Nucleic acid constructs containing UTRs and uses thereof

Abstract

Regarding the nucleic acid construct containing UTR and its use, specifically, UTR can significantly improve the expression efficiency of the target gene in the nucleic acid construct. The nucleic acid construct can contain, for example, a nucleic acid sequence expressing human hepatocyte growth factor (hHGF) for gene therapy drugs used for diseases such as critical limb ischemia and diabetic foot.

Description

[Technical field]

[0001] This disclosure claims priority to a Chinese patent application filed on May 13, 2022, bearing application number CN202210522056.8 and entitled "Nucleic Acid Construct Containing UTR and Use Thereof," the entire contents of which are incorporated herein by reference.

[0002] The present disclosure belongs to the field of nucleic acids and relates to a nucleic acid construct comprising a UTR and its use for preventing or treating diseases. The nucleic acid construct may comprise a nucleic acid sequence expressing human hepatocyte growth factor (hHGF) for use in gene therapy drugs for diseases such as critical limb ischemia and diabetic foot. [Background technology]

[0003] Gene therapy and gene vaccination can provide highly specific and personalized treatment and prevention strategies for a variety of diseases, including genetic disorders, autoimmune diseases, cancer and tumor-related diseases, and inflammatory diseases.

[0004] Both DNA and RNA can be used for gene therapy or gene vaccination. DNA is stable and easy to handle, but insertion of DNA fragments into the patient's genome carries the risk of causing mutation events (e.g., loss of function of damaged genes). RNA can avoid undesired genome integration, but RNA enzymes are ubiquitous, making it susceptible to degradation. Therefore, it is necessary to improve the stability of RNA to accumulate the protein products encoded by it in the body and achieve treatment or prevention against diseases, while maintaining the integrity of RNA structure and function during storage and administration. It has been found that naturally occurring eukaryotic mRNA molecules contain stabilizing elements, such as untranslated regions (UTRs) at their 5' and 3' ends, and other structural features such as a 5' cap structure and a 3' polyadenylic acid tail. The 5' UTR and 3' UTR are premature mRNA elements, and structural features specific to mature mRNAs (e.g., 5' cap and 3' polyadenylic acid tail) are added to the transcribed (premature) mRNA during mRNA processing. Regarding the correlation between UTR and mRNA stability, studies have shown that the 3'UTR of α-globin mRNA is an important factor in the stability of α-globin mRNA (Nancy D Rodgers et al, RNA. 2002 Dec;8(12):1526~37; Z Wang et al, Mol Cell Biol. 1999 Jul;19(7):4552~60.).

[0005] Peripheral artery disease (PAD) refers to a series of non-coronary artery syndromes caused by structural and functional disorders of the arteries of the limbs, organs and brain, characterized by stenosis, occlusion and tumor-like lesions occurring in the non-coronary blood circulation, affecting the aorta and branch arteries. Among PAD, lower limb ischemic disease is the most common clinically, and the main etiologies include atherosclerosis obliterans (ASO), diabetic artery obliterans (DAO) and thromboangiitis obliterans (TAO). Critical limb ischemia (CLI) is the final stage of ASO development, and diabetic foot ulcer (DFU) is a type of DAO. Both are peripheral vascular disorders, characterized by lower limb pain, ulcers and necrosis due to stenosis or occlusion of lower limb blood vessels and insufficient distal blood perfusion. Currently, the most effective treatment for CLI and DFU is revascularization by surgery or endovascular intervention, but more than 40% of patients do not meet the requirements for revascularization due to age, comorbidities, etc., and can only be treated conservatively with medication. Drug therapy can only delay the progression of the disease, but cannot cure it.

[0006] Hepatocyte growth factor (HGF) is a multifunctional stromal-derived growth factor that binds to the cell membrane surface receptor c-met, induces phosphorylation of intracellular tyrosine residues, recruits adaptor proteins, promotes kinase activity, and activates downstream signaling pathways. HGF is an important regulator of processes such as embryonic development, tissue and organ regeneration, wound healing, and angiogenesis, and can promote the proliferation and migration of endothelial cells and smooth muscle cells, promote the reconstruction of the microvascular network in ischemic areas, and inhibit cell apoptosis. Research results show that delivery of hHGF-cDNA by intramuscular injection of naked plasmid can effectively promote blood flow perfusion in the ischemic hindlimb of mice and rabbits (Y Taniyama et al.Therapeutic angiogenesis induced byhumanhepatocyte growth factor gene in rat and rabbithindlimb ischemia models:preclinical study for treatment of peripheral arterial disease.Gene Ther .2001 Feb;8(3):181-9). Clinical data shows that injecting naked plasmid into the calf muscle can promote wound healing in patients (S Cui et al. Clinical Safety and Preliminary Efficacy of Plasmid pUDK-HGF Expressing Human Hepatocyte Growth Factor (HGF) in Patients with Critical Limb Ischemia. Eur J Vasc Endovasc Surg. 2015 Oct; 50 (4): 494-501.). However, naked plasmid drug delivery has shortcomings such as low transfection efficiency in vivo, high dosage burden, high risk of DNA integration, and high treatment cost. Moreover, clinical data shows that the toe-brachial pressure index, transcutaneous oxygen tension, and amputation rate have not been improved, so there is still a lot of room for improvement in the drug.

[0007] The present disclosure provides an mRNA that includes a novel structure of 5'UTR and 3'UTR, which reduces premature degradation of the mRNA or stabilizes the degradation of the mRNA, but does not impair or even enhances protein translation efficiency. The above mRNA has higher stability and can be used for gene therapy or gene vaccination. The present disclosure also provides an mRNA capable of expressing human hepatocyte growth factor (hHGF) and its lipid nanoparticle (LNP) delivery system, which can realize efficient and rapid transformation of exogenous hHGF protein in vivo, has the advantages of no integration risk and being easy to mass-produce industrially, is a more desirable treatment regimen than naked plasmid, and can be used as a gene therapy drug for various diseases such as CLI and DFU. Summary of the Invention

[0008] The present disclosure provides a nucleic acid construct comprising at least one nucleic acid element that can regulate the expression of a target gene, and the nucleic acid element is UTR.The nucleic acid construct can also comprise one or more target genes, and the target gene is, for example, HGF.

[0009] nucleic acid construct The present disclosure relates to (a) an open reading frame (ORF); (b) a untranslated region (UTR),

[0010] In some embodiments, the nucleic acid construct is a DNA molecule, and in some embodiments, the nucleic acid construct is an RNA molecule (eg, mRNA).

[0011] In some embodiments, the ORF is a polynucleotide sequence that encodes a target gene.

[0012] In some embodiments, the target gene is heterologous. In other embodiments, the target gene is endogenous. In some embodiments, the target gene is one or more (e.g., two, three, four).

[0013] In some embodiments, the UTR is a UTR from the genes ACTG1, ATP6V0B, ATP6V0E1, CFL1, COX4I1, CTSB, FAM166A, NDUFB9, CHCHD10, SLC38A2, NDUFA11, NDUFV3, PRDX5, GUK1, IAH1, ABHD16A, SLC25A39, ATPIF1, ANAPC11, CCDC12, MRPL14, or APOA1BP. In some embodiments, the gene is a human gene.

[0014] In some embodiments, the UTR is a 3' untranslated region element (3'UTR) or a 5' untranslated region element (5'UTR).

[0015] In some embodiments, the 3'UTR and 5'UTR are from the same or different origins, e.g., from the same or different genes. For example, the 3'UTR is from gene ACTG1, and the 5'UTR is from gene ACTG1. Also, for example, the 3'UTR is from gene CTSB, and the 5'UTR is from gene CHCHD10. In some embodiments, the 5'UTR and 3'UTR are from the same or different species.

[0016] In some embodiments, the 5'UTR is located upstream of the ORF. In some embodiments, the 5'UTR in the nucleic acid construct is located at the 5' end of the ORF. In some embodiments, the 3'UTR is located downstream of the ORF. In some embodiments, the 3'UTR in the nucleic acid construct is located at the 3' end of the ORF.

[0017] In some embodiments, the nucleic acid construct comprises: (a) an open reading frame (ORF); (b-1) a 3'UTR that is a 3'UTR derived from the gene ACTG1, ATP6V0B, ATP6V0E1, CFL1, COX4I1, CTSB, FAM166A, or NDUFB9, and (b-2) a 5'UTR that is a 5'UTR derived from the genes ACTG1, ATP6V0B, ATP6V0E1, CFL1, COX4I1, CTSB, FAM166A, NDUFB9, CHCHD10, SLC38A2, NDUFA11, NDUFV3, PRDX5, GUK1, IAH1, ABHD16A, SLC25A39, ATPIF1, ANAPC11, CCDC12, MRPL14 or APOA1BP.

[0018] In some embodiments, the nucleic acid construct comprises: (a) an open reading frame (ORF); (b-1) a 3'UTR that is a 3'UTR derived from the gene CTSB, FAM166A, or NDUFB9, and (b-2) a 5'UTR that is a 5'UTR derived from the gene ACTG1, CHCHD10, or NDUFA11.

[0019] In some embodiments, the nucleic acid construct (e.g., DNA or RNA molecule), wherein the 3'UTR is a 3'UTR from gene ACTG1 and comprises a sequence set forth in SEQ ID NO:1 or SEQ ID NO:58, or having identity to any one of them; The 3'UTR is a 3'UTR derived from the gene ATP6V0B and comprises a sequence as set forth in SEQ ID NO: 2, 3 or SEQ ID NO: 59, 60, or a sequence having identity to any one of them; The 3'UTR is a 3'UTR derived from the gene ATP6V0E1 and comprises a sequence as set forth in SEQ ID NO: 4, 5 or SEQ ID NO: 61, 62 or a sequence having identity to any one of them; The 3'UTR is a 3'UTR derived from the gene CFL1 and comprises a sequence as set forth in SEQ ID NO: 6, 7, 8 or SEQ ID NO: 63, 64, 65 or a sequence having identity to any one of them; The 3'UTR is a 3'UTR derived from the gene COX4I1 and comprises a sequence as set forth in SEQ ID NO: 9, 10, 11 or SEQ ID NO: 66, 67, 68 or a sequence having identity to any one of them; The 3'UTR is a 3'UTR derived from the gene CTSB and comprises a sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 69 or a sequence having identity to any one of them; The 3'UTR is a 3'UTR derived from gene FAM166A and comprises a sequence as set forth in SEQ ID NO: 13 or SEQ ID NO: 70 or has identity to any one of them; or The 3'UTR is a 3'UTR derived from gene NDUFB9 and comprises a sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 71 or having identity to any one of them.

