is the normal nucleotide sequence of a structural gene, with the potential to encode a protein or polypeptide, starting with an initiation codon and ending with a termination codon, without a stop codon interrupting translation. On a strand of mRNA, the ribosome begins translation at the initiation codon, synthesizes and extends the polypeptide chain along the RNA sequence, and terminates the extension of the polypeptide chain when a termination codon is encountered.

[0040] As used herein, the term “identity” refers to the ratio, expressed as a percentage, of mismatched nucleotides to the total number of nucleotides in a homologous region. For example, a 20-base oligonucleotide that hybridizes to a homologous region (site) in a target genome with two mismatches is said to have 90% identity to that region.

[0041] As used herein, the term “5’ UTR” generally refers to the sequence of an mRNA molecule between the 5’ end and the translation initiation codon, which is capable of recruiting ribosomal complexes and initiating translation of the mRNA. The 5’ UTR includes the 5’ UTR region structure on the mRNA or the structure corresponding to the coding sequence on the DNA template. The 5’ UTR regulates processes such as post-transcriptional modification, formation and stability of the translation initiation complex, etc. by interacting with transcription factors, ribosomes, and other transcriptional regulatory proteins. The sequence design and optimization of this region is critical for improving post-transcriptional modification and efficiency of protein expression. The 5’ UTR sequence involved in the present disclosure has a nucleotide sequence selected from the group consisting of the nucleotide sequence set forth in one of SEQ ID NOs: 1-2 or a complement thereof, or a nucleotide sequence having at least 80% identity to the nucleotide sequence set forth in one of SEQ ID NOs: 1-2 or a complement thereof.

[0042] As used herein, the term “3’ UTR” refers to the sequence of an mRNA between the stop codon of the polypeptide-encoding sequence and the poly(A) sequence. The 3’ UTR can regulate translation of the mRNA by interacting with mRNA-binding proteins, miRNAs, etc. The 3’ UTR includes the 3’ UTR region structure on the mRNA or the structure corresponding to the coding sequence on the DNA template. It is closely related to post-transcriptional modification and mRNA stability. The sequence and structural features of the 3’ UTR can affect the stability of the mRNA, scanning of the ribosome, and formation of the translation termination complex, etc., thereby affecting the expression level of the protein. The 3’ UTR element involved in the present disclosure comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence set forth in one of SEQ ID NOs: 4-5 or a complement thereof, or a nucleotide sequence having at least 80% identity to the nucleotide sequence set forth in one of SEQ ID NOs: 4-5 or a complement thereof.

[0043] As used herein, the term “complement” refers to one nucleic acid forming hydrogen bonds with another nucleic acid sequence by traditional Watson-Crick or other non-traditional types.

[0044] As used herein, the term "poly-A sequence" includes the poly-A sequence structure on mRNA or the structure corresponds to the coding sequence on the DNA template. The addition of poly-A sequence helps 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 can also contain non-adenine nucleotides. In any form, as long as it is functionally equivalent to the traditional poly-A sequence, i.e. it can provide similar biological functions to the traditional poly-A sequence, such as affecting the stability, translation efficiency or ribosome binding of mRNA, etc. It is recognized as a poly-A sequence. This includes but is not limited to known variants such as human growth hormone (hGH) poly-A sequence and monkey virus 40 (SV40) poly-A sequence, which can differ in nucleotide composition but are functionally equivalent to traditional poly-A sequences. In the present disclosure, 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 term "5' cap structure" includes the meaning of the 5' cap structure present on natural mRNA and its analogs. The 5' cap structure on natural mRNA refers to the methylation of guanosine acid connected to the 5' terminal nucleotide of RNA through pyrophosphate, forming a 5', 5'-triphosphate linkage. The 5' cap structure usually has three types (m7G5'ppp5'Np, m7G5'ppp5'NmpNp, m7G5'ppp5'NmpNmpNp), respectively called Cap0, Cap1 and Cap2. Cap0 refers to the ribose of the terminal nucleotide being unmethylated, Cap1 refers to the ribose of the terminal one nucleotide being methylated, and Cap2 refers to the ribose of the terminal two nucleotides being methylated. The method of capping mRNA molecules is known in the art. The 5' cap structure of the aforementioned mRNA molecules can be added after the mRNA molecules are obtained by chemical synthesis or in vitro transcription using an enzymatic reaction (for example, by using a commercial kit containing vaccinia capping enzyme and mRNA cap structure 2'-O-methyltransferase). However, mRNA with cap structure can also be produced by directly incorporating nucleotide analogs with cap structure as the first nucleotide into the transcript during in vitro transcription.

