CN118516381B - MRNA encoding luciferase and uses thereof - Google Patents

Abstract

The invention belongs to the field of biotechnology and medicine, and discloses mRNA (messenger ribonucleic acid) for encoding luciferase and application thereof. The mRNA encoding the luciferase comprises an ORF sequence encoding the luciferase, which is the nucleotide sequence shown in one of SEQ ID NOS.7-13. The present disclosure provides a novel mRNA encoding luciferase, which has stable structure, is not easily degraded, has high translation efficiency, has high protein yield, has high bioavailability and safety, can be used as a reporter gene by reasonably optimizing a nucleic acid sequence of a functional region, and has good potential.

Description

mRNA encoding luciferase and its application

Technical Field

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

Background Art

In recent years, mRNA technology has shown unprecedented potential in the field of biomedicine, especially in vaccine development and genetic disease treatment. In the global response to the Sars-CoV-2 epidemic, mRNA technology has played a key role in vaccine development. The rapid design and production of mRNA vaccines targeting specific viral antigens has demonstrated the great value and potential of mRNA technology in public health emergencies. In particular, by chemically modifying mRNA molecules, such as replacing uracil with N1-methylpseudouridine (N1-Methyl-Pseudouridine, m1Ψ) or 5-methoxy-uridine (5-Methoxy-Uridine, 5moU), the immunogenicity of mRNA is significantly reduced, making mRNA-based vaccines and enzyme replacement therapy 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.

However, the application of mRNA still faces many challenges, and efficient delivery is one of them. Since all kinds of nucleases are prevalent outside and inside cells, it is difficult for unprotected mRNA molecules to maintain stability in the body. In order to effectively deliver mRNA to target cells, it is often necessary to rely on the encapsulation of carrier materials such as lipid nanoparticles. These added ingredients sometimes trigger an immune response in the human body. Therefore, the key is how to deliver the least amount of mRNA while ensuring that it can be translated into target proteins in the body for a long time and in large quantities to maximize the therapeutic effect. Therefore, 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.

At present, the optimization of mRNA sequences is mainly focused on adjusting the Codon Adaptation Index (CAI) to match the antisense codon-tRNA abundance in the host (human or other model organisms) cells and increase the translation rate. In addition, by changing the base pairing inside the mRNA, namely Watson-Crick base pairing, its secondary structure can be adjusted, thereby affecting the stability of the mRNA molecule. However, although this method is theoretically feasible, existing technologies still face limitations in exhausting the possibilities of mRNA sequence design, exploring the boundaries of its expression level and stability, and finding the optimal balance point.

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 carried out to indicate the delivery efficiency, distribution and metabolism of lipid nanoparticles. The usual practice includes encapsulating mRNA containing the firefly luciferase reporter gene together with the luciferin substrate into lipid nanoparticles (LNPs), injecting them into animals, and detecting the luminescence intensity by imaging technology to evaluate their delivery efficiency, distribution and metabolic status. In the experimental design, in order to ensure the experimental effect 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 to ensure the therapeutic effect and patient safety. This is related to reducing potential immune responses and side effects, and 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 the key to improving efficacy and reducing treatment risks. On this basis, developing firefly luciferase mRNA sequences that can produce higher protein production will greatly enhance the economic benefits and scientific value of the experiment.

Summary of the invention

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

The present invention provides an mRNA encoding luciferase, which comprises an ORF sequence encoding luciferase, wherein the ORF sequence is a nucleotide sequence shown in one of SEQ ID NOs: 7-13.

Furthermore, the mRNA also includes 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, and the 3'UTR is a nucleotide sequence as shown in one of SEQ ID NOs: 4-5.

In one of 4-5, in a specific embodiment, the mRNA 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,

The mRNA comprises, in 5' to 3' order: 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,

The mRNA 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: 9, and a 3'UTR as shown in SEQ ID NO: 4; or,

The mRNA 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: 10, and a 3'UTR as shown in SEQ ID NO: 4; or,

The mRNA 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: 11, and a 3'UTR as shown in SEQ ID NO: 4; or,

The mRNA 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:12, and a 3'UTR as shown in SEQ ID NO:5; or,

The mRNA 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:13, and a 3'UTR as shown in SEQ ID NO:5; or,

The mRNA 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:11, and a 3'UTR as shown in SEQ ID NO:5; or,

The mRNA 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:10, and a 3'UTR as shown in SEQ ID NO:5.

