Viral RNA element for increasing gene expression and screening of sequence variants thereof

Abstract

The present invention relates to a viral RNA element for increasing gene expression and screening of sequence variants thereof. A regulatory element according to one embodiment can increase RNA stability or mRNA translation of a transcript of a target gene, thereby increasing the expression level of a target protein, and can be usefully employed in systems requiring precise control of gene expression, such as gene therapy, vaccine development, production of protein therapeutics, and the like.

Description

[0001] specification

[0002] Title of Invention: Viral RNA Element Increasing Gene Expression and Screening of Sequence Variants thereof

[0003] Technology field

[0004] The present invention relates to a viral RNA element that increases gene expression and screening of sequence variations thereof.

[0005] Background alcohol

[0006] Research on vertebrate viruses is crucial due to the potential for cross-species transmission that could cause novel infectious diseases in humans. These viruses can infect multiple host species and possess unique RNA regulatory elements that enable replication and survival in different biological environments (Non-patent Literature 0001 (Roche et al., 2018)). These regulatory elements are key factors that enable viruses to adapt to and survive in new hosts, which constitutes an essential process in the emergence of zoonotic diseases. By understanding these viral regulatory elements, it is possible to predict viruses that may pose a threat to the human body and develop strategies to control or prevent outbreaks (Non-patent Literature 0002 (Menachery et al., 2015)), which is an important approach for strengthening global health security in responding to novel infectious diseases.

[0007] Despite this importance, major findings to date have been made through deep, low-throughput analysis of pathogenic viruses, which represents only a tiny fraction of the entire virome. Given the vast diversity of viral sequences revealed by recent metagenomics research, an efficient strategy for functionally characterizing them is essential (Non-patent Literature 0003 (Simmonds et al., 2017)). To address this issue, screening of viral genomes infecting humans was performed using the massively paral lei reporter assay (MPRA) method, and elements regulating mRNA were identified (Non-patent Literature 0004 (Seo et al., 2023)).

[0008] The size of regulatory elements is a critical factor in the design of mRNA-based therapeutics. If the element length is excessively long, the overall size of the mRNA construct increases, which can reduce loading efficiency within carriers such as lipid nanoparticles (LNPs) and lead to complexity in the manufacturing process. Particularly when delivering mRNA using viral vectors such as adeno-associated viruses (AAVs), miniaturization of regulatory elements is an essential technical challenge due to the limited cargo capacity of the vector. Therefore, there is a need to discover regulatory elements that are the smallest functional units capable of maximizing vector insertion efficiency by minimizing the length of the insertion sequence during mRNA design.

[0009] Accordingly, the inventors of the present invention systematically identified and characterized regulatory elements present in various vertebrate viruses by screening a novel large-scale virus library using the massively parel lei reporter assay (MPRA) technique. Furthermore, they sought to minimize the size of regulatory elements by utilizing the high nucleotide conservation within the viral genera. These regulatory elements can serve as a foundation for future biotechnological applications, such as nucleic acid-based therapeutics. Prior Art Literature

[0010] Non-patent literature

[0011] (Non-Patent Document 0001) Roche, B., Brout in, H., & Simard, F. (2018). Ecology and Evolution of Infectious Diseases: Pathogen Control and Pub 1 ic Health Management in Low-Income Countries. Oxford University Press.

[0012] (비 특허 문헌 0002) Menachery , VD , Yount , BL , Debbink, K. , Agnihothram, S. , Gral inski , LE , Plante, J . A. , Graham, R.L. , Scobey, T. , Ge, A SARS-1 ike cluster of circulating bat coronaviruses shows potential for human emergence. Nature Medicine, 21(12) . ht tps: / / do i . org / 10.1038 / nm .3985

[0013] (비 특허 문헌 0003) Simmonds, P., Adams, M. J., Benko, M., Breitbart, M., Brister, J. R., Carstens, E. B., Davison, A. J., Delwart, E., Gorbal enya, A. E., Harrach, B., Hull, R., King, A. M. Q., Koonin, E. V., Krupovic, M., Kuhn, J. H., Lefkowitz, E. J., Nibert, M. L., Orton, R., Roossinck, M. J., ... Zerbini, F. M. (2017). Consensus statement: Virus t axonomy in the age of metagenomics. Nature Reviews. Microbiology, 15(3) , 161-168.

[0014] (Non-patent literature 0004) Seo, JJ, Jung, S.— J., Yang, J., Choi, D.— E., & Kim, VN (2023). Functional viromic screens uncover regulatory RNA elements. Cel l, 186(15), 3291-3306. e21.

[0015] Detailed description of the invention

[0016] Technical challenges

[0017] One objective of the present invention is to provide a control element comprising the following structure:

[0018] (i) 1st loop;

[0019] (ii) 1st stem connected to the 1st loop ;

[0020] (iii) a second loop connected to the first stem; and

[0021] (iv) The second stem connected to the second loop, where, the first loop is GTHRN 3 Includes the R sequence,

[0022] The first stem is GN X N 2 Sequence and N that binds complementarily to it 4 N 5 It includes a C sequence, and the second loop includes a TMM sequence,

[0023] The second stem includes CC sequences and GG sequences, and

[0024] The above view is A, T, or Benevolence, and

[0025] The above R is G or A, and

[0026] The above 'Nieun' is A, T, G, or In, and

[0027] The above M is C or A, and

[0028] N 1 and N 5 and N2 and N 4 Each forms a base pair.

[0029] Another object of the present invention is to provide a regulatory element comprising a sequence of nucleotides of sequence number 1 or 2, or a sequence of nucleotides having at least 80% identity with it. Yet another object of the present invention is to provide a regulatory element for RNA stability or mRNA translation enhancement.

[0030] Another objective of the present invention is to provide regulatory elements derived from the viral genome or fragments thereof.

[0031] Another objective of the present invention is to provide a construct, vector, or recombinant host cell comprising a target gene; and said regulatory element.

[0032] Another objective of the present invention is to provide an AAV (Adeno-Associated Virus) vector containing the above-mentioned control element.

[0033] Another objective of the present invention is to provide a composition comprising the above construct, vector, or recombinant host cell.

[0034] Another objective of the present invention is to provide a method for manufacturing the above-mentioned construct, vector, recombinant host cell, or composition.

[0035] Another objective of the present invention is to provide a method for increasing RNA stability and / or mRNA translation of a target gene, comprising the step of inserting or linking the regulatory element to a UTR of the target gene.

[0036] Another objective of the present invention is to provide a use for increasing RNA stability and / or mRNA translation of the above construct, vector, recombinant host cell, or composition.

[0037] Another objective of the present invention is to provide a use for the production of the above-mentioned construct, vector, recombinant host cell, or mRNA construct of the composition or a target protein.

[0038] Another objective of the present invention is to provide a method for preventing, improving, or treating a disease comprising the step of administering the above-mentioned construct, vector, recombinant host cell, or composition to an individual in need thereof.

[0039] Another objective of the present invention is to provide a use for the prevention or treatment of disease of the above-mentioned construct, vector, recombinant host cell, or composition. Technical solution

[0040] Each description and embodiment disclosed herein may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of this application. Furthermore, the scope of this application is not to be limited by the specific descriptions provided below. Additionally, a person skilled in the art can recognize or identify numerous equivalents to the specific embodiments of this application described herein using only ordinary experiment. Moreover, such equivalents are intended to be included herein.

[0041] The present invention provides a regulatory element capable of increasing RNA stability and / or mRNA translation. Since the regulatory element can increase RNA stability and / or mRNA translation, it may be suitable for increasing protein production in a construct containing a target gene.

[0042] The regulatory elements of the present invention may be derived from a viral genome or a fragment thereof.

[0043] One aspect of the present invention provides a control element comprising the following structure:

[0044] (i) 1st loop;

[0045] (ii) 1st stem connected to the 1st loop ;

[0046] (iii) a second loop connected to the first stem; and

[0047] (iv) The second stem connected to the second loop,

[0048] Here, the first loop is GTHRN 3 Includes the R sequence,

[0049] The first stem is GN X N 2 Sequence and N that binds complementarily to it 4 N 5 It includes a C sequence, and the second loop includes a TMM sequence,

[0050] The second stem includes CC sequences and GG sequences, and

[0051] The above view is A, T, or Benevolence, and

[0052] The above R is G or A, and

[0053] The above 'Nieun' is A, T, G, or In, and

[0054] The above M is C or A, and

[0055] N 1 and N 5 and N 2 and N 4 Each forms a base pair.

