Expression cassette comprising CYBA-derived untranslated region and use thereof

Abstract

The present invention relates to a novel CYBA-derived 5'-untranslated region sequence, an expression cassette comprising same, and use thereof. An expression cassette comprising a CYBA-derived 5'-untranslated region and / or a CYBA-derived 3'-untranslated region according to an aspect can improve the expression level of a target protein. Thus, by using the expression cassette, the target protein can be stably expressed with high efficiency in vivo or ex vivo.

Description

Expression cassette comprising a CYBA-derived non-translated region and uses thereof

[0001] The present invention relates to a novel CYBA-derived 5'-untranslated region sequence, an expression cassette comprising the same, and uses thereof.

[0002] Protein production in microbial and animal cells plays a crucial role in biotechnology. This is particularly crucial for mass production of proteins for industrial use. While host cells are being modified to increase or enhance protein production and expression within the host cells, unmet needs remain.

[0003] Nucleic acid-based gene therapy and vaccine development technologies, unlike those used in small animals, have shown insufficient transcription and translation efficiency in humans, resulting in insufficient therapeutic efficacy. Enhancing the intracellular and extracellular production and expression of proteins for disease treatment or antigenic proteins for vaccines against infectious diseases is essential for achieving the goals of nucleic acid-based therapeutics and preventives. The purpose of the present invention is to develop strategies for achieving enhanced efficacy in the development of therapeutics and preventives using artificial nucleic acid molecules.

[0004] Furthermore, in the fields of nucleic acid-based gene therapy and vaccine development, securing mRNA sequences with high stability and translation efficiency is becoming a prerequisite for both DNA and mRNA-based therapeutic and preventive applications. In DNA-based therapies, the accidental insertion of injected DNA fragments into the patient's genome carries the potential risk of causing unexpected diseases by abnormally lowering or increasing the function of the inserted gene. In this context, various mRNA-based therapies, while somewhat less stable than DNA but free of the potential risks associated with exogenous DNA injection, are being proposed. However, their low stability poses numerous challenges in ensuring that mRNA is translated into a therapeutically viable protein.

[0005] Therefore, in order to overcome these limitations, the present invention discovered a novel UTR sequence and developed a novel target protein expression cassette including the UTR sequence.

[0006] One aspect provides an expression cassette comprising a CYBA (Homo sapiens cytochrome b-245 alpha chain)-derived 5'-untranslated region (UTR) sequence.

[0007] Another aspect provides an expression vector comprising the above expression cassette.

[0008] Another aspect provides a transformant comprising the expression vector.

[0009] Another aspect provides a method for producing a target protein, comprising the steps of culturing the transformant to produce a target protein; and recovering the produced target protein from the transformant or culture medium.

[0010] Another aspect provides a composition for a vaccine or therapeutic agent comprising the expression cassette.

[0011] Another aspect provides a polynucleotide molecule comprising the sequence of SEQ ID NO: 2.

[0012] Another aspect provides a polynucleotide molecule comprising the sequence of SEQ ID NO: 27.

[0013] One aspect is to provide an expression cassette comprising a CYBA (Homo sapiens cytochrome b-245 alpha chain)-derived 5'-untranslated region (UTR) sequence.

[0014] The term "expression cassette" in this specification means a unit cassette capable of expressing / producing and / or secreting a target protein, including any combination of one or more genes and sequences that regulate their expression, such as various cis-acting transcriptional regulatory elements.

[0015] The term "untranslated region (UTR)" in this specification, also called an untranslated region, refers to a portion of an mRNA chain that does not serve as a template for a protein gene, i.e., an untranslated portion. Generally, the UTR exists at the 5' end and 3' end as the 5'-UTR or 3'-UTR, respectively.

[0016] The above CYBA-derived 5`-untranslated region sequence may include a wild-type sequence or a mutant sequence of the CYBA-derived 5`-untranslated region, and specifically may include a mutant sequence of the CYBA-derived 5`-untranslated region.

[0017] In one embodiment, the wild-type sequence of the CYBA-derived 5'-untranslated region may include the sequence of SEQ ID NO: 1, and specifically may consist of the sequence of SEQ ID NO: 1.

[0018] The mutant sequence of the CYBA-derived 5'-untranslated region may be one in which one or more bases or nucleotides are substituted, added, and / or removed from the wild-type sequence of the CYBA-derived 5'-untranslated region.

[0019] In one embodiment, the mutant sequence of the CYBA-derived 5'-untranslated region may include the sequence of SEQ ID NO: 2, and may specifically consist of the sequence of SEQ ID NO: 2.

[0020] In one embodiment, the mutant sequence of the CYBA-derived 5'-untranslated region may include one or more sequences selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO: 25, and specifically may be composed of one or more sequences selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO: 25.

[0021] The sequences of SEQ ID NOs 1 to 25 mentioned above are described in the table below (in the table below, V means G, C, or A; N means A, T, G, or C; D means G, A, or T; B means G, T, or C; H means A, C, or T). Meanwhile, the sequences of SEQ ID NOs 1 to 25 may be a DNA sequence or an mRNA sequence, and in the case of an mRNA sequence, T (thymine) may be substituted with U (uracil).

[0022] 구분5' UTR 서열 (5`-3`)서열번호WTCGC GCC TAG CAG TGT CCC AGC CGG GTT CGT GTC GCC1변이서열 일반식VGCNCC TAG CAGNGTDCCBGCNGGHTTDGTNTCNCC2KT-17CGC GCC TAG CAG TGT TCC GGC GGG ATT GGT TTC GCC3KT-44GGC GCC TAG CAG AGT GCC CGC CGG TTT AGT GTC GCC4SB-3GGC TCC TAG CAG CGT ACC CGC GGG TTT GGT ATC ACC5SB-8GGC ACC TAG CAG TGT TCC TGC GGG CTT TGT GTC ACC6CA-3CGC GCC TAG CAG TGT GCC GGC GGG TTT TGT GTC GCC7CA-10CGC GCC TAG CAG TGT GCC GGC GGG ATT TGT CTC GCC8CA-16GGC GCC TAG CAG GGT TCC CGC TGG TTT AGT TTC ACC9LCA-2GGC TCC TAG CAG TGT TCC CGC TGG ATT AGT GTC GCC10LCA-5GGC TCC TAG CAG GGT TCC TGC GGG TTT GGT CTC ACC11LCA-7GGC CCC TAG CAG GGT GCC GGC CGG TTT GGT GTC ACC12HS-1GGC GCC TAG CAG TGT TCC CGC GGG TTT GGT GTC GCC13HS-2CGC TCC TAG CAG GGT TCC GGC GGG CTT GGT GTC GCC14HS-4GGC TCC TAG CAG TGT ACC TGC GGG TTT TGT TTC TCC15HS-6CGC GCC TAG CAG TGT GCC GGC AGG TTT AGT GTC TCC16HS-8AGC ACC TAG CAG AGT TCC TGC TGG ATT AGT ATC ACC17HS-10GGC TCC TAG CAG AGT GCC CGC GGG ATT GGT GTC GCC18HS-13GGC GCC TAG CAG CGT TCCGGC GGG TTT GGT ATC GCC19HS-15GGC TCC TAG CAG GGT ACC CGC TGG CTT TGT GTC TCC20HS-16GGC GCC TAG CAG AGT TCC TGC TGG ATT TGT CTC GCC21HS-18GGC ACC TAG CAG GGT ACC GGC GGG CTT TGT GTC CCC22HS-20AGC CCC TAG CAG GGT GCC TGC AGG TTT GGT TTC ACC23HS-22GGC TCC TAG CAG AGT GCC TGC GGG CTT TGT GTC ACC24HS-24CGC GCC TAG CAG GGT GCC GGC AGG TTT GGT CTC GCC25

[0023]

[0024] The above expression cassette may additionally comprise a CYBA-derived 3'-untranslated region sequence.