In another embodiment, 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; and the poly A sequence comprises 20-500 adenine nucleotides.

In another embodiment, 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.

The present invention provides a nucleotide molecule encoding the above mRNA.

The present invention also provides a vector comprising the above nucleotide molecule.

The present invention also provides a cell, which comprises the above mRNA, the above nucleotide molecule, or the above vector.

The present invention further provides a method for preparing luciferase, which comprises culturing the above-mentioned cell to obtain luciferase.

The present invention also provides use of the above mRNA or the above vector as a reporter gene.

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

The beneficial effects of the present disclosure are at least as follows:

The present disclosure provides a novel mRNA encoding luciferase. By rationally optimizing the nucleic acid sequence of the functional region, the mRNA encoding luciferase has a higher translation efficiency, thereby increasing the output of luciferase protein. The mRNA encoding luciferase of the present disclosure has an effect comparable to or even better than the industry standard in terms of aggregate control, stability, bioavailability and safety, so that the mRNA encoding luciferase has broad application prospects in the field of gene drug development.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the sequence information of an exemplary luciferase mRNA molecule.

FIG. 2 is an exemplary capillary electrophoresis detection result of luciferase mRNA molecules.

FIG3 shows the relative chemiluminescence intensity of luciferase expressed by candidate mRNA molecules at different times.

FIG. 4 is a diagram showing ratiometric analysis of candidate mRNA molecules using ratiometric size exclusion chromatography.

DETAILED DESCRIPTION

I. Definition and Explanation

In the present disclosure, unless otherwise stated, 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 disclosure, the definitions and explanations of the relevant 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 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.

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.

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 this 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.

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.

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.

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.

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.

As used herein, the term "5'UTR" generally refers to the sequence between the 5' end of the mRNA molecule and the translation start codon, which can recruit the ribosome complex and initiate the translation of the 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 by interacting with transcription factors, ribosomes and other transcription regulatory proteins. The sequence design and optimization of this region are crucial to 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 NO: 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 NO: 1-2 or its complementary sequence.

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 disclosure 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.

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.

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 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.

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 methylated guanylate 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), respectively referred to as Cap0, Cap1 and Cap2. Cap0 refers to the ribose of the terminal nucleotide is not methylated, Cap1 refers to the ribose methylation of one nucleotide at the end, and Cap2 refers to the ribose methylation of both nucleotides at the end. 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 chemical synthesis or in vitro transcription to obtain the mRNA molecule (e.g., 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.

As used herein, the term "chemical modification" or "chemically modified" refers to the 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.

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.

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

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.

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.

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.

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.

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.

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.

II. Detailed description of specific implementation plan

In one aspect, the present invention provides an mRNA encoding luciferase, which includes an ORF sequence encoding firefly luciferase, wherein the ORF sequence is a nucleotide sequence shown in one of SEQ ID NOs: 7-13 or a nucleotide sequence having at least 80% identity thereto.

In some embodiments, the above-mentioned mRNA also includes 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 its complementary sequence, or a nucleotide sequence that has at least 80% identity with the nucleotide sequence as shown in one of SEQ ID NOs: 1-2 or its complementary sequence; the above-mentioned 3'UTR is a nucleotide sequence as shown in one of SEQ ID NOs: 4-5 or its complementary sequence, or a nucleotide sequence that has at least 80% identity with the nucleotide sequence as shown in one of SEQ ID NOs: 1-2 or its complementary sequence.

In some optional 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.

In some embodiments, the mRNA 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,

The mRNA comprises, in 5' to 3' order: the 5'UTR shown in SEQ ID NO: 1, the CDS shown in SEQ ID NO: 8, and the 3'UTR shown in SEQ ID NO: 4; or,

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

The mRNA comprises, in 5' to 3' order: the 5'UTR shown in SEQ ID NO: 1, the ORF sequence shown in SEQ ID NO: 10, and the 3'UTR shown in SEQ ID NO: 4; or,

The mRNA comprises, in 5' to 3' order: the 5'UTR shown in SEQ ID NO: 1, the ORF sequence shown in SEQ ID NO: 11, and the 3'UTR shown in SEQ ID NO: 4; or,

The mRNA comprises, in 5' to 3' order: the 5'UTR shown in SEQ ID NO:2, the ORF sequence shown in SEQ ID NO:12, and the 3'UTR shown in SEQ ID NO:5; or,

The mRNA comprises, in 5' to 3' order: the 5'UTR shown in SEQ ID NO:2, the ORF sequence shown in SEQ ID NO:13, and the 3'UTR shown in SEQ ID NO:5; or,

The mRNA comprises, in 5' to 3' order: the 5'UTR shown in SEQ ID NO:2, the ORF sequence shown in SEQ ID NO:11, and the 3'UTR shown in SEQ ID NO:5; or,

The mRNA 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:10, and a 3'UTR as shown in SEQ ID NO:5.