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

[0047] As used herein, the term "pharmaceutically acceptable carrier" refers to one or more compatible solid, semi-solid, liquid or gel fillers, which are suitable for human or animal use, and must be of sufficient purity and low toxicity. "Compatible" means that the components of the pharmaceutical composition and the active ingredients of the drug are mutually tolerable and admix without any significant degradation or undesired interactions. In the present invention, the aforementioned pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. Examples of pharmaceutically acceptable carriers are physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, such as salts, buffers, sugars, antioxidants, aqueous or non-aqueous carriers, preservatives, wetting agents, surfactants or emulsifiers, or combinations thereof. The amount of pharmaceutically acceptable carrier in the 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.

[0048] As used herein, the term "lipid nanoparticle" refers to a particle containing a lipid component, having a nanoscale size.

[0049] As used herein, the term "ionizable cationic lipid" refers to a lipid molecule that is capable of carrying a positive charge under physiological pH conditions. As an ionizable cationic lipid is an amino lipid.

[0050] As used herein, the term "neutral lipid" refers to a lipid molecule that is not charged under certain pH conditions, for example 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), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), preferably DSPC and / or DOPE.

[0051] As used herein, the term "structural lipid" refers to a lipid that enhances nanoparticle stability by filling the gaps between lipids, often seen with steroids. Steroids are compounds having a cyclopentanoperhydrophenanthrene class of carbon skeleton, in a preferred embodiment, the steroid is selected from one or more of cholesterol, beta-sitosterol, coprostanol, fecosterol, brassicasterol, ergosterol, tomatidine, ursolic acid, alpha-tocopherol, stigmasterol, avenasterol, ergocalciferol, or campesterol, preferably cholesterol and / or beta-sitosterol, more preferably cholesterol.

[0052] As used herein, the term "polymeric lipid" refers to a molecule containing a polymeric moiety and a lipid moiety. In some embodiments, the polymeric lipid is a polyethylene glycol (PEG) lipid. Other lipids capable of reducing aggregation, such as the product of coupling a compound having no charge, hydrophilic, steric hindering moiety to a lipid, 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, PEG-modified dialkylglycerol. Optionally, the PEGylated lipid comprises a PEG moiety of about 1000 Da to about 20 kDa, preferably a PEG moiety of about 1000 Da to about 5000 Da. 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, DSPE-PEG5000, preferably DMG-PEG2000.

[0053] As used herein, the term "vector" refers to a piece of DNA extracted from a virus, plasmid, or cell of a higher organism into which a foreign DNA segment can be or has been inserted for cloning and / or expression purposes. In certain embodiments, a vector can be stably maintained in an organism. Vectors can comprise, for example, an origin of replication, a selectable marker or 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, linearized plasmid fragments), plasmid vectors, viral vectors, cosmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and the like.

[0054] As used herein, the terms "cell" and "host cell" are used interchangeably herein to refer to a cell that expresses or is capable of expressing a sequence to be expressed. The host cells of the present application express polynucleotides that encode polypeptides or RNAs having a variety of uses, including biotechnological, molecular biological, and clinical applications. Host cells include prokaryotic or eukaryotic cells, and examples of suitable host cells in the present application include, but are not limited to, bacterial, yeast, insect, animal, and mammalian cells.

[0055] II. DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

[0056] In one aspect, the present application provides an mRNA encoding luciferase, comprising an ORF sequence encoding firefly luciferase, the ORF sequence being a nucleotide sequence as set forth in one of SEQ ID NOs: 7-13 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 set forth in one of SEQ ID NOs: 1-2 or a complement thereof, or a nucleotide sequence having at least 80% identity to the nucleotide sequence as set forth in one of SEQ ID NOs: 1-2 or a complement thereof; and the 3' UTR is a nucleotide sequence as set forth in one of SEQ ID NOs: 4-5 or a complement thereof, or a nucleotide sequence having at least 80% identity to the nucleotide sequence as set forth in one of SEQ ID NOs: 1-2 or a complement thereof.