[0056] In one specific example, the control element includes a third loop connected to the second stem, and the third loop may include a G sequence on one side of the loop and an AA sequence on the other side of the loop.

[0057] In one specific example, the control element comprises a third stem connected to the third loop, and the third stem may comprise 1 to 5 base pairs.

[0058] In one specific example, the control element may include the structure of Formula I or Formula II below:

[0059] [Equation I]

[0060] 5'-[Stage 2 A]-[Loop 2 A]-[Stage 1 A]-[Loop 1]-[Stage 1 B]-[Loop 2 B]-[Stage 2 B]- 3',

[0061] [Equation II]

[0062] 5'-[3rd Loop A]-[2nd Stem A]-[2nd Loop A]-[1st Stem A]-[1st Loop]-[1st Stem B]-[2nd Loop B]-[2nd Stem B]-[3rd Loop B]- 3',

[0063] In the above Equation I or Equation II,

[0064] The first loop is GTHRN 3 Includes the R sequence,

[0065] First Stem A and First Stem B combine complementarily to form the first stem, and First Stem A is GN X N 2 It includes a sequence, and the first stem B is N 4 N 5 It includes the C sequence, and the second loop A includes the TMM, CKTMM, or CCKTMM sequence, and the second loop B includes the A sequence or does not include nucleotides,

[0066] Second Stem A and Second Stem B combine complementarily to form a second stem, Second Stem A includes a CC, ACC, or CCT sequence, and Second Stem B includes a GG, GGT, or AGG sequence, and

[0067] In the above Equation II,

[0068]

[0069] The third loop A contains a G, GA, GAT, or GATC sequence, and the third loop B contains an AA, or TAA sequence.

[0070] In one specific example, the first loop may be composed of 1 to 10, 2 to 9, 3 to 8, or 4 to 7 nucleotides, and preferably may be composed of 6 nucleotides.

[0071] In one embodiment, the first stem may be composed of 1 to 5 or 2 to 4 base pairs, preferably 3 base pairs. The first stem A and / or B may each be composed of 1 to 5 or 2 to 4 nucleotides, preferably 3 nucleotides.

[0072] In one embodiment, the second loop may be composed of 1 to 10, 2 to 9, 3 to 8, 3 to 7, or 3 to 6 nucleotides. The second loop A may be composed of 1 to 10, 2 to 9, 3 to 8, 3 to 7, or 3 to 6 nucleotides. The second loop may be composed of 5, 4, 3, 2, or 1 nucleotide, or may not contain any nucleotides. In one embodiment, the second stem may be composed of 1 to 5, 2 to 4, or 2 to 3 base pairs. The above second stem A and / or may each consist of 1 to 5, 2 to 4, or 2 to 3 nucleotides.

[0073] In one specific example, the third loop may be composed of 1 to 10, 2 to 9, or 3 to 7 nucleotides. The third loop A may be composed of 1 to 10, 1 to 9, 1 to 8, 1 to 7, or 1 to 6 nucleotides. The third loop may be composed of 1 to 5, 1 to 4, or 1 to 3 nucleotides.

[0074] The regulatory element of the present invention may comprise or be composed of a fragment consisting of a sequence of nucleotides of 1 to 200 nucleotides or less derived from the viral genome. Specifically, the regulatory elements are fragments of the viral genome, comprising 200, 199, 198, 197, 196, 195, 194, 193, 192, 191, 190, 189, 188, 187, 186, 185, 184, 183, 182, 181, 180, 179, 178, 177, 176, 175, 174, 173, 172, 1, 170, 169, 168, 167, 166, 165, 164, 163, 162, 161, 160, 159, 158, 157, 156, 155, 154, 153, 152, 151, 150, 149, 148, 147, 146, 145, 144, 143, 142, 141, 140, 139, 138, 137, 136, 135, 134, 133, 132, 131, 130, 129, 128, 127, 126, 125, 124, 123, 122, 121, 120, 119, 118, 117, 116, 115, 114, 113, 112, 111, 110, 109, 108, 107, 106, 105, 104, 103, 102, 101, 100, 99, 98 97, 96, 95, 94, 93, 92 / fl, 9171 90 / fl, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74,73, 72, spirit, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, It may contain or consist of a fragment consisting of a sequence of 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 nucleotides.

[0075] The control element of the present invention may comprise or be composed of the nucleotide sequence of CCCKTMMGN^^THRN^N^^AGG (Sequence No. 1); or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the same.

[0076] The control element of the present invention may comprise or be composed of the nucleotide sequence of CCCKTMMGN^^THRN^N^^AGGTAA (SEQ No. 2); or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the same.

[0077] The single letter of a base used herein refers to the following bases in accordance with standard abbreviation rules in the field of biochemistry:

[0078] A: Adenine, G: Guanine, C: Cytosine, T: Thymine, U: Uracil.

[0079] The above-mentioned G, T, or U; or a nucleotide analog thereof; the above-mentioned M is C or A; or a nucleotide analog thereof; the above-mentioned H is A, T, U, or C; or a nucleotide analog thereof; the above-mentioned R is G or A; or a nucleotide analog thereof; Y is T, U, or C; the above-mentioned N 1 , N 2 , N 3 , N 4 and N 5 are each a nucleotide selected from A, U, T, G, and C or a nucleotide analog thereof, said R may be a purine or an analog thereof, and said N 1 and N 5 And N 2 and N 4 It can be a base that can form base pairings with each other.

[0080] In one specific example, the regulatory element is (i) a fragment of a picornavirus 1 (Eel picornavirus 1) gene (NCBI Reference Sequence: NC_022332.1), wherein the fragment comprises a nucleotide sequence of sequence number 1 or 2; or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with the same;

[0081] (ii) a fragment of the Perch picornavirus M9 / 2015 / HUN gene (NCBI Reference Sequence: MW590713.1), wherein the fragment comprises a nucleotide sequence of sequence number 1 or 2; or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity therewith;

[0082] (iii) a fragment of the gene of Wenling lepidotr igla picornavirus strain YXMC111438 (NCBI Reference Sequence: MG600079.1), wherein the fragment comprises the nucleotide sequence of Sequence No. 1 or 2; or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the same;

[0083] (iv) a fragment of the Wuhan carp picornavirus strain DSYC18088 gene (NCBI Reference Sequence: MG600066.1), wherein the fragment comprises the nucleotide sequence of sequence number 1 or 2; or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity therewith;

[0084] (v) A fragment of the gene of Yancheng osbecks grenadier anchovy picornavirus strain XFXMC15460 (NCBI Reference Sequence: MG600099.1), wherein said fragment comprises the nucleotide sequence of SEQ ID NO. 1 or 2; or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity therewith; or

[0085] (vi) A fragment of the Potamipivirus A isolate TSPV gene (NCBI Reference Sequence: NC_075994.1), wherein the fragment comprises a sequence of nucleotides of sequence number 1 or 2; or may comprise a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the same.

[0086] The control element of the present invention may comprise or be composed of a sequence of nucleotides of sequence no. 3; or a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the same.

[0087] In one specific example, the regulatory element is a fragment of the Eel picornavirus 1 gene (NCBI Reference Sequence: NC_022332.1), wherein the fragment comprises a sequence of nucleotides of sequence number 3; or may comprise or consist of a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with the same.

[0088] The control element of the present invention may comprise or be composed of the base sequence of sequence number 4; or at least a base sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the same.

[0089] In one specific example, the regulatory element is a fragment of the Eel picornavirus 1 gene (NCBI Reference Sequence: NC_022332.1), wherein the fragment comprises a sequence of nucleotides of sequence number 4; or may comprise or consist of a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with the same.

[0090] The control element of the present invention may comprise or be composed of a base sequence of sequence number 5; or a base sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the same.