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 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.

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

In some embodiments, the uracil in the above mRNA molecules has a chemical modification. In some embodiments, the chemical modification is at the 5-position of uracil. Preferably, the chemical modification is N1-methyl pseudouridine. More preferably, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the uracil in the above mRNA molecules has a chemical modification, and the chemical modification is N1-methyl pseudouridine.

The present invention provides a nucleotide molecule encoding the above mRNA.

The present invention also provides a vector comprising the above nucleotide molecule.

The present invention also provides a cell, which comprises the above mRNA, the above nucleotide molecule, or the above vector.

The present invention further provides a method for preparing luciferase, which comprises culturing the above cells to obtain firefly luciferase.

The present invention also provides use of the above mRNA or the above vector as a reporter gene.

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

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

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.

The present invention is described below through specific implementation modes in order to better understand the present invention, but it does not constitute a limitation of the present invention.

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 present disclosure may include embodiments having a combination of all or some of the described features.

Example 1: Construction of a vector containing firefly luciferase mRNA sequence

In order 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) that has been reported in the applicant's previous patent application (CN202410354542.2 CN202410354702.3) and can effectively improve the translation efficiency was selected for sequence design and screening. For the firefly luciferase protein determined by the amino acid sequence (SEQ ID NO: 14), the conservative codon sequence was screened out by column alignment, and the technology in the field of natural language processing and RNA secondary structure prediction model (such as CDSfold, Ribotree) were further used to assign weights and identify stable mRNA sequences. The two parameters of CAI and MFE were balanced to screen out a batch of mRNA sequences that may achieve higher protein production. These mRNA sequences were obtained through 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, the candidate firefly luciferase mRNA sequence was obtained (see Table 2). The control firefly luciferase mRNA sequence (ORF sequence is SEQ ID NO: 10) was derived from the firefly luciferase reporter gene sequence CleanCap® FLuc mRNA - (L-7602) publicly provided by Trilink and matched with the 5'UTR (SEQ ID NO: 3) and 3'UTR sequence (SEQ ID NO: 6) from Modern mRNA1273 expression vector, complete sequence (GenBank: OR134578.1).

Table 1. UTR sequence information

Table 2. Candidate firefly luciferase mRNA sequence structure

To test candidate mRNA sequences, 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) was synthesized in vitro and cloned into an in vitro transcription vector (pIVTRup, Addgene plasmid #101362).

Example 2: mRNA molecule length and integrity detection

The vector obtained in Example 1 is linearized, and T7-RNA polymerase is used to perform in vitro transcription to produce mRNA molecules, and a 5' cap structure is 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, directly producing an mRNA molecule with a Cap1 structure. Figure 1 shows the sequence of an exemplary mRNA containing a firefly luciferase mRNA molecule of the present invention. In order to produce plasmids of other candidate mRNAs in Table 2, the 5'UTR sequence (underlined)-ORF sequence encoding firefly luciferase (bold)-3'UTR sequence (underlined) in Figure 1 can be replaced with other candidate sequences, which are also synthesized and cloned into the vector, and other sequence elements remain unchanged.

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 the length and integrity of the mRNA molecules (the values were derived from the area under the curve of the expected length fragments). If they met the requirements (see Figure 2), they could be used for subsequent testing of different candidate sequences.

Example 3: Verification of in vitro expression level of firefly luciferase mRNA sequence

In vitro experiments were performed to test whether the candidate firefly luciferase mRNA sequences generated by the algorithm had higher protein output, as follows:

Mammalian cells 293T were co-transfected with the same amount of the candidate mRNA molecules and Renilla luciferase mRNA molecules described above. The chemiluminescent light absorption value of firefly luciferase was detected at several time points after transfection, representing its protein expression amount; the chemiluminescent light absorption value of Renilla luciferase represented the transfection efficiency of the group. The light absorption value reading of firefly luciferase 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 firefly luciferase mRNA molecule.