[0058] In some alternative embodiments, the 3' UTR further comprises a 3' UTR or a complement thereof into which an additional sequence is inserted; preferably the additional sequence is a miRNA binding site; more preferably the additional sequence is selected from the group consisting of a full-length microRNA reverse complement or a seed sequence reverse complement thereof; preferably the full-length microRNA reverse complement has a length of 19-25 nt; preferably the seed sequence reverse complement has a length of 7-8 nt; preferably the 3' UTR sequence further comprises a 3' UTR sequence or a complement thereof into which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional sequences are inserted.

[0059] In some embodiments, the mRNA comprises, in the 5' to 3' order: a 5' UTR as set forth in SEQ ID NO: 1, an ORF sequence as set forth in SEQ ID NO: 7, and a 3' UTR as set forth in SEQ ID NO: 4; or,

[0060] In some embodiments, the mRNA comprises, in the 5' to 3' order: a 5' UTR as set forth in SEQ ID NO: 1, a CDS as set forth in SEQ ID NO: 8, and a 3' UTR as set forth in SEQ ID NO: 4; or,

[0061] the mRNA comprises, in 5' to 3' order, a 5' UTR of SEQ ID NO: 1, an ORF sequence of SEQ ID NO: 9, and a 3' UTR of SEQ ID NO: 4; or,

[0062] the mRNA comprises, in 5' to 3' order, a 5' UTR of SEQ ID NO: 1, an ORF sequence of SEQ ID NO: 10, and a 3' UTR of SEQ ID NO: 4; or,

[0063] the mRNA comprises, in 5' to 3' order, a 5' UTR of SEQ ID NO: 1, an ORF sequence of SEQ ID NO: 11, and a 3' UTR of SEQ ID NO: 4; or,

[0064] the mRNA comprises, in 5' to 3' order, a 5' UTR of SEQ ID NO: 2, an ORF sequence of SEQ ID NO: 12, and a 3' UTR of SEQ ID NO: 5; or,

[0065] the mRNA comprises, in 5' to 3' order, a 5' UTR of SEQ ID NO: 2, an ORF sequence of SEQ ID NO: 13, and a 3' UTR of SEQ ID NO: 5; or,

[0066] the mRNA comprises, in 5' to 3' order, a 5' UTR of SEQ ID NO: 2, an ORF sequence of SEQ ID NO: 11, and a 3' UTR of SEQ ID NO: 5; or,

[0067] the mRNA comprises, in 5' to 3' order, a 5' UTR of SEQ ID NO: 2, an ORF sequence of SEQ ID NO: 10, and a 3' UTR of SEQ ID NO: 5.

[0068] In some alternative embodiments, the mRNA 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, preferably a 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.

[0069] 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. Preferably, the chemical modification is N1-methylpseudouridine.

[0070] In some embodiments, the uracil in the mRNA molecule has a chemical modification. In some embodiments, the chemical modification is at the 5-position of the uracil. Preferably, the chemical modification is N1-methylpseudouridine. More preferably, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the uracil in the mRNA molecule has the chemical modification, which is N1-methylpseudouridine.

[0071] The present application provides a nucleotide molecule encoding the mRNA.

[0072] The present application also provides a vector comprising the nucleotide molecule.

[0073] The present application also provides a cell comprising the mRNA, the nucleotide molecule, or the vector.

[0074] The present application further provides a method of preparing luciferase by culturing the cell to obtain firefly luciferase.

[0075] The present application also provides the use of the mRNA or the vector as a reporter gene.

[0076] In some embodiments, the above mRNA or the above vector is used in detecting gene expression level, in vivo bioluminescence imaging, in vitro bioluminescence imaging cell tracking, high-throughput screening, safety and toxicology testing, or environmental monitoring.

[0077] The present application finally provides a lipid nanoparticle comprising the above mRNA.

[0078] In some preferred embodiments, the above lipid nanoparticle further comprises one or more lipid moieties selected from the group consisting of: an ionizable cationic lipid, a structural lipid, a neutral lipid, or a polymeric lipid.

[0079] The present application is described below by way of specific embodiments in order to better understand the present application, but does not constitute a limitation on the present application.

[0080] For the purpose of clarity and brevity, features are described herein as part of the same or separate embodiments, however, it will be appreciated that the scope of the present disclosure can include embodiments having combinations of all or some of the described features.

