Method for selecting exogenous mRNA that can persist in cells for a long period of time

Abstract

The present invention relates to a method for selecting and preparing mRNA sequences with improved intracellular stability that can persist in cells for long periods of time. According to one embodiment of the method, a mutation is introduced into the base sequence encoding a target protein, without causing any change in the amino acid sequence of the protein, and an mRNA sequence with improved intracellular stability can be selected and produced. This method has the advantage of being able to improve the expression level of a target protein in vivo.

Description

[Technical field]

[0001] The present invention relates to a method for selecting and producing mRNA sequences with improved intracellular stability that can persist in cells for extended periods of time. [Background technology]

[0002] Recently, mRNA has been expected to be a promising new drug, rather than simply a substance that transmits genetic information. By using synthetic mRNA, which is structurally similar to natural RNA, the desired protein is temporarily produced and exhibits the desired activity. Currently, mRNA-based cancer immunotherapy treatments and infectious disease vaccines are in clinical trials.

[0003] nucleic acid code The concept of the drug was first proposed by Wolff et al. when they injected in vitro transcription (IVT) mRNA or plasmid DNA (pDNA) into the skeletal muscles of mice. code It started by showing that the protein expressed by the IVT mRNA is expressed in the nucleus. Conceptually, there are several important differences between IVT mRNA-based therapeutic methods and other nucleic acid-based therapeutics. The IVT mRNA does not need to enter the nucleus to function, and once introduced into the cytoplasm by various methods, the mRNA is immediately translated. On the other hand, DNA therapeutics must access the nucleus to be transcribed into RNA, and their function requires nuclear membrane breakdown during cell division. Therefore, unlike DNA and viral vectors, the IVT mRNA-based therapeutic has the advantage that there is no possibility of insertional mutagenesis because it is not integrated into the genome.

[0004] In order to use mRNA as a drug, it is first necessary to understand the regulation of relative stability of mRNA, which is an important step in intracellular gene expression. The stability of the mRNA plays an important role in determining not only the expression level but also the production rate of the gene product. In general, many proteins with short life spans are produced by short-lived mRNA. codeStable proteins such as α-globin in blood cells are code mRNAs that contain nonsense codon mutations are known to have fairly long half-lives, and cells use surveillance mechanisms to determine and shorten the half-lives of mRNAs that contain nonsense codon mutations. Thus, such changes in mRNA stability can have far-reaching consequences on cellular response and function.

[0005] Therefore, in order to improve the function of an mRNA therapeutic agent, it is necessary to improve its in vivo stability. In the present invention, a method has been developed for selecting and producing mRNA with improved intracellular stability. Summary of the Invention [Problem to be solved by the invention]

[0006] In one embodiment, (A) a target protein is modified to a degree that does not cause any change in the amino acid sequence of the target protein. code The present invention provides a method for selecting an mRNA sequence with improved intracellular stability, comprising the steps of: (A) designing a template sequence including a sequence with a mutation introduced into a base sequence to be selected; (B) preparing a primer based on the template sequence designed in the above step; (C) using the prepared primer sequence to prepare a library including various sequences with mutations introduced therein; and (D) selecting an mRNA sequence with improved intracellular stability from the prepared library. [Means for solving the problem]

[0007] In one embodiment, (A) a target protein is modified to a degree that does not cause any change in the amino acid sequence of the target protein. codeThe present invention provides a method for selecting an mRNA sequence with improved intracellular stability, comprising the steps of: (A) designing a template sequence including a sequence with a mutation introduced into a base sequence to be selected; (B) preparing a primer based on the template sequence designed in the above step; (C) preparing a library including various sequences with mutations using the above prepared primer sequence; and (D) selecting a sequence with improved intracellular stability from the above prepared library.

[0008] The method includes: code The present invention relates to the creation of sequences with improved intracellular stability among various base sequences that can be used to generate target proteins. Specifically, the method involves the creation of target proteins by modifying the target proteins without altering the amino acid sequence of the target proteins. code It also includes selecting a sequence having excellent intracellular stability from among a variety of sequences in which mutations have been introduced into the wild-type base sequence.

[0009] The sequence with improved intracellular stability also means a sequence that is less degraded by ribonuclease (RNase) in vivo / cells and / or has an excellent expression level of a target protein. The sequence may be a nucleic acid sequence, such as a DNA sequence or an RNA sequence, and more specifically, a sequence that is capable of expressing a target protein. code It is also a cDNA or mRNA sequence that corresponds to the sequence.

[0010] The term "target protein" as used herein refers to a protein that a person skilled in the art wishes to produce, and refers to any protein that can be expressed by introducing the cDNA or mRNA selected using the method of the present invention into a host cell.

[0011] The target protein or code The mRNA can also be used as an anti-cancer immunotherapeutic agent, a cancer treatment vaccine, an infectious disease vaccine, a protein replacement therapeutic agent, an allergy therapeutic agent, or an immune disease therapeutic agent.

[0012] As used herein, the term " code "Sequence" means a double-stranded DNA sequence or a double-stranded mRNA sequence that, when placed under the control of appropriate regulatory sequences, is transcribed and interpreted as a polypeptide in vitro or in a living cell; code " or " code It is also used interchangeably with "array". code The boundaries of the sequence are determined by a start codon at the 5' (amino) terminus and a stop codon at the 3' (carboxyl) terminus. code The sequences can include prokaryotic sequences, cDNA sequences derived from eukaryotic mRNA, genomic DNA sequences derived from eukaryotic (eg, mammalian) DNA, and even synthetic DNA sequences.

[0013] The target protein code The nucleotide sequence may be a cDNA sequence consisting of only the exons necessary for the translation of mRNA.

[0014] In the step (A), introducing a mutation into a target protein code The same amino acid is contained in the whole or part of the base sequence. code Specifically, N (A, G, C, or T), R (A or G), Y (C or T), or H (A, C, or T) can be introduced into the third base of the codon. Thus, any base that is not specifically specified is introduced into the third base of each codon, and many base sequences are created depending on the combination of the introduced bases, through which the same amino acid sequence can be generated. code It is possible to create a library of sequences that match the above criteria.

[0015] The term "codon" as used herein refers to a triplet code of mRNA, in which a triplet sequence of mRNA is assembled to form one amino acid, and in the process, the triplet combination is integrated. codeIn order to be interpreted by information, it is called a triplet code and is expressed in units called codons.

[0016] As used herein, the term "redundancy codon" refers to a codon that encodes the same amino acid. code The term synonymous codon refers to a set of codons that encode 20 amino acids. There are 64 types of codons in total, excluding the three termination codons. code One amino acid code There are at least one codon that encodes 1 or 6 amino acids. Specifically, the former include methionine and tryptophan, and the latter include leucine, arginine, and serine. The remaining 15 amino acids are encoded by codons. code The number of codons that can be used to make a protein can vary from two, three, and four. code This means that there are an infinite number of types of mRNA that can be produced.