[0020] In some embodiments, the nucleic acid construct (e.g., DNA or RNA molecule), wherein the 5'UTR is a 5'UTR from gene ACTG1 and comprises a sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 72, or has identity to any one of them; The 5'UTR is a 5'UTR derived from the gene ATP6V0B and comprises a sequence as set forth in SEQ ID NO: 16, 17 or SEQ ID NO: 73, 74 or a sequence having identity to any one of them; The 5'UTR is a 5'UTR derived from the gene ATP6V0E1 and comprises a sequence as set forth in SEQ ID NO: 18, 19 or SEQ ID NO: 75, 76 or a sequence having identity to any one of them; The 5'UTR is a 5'UTR derived from the gene CFL1 and comprises a sequence as set forth in SEQ ID NO: 20, 21, 22 or SEQ ID NO: 77, 78, 79 or a sequence having identity to any one of them; The 5'UTR is a 5'UTR derived from the gene COX4I1 and comprises a sequence as set forth in SEQ ID NO: 23, 24, 25 or SEQ ID NO: 80, 81, 82 or a sequence having identity to any one of them; The 5'UTR is a 5'UTR derived from the gene CTSB and comprises a sequence set forth in SEQ ID NO: 26 or SEQ ID NO: 83 or a sequence having identity to any one of them; The 5'UTR is a 5'UTR derived from gene FAM166A and comprises a sequence set forth in SEQ ID NO: 27 or SEQ ID NO: 84 or a sequence having identity to any one of them; The 5'UTR is a 5'UTR derived from the gene NDUFB9 and comprises a sequence set forth in SEQ ID NO: 28 or SEQ ID NO: 85 or a sequence having identity to any one of them; The 5'UTR is a 5'UTR derived from gene CHCHD10 and comprises a sequence as set forth in SEQ ID NO: 29, 30 or SEQ ID NO: 86, 87 or a sequence having identity to any one of them; The 5'UTR is a 5'UTR derived from gene SLC38A2 and comprises a sequence as set forth in SEQ ID NO: 31 or SEQ ID NO: 88 or a sequence having identity to any one of them; The 5'UTR is a 5'UTR derived from gene NDUFA11 and comprises a sequence set forth in SEQ ID NO: 32 or SEQ ID NO: 89 or a sequence having identity to any one of them; The 5'UTR is a 5'UTR derived from gene NDUFV3 and comprises a sequence set forth in SEQ ID NO: 33 or SEQ ID NO: 90 or having identity to any one of them; The 5'UTR is a 5'UTR derived from the gene PRDX5 and comprises the sequence shown in SEQ ID NO: 34 or SEQ ID NO: 91; The 5'UTR is a 5'UTR derived from the gene GUK1 and comprises a sequence as set forth in SEQ ID NO: 35, 36, 37 or SEQ ID NO: 92, 93, 94 or a sequence having identity to any one of them; The 5'UTR is a 5'UTR derived from gene IAH1 and comprises a sequence set forth in SEQ ID NO: 38 or SEQ ID NO: 95 or a sequence having identity to any one of them; The 5'UTR is a 5'UTR derived from gene ABHD16A and comprises a sequence set forth in SEQ ID NO: 39 or SEQ ID NO: 96 or a sequence having identity to any one of them; The 5'UTR is a 5'UTR derived from gene SLC25A39 and comprises a sequence as set forth in SEQ ID NO: 40 or SEQ ID NO: 97 or a sequence having identity to any one of them; The 5'UTR is a 5'UTR derived from the gene ATPIF1 and comprises a sequence set forth in SEQ ID NO: 41 or SEQ ID NO: 98 or a sequence having identity to any one of them; The 5'UTR is a 5'UTR derived from the gene ANAPC11 and comprises a sequence as set forth in SEQ ID NO: 42, 43 or SEQ ID NO: 99, 100, or a sequence having identity to any one of them; The 5'UTR is a 5'UTR derived from gene CCDC12 and comprises a sequence set forth in SEQ ID NO: 44 or SEQ ID NO: 101 or having identity to any one of them; The 5'UTR is a 5'UTR derived from gene MRPL14 and comprises a sequence as set forth in SEQ ID NO: 45 or SEQ ID NO: 102 or has identity to any one of them; or The 5'UTR is a 5'UTR derived from the gene APOA1BP and comprises a sequence shown in SEQ ID NO: 46, 47 or SEQ ID NO: 103, 104 or a sequence having identity to any one of them.

[0021] In some embodiments, a nucleic acid construct (e.g., a DNA or RNA molecule) is provided that comprises: (a) an open reading frame (ORF); (b-1) 3'UTR, and (b-2) 5'UTR, The 3'UTR and 5'UTR are any one of the following combinations: 1) the 3'UTR comprises a sequence shown in SEQ ID NO: 1 or SEQ ID NO: 58, or a sequence having identity to any one of them, and the 5'UTR comprises a sequence shown in any one of SEQ ID NOs: 15 to 47, or any one of SEQ ID NOs: 72 to 104, or a sequence having identity to any one of them; 2) the 3'UTR comprises a sequence shown in SEQ ID NO: 2 or SEQ ID NO: 59, or a sequence having identity to any one of them, and the 5'UTR comprises a sequence shown in any one of SEQ ID NOs: 15 to 47, or any one of SEQ ID NOs: 72 to 104, or a sequence having identity to any one of them; 3) the 3'UTR comprises a sequence shown in SEQ ID NO: 3 or SEQ ID NO: 60, or a sequence having identity to any one of them, and the 5'UTR comprises a sequence shown in any one of SEQ ID NOs: 15 to 47, or any one of SEQ ID NOs: 72 to 104, or a sequence having identity to any one of them; 4) the 3'UTR comprises a sequence shown in SEQ ID NO: 4 or SEQ ID NO: 61, or a sequence having identity to any one of them, and the 5'UTR comprises a sequence shown in any one of SEQ ID NOs: 15 to 47, or any one of SEQ ID NOs: 72 to 104, or a sequence having identity to any one of them; 5) the 3'UTR comprises a sequence shown in SEQ ID NO: 5 or SEQ ID NO: 62, or a sequence having identity to any one of them, and the 5'UTR comprises a sequence shown in any one of SEQ ID NOs: 15 to 47, or any one of SEQ ID NOs: 72 to 104, or a sequence having identity to any one of them; 6) The 3'UTR comprises a sequence shown in SEQ ID NO: 6 or SEQ ID NO: 63, or a sequence having identity to any one of them, and the 5'UTR comprises a sequence shown in any one of SEQ ID NOs: 15 to 47, or any one of SEQ ID NOs: 72 to 104, or a sequence having identity to any one of them; 7) The 3'UTR comprises a sequence shown in SEQ ID NO: 7 or SEQ ID NO: 64, or a sequence having identity to any one of them, and the 5'UTR comprises a sequence shown in any one of SEQ ID NOs: 15 to 47, or any one of SEQ ID NOs: 72 to 104, or a sequence having identity to any one of them; 8) The 3'UTR comprises a sequence shown in SEQ ID NO: 8 or SEQ ID NO: 65, or a sequence having identity to any one of them, and the 5'UTR comprises a sequence shown in any one of SEQ ID NOs: 15 to 47, or any one of SEQ ID NOs: 72 to 104, or a sequence having identity to any one of them; 9) The 3'UTR comprises a sequence shown in SEQ ID NO: 9 or SEQ ID NO: 66, or a sequence having identity to any one of them, and the 5'UTR comprises a sequence shown in any one of SEQ ID NOs: 15 to 47, or any one of SEQ ID NOs: 72 to 104, or a sequence having identity to any one of them; 10) The 3'UTR comprises a sequence shown in SEQ ID NO: 10 or SEQ ID NO: 67, or a sequence having identity to any one of them, and the 5'UTR comprises a sequence shown in any one of SEQ ID NOs: 15 to 47, or any one of SEQ ID NOs: 72 to 104, or a sequence having identity to any one of them; 11) The 3'UTR comprises a sequence shown in SEQ ID NO: 11 or SEQ ID NO: 68, or a sequence having identity to any one of them, and the 5'UTR comprises a sequence shown in any one of SEQ ID NOs: 15 to 47, or any one of SEQ ID NOs: 72 to 104, or a sequence having identity to any one of them; 12) The 3'UTR comprises a sequence shown in SEQ ID NO: 12 or SEQ ID NO: 69, or a sequence having identity to any one of them, and the 5'UTR comprises a sequence shown in any one of SEQ ID NOs: 15 to 47, or any one of SEQ ID NOs: 72 to 104, or a sequence having identity to any one of them; 13) The 3'UTR comprises a sequence shown in SEQ ID NO: 13 or SEQ ID NO: 70, or a sequence having identity to any one of them, and the 5'UTR comprises a sequence shown in any one of SEQ ID NOs: 15 to 47, or any one of SEQ ID NOs: 72 to 104, or a sequence having identity to any one of them; 14) The 3'UTR comprises a sequence shown in SEQ ID NO: 14 or SEQ ID NO: 71, or a sequence having identity to any one of them, and the 5'UTR comprises a sequence shown in any one of SEQ ID NOs: 15 to 47, or any one of SEQ ID NOs: 72 to 104, or a sequence having identity to any one of them; 15) The 3'UTR comprises a sequence shown in any one of SEQ ID NOs: 1 to 14 or any one of SEQ ID NOs: 58 to 71, or a sequence having identity thereto, and the 5'UTR comprises a sequence shown in SEQ ID NO: 15 or SEQ ID NO: 72, or a sequence having identity thereto; 16) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or any one of SEQ ID NOs: 58 to 71, or a sequence having identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 16 or SEQ ID NO: 73, or a sequence having identity thereto; 17) The 3'UTR comprises a sequence shown in any one of SEQ ID NOs: 1 to 14 or any one of SEQ ID NOs: 58 to 71, or a sequence having identity thereto, and the 5'UTR comprises a sequence shown in SEQ ID NO: 17 or SEQ ID NO: 74, or a sequence having identity thereto; 18) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or any one of SEQ ID NOs: 58 to 71, or a sequence having identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 18 or SEQ ID NO: 75, or a sequence having identity thereto; 19) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or any one of SEQ ID NOs: 58 to 71, or a sequence having identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 19 or SEQ ID NO: 76, or a sequence having identity thereto; 20) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or any one of SEQ ID NOs: 58 to 71, or a sequence having identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 20 or SEQ ID NO: 77, or a sequence having identity thereto; 21) The 3'UTR comprises a sequence shown in any one of SEQ ID NOs: 1 to 14 or any one of SEQ ID NOs: 58 to 71, or a sequence having identity thereto, and the 5'UTR comprises a sequence shown in SEQ ID NO: 21 or SEQ ID NO: 78, or a sequence having identity thereto; 22) The 3'UTR comprises a sequence shown in any one of SEQ ID NOs: 1 to 14 or any one of SEQ ID NOs: 58 to 71, or a sequence having identity thereto, and the 5'UTR comprises a sequence shown in SEQ ID NO: 22 or SEQ ID NO: 79, or a sequence having identity thereto; 23) The 3'UTR comprises a sequence shown in any one of SEQ ID NOs: 1 to 14 or any one of SEQ ID NOs: 58 to 71, or a sequence having identity thereto, and the 5'UTR comprises a sequence shown in SEQ ID NO: 23 or SEQ ID NO: 80, or a sequence having identity thereto; 24) The 3'UTR comprises a sequence shown in any one of SEQ ID NOs: 1 to 14 or any one of SEQ ID NOs: 58 to 71, or a sequence having identity thereto, and the 5'UTR comprises a sequence shown in SEQ ID NO: 24 or SEQ ID NO: 81, or a sequence having identity thereto; 25) The 3'UTR comprises a sequence shown in any one of SEQ ID NOs: 1 to 14 or any one of SEQ ID NOs: 58 to 71, or a sequence having identity thereto, and the 5'UTR comprises a sequence shown in SEQ ID NO: 25 or SEQ ID NO: 82, or a sequence having identity thereto; 26) The 3'UTR comprises a sequence shown in any one of SEQ ID NOs: 1 to 14 or any one of SEQ ID NOs: 58 to 71, or a sequence having identity thereto, and the 5'UTR comprises a sequence shown in SEQ ID NO: 26 or SEQ ID NO: 83, or a sequence having identity thereto; 27) The 3'UTR comprises a sequence shown in any one of SEQ ID NOs: 1 to 14 or any one of SEQ ID NOs: 58 to 71, or a sequence having identity thereto, and the 5'UTR comprises a sequence shown in SEQ ID NO: 27 or SEQ ID NO: 84, or a sequence having identity thereto; 28) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or any one of SEQ ID NOs: 58 to 71, or a sequence having identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 28 or SEQ ID NO: 85, or a sequence having identity thereto; 29) The 3'UTR comprises a sequence shown in any one of SEQ ID NOs: 1 to 14 or any one of SEQ ID NOs: 58 to 71, or a sequence having identity thereto, and the 5'UTR comprises a sequence shown in SEQ ID NO: 29 or SEQ ID NO: 86, or a sequence having identity thereto; 30) The 3'UTR comprises a sequence shown in any one of SEQ ID NOs: 1 to 14 or any one of SEQ ID NOs: 58 to 71, or a sequence having identity thereto, and the 5'UTR comprises a sequence shown in SEQ ID NO: 30 or SEQ ID NO: 87, or a sequence having identity thereto; 31) The 3'UTR comprises a sequence shown in any one of SEQ ID NOs: 1 to 14 or any one of SEQ ID NOs: 58 to 71, or a sequence having identity thereto, and the 5'UTR comprises a sequence shown in SEQ ID NO: 31 or SEQ ID NO: 88, or a sequence having identity thereto; 32) The 3'UTR comprises a sequence shown in any one of SEQ ID NOs: 1 to 14 or any one of SEQ ID NOs: 58 to 71, or a sequence having identity thereto, and the 5'UTR comprises a sequence shown in SEQ ID NO: 32 or SEQ ID NO: 89, or a sequence having identity thereto; 33) The 3'UTR comprises a sequence shown in any one of SEQ ID NOs: 1 to 14 or any one of SEQ ID NOs: 58 to 71, or a sequence having identity thereto, and the 5'UTR comprises a sequence shown in SEQ ID NO: 33 or SEQ ID NO: 90, or a sequence having identity thereto; 34) The 3'UTR comprises a sequence shown in any one of SEQ ID NOs: 1 to 14 or any one of SEQ ID NOs: 58 to 71, or a sequence having identity thereto, and the 5'UTR comprises a sequence shown in SEQ ID NO: 34 or SEQ ID NO: 91, or a sequence having identity thereto; 35) The 3'UTR comprises a sequence shown in any one of SEQ ID NOs: 1 to 14 or any one of SEQ ID NOs: 58 to 71, or a sequence having identity thereto, and the 5'UTR comprises a sequence shown in SEQ ID NO: 35 or SEQ ID NO: 92, or a sequence having identity thereto; 36) The 3'UTR comprises a sequence shown in any one of SEQ ID NOs: 1 to 14 or any one of SEQ ID NOs: 58 to 71, or a sequence having identity thereto, and the 5'UTR comprises a sequence shown in SEQ ID NO: 36 or SEQ ID NO: 93, or a sequence having identity thereto; 37) The 3'UTR comprises a sequence shown in any one of SEQ ID NOs: 1 to 14 or any one of SEQ ID NOs: 58 to 71, or a sequence having identity thereto, and the 5'UTR comprises a sequence shown in SEQ ID NO: 37 or SEQ ID NO: 94, or a sequence having identity thereto; 38) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or any one of SEQ ID NOs: 58 to 71, or a sequence having identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 38 or SEQ ID NO: 95, or a sequence having identity thereto; 39) The 3'UTR comprises a sequence shown in any one of SEQ ID NOs: 1 to 14 or any one of SEQ ID NOs: 58 to 71, or a sequence having identity thereto, and the 5'UTR comprises a sequence shown in SEQ ID NO: 39 or SEQ ID NO: 96, or a sequence having identity thereto; 40) The 3'UTR comprises a sequence shown in any one of SEQ ID NOs: 1 to 14 or any one of SEQ ID NOs: 58 to 71, or a sequence having identity thereto, and the 5'UTR comprises a sequence shown in SEQ ID NO: 40 or SEQ ID NO: 97, or a sequence having identity thereto; 41) The 3'UTR comprises a sequence shown in any one of SEQ ID NOs: 1 to 14 or any one of SEQ ID NOs: 58 to 71, or a sequence having identity thereto, and the 5'UTR comprises a sequence shown in SEQ ID NO: 41 or SEQ ID NO: 98, or a sequence having identity thereto; 42) The 3'UTR comprises a sequence shown in any one of SEQ ID NOs: 1 to 14 or any one of SEQ ID NOs: 58 to 71, or a sequence having identity thereto, and the 5'UTR comprises a sequence shown in SEQ ID NO: 42 or SEQ ID NO: 99, or a sequence having identity thereto; 43) The 3'UTR comprises a sequence shown in any one of SEQ ID NOs: 1 to 14 or any one of SEQ ID NOs: 58 to 71, or a sequence having identity thereto, and the 5'UTR comprises a sequence shown in SEQ ID NO: 43 or SEQ ID NO: 100, or a sequence having identity thereto; 44) The 3'UTR comprises a sequence shown in any one of SEQ ID NOs: 1 to 14 or any one of SEQ ID NOs: 58 to 71, or a sequence having identity thereto, and the 5'UTR comprises a sequence shown in SEQ ID NO: 44 or SEQ ID NO: 101, or a sequence having identity thereto; 45) The 3'UTR comprises a sequence shown in any one of SEQ ID NOs: 1 to 14 or any one of SEQ ID NOs: 58 to 71, or a sequence having identity thereto, and the 5'UTR comprises a sequence shown in SEQ ID NO: 45 or SEQ ID NO: 102, or a sequence having identity thereto; 46) The 3'UTR comprises a sequence shown in any one of SEQ ID NOs: 1 to 14 or any one of SEQ ID NOs: 58 to 71, or a sequence having identity thereto, and the 5'UTR comprises a sequence shown in SEQ ID NO: 46 or SEQ ID NO: 103, or a sequence having identity thereto, or 47) The 3'UTR comprises a sequence shown in any one of SEQ ID NOs: 1 to 14 or any one of SEQ ID NOs: 58 to 71, or a sequence having identity to any one of them, and the 5'UTR comprises a sequence shown in SEQ ID NO: 47 or SEQ ID NO: 104, or a sequence having identity to any one of them.