[0091] In one specific example, the regulatory element is a fragment of the Eel picornavirus 1 gene (NCBI Reference Sequence: NC_022332.1), wherein the fragment comprises a sequence of nucleotides of sequence number 5; or may comprise or consist of a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with the same.

[0092] The control element of the present invention may comprise or be composed of a sequence of nucleotides of sequence no. 6; or a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the same.

[0093] In one specific example, the regulatory element is a fragment of the Eel picornavirus 1 gene (NCBI Reference Sequence: NC_022332.1), wherein the fragment comprises a sequence of nucleotides of sequence number 6; or may comprise or consist of a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with the same.

[0094] The control element of the present invention may comprise or be composed of a base sequence of sequence number 7; or a base sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the same.

[0095] In one specific example, the control element is a fragment of the Perch picornavirus M9 / 2015 / HUN gene (NCBI Reference Sequence: MW590713.1), wherein the fragment comprises a sequence of nucleotides of sequence number 7; or may comprise or consist of a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with the above.

[0096] The control element of the present invention may comprise or be composed of a sequence of nucleotides of sequence no. 8; or a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the same.

[0097] In one specific example, the control element is a fragment of the Perch picornavirus M9 / 2015 / HUN gene (NCBI Reference Sequence: MW590713.1), wherein the fragment comprises a sequence of nucleotides of sequence number 8; or may comprise or consist of a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with the above.

[0098] The regulatory element of the present invention may comprise or be composed of the nucleotide sequence of sequence no. 9; or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity therewith. In one embodiment, the regulatory element is a fragment of the Perch picornavirus M9 / 2015 / HUN gene (NCBI Reference Sequence: MW590713.1), wherein the fragment comprises the nucleotide sequence of sequence no. 9; Or it may include or be composed of a sequence of bases having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with this.

[0099] The control element of the present invention may comprise or be composed of a sequence of nucleotides of sequence no. 10; or a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the same.

[0100] In one specific example, the regulatory element is a fragment of the gene (NCBI Reference Sequence: MG600079.1) of the Wen 1 ing lepidotr igla picornavirus strain YXMC111438; said fragment comprises a sequence of nucleotides of sequence number 10; or may comprise or consist of a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with the same. The control element of the present invention may comprise or be composed of a sequence of nucleotides of sequence no. 11; or a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the same.

[0101] In one specific example, the regulatory element is a fragment of the gene (NCBI Reference Sequence: MG600079.1) of the Wen 1 ing lepidotr igla picornavirus strain YXMC111438; said fragment comprises the sequence of nucleotides of sequence number 11; or may comprise or consist of a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with the same. The control element of the present invention may comprise or be composed of a sequence of nucleotides of sequence no. 12; or a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the same.

[0102] In one specific example, the regulatory element is a fragment of the gene (NCBI Reference Sequence: MG600079.1) of the Wen 1 lepidotr igla picornavirus strain YXMC111438; said fragment comprises the nucleotide sequence of Sequence No. 12; Or it may comprise or be composed of a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity. The control element of the present invention is the sequence of nucleotides of SEQ ID NO. 13; Or it may include or be composed of a sequence of bases having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with this.

[0103] In one specific example, the control element is a fragment of the Wuhan carp picornavirus strain DSYC18088 gene (NCBI Reference Sequence: MG600066.1), wherein the fragment comprises a sequence of nucleotides of sequence number 13; or may comprise or consist of a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the above.

[0104] The control element of the present invention may comprise or be composed of a sequence of nucleotides of sequence no. 14; or a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the same.

[0105] In one specific example, the control element is a fragment of the Wuhan carp picornavirus strain DSYC18088 gene (NCBI Reference Sequence: MG600066.1), wherein the fragment comprises a sequence of nucleotides of sequence number 14; or may comprise or consist of a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with the above.

[0106] The control element of the present invention may comprise or be composed of a sequence of nucleotides of sequence no. 15; or a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the same.

[0107] In one specific example, the control element is a fragment of the Wuhan carp picornavirus strain DSYC18088 gene (NCBI Reference Sequence: MG600066.1), wherein the fragment comprises a sequence of nucleotides of sequence number 15; or may comprise or consist of a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with the above.

[0108] The control element of the present invention may comprise or be composed of a sequence of nucleotides of sequence number 16; or a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the same.

[0109] In one specific example, the control element is a fragment of the Yancheng osbecks grenadier anchovy picornavirus strain XFXMC15460 gene (NCBI Reference Sequence: MG600099.1), wherein the fragment comprises the nucleotide sequence of sequence number 16; or may comprise or consist of a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the above.

[0110] The control element of the present invention may comprise or be composed of a sequence of nucleotides of sequence no. 17; or a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the same.

[0111] In one specific example, the regulatory element is a fragment of the Yancheng osbecks grenadier anchovy picornavirus strain XFXMC15460 gene (NCBI Reference Sequence: MG600099.1), wherein the fragment comprises the nucleotide sequence of sequence number 17; or may comprise or consist of a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the above.

[0112] The control element of the present invention may comprise or be composed of a sequence of nucleotides of sequence no. 18; or a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the same.

[0113] In one specific example, the control element is a fragment of the Yancheng osbecks grenadier anchovy picornavirus strain XFXMC15460 gene (NCBI Reference Sequence: MG600099.1), said fragment comprising the nucleotide sequence of sequence number 18; or may comprise or be composed of a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the above.

[0114] The control element of the present invention may comprise or be composed of a sequence of nucleotides of sequence number 19; or a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the same.

[0115] In one specific example, the regulatory element is a fragment of the Potamipivirus A isolate TSPV gene (NCBI Reference Sequence: NC_075994.1), wherein the fragment comprises the sequence of nucleotides of sequence number 19; or may comprise a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with the above, or may be composed of the above.

[0116] The control element of the present invention may comprise or be composed of a sequence of nucleotides of sequence no. 20; or a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the same.

[0117] In one specific example, the regulatory element is a fragment of the Potamipivirus A isolate TSPV gene (NCBI Reference Sequence: NC_075994.1), wherein the fragment comprises the sequence of nucleotides of sequence number 20; or may comprise a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with the above, or may be composed of the above.

[0118] The control element of the present invention may comprise or be composed of a sequence of nucleotides of sequence no. 21; or a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the same.

[0119] In one specific example, the regulatory element is a fragment of the Potamipivirus A isolate TSPV gene (NCBI Reference Sequence: NC_075994.1), wherein the fragment comprises the nucleotide sequence of Sequence No. 21; or may comprise or be composed of a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the above.

[0120] The control element of the present invention may comprise or be composed of a sequence of nucleotides of sequence no. 22; or a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the same.

[0121] In one specific example, the regulatory element is a fragment of the Potamipivirus A isolate TSPV gene (NCBI Reference Sequence: NC_075994.1), wherein the fragment comprises the nucleotide sequence of Sequence No. 22; or may comprise or be composed of a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the above.

[0122] The control element of the present invention may comprise or be composed of a sequence of nucleotides of sequence no. 23; or a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the same.

[0123] In one specific example, the regulatory element is a fragment of the Eelpicornavirus 1 gene (NCBI Reference Sequence: NC_022332.1), said fragment comprising the sequence of nucleotides of sequence number 23; or may comprise or be composed of a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the same.

[0124] The control element of the present invention may comprise or be composed of a sequence of nucleotides of sequence no. 24; or a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the same.

[0125] In one specific example, the control element is a fragment of the Yancheng osbecks grenadier anchovy picornavirus strain XFXMC15460 gene (NCBI Reference Sequence: MG600099.1), said fragment comprising the sequence of nucleotides of sequence number 24; or may comprise or be composed of a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the above.

[0126] The control element of the present invention may comprise or be composed of a sequence of nucleotides of sequence no. 25; or a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the same.

[0127] In one specific example, the control element is a fragment of the Yancheng osbecks grenadier anchovy picornavirus strain XFXMC15460 gene (NCBI Reference Sequence: MG600099.1), said fragment comprising the nucleotide sequence of sequence number 25; or may comprise or be composed of a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the above.