[0017] In the step (A), the target protein is modified so as not to alter the amino acid sequence of the target protein. code In order to provide diversity to the base sequence, each amino acid is code The codons that are modified are as follows: uuY for phenylalanine, uuR or cuN for leucine, auH for isoleucine, guN for valine, ucN and agY for serine, ccN for proline, acN for threonine, gcN for alanine, uaY for tyrosine, caY for histidine, caR for glutamine, aaY for asparagine, aaR for lysine, gaY for aspartic acid, gaR for glutamic acid, ugY for cysteine, cgN or agR for arginine, ggN for glycine. Also, tryptophan, the start codon methionine, and the stop codon may not be mutated.

[0018] The template sequence designed in step (A) may further include one or more selected from the group consisting of a promoter, a untranslated region (UTR), a 5' cap, and a 3' polyadenylic acid sequence (poly-A), and may further include components that regulate the expression of the target protein.

[0019] The term "promoter" as used herein refers to a portion of DNA that is involved in the binding of RNA polymerase to initiate transcription. In general, it is adjacent to and located upstream of a target gene, and is a site to which RNA polymerase or a transcription factor, a protein that induces RNA polymerase, binds, and can induce the enzyme or protein to be located at the correct transcription start site. That is, it is located at the 5' site of the gene to be transcribed on the sense strand, and has a gene sequence specified to induce RNA polymerase to bind to the site directly or via a transcription factor to initiate mRNA synthesis for the target gene.

[0020] The promoter may be constitutive or inducible among general promoters, and may include, but is not limited to, lac, tac, T3 and T7 promoters in prokaryotic cells, simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) promoter, such as the HIV long terminal repeat (LTR) promoter, Moloney virus, cytomegalovirus (CMV), Epstein-Barr virus (EBV), Rous sarcoma virus (RSV) promoter, as well as β-actin promoter, human hemoglobin, human muscle creatine, human metallothionein promoter, etc. in eukaryotic cells.

[0021] The term "untranslated region (UTR)" as used herein refers to a portion of an mRNA chain that does not serve as a template for a protein gene, i.e., a portion that is not translated. Generally, the UTR includes 5'-UTR and 3'-UTR.

[0022] As used herein, the term "5' cap" or "terminal cap" refers to a 7-methylguanosine (7mG) cap and an ARCA (anti-reverse cap analog) chemically attached to the terminal 5' nucleotide of an RNA molecule.

[0023] As used herein, the term "polyadenylic acid sequence (poly-A)" or "polyadenylic acid tail" refers to a series of contiguous adenylic acids (polyadenylate) that are added to the 3' end of an RNA molecule, such as mRNA, after it has been transcribed.

[0024] The promoter, untranslated region (UTR), 5' cap and / or 3' polyadenylic acid sequence (poly-A) contained in the template are used to encode the target protein. code It can be operably linked to a sequence that

[0025] As used herein, the term "operably linked" refers to a nucleic acid expression control sequence and a protein or peptide of interest that are linked together so as to carry out their general function. code A functional linkage means a state in which a nucleic acid sequence that encodes a protein or peptide is functionally linked to a promoter. code a nucleic acid sequence corresponding to the code The operably linked sequence may affect the expression of the sequence. The operably linked sequence may be produced by using recombinant gene technology known in the art, and site-specific DNA cleavage and ligation may be performed using enzymes or the like generally known in the art.

[0026] The 5' untranslated region, promoter, and target protein code The sequence, the 3' untranslated region and the polyadenylic acid sequence are linked in the above-mentioned order to constitute a template, and may be linked directly to each other or indirectly via a linker.

[0027] The linker is not particularly limited at this time as long as it does not affect the expression of the target protein, and may be composed of, for example, 3 to 21 nucleic acids.

[0028] The step (A) may further include setting the designed template sequence into one or more regions, specifically, dividing the designed template sequence into two or more regions. The template sequence designed in the step (A) is divided into two or more regions, specifically, the template sequence is divided into 10 to 1,000 bp sizes, and two or more regions are set, and the divided regions can be named from the 5' end as the 1st region to the nth region (n is an integer of 2 or more). The sizes of the divided regions may be the same or different.

[0029] The size of the aforementioned divided region may be 10 to 1,000 bp, for example, 10 to 1,000 bp, 10 to 900 bp, 10 to 800 bp, 10 to 700 bp, 10 to 600 bp, 10 to 500 bp, 10 to 400 bp, 10 to 300 bp, 10 to 200 bp, 10 to 180 bp, 10 to 150 bp, 10 to 140 bp, 10 to 130 bp, 10 to 120 bp, 10 to 110 bp, 10 to 100 bp, 30 to 1,000 bp, 30 to 900bp, 30 to 800bp, 30 to 700bp, 30 to 600bp, 30 to 500bp, 30 to 400bp, 30 to 300bp, 30 to 200bp, 30 to 180bp, 30 to 150bp, 30 to 140bp, 30 to 130bp, 30 to 120bp, 30 to 110bp, 30 to 100bp, 50 to 1,000bp, 50 to 900bp, 50 to 800bp, 50 to 700bp, 50 to 600bp, 50 to 500bp, 50 to 4 00bp, 50 to 300bp, 50 to 200bp, 50 to 180bp, 50 to 150bp, 50 to 140bp, 50 to 130bp, 50 to 120bp, 50 to 110bp, 50 to 100bp, 60 to 1,000bp, 60 to 900bp, 60 to 800bp, 60 to 700bp, 60 to 600bp, 60 to 500bp, 60 to 400bp, 60 to 300bp, 60 to 200bp, 60 to 180bp, 60 to 150bp, 60 to 1 40bp, 60 to 130bp, 60 to 120bp, 60 to 110bp, 60 to 100bp, 70 to 1,000bp, 70 to 900bp, 70 to 800bp, 70 to 700bp, 70 to 600bp, 70 to 500bp, 70 to 400bp, 70 to 300bp, 70 to 200bp, 70 to 180bp, 70 to 150bp, 70 to 140bp, 70 to 130bp, 70 to 120bp, 70 to 110bp, 70 to 100bp, 80 to 1,000bp, 80 to 900bp, 80 to 800bp, 80 to 700bp, 80 to 600bp, 80 to 500bp, 80 to 400bp, 80 to 300bp, 80 to 200bp, 80 to 180bp, 80 to 150bp, 80 to 140bp, 80 to 130bp, 80 to 120bp, 80 to 110bp, 80 to 100bp, 90 to 1, 000bp, 90 to 900bp, 90 to 800bp, 90 to 700bp, 90 to 600bp, 90 to 500bp, 90 to 400bp, 90 to 300bp, 90 to 200bp, 90 to 180bp, 90 to 150bp, 90 to 140bp, 90 to 130bp, 90 to 120bp, 90 to 110bp, or 90 to 100bp.