[0022] In some embodiments, a nucleic acid construct (e.g., a DNA or RNA molecule) is provided that comprises: (a) an open reading frame (ORF); (b-1) 3'UTR, and (b-2) 5'UTR, the 3'UTR is a 3'UTR derived from a gene CTSB, FAM166A or NDUFB9, and the 5'UTR is a 5'UTR derived from a gene ACTG1, CHCHD10 or NDUFA11; For example, the 3'UTR comprises any one of SEQ ID NOs: 12, 13, 14, or any one of SEQ ID NOs: 69, 70, 71, or a sequence having identity to any one of them, and the 5'UTR comprises any one of SEQ ID NOs: 15, 29, 30, 32, or any one of SEQ ID NOs: 72, 86, 87, 89, or a sequence having identity to any one of them; For example, the 3'UTR comprises a sequence shown in SEQ ID NO: 12 or SEQ ID NO: 69 or has an identity thereto, and the 5'UTR comprises a sequence shown in SEQ ID NO: 15 or SEQ ID NO: 72 or has an identity thereto, The 3'UTR comprises a sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 70, or a sequence having identity thereto; and the 5'UTR comprises a sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 72, or a sequence having identity thereto; The 3'UTR comprises a sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 71, or a sequence having identity thereto; and the 5'UTR comprises a sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 72, or a sequence having identity thereto; The 3'UTR comprises a sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 69, or a sequence having identity thereto, and the 5'UTR comprises a sequence set forth in SEQ ID NO: 29 or SEQ ID NO: 86, or a sequence having identity thereto; The 3'UTR comprises a sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 70, or a sequence having identity thereto; and the 5'UTR comprises a sequence set forth in SEQ ID NO: 29 or SEQ ID NO: 86, or a sequence having identity thereto; The 3'UTR comprises a sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 71, or a sequence having identity thereto; and the 5'UTR comprises a sequence set forth in SEQ ID NO: 29 or SEQ ID NO: 86, or a sequence having identity thereto; The 3'UTR comprises a sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 69, or a sequence having identity thereto, and the 5'UTR comprises a sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 87, or a sequence having identity thereto; The 3'UTR comprises a sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 70, or a sequence having identity thereto, and the 5'UTR comprises a sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 87, or a sequence having identity thereto; The 3'UTR comprises a sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 71, or a sequence having identity thereto; and the 5'UTR comprises a sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 87, or a sequence having identity thereto; The 3'UTR comprises a sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 69, or a sequence having identity thereto, and the 5'UTR comprises a sequence set forth in SEQ ID NO: 32 or SEQ ID NO: 89, or a sequence having identity thereto; the 3'UTR comprises a sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 70, or a sequence having identity thereto, and the 5'UTR comprises a sequence set forth in SEQ ID NO: 32 or SEQ ID NO: 89, or a sequence having identity thereto; or The 3'UTR comprises a sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 71 or having identity to any one of them, and the 5'UTR comprises a sequence set forth in SEQ ID NO: 32 or SEQ ID NO: 89 or having identity to any one of them.

[0023] In the above embodiments, "having identity" refers to having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity, and ranges between any two of the above numbers, including integers and decimals, for example, "having at least 90% identity" or "having at least 95% identity."

[0024] In some embodiments, the 3'UTR and / or 5'UTR are mutants of the 3'UTR and / or 5'UTR, e.g., truncations, nucleotide mutants, which still retain a similar activity or function as the 3'UTR and / or 5'UTR of the present disclosure, e.g., the ability to regulate a target gene expressed protein encoded by an ORF.

[0025] In some embodiments, the nucleic acid construct provided by the present disclosure comprises: (c) further comprising a polyadenylic acid (poly-A) tail.

[0026] In some specific embodiments, the poly-A tail in the nucleic acid construct is located downstream of the 3'UTR. In some specific embodiments, the poly-A tail in the nucleic acid construct is located at the 3' end of the 3'UTR. In some specific embodiments, the poly-A tail is at the 3' end of the nucleic acid construct. In some specific embodiments, the length of the poly-A tail is at least about 50, 100, 150, 200, 300, 400, 500 nucleotides.

[0027] In some specific embodiments, the poly-A tail is selected from A120, A30L70, HGH polyA, SV40polyA, BGH polyA, rbGlob polyA, or SV40late polyA. For example, the A30L70 is the sequence shown in SEQ ID NO:52.

[0028] In some embodiments, the nucleic acid construct provided by the present disclosure, wherein the target gene (i.e., open reading frame ORF) to be expressed is hepatocyte growth factor (HGF), an antibody or antigen-binding fragment thereof, such as an antibody or antigen-binding fragment thereof that binds to a tumor antigen, an antibody or antigen-binding fragment thereof that binds to a viral antigen, etc.

[0029] In some specific embodiments, the polypeptide or protein encoded by the ORF is a fluorescent protein or luciferase, for example the sequence shown in SEQ ID NO:126.

[0030] In some specific embodiments, the HGF is human hepatocyte growth factor (hHGF).

[0031] In some specific embodiments, the coding sequence of hHGF is selected from the group consisting of 1) to 3) below, i.e. 1) a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 109 or a codon-optimized nucleic acid sequence; 2) A DNA sequence represented by SEQ ID NOs: 110 to 113 or having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto; 3) An RNA sequence represented by any one of SEQ ID NOs: 128 to 131 or having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.

[0032] In some specific embodiments, the nucleic acid construct expressing the above-mentioned HGF as a target gene comprises any one of SEQ ID NOs: 115, 116, and 127 or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.

[0033] In some specific embodiments, the antibody or antigen-binding fragment thereof is an anti-PD-1 antibody or antigen-binding fragment thereof.

[0034] In some specific embodiments, the coding sequence of the anti-PD-1 antibody or antigen-binding fragment thereof is selected from the group consisting of the following 1) to 4), i.e. 1) a nucleic acid sequence encoding a heavy chain amino acid sequence shown in SEQ ID NO: 117 or a codon-optimized nucleic acid sequence, and a nucleic acid sequence encoding a light chain amino acid sequence shown in SEQ ID NO: 118 or a codon-optimized nucleic acid sequence; 2) A nucleic acid sequence or a codon-optimized nucleic acid sequence encoding HCDR1, HCDR2 and HCDR3 in the heavy chain amino acid sequence shown in SEQ ID NO: 117, and a nucleic acid sequence or a codon-optimized nucleic acid sequence encoding LCDR1, LCDR2 and LCDR3 in the light chain amino acid sequence shown in SEQ ID NO: 118, wherein the CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system, for example, the Kabat numbering system; 3) a DNA sequence as set forth in SEQ ID NO: 119 or having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, and a DNA sequence as set forth in SEQ ID NO: 120 or having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto; 4) A DNA sequence as set forth in SEQ ID NO: 121 or having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and a DNA sequence as set forth in SEQ ID NO: 122 or having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.