[0128] The control element of the present invention may comprise or be composed of a sequence of nucleotides of sequence number 26; or a sequence of nucleotides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the same.

[0129] In one specific example, the control element is a fragment of the Yancheng osbecks grenadier anchovy picornavirus strain XFXMC15460 gene (NCBI Reference Sequence: MG600099.1), said fragment comprising the nucleotide sequence of sequence number 26; or may comprise or consist of a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity with the above.

[0130] The control element of the present invention may include one or more base sequences of any one of sequence numbers 3 to 26.

[0131] In one specific example, the control element may induce an increase in the length of the poly (A)tail, an increase in the stability of the poly (A)tail, or both.

[0132] In one specific example, the regulatory element may be a regulatory element for RNA stability or mRNA translation increase.

[0133] In one specific example, the regulatory element may increase the stability or translation efficiency of RNA or mRNA containing one or more modified nucleotides or nucleotide analogs. The modification may occur at the base, sugar, or binding site between nucleosides of the nucleotide, but is not limited thereto. Specifically, the modified nucleoside may include structures such as ☐ (pseudouridine), ml☐ (N1-methylpseudouridine), m5C (5-methylcytidine), or mo5U (5-methoxyuridine), but is not limited thereto and may include various other chemically modified nucleosides. The modification of the sugar and backbone may be phosphorothioate, LNA (locked nucleotide), or PMO (phosphodiamide morphol ionol igomer), but is not limited thereto and may include various other chemical modifications.

[0134] The regulatory element of the present invention can increase the stability of the error or induce an improvement in the translation efficiency of the mRNA even when applied to RNA or mRNA into which the modified nucleotide as described above has been introduced.

[0135] Even if the above is described as "a regulatory element comprising a sequence of nucleotides of a specific sequence number" or "a regulatory element consisting of a sequence of nucleotides of a specific sequence number," it is obvious that a regulatory element having a sequence of nucleotides with some sequences modified may also be used in the above, provided that it has the same or equivalent function as the regulatory element consisting of the sequence of nucleotides of the said sequence number. The above "modification" means, but is not limited to, substitution, deletion, and / or insertion of nucleotides.

[0136] For example, it is obvious that a control element having the same or equivalent function as the control element above, in which a meaningless sequence is added to the inside or end of the control element sequence of the corresponding sequence number, or in which a part of the sequence inside or end of the control element sequence of the corresponding sequence number is deleted, also falls within the scope of the present invention.

[0137] In one specific example, the control element of the present invention may include a sequence of bases in which one or more bases are modified in the sequence of bases of each sequence number. Specifically, the control elements of the present invention are 50 or fewer, 49 or fewer, 48 or fewer, 47 or fewer, 46 or fewer, 45 or fewer, 44 or fewer, 43 or fewer, 42 or fewer, 41 or fewer, 40 or fewer, 39 or fewer, 38 or fewer, 37 or fewer, 36 or fewer, 35 or fewer, 34 or fewer, 33 or fewer, 32 or fewer, 31 or fewer, 30 or fewer, 29 or fewer, 28 or fewer, 27 or fewer, 26 or fewer, 25 or fewer, 24 or fewer, 23 or fewer, 22 or fewer, 21 or fewer, 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, in the nucleotide sequence of each sequence number. It may include a sequence of bases modified with 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer bases.

[0138] In one specific example, the control element may comprise a sequence of bases in which a base is modified at any one or more positions selected from the group consisting of 71 to 78, 80, 83 to 85, 87, 88, 90, 92, 93, 96 to 98, 101, 102, 106, 108, 109, 112, 116 to 119, 129, 130, 135, 142, 144 to 146, 148, and 150 in the sequence of bases of sequence no. 3.

[0139] In one specific example, the substitution may include a substitution of A, G, C, U, or T (substitution with different bases) of at least one of the bases corresponding to positions 71 to 78, 80, 83 to 85, 87, 88, 90, 92, 93, 96 to 98, 101, 102, 106, 108, 109, 112, 116 to 119, 129, 130, 135, 142, 144 to 146, 148, and 150 in the sequence of bases of sequence number 3. In this specification, the term “corresponding” is used to determine the position / identity of a base within a given base sequence. The bases within the base sequence are designated using a canonic numbering system based on a reference base sequence.

[0140] For example, the base sequence of sequence number 4 is identical to the base sequence from the 103rd to the 124th position of the base sequence of sequence number 3, but the base "corresponding" to the 103rd position in the base sequence of sequence number 3 may be the base at the 1st position in sequence number 4. Therefore, in any base sequence, the base "corresponding" to the 103rd position in sequence number 3 may not necessarily be the 103rd position, and a person skilled in the art can easily understand the "corresponding" bases by comparing the two base sequences.

[0141] Homology and identity refer to the degree of relationship between two given base sequences and can be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0142] The calculation of homology / identity (%) between two base sequences can be performed by arranging the two sequences for optimal comparison. Preferably, the length of the sequence arranged for comparison purposes may be at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or substantially 100% of the length of the reference sequence. Then, the bases at corresponding sites are compared with each other. If a base located at a site in the first sequence is identical to a base at a corresponding site in the second sequence, the two sequences have identity at that site. The identity (%) of the two sequences is a function of the number of sites with common bases in the two sequences, taking into account the number and length of the gaps that must be introduced for optimal alignment between the two sequences. Comparison between two sequences and determination of identity (%) can be performed through mathematical algorithms.

[0143] Whether any two sequences have homology, similarity, or identity can be determined using known computer algorithms, such as the "FASTA" program, with default parameters, for example, as in Pearson et al. (1988) [Proc. Nat l. Acad. Sci. USA 85]: 2444. Alternatively, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.), as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) (Needleman and Wunsch, 1970, J. Mol. Biol.

[0144] 48: 443-453) can be used to determine this (including GCG program packages (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.] Academic Press, San Diego, 1994, and [CARILLO ETA / .] (1988) SIAM J Applied Math 48: 1073). For example, homology, similarity, or identity of sequences can be determined using BLAST from the National Center for Biotechnology Information Database, or ClustalW.

[0145] Another aspect of the present invention provides a construct comprising a target gene and the regulatory element. The regulatory element is as described above.

[0146] As used in this application, the term "construct" may be understood as DNA or RNA that does not occur naturally. That is, the construct may be understood as an artificial nucleic acid molecule or a non-natural nucleic acid molecule, and may be planned or generated by genetic engineering methods or chemical synthesis methods. The construct may include at least one of the aforementioned regulatory elements and at least one open reading frame. The construct may be a DNA molecule, an RNA molecule, or a hybrid molecule comprising DNA and RNA portions.

[0147] In one specific example, the construct may be a construct comprising a UTR of a target gene and a regulatory element. Within the construct, the regulatory element may be inserted into the UTR of the target gene or linked to the UTR of the target gene in the 5' or 3' direction, and the manner of insertion or linkage is not limited. Specifically, the insertion may be positioning the regulatory element in the 5' UTR or 3' UTR region of the target gene, but is not limited thereto. The linkage may include the regulatory element being directly linked to the UTR of the target gene or being linked by including an additional sequence of nucleotides between the regulatory element and the UTR, but is not limited thereto. The target gene and the regulatory element within the construct may be of heterologous origin. The target gene may be derived from a gene different from the regulatory element and may not naturally combine.

[0148] In one specific example, the construct may additionally include one or more barcode sequences, forward adapter sequences, reverse adapter sequences, poly(A)tail sequences, or combinations thereof, but is not limited thereto.

[0149] In one specific example, the construct may additionally include a promoter sequence, wherein the target gene may be operablely linked to the promoter sequence, but is not limited thereto. The term "operably linked" above means that the gene sequence is functionally linked to the promoter sequence that initiates and mediates the transcription of the target gene.

[0150] In one specific example, the construct may include, but is not limited to, 5' repeat sequences and 3' repeat sequences of a virus selected from the group consisting of adeno-associated viruses, adenoviruses, alphaviruses, retroviruses (e.g., gamma retroviruses, and lentiviruses), parvoviruses, herpesviruses, and SV40.