The specific experimental procedure for determining the luciferase chemiluminescence absorbance value for translation efficiency is as follows:

293T human embryonic kidney cells were seeded in a 48-well plate at a density of 5×10 ⁴ 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 firefly luciferase, 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 medium was aspirated and replaced with complete medium.

18, 24, and 40 hours after transfection, the medium was aspirated, 100 μL of lysis buffer (Promega) was added, and lysis was carried out at room temperature for 5 minutes. Luciferase activity was measured in relative light units (RLU) in a multiwell plate reader (Agilent). The activity of firefly luciferase was measured sequentially from a single sample in the luciferase assay. 20 μL of lysis buffer was aspirated, 50 μL of buffer containing firefly luciferase substrate was added, the plate was shaken to mix, and the firefly luciferin light absorption value was detected. Further, a buffer containing the reaction stop solution and Renilla luciferase substrate was added, the plate was shaken to mix, and the Renilla luciferin light absorption value was detected.

After subtracting the background group light absorption value from the firefly luciferin light absorption value and the sea renilla luciferin light absorption value, the two are divided to obtain the relative value that can show the amount of firefly luciferase produced after normalization of transfection efficiency and cell number. The relative values of different candidate mRNA molecules at different times are analyzed and curve fitting is performed, as shown in Figure 3.

As can be seen from FIG3 , at 18, 24 and 40 hours after transfection, the candidate firefly luciferase mRNA has a higher protein yield, indicating that the protein expression efficiency of the candidate firefly luciferase mRNA is high.

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

The candidate firefly luciferase mRNA sequences generated by the algorithm were tested in vivo to determine whether they had higher protein production and acceptable safety, as follows:

The candidate mRNA molecules are purified by Oligo d(T) to remove byproducts such as dsRNA. Suitable lipid materials, including cationic lipids (such as DOTAP or DOTMA), co-lipids (such as phosphatidylcholine or phosphatidylpropanol), cholesterol, and PEGylated lipids, are selected and dissolved in a suitable organic solvent (such as ethanol or chloroform). The solvent is removed by a rotary evaporator to form a lipid film. Subsequently, the lipid film is hydrated in a buffer solution to form multilayer lipid vesicles (liposomes). The synthesized mRNA is dissolved in a suitable buffer. Under certain pH and ionic strength conditions, the mRNA solution is slowly added to the lipid vesicle suspension, and the mRNA and lipid vesicles are fully mixed by gentle stirring. The encapsulated LNPs are centrifuged to remove unencapsulated mRNA and excess lipids. After that, they are further purified by gel permeation chromatography (Size Exclusion Chromatography, SEC) to ensure that the prepared LNPs have high purity and uniform particle size.

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

Blood samples from three mice that were intravenously injected were taken for analysis of biochemical indices, including but not limited to alkaline phosphatase (ALP), creatinine (Cr), urea (Urea), aspartate aminotransferase (AST) and alanine aminotransferase (ALT), to evaluate possible systemic toxic reactions. The test results are shown in Table 3, showing the average values of biochemical indices of two candidate mRNA molecules (SEQ ID NO: 7, 10) and control mRNA molecules (SEQ ID NO: 16) in three mice 24 hours after injection. The units and healthy ranges corresponding to each test indicator are marked in brackets. Overall, these serum toxicity results indicate that the candidate mRNA molecules are safe in animals, with no obvious liver or kidney damage, and are comparable to the safety of the control mRNA molecules.

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

After completing the serum toxicity test, mice with two different dosing methods were subjected to in vivo imaging tests. Specifically, about 10-15 minutes before imaging, the prepared luciferin was administered to the animal by intraperitoneal injection. Luciferin is an essential substrate for detecting the activity of cells expressing luciferase in vivo. Usually, for mice, the dose of luciferin is about 150 mg/kg. Before starting the test, isoflurane gas was used for anesthesia, and the anesthetized animal was placed on the platform of the imaging device, and the observation area was unobstructed for imaging. The obtained images were analyzed using the software provided by the imaging system, 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. Depending on the different dosing methods, the expression area was detected differently. The intravenous administration group mainly observed the liver where LNP was most easily enriched for detection, and the luminescence of the candidate mRNA molecule was significantly higher than that of the control group. The luminescence intensity of the liver and muscle tissues was detected in the intramuscular injection group. The results showed that the luminescence intensity in the liver was 2-3 times higher than that of the control group, and the luminescence intensity in the muscle tissue was slightly higher than that of the control group. This indicates that the candidate mRNA molecule has a higher protein output.