[0035] In some specific embodiments, the nucleic acid construct expressing the anti-PD-1 antibody or antigen-binding fragment thereof comprises a sequence set forth in SEQ ID NOs:124 and 125, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity to SEQ ID NOs:124 and 125.

[0036] In some embodiments, the nucleic acid construct provided by the present disclosure is DNA or RNA, for example, mRNA.

[0037] In some embodiments, the nucleic acid construct (DNA or RNA) provided by the present disclosure comprises, in the 5' to 3' direction, the following 1) to 6), i.e. 1) 5'UTR and ORF, 2) ORF and 3'UTR, 3) 5'UTR, ORF and 3'UTR, 4) 5'UTR, ORF, 3'UTR and poly-A tail, 5) 5'UTR, ORF and poly-A tail, 6) Contains one of the following: ORF, 3'UTR and poly-A tail; The 5'UTR and 3'UTR may be derived from the same or different genes.

[0038] In some embodiments, the nucleic acid construct (DNA) provided by the present disclosure comprises, in the 5' to 3' direction, the following 1) to 4), i.e. 1) 5'UTR and ORF, 2) ORF and 3'UTR, 3) 5'UTR, ORF and 3'UTR, 4) Contains one of the following: 5'UTR, ORF, 3'UTR and poly-A tail; The 5'UTR and 3'UTR may be derived from the same or different genes.

[0039] In some specific embodiments, the 5'UTR in 1), 3), and 4) above comprises a nucleotide sequence shown in any one of SEQ ID NOs: 15 to 47, or is the above amino acid sequence. In some specific embodiments, the ORF in 1) to 4) above comprises a nucleotide sequence shown in SEQ ID NO: 110 or a codon-optimized nucleotide sequence thereof (e.g., SEQ ID NOs: 111 to 113), or is the above amino acid sequence. In some specific embodiments, the ORF in 1) to 4) above comprises a nucleotide sequence shown in SEQ ID NOs: 119 and 120, or is the above amino acid sequence. In some specific embodiments, the 3'UTR in 2) to 4) above comprises a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 14, or is the above amino acid sequence.

[0040] In some embodiments, the nucleic acid construct (RNA or mRNA) provided by the present disclosure comprises: (d) further comprising a 5' cap structure (5'Cap).

[0041] In some specific embodiments, the 5' Cap structure in the RNA molecule is located upstream of the 5' UTR. In some embodiments, the 5' Cap structure in the RNA molecule is located at the 5' end of the 5' UTR. In some embodiments, the 5' cap structure is a cap structure known to those skilled in the art, such as Cap0 (methylation of the first nucleobase, e.g., m 7 GpppN), Cap1(m 7Additional methylation of the ribose of the adjacent nucleotide of GpppN, e.g., m 7 G(5')ppp(5')(2'OMeA)pG), Cap2(m 7 GpppN, the third ribose methylation downstream), Cap3 (m 7 Additional methylation of the ribose at the third nucleotide downstream of GpppN), Cap4 (m 7 These include: additional methylation of the ribose of the fourth nucleotide downstream of GpppN), ARCA (reverse inverted cap analog), modified ARCA (e.g., phosphorothioate-modified ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine and 2-azido-guanosine.

[0042] In some specific embodiments, the 5'Cap structure (eg, Cap0 or Cap1) is formed by chemical RNA synthesis or RNA in vitro transcription (co-transcriptional capping).

[0043] In some specific embodiments, the 5'-cap structure (e.g., Cap0 or Cap1) is formed by enzymatic capping using a capping enzyme (e.g., cowpox virus capping enzyme and / or a cap-dependent 2'-O methyltransferase). In some embodiments, an immobilized capping enzyme is used to add the 5' cap structure (Cap0 or Cap1). The capping methods and means in WO2016 / 193226 are incorporated herein in their entirety.

[0044] In some specific embodiments, the 5' Cap is ARCA, 3'-O-Me-m 7 G(5')ppp(5')G,m 7 G(5')ppp(5')(2'OMeA)pU,m 7 Gppp(A2'O-MOE)pG,m 7 G(5')ppp(5')(2'OMeA)pG,m 7 G(5')ppp(5')(2'OMeG)pG,m7 (3'OMeG)(5')ppp(5')(2'OMeG)pG or m 7 (3'OMeG)(5')ppp(5')(2'OMeA)pG. In some specific embodiments, the 5'Cap is selected from 3'-O-Me-m 7 G(5')ppp(5')G or m 7 G(5')ppp(5')(2'OMeA)pG.

[0045] In some embodiments, the nucleic acid construct (RNA or mRNA) provided by the present disclosure comprises, in the 5' to 3' direction, the following 1) to 5): 1) 5'UTR and ORF, 2) ORF and 3'UTR, 3) 5'UTR, ORF and 3'UTR, 4) 5'UTR, ORF, 3'UTR and poly-A tail, 5) Contains any one of 5'Cap, 5'UTR, ORF, 3'UTR, and a poly-A tail; The 5'UTR and 3'UTR may be derived from the same or different genes.

[0046] In some specific embodiments, the 5'UTR in 1), 3), 4), and 5) above comprises a nucleotide sequence shown in any one of SEQ ID NOs: 72 to 104, or is the above amino acid sequence. In some specific embodiments, the ORF in 1) to 5) above comprises a nucleotide sequence shown in any one of SEQ ID NOs: 128 to 131, or is the above amino acid sequence. In some specific embodiments, the 3'UTR in 2) to 5) above comprises a nucleotide sequence shown in any one of SEQ ID NOs: 58 to 71, or is the above amino acid sequence. In some specific embodiments, the structure is Cap0, Cap1 (e.g., m 7 G(5')ppp(5')(2'OMeA)pG), Cap2, Cap3, Cap4, and ARCA.

[0047] In some embodiments, the present disclosure provides a nucleic acid construct (DNA) that includes, in the 5' to 3' direction, a 5'UTR, an ORF, and a 3'UTR, and optionally further includes a poly-A tail in the 3' direction. In some embodiments, the 5'UTR includes a nucleotide sequence set forth in any one of SEQ ID NOs: 15 to 47, or is the amino acid sequence described above, the ORF includes a nucleotide sequence set forth in SEQ ID NO: 110 or a codon-optimized nucleotide sequence thereof (e.g., SEQ ID NOs: 111 to 113), or is the amino acid sequence described above, and the 3'UTR includes a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 14, or is the amino acid sequence described above. In some embodiments, the 5'UTR comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 15 to 47, or is the amino acid sequence set forth above, the ORF comprises a nucleotide sequence set forth in SEQ ID NOs: 119 and 120, or is the amino acid sequence set forth above, and the 3'UTR comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 14, or is the amino acid sequence set forth above.

[0048] In some embodiments, the present disclosure provides a nucleic acid construct (RNA or mRNA) that includes, in the 5' to 3' direction, a 5'UTR, an ORF, and a 3'UTR, and optionally further includes a poly-A tail in the 3' direction. In some embodiments, the 5'UTR includes a nucleotide sequence set forth in any one of SEQ ID NOs: 72-104, or is the amino acid sequence, the ORF includes a nucleotide sequence set forth in any one of SEQ ID NOs: 128-131, or is the amino acid sequence, the 3'UTR includes a nucleotide sequence set forth in any one of SEQ ID NOs: 58-71, or is the amino acid sequence, and the poly-A tail includes 120 consecutive adenyl acids or the nucleotide sequence set forth in SEQ ID NO: 52, or is the amino acid sequence. In some specific embodiments, the nucleic acid construct includes a nucleotide sequence set forth in SEQ ID NO: 115, 116, or 127.

[0049] In some embodiments, the 5'UTR comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 72-104 or is the amino acid sequence, the ORF comprises a nucleotide sequence set forth in SEQ ID NOs: 121 and 122 or is the amino acid sequence, the 3'UTR comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 58-71 or is the amino acid sequence, and the poly-A tail comprises 120 consecutive adenyl acids or the nucleotide sequence set forth in SEQ ID NO: 52 or is the amino acid sequence. In some specific embodiments, the nucleic acid construct comprises a nucleotide sequence set forth in SEQ ID NO: 124 or 125.

[0050] In some embodiments, in any one of the above nucleic acid constructs (DNA or RNA molecules), the UTR is for improving the expression level of the protein of the ORF. Exemplarily, in some specific embodiments, the 5'UTR represented by any one of SEQ ID NOs: 15 to 47, 72 to 104 in the present disclosure has an improved expression level of the target protein of the ORF compared to the 5'UTR represented by SEQ ID NO: 48 or 50. In some specific embodiments, the 3'UTR represented by any one of SEQ ID NOs: 1 to 14, 58 to 71 in the present disclosure has an improved expression level of the target protein of the ORF compared to the 5'UTR represented by SEQ ID NO: 49 or 51. In some specific embodiments, the combination of 5'UTR represented by any one of SEQ ID NOs: 15 to 47, 72 to 104 in the present disclosure and 3'UTR represented by any one of SEQ ID NOs: 1 to 14, 58 to 71 in the present disclosure has an improved expression level of the target protein of the ORF compared to the combination of 5'UTR and 3'UTR represented by SEQ ID NOs: 48 and 49. In some specific embodiments, the combination of 5'UTR represented by any one of SEQ ID NOs: 15 to 47, 72 to 104 in the present disclosure and 3'UTR represented by any one of SEQ ID NOs: 1 to 14, 58 to 71 in the present disclosure has an improved expression level of the target protein of the ORF compared to the combination of 5'UTR and 3'UTR represented by SEQ ID NOs: 50 and 51.

[0051] In some embodiments, the expression level of a nucleic acid construct of the present disclosure that expresses a target protein is about 1 to about 20 times that of a BioN vector, for example, about 1 time, about 1.1 times, about 1.2 times, about 1.3 times, about 1.4 times, about 1.5 times, about 1.6 times, about 1.7 times, about 1.8 times, about 1.9 times, about 2 times, about 2.1 times, about 2.3 times, about 2.5 times, about 2.8 times, about 3 times, about 3.2 times, about 3.4 times, about 3.8 times, about 4 times, about 4.5 times, about 5 times, about 5.2 times, about 5.5 times, about 5.8 times, about 6 times, about 7 times, about 8 times, about 10 times, about 12 times, about 15 times, etc. In some specific embodiments, the nucleic acid construct of the present disclosure is an mRNA molecule that expresses a target protein, and the expression level of the mRNA molecule that expresses the target protein is about 1 to about 20 times that of the BioN vector. In some specific embodiments, the BioN vector includes a 5'UTR shown in SEQ ID NO: 50 or 107, and / or a 3'UTR shown in SEQ ID NO: 51 or 108.

[0052] In some embodiments, the expression level of a nucleic acid construct of the present disclosure that expresses a target protein is about 1 to about 20 times that of a Mod vector, for example, about 1 time, about 1.1 times, about 1.2 times, about 1.3 times, about 1.4 times, about 1.5 times, about 1.6 times, about 1.7 times, about 1.8 times, about 1.9 times, about 2 times, about 2.1 times, about 2.3 times, about 2.5 times, about 2.8 times, about 3 times, about 3.2 times, about 3.4 times, about 3.8 times, about 4 times, about 4.5 times, about 5 times, about 5.2 times, about 5.5 times, about 5.8 times, about 6 times, about 7 times, about 8 times, about 10 times, about 12 times, about 15 times, etc. In some specific embodiments, the nucleic acid construct of the present disclosure is an mRNA molecule that expresses a target protein, and the expression level of the target protein of the mRNA molecule is about 1 to about 20 times that of the BioN vector. In some specific embodiments, the Mod vector includes a 5'UTR shown in SEQ ID NO: 48 or 105, and / or a 3'UTR shown in SEQ ID NO: 49 or 106.