[0025] The above CYBA-derived 3'-untranslated region sequence may include a wild-type sequence or a mutant sequence of the CYBA-derived 3'-untranslated region.

[0026] In one embodiment, the wild-type sequence of the CYBA-derived 3'-untranslated region may comprise the sequence of SEQ ID NO: 26, and specifically may consist of the sequence of SEQ ID NO: 26.

[0027] The mutant sequence of the CYBA-derived 3'-untranslated region may be one in which one or more bases or nucleotides are substituted, added, and / or removed from the wild-type sequence of the CYBA-derived 3'-untranslated region.

[0028] In one embodiment, the mutant sequence of the CYBA-derived 3'-untranslated region may include the sequence of SEQ ID NO: 27, and specifically may consist of the sequence of SEQ ID NO: 27.

[0029] In one embodiment, the mutant sequence of the CYBA-derived 3'-untranslated region may include one or more sequences selected from the group consisting of SEQ ID NO: 28 to SEQ ID NO: 50, and specifically may be composed of one or more sequences selected from the group consisting of SEQ ID NO: 28 to SEQ ID NO: 50.

[0030] The sequences of SEQ ID NOs 26 to 50 mentioned above are described in the table below. Meanwhile, the sequences of SEQ ID NOs 26 to 50 may be a DNA sequence or an mRNA sequence, and in the case of an mRNA sequence, T (thymine) may be substituted with U (uracil).

[0031] 구분3' UTR 서열 (5`-3`)서열번호WTCCT CGC CCC GGA CCT GCC CTC CCG CCA GGT GCA CCC ACC TGC AAT AAA TGC AGC GAA GCC GGG A26변이서열 일반식NCTNGCNCCNGANCTNCCNTC CNG CNA GNT GCNCCCNCCNGCNAT AAA TGC AGC GAA GCC GGG A27KT-17GCT AGC GCC CGA GCT CCC ATC CAG CGA GCT GCG CCC CCC AGC GAT AAA TGC AGC GAA GCC GGG A28KT-44CCT CGC CCC AGA ACT CCC ATC CTG CAA GAT GCG CCC CCC CGC TAT AAA TGC AGC GAA GCC GGG A29SB-3CCT TGC ACC CGA ACT CCC ATC CAG CGA GCT GCC CCC ACC CGC AAT AAA TGC AGC GAA GCC GGG A30SB-8ACT CGC CCC TGA GCT CCC GTC CGG CTA GCT GCG CCC CCC CGC CAT AAA TGC AGC GAA GCC GGG A31CA-3TCT AGC GCC TGA GCT ACC ATC CTG CGA GTT GCC CCC TCC TGC GAT AAA TGC AGC GAA GCC GGG A32CA-10CCT AGC CCC AGA TCT CCC TTC CTG CTA GCT GCG CCC ACC GGC CAT AAA TGC AGC GAA GCC GGG A33CA-16ACT AGC CCC AGA ACT ACC TTC CGG CTA GAT GCG CCC TCC AGC CAT AAA TGC AGC GAA GCC GGG A34LCA-2ACT CGC ACC AGA ACT CCC ATC CAG CAA GAT GCC CCC CCC CGC CAT AAA TGC AGC GAA GCC GGG A35LCA-5TCT CGC CCC CGA GCT TCC TTC CAG CTA GTT GCC CCC ACC TGC CAT AAATGC AGC GAA GCC GGG A36LCA-7TCT GGC GCC AGA ACT ACC GTC CAG CTA GCT GCG CCC GCC CGC AAT AAA TGC AGC GAA GCC GGG A37HS-1ACT CGC GCC CGA GCT ACC GTC CGG CAA GAT GCC CCC GCC GGC AAT AAA TGC AGC GAA GCC GGG A38HS-2CCT TGC CCC TGA ACT ACC ATC CTG CAA GTT GCC CCC CCC AGC AAT AAA TGC AGC GAA GCC GGG A39HS-4ACT CGC CCC TGA ACT ACC ATC CAG CAA GTT GCC CCC GCC CGC AAT AAA TGC AGC GAA GCC GGG A40HS-6GCT GGC CCC TGA GCT ACC TTC CAG CTA GCT GCG CCC CCC CGC AAT AAA TGC AGC GAA GCC GGG A41HS-8CCT GGC CCC CGA ACT CCC ATC CGG CTA GAT GCC CCC CCC TGC AAT AAA TGC AGC GAA GCC GGG A42HS-10CCT CGC CCC GGA CCT GCC CTC CCG CCA GGT GCA CCC ACC CGC AAT AAA TGC AGC GAA GCC GGG A43HS-13ACT AGC TCC TGA GCT CCC ATC CTG CAA GCT GCC CCC TCC CGC AAT AAA TGC AGC GAA GCC GGG A44HS-15CCT CGC ACC AGA ACT TCC GTC CGG CAA GCT GCC CCC GCC GGC AAT AAA TGC AGC GAA GCC GGG A45HS-16GCT CGC ACC CGA GCT CCC ATC CGG CGA GTT GCT CCC GCC AGC AAT AAA TGC AGC GAA GCC GGG A46HS-18CCT CGC CCC GGA CCT GCC CTC CCG CCA GGT GCA CCC CCC TGCAAT AAA TGC AGC GAA GCC GGG A47HS-20CCT AGC CCC AGA TCT TCC GTC CAG CTA GCT GCT CCC CCC TGC AAT AAA TGC AGC GAA GCC GGG A48HS-22GCT AGC TCC AGA TCT CCC GTC CAG CTA GCT GCT CCC ACC CGC AAT AAA TGC AGC GAA GCC GGG A49HS-24CCT CGC CCC GGA CCT GCC CTC CCG CCA GGT GCA CCC ACC TGC AAT AAA TGC AGC GAA GCC GGG A50

[0032]

[0033] In one embodiment, the expression cassette may comprise a 5'-untranslated region comprising the sequence of SEQ ID NO: 2 and a 3'-untranslated region comprising the sequence of SEQ ID NO: 27.

[0034] In one embodiment, the expression cassette may comprise a 5'-untranslated region comprising one of the sequences of SEQ ID NOs: 3 to 25 and a 3'-untranslated region comprising one of the sequences of SEQ ID NOs: 28 to 50.

[0035] In one embodiment, the expression cassette may be one or more of the following expression cassettes comprising a combination of a 5'-untranslated region and a 3'-untranslated region:

[0036] 1) a 5'-untranslated region comprising the sequence of SEQ ID NO: 3 and a 3'-untranslated region comprising the sequence of SEQ ID NO: 28,

[0037] 2) a 5'-untranslated region comprising the sequence of SEQ ID NO: 4 and a 3'-untranslated region comprising the sequence of SEQ ID NO: 29;

[0038] 3) a 5'-untranslated region comprising the sequence of SEQ ID NO: 5 and a 3'-untranslated region comprising the sequence of SEQ ID NO: 30;

[0039] 4) A 5'-untranslated region comprising the sequence of SEQ ID NO: 6 and a 3'-untranslated region comprising the sequence of SEQ ID NO: 31;

[0040] 5) A 5'-untranslated region comprising the sequence of SEQ ID NO: 7 and a 3'-untranslated region comprising the sequence of SEQ ID NO: 32,

[0041] 6) A 5'-untranslated region comprising the sequence of SEQ ID NO: 8 and a 3'-untranslated region comprising the sequence of SEQ ID NO: 33,

[0042] 7) A 5'-untranslated region comprising the sequence of SEQ ID NO: 9 and a 3'-untranslated region comprising the sequence of SEQ ID NO: 34,

[0043] 8) A 5'-untranslated region comprising the sequence of SEQ ID NO: 10 and a 3'-untranslated region comprising the sequence of SEQ ID NO: 35,

[0044] 9) A 5'-untranslated region comprising the sequence of SEQ ID NO: 11 and a 3'-untranslated region comprising the sequence of SEQ ID NO: 36,

[0045] 10) A 5'-untranslated region comprising the sequence of SEQ ID NO: 12 and a 3'-untranslated region comprising the sequence of SEQ ID NO: 37,