[0030] The ends of each of the divided regions may overlap each other, so that the ends of each of the divided regions may have overlapping portions with the ends of both regions. The ends of each of the divided regions may overlap each other in a region of 1 to 100 bp in size, for example, the 5' end of the second region may overlap with the 3' end of the first region, and the 3' end may overlap with the 5' end of the third region.

[0031] The size of the overlapping region may be 1 to 100 bp, for example, 1 to 100 bp, 1 to 99 bp, 1 to 90 bp, 1 to 81 bp, 1 to 72 bp, 1 to 63 bp, 1 to 54 bp, 1 to 45 bp, 1 to 36 bp, 1 to 30 bp, 1 to 27 bp, 1 to 24 bp, 1 to 21 bp, 1 to 18 bp, 1 to 15 bp, 1 to 12 bp, 1 to 9 bp, 3 to 100 bp, 3 to 99 bp, 3 to 90 bp, 3 to 81 bp, 3 to 72 bp, 3 6 to 63bp, 3 to 54bp, 3 to 45bp, 3 to 36bp, 3 to 30bp, 3 to 27bp, 3 to 24bp, 3 to 21bp, 3 to 18bp, 3 to 15bp, 3 to 12bp, 3 to 9bp, 6 to 100bp, 6 to 99bp, 6 to 90bp, 6 to 81bp, 6 to 72bp, 6 to 63bp, 6 to 54bp, 6 to 45bp, 6 to 36bp, 6 to 30bp, 6 to 27bp, 6 to 24bp, 6 to 21bp, 6 to 18bp, 6 etc. 15bp, 6 to 12bp, 6 to 9bp, 9 to 100bp, 9 to 99bp, 9 to 90bp, 9 to 81bp, 9 to 72bp, 9 to 63bp, 9 to 54bp, 9 to 45bp, 9 to 36bp, 9 to 30bp, 9 to 27bp, 9 to 24bp, 9 to 21bp, 9 to 18bp, 9 to 15bp, 9 to 12bp, 12 to 100bp, 12 to 99bp, 12 to 90bp, 12 to 81bp, 12 to 72bp, 12 to 63 ...63bp, 12 to 63bp, 12 to 63bp, 12 to 63bp, 12 to 63bp, 12 to 63bp, 12 to 63bp, 12 to 63bp, 12 to 63bp, 12 to 63bp, 12 to 63bp, 12 to 63bp, 12 to 63bp, 12 to 63bp, 12 to 63bp, 12 to 63bp, or 54bp, 12 to 45bp, 12 to 36bp, 12 to 30bp, 12 to 27bp, 12 to 24bp, 12 to 21bp, 12 to 18bp, 12 to 15bp, 15 to 100bp, 15 to 99bp, 15 to 90bp, 15 to 81bp, 15 to 72bp, 15 to 63bp, 15 to 54bp, 15 to 45bp, 15 to 36bp, 15 to 30bp, 15 to 27bp, 15 to 24bp, 15 to 21bp, or 15 to 18bp.

[0032] In addition, the overlapping portions of each of the divided regions may be non-mutated, in order to induce complementary binding of primer-primer or primer-PCR product when a PCR reaction is subsequently performed using primers synthesized in the divided regions.

[0033] In the step (B), a target protein is generated based on a template sequence into which a mutation has been introduced. code The step of designing and preparing primer sequences for preparing a library containing a variety of sequences corresponding to the above-mentioned sequences may include a step of synthesizing primers based on the sequences of the divided regions of the template.

[0034] Specifically, the step (B) synthesizes a primer based on the sequence of the divided region of the template or its complementary sequence, and the primer is synthesized to include the sequence of the divided region or its complementary sequence, and specifically, the sequence of the divided region or its complementary sequence may constitute a primer sequence. The synthesized primers may be named 1st primer to nth primer (n is an integer of 2 or more).

[0035] In addition, the primer synthesis step includes synthesizing the remaining regions (regions 2 to n) excluding the first region (region 1) from the 5' end using complementary sequences as primers. Specifically, the remaining primers excluding the first primer among the synthesized primers may be synthesized based on the complementary sequences of the divided regions.

[0036] In the step (C), a target protein containing various mutations is isolated using the primer sequences prepared in the step (B). codeSpecifically, the step of preparing a library containing a variety of sequences corresponding to the above-mentioned sequences may include a step of continuously performing PCR reactions using the above-mentioned synthesized primers and / or PCR products. In the case of a template divided into n regions, the continuous PCR reactions may be continuously performed (n-1) times. The continuous PCR reactions may be named from the first PCR reaction to the (n-1)th PCR reaction.

[0037] For example, the first PCR reaction of the sequential PCR reactions uses a first primer and a second primer to perform a PCR reaction without a template, and the 3' ends (overlapping parts of the template) of the first primer and the second primer complementarily bind to each other to act as their respective templates, thereby allowing the PCR reaction to proceed. Next, the second PCR reaction uses a first primer and a third primer to perform a PCR reaction using the product of the first PCR reaction as a template, and the product of the first PCR reaction and the 3' end (overlapping part of the template) of the third primer complementarily bind to each other to allow the PCR reaction to proceed.

[0038] In the above manner, PCR reactions are performed successively, and finally, the first PCR reaction is performed using the first primer and the second primer (the 3' end of the first primer and the 3' end of the second primer correspond to the overlapped portion of the template and bind complementary to each other), and the (n-1)th PCR reaction is performed sequentially using the first primer and the nth primer and the product of the (n-2)th PCR reaction as a template (the 3' end of the product of the (n-2)th PCR reaction and the 3' end of the nth primer correspond to the overlapped portion of the template and bind complementary to each other) (n is an integer of 3 or more), and the final product of the PCR reaction is a library containing various sequences based on the template designed in step (A) (Figure 4). If the template designed in the above step is divided into two regions, the library can be prepared only by the first PCR reaction performed using the first and second primers.

[0039] The library contains a target protein. code The target protein is also detected by the DNA or RNA sequence. code The sequence may further include one or more selected from the group consisting of a promoter, an untranslated region (UTR), a 5' cap, and a 3' polyadenylic acid sequence (poly-A).

[0040] The step (D) is to select a target protein having improved intracellular stability from the library prepared as described above. code D-1) selecting an mRNA sequence that is capable of encoding a target protein contained in the library; code A DNA sequence that encodes a nucleotide sequence for a gene that is capable of expressing a gene for a gene, or an RNA sequence derived from the DNA sequence, is introduced into a cell. Transfection D-2) the aforementioned step of Transfection Extraction of target proteins with excellent intracellular stability from cultured cells code D-3) obtaining an RNA sequence corresponding to the target protein from the obtained RNA sequence. codeand synthesizing DNA corresponding to the nucleic acid sequence to produce a library.