[0053] In some embodiments, the nucleic acid construct of the present disclosure expresses a target protein (e.g., hHGF protein) about 0.5 to 1.5 hours after delivery into the subject's body. In some embodiments, the nucleic acid construct of the present disclosure reaches a peak expression about 2 to 10 hours (e.g., about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 10 hours, etc.) after delivery into the subject's body. In some specific embodiments, the nucleic acid construct of the present disclosure is an mRNA molecule that expresses a target protein, for example, the nucleotide construct is an mRNA molecule that expresses an hHGF protein.

[0054] In some embodiments, the nucleic acid constructs of the present disclosure are delivered to a subject and have higher pharmacokinetic properties than Collategene plasmids. In some specific embodiments, the nucleic acid constructs are mRNA molecules, and the mRNA molecules have higher pharmacokinetic properties (e.g., Cmax, AUC 0-inf , MRT 0-inf ) is improved.

[0055] In some embodiments, the nucleic acid construct of the present disclosure (e.g., m-A16-B12 (hHGF) expressing hHGF protein) can improve the blood flow perfusion ratio of the ischemic lower limb of a subject. Illustratively, lower limb ischemic mice injected with m-A16-B12 (hHGF) at 50 ng / mouse or 500 ng / mouse can achieve an improved blood flow perfusion ratio. Among them, lower limb ischemic mice injected with m-A16-B12 (hHGF) at about 500 ng / mouse can restore blood flow perfusion to about 90% or more.

[0056] In some embodiments, the nucleic acid construct of the present disclosure (e.g., m-A16-B12 (hHGF) expressing hHGF protein) can ameliorate ischemic necrosis in a subject with hindlimb ischemia. Illustratively, the hindlimb of a mouse with hindlimb ischemia injected with m-A16-B12 (hHGF) at 50 ng / mouse or 500 ng / mouse can maintain good integrity and no necrosis of the hindlimb can be observed.

[0057] In some embodiments, the nucleic acid construct of the present disclosure (e.g., m-A16-B12 (hHGF) expressing hHGF protein) can promote angiogenesis in ischemic hindlimb muscles of each group. Illustratively, when m-A16-B12 (hHGF) is administered at 50 ng / mouse or 500 ng / mouse for treatment, angiogenesis in ischemic hindlimb muscles of each group can be significantly promoted, and the number of newly formed blood vessels is statistically significantly different from that of the PBS group (p<0.05).

[0058] In some embodiments, the nucleic acid constructs of the present disclosure (e.g., m-A16-B12(hHGF) expressing hHGF protein) can improve wound healing in diabetic subjects. Illustratively, in Db / Db diabetic mouse models injected with m-A16-B12(hHGF) at 50ng / mouse, 200ng / mouse and 500ng / mouse, the wounds of the mice are well healed. Among them, on the 14th day, the wound healing rate of m-A16-B12(hHGF) at a dose of 50ng / mouse can reach 66%, and the wound healing rate of m-A16-B12(hHGF) at doses of 200ng / mouse and 500ng / mouse can reach 100% wound healing.

[0059] The mRNA provided by the present disclosure contains novel structures of 5'UTR and 3'UTR, which reduces early degradation of the mRNA or stabilizes degradation, but does not impair or even enhance protein translation efficiency. The above mRNA has higher stability and can be used for gene therapy or gene vaccination. The present disclosure also provides an mRNA capable of expressing human hepatocyte growth factor (hHGF) and its lipid nanoparticle (LNP) delivery system, which can realize efficient and rapid transformation of exogenous hHGF protein in vivo, has the advantages of no integration risk and being easy to mass-produce industrially, is a more desirable treatment regimen than naked plasmids, and can be used as a gene therapy drug for various diseases such as CLI and DFU.

[0060] Polynucleotides The disclosure further provides an isolated polynucleotide comprising: (a) an open reading frame (ORF). Illustratively, the ORF encodes a hepatocyte growth factor (HGF), e.g., human hepatocyte growth factor (hHGF).

[0061] In some embodiments, the coding sequence of the hHGF is selected from the group consisting of the following 1) to 3), i.e. 1) a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 109 or a codon-optimized sequence thereof; 2) A DNA sequence represented by any one of SEQ ID NOs: 110 to 113 or having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto; 3) An RNA sequence represented by any one of SEQ ID NOs: 128 to 131 or having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.

[0062] In some specific embodiments, the 5' end of the polynucleotide may include any 5' UTR provided by the present disclosure, and / or the 3' end of the polynucleotide may include any 3' UTR provided by the present disclosure.

[0063] In some specific embodiments, the 5' end of the polynucleotide may include any 5' Cap provided by the present disclosure, and / or the 3' end of the polynucleotide may include any poly-A tail provided by the present disclosure.

[0064] In some embodiments, the polynucleotide is RNA, such as mRNA.

[0065] qualification To further improve the stability of the RNA or polynucleotide of the present disclosure in relation to protein expression, the RNA or polynucleotide may further include one or more modifications (including chemical modifications), such as backbone modifications, sugar modifications, base modifications, and / or lipid modifications. In some embodiments, the RNA or polynucleotide is uniformly modified with a particular modification (e.g., completely modified throughout the entire sequence). For example, the RNA can be uniformly modified with pseudouridine (e.g., N1-methylpseudouridine) so that each U in the sequence is a pseudouridine.

[0066] The backbone modification of the present disclosure refers to a chemical modification of the backbone phosphate of the nucleotide contained in the RNA or polynucleotide of the present disclosure. In some embodiments, the backbone modification includes, but is not limited to, the complete replacement of the unmodified phosphate moieties in the backbone with modified phosphate, for example, the backbone phosphate group can be modified by replacing one or more oxygen atoms with different substituents. In some embodiments, the modified phosphate includes, but is not limited to, phosphorothioate, phosphite selenate, borane phosphate, borane phosphate, hydrogen phosphonate, phosphoramidate, alkyl or aryl phosphonate, and phosphate triester.

[0067] Sugar modifications according to the present disclosure refer to chemical modifications of the sugars of nucleotides contained in an RNA or polynucleotide of the present disclosure. In some embodiments, the sugar modifications include, but are not limited to, modifying or replacing the 2' hydroxyl (OH) group of an RNA molecule with a number of different "oxy" or "deoxy" substituents. In some embodiments, the "oxy" modifications include, but are not limited to, substitution modifications such as alkoxy, aryloxy, polyethylene glycol (PEG), etc. In some embodiments, the "deoxy" modifications include, but are not limited to, modifications of hydrogen, amino groups (e.g., NH2, alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acids).

[0068] The base modification of the present disclosure refers to a chemical modification of the base portion of a nucleotide contained in an RNA or polynucleotide of the present disclosure. In some embodiments, the base modification includes adenine, guanine, cytosine, and uracil modifications in the nucleotide. For example, the nucleosides and nucleotides described herein may be chemically modified on the surface of the major groove. In some embodiments, the chemical modification of the major groove may include an amino group, a thiol group, an alkyl group, or a halogen group. In some embodiments, the base modification includes, but is not limited to, a modification with pseudouridine, 1-methyl-pseudouridine, 5-azacytidine, 5-methylcytosine-5'-triphosphate, or 2-methoxyadenine. In some embodiments, the base modification is a pseudouridine modification. For example, an RNA can be uniformly modified with pseudouridine such that each U in the sequence is a pseudouridine.

[0069] The lipid modification of the present disclosure refers to the lipid modification contained in the RNA or polynucleotide of the present disclosure. In some embodiments, the lipid modification includes, but is not limited to, the covalent attachment of the RNA or polynucleotide of the present disclosure to at least one linker, and the covalent attachment of the corresponding linker to at least one lipid. In some embodiments, the lipid modification includes, but is not limited to, the covalent attachment (without linker) of the RNA or polynucleotide of the present disclosure to at least one lipid.

[0070] UTRs UTRs (5'UTR and / or 3'UTR) may be provided as flanking regions in a nucleic acid construct, RNA or polynucleotide molecule of the present disclosure. The UTRs may be homologous or heterologous to the coding region in a nucleic acid construct, RNA or polynucleotide molecule of the present disclosure. The flanking regions may comprise one or more 5'UTR and / or 3'UTR, said UTRs may be of the same or different sequences. Any portion of the flanking regions may be codon optimized. Any portion of the flanking regions may independently comprise one or more different structural or chemical modifications before and / or after codon optimization.

[0071] To alter one or more properties of the nucleic acid construct, RNA, or polynucleotide of the present disclosure, UTRs heterologous to the ORF of the present disclosure are introduced or engineered into the nucleic acid construct, RNA, or polynucleotide of the present disclosure. The recombinant nucleic acid construct, RNA, or polynucleotide is then administered to a cell, tissue, or organism, and the beneficial effect of the heterologous UTR on the RNA or polynucleotide of the present disclosure is evaluated by measuring the results, such as protein level, localization, and / or half-life. In some embodiments, the UTR comprises a wild-type UTR or a mutant thereof, the UTR mutant comprising one or more nucleotides added or removed, including A, T, C, or G at the end. In some embodiments, the UTR mutant also comprises codon optimization or modification performed in any manner. In some embodiments, the UTR mutant also comprises a derived sequence of any embodiment of the present disclosure, for example, a portion of nucleotides are point mutated based on the native UTR sequence, and the expression level or stability of the target gene by the mutated mutant is maintained or improved. The detection methods for the expression level and stability of the above target genes are common in the field, for example, the detection methods in Examples 3 and 4 of the present disclosure.

[0072] vector The present disclosure further provides a vector comprising any one of the above described nucleic acid constructs, RNA or polynucleotides. Wherein, the nucleic acid constructs, RNA or polynucleotides may be present in and / or part of a vector, such as a plasmid, cosmid, YAC or viral vector. The vector may be an expression vector, i.e. a vector that allows expression of a polypeptide encoded by the nucleic acid construct, RNA or polynucleotide. Such an expression vector usually comprises at least one nucleic acid according to the present disclosure, operably linked to one or more suitable expression control elements (e.g. promoter, terminator, etc.). It is common knowledge of the skilled artisan to select the above elements and their sequences for expression in a particular host. Regulatory and other elements useful or necessary for expression of the encoded polypeptide according to the present disclosure are, for example, promoters, terminators, selection markers, leader sequences, reporter genes, etc.

[0073] In some embodiments, the vector is a therapeutic vector capable of expressing a target gene of the present disclosure (e.g., HGF), such as a plasmid (e.g., a naked plasmid), an adenoviral vector, an adeno-associated viral vector, and a lentiviral vector.

[0074] The nucleic acid constructs of the present disclosure can be prepared or obtained by known methods (e.g., automated DNA synthesis and / or recombinant DNA techniques) based on the nucleotide sequence information of the present disclosure, and / or can be isolated from a suitable natural source.

[0075] In some embodiments, the vector of the present disclosure further comprises a promoter, e.g., the promoter is at the 5' end of the nucleic acid construct 5'UTR, e.g., the promoter is a T7 promoter, a T7 lac promoter, a Tac promoter, a Lac promoter, a Trp promoter.

[0076] host cell The present disclosure further provides a host cell comprising any one of the nucleic acid constructs, RNA, or polynucleotides described above. In some embodiments, the cell is capable of expressing a polypeptide encoded by one or more of the nucleic acid constructs, RNA, or polynucleotides of the present disclosure. In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0077] Bacterial cells include, for example, cells of gram-negative strains (e.g., Escherichia coli, Proteus and Pseudomonas strains) and gram-positive strains (e.g., Bacillus, Streptomyces, Staphylococcus and Lactococcus strains).

[0078] Fungal cells include, for example, cells of species of Trichoderma, Neurospora, and Aspergillus, or cells of species of Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0079] Mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0080] However, the present disclosure may be used with amphibian cells, insect cells, plant cells and any other cells known in the art for expressing heterologous proteins.