[0046] 11) A 5'-untranslated region comprising the sequence of SEQ ID NO: 13 and a 3'-untranslated region comprising the sequence of SEQ ID NO: 38,

[0047] 12) A 5'-untranslated region comprising the sequence of SEQ ID NO: 14 and a 3'-untranslated region comprising the sequence of SEQ ID NO: 39,

[0048] 13) A 5'-untranslated region comprising the sequence of SEQ ID NO: 15 and a 3'-untranslated region comprising the sequence of SEQ ID NO: 40,

[0049] 14) A 5'-untranslated region comprising the sequence of SEQ ID NO: 16 and a 3'-untranslated region comprising the sequence of SEQ ID NO: 41,

[0050] 15) A 5'-untranslated region comprising the sequence of SEQ ID NO: 17 and a 3'-untranslated region comprising the sequence of SEQ ID NO: 42,

[0051] 16) A 5'-untranslated region comprising the sequence of SEQ ID NO: 18 and a 3'-untranslated region comprising the sequence of SEQ ID NO: 43,

[0052] 17) A 5'-untranslated region comprising the sequence of SEQ ID NO: 19 and a 3'-untranslated region comprising the sequence of SEQ ID NO: 44,

[0053] 18) A 5'-untranslated region comprising the sequence of SEQ ID NO: 20 and a 3'-untranslated region comprising the sequence of SEQ ID NO: 45,

[0054] 19) A 5'-untranslated region comprising the sequence of SEQ ID NO: 21 and a 3'-untranslated region comprising the sequence of SEQ ID NO: 46,

[0055] 20) A 5'-untranslated region comprising the sequence of SEQ ID NO: 22 and a 3'-untranslated region comprising the sequence of SEQ ID NO: 47,

[0056] 21) A 5'-untranslated region comprising the sequence of SEQ ID NO: 23 and a 3'-untranslated region comprising the sequence of SEQ ID NO: 48,

[0057] 22) a 5'-untranslated region comprising the sequence of SEQ ID NO: 24 and a 3'-untranslated region comprising the sequence of SEQ ID NO: 49, and

[0058] 23) A 5'-untranslated region comprising the sequence of SEQ ID NO: 25 and a 3'-untranslated region comprising the sequence of SEQ ID NO: 50.

[0059] The above expression cassette has the characteristic of being able to improve stability in vivo or in vitro and / or increase expression efficiency of a target protein, thereby stably and efficiently expressing a target protein in vivo or in vitro.

[0060] In one embodiment, the expression cassette may have improved stability in vivo or in vitro and / or enhanced expression efficiency of the target protein compared to an expression cassette comprising a wild-type sequence of a CYBA-derived 5'-untranslated region.

[0061] In one embodiment, the expression cassette may have improved stability in vivo or in vitro and / or enhanced expression efficiency of the target protein compared to an expression cassette comprising a wild-type sequence of a CYBA-derived 5'-untranslated region and a wild-type sequence of a 3'-untranslated region.

[0062] The above expression cassette may additionally comprise a sequence encoding a target protein.

[0063] The term "target protein" in this specification refers to a protein that a person skilled in the art wishes to express, and refers to any protein that can be expressed in a host cell or a target organism by inserting a polynucleotide sequence encoding the protein into the expression cassette, an expression vector containing the same, or the expression cassette or expression vector.

[0064] The above target protein or the polynucleotide sequence (cDNA or mRNA) encoding the same may be used as an anticancer immunotherapy agent, a cancer treatment vaccine, an infectious disease vaccine, a protein replacement treatment agent, an allergy treatment agent, or an immune disease treatment agent, and specifically may be an effective ingredient of the treatment agent or an antigen / immunogen of the vaccine.

[0065] The above expression cassette may be a polynucleotide sequence or a polynucleotide molecule.

[0066] The term "polynucleotide" in this specification refers to a polymeric substance in which nucleotides are bound, and may mean DNA or RNA that encodes genetic information.

[0067] The above expression cassette may be in the form of a nucleic acid, and may be composed of DNA, RNA, or a modified form thereof, for example, cDNA, mRNA, or a modified form thereof. If the expression cassette is DNA or a modified DNA, it may be composed of A (Adenine), T (Thymine), G (Guanine), C (Cytosine), and / or a modified form thereof, and if the expression cassette is RNA or a modified RNA, it may be composed of A, U (Uridine), G, C, and / or a modified form thereof. For example, the modified DNA or RNA may be 5-methylcytidine (5mC), N6-methyladenosine (m6A), 3,2'-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2' fluorouridine, pseudouridine, 2'-O-methyluridine (Um), 2' deoxyuridine (2' dU), 4-thiouridine (s4U), 5-methyluridine (m5U), 2'-O-methyladenosine (m6A), N6,2'-O-dimethyladenosine (m6Am), N6,N6,2'-O-trimethyladenosine (m62Am), 2'-O-methylcytidine (Cm), 7-methylguanosine (m7G), 2'-O-methylguanosine (Gm), It may include at least one selected from the group consisting of N2,7-dimethylguanosine (m-2,7G), N2,N2,7-trimethylguanosine (m-2,2,7G) and N1-methyl-pseudouridine.

[0068] Accordingly, when the expression cassette is in the form of RNA, T of the polynucleotide sequence expressing the expression cassette in the present specification may be expressed by being replaced with U, and when the expression cassette is in the form of modified DNA or modified RNA, A, T, G, C and U of the polynucleotide sequence expressing the expression cassette in the present specification may be expressed by being replaced with their modified forms, respectively.

[0069] The above expression cassette may be composed of DNA, RNA or a modified form thereof, for example, cDNA, mRNA or a modified form thereof.

[0070] The expression cassette may further include, inside or outside the expression cassette, various factors that can assist in efficient expression of the target protein, such as a signal peptide or a sequence encoding the signal peptide, a promoter, a transcription enhancer, a terminator, an initiator, an untranslated region, a His-tag, a protease recognition site, and one or more components that regulate expression of the target protein.

[0071] The term "signal peptide" as used herein refers to a short peptide (5 to 30 amino acids) present at the N-terminus of a newly synthesized protein, which directs the protein to be secreted to a designated location via the secretory pathway. The signal peptide may be used interchangeably with "signal peptide" or "secretory peptide."

[0072] The above expression cassette may further comprise one or more selected from the group consisting of a 5'-cap, a promoter, and a poly-A tail (polyadenylation tail), and specifically may comprise all of the 5'-cap, the promoter, and the poly-A tail.

[0073] The above 5'-cap can be a known 5'-capping composition / method (e.g., 7-methylguanylate cap (m)) that can improve the in vivo stability of the expression cassette and / or improve the expression efficiency of the target protein. 7 G)) or its variants may be used without limitation.

[0074] The poly(A) tail or its variants may be any known poly(A) tail or its variants without limitation as long as it can improve the in vivo stability of the expression cassette and / or improve the expression efficiency of the target protein.

[0075] The CYBA-derived 5'-untranslated region sequence, the CYBA-derived 3'-untranslated region sequence, and the polynucleotide sequence encoding the target protein of the above expression cassette, as well as the signal peptide, 5'-cap, promoter, and / or poly-A tail sequence that may be additionally included, may be operably linked.

[0076] The term "operably linked" as used herein refers to a state in which a nucleic acid expression control sequence and a nucleic acid sequence encoding a desired protein or peptide are functionally linked to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein or peptide are operably linked to affect the expression of the coding sequence. The operably linked sequence with the expression vector can be produced using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes generally known in the art.

[0077] The above expression cassette may be connected in the order of a 5'-untranslated region, a polynucleotide sequence encoding a target protein, and a 3'-untranslated region in the 5' to 3' direction, and when a signal peptide, a 5'-cap, a promoter, and / or a poly-A tail sequence is additionally included, the expression cassette may be connected in the order of a 5'-cap, a promoter, a 5'-untranslated region, a sequence encoding a signal peptide, a polynucleotide sequence encoding a target protein, a 3'-untranslated region, and / or a poly-A tail sequence. In addition, the above configurations may be directly connected to each other or may be indirectly connected via a linker.