[0041] The DNA sequences contained in the library are designed to amplify the target protein without altering the amino acid sequence of the target protein. code It is a collection of many template sequences containing diverse cDNA sequences that

[0042] As mentioned above Transfection The sequences used for the synthesis can be DNA sequences contained in the library or RNA sequences generated therefrom. The RNA sequences can be transcribed from sequences contained in the library, in particular, obtained via an in vitro transcription (IVT) reaction.

[0043] As used herein, the term " Transfection For the purposes of the present invention, the term "transfection" refers to introducing the library sequence, an expression vector containing the sequence, or an RNA sequence transcribed from the sequence into a host cell, and expressing the nucleic acid molecule of the library sequence in the host cell, and may be used interchangeably with "transduction" or "transformation." The nucleic acid molecule may also be used to express a target protein. code The library sequences may be in the form of DNA and RNA, in the form of template sequences that contain sequences that are capable of being introduced into a host cell and expressed.

[0044] Suitable host cells for introduction of the library sequences can be prokaryotic cells such as Escherichia coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis or Staphylococcus sp., fungi such as Aspergillus sp., yeasts such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp. or Neurospora crassa, other lower eukaryotic cells, or higher eukaryotic cells such as plant or insect cells. Mammalian cells may also be used, including, but not limited to, monkey kidney cells 7 (COS7), NSO cells, SP2 / 0, Chinese hamster ovary (CHO), W138, baby hamster kidney (BHK), MDCK, myeloma cell lines, HeLa cells, HuT78 cells, and HEK293 cells.

[0045] As mentioned above Transfection The methods include any method of introducing nucleic acid into an organism, cell, tissue, or organ, and can be performed by selecting standard techniques appropriate to the host cell known in the art, and can include, for example, electroporation, protoplast fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, agitation using silicon carbide fibers, Agrobacterium-mediated transformation, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, lithium acetate-DMSO method, lipofectamine, and desiccation / quenching-mediated transformation methods.

[0046] The term "expression vector" as used herein refers to a recombinant vector that can be introduced into a suitable host cell to express a target protein, and refers to a genetic construct that contains essential regulatory elements operably linked to express a gene insert. The term "operably linked" means that a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding a target protein are functionally linked to each other so as to perform a general function. The operably linked recombinant vector is produced using recombinant gene technology well known in the art, and site-specific DNA cleavage and ligation can be easily performed using enzymes generally known in the art.

[0047] A suitable expression vector of the present invention may contain, in addition to expression regulatory elements such as a promoter, an initiation codon, a stop codon, a polyadenylation signal, and an enhancer, a signal sequence for membrane targeting or secretion. The initiation codon and the stop codon are generally considered to be part of the nucleotide sequence encoding the protein of interest, and must be functional in an individual when the gene construct is administered and must be in frame with the coding sequence.

[0048] The expression vector may also contain a selectable marker for selecting host cells containing the vector. The selectable marker is used to select cells transformed with the vector, and a marker that confers a selectable phenotype such as drug resistance, auxotrophy, resistance to cytotoxic agents, or surface protein expression may be used. In an environment treated with a selective agent, only cells expressing the selectable marker survive, so that transformed cells can be selected. In addition, when the vector is a replicable expression vector, it may contain a replication origin, which is a specific nucleic acid sequence from which replication is initiated.

[0049] Various types of vectors such as plasmids, viruses, cosmids, etc. can be used as recombinant expression vectors for inserting foreign genes. The type of recombinant vector is not particularly limited as long as it expresses a desired gene in various host cells, including prokaryotic and eukaryotic cells, and performs the function of producing a desired protein. Specifically, a promoter that exhibits strong activity and a vector that can mass-produce a foreign protein in a form similar to that in the natural state while retaining strong expression power can be used.

[0050] In the present invention, a variety of host and vector combinations can be used to express the target protein. Expression vectors suitable for eukaryotic hosts can include, but are not limited to, expression regulatory sequences derived from SV40, bovine papilloma virus, adenovirus, adeno associated virus, cytomegalovirus, and retrovirus. Expression vectors that can be used for bacterial hosts can include, but are not limited to, bacterial plasmids from Escherichia coli, including pET, pRSET, pBluescript, pGEX2T, pUC vectors, T vector system, colE1, pCR1, pBR322, pMB9, or derivatives thereof; plasmids with a broader host range, such as RP4; phage DNA, such as phage lambda derivatives, such as λgt10, λgt11, or NM989; and other DNA phages, such as M13 and filamentous single-stranded DNA phages. For yeast cells, plasmids such as 2°C or derivatives thereof may be used, and for insect cells, plasmids such as pVL941 may be used.

[0051] The step D-1) is Transfection The method may further include a step of determining the expression level of the protein of interest in the cells thus obtained, which may include determining the expression level of the protein of interest or the expression level of the protein of interest. codeIt can also measure whether or not a sequence associated with a gene is expressed, or the degree of expression of that sequence. Transfection It can be used to check whether the following is done correctly.

[0052] The method for measuring the expression level of the protein may be western blotting, enzyme linked immunosorbent assay (ELISA), radioimmune assay (RIA), radial immunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence, immunochromatography, fluorescence activated cell sorter analysis (FACS analysis), or protein chip technology.

[0053] The target protein code The method for measuring the expression level of a target sequence, specifically, mRNA or gene, can be reverse transcriptase polymerase reaction (RT-PCR), competitive reverse transcriptase polymerase reaction (competitive RT-PCR), real time quantitative reverse transcriptase polymerase reaction (real time quantitative RT-PCR), quantitative RT-PCR, RNase protection method, northern blotting or DNA chip technology.

[0054] The step of evaluating the expression level of the target protein may utilize a preparation capable of measuring the expression level. The aforementioned "preparation capable of measuring the expression level" refers to a specific protein or code Specifically, the term "target protein" refers to a molecule that can be used to confirm the expression level of a gene that is a target protein or a gene that is a target protein. code The term also includes, but is not limited to, preparations capable of detecting and / or amplifying genes associated with a gene.

[0055] As used herein, the term "specific protein or code The term "preparation capable of detecting a gene corresponding to a specific gene or protein" refers to a preparation that can specifically bind to and recognize the specific gene or protein, or that can detect and amplify the specific gene or protein, and the term "preparation capable of amplifying a specific gene or protein" refers to a preparation that can repeatedly replicate the specific gene or protein and increase its number, and may refer to, for example, a primer that can specifically amplify a polynucleotide containing the gene, or a probe that can specifically bind to the gene, but is not limited thereto.