[0081] Method of production or preparation The present disclosure provides methods for preparing the nucleic acid constructs, RNA or polynucleotides of the disclosure, and methods for preparing the encoded polypeptides.

[0082] Methods and reagents for preparing and producing nucleic acid constructs, RNA or polynucleotides, and their encoded polypeptides, such as particular suitable vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, culture conditions, etc., are known in the art. Similarly, protein isolation and purification techniques in methods applied to produce the encoded polypeptides of the present disclosure are known to those of skill in the art.

[0083] In some embodiments, the method for preparing the nucleic acid construct or polynucleotide comprises culturing the host cell and recovering the produced nucleic acid construct or polynucleotide from the culture. The nucleic acid construct or polynucleotide according to the present disclosure, and its encoded polypeptide, may also be obtained by other production methods known in the art, for example chemical synthesis, including solid-phase or liquid-phase synthesis.

[0084] In some embodiments, the method for preparing an RNA molecule includes preparing a nucleic acid construct or vector, and performing reverse transcription with the nucleic acid construct or vector to obtain an RNA molecule. In some specific embodiments, the method further includes adding a 5' Cap to the 5' end of the RNA molecule.

[0085] In some embodiments, the RNA may further include one or more modifications (including chemical modifications), such as backbone modifications, sugar modifications, base modifications, and / or lipid modifications. In some embodiments, the RNA or polynucleotide is uniformly modified with a particular modification (e.g., completely modified throughout the entire sequence). For example, the RNA can be uniformly modified with pseudouridine (e.g., N1-methylpseudouridine) such that each U in the sequence is a pseudouridine (e.g., N1-methylpseudouridine).

[0086] delivery vehicle The present disclosure further provides a delivery vehicle comprising any one of the nucleic acid constructs, any of the RNA molecules, polynucleotides, or vectors described above, wherein the delivery vehicle is a cationic lipid delivery particle. In some embodiments, the particle is a nanoparticle. In some embodiments, the delivery vehicle is a nanolipid particle. The nucleic acid construct, RNA molecule polynucleotide or vector of the present disclosure can be delivered into a cell and / or into the body using any type of nanolipid particle in the art, and exemplary nanolipid particles include, but are not limited to, the lipid particles disclosed in WO2017075531, WO2018081480A1, WO2017049245A2, WO2017099823A1, WO2022245888Al, WO2022150717A1, CN101291653A, CN102119217A, WO2011000107A1, CN107028886A, US9868692B2, all of which are incorporated herein by reference.

[0087] In some embodiments, the delivery vehicle comprises a nanolipid particle as set forth in US9868692B2, such as an ionizable lipid having the formula B (also referred to as SM-102). In some embodiments, the delivery vehicle comprises a nanolipid particle, a phospholipid, a structured lipid, and / or a PEG lipid. Illustratively, the lipid components contained in the delivery vehicle comprise about 50 mol% of the ionizable lipid (e.g., SM-102), about 10 mol% of a phospholipid (e.g., DSPC), about 38.5 mol% of a structured lipid (e.g., cholesterol), and about 1.5 mol% of a PEG lipid (e.g., PEG-DMG).

[0088] Pharmaceutical Compositions The present disclosure further provides a pharmaceutical composition comprising any of the nucleic acid constructs, any of the RNA molecules, any of the polynucleotides, any of the vectors, and / or any of the delivery vehicles described herein, and a pharma- ceutically acceptable carrier, diluent or excipient, specifically, the pharmaceutical composition is a solid formulation, an injectable formulation, a topical formulation, a spray, a liquid formulation or a combination formulation.

[0089] Product or Reagent Kit The present disclosure provides an article of manufacture or a reagent kit comprising any one of the nucleic acid constructs described above, any one of the RNA molecules described above, any one of the polynucleotides described above, any one of the vaccines described above, any one of the vectors described above, any one of the delivery vehicles described above, and / or any one of the pharmaceutical compositions described above, which can be used to provide a related detection or diagnostic use.

[0090] Methods and pharmaceutical uses for treating and / or preventing diseases The present disclosure further provides a method of treating and / or preventing a disease, comprising administering to a subject in need thereof a therapeutically and / or prophylactically effective amount of any of the above nucleic acid constructs, any of the above RNA molecules, any of the above polynucleotides, any of the above vectors, any of the above delivery vehicles, any of the above pharmaceutical compositions, and / or any of the above products or reagent kits.

[0091] The present disclosure also provides the use of any of the above nucleic acid constructs, any of the above RNA molecules, any of the above polynucleotides, any of the above vectors, any of the above delivery vehicles, any of the above pharmaceutical compositions, and / or any of the above articles of manufacture or reagent kits for the preparation of a medicament for treating and / or preventing a disease.

[0092] In some embodiments, the disease is one in which the subject can benefit from the activity of native hHGF.

[0093] In some embodiments, the disease is selected from ischemic disease, metabolic syndrome, diabetes and its complications, restenosis, and nerve damage. In some specific embodiments, the disease is ischemic disease, such as coronary artery disease (CAD) or peripheral artery disease (PAD), such as myocardial infarction or lower limb arterial ischemia. In some specific embodiments, the disease is diabetes or its complications, such as diabetic peripheral neuropathy. In some specific embodiments, the disease is restenosis, such as postoperative restenosis and postperfusion restenosis. In some specific embodiments, the disease is nerve damage, such as neurodegenerative diseases (e.g., amyotrophic lateral sclerosis (ALS), Parkinson's disease, dementia), traumatic nerve injury, and peripheral neuropathy (e.g., diabetic peripheral neuropathy). In some specific embodiments, the disease is selected from lower limb arterial ischemia, myocardial infarction, and diabetic peripheral neuropathy.

[0094] In some embodiments, the disease is selected from peripheral arterial disease (PAD), atherosclerosis obliterans (ASO), diabetic artery obliterans (DAO), and thrombotic angiitis obliterans (TAO). In some embodiments, the disease is selected from limb ischemia (e.g., lower limb ischemia, critical limb ischemia (CLI)), and diabetic foot ulcer (DFU).

[0095] In some embodiments, there is provided a method of promoting growth and / or migration of endothelial cells (e.g., umbilical vein endothelial cells), comprising administering to an endothelial cell or a subject in need thereof an effective amount of any of the polynucleotides, any of the vectors, any of the delivery vehicles, any of the pharmaceutical compositions, and / or any of the products or reagent kits disclosed herein. Also provided is a pharmaceutical use relating to the preparation of a medicament for promoting growth and / or migration of endothelial cells.

[0096] In some embodiments, a method of promoting angiogenesis is provided, comprising administering to an endothelial cell or a subject in need thereof an effective amount of any of the polynucleotides, any of the vectors, any of the delivery vehicles, any of the pharmaceutical compositions, and / or any of the products or reagent kits of the present disclosure. In some embodiments, the angiogenesis is microangiogenesis. Also provided is a pharmaceutical use related to the preparation of an agent for promoting angiogenesis (e.g., microangiogenesis).

[0097] Beneficial effects The nucleic acid construct, vector, delivery vehicle, pharmaceutical composition, product, or reagent kit provided by the present disclosure, in which the target gene is HGF, can be advantageously used for one or more of promoting endothelial cell growth and / or migration, promoting angiogenesis (e.g., microvascular) neovascularization, treating ischemic diseases such as coronary artery disease (CAD) or peripheral arterial disease (PAD), such as limb ischemia (e.g., lower limb ischemia, critical limb ischemia), treating metabolic syndrome and diabetes and its complications (e.g., diabetic peripheral neuropathy, diabetic foot), inhibiting restenosis, and promoting repair of nerve damage (e.g., neurodegenerative diseases, traumatic nerve injury, peripheral neuropathy). [Brief description of the drawings]