[0151] In one specific example, the construct may be an mRNA construct. The mRNA construct may additionally include, but is not limited to, a sequence of a 5' UTR, a 3' UTR, a poly(A)tail, or a combination thereof.

[0152] In the present application, the target gene may be a gene of a reporter, protein, physiologically active peptide, antigen, or antibody or a fragment thereof; or one or more selected from the group consisting of antisense oligonucleotides, mRNA, dsRNA, shRNA, miRNA, siRNA, gRNA, saRNA, IncRNA, t aRNA, ribozymes, ncRNA, exosoma 1RNA, and aptamers, but the type is not limited as long as RNA stability and / or translation of mRNA can be increased by the regulatory element of the present application.

[0153] In one specific example, the reporter may be, but is not limited to, luciferase, fluorescent protein, beta-galactosidase, chloramphenicol acetyltransferase, or equorin.

[0154] In one specific example, the physiologically active polypeptide may be a hormone, cytokine, cytokine binding protein, enzyme, growth factor, or insulin, but is not limited thereto.

[0155] In one specific example, the antigen may be a vaccine antigen, a cancer-related antigen, or an allergy antigen, but is not limited thereto.

[0156] Another aspect provides a vector containing the above construct or a pool of the above vector.

[0157] As used in this application, the term “vector” means a gene product containing a base sequence encoding a target protein or a target gene operably linked to a suitable regulatory sequence to enable the expression of the target protein within a suitable host. The regulatory sequence may include, but is not limited to, a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence coding for a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. After being introduced into a suitable host cell, the vector may be replicated or function independently of the host genome, or may be incorporated into the genome itself.

[0158] The vector used in this application is not particularly limited as long as it is expressible within a host cell, and any vector known in the art can be introduced into a host cell. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in their natural or recombinant state.

[0159] In one specific example, the vector may be an AAV (Adeno-Assisted Virus) vector.

[0160] AAV is divided into several serotypes based on differences in capsid proteins, including AAV1, AAV2, AAV5, AAV8, AAV9, and AAV-DJ, but is not limited to these.

[0161] Structural differences in capsid proteins among AAV serotypes determine binding affinity with specific cell receptors, which can ultimately have a decisive impact on tissue-specific transduction efficiency. Therefore, by selecting the optimal serotype, off-target effects can be minimized while maximizing the efficiency of gene delivery to target tissues. As used in this application, the term "host cell" includes all cells capable of expressing a target protein and encompasses cells that have undergone natural or artificial genetic modification. Furthermore, the host cells include eukaryotic and prokaryotic cells, and specifically may be eukaryotic cells or cells derived from mammals (e.g., humans), but are not limited thereto.

[0162] In this application, the method of introducing a construct or vector into a cell includes any method of introducing nucleic acid into a cell (e.g., transfection or transformation), and depending on the cell, a suitable standard technique as known in the art may be selected and performed. Examples include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, lipid nanoparticle method, and lithium acetate-DMSO method.

[0163] Another aspect provides a composition comprising the above-described construct, vector, or recombinant host cell. The construct, vector, recombinant host cell, or composition comprising these of the present application may express a target protein in vitro, in vivo, or ex vivo. In one embodiment, when the composition is administered to an individual, the target protein may be provided to the individual by the construct, vector, or recombinant host cell, and thus may exhibit a preventive or therapeutic effect against a disease (e.g., an infectious disease) depending on the use of the provided target protein. Accordingly, the composition may be a pharmaceutical composition, but is not limited thereto.

[0164] In one specific example, the construct or target protein of the present application may be produced in vitro, in vivo, or ex vivo using the construct, vector, or recombinant host cell. Accordingly, the composition may be a composition for producing the construct or target protein of the present application, but is not limited thereto.

[0165] For example, if the target protein is a vaccine antigen, the construct, vector, recombinant host cell, or composition itself may be used as a vaccine, or a vaccine antigen may be manufactured using these.

[0166] Another aspect provides a method for preventing, improving, or treating a disease comprising the step of administering the above construct, vector, recombinant host cell, or composition to an individual in need thereof.

[0167] Another aspect provides the use of the above construct, vector, recombinant host cell, or composition for the prevention or treatment of disease.

[0168] Another aspect provides a method for producing a target protein comprising the steps of: culturing the recombinant host cell; and recovering the target protein.

[0169] The method for producing a target protein using the recombinant host cell in the present application can be carried out using methods widely known in the art. Specifically, the above culture may be cultured continuously in a batch process, a fed batch, or a repeated fed batch process, but is not limited thereto. The culture medium used for the culture can be appropriately selected by a person skilled in the art depending on the host cell. Specifically, the recombinant host cell of the present application may be cultured in a conventional medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid, and / or vitamin, etc., under aerobic or anaerobic conditions while controlling the temperature, pH, etc.

[0170] The method for preparing the target protein described above may further include additional processes after the culture step. The additional processes may be appropriately selected depending on the intended use of the target protein.

[0171] Specifically, the method for producing the target protein may include a step of recovering the target protein from one or more substances selected from the recombinant host cell, the dried product of the recombinant host cell, the extract of the recombinant host cell, the culture of the recombinant host cell, the supernatant of the culture, and the lysate of the recombinant host cell after the culture step. The method may additionally include a step of lysing the recombinant host cell prior to or simultaneously with the recovery step. The lysing of the recombinant host cell may be carried out by methods commonly used in the art to which this application belongs, for example, by a lysis buffer solution, a sonicator, heat treatment, and a flux presser. Furthermore, the lysis step may include, but is not limited to, enzymatic reactions such as cell wall / membrane degrading enzymes, nucleases, nucleotransferases, and / or proteolytic enzymes.

[0172] In the present application, the dried product of the recombinant host cell may be prepared by drying the cell that has accumulated the target substance, but is not limited thereto.

[0173] In the present application, the extract of a recombinant host cell may refer to the material remaining after separating the cell wall / cell membrane from the cell. Specifically, it may refer to the remaining components excluding the cell wall / cell membrane from the components obtained by lysing the cell. The cell extract contains a target protein, and components other than the target protein may include one or more components selected from cell proteins, carbohydrates, nucleic acids, and fibers, but are not limited thereto.

[0174] In the present application, the recovery step may recover the target protein using a suitable method known in the art (e.g., centrifugation, filtration, anion exchange chromatography, crystallization, and HPLC, etc.).

[0175] In the present application, the recovery step may include a purification process. The purification process may involve separating only the target protein from cells and purifying it. Through the purification process, a purely purified target protein may be produced.

[0176] Another aspect provides use for the preparation of the mRNA construct or target protein of the above construct, vector, recombinant host cell, or composition.

[0177] Another aspect provides a method for increasing RNA stability and / or mRNA translation of a target gene, comprising the step of inserting or linking the above regulatory element to a UTR of the target gene.

[0178] Another aspect provides use for increasing RNA stability and / or mRNA translation of the above construct, vector, recombinant host cell, or composition.

[0179] Another aspect is a method for preparing an mRNA construct, comprising the steps of: transcribing the above construct or vector in vitro; and recovering the transcribed mRNA construct.

[0180] The above transfer method and recovery method may utilize suitable methods known in the relevant technical field.

[0181] In one specific example, the method may further include, but is not limited to, a step of treating with DNase I after transcription to remove the contents of the construct or vector used as a template; and / or a washing step.

[0182] Effects of the invention

[0183] A regulatory element according to one specific example can increase RNA stability or mRNA translation, which are transcription products of a target gene, thereby increasing the expression level of a target protein. Accordingly, the regulatory element of the present invention can be usefully utilized in systems requiring precise control of gene expression, such as gene therapy, vaccine development, and the production of protein therapeutics.

[0184] Brief explanation of the drawing

[0185] Figure 1 shows the alignment of sequences that are conserved and commonly appear in the Q20 element and its homologs. Regions corresponding to Q20_22 (22nt) and Q20_25 (25nt) are indicated by solid and dotted boxes, respectively. The consensus sequence at the top of the figure represents the common sequence of the Q20 element and its homologs, while the elements listed at the bottom are distinguished from the consensus sequence by different colors. The NCBI serial number and genomic position of each sequence are indicated on the left side of the sequence.