[0078] The above linker is not particularly limited as long as it enables expression and extracellular secretion of the target protein of the expression cassette, and may be composed of, for example, 1 to 50 nucleic acids.

[0079] The above expression cassette may further include a polynucleotide encoding His, GST or intein upstream or downstream of the polynucleotide sequence within the expression cassette to facilitate detection, expression level confirmation or purification of the expressed target protein, and specifically, may include a polynucleotide encoding 3 to 15 histidines (His) at the 3'-terminal end of the sequence encoding the target protein of the expression cassette.

[0080] The above expression cassette may further include a polynucleotide encoding a cleavage recognition site of a protein-decomposing enzyme, including trypsin, enterokinase, or thrombin, upstream or downstream of the polynucleotide sequence in the expression cassette to facilitate detection, expression level confirmation, or purification of the expressed target protein, and specifically, may include a polynucleotide encoding a thrombin cleavage recognition site at the 3'-end of the sequence encoding the target protein of the expression cassette.

[0081] The above expression cassette can be used for various purposes, and specifically, it can be used for the production of a target protein, therapeutic use, or vaccine use. In the case of therapeutic / vaccine use, the therapeutic / vaccine can be a gene therapy / vaccine such as a DNA therapeutic / vaccine, RNA therapeutic / vaccine, or mRNA therapeutic / vaccine. Furthermore, the expression cassette for therapeutic / vaccine use can be used for intracellular / in vivo delivery and / or expression of a gene vaccine / therapeutic agent.

[0082] The term "vaccine" in this specification refers to a biological preparation containing an antigen that provides immunity to a living organism, and refers to an immunogen or antigenic substance that creates immunity in a living organism by administering it to a human or animal to prevent infection and / or infectious disease.

[0083] The target protein may be an antigen / immunogen, a therapeutically effective substance and / or a fragment thereof, and the expression cassette may comprise a polynucleotide sequence encoding the antigen / immunogen, the therapeutically effective substance and / or a fragment thereof.

[0084] The above therapeutic / vaccine use expression cassette may be for delivering a sequence encoding an antigen gene or an effective ingredient of a genetic vaccine such as an RNA therapeutic / vaccine, an RNA therapeutic / vaccine, or a DNA therapeutic / vaccine into the body to effectively express the antigen or effective ingredient in the body, and the target protein of the therapeutic / vaccine use expression cassette may be a sequence encoding an antigen for inducing an immune response or an effective ingredient capable of exhibiting therapeutic efficacy.

[0085] According to one embodiment, the expression cassette can improve the expression level of the target protein, so that the expression cassette including an antigen or a therapeutically effective ingredient as the target protein or an expression vector including the same can not only effectively deliver the antigen sequence or the effective ingredient coding sequence into the body as a genetic vaccine / therapeutic agent, but also induce an effective expression level increase of the antigen or the therapeutically effective ingredient, thereby more effectively inducing a vaccine immune response or therapeutic efficacy, such as a neutralizing antibody formation induction response.

[0086] The above antigens are antigens of Coronavirus, antigens of Japanese encephalitis virus, antigens of Heamophilus influenzae type B (HIB), antigens of Zika virus, antigens of Pseudomonas aeruginosa, antigens of pertussis, antigens of tuberculosis, antigens of anthrax, antigens of Hepatitis A virus (HAV), antigens of Hepatitis B virus (HBV), antigens of Hepatitis C virus (HCV), antigens of human immunodeficiency virus (HIV), antigens of Herpes simplex virus (HSV), antigens of Neisseria meningitidis, antigens of Corynebacterium diphtheria, antigens of Bordetella pertussis, antigens of Clostridium tetani, and antigens of HPV (human papillomavirus). virus), an antigen of Varicella virus, an antigen of Enterococci, an antigen of Staphylococcus aureus, an antigen of Klebsiella pneumonia, an antigen of Acinetobacter baumannii, an antigen of Enterobacter, an antigen of Helicobacter pylori, an antigen of malaria, an antigen of dengue virus, an antigen of Orientia tsutsugamushi, an antigen of severe fever with thrombocytopenia syndrome Bunyavirus (SFTS Bunyavirus), an antigen of influenza virus, an antigen of Ebola virus, and an antigen of Streptococcus pneumoniae, but is not limited thereto.

[0087] If the vaccine is for preventing or treating an infection or infectious disease caused by the bacteria and / or virus, the vaccine may be selected from the group consisting of a coronavirus vaccine, a Japanese encephalitis vaccine, a Hemophilus influenzae type B vaccine, a MERS vaccine, a Zika vaccine, a Pseudomonas aeruginosa vaccine, a cancer vaccine, a tuberculosis vaccine, anthrax vaccine, HAV vaccine, HBV vaccine, HCV vaccine, HIV vaccine, herpes simplex vaccine, meningitis vaccine, diphtheria vaccine, pertussis vaccine, tetanus vaccine, varicella vaccine, multidrug-resistant bacteria vaccine, enterococcus vaccine, staphylococcus vaccine, Klebsiella pneumoniae vaccine, Acinetobacter vaccine, Enterobacter vaccine, Helicobacter vaccine, malaria vaccine, dengue vaccine, tsutsugamushi vaccine, severe fever with thrombocytopenia syndrome vaccine, SARS vaccine, Ebola vaccine, influenza vaccine, COVID-19 vaccine, and pneumococcal vaccine. It is not limited to this.

[0088]

[0089] Another aspect is to provide an expression vector comprising the expression cassette. The same parts described above also apply to the expression vector.

[0090] The term "expression vector" as used herein refers to a recombinant vector capable of expressing a target protein when introduced into a suitable host cell or living organism, and a genetic construct containing essential regulatory elements operably linked to enable expression of the gene insert. The term "operably linked" means that a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding the target protein are functionally linked to perform a general function. The operably linked vector can be produced using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be easily performed using enzymes generally known in the art.

[0091] Suitable expression vectors of the present invention may include signal sequences for membrane targeting or secretion, in addition to expression control elements such as a promoter, initiation codon, termination codon, polyadenylation signal, and enhancer. The initiation and termination codons are generally considered to be part of the nucleotide sequence encoding the protein of interest, and must be functional in a subject when the genetic construct is administered, and must be in frame with the coding sequence. Common promoters can be constitutive or inducible and include, but are not limited to, the lac, tac, T3 and T7 promoters in prokaryotes, and the simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoters, human immunodeficiency virus (HIV), e.g., the long terminal repeat (LTR) promoter of HIV, Moloney virus, cytomegalovirus (CMV), Epstein-Barr virus (EBV), Rous sarcoma virus (RSV) promoters, as well as the β-actin promoter, human hemoglobin, human muscle creatine and human metallothionein promoters in eukaryotes.

[0092] Additionally, the expression vector may include a selectable marker for selecting host cells containing the vector. The selectable marker is used to select cells transformed with the vector, and markers that confer selectable phenotypes such as drug resistance, nutrient requirements, cytotoxic agent resistance, or expression of surface proteins may be used. In an environment treated with a selective agent, only cells expressing the selectable marker survive, allowing the transformed cells to be selected. Furthermore, if the vector is a replicable expression vector, it may include a replication origin, which is a specific nucleic acid sequence where replication begins.

[0093] Recombinant expression vectors for inserting foreign genes can take many forms, including plasmids, viruses, and cosmids. The type of recombinant vector is not particularly limited, as long as it can express the desired gene and produce the desired protein in various host cells, both prokaryotic and eukaryotic. However, vectors that possess a highly active promoter and strong expression capacity, while also being capable of mass-producing foreign proteins in a form similar to that of the native form, are particularly useful.