[0098] [Figure 1A]Schematic diagram of vector construction. FIG. 1A is a schematic diagram of constructing a 5'UTR element screening vector, and the control is an mRNA sequence containing 5'UTR and 3'UTR elements manufactured by Moderna, i.e., 5'UTR-Fluc-α globin 3'UTR-120A (abbreviated as Mod.), whose 5'-UTR is an artificial nucleic acid sequence, and whose 3'UTR is derived from human α globin mRNA. When constructing a 5'UTR screening vector, the 5'UTR region is replaced with a suitable enzyme cleavage site. PmeI is a linearization enzyme cleavage site. FIG. 1B is a schematic diagram of constructing a 3'UTR element screening vector, and the control is Mod., and when constructing a 3'UTR screening vector, the 3'UTR region is replaced with a suitable enzyme cleavage site. Figure 1C is a schematic diagram of constructing a combination screening vector of 5' and 3' UTR elements, and the control is Mod. When constructing a combination screening vector of 5' UTR and 3' UTR elements, the 5' and 3' UTR regions are replaced with suitable enzyme cleavage sites or the entire gene is synthesized. [Figure 1B] Schematic diagram of vector construction. FIG. 1A is a schematic diagram of constructing a 5'UTR element screening vector, and the control is an mRNA sequence containing 5'UTR and 3'UTR elements manufactured by Moderna, i.e., 5'UTR-Fluc-α globin 3'UTR-120A (abbreviated as Mod.), whose 5'-UTR is an artificial nucleic acid sequence, and whose 3'UTR is derived from human α globin mRNA. When constructing a 5'UTR screening vector, the 5'UTR region is replaced with a suitable enzyme cleavage site. PmeI is a linearization enzyme cleavage site. FIG. 1B is a schematic diagram of constructing a 3'UTR element screening vector, and the control is Mod., and when constructing a 3'UTR screening vector, the 3'UTR region is replaced with a suitable enzyme cleavage site. Figure 1C is a schematic diagram of constructing a combination screening vector of 5' and 3' UTR elements, and the control is Mod. When constructing a combination screening vector of 5' UTR and 3' UTR elements, the 5' and 3' UTR regions are replaced with suitable enzyme cleavage sites or the entire gene is synthesized. [Figure 1C]Schematic diagram of vector construction. FIG. 1A is a schematic diagram of constructing a 5'UTR element screening vector, and the control is an mRNA sequence containing 5'UTR and 3'UTR elements manufactured by Moderna, i.e., 5'UTR-Fluc-α globin 3'UTR-120A (abbreviated as Mod.), whose 5'-UTR is an artificial nucleic acid sequence, and whose 3'UTR is derived from human α globin mRNA. When constructing a 5'UTR screening vector, the 5'UTR region is replaced with a suitable enzyme cleavage site. PmeI is a linearization enzyme cleavage site. FIG. 1B is a schematic diagram of constructing a 3'UTR element screening vector, and the control is Mod., and when constructing a 3'UTR screening vector, the 3'UTR region is replaced with a suitable enzyme cleavage site. Figure 1C is a schematic diagram of constructing a combination screening vector of 5' and 3' UTR elements, and the control is Mod. When constructing a combination screening vector of 5' UTR and 3' UTR elements, the 5' and 3' UTR regions are replaced with suitable enzyme cleavage sites or the entire gene is synthesized. [Diagram 2] The present disclosure shows the results of evaluating the effects of various 3'UTR elements in various cell lines. mRNAs containing various 3'UTR elements were transfected into HEK293, HeLa and A549 cells, and luciferase expression was detected 24 hours after transfection to evaluate the effect of the 3'UTR sequence on protein expression levels. Mod. was used as a control, and the expression level of Mod. was set to 1. The results showed that the effect of the 3'UTR elements on protein expression levels was consistent in various cell lines. [Diagram 3] The results of the effect of various 3'UTR elements of the present disclosure on mRNA expression efficiency. The mRNAs containing various 3'UTR elements were delivered to HEK293 cells by lipofection, and the expression levels of luciferase were detected 6 h, 24 h, 48 h and 72 h after transfection. Mod. was used as a control, and its expression level was set to 1. The results showed that the 3'UTR elements numbered B9, B10, B12, B13 and B14 can significantly improve the expression amount of protein. [Figure 4]The results of the effect of various 5'UTR elements of the present disclosure on mRNA expression efficiency. mRNA containing various 5'UTR elements was delivered to HEK293 cells by lipofection, and the expression level of luciferase was detected 6 h, 24 h, 48 h and 72 h after transfection. Mod. was used as a control, and its expression level was set to 1. The results showed that the 5'UTR elements numbered A1, A3 to A7, and A9 to A14 significantly improved the expression level of protein. [Diagram 5] The results of the effect of various 5'UTR elements of the present disclosure on mRNA expression efficiency. The mRNAs containing various 5'UTR elements were delivered to HEK293 cells by lipofection, and the expression levels of luciferase were detected 6 h, 24 h, 48 h and 72 h after transfection. Mod. was used as a control, and its expression level was set to 1. The results showed that the 5'UTR elements numbered A15, A16, A18-A19, A21, A24, A27, A28, and A30-A33 can significantly improve the expression level of protein. [Figure 6] The results of the effect of various 5'UTR elements of the present disclosure on mRNA expression efficiency. The mRNAs containing various 5'UTR elements were transfected into HEK293 cells by lipofection, and the expression levels of luciferase were detected 6 h, 24 h and 48 h after transfection. BioN. was used as a control, and its expression level was set to 1. The results showed that the 5'UTR elements numbered A1, A15, A16 and A18 can significantly improve the expression level of protein compared to the control nucleic acid molecule. [Figure 7]The results show that the combination of UTR elements of the present disclosure affects the mRNA expression efficiency. The mRNAs containing various 5'UTR and 3'UTR elements were transfected into HEK293 cells by lipofection, and the expression levels of luciferase were detected 6 h, 24 h, 48 h and 72 h after transfection. Mod. was used as a control, and its expression level was set to 1. The results show that the combination of 5'UTR elements numbered A1, A15, A16, and A18 and 3'UTR elements numbered B12, B13, and B14 can all significantly improve the expression level of protein. [Figure 8A] The results show that the combination of UTR elements of the present disclosure affects the expression efficiency of various target proteins. Figure 8A shows the regulation of hHGF expression level in HEK293 cells by mRNAs containing the screened 5'UTR and 3'UTR elements of the present disclosure, and the results show that the combination of 5'UTR elements numbered A1, A15, A16, and A18 and 3'UTR elements numbered B12, B13, and B14 can significantly improve the expression level of hHGF compared to the control Mod. Figure 8B shows the regulation of anti-PD-1 antibody expression level in HEK293 cells by mRNAs containing the screened 5'UTR and 3'UTR elements of the present disclosure, and the results show that the combination of UTRs A1-B12 and A15-B12 can significantly improve the expression level of anti-PD-1 antibody compared to the control Mod. [Figure 8B]The results show that the combination of UTR elements of the present disclosure affects the expression efficiency of various target proteins. Figure 8A shows the regulation of hHGF expression level in HEK293 cells by mRNAs containing the screened 5'UTR and 3'UTR elements of the present disclosure, and the results show that the combination of 5'UTR elements numbered A1, A15, A16, and A18 and 3'UTR elements numbered B12, B13, and B14 can significantly improve the expression level of hHGF compared to the control Mod. Figure 8B shows the regulation of anti-PD-1 antibody expression level in HEK293 cells by mRNAs containing the screened 5'UTR and 3'UTR elements of the present disclosure, and the results show that the combination of UTRs A1-B12 and A15-B12 can significantly improve the expression level of anti-PD-1 antibody compared to the control Mod. [Figure 9] 1 is a schematic diagram of the experimental results of the expression level of hHGF protein in mouse muscle tissue of m-A16-B12 (hHGF) and control plasmid of the present disclosure over time. As a result, injection of m-A16-B12 (hHGF) and Collategene plasmid can effectively express hHGF protein, and 1 hour after intramuscular injection of m-A16-B12 (hHGF), hHGF expression peaks 6 hours after injection, showing dose-dependency, and low doses of m-A16-B12 (hHGF) can reach the same level as Collategene expression AUCinf (hr*pg / mg protein). [Figure 10A]The results of the therapeutic effect of m-A16-B12 (hHGF) and control plasmid of the present disclosure on a mouse model of hindlimb ischemia. Figure 10A is a schematic diagram showing the research plan of the effect of m-A16-B12 (hHGF) and Collategene plasmid on blood flow perfusion in a mouse model of hindlimb ischemia and a photograph of the experimental results. Figure 10B is a statistical result showing the blood flow perfusion ratio of a mouse model of hindlimb ischemia with m-A16-B12 (hHGF) and Collategene plasmid. As a result, in the case of treatment with 50ng / mouse, 500ng / mouse m-A16-B12 (hHGF) and 200ng / mouse Collategene naked plasmid, the blood flow perfusion ratio of each mouse's ischemic lower limb was significantly improved compared to the control group, and the blood flow perfusion ratio of the ischemic lower limb was gradually restored over time. The 50ng / mouse m-A16-B12 (hHGF) group showed a blood flow recovery effect similar to that of the 200ng / mouse Collategene naked plasmid group, and the 500ng / mouse m-A16-B12 (hHGF) group showed a blood flow recovery effect significantly better than that of the 200ng / mouse Collategene naked plasmid group, and the blood flow perfusion could be restored to more than 90%. [Figure 10B]The results of the therapeutic effect of m-A16-B12 (hHGF) and control plasmid of the present disclosure on a mouse model of hindlimb ischemia. Figure 10A is a schematic diagram showing the research plan of the effect of m-A16-B12 (hHGF) and Collategene plasmid on blood flow perfusion in a mouse model of hindlimb ischemia and a photograph of the experimental results. Figure 10B is a statistical result showing the blood flow perfusion ratio of a mouse model of hindlimb ischemia with m-A16-B12 (hHGF) and Collategene plasmid. As a result, in the case of treatment with 50ng / mouse, 500ng / mouse m-A16-B12 (hHGF) and 200ng / mouse Collategene naked plasmid, the blood flow perfusion ratio of each mouse's ischemic lower limb was significantly improved compared to the control group, and the blood flow perfusion ratio of the ischemic lower limb was gradually restored over time. The 50ng / mouse m-A16-B12 (hHGF) group showed a blood flow recovery effect similar to that of the 200ng / mouse Collategene naked plasmid group, and the 500ng / mouse m-A16-B12 (hHGF) group showed a blood flow recovery effect significantly better than that of the 200ng / mouse Collategene naked plasmid group, and the blood flow perfusion could be restored to more than 90%. [Figure 11A] The results of the therapeutic effect of m-A16-B12 (hHGF) and control plasmid of the present disclosure on a mouse model of hindlimb ischemia. FIG. 11A shows the scoring criteria and representative photos of different degrees of hindlimb necrosis in a mouse model of hindlimb ischemia. FIG. 11B shows the statistical results showing the effect of m-A16-B12 (hHGF) and Collategene plasmid on hindlimb necrosis in a mouse model of hindlimb ischemia. The experimental results showed that in the treatment groups injected with m-A16-B12 (hHGF) and Collategene naked plasmid, the hindlimb of each mouse was able to maintain good integrity and no necrotic state of the hindlimb occurred. [Figure 11B]The results of the therapeutic effect of m-A16-B12 (hHGF) and control plasmid of the present disclosure on a mouse model of hindlimb ischemia. FIG. 11A shows the scoring criteria and representative photos of different degrees of hindlimb necrosis in a mouse model of hindlimb ischemia. FIG. 11B shows the statistical results showing the effect of m-A16-B12 (hHGF) and Collategene plasmid on hindlimb necrosis in a mouse model of hindlimb ischemia. The experimental results showed that in the treatment groups injected with m-A16-B12 (hHGF) and Collategene naked plasmid, the hindlimb of each mouse was able to maintain good integrity and no necrotic state of the hindlimb occurred. [Figure 12] Representative photographs and statistical results of the effect of m-A16-B12 (hHGF) and control plasmid of the present disclosure on angiogenesis in a mouse model of hindlimb ischemia. As a result, it was shown that treatment with 50 ng / mouse or 500 ng / mouse of m-A16-B12 (hHGF) and 200 ng / mouse of Collategene naked plasmid can significantly promote angiogenesis in ischemic hindlimb muscles. [Figure 13] The present disclosure shows the treatment plan, representative photographs and statistical results of the effect of m-A16-B12 (hHGF) and control plasmid on wounds in a Db / Db mouse model with total cortical injury. The results showed that the wounds of mice treated with 50ng / mouse, 200ng / mouse and 500ng / mouse of m-A16-B12 (hHGF) and 200μg / mouse of Collategene naked plasmid were better healed than those of the control group, and the promotion of wound healing by m-A16-B12 (hHGF) was significantly superior to that of 200μg / mouse of Collategene naked plasmid. [Figure 14] Representative photographs of the effect of m-A16-B12 (hHGF) and control plasmid of the present disclosure on tissue reconstitution in a Db / Db mouse model of total cortical injury. The results showed that treatment with m-A16-B12 (hHGF) achieved total epithelial coverage of the wound epithelium in each group of mice, achieving tissue reconstitution. On the other hand, neither the control group nor the 200 μg / mouse Collategene naked plasmid group achieved epithelial reconstitution, and collagen abnormally proliferated in the wound area, resulting in a disorganized structure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0099] term In order that the present disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art.

[0100] The term "HGF" in the present disclosure covers natural or wild-type HGF and its homologues from different species, including naturally occurring human hepatocyte growth factor (hHGF) and variants thereof having biological activity. The amino acid sequence of natural or wild-type hHGF is conveniently available from various common databases (e.g., GenBank database). For example, the amino acid sequence of natural hHGF can be found in the GenBank database under accession number: NP_000592.3. For example, the present disclosure also provides hHGF whose amino acid sequence is shown in SEQ ID NO: 109, whose DNA sequence is shown in SEQ ID NO: 110, and whose codon-optimized sequence is shown in SEQ ID NOs: 111-113. HGF has multiple biological activities, including, but not limited to, one or more of the following activities: (1) promoting endothelial cell growth and / or migration, (2) promoting angiogenesis (e.g., microvascular) and / or (3) promoting repair of nerve damage (e.g., peripheral neuropathy such as diabetic peripheral neuropathy). Thus, HGF has many potential uses, including, but not limited to, (1) promoting endothelial cell growth and / or migration, (2) promoting angiogenesis (e.g., microvascular), (3) treating ischemic diseases, such as coronary artery disease (CAD) or peripheral arterial disease (PAD), e.g., lower limb arterial ischemia, (4) treating metabolic syndrome and diabetes and its complications (e.g., diabetic peripheral neuropathy), (5) inhibiting restenosis, and (6) promoting repair of nerve damage (e.g., neurodegenerative diseases, traumatic nerve injury, peripheral neuropathy). Thus, examples of the term "diseases that can benefit from the activity of native hHGF" include, but are not limited to, the above-mentioned diseases, such as ischemic diseases, metabolic syndrome, diabetes and its complications, restenosis, and nerve damage.

[0101] "Nucleic acid" or "nucleotide" includes RNA, DNA and cDNA molecules. It should be understood that due to the degeneracy of the genetic code, a large number of nucleotide sequences that code for a given protein can be produced. The term nucleic acid may be used interchangeably with the term "polynucleotide." An "oligonucleotide" is a short-stranded nucleic acid molecule. A "primer" is an oligonucleotide, whether naturally occurring in purified restriction enzyme digestion or produced synthetically, that can act as a synthesis initiator when placed under conditions that induce the synthesis of a primer extension product complementary to the nucleic acid strand (i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase, and at a suitable temperature and pH). To maximize amplification efficiency, the primer is preferably single-stranded, but may also optionally be double-stranded. If double-stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is a deoxyribonucleotide. The primer must be long enough to trigger the synthesis of an extension product in the presence of the inducing agent. The exact length of the primer is determined by many factors, including temperature, source of primer and the method used.