Table 4. Luciferase and luciferin luminescence intensity test results of mice in the intravenous injection group and the intramuscular injection group

Example 5: Detection of the ratio of candidate firefly luciferase mRNA aggregates

The aggregate ratio of the candidate firefly luciferase mRNA sequence was analyzed in detail by size exclusion chromatography (SEC). This data not only reflects the aggregation state of the candidate mRNA, but also indicates the stability and availability of the candidate mRNA sequence in practical applications, thereby determining its efficiency and safety in biomedical applications. Larger aggregates may affect the bioavailability and safety of mRNA, reducing the safety and effectiveness of mRNA therapy by causing nonspecific immune responses or affecting delivery efficiency.

Size exclusion chromatography is a technique commonly used to separate macromolecules or particles, and is particularly suitable for evaluating the distribution of monomers and aggregates in RNA samples. First, the candidate mRNA sequence is dissolved in a suitable buffer to ensure 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 selection of the chromatographic column is based on its ability to effectively resolve different molecular sizes to ensure that monomers and aggregates of various sizes of mRNA can be distinguished. During the process of the sample passing through the chromatographic column and being eluted, the absorption of 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 chromatographic column. The proportion of each component (monomer mRNA and different aggregates) is analyzed and determined based on the elution time of each molecule and the area of the elution peak. The test results are shown in Figure 4 and Table 5. Figure 4 and Table 5 show the percentages of monomer mRNA, primary aggregates (aggregate component 1), secondary aggregates (aggregate component 2), and tertiary aggregates (aggregate component 3) in each sample. Monomer mRNA also dominates the mRNA molecules in the control group. This provides a benchmark for comparison, showing that candidate mRNA sequences perform as well or better than industry standards in aggregate control.

Table 5. Detection results of candidate firefly luciferase mRNA aggregate ratios

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 group firefly luciferase mRNA, indicating that the candidate firefly luciferase mRNA has higher biological availability and safety.

Claims (9)

1. MRNA encoding a luciferase, characterized in that it comprises an ORF sequence encoding a luciferase, said ORF sequence being a nucleotide sequence set forth in one of SEQ ID NOS: 7-10;

The mRNA also includes a 5'utr and a 3' utr;

The mRNA comprises in 5 'to 3' order: a 5'UTR shown in SEQ ID NO. 1, an ORF sequence shown in SEQ ID NO. 7 and a 3' UTR shown in SEQ ID NO. 4; or alternatively, the first and second heat exchangers may be,

The mRNA comprises in 5 'to 3' order: a 5'UTR shown in SEQ ID NO. 1, a CDS shown in SEQ ID NO. 8 and a 3' UTR shown in SEQ ID NO. 4; or alternatively, the first and second heat exchangers may be,

The mRNA comprises in 5 'to 3' order: a 5'UTR shown in SEQ ID NO. 1, an ORF sequence shown in SEQ ID NO. 9 and a 3' UTR shown in SEQ ID NO. 4; or alternatively, the first and second heat exchangers may be,

The mRNA comprises in 5 'to 3' order: 5'UTR shown in SEQ ID NO.1, ORF sequence shown in SEQ ID NO. 10 and 3' UTR shown in SEQ ID NO. 4.

2. The mRNA of claim 1, wherein said 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; the poly-a sequence comprises 20-500 adenine nucleotides.

3. The mRNA of claim 2, wherein the mRNA comprises at least one chemical modification selected from at least one of pseudouridine, N1-methyl pseudouridine, N1-ethyl pseudouridine, 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-dihydro-pseudouridine, 2-thio-dihydro-uridine, 2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydro-pseudouridine or 5-methoxy-uridine, and 2' -O-methyluridine.

4. A nucleotide molecule encoding the mRNA of any one of claims 1-3.

5. A vector comprising the nucleotide molecule of claim 4.

6. A cell comprising the mRNA of any one of claims 1-3, the nucleotide molecule of claim 4, or the vector of claim 5.

7. A method for preparing luciferase, characterized in that it comprises obtaining firefly luciferase by culturing the cell of claim 6.

8. Use of an mRNA according to any one of claims 1 to 3 or a nucleotide molecule according to claim 4 or a vector according to claim 5 as a reporter gene.

9. A lipid nanoparticle comprising the mRNA of any one of claims 1-3.