[0094] To express the target protein of the present invention, a variety of combinations of hosts and vectors can be utilized. Suitable expression vectors for eukaryotic hosts include, but are not limited to, expression regulatory sequences derived from SV40, bovine papillomavirus, adenovirus, adeno-associated virus, cytomegalovirus, and retroviruses. Expression vectors that can be used in bacterial hosts include, but are not limited to, bacterial plasmids obtained from Escherichia coli, including pET, pRSET, pBluescript, pGEX2T, pUC vectors, col E1, pCR1, pBR322, pMB9 or derivatives thereof, plasmids having a wider 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. In yeast cells, a second-order plasmid or derivatives thereof can be used, and in insect cells, pVL941 can be used, etc.

[0095]

[0096] Another aspect is to provide a transformant comprising the expression vector. The same parts as described above also apply to the transformant.

[0097] As used herein, the term "transformation" refers to introducing, for the purposes of the present invention, the target protein expression cassette or the expression vector containing the target protein expression cassette into a host cell so that the nucleic acid molecule of the expression cassette or the expression vector can be expressed in the host cell. It also includes the case where the target protein expression cassette can be integrated into a specific location by additionally inserting a sequence identical to a portion of the host cell's chromosome on both sides of the target protein expression cassette, thereby achieving expression and secretion of the target protein. The transformed expression cassette or expression vector includes both, whether it is inserted into the chromosome of the host cell or located outside the chromosome, as long as it can be expressed in the host cell. In addition, the nucleic acid molecule can include DNA and RNA in the form of a sequence encoding the target protein. The expression cassette or vector may be introduced in any form as long as it can be introduced into the host cell and expressed. For example, an expression cassette, which is a genetic construct containing all the elements necessary for self-expression, may additionally include a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replication. In addition, the expression cassette may be introduced into a host cell in its own form and operably linked to a sequence necessary for expression in the host cell.

[0098] The term "transformant" in this specification may be a host cell into which the expression cassette or expression vector can be introduced, and may be a transformant other than a human.

[0099] A host cell suitable for introduction of the above expression cassette or expression vector may be a prokaryotic cell such as Escherichia coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis or Staphylococcus sp. In addition, it may be a fungus such as Aspergillus sp., a yeast such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp. or Neurospora crassa, other lower eukaryotic cells, or a cell of a higher eukaryote such as a plant or insect cell. In addition, it may be a mammalian cell, and specifically, monkey kidney cells 7 (COS7: monkey kidney cells) cells, NSO cells, SP2 / 0, Chinese hamster ovary (CHO: Chinese hamster ovary) cells, W138, baby hamster kidney (BHK: baby hamster kidney) cells, MDCK, myeloma cell lines, HeLa cells, HuT 78 cells, or HEK293 cells may be used, but is not limited thereto.

[0100] The transformation method of the present invention includes any method for introducing a nucleic acid into an organism, cell, tissue, or organ, and can be performed by selecting an appropriate standard technique according to the host cell known in the art. Specifically, the transformation method includes, but is not limited to, electroporation, protoplast fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, stirring using silicon carbide fibers, Agrobacterium-mediated transformation, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, lithium acetate-DMSO method, lipofectamine, and desiccation / inhibition-mediated transformation methods.

[0101]

[0102] Another aspect provides a polynucleotide molecule comprising the expression cassette. The same parts described above also apply to the polynucleotide molecule.

[0103]

[0104] Another aspect provides a composition for producing a target protein, comprising the expression cassette or a polynucleotide molecule comprising the same. The same aspects described above also apply to the composition.

[0105] In the above composition, the expression cassette may be included in various forms, and specifically, may be included in the form of the expression cassette, an expression vector including the expression cassette, or a transformant including the expression vector.

[0106]

[0107] Another aspect provides a method for producing a target protein, comprising: culturing the transformant to produce a target protein; and recovering the produced target protein from the transformant or culture medium. The same portions described above also apply to the method.

[0108] The term "cultivation" as used herein refers to growing microorganisms or cells under appropriately artificially controlled environmental conditions. The method for producing a target protein using the transformant of the present invention can be performed using methods widely known in the art. Specifically, the cultivation can be performed continuously in a batch process, fed batch, or repeated fed batch process, but is not limited thereto.

[0109] The medium used for the above culture must suitably meet the requirements of the transformant. Can be used sugar sources include, but are not limited to, sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, cellulose; oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil; fatty acids such as palmitic acid, stearic acid, linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as gluconic acid, acetic acid, and pyruvic acid. These substances can be used individually or as a mixture. Can be used nitrogen sources include, but are not limited to, peptone, yeast extract, meat juice, malt extract, corn steep liquor, soybean meal, and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. Nitrogen sources can also be used individually or as a mixture. Potential sources of growth media include, but are not limited to, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or their corresponding sodium-containing salts. Additionally, the culture medium may contain metal salts, such as magnesium sulfate or iron sulfate, necessary for growth. Finally, in addition to the above substances, essential growth materials, such as amino acids and vitamins, may be used. Appropriate precursors may also be incorporated into the culture medium. The aforementioned raw materials may be added to the culture in a batch or continuous manner during the culture process in an appropriate manner. These various culture methods may utilize known techniques.

[0110] Additionally, the pH of the culture can be adjusted by appropriately using basic compounds such as sodium hydroxide, potassium hydroxide, and ammonia, or acid compounds such as phosphoric acid or sulfuric acid. Furthermore, foaming can be suppressed by using antifoaming agents such as fatty acid polyglycol esters. Oxygen or an oxygen-containing gas (e.g., air) can be injected into the culture to maintain an aerobic state. The temperature of the culture can usually be between 20°C and 45°C, preferably between 25°C and 40°C, but can be changed depending on the conditions, but is not limited thereto.

[0111] Methods for recovering the target protein or product from the transformant or culture / culture medium may include methods known in the art, such as centrifugation, filtration, anion exchange chromatography, crystallization, and HPLC, but are not limited to these examples.

[0112] The above recovery step may include a purification process, and a person skilled in the art may select and utilize one of several known purification processes as needed.

[0113]

[0114] Another aspect provides a composition for a vaccine or therapeutic agent comprising the polynucleotide molecule or expression cassette. The same aspects described above also apply to the composition.

[0115] The above composition for a vaccine or therapeutic agent may be a composition for delivering and / or expressing a target protein, a polynucleotide molecule encoding the same, and / or the expression cassette, and specifically may be a composition for delivering and / or expressing the same in vivo or in a cell.

[0116] The above vaccine or therapeutic agent may be a genetic vaccine or a genetic therapeutic agent.

[0117] The target protein may be an antigen / immunogen, a therapeutically effective substance and / or a fragment thereof, and the expression cassette may comprise a polynucleotide sequence encoding the antigen / immunogen, the therapeutically effective substance and / or a fragment thereof.

[0118] The above-mentioned vaccine or therapeutic composition may be a pharmaceutical composition and may additionally include a pharmaceutically acceptable excipient, diluent, or carrier. The term "pharmaceutically acceptable excipient, diluent, or carrier" may refer to an excipient, diluent, or carrier that does not stimulate a living organism and does not inhibit the biological activity and properties of the compound to be injected. Here, "pharmaceutically acceptable" means that it does not inhibit the activity of the active ingredient and does not exhibit toxicity beyond what the target of application (prescription) can tolerate.

[0119] Suitable carriers for vaccines or therapeutic agents are well known to those skilled in the art and include, but are not limited to, proteins, sugars, etc. The carriers may be aqueous or non-aqueous solutions, suspensions, or emulsions.

[0120] The above vaccine or therapeutic composition may be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or unit dose ampoules or multiple dose injections, respectively, according to conventional methods. When formulating the above vaccine composition, it may be prepared by adding diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, or surfactants that are commonly used.