[0056] In the above step, in order to effectively evaluate the expression level of the target protein, code Compositions that specifically bind to a gene or mRNA that encodes a target gene, including a primer, a probe, a nucleotide, an antibody or an antigen-binding fragment thereof, a ligand, a receptor, a protein, or a combination thereof, can be used.

[0057] The term "primer" as used herein refers to a nucleic acid sequence having a free 3' hydroxyl group at its 3' end, capable of forming base pairs with a template complementary to a specific base sequence and acting as an initiation site for template strand copying. The primer can initiate DNA synthesis in the presence of a polymerization reagent (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside polyphosphates in an appropriate buffer solution and temperature. The PCR conditions and the lengths of the sense primer and antisense primer can be appropriately selected by techniques known in the art.

[0058] The term "probe" as used herein means a nucleic acid fragment such as RNA or DNA that can specifically bind to a target nucleic acid, for example, mRNA, and may be labeled to confirm the presence, content, and expression level of a specific mRNA. The probe may be prepared in the form of an oligonucleotide probe, a single strand DNA probe, a double strand DNA probe, or an RNA probe. The selection of an appropriate probe and the hybridization conditions may be appropriately selected according to techniques known in the art. The probe may have a length of 10 to 100 nt, 15 to 100 nt, 10 to 80 nt, 10 to 50 nt, 10 to 30 nt, 10 to 20 nt, 15 to 80 nt, 15 to 50 nt, 15 to 30 nt, 15 to 20 nt, 20 to 100 nt, 20 to 80 nt, 20 to 50 nt, or 20 to 30 nt.

[0059] The primer or the probe may be chemically synthesized using phosphoramidite solid support synthesis or other widely known methods. Such nucleic acid sequences may also be modified through a variety of methods known in the art. Examples of such modifications may include methylation, capping, substitution of one or more natural nucleotides with their analogs, or modifications between nucleotides, such as modifications to uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidites, carbamates) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). The primer or the probe may also be modified with a label capable of providing a detectable signal, directly or indirectly. Examples of such labels may include radioisotopes, fluorescent molecules, or biotin.

[0060] The term "antibody" as used herein is a term known in the art and refers to a specific immunoglobulin directed against an antigenic site, said antibody being capable of specifically binding to a protein of interest or a fragment thereof, said fragment being an immunogenic fragment and referring to a protein fragment having one or more epitopes that can be recognized by an antibody against the protein.

[0061] The step D-1) Transfection After that, Transfection The method further includes culturing the cells obtained by the method. Specifically, the method further includes culturing the cells obtained by the method, the cells being cultured to select a sequence having improved stability in a cell or in vivo. code The method may include culturing the cells into which the sequence has been introduced.

[0062] The culturing step can be, for example, culturing for 12 to 96 hours, 12 to 84 hours, 12 to 72 hours, 12 to 60 hours, 12 to 48 hours, 12 to 36 hours, 12 to 24 hours, 24 to 96 hours, 24 to 84 hours, 24 to 72 hours, 24 to 60 hours, 24 to 48 hours, 24 to 36 hours, 36 to 96 hours, 36 to 84 hours, 36 to 72 hours, 36 to 60 hours, 36 to 48 hours, 48 ​​to 96 hours, 48 ​​to 84 hours, 48 ​​to 72 hours, 48 ​​to 60 hours, 60 to 96 hours, 60 to 84 hours, 60 to 72 hours, 72 to 96 hours, or 72 to 84 hours.

[0063] As mentioned above Transfection During subsequent cell culture, among the various RNA sequences introduced into the cells or the RNA sequences translated from the DNA sequences, sequences with low in vivo stability are degraded within the cells and cannot exist or their expression levels are reduced, and only sequences with high intracellular stability remain or their expression levels are increased. Therefore, in the next step, D-2), Transfection If RNA is obtained from cells that have been modified, the stability within a cell or in a living organism will be improved, and an RNA sequence that has not been degraded within a cell or has an expression level that is improved compared to a wild-type sequence that has not been mutated can be obtained. In addition, by synthesizing DNA sequences from the obtained RNA sequences, a library (secondary library) can be constructed that is selected and composed of sequences with excellent intracellular stability.

[0064] The step (D) may be carried out by repeating the steps (D-1) to (D-3) one or more times. Specifically, the DNA sequences contained in the secondary library prepared in the above steps or the RNA sequences prepared therefrom are further introduced into cells. Transfection Let, Transfection A more purified library (tertiary library) can be produced from the cells that have been cultured. By repeating the above steps D-1) to D-3) one or more times, twistIt is possible to obtain libraries of sequences with excellent intracellular stability, which can provide advantageous effects in selecting and producing mRNA sequences with improved intracellular stability.

[0065] The step (D) comprises: D-4) isolating a target protein contained in the library; code D-5) cloning the DNA sequence into a vector, and D-6) injecting the vector or RNA produced therefrom into a cell. Transfection D-6) the aforementioned step of Transfection Extract the target protein from the cultured cells code and obtaining and selecting RNA that is

[0066] Specifically, the library obtained through the step D-3) contains the same amino acid sequence. code Each of these base sequences is isolated and then transferred to the cell. Transfection The method may include cloning the DNA sequences contained in the library into a vector to produce a recombinant vector that contains only one type of sequence per vector. Transfection with The engineered cells are capable of expressing only one sequence.

[0067] Therefore, in the step D-6) Transfection The cells (clones) are divided into one type per cell. code Although it may contain sequences (DNA or RNA), the RNA obtained from said cells (clones) may yield one type of sequence per cell (clone).

[0068] The step D-6) Transfection The cells that are selected are also selected cells, specifically those that express the target protein or code The cells that have a high expression level of the sequence can be selected. TransfectionThe control group may be a wild-type gene sequence in which no mutation has been introduced, and the control group may be a wild-type gene sequence in which no mutation has been introduced.

[0069] The step (D) may further include a step of D-7) determining the sequence of the obtained RNA and the DNA synthesized therefrom.