[0081] Example 1: Vector construction comprising a firefly luciferase mRNA sequence

[0082] To achieve the effect of improving the production of firefly luciferase protein, the UTR combination (5' UTR SEQ ID NO: 1-2, 3' UTR SEQ ID NO: 4-5, see Table 1) reported in the applicant's previous patent application (CN202410354542.2 CN202410354702.3) which can effectively improve the translation efficiency is selected for sequence design and screening. For the amino acid sequence (SEQ ID NO: 14) of the firefly luciferase protein, the conservative codon sequence is screened out by alignment, and further the technology in the field of natural language processing and the RNA secondary structure prediction model (such as CDSfold, Ribotree) are used to assign weights and identify stable mRNA sequences. And balance the two parameters of CAI and MFE, from which a batch of mRNA sequences that can achieve higher protein production are screened out. These mRNA sequences are subjected to steps such as gene synthesis, in vitro transcription template preparation, plasmid linearization, IVT mRNA synthesis, purification and quality inspection to obtain mRNA molecules, which are used for in vitro cell experiments. Finally, the candidate firefly luciferase mRNA sequence (see Table 2) is obtained. As a control, the firefly luciferase mRNA sequence (ORF sequence is SEQ ID NO: 10) is derived from the firefly luciferase reporter gene sequence CleanCap® FLuc mRNA - (L-7602) provided by Trilink, which is matched with the 5' UTR (SEQ ID NO: 3) and 3' UTR sequence (SEQ ID NO: 6) from the Modern mRNA1273 expression vector, complete sequence (GenBank: OR134578.1).

[0083] Table 1. UTR sequence information

[0084]

[0085] Table 2. Candidate firefly luciferase mRNA sequence structure composition

[0086]

[0087] To test the candidate mRNA sequence, a nucleic acid fragment containing a T7 promoter, a 5' UTR, an ORF sequence encoding firefly luciferase (Table 2), a 3' UTR, a poly(A) sequence containing 120 A nucleotide residues, and a type IIS restriction endonuclease cleavage site (corresponding to genes FLuc-1 to FLuc-9) is synthesized in vitro and cloned into an in vitro transcription vector (pIVTRup, Addgene plasmid #101362).

[0088] Example 2: mRNA molecule length and integrity detection

[0089] The vector obtained in Example 1 was linearized and in vitro transcribed to produce mRNA molecules using T7-RNA polymerase with 5' cap structure added simultaneously. The 5' cap structure was added by co-transcriptional capping, in which the cap analog was incorporated as the first nucleotide into the transcript during in vitro transcription, directly producing mRNA molecules with Cap1 structure. Figure 1 The sequence of an exemplary mRNA comprising the firefly luciferase mRNA molecule of the present application is shown. To produce the plasmids of other candidate mRNAs in Table 2, Figure 1 The 5' UTR sequence (underlined) - the ORF sequence encoding firefly luciferase (bold) - the 3' UTR sequence (underlined) in Table 2 can be replaced by other candidate sequences, which are also synthesized and cloned into the vector, and other sequence elements remain unchanged.

[0090] The mRNA molecules thus obtained were purified and resuspended in water. The mRNA molecules were quality checked using a 5200 TapeStation (Agilent) to detect the length and integrity of the mRNA molecules (the value is derived from the area under the curve of the expected length fragment) to meet the requirements (see Figure 2 ), which can be used for subsequent testing of different candidate sequences.

[0091] Example 3: In vitro expression level verification of firefly luciferase mRNA sequence

[0092] To determine whether the candidate firefly luciferase mRNA sequences generated by the algorithm have higher protein output, the following method was used:

[0093] The same amount of the above candidate mRNA molecules and sea renilla luciferase mRNA molecules were co-transfected into mammalian cells 293T. At several time points after transfection, the chemiluminescence light absorption value of firefly luciferase was detected, representing its protein expression level; the chemiluminescence light absorption value of sea renilla luciferase was detected, representing the transfection efficiency of the group. The light absorption value reading of firefly luciferase was divided by the light absorption value reading of sea renilla luciferase to obtain the relative light unit of the group. Each candidate mRNA molecule was detected to obtain the corresponding relative light unit in this experiment, which can be used to indicate the protein expression level of the candidate firefly luciferase mRNA molecule.

[0094] The specific experimental process of luciferase chemiluminescence light absorption value for determining translation efficiency is as follows:

[0095] 293T human embryonic kidney cells were seeded at 5 x 10 4Cells were seeded at a density of 1 cell per well in 48-well plates. The next day, cells were washed in Opti-MEM and then co-transfected with 2 μl of Lipofectamine 2000, 200 ng / well of the candidate mRNA molecule encoding firefly luciferase and 100 ng / well of mRNA molecule encoding Renilla luciferase in Opti-MEM. Cells without any RNA molecule were used as background group. After 6 hours of transfection, the mixed medium was removed and replaced with complete medium.