[0186] Figure 2 is a heatmap illustrating the results of a mutagenesis experiment confirming the effect on Q20 function when substitution mutations were introduced to the bases corresponding to each position in Q20_22. The dot brackets below the base column at the top of the figure represent the predicted secondary structure of Q20, and the brackets indicate sequences predicted to form base pairs. The numbers at the bottom of the X-axis (Position in ti le) indicate the relative position of the corresponding base within Q20(l-197), and the numbers in parentheses indicate the actual position within the entire viral genome. WT expression represents the level of protein expression in the reporter containing Q20.

[0187] Figure 3 shows the substitution of each base (X-axis) for positions 103 through 124 in the base sequence of Q20. 0 This is a bar graph showing the effect on the expression score (Y-axis). The top of the panel displays the relative position at Q20(1-197) and the base sequence of ", and the gray dotted line indicates the expression score of Q20C1-197). Figure 4 is a diagram showing the importance of the base pair structure and its position as a result of the base pairing mutagenesis experiment at Q20_22. The numbers written alongside the secondary structure of Q20 indicate the relative position of the corresponding base at Q20. In the secondary structure diagram, the AExp value is represented by the thickness of the line, and the exact numerical values ​​are summarized in the table on the right.

[0188] Figure 5 shows the results of measuring the activity of the Q20 fragment. (A) shows the results of confirming the luciferase activity of in vitro transcribed firefly mRNA constructs by inserting each element into the 3' UTR, and (B) shows the alignment of the elements used in the experiment. Figure 6 shows the predicted secondary structures of Q20, Q20hl, Q20h2, Q20h4, Q20h5, and Q20h6. Arrows indicate Q20_22nt, Q20_40nt, and Q20_79nt, and arrows indicate the parts corresponding to their homologs in each similar column (Q20hl, 2, 4, 5, and 6).

[0189] Figure 7 (A) shows the results of confirming the gene expression-enhancing effect of Q20.40 analogues through luciferase activity analysis. (B) shows the elements used in the experiment aligned based on Q20_22. Bases identical to Q20_22 are represented by a dot (.).

[0190] Figure 8 shows the activity of the Q20 fragment and its variants. (A) shows the results of confirming the gene expression effects of the Q20_22 mutant and the Q20h6_22 element, along with Q20_25 and Q20_22, through luciferase activity analysis. (B) shows the elements used in the experiment aligned based on Q20_22. Bases identical to Q20_22 are represented by a dot (.).

[0191] Figure 9 shows the activity of the Q20 fragment and its variant. (A) shows the results confirming the gene expression-enhancing effect of Q20h5_38 and fragments with modified sequences of Q20h5_38 together through luciferase activity analysis. (B) shows the elements used in the experiment aligned with respect to Q20_22. Bases identical to Q20_22 are represented by a dot (.).

[0192] Figure 10 is a figure showing the sequence similarity of the fragment sequences used in the experiment. Sequences identical to Q20.22 are represented by a dot ( .).

[0193] Figure 11 is a figure showing the activity of Q20 and fragments of its analogs through luciferase activity analysis.

[0194] Figure 12 is a figure showing the activity of Q20 and its analogues through luciferase activity analysis.

[0195] Figure 13 is a figure showing the activity of Q20 and its analogs in a plasmid-based dual-luciferase reporter system.

[0196] Form for carrying out the invention

[0197] Preferred embodiments are presented below to aid in understanding the present invention. However, the following embodiments are provided merely to facilitate a better understanding of the invention and do not limit the scope of the invention. Since various modifications can be made to the embodiments, the embodiments are not limited to those disclosed below but can be implemented in various forms.

[0198] Terms or words used in the specification and claims of the present invention are not limited to their ordinary or dictionary meanings, and must be interpreted in a meaning and concept consistent with the technical spirit of the present invention, based on the principle that the inventor may appropriately define the concept of the terms to best describe his invention. Throughout the specification of the present invention, when a part is described as "comprising" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0199] Throughout the specification of the present invention, "A and / or B" means A or B, or A and B.

[0200] Example of implementation

[0201] Example 1. Analysis Method

[0202] The analysis of experimental results related to the following embodiments was performed in the following manner.

[0203] 1.1 Data Analysis

[0204] For all samples, sequenced reads were aligned to each oligonucleotide sequence using bowtie2.2.652 with -local parameter conditions. The aligned reads were filtered according to the following criteria:

[0205] - Perfect match > 145;

[0206] - Insertion or deletion < 1.

[0207] For polysome analysis, samples were used in which a library containing oligo-pools cloned into the 3' untranslated region of the EGFP gene was expressed in HCT116 cell lines, and each fraction of the polysome (free mRNA, monosome, and light / medium / heavy polysome) was sequenced. After performing a variance stabilizing transformation using DESeq2, the average value of the five fractions was subtracted from the value of each fraction to calculate the relative distance between each fraction. The calculated relative distances (z-scores) were used for hierarchical clustering analysis using the scipy module.

[0208] For TENT4 screening and mutation experiments, the oligopool was cloned into the 3' untranslated region of the GFP gene and integrated into the genome of HEK293T cells. Cells were classified into three or four segments based on GFP expression levels using FACS, and elements were amplified and sequenced from the genomic sample of each segment. The expression level (Expression score or Exp score) for each segment was calculated as a weighted sum. Weights were calculated using the relative median FITC value measured during the sorting process. Statistical tests were performed using limma.

[0209] 1.2 Element Selection Criteria (Cutoff for element selection) Screening criteria for HCT116 cell line:

[0210] - Count _HP (z-score) > 0.2;

[0211] Count _HP (z-score) represents the relative distance of the Heavy polysome fraction.

[0212] - Log2(HP / Free) > 0.3 and FDR < 0.01;

[0213] HP: Heavy polysome, Free: free mRNA fraction 1 He does.

[0214] - log2(RNA / DNA) > 0.4 and FDR < 0.01.

[0215] Screening criteria for the HEK293T cell line:

[0216] - FDR(T) < 0.01;

[0217] - FDR(TT) < 0.05

[0218] - Fold T > 50

[0219] Here, T is the result of sequencing cells with increasing expression after classifying them once using FACS, and TT is the result of classifying and sequencing cells with high expression using FACS in the same way as T.

[0220] 1.3 Mutation (Mutagenesis) Screening Analysis

[0221] High-throughput mutation analysis was performed to precisely analyze the sequence characteristics and secondary structural correlations that determine the activity of the Q20 element. A library containing single base substitutions and single deletions for each base of the Q20 was constructed. In particular, to evaluate the effect of whether the secondary structure is maintained, compensatory mutations of complementary paired regions were included. Using the library, screening was performed in the same manner as in Example 1.1 to calculate the expression score (Expression score or Exp score) for each mutation.

[0222] A Exp was defined as follows:

[0223] - The value obtained by subtracting the average expression amount of unpaired nucleotides (excluding GU pairs) (Exp score) from the average expression amount of paired nucleotides (AU / GC / CG / UA) (Exp score).

[0224] Example 2. Identification of virus-derived Q20 and Q20 homologs elements

[0225] We aimed to discover a regulatory element that can improve the expression efficiency and stability of mRNA from the viral genome. Specifically, we designed an oligonucleotide containing nucleotides 7301 through 7497 of the full sequence of the Eel picornavirus 1 gene (NCBI Reference Sequence: NC_022332.1) and named it the 'Q20 (sequence number 3)' element.