[0121] When the above vaccine or therapeutic composition is prepared as a parenteral formulation, it can be formulated in the form of injections, transdermal administration, nasal inhalants, and suppositories using a suitable carrier according to a method known in the art. When formulated as an injection, suitable carriers include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof, and preferably, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, sterile water for injection, and isotonic solutions such as 5% dextrose can be used. When formulated as a transdermal administration, it can be formulated in the form of ointments, creams, lotions, gels, external solutions, pastes, liniments, aerosols, etc. In the case of nasal inhalation, it can be formulated in the form of an aerosol spray using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, or carbon dioxide, and in the case of formulating it as a suppository, the base can be witepsol, tween 61, polyethylene glycol, cacao butter, laurin butter, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene stearate, sorbitan fatty acid ester, etc.

[0122] The route of administration of the above vaccine or therapeutic composition may be through any general route as long as it can reach the target tissue, and specifically, the vaccine composition may be selected from the group consisting of compositions for intramuscular administration, subcutaneous administration, intraperitoneal administration, intravenous administration, oral administration, dermal administration, ocular administration, and intracerebral administration.

[0123] The above composition for vaccine or therapeutic agent can be administered in a pharmaceutically effective amount, wherein the term "pharmaceutically effective amount" means an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention, and the effective dosage level can be determined according to factors including the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the time of administration of the composition of the present invention used, the route of administration and the excretion rate, the treatment period, the drug used in combination with or concurrently with the composition of the present invention used, and other factors well known in the medical field.

[0124] The dosage of the above-mentioned vaccine or therapeutic composition can be determined by a person skilled in the art in consideration of the purpose of use, the degree of toxicity of the disease, the patient's age, weight, sex, medical history, or the type of substance used as the active ingredient. For example, the vaccine composition of the present invention can be administered at about 0.1 ng to about 1,000 mg / kg, preferably 1 ng to about 100 mg / kg, per adult, and the frequency of administration of the composition of the present invention is not particularly limited thereto, but can be administered once a day or administered in divided doses several times. The dosage or frequency of administration does not limit the scope of the present invention in any way.

[0125]

[0126] Another aspect is to provide a polynucleotide molecule comprising the sequence of SEQ ID NO: 2. The same parts as described above also apply to the polynucleotide molecule.

[0127] The polynucleotide molecule may be in the form of a nucleic acid, consisting of DNA, RNA or a modified form thereof, for example, cDNA, mRNA or a modified form thereof.

[0128] The polynucleotide molecule may comprise a CYBA-derived 5'-untranslated region sequence.

[0129] In one embodiment, the polynucleotide molecule may comprise one or more selected from the group consisting of SEQ ID NOs: 3 to 25.

[0130]

[0131] Another aspect is to provide a polynucleotide molecule comprising the sequence of SEQ ID NO: 27. The same parts as described above also apply to the polynucleotide molecule.

[0132] The polynucleotide molecule may be in the form of a nucleic acid, consisting of DNA, RNA or a modified form thereof, for example, cDNA, mRNA or a modified form thereof.

[0133] The polynucleotide molecule may comprise a CYBA-derived 3'-untranslated region sequence.

[0134] In one embodiment, the polynucleotide molecule may comprise one or more selected from the group consisting of SEQ ID NOs: 28 to 50.

[0135] According to an expression cassette comprising a CYBA-derived 5'-untranslated region and / or a CYBA-derived 3'-untranslated region according to an aspect, the expression level of a target protein can be improved, and the use of the expression cassette has the advantage of being able to stably express a target protein with high efficiency in vivo or in vitro.

[0136] Figure 1 is a diagram showing the expression cassette configuration of Nat-GL and 5CYB-GL.

[0137] Figure 2 is a diagram showing the structure of an expression cassette comprising a CYBA-derived 5'-untranslated region and / or a 3'-untranslated region.

[0138] Figure 3 is a drawing showing the amount of target protein expressed after introduction of the expression cassette into cells, confirmed by Western blotting.

[0139] Figure 4 is a schematic diagram showing an expression cassette into which a CYBA-derived UTR sequence and its mutant sequences are introduced.

[0140] Figures 5 to 9 are diagrams confirming the expression level of an expression cassette containing a CYBA-derived UTR mutant sequence.

[0141] Figure 10 is a diagram showing the results of comparing the expression level of an expression cassette containing a CYBA-derived UTR variant sequence with an expression cassette containing a known UTR sequence.

[0142] The present invention will be described in more detail through the following examples. However, these examples are provided for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0143]

[0144] The present invention relates to an expression cassette comprising a novel CYBA (Homo sapiens cytochrome b-245 alpha chain)-derived 5'-untranslated region (5'-UTR) and / or 3'-untranslated region (3'-UTR). Hereinafter, novel 5'-UTR and 3'-UTR sequences were discovered, and the target protein expression efficiency of an expression cassette comprising the same was evaluated.

[0145]

[0146] Example 1: Construction of an expression cassette with a CYBA-derived 5'-UTR

[0147] To construct an expression cassette containing the CYBA 5'-UTR wild-type sequence and evaluate its target protein expression level, the following experiments were performed. Meanwhile, experiments were performed using Gaussian luciferase, a reporter protein, as an example of the target protein.

[0148]

[0149] 1.1: Creating an expression cassette

[0150] To construct an expression cassette incorporating a sequence encoding Gaussian luciferase as an example of a target protein, the following experiments were performed.

[0151] Specifically, mRNA was produced through in vitro transcription (IVT) using a sequence encoding wild-type Gaussian Luciferase (Nat-GL) containing the 5'-UTR, 3'-UTR, and open reading frame (ORF) of Gaussian Luciferase (SEQ ID NO: 51) and a sequence (5CYB-GL) (SEQ ID NO: 52) in which the 5'-UTR of Nat-GL is replaced with the wild-type CYBA 5'-UTR, respectively, as templates. The composition of the expression cassette is specifically shown in Fig. 1.

[0152] Specifically, in order to obtain PCR products using the sequences of the above sequence numbers 51 and 52 as templates, they were mixed under the conditions of Table 3 below, and polymerase #R051A from Takara was used.

[0153] Ingredients Capacity DNA template 10 ng 1 μl 10 μM forward primer 1 μl 10 μM reverse primer 1 μl 5x Phusion buffer 4 μl 10 mM dNTP 0.4 μl Polymerase 0.2 μl 3'DW 12.4 μl Total 20 μl

[0154] The PCR reaction conditions for the above mixed sample were performed as shown in Table 4 below.

[0155] Step Temperature (℃) Time Cycle Initial denaturation 982 min 1 Denaturation 9830 sec 25 Annealing 6030 sec Extention 721 min Final Extention 725 min 1

[0156] Next, DNA strands that served as templates for in vitro transcription from the PCR reaction products were identified through agarose gel electrophoresis, and then purified and separated from the agarose gel using Qiagen's #28706 kit. Using this as a template, mRNA was synthesized using NEB's #E2060. The composition of the reaction mixture for in vitro transcription is shown in Table 5 below.

[0157] Ingredients (μl) 2X Premix 10 T7 polymerase 2 RNase inhibitor 1 Pseudo-UTP (10 mM) 2.5 5'-Methyl-CTP (10 mM) 2.5 DNA template (10 ng / ul) 2 Total 20

[0158] After mixing under the conditions described above, the mixture was reacted at 37°C for 3 hours, and in order to remove the template DNA present in the reacted sample, DNase I (#E2060) was treated and the mixture was reacted at 37°C for 30 minutes to decompose the template DNA.

[0159] Next, poly-A tailing was performed on the mRNA synthesized using NEB's #E2060 under the following conditions.

[0160] Ingredient volume (㎕) DW 20 IVT reaction 20 10 X Poly(A) polymerase reaction buffer 5 Poly(A) polymerase 5 Total 50

[0161] To purify mRNA produced after in vitro transcription, Qiagen's #74204 kit was used, and the concentration of the final product, mRNA, was confirmed using BioTek's Synergy HTX Multi-Mode Reader after the purification.