[0070] Determining the sequence refers to confirming sequence information by analyzing the base sequence of the target sequence, and may be performed using a method known in the art, such as Sanger sequencing, Maxam-Gilbert sequencing, or next generation sequencing. Effect of the Invention

[0071] According to one embodiment of the method, the amino acid sequence of the target protein is modified without causing any change thereto. code By introducing a mutation into a base sequence that is a target of the gene expression, an mRNA sequence with improved intracellular stability can be selected and prepared. The above method has the advantage of being able to improve the expression level of a target protein in vivo. [Brief description of the drawings]

[0072] [Figure 1] FIG. 1 is a schematic diagram showing the steps of a method for selecting or creating an mRNA sequence with improved intracellular stability. [Diagram 2] FIG. 1 is a schematic diagram showing the steps of a method for selecting or creating an mRNA sequence with improved intracellular stability. [Diagram 3] FIG. 1 is a schematic diagram showing the process of mutagenesis of a target protein (e.g., Gaussia luciferase) and the process of primer design. [Figure 4] FIG. 1 is a schematic diagram illustrating the synthesis of a mutagenesis template via sequential PCR reactions. [Diagram 5] FIG. 1 shows the process of performing sequential PCR reactions using the designed / constructed primers. [Figure 6] FIG. 1 is a diagram comparing the expression levels of target proteins of candidate mRNA sequences selected via the method of the present invention. [Figure 7] FIG. 1 is a diagram comparing the secondary structure of mRNA sequences with excellent intracellular stability selected by the method of the present invention with a control group. [Figure 8] This figure compares the expression levels of sequences encoding COVID19 antigen proteins with excellent intracellular stability selected using HeLa cells through the method of the present invention. [Figure 9] This figure compares the expression levels of sequences encoding COVID19 antigen proteins with excellent intracellular stability selected using HEK293T cells through the method of the present invention. [Figure 10] This figure compares the expression levels of sequences encoding recombinant proteins including COVID19 antigen proteins with excellent intracellular stability selected using HeLa cells through the method of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0073] The present invention will be described in more detail below with reference to examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples. EXAMPLES

[0074] Example 1: Template design and library construction for maximizing mRNA codon mutations In order to explain the method of the present invention for selecting and producing mRNA with improved stability, the gene encoding Gaussia luciferase (GL), a well-known secreted luciferase enzyme, was used. code The sequences were used as representative examples. codeThe sequence was determined from the Gaussia princeps luciferase gene in the cloning vector pCMV-GLuc_2 (New England Biolabs, USA). code The sequence is shown in Fig. 1. At the 5' end of the gene sequence, a T7 promoter is located, enabling in vitro transcription (IVT) and a UTR known to increase mRNA stability. code The sequences were inserted into the 5'-end and 3'-end of the gene sequence, respectively. A polyadenylation sequence was placed at the 3'-end, so that it could be used immediately as mRNA after in vitro transcription (IVT). The template sequence containing the wild-type Gaussia luciferase gene sequence without the mutation (wild-type Gaussia luciferase (GL) gene template) was represented by the sequence of SEQ ID NO:1.

[0075] Next, the Gaussia luciferase gene of the sequence of SEQ ID NO: 1 code The amino acid sequence of the protein translated from the open reading frame (ORF) sequence was maintained, while the third base of the codon was diversified to introduce a mutation into the mRNA codon. codeIn order to design and describe in detail the mutant sequences to be introduced into the sequence, the ambiguity codes nomenclature of the International Union of Pure and Applied Chemistry (IUPAC), which is commonly used by those skilled in the art, is used and expressed as follows: N means adenine (A), guanine (G), C (cytosine) or T (thymine); R means adenine (A) or guanine (G); Y means C (cytosine) or T (thymine); H means adenine (A), C (cytosine) or T (thymine); D means adenine (A), guanine (G) or T (thymine). In the mRNA sequence of Gaussia luciferase in which a mutation has been introduced into the third base of the codon using the above-mentioned multiple meaning code, phenylalanine is represented as uuY, leucine as uuR or cuN, isoleucine as auH, valine as guN, serine as ucN or agY, proline as ccN, threonine as acN, alanine as gcN, tyrosine as uaY, histidine as caY, glutamine as caR, asparagine as aaY, lysine as aaR, aspartic acid as gaY, glutamic acid as gaR, cysteine ​​as ugY, arginine as cgN and agR, and glycine as ggN. In addition, methionine is represented as atg, tryptophan as tgg, and there is only one codon. In SEQ ID NO: 1, the sequence was designed to include the above-mentioned codon mutation. code The template containing the sequence (mutated Gaussia luciferase (GL) gene template) is shown in SEQ ID NO: 2, and the sequences of SEQ ID NOs: 1 and 2 above are set forth in Table 1 below (the sequence of the open reading frame is written in lower case letters, and non-mutated portions of the open reading frame are underlined).

[0076] [Table 1]

[0077] Next, in order to prepare the template DNA sequence of SEQ ID NO: 2 based on the sequence of SEQ ID NO: 1, first, the entire sequence of SEQ ID NO: 2 containing the above-mentioned approximately 760 base pairs was code The sequence and its complementary sequence were divided into approximately 75 to 135 bases in size, and each divided region was synthesized (Figure 3). Information on the synthesized oligonucleotide sequences is shown in Table 2 below.

[0078] [Table 2]

[0079] Next, sequential PCR reactions were performed using the synthesized oligonucleotide sequences as primers (Figure 4). First, PCR reactions were performed using PM_Rpic_GL_F1 (SEQ ID NO: 3) and PM_Rpic_GL_R1 (SEQ ID NO: 4) as primers. In this case, the 3'-end portion of PM_Rpic_GL_F1 and the 3'-end portion of PM_Rpic_GL_R1 were complementary to each other to perform PCR reactions, and as a result of the reaction, a sequence corresponding to a partial region of SEQ ID NO: 2 was generated as a product (SEQ ID NO: 12).

[0080] Next, a secondary PCR reaction is carried out using the sequence of SEQ ID NO: 12 as a template and PM_Rpic_GL_F1 (SEQ ID NO: 3) and PM_Rpic_GL_R2 (SEQ ID NO: 5) as primers, whereby the 3'-end portion of SEQ ID NO: 12 and the 3'-end portion of PM_Rpic_GL_R2 are complementary to each other to produce a product of SEQ ID NO: 13. In this manner, the product of the previous PCR reaction is used as a template to carry out successive PCR reactions, thereby finally producing the sequence of SEQ ID NO: 2 (FIG. 5). The sequence information of the template, primers, and products of the above-mentioned successive PCR reactions is shown in Table 3 below.

[0081] [Table 3]

[0082] The final product of the above-mentioned sequential PCR reaction is a library containing various combinations of mutant codons, and specifically, the mutant codons introduced into the final product SEQ ID NO: 18 correspond to a total of 160, with 83 Ns in which 4 types of mutations are induced, 10 Hs in which 3 types of mutations are induced, and 31 and 36 Ys and Rs in which 2 types of mutations are induced, respectively, so that a library containing sequences containing approximately 8.14x10^74 mutant codons, which is (4^83)x(3^10)x(2^31)x(2^36), can be prepared. Therefore, by identifying sequences that can interact with a stabilized structure or a stabilizing factor in the library sequences, mRNA sequences with improved stability can be selected.

[0083] Example 2: In vitro transcription (IVT) and purification steps for the production of mutated mRNA In order to prepare mRNA from various mutant sequences contained in the library prepared in Example 1 through in vitro transcription (IVT), the following experiment was carried out.