[0226] In addition, we discovered additional regulatory elements as Q20 homologs that can improve mRNA expression efficiency and stability, similar to the Q20 element. Specifically, the designed Q20 homologs are as follows:

[0227] (1) Q20hl (Sequence No. 7): An oligonucleotide comprising nucleotides 7542 through 7738 of the full sequence of the Perch picornavirus M9 / 2015 / HUN gene (NCBI Reference Sequence: MW590713.1),

[0228] (2) Q20h2 (Sequence No. 10): An oligonucleotide containing nucleotides at positions 7322 through 7518 of the full-length sequence of the Wenling lepidotr igla picornavirus strain YXMC111438 gene (NCBI Reference Sequence: MG600079.1),

[0229] (3) Q20h4 (Sequence No. 13): An oligonucleotide comprising nucleotides 7585 through 7781 of the full sequence of the Wuhan carp picornavirus strain DSYC18088 gene (NCBI Reference Sequence: MG600066.1),

[0230] (4) Q20h5 (Sequence No. 16): An oligonucleotide comprising nucleotides 7506 through 7702 of the full-length sequence of the Yancheng osbecks grenadier anchovy picornavirus strain XFXMC15460 gene (NCBI Reference Sequence: MG600099.1), and

[0231] (5) Q20h6 (Sequence No. 19): An oligonucleotide comprising nucleotides 8296 through 8492 of the full sequence of the Potamipivirus A isolate TSPV gene (NCBI Reference Sequence: NC_075994.1). Example 3. Confirmation of the common column of Q20 and Q20 homologs elements.

[0232] As a result of analyzing the base sequences of Q20 and Q20 homologs elements, it was confirmed that oligonucleotides containing nucleotides corresponding to the 103rd to 124th or 103rd to 127th positions in the Q20 base sequence are conserved and commonly appear in the Q20 element and its homologs (Fig. 1). These were named Q20.22 and Q20_25, respectively.

[0233] Example 4. Identification of mutations with protein expression enhancement effects through Mutagenesis analysis

[0234] Through Mutagenesis experiments, the effect on Q20 function was confirmed when mutations were introduced to the bases corresponding to each position in Q20_22. Specifically, it was determined whether replacing the bases corresponding to each position in Q20_22 with other bases enhanced protein expression levels, and a higher Exp score (expression score) value suggests an enhanced protein expression effect. In the heatmap of Figure 2, darker colors and lower values ​​indicate that the element substitution mutations do not function properly; this implies that the corresponding mutation is not allowed at that position and that those bases are important for Q20 activity.

[0235] As shown in Figure 3, the effect of substitution with each base on the expression score for individual positions of Q20_22 was confirmed, and the effect of enhancing protein expression amount according to base substitution from the 103rd base (Q20 pos 103) to the 124th base (Q20 pos 124) relative to Q20 was confirmed.

[0236] Additionally, base pairing mutagenesis experiments were conducted by substituting base pairs at corresponding positions of the Q20_22 bases with various combinations. The difference in expression levels between when a mutation preserving base pairs was introduced and when a mutation not preserving base pairs was introduced was calculated and expressed as AExp (Fig. 4). A higher AExp value suggests that base pairing at the corresponding position is important for the Q20 effect. The 111th and 120th bases, and the 112th and 119th bases, were identified as base pairs with high AExp values ​​of 0.2 or higher.

[0237] From these results, it can be seen that the base pairs between positions 112 and 119 and between positions 111 and 120 are important.

[0238] Through mutagenesis analysis, core sequences reflecting allowed mutations in Q20_22 were defined and named Q20core_22 (sequence number 1) and Q20core_25 (sequence number 2).

[0239] Example 5. Confirmation of the gene expression enhancement effect of Q20 and Q20 homologs elements. Q20 and Q20 homologs and their fragments were inserted into the 3' UTR of a luciferase reporter construct to prepare a firefly mRNA construct transcribed in vitro. After transfection into HCT116 cell lines, luciferase activity was measured 72 hours later. As a control (ctrl), a firefly mRNA construct without additional elements inserted into the 3' UTR was used.

[0240] Each value was normalized to the luciferase activity of the reni l la mRNA construct transfected together.

[0241] The higher the luciferase activity, the better the effect of enhancing gene expression.

[0242] Experimental results confirmed that Q20_25 and Q20_22, fragments of the Q20 element, and Q20h2_38 and Q20h5_38, sequences of Q20_40, all had a gene expression-enhancing effect (Figs. 5, 7).

[0243] As shown in Figure 7, it is shown that similar sequences of Q20_40 can also enhance gene expression. In particular, since activity is maintained even when A at position 116 is substituted with G, it suggests that purine bases (A or G) are allowed at that position.

[0244] As shown in Figure 6, the secondary structure of Q20 and its similar sequences was predicted. As a result, for Q20, arrows were used to indicate the regions corresponding to Q20_22 (22nt), Q20_40 (40nt), and Q20_79 (79nt). For the similar sequences (Q20hl, Q20h2, Q20h4, Q20h5, Q20h6), arrows were used to indicate the regions that are homologous to Q20_22, Q20_40, and Q20_79, respectively. In Q20h2, the sequence homologous to Q20_40 is Q20h2_38.

[0245] It was confirmed that Q20_22m2, a mutation in which the 112th and 119th bases of Q20_22 maintain a base pair, and Q20h6_22, a 22-base fragment of Q20h6, both have a gene expression-enhancing effect (Fig. 8).

[0246] These experimental results show that even if positions 112 and 119 of Q20_22 are substituted, the function of Q20 is maintained as long as the base pair between those two positions is maintained. In addition, it shows that the activity is maintained even though Q20_22 and Q20h6_22 show differences in the two bases (bases at positions 106 and 116 relative to Q20).

[0247] We confirmed the gene expression-enhancing effects of fragments with modified sequences of Q20h5_38, along with Q20h5_38, a 38-base fragment of Q20h5. Q20h5_38m2 is a fragment in which the core 22nt sequence of Q20h5 is replaced with Q20_22m2 of Fig. 8, and Q20h5_38s3 and s4 are sequences in which the 5 bases at the 5' and 3' ends of Q20h5_38 (a total of 10 bases) that are thought to form base pairs are replaced with other bases capable of maintaining base pairs.

[0248] It was confirmed that all of these showed higher luciferase activity compared to the control group, indicating an effect of enhancing gene expression (Fig. 9).

[0249] In particular, since Q20h5 is active even though it has a group instead of A at position 116, it can be seen that a purine base is allowed at that position.

[0250] The sequences of the fragment sequences used in the experiment were compared and summarized comprehensively. (Fig. 10)

[0251] Additionally, to confirm the activity of fragments of Q20 and its analogs, homolog sequences of Q20_79 were inserted into the 3' UTR of a luciferase reporter construct, as in the experiment above, and the luciferase activity of in vitro transcribed firefly mRNA constructs was measured. Q20 refers to a 197nt-long fragment containing Q20_79.

[0252] The luciferase activity of Q20, Q20_79, and homolog sequences of Q20_79 (Q20hl_89, Q20h2_67, Q20h4_80, Q20h5_66, Q20h6_84) was confirmed, and it was confirmed that all of them have a gene expression-enhancing effect (Fig. 11).

[0253] The luciferase activity of Q20 and homolog sequences (197nt; Q20hl, 2, 3, 4, 5, 6) was confirmed, and it was verified that all of them had a gene expression-enhancing effect (Fig. 12). In addition, the activity of firefly luciferase was measured in a plasmid-based dual-luciferase reporter system and normalized to the activity of renilla luciferase. Firefly and renilla luciferases are expressed in the same plasmid, and each element was inserted into the 3' UTR of firefly luciferase, and experiments were conducted in HCT116 cells.

[0254] As a result of the experiment, it was confirmed that both Q20 and its homolog sequences had high luciferase activity (Fig. 13).

[0255] Example 6. Confirmation of importance for each base

[0256] Based on the mutagenesis and luciferase activity analysis data of Examples 2 to 5, base substitutions at specific positions within the Q20 element showed substitution tolerance, while functional loss was observed even with a single base change in a specific core region.

[0257] By comprehensively analyzing the data, an optimized Q20 core sequence with lengths of 22nt and 25nt that maintained the expression enhancement activity while ensuring sequence flexibility was established. This sequence was designed to introduce hybrid bases at specific positions to enable the design of various mutant libraries, and its composition is the same as SEQ ID NO: 1 and SEQ ID NO: 2 below.

[0258] SEQ ID NO: 1: CCCKTMMGN 1 N2GTHRN3RN 4 N 5 CAGG

[0259] SEQ ID NO: 2: CCCKTWWGTHRWLGGTAA

[0260] (K: [G / T], M: [C / A], H: [A / T / C], R: [G / A], N: [A / T / G / this (N 1 and N 5 , Ding and N 4 are bases that form base pairs with each other ))

[0261] The sequences of the elements described in the present invention are as shown in Table 1 below.