[0162]

[0163] 1.2: Introduction of the expression cassette into cells

[0164] For the intracellular introduction (transfection) of polynucleotide sequences including the expression cassette constructed in Example 1.1, HeLa cells were subcultured in DMEM (Sigma-Aldrich) medium containing 10% non-synthesized FBS, 100 units / mL penicillin, and 100 mg / mL streptomycin at 37°C and 5% CO2. 24 hours before transfection, cells were seeded at a density of about 1 x 10 5 / ㎖ were suspended in DMEM medium containing 10% non-synthesized FBS and seeded in 6-well plates at 1 ㎖ each. For transfection 24 hours after seeding, Opti-MEM (Gibco, #31985-062) and Invitrogen's #LMRNA015 (Lipofectamine TM Messenger MAX TM Using a transfection reagent, 1 μg of each mRNA synthesized from the sequences of SEQ ID NOs. 1 to 3 in Example 1.1 was incubated according to the manufacturer's instructions and then treated on cells.

[0165] After transfection, the original cell culture medium was collected every 24 hours and stored at 4℃. After recovering the original cell culture medium, new cell culture medium was added to each well. In this way, cell culture samples were collected at 24, 48, and 72 hours after transfection. For the final sample (72 hours after transfection), the cell culture medium and cells were collected in PBS. Each sample was prepared using Promega's E1980 kit. For activity measurement, the original sample was diluted to 1 / 4 by adding PBS, and the 1X luciferase assay buffer was mixed at a ratio of 1:10, and the luciferase activity was confirmed using a spectrophotometer.

[0166]

[0167] 1.3: Confirming the target protein expression level and extracellular secretion efficiency of the expression cassette.

[0168] As a result of confirming the Gaussian luciferase expression level and extracellular secretion efficiency of the cells transfected in the above Example 1.2, it was confirmed that even when the 5'-UTR of the wild-type Gaussian luciferase expression cassette was replaced with the wild-type CYBA 5'-UTR, the expression level of the target protein Gaussian luciferase was not affected or the expression level was slightly increased (Table 7).

[0169] 24h cell culture medium 48h cell culture medium 72h cell culture medium Nat-GL5CYB-GLNat-GL5CYB-GLNat-GL5CYB-GL1234,002241,305189,768200,285106,110125,9912219,171228,991188,430200,695104,229124,5863233,8 57241,969192,889204,196108,153128,972Average229,010237,422190,362201,725106,164126,516Deviation8,5217,3082,2882,1491,9622,240Ratio11.040.830.880.460.55

[0170] Based on the above results, it can be seen that an expression cassette containing a CYBA-derived 5'-UTR sequence can express a target protein.

[0171]

[0172] Example 2: Confirmation of the effect of expression cassettes according to the 3'-untranslated region.

[0173]

[0174] 2.1: Construction of expression cassettes and introduction into cells

[0175] To evaluate the target protein expression efficiency of the expression cassette by introducing a CYBA-derived 3'-UTR sequence, the following experiments were performed.

[0176] Specifically, we aimed to construct an expression cassette containing a 5'-UTR, a 3'-UTR, and an ORF. First, a Gaussian luciferase-derived signal peptide coding sequence was used as an example of a signal peptide, and a portion (amino acids 322-524) of the surface glycoprotein sequence of SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2, NCBI Reference accession MW110903.1) was used as an example of a target protein. In addition, as untranslated regions, the wild-type CYBA 5'-UTR (5CYB), the wild-type CYBA 3'-UTR (3CYB), and the 5'-UTR or 3'-UTR sequence derived from the Romboutsia sp. CE17 chromosome (complete genome Sequence ID: CP051144.1) were used (designated 5ORI or 3ORI, respectively). An expression cassette was designed using the above sequences, and its detailed configuration is shown in Fig. 3. Meanwhile, a His-tag was included for effective detection of the target protein.

[0177] Using the above expression cassette sequences as templates, mRNA was produced in the same manner as in Examples 1.1 and 1.2 and introduced into cells.

[0178]

[0179] 2.2: Confirming the target protein expression level and extracellular secretion efficiency of the expression cassette

[0180] To confirm the expression level of the target protein, SARS-CoV-2 glycoprotein, in the cells transfected in Example 2.1 above, Western blotting was performed.

[0181] Specifically, 24 hours after transfection in Example 2.1, the cell culture medium and cells were washed with cold PBS, and then collected with RIPA buffer (50 mM Tris-HCl [pH 8.0], 1% NP-40, 150 mM NaCl, 0.1% SDS, 0.5% deoxycholate). The cell solution dissolved in RIPA buffer was centrifuged (15,000 RPM) at 4°C for 10 minutes to recover the supernatant, and 25 μl of 5X sample buffer (250 mM Tris-HCl (pH 6.8), 25% glycerol, 2% SDS, 14.4 mM beta-mercaptoethanol, 0.1% bromophenol blue) was added to 100 μl of the supernatant. The mixed sample was heated at 100°C for 5 minutes, and the supernatant was recovered by centrifugation (15,000 RPM) at 4°C for 10 minutes. 50 μl of the supernatant was subjected to SDS PAGE gel electrophoresis for Western blot analysis. Anti-His antibody (Cell Signaling Technology, #12698) and anti-GAPDH antibody (Santacruz, SC-1616) were used for Western blot analysis, and HRP conjugated anti-rabbit IgG (Santacruz, SC-2357) and HRP conjugated anti-mouse IgG (Santacruz, SC-516102) were used as secondary antibodies. The reacted proteins were purchased from Thermo Scientific. TM SuperSignal of the company TM After treatment with West Pico PLUS Chemiluminescent Substrate, #34580, the protein was visualized using a Bio-rad chimidoc imaging system. The visualized protein amount was analyzed using Quantity One® software provided by Bio-rad.

[0182] As a result, when comparing 5ORI-3CYB and 5CYB-3CYB, it was confirmed that the expression level of the target protein of 5CYB-3CYB, which has a 5'-untranslated region derived from CYBA, increased 1.6-fold compared to 5ORI-3CYB. In addition, when comparing 5ORI-3ORI and 5CYB-3ORI, it was confirmed that the expression level of the target protein of 5CYB-3ORI, which has a 5'-untranslated region derived from CYBA, increased 2.5-fold (Fig. 3).

[0183] In summary of the above results, it was confirmed that expression cassettes containing 5'-untranslated regions and / or 3'-untranslated regions derived from CYBA can effectively express a target protein, and it can be seen that the results are greatly influenced by the 5'-UTR rather than the 3'-UTR.

[0184]

[0185] Example 3: Confirmation of the effect of expression cassettes according to the mutant sequence of CYBA-derived UTR sequence.

[0186] To evaluate the target protein expression level (mRNA translation efficiency) of an expression cassette in which mutant sequences of 5'-UTR and 3'-UTR sequences derived from CYBA were introduced as novel non-translated region sequences, the following experiments were performed.

[0187]

[0188] 3.1: Evaluation of the expression level of expression cassettes with mutant sequences of CYBA-derived UTR sequences (1)

[0189] Specifically, as described in Fig. 4, one or more mutations were introduced into the CYBA-derived 5'-UTR or 3'-UTR wild-type sequence, and an expression cassette was constructed based on the mutant sequence. The specific base sequences of the CYBA-derived 5'-UTR sequence and its mutant sequences and the CYBA-derived 3'-UTR sequence and its mutant sequences are described in Tables 1 and 2, and information on expression cassettes comprising combinations of the 5'-UTR sequence and the 3'-UTR sequence is described in the table below.