[0096] After 18, 24 and 40 hours of transfection, the medium was removed and 100 μL of lysis buffer (Promega) was added and lysed for 5 minutes at room temperature. Luciferase activity was measured in relative light units (RLU) in a multi-well plate reader (Agilent). Firefly luciferase activity was measured sequentially from a single sample in the luciferase assay. 20 μL of lysate was removed and 50 μL of buffer containing firefly luciferase substrate was added, mixed by shaking the plate and the firefly luciferase light absorbance was measured. Further, buffer containing reaction stop solution and Renilla luciferase substrate was added, mixed by shaking the plate and the Renilla luciferase light absorbance was measured.

[0097] After subtracting the background group light absorbance from the firefly luciferase light absorbance and Renilla luciferase light absorbance respectively, the two values were divided to obtain a relative value that was normalized for transfection efficiency and cell number and that could reflect the amount of firefly luciferase produced. The relative values of different candidate mRNA molecules at different times were analyzed and curve fitting was performed as shown in Figure 3 .

[0098] From Figure 3 it can be seen that the candidate firefly luciferase mRNA has a high protein yield at 18, 24 and 40 hours after transfection, indicating that the protein expression efficiency of the candidate firefly luciferase mRNA is high.

[0099] Example 4: In vivo expression level and safety verification of firefly luciferase mRNA sequence

[0100] To test whether the candidate firefly luciferase mRNA sequence generated by the algorithm has higher protein output and qualified safety in vivo experiments, the following method was used:

[0101] The candidate mRNA molecules are purified by Oligo d(T) to remove the byproducts such as dsRNA. The appropriate lipid materials are selected, including cationic lipids (such as DOTAP or DOTMA), helper lipids (such as phosphatidylcholine or phosphatidylpropanol), cholesterol, and PEGylated lipids, dissolved in an appropriate organic solvent (such as ethanol or chloroform), and the solvent is removed by a rotary evaporator to form a lipid film. The lipid film is then hydrated in a buffer solution to form multilayered liposomes. The synthesized mRNA is dissolved in an appropriate buffer. The mRNA solution is slowly added to the liposome suspension under certain pH and ionic strength conditions, and the mRNA is mixed with the liposomes by gentle stirring. The encapsulated LNP is removed from the unencapsulated mRNA and excess lipids by centrifugation. The prepared LNP is further purified by size exclusion chromatography (SEC) to ensure high purity and uniform particle size.

[0102] Healthy mice are selected as animal models, and a one-week adaptation period is given to the mice before the experiment to eliminate the influence of environmental changes on the experimental results. The encapsulated mRNA-LNP is administered by intravenous injection (IV, 0.5 mg / kg) and intramuscular injection (IM, 0.1 mg / kg). The behavior and physiological response of the mice after administration are observed, and any abnormal performance is recorded. Serum toxicity detection and in vivo imaging detection are performed 24 hours after administration.

[0103] Three blood samples are taken from the mice administered by intravenous injection for biochemical index analysis, including but not limited to alkaline phosphatase (ALP), creatinine (Cr), urea, aspartate aminotransferase (AST), and alanine aminotransferase (ALT), to evaluate possible systemic toxicity. The test results are shown in Table 3, which shows the average values of the biochemical indexes of the three mice after 24 hours of injection of the two candidate mRNA molecules (SEQ ID NO: 7, 10) and the control mRNA molecule (SEQ ID NO: 16). The units corresponding to each test index and the healthy range are indicated in parentheses. Overall, these serum toxicity results indicate that the candidate mRNA molecules have good safety in the animal body, without obvious liver or kidney damage, and have comparable safety to the control mRNA molecule.