[0084] Specifically, an mRNA production kit, HiScribe T7 ARCA mRNA kit with tailing from New England Biolabs, USA, was used to perform the in vitro transcription (IVT) reaction. First, 1 μg of the library mixture containing the mutant DNA prepared in Example 1, 10 μl of 2x ARCA / NTP Mix, and 2 μl of T7 RNA Polymerase Mix were mixed, and the remaining amount was distilled water to form a final reaction mixture of 20 μl, which was then reacted at 37° C. for 30 minutes to perform the in vitro transcription (IVT) reaction. After the reaction was completed, 2 μl of DNase enzyme was added and the mixture was treated at 37° C. for 15 minutes to remove any residual DNA. After DNA removal was completed, 5 μl of Poly(A) polymerase Reaction Buffer and 5 μl of Poly(A) polymerase were mixed with 20 μl of the above IVT reaction mixture to perform the reaction of adding polyadenylic acid tails, and the remainder was added with distilled water to make a final reaction mixture of 50 μl, which was reacted at 37° C. for 30 minutes. The final IVT mRNA was purified using an RNA purification kit.

[0085] Example 3: Delivery of IVT mRNA to host cells Transfection The IVT mRNA prepared in Example 2 was transfected into the human cell line HEK293T (ATCC, CRL-3216). Transfection In order to express the gene by the above reaction, the following experiment was carried out.

[0086] Specifically, HEK293 cells were first cultured in a T75 flask in MEM medium (#11095-080 (Gibco)) supplemented with 10% FBS (fetus bovine serum) (#16000-028 (Gibco)) and 1% ABAM (#15240-O13 (Gibco BRL)) for 3 days, washed with 1x PBS buffer solution, treated with trypsin, and separated at the bottom of the culture vessel. The separated cells were centrifuged, the supernatant was discarded, and the cells were redispersed in 1x PBS buffer solution. Approximately 5x10 cells were then added to the electroporation solution. 5The cells were resuspended at a concentration of 1000 cells / ml, and approximately 20 μg of IVT mRNA was added to 1 ml of the cell suspension. The cells were then mixed and stored in a neon buffer (Thermo Fisher Scientific, USA). Transfection Using the Neon Transfection System Transfection The electroporation conditions were according to the manufacturer's protocol. Transfection The cells were then dispersed in a 6-well microplate and cultured in a 5% CO2 cell incubator.

[0087] Example 4: Transfection Isolation / purification of mRNA with excellent biostable properties from cultured cells The product prepared in Example 3 Transfection In order to obtain and purify an mRNA sequence having excellent intracellular stability in cells, the following experiment was carried out.

[0088] Specifically, using the electroporation method in Example 3 Transfection After that, the cells were cultured for 72 hours to select RNA sequences with improved intracellular stability, and then the cultured cells and the cell culture medium were harvested. code The array to be Transfection To confirm whether the incubation was successful and the target protein was expressed, the expression level of Gaussia luciferase secreted in the harvested cell culture medium was measured. 50 μl of BioLux GLuc Substrate and 800 μl of BioLux GLuc Stabilizer were added to 5 ml of BioLux GLuc Assay Buffer per 100 samples to prepare a GLuc assay solution. 10 μl of the culture medium collected at each time point was placed in a 96-well microplate, 50 μl of GLuc assay solution was added per well, and the wells were incubated for 35 to 40 seconds, after which the luminescence was measured using a luminometer.

[0089] Next, TransfectionAfter that, the cells were cultured for 72 hours, and intracellular RNA was isolated and purified by a method well known to those skilled in the art.

[0090] Example 5: Amplification of cDNA from purified mRNA using RT-PCR method Example 4 Transfection In order to synthesize / amplify cDNA from the mRNA obtained in the cultured cells, the following experiment was carried out.

[0091] Specifically, cDNA was synthesized by RT-PCR using reverse transcriptase using RNA as a template, and the cDNA was amplified by general polymerase chain reaction (PCR). First, to perform RT-PCR, 100ng Random Hexamer, 2μl 10x Reverse Transcriptase Buffer, 10mM MDTT, 10mM dNTP, and 250 units of reverse transcriptase were added to 2μg RNA purified in Example 4, and the final volume was adjusted to 20μl with distilled water. The reaction product was incubated at 70°C for 10 minutes to modify the RNA, and reverse transcription was performed at 42°C for 1 hour, and the reverse transcriptase was inactivated at 80°C for 15 minutes. The cDNA product was further subjected to polymerase chain reaction using primers of SEQ ID NOs: 10 and 11 to amplify, separate, and purify DNA fragments for preparing IVT mRNA. The DNA fragments contain various mutant sequences, and can be considered as a secondary library. The steps of Examples 3 to 5 above can be repeated several times to produce tertiary libraries, quaternary libraries, etc., and the more times the steps are repeated, the more likely it is that a library composed of RNA sequences with improved intracellular stability can be obtained.

[0092] Example 6: IVT mRNA production in amplified cDNA and cells Transfection The DNA fragments isolated / purified in Example 5 were used to prepare IVT mRNA by the method of Example 2, and then transfected again into HEK293T cells. Transfection As a control, IVT mRNA was prepared using the template sequence of SEQ ID NO: 1 without any mutations, and the results were analyzed in the same manner. Transfection After 24, 48, and 72 hours, Transfection The secretory luciferase expression level in the cells was compared and measured over time. code It was confirmed that the sequence of the luciferase mRNA was more stable than the wild-type luciferase mRNA without the mutation, which was used as a control. As a result, it was confirmed that the expression level of the Gaussia luciferase protein was significantly increased along with the increase in translation efficiency. RNA was isolated and purified from cells in which the stability and expression level were significantly increased.

[0093] Example 7: Cloning of cDNA synthesized from IVT mRNA and evaluation of IVT mRNA reproduction and stability using the same For all RNA or mRNA obtained from cells in which the stability and expression levels were significantly increased as confirmed in Example 6, cDNA was amplified by the above-mentioned RT-PCR method using primers of SEQ ID NOs: 10 and 11. The amplified cDNA at this stage contains a mixture of cDNA reverse transcribed from various types of stabilized mRNA, and can be considered as a further refined library. Therefore, a specific sequence of stabilized mRNA was selected from the various sequences described above, codeTo analyze the sequences and test the degree of stabilization, each corresponding cDNA sequence must be cloned into a T vector system for E. coli. For this purpose, a T&A cloning vector from Yeastern Biotech Co., Ltd., Taiwan, which is well known to those skilled in the art, was used to isolate and purify the plasmid vector DNA from the clones into which each cDNA was inserted, and then an in vitro transcription (IVT) reaction was carried out for each clone using the method described above to prepare IVT mRNA. The selected and prepared IVT mRNA and the control IVT mRNA were then electroporated into HEK293T cells for each clone using the method described above. Transfection The culture medium was collected at regular intervals, and the luciferase expression level was analyzed. Clones showing superior luciferase expression levels compared to the control group were selected.