[0262] 【Table 11

[0263] Sequence Q20 Q20

[0264] Length Accession Virus Virus Number Type Criteria Criteria nu Criteria START END mber START END 1 Q20core_22 22 103 124 — 7301 7497 2 Q20core_25 25 103 127 — 7301 7497 3 Q20 197 1 197 NC_022332.1 7301 7497 4 Q20.22 22 103 124 NC_022332.2 7403 7424 5 Q20.25 25 103 127 NC_022332.3 7403 7427 6 Q20.40 40 94 133 NC_022332.5 7394 7433 7 Q20hl 197 1 197 MW590713.1 7542 7738

[0265]

[0266] 8 Q20hl_89 89 61 149 MW590713.1 7602 7690 9 Q20hl_38 38 88 125 MW590713.1 7629 7666 10 Q20h2 197 1 197 MG600079.1 7319 7518 11 Q20h2_67 67 78 144 MG600079.1 7396 7465 12 Q20h2_38 38 95 132 MG600079.1 7413 7453 13 Q20h4 197 1 197 MG600066.1 7582 7781 14 Q20h4_80 80 102 181 MG600066.1 7683 7765 15 Q20h4_38 38 125 162 MG600066.1 7706 7746 16 Q20h5 197 1 197 MG600099.1 7503 7702 17 Q20h5_66 66 79 144 MG600099.1 7581 7649 18 Q20h5_38 38 94 131 MG600099.1 7596 7636 19 Q20h6 197 1 197 NC_075994.1 8293 8492 20 Q20h6_84 84 57 140 NC_075994.1 8349 8435 21 Q20h6_39 39 82 120 NC_075994.1 8374 8415 22 Q20h6_22 22 90 111 NC_075994.1 8382 8406 23 Q20_22m2 22 103 124 — 8395 8419 24 Q20h5_38m2 38 94 131 — 8386 8426 25 Q20h5_38s3 38 94 131 — 8386 8426 26 Q20h5_38s4 38 94 131 — 8386 8426

[0267]

[0268] From the foregoing description, those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without altering its technical concept or essential features. In this regard, the experimental examples and embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as including all modifications or variations derived from the meaning and scope of the claims set forth below and their equivalents, rather than from the detailed description above.

Claims

Scope of the claim

1. Control element including the following structure: (i) 1st loop; (ii) 1st stem connected to the 1st loop ; (iii) a second loop connected to the first stem; and (iv) The second stem connected to the second loop, Here, the first loop is GTHRN 3 Includes R sequence, The first stem is GN X N 2 Sequence and N that binds complementarily to it 4 N 5 It includes a C sequence, and the second loop includes a TMM sequence, The second stem includes CC sequences and GG sequences, and The above view is A, T, or Benevolence, and The above R is G or A, and The above 'Nieun' is A, T, G, or In, and The above M is C or A, and N 1 and N 5 and N 2 and N 4 Each forms a base pair.

2. In claim 1, the control element is It includes a third loop connected to the second stem, and The above third loop is a regulatory element comprising a G sequence on one side of the loop and an AA sequence on the other side of the loop.

3. In claim 2, the adjustment element is It includes a third stem connected to the third loop above, and The above third stem is a regulatory element comprising 1 to 5 base pairs.

4. In claim 1, the control element is a control element comprising the structure of the following formula I or formula II: [Equation I] 5'-[Stage 2 A]-[Loop 2 A]-[Stage 1 A]-[Loop 1]-[Stage 1 B]-[Loop 2 B]-[Stage 2 B]- 3', [Equation II] 5'-[3rd Loop A]-[2nd Stem A]-[2nd Loop A]-[1st Stem A]-[1st Loop]-[1st Stem B]-[2nd Loop B]-[2nd Stem B]-[3rd Loop B]- 3', in the above Equation I or Equation II, , The first loop is GTHRN 3 Includes R sequence, First Stem A and First Stem B combine complementarily to form the first stem, and First Stem A is GN X N 2 It includes a sequence, and the first stem B is N 4 N 5 It includes the C sequence, and the second loop A includes the TMM, CKTMM, or CCKTMM sequence, and the second loop B includes the A sequence or does not include nucleotides, Second Stem A and Second Stem B combine complementarily to form a second stem, Second Stem A includes a CC, ACC, or CCT sequence, and Second Stem B includes a GG, GGT, or AGG sequence, and In the above Equation II, , The third loop A contains a G, GA, GAT, or GATC sequence, and the third loop B contains an AA, or TAA sequence.

5. A control element comprising the base sequence of sequence number 1 or 2, or a base sequence having at least 80% identity with the same as that of claim 1.

6. In claim 5, (i) A fragment of the Eel picornavirus 1 gene (NCBI Reference Sequence: NC_022332.1), wherein the fragment comprises the nucleotide sequence of Sequence No. 1 or 2; or a nucleotide sequence having at least 80% identity with the same; (ii) a fragment of the Perch picornavirus M9 / 2015 / HUN gene (NCBI Reference Sequence: MW590713.1), wherein the fragment comprises a nucleotide sequence of sequence number 1 or 2; or a nucleotide sequence having at least 80% identity with the same; ( iii ) One-ring Lepidotriglycerin virus strain YXMC A fragment of the gene 111438 (Wenl ing lepidotr igla picornavirus strain YXMC111438) (NCBI Reference Sequence: MG600079.1), wherein the fragment comprises the nucleotide sequence of Sequence No. 1 or 2; or a nucleotide sequence having at least 80% identity with the same; (iv) a fragment of the gene of Wuhan carp picornavirus strain DSYC18088 (NCBI Reference Sequence: MG600066.1), wherein the fragment comprises the nucleotide sequence of sequence number 1 or 2; or a nucleotide sequence having at least 80% identity with the same; (v) A fragment of the gene of Yancheng osbecks grenadier anchovy picornavirus strain XFXMC15460 (NCBI Reference Sequence: MG600099.1), wherein the fragment comprises the nucleotide sequence of SEQ No. 1 or 2; or a nucleotide sequence having at least 80% identity with the same; or (vi) A fragment of the Potamipivirus A isolate TSPV gene (NCBI Reference Sequence: NC_075994.1), wherein the fragment comprises a sequence of nucleotides of sequence number 1 or 2; or a regulatory element comprising a sequence of nucleotides having at least 80% identity with the same.

7. A control element according to claim 1, wherein the base sequence comprises one or more base sequences of any one of sequence numbers 3 to 26.

8. In claim 1, the regulating element is a regulating element that induces an increase in the length of the poly (A)tail, an increase in the stability of the poly (A)tail, or both.

9. In claim 1, the regulatory element is a regulatory element for increasing RNA stability or mRNA translation.

10. A regulatory element according to claim 9, wherein the RNA or mRNA is an RNA or mRNA comprising one or more modified nucleotides.

11. A construct comprising a target gene; and a regulatory element of any one of claims 1 to 10.

12. The construct of claim 11, wherein the target gene is a gene of a reporter, protein, physiologically active peptide, antigen, or antibody or a fragment thereof; or one or more selected from the group consisting of antisense oligonucleotides, mRNA, dsRNA, shRNA, miRNA, siRNA, gRNA, saRNA, IncRNA, taRNA, ribozymes, ncRNA, exosoma 1 RNA, and aptamers.

13. In claim 11, the construct is an mRNA construct, the construct

14. A vector comprising the construct of claim 11.

15. In claim 14, the vector is an AAV (Adeno-Associated Virus) vector.

16. A recombinant host cell comprising the construct of claim 11 or a vector comprising said construct.

17. A composition comprising the construct of Claim 11; a vector comprising the construct; or a recombinant host cell comprising the construct or the vector.

18. The composition of Claim 17, wherein the composition is for the prevention or treatment of a disease; or for the manufacture of an mRNA construct or a protein coding for a target gene.

19. A method for increasing RNA stability or mRNA translation of a target gene, comprising the step of inserting or linking a regulatory element of any one of claims 1 to 10 into a UTR of the target gene.

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