[0104] Table 3. Serum toxicity test results of mice in the intravenous injection group

[0105]

[0106] After the serum toxicity test was completed, the mice of the two different administration methods were subjected to in vivo imaging detection. Specifically, about 10-15 minutes before imaging, the prepared luciferin was administered to the animals by intraperitoneal injection. Luciferin is an essential substrate for detecting the activity of luciferase-expressing cells in vivo. Generally, for mice, the dose of luciferin is about 150 mg / kg. Before starting the test, isoflurane gas was used for anesthesia, and the anesthetized animals were placed on the platform of the imaging device, and the observation area was not covered, and imaging was performed. The software provided by the imaging system was used to analyze the obtained images, and the image data was analyzed to determine the luminescence intensity, thereby indicating the expression level of luciferase. The results are shown in Table 4. According to different administration methods, the detected expression regions are different. The intravenous injection administration group mainly observed the liver where LNP is most easily enriched for detection, and the luminescence intensity of the candidate mRNA molecule was significantly higher than that of the control group. The luminescence intensity of the muscle injection group was detected in the liver and muscle tissue. The results show that the luminescence intensity in the liver is 2-3 times higher than that of the control group, and the luminescence intensity in the muscle tissue is slightly higher than that of the control group. It shows that the candidate mRNA molecule has higher protein output.

[0107] Table 4. Luciferase luciferin luminescence intensity detection results of mice in intravenous injection group and muscle injection group

[0108]

[0109] Example 5: Detection of the proportion of candidate firefly luciferase mRNA aggregates

[0110] The proportion of aggregates of the candidate firefly luciferase mRNA sequence was analyzed in detail by size exclusion chromatography (SEC). This data reflects the aggregation state of the candidate mRNA, and also indicates the stability and usability of the candidate mRNA sequence in actual application, thereby determining its efficiency and safety in biomedical applications. Larger aggregates can affect the bioavailability and safety of mRNA, reducing the safety and effectiveness of mRNA therapy by causing non-specific immune reactions or affecting delivery efficiency.

[0111] Size exclusion chromatography is a technique commonly used to separate large molecules or particles, and is particularly useful for assessing the distribution of monomers and aggregates in an RNA sample. First, the candidate mRNA sequence is dissolved in an appropriate buffer, ensuring complete dissolution and dispersion of the mRNA. The buffer containing the candidate mRNA molecules is added to a pre-equilibrated size exclusion chromatography column. The choice of column is based on its ability to effectively distinguish between different molecular sizes, ensuring that monomeric mRNA and aggregates of various sizes can be distinguished. During the passage of the sample through the column and elution, the absorption of the RNA is monitored in real time using a UV detector. As the eluent flows, mRNA molecules of different sizes are separated due to their different retention times in the column. The proportion of each component (monomeric mRNA and different aggregates) is analyzed and determined based on the elution time of each molecule and the area of the elution peak. The results of the detection are shown in Table 5. Figure 4 and Table 5. Figure 4 Table 5 shows the percentage of monomeric mRNA, primary aggregates (aggregate component 1), secondary aggregates (aggregate component 2), and tertiary aggregates (aggregate component 3) in each sample. Monomeric mRNA is also dominant in the mRNA molecules of the control group. This provides a benchmark for comparison, showing that the candidate mRNA sequence has comparable or even superior effects on aggregate control compared to industry standards.

[0112] Table 5. Detection results of the proportion of candidate firefly luciferase mRNA aggregates

[0113]

[0114] The proportion of monomeric mRNA in the candidate firefly luciferase mRNA is greater than 90%, which is much higher than the proportion of monomeric mRNA in the control firefly luciferase mRNA. This indicates that the candidate firefly luciferase mRNA has higher bioavailability and safety.

Claims

1. An mRNA encoding luciferase, characterized in that, which comprises an ORF sequence encoding luciferase, the ORF sequence being a nucleotide sequence as set forth in SEQ ID NO: 10 or SEQ ID NO: 12; the mRNA further comprises a 5' UTR and a 3' UTR; and the 5' UTR is a nucleotide sequence as set forth in SEQ ID NO: 2 and the 3' UTR is a nucleotide sequence as set forth in SEQ ID NO:

5.

2. The mRNA of claim 1, wherein the mRNA further comprises a 5' cap structure and / or a poly-A sequence.

3. The mRNA of claim 2, wherein 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.

4. The mRNA of claim 1, wherein the mRNA 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.

5. A nucleic acid molecule encoding the mRNA of any one of claims 1 to 4.

6. A vector comprising the nucleic acid molecule of claim 5.

7. A cell comprising the mRNA of any one of claims 1 to 4, the nucleic acid molecule of claim 5 or the vector of claim 6.

8. A method of preparing luciferase, characterized by, which is obtained by culturing the cell of claim 7.

9. Use of the mRNA of any one of claims 1 to 4 or the nucleic acid molecule of claim 5 or the vector of claim 6 as a reporter gene.

10. A lipid nanoparticle comprising the mRNA of any one of claims 1 to 4.

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