[0094] Example 8: Stability-confirmed mutations code Codon evaluation and determination As mentioned above, the clones selected as showing superior expression levels were code The sequences were confirmed by sequencing methods well known to those skilled in the art. code Compare sequences and find common code The sequence can be derived or novel significant structures can be analyzed via RNA tertiary structure prediction programs (http: / / rna.tbi.univie.ac.at / cgi-bin / RNAWebSuite / RNAfold.cgi). It is also possible to predict or discover proteins that can bind to the RNA tertiary structure and regulate the stability of the mRNA.

[0095] Specifically, in Example 7, several dozen types of mRNA were obtained through a series of Gaussia luciferase IVT mRNA reproduction and stability evaluation processes. code The sequences were selected, and eight of the clones code The sequences are summarized in SEQ ID NOs: 19 to 26. codeThe sequence (SEQ ID NO: 1) was compared with the sequence of each clone, and the results of the similarity comparison showed that, in the absence of changes in the amino acid sequence, code The sequence similarity was analyzed to range from 79.0% to 82.6% (Table 4).

[0096] [Table 4]

[0097] In addition, among the selected clones, RPIC_GL_15 Transfection It was confirmed that the expression level and stability was much higher than that of the control group not only at 24 hours but also at 48 and 72 hours (Figure 6), indicating that RPIC_GL_15 has excellent intracellular stability in various mutant sequences. Next, the RNA structure of RPIC_GL_15 was determined using the algorithm program for determining RNA structure mentioned in the previous step. code Sequence analysis revealed that the first code The secondary structure of the sequence was very different from that of the original sequence (Figure 7). Based on the above results, it can be seen that the process of the present invention can select cDNA or mRNA that is structurally different in cells but has significantly improved stability, and that the selected sequence can be used to improve protein production.

[0098] Example 9: Use of the method to select and generate mRNA with improved intracellular stability In order to confirm whether the mRNA selection method of the present invention, which has been confirmed to have excellent effects in the above examples, can be applied to target proteins other than Gaussia luciferase, the following experiment was performed.

[0099] First, the Covid19 antigen protein is used as the target protein, and the Covid19 antigen protein is code Specifically, we selected mRNA with improved intracellular stability for the Covid19 antigen protein without mutations. code array but Using the included template (SEQ ID NO: 27), the mRNA with improved intracellular stability was effectively selected through the mRNA selection method described in the previous Example. As a result, the target protein was identified by the mRNA selection method without changing the amino acid sequence of the target protein. code It was confirmed that, among sequences in which mutations were introduced into the base sequence, #57 (sequence number 30) and #114 (sequence number 34) had superior intracellular stability to the wild-type sequence (WT), and the selected sequences showed identical results in HeLa cells and HEK293T cells (Figures 8 and 9).

[0100] In addition, a recombinant protein containing a Covid19 antigen protein and an additional peptide was used as the target protein, and the above-mentioned method was used to select mRNA with improved intracellular stability. As a result, the target protein was found to be able to be expressed in a manner similar to the above-mentioned results, without changing the amino acid sequence of the target protein. code It was confirmed that, among the sequences in which mutations were introduced into the base sequence, #8 (sequence number 36) and #29 (sequence number 39) had superior intracellular stability to the wild-type sequence (WT) ( FIG. 10 ).

[0101] Based on the above results, it can be seen that the mRNA selection method of the present invention can effectively select sequences with improved intracellular stability, regardless of the type and sequence of the target protein.

[0102] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical concept or essential features of the present invention. Therefore, the above-described embodiments should be understood to be illustrative in all respects and not restrictive.

Claims

1. (A) designing a template sequence including a sequence in which a mutation has been introduced into a nucleotide sequence encoding a target protein, without causing any change in the amino acid sequence of the target protein; (B) preparing a primer based on the template sequence designed in the previous step; (C) preparing a library containing a variety of sequences into which mutations have been introduced using the prepared primer sequences; (D) selecting sequences with improved intracellular stability from the library prepared as above, The step (C) comprises sequentially carrying out a PCR reaction using the primer sequences and PCR products prepared in the step (B).

2. The method according to claim 1, wherein in step (A), the introduction of a mutation comprises introducing a redundancy codon encoding the same amino acid into the entire region or a partial region of a base sequence encoding a target protein.

3. The method according to claim 1, wherein in step (A), the introduction of a mutation comprises introducing N (A, G, C, or T), R (A or G), Y (C or T), or H (A, C, or T) into the third base of a codon in the entire region or a portion of a base sequence encoding a target protein.

4. The method of claim 1, wherein the template sequence designed in step (A) further comprises at least one selected from the group consisting of a promoter, an untranslated region (UTR), a 5' cap, and a 3' polyadenylic acid sequence (poly-A).

5. The step (A) may further include dividing the designed template sequence into two or more regions; The method of claim 1 , wherein the ends of each of said divided sections overlap one another.

6. The method of claim 5 , wherein the overlapping portions of each of the divided segments are free of mutations.

7. Dividing the designed template sequence into two or more regions includes dividing the template sequence into regions of 10 to 1,000 bp in size; The method of claim 5, wherein each of the divided regions overlaps with each other at 1 to 100 bp regions at the ends.

8. The step (B) comprises: The method of claim 1, further comprising synthesizing the sequence of the divided section of the designed template sequence, or a sequence complementary thereto, as a primer.

9. The method according to claim 8, wherein the primer synthesis step comprises synthesizing a complementary sequence as a primer for the remaining region except for the first region from the 5' end.

10. The step (D) comprises: D-1) transfecting a cell with DNA encoding a target protein contained in the library, or RNA prepared therefrom; D-2) obtaining RNA encoding a protein of interest from the transfected cells; D-3) synthesizing DNA encoding a target protein from the obtained RNA to prepare a library.

11. The method of claim 10, wherein step (D) comprises repeating steps D-1) to D-3) one or more times.

12. The step (D) comprises: D-4) cloning the DNA encoding the target protein contained in the library into a vector; D-5) transfecting the vector or RNA produced therefrom into a cell; D-6) obtaining RNA encoding a protein of interest from the transfected cells.

13. The step (D) comprises: D-7) The method of claim 12, further comprising determining the sequence of the obtained RNA and DNA synthesized therefrom.

14. The method according to claim 1, wherein the target protein or the mRNA encoding the target protein is used as an anti-cancer immunotherapeutic agent, a cancer therapeutic vaccine, an infectious disease vaccine, a protein replacement therapeutic agent, an allergy therapeutic agent, or an immune disease therapeutic agent.

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