[0190] No. Expression cassette name 5`-UTR3`-UTR1WT SEQ ID NO. 1 SEQ ID NO. 262KT-17 SEQ ID NO. 3 SEQ ID NO. 283KT-44 SEQ ID NO. 4 SEQ ID NO. 294SB-3 SEQ ID NO. 5 SEQ ID NO. 305SB-8 SEQ ID NO. 6 SEQ ID NO. 316CA-3 SEQ ID NO. 7 SEQ ID NO. 327CA-10 SEQ ID NO. 8 SEQ ID NO. 338CA-16 SEQ ID NO. 9 SEQ ID NO. 349LCA-2 SEQ ID NO. 10 SEQ ID NO. 3510LCA-5 SEQ ID NO. 11 SEQ ID NO. 3611LCA-7 SEQ ID NO. 12 SEQ ID NO. 3712HS-1 SEQ ID NO. 13 SEQ ID NO. 3813HS-2 SEQ ID NO. 14 SEQ ID NO. 3914HS-4 SEQ ID NO. 15 SEQ ID NO 4015HS-6 SEQ ID NO: 16 4116HS-8 SEQ ID NO: 17 4217HS-10 SEQ ID NO: 18 4318HS-13 SEQ ID NO: 19 4419HS-15 SEQ ID NO: 20 4520HS-16 SEQ ID NO: 21 4621HS-18 SEQ ID NO: 22 4722HS-20 SEQ ID NO: 23 4823HS-22 SEQ ID NO: 24 4924HS-24 SEQ ID NO: 25 50

[0191]

[0192] In addition, as examples of target proteins to be introduced into the above expression cassette, proteins derived from various viruses (e.g., coronavirus, etc.) or combinations thereof were used, and experiments were performed based on the method described in Example 1. Meanwhile, a His-tag was included for effective detection of the target protein.

[0193] First, as an example of a target protein, the target protein expression level of the expression cassette into which the sequence encoding the COVID-19 antigen protein was introduced was evaluated, and it was confirmed that among the eight candidate expression cassettes, the KT-17 and KT-44 expression cassettes showed significantly superior target protein expression levels compared to other expression cassettes including the wild type (Fig. 5).

[0194] Next, as an example of a target protein, the target protein expression level of the expression cassettes into which the sequence encoding the COVID-19 antigen protein and the PD1 protein was introduced was evaluated, and it was confirmed that among the seven candidate expression cassettes, the SB-3 and SB-8 expression cassettes showed significantly superior target protein expression levels compared to other expression cassettes including the wild type (Fig. 6).

[0195] Next, as an example of a target protein, the target protein expression level of the expression cassettes into which the sequence encoding the COVID-19 antigen protein and SIRPα protein was introduced was evaluated, and it was confirmed that among the seven candidate expression cassettes, the CA-3, CA10, and CA-16 expression cassettes showed significantly superior target protein expression levels compared to other expression cassettes including the wild type (Fig. 7).

[0196] Next, as an example of a target protein, the target protein expression level of the expression cassettes into which sequences encoding the COVID-19 antigen protein, PD1, and SIRPα proteins were introduced was evaluated, and it was confirmed that among the 10 candidate expression cassettes, the LCA-2, LCA-5, and LCA-7 expression cassettes showed significantly superior target protein expression levels compared to other expression cassettes including the wild type (Fig. 8).

[0197] Next, as an example of a target protein, the target protein expression level of the expression cassette into which the sequence encoding the Gaussian luciferase protein was introduced was evaluated, and it was confirmed that among the 28 candidate expression cassettes, HS-1, HS-2, HS-4, HS-6, HS-8, HS-10 HS-13, HS-15, HS-16, HS-18, HS-20, HS-22, and HS-24 showed a significantly superior target protein expression level compared to other expression cassettes including the wild type (Fig. 9).

[0198] Based on the above results, it can be seen that in the case of an expression cassette including a novel 5'-untranslated region and a 3'-untranslated region discovered in the present invention, the translation efficiency of mRNA is increased, thereby increasing the expression level of the target protein introduced into the expression cassette.

[0199]

[0200] 3.2: Evaluation of the expression level of expression cassettes with mutant sequences of CYBA-derived UTR sequences (2)

[0201] To further evaluate the efficiency of the expression cassette that showed a significantly superior expression level in Example 3.1 above, the following experiments were performed.

[0202] Specifically, to evaluate the effect of the CYBA-derived UTR mutant sequence discovered in the present invention on the translation efficiency of mRNA, the expression efficiency of the target protein of an expression cassette including a previously known UTR sequence and an expression cassette including the CYBA-derived UTR mutant sequence discovered in the present invention was compared. As an example of an expression cassette including the CYBA-derived UTR mutant sequence of the present invention, LCA-2 was used, and as known UTR sequences, an expression cassette including the BNT sequence and the RNA1273 sequence (BNT and RNA1273, respectively) was used, and a control (NC) and a CYBA-derived UTR wild-type sequence (CYB) were included for comparison. In addition, firefly luciferase was used as an example of a target protein.

[0203] Distinct UTR sequence (5`-3`) Sequence number BNT- 5`UTRAGG GAG AAT AAA CTT GTA TTC TTC TGG TCC CCA CAG ACT CAG AGA GAA CCC GCC ACC53RNA1273- 5`UTRAGG AAA TAA GAG AGA AAA GAA GAG TAA GAA GAA ATA TAA GAC CCC GGC GCC GCC ACC54BNT- 3`UTRCTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTGCAATAAAACG AAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGCGCGAAGCCGGGAAATATT55RNA12733-3`UTRGCTGGAGCCTCGG TGGCCTAGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCGTACCCCCGTGTCTTTTGAATAAAGTCTGAGTGGGCGGCAGCGAAGCCGGGAAATATT56

[0204]

[0205] After synthesizing mRNAs having different 5' and 3' UTR sequences under in vitro conditions, they were introduced into cells and luciferase activity was measured 24 hours later.

[0206] As a result of the above experiment, it was confirmed that the expression cassette including LCA2, a CYBA-derived UTR mutant sequence discovered in the present invention, showed significantly superior expression of the target protein, luciferase, compared to the expression cassette (CYB) including the CYBA-derived UTR wild-type sequence and other expression cassettes, and it was confirmed that it showed a high expression level compared to the expression cassette including the combination of the known UTR sequences, BNT and RNA1273 UTR sequences (Fig. 10).

[0207] Based on the above results, it can be seen that an expression cassette including the CYBA-derived UTR mutant sequence of the present invention can significantly improve the translation efficiency of mRNA, and an excellent expression cassette can be produced when the CYBA-derived UTR mutant sequence is used.

[0208]

[0209] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. An expression cassette containing a CYBA (Homo sapiens cytochrome b-245 alpha chain)-derived 5'-untranslated region (UTR) sequence.

2. An expression cassette according to claim 1, wherein the CYBA-derived 5'-untranslated region sequence comprises the sequence of SEQ ID NO:

2.

3. An expression cassette according to claim 1, wherein the CYBA-derived 5'-untranslated region sequence comprises at least one sequence selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO:

25.

4. An expression cassette according to claim 1, wherein the expression cassette further comprises a CYBA-derived 3'-untranslated region sequence.

5. In claim 4, the expression cassette comprises a CYBA-derived 3'-untranslated region sequence having the sequence of SEQ ID NO:

27.

6. In claim 4, the expression cassette comprises a CYBA-derived 3'-untranslated region sequence comprising at least one sequence selected from the group consisting of SEQ ID NO: 28 to SEQ ID NO:

50.

7. An expression cassette according to claim 1, wherein the expression cassette further comprises a sequence encoding a target protein.

8. An expression cassette according to claim 1, wherein the expression cassette further comprises at least one selected from the group consisting of a 5'-cap, a promoter, and a poly-A tail (polyadenylation tail).

9. An expression cassette according to claim 1, wherein the expression cassette is for improving the expression efficiency of a target protein.

10. An expression cassette according to claim 1, wherein the expression cassette is for producing a target protein, for a therapeutic agent, or for a vaccine.

11. An expression vector comprising an expression cassette according to any one of claims 1 to 10.

12. A transformant comprising the expression vector of claim 11.

13. A step of culturing the transformant of claim 12 to produce the target protein; and A step of recovering the produced target protein from the transformant or culture medium. A method for producing a target protein, comprising:

14. A polynucleotide molecule comprising the sequence of sequence number 2.

15. A polynucleotide molecule according to claim 14, wherein the polynucleotide molecule comprises one or more sequences selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO: 25.

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