[0102] "Vector" or "expression vector" refers to a replicon, such as a plasmid, bacmid, phage, virus, virion, or cosmid, that can be linked to another DNA segment, i.e., an "insert," to effect replication of the linked segment in a cell. A vector may be a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector may be viral or non-viral in origin and / or final form, such as the PUC57 DNA vector used herein. The term "vector" covers any genetic element that is capable of replicating and transferring genetic sequences to a cell when associated with the appropriate control elements. In some embodiments, a vector may be an expression vector or a recombinant vector.

[0103] "Promoter" refers to any nucleic acid sequence that regulates the expression of another nucleic acid sequence by driving the transcription of that nucleic acid sequence, which may be a heterologous target gene encoding a protein or RNA. Promoters may be constitutive, inducible, inhibitable, tissue-specific, or any combination thereof. A promoter is a control region of a nucleic acid sequence, by which the initiation and rate of transcription of the remainder of the nucleic acid sequence is controlled.

[0104] "Gene" refers to a segment of DNA involved in the production of a polypeptide chain, which may or may not include preceding and following coding regions, such as 5' untranslated (5'UTR) or "leader" sequences and 3'UTR or "untranscribed tail region" sequences, as well as inserted sequences (introns) between each coding segment (exon).

[0105] "Recombinant" refers to a polynucleotide that is the product of various combinations of cloning, restriction, or ligation steps, as well as other processes that result in a construct that is distinct and / or different from naturally occurring polynucleotides.

[0106] "Introducing" means "transfection," "transformation," or "transduction" in the context of inserting a nucleic acid sequence into a cell, and includes the incorporation of a reference nucleic acid sequence into a eukaryotic or prokaryotic cell, where the nucleic acid sequence can be integrated into the cell's genome (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted to autonomous replication, or expressed transiently (e.g., transfection of mRNA).

[0107] "Nucleic acid construct" refers to a single-stranded or double-stranded nucleic acid molecule. In some embodiments, a nucleic acid construct is a DNA molecule that has been modified or synthesized to include a nucleic acid segment in a manner that does not occur in nature, the nucleic acid molecule including one or more control sequences or regulatory elements. In some embodiments, a nucleic acid construct is an RNA molecule formed after transcription of DNA. In the context of the present disclosure, a nucleic acid construct comprises a recombinant nucleotide sequence, which consists essentially of, and optionally includes, one, two, three or more isolated nucleotide sequences: a 5'UTR, an open reading frame (ORF) and a 3'UTR. In embodiments relating to a construct that includes two or more sequences, the sequences are operably linked to each other in the construct.

[0108] "Derived sequence" refers to a nucleotide sequence that is highly homologous to the UTR sequence of the present disclosure (e.g., has at least 80%, 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 100% identity to the UTR sequence of the present disclosure) and still retains a similar functional activity to the UTR sequence of the present disclosure. In some embodiments, the derived sequence is a nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotides based on the native UTR sequence. In some embodiments, the derived sequence is a nucleotide sequence obtained by truncating the native UTR sequence.

[0109] "Operably linked" is defined herein as the following structure: the control sequences, a promoter sequence and / or a 5'UTR sequence, are appropriately positioned relative to a coding DNA sequence such that the control sequences direct the transcription of the coding sequence and the translation of mRNA into a polypeptide sequence encoded by the coding DNA.

[0110] "Open reading frame" is abbreviated as "ORF" and refers to a segment or region of a nucleic acid sequence that encodes a polypeptide. An ORF contains consecutive, non-overlapping, in-frame codons that begin with an initiation codon in an mRNA sequence and end with a termination codon, and is translated by the ribosome.

[0111] "Endogenous" refers to any substance that originates from or is produced within an organism, cell, tissue, or system.

[0112] "Exogenous" refers to any substance introduced or produced from outside an organism, cell, tissue, or system.

[0113] "Sequence of identity" or "sequence identity" refers to sequence identity at the nucleotide or amino acid level between genes or proteins, respectively. "Sequence of identity" or "sequence identity" is a measure of identity at the amino acid level between proteins and between nucleic acids at the nucleotide level. Sequence identity of proteins can be determined by comparing the amino acid sequence at a given position in each sequence when aligning the sequences. Similarly, sequence identity of nucleic acids can be determined by comparing the nucleotide sequence at a given position in each sequence when aligning the sequences. Methods for aligning sequences for comparison are well known in the art, and such methods include GAP, BESTFIT, BLAST, FASTA, and TFASTA. In some embodiments, the method for aligning sequences is BLAST, and the BLAST algorithm calculates the percentage of sequence identity to statistically analyze the similarity between two sequences. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (NCBI) website.

[0114] "Homology" or "homology" is defined as the percentage of nucleotide residues that match the nucleotide residues in the corresponding sequence in the target chromosome after aligning the sequences and, if necessary, introducing gaps to achieve the maximum sequence identity percentage. The alignment of the homology percentage of nucleotide sequences can be achieved by various methods within the scope of the art, for example, by publicly available computer software such as BLAST, BLAST-2, ALIGN, ClustalW2 or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences to be compared. In some embodiments, for example, a nucleic acid sequence (e.g., a DNA sequence) of a homologous arm is considered to be "homologous" if it is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the corresponding native or unedited nucleic acid sequence (e.g., genomic sequence) of the host cell.

[0115] A "substitution" is defined as a change in an amino acid or nucleotide sequence that occurs by replacing one or more amino acids or nucleotides with different amino acids or nucleotides, respectively, compared to the amino acid or nucleotide sequence of a reference polypeptide. If the substitution is conservative, the amino acid substituted in the polypeptide has similar structure or chemical properties (e.g., charge, polarity, hydrophobicity, etc.) as the substituted amino acid. In some embodiments, a polypeptide variant may have "non-conservative" changes, in which the substituted amino acid differs in structure and / or chemical properties.

[0116] A "deletion" is defined as a change in an amino acid or nucleotide sequence such that one or more amino acids or nucleotide residues, respectively, are deleted compared to a reference polypeptide amino acid or nucleotide sequence. In the case of a polypeptide or polynucleotide sequence, a deletion may affect the deletion of 2, 5, 10, up to 20, up to 30, or up to 50 or more amino acids or nucleotide residues, taking into account the length of the modified polypeptide or polynucleotide sequence.

[0117] An "insertion" or "addition" refers to a change in amino acid or nucleotide sequence that results in the addition of one or more amino acid or nucleotide residues, respectively, compared to the amino acid or nucleotide sequence of a reference polypeptide. An "insertion" usually refers to the addition of one or more amino acid residues (or nucleotide residues in a polynucleotide) to the amino acid sequence of a polypeptide, although an "addition" can also refer to an insertion or an amino acid residue added to the N- or C-terminus of a polypeptide (or a nucleotide residue added to the 5' or 3' terminus of a polynucleotide). In the case of a polypeptide or polynucleotide sequence, an insertion or addition can be up to 10, up to 20, up to 30, up to 50 or more amino acids (or nucleotide residues).

[0118] "Codon optimization" refers to replacing codons present in a target sequence that are generally rare in highly expressed genes of a given species with codons commonly found in highly expressed genes of this species, such that the codons before and after the replacement code for the same amino acid. Each species exhibits specific preferences for certain codons for specific amino acids. Codon bias (differences in codon usage between organisms) is usually associated with the translation efficiency of messenger RNA (mRNA), which is believed to depend in particular on the properties of the codons being translated and on the availability of specific transfer RNA (tRNA) molecules. The dominance of the selected tRNA in a cell is generally a reflection of the codons most commonly used in peptide synthesis. Thus, based on codon optimization, a gene can be modified for optimal gene expression in a given organism. Thus, optimal codons are selected according to the codon usage preferences of the host genome.

[0119] A "cell" or "host cell" includes any cell type susceptible to transformation, transfection, transduction, etc., with a nucleic acid construct or vector according to the present disclosure. As non-limiting examples, a host cell may be an isolated primary cell, a pluripotent stem cell, a CD34+ cell, an induced pluripotent stem cell, or any one of a number of immortalized cell lines (e.g., HepG2 cells). Alternatively, a host cell may be an in situ or in vivo cell in a tissue, organ, or organism.

[0120] "Treatment" refers to, for example, administering an oral or topical therapeutic agent comprising a composition of any one of the nucleic acid constructs disclosed herein to a patient, the patient having one or more disease symptoms, the therapeutic agent being known to have a therapeutic effect on those symptoms. Typically, the patient or population being treated is administered an amount of therapeutic agent that effectively relieves one or more disease symptoms, thereby inducing the resolution of those symptoms or inhibiting those symptoms from progressing to any clinically measurable extent. The amount of therapeutic agent that effectively relieves any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on a variety of factors, including the disease state, age and weight of the patient, and the ability of the drug to produce the required therapeutic effect in the patient. Reduction of disease symptoms can be assessed by any clinical detection method commonly used by physicians or other professional health care providers to assess the severity or progression of the condition.

[0121] An "effective amount" or "pharmaceutical effective amount" includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical disease. An effective amount also refers to an amount sufficient to permit or facilitate diagnosis. The effective amount used in a particular patient or veterinary subject can vary depending on factors such as, for example, the condition being treated, the overall health of the patient, the route and dose of administration, and the severity of side effects. An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects. In some embodiments, an "effective amount" is a dose of RNA effective to generate an antigen-specific immune response.

[0122] The term "pharmacologically acceptable" means that the therapeutic agents, materials, compositions and / or dosage forms are, within the scope of reasonable medical judgment, applicable for contact with the tissues of a patient without undue toxicity, irritation, allergic response or other problem or complication, and are effective for the desired use, with a reasonable benefit / risk ratio.

[0123] "Polypeptide" and "protein" have the same meaning and may be used interchangeably.

[0124] "Subject" refers to a mammal, including, but not limited to, humans, rodents (mice, rats, guinea pigs), dogs, horses, cows, cats, pigs, monkeys, chimpanzees, etc. Preferably, the subject is a human. Working Example

[0125] The present disclosure will be further described below with reference to examples, but these examples do not limit the scope of the present disclosure. Experimental methods for which specific conditions are not specified in the examples of the present disclosure generally follow standard conditions, such as those in the Cold Spring Harbor Antibody Technology Experiment Manual and the Molecular Cloning Manual, or conditions suggested by raw material or product manufacturers. Reagents for which a specific source is not specified are standard commercially available reagents. Example 1. Screening of 5'UTR and 3'UTR

[0126] In order to screen for 3'-untranslated region elements (3'UTR elements) and 5'-untranslated region elements (5'UTR elements) capable of improving protein expression efficiency, this example screened UTR sequences in housekeeping gene mRNAs.

[0127] First, 301 candidate human housekeeping genes, including Abhd16a, were determined from the database (https: / / esbl.nhlbi.nih.gov / helixweb / Database / NephronRNAseq / Housekeeping_Genes.html). Then, the expression levels of the above genes were analyzed by bioinformatics methods to rank the expression levels, and gene transcripts and UTR sequence information were obtained. Furthermore, UTR elements were screened according to the gene expression level and UTR sequence length to obtain multiple candidate UTR sequences, from which 33 5'UTRs numbered A1 to A33 and 14 3'UTRs numbered B1 to B14 were screened to obtain the origins and sequence numbers of the genes, as shown in Tables 1 and 2.

[0128] Table 1. New 3'UTRs obtained by screening [Table 1] Table 2. New 5'UTRs obtained by screening [Table 2] Example 2. Preparation of mRNA

[0129] In order to obtain mRNA stably expressing the 5'UTR and 3'UTR elements, the UTRs obtained by screening in Example 1 were constructed into a DNA vector for in vitro transcription. The vector contains a T7 promoter, a sequence (SEQ ID NO: 126) encoding Firefly Luciferase (Fluc) as an open reading frame (ORF), and a polyadenylic acid (polyA) sequence followed by a restriction site for linearization vector. The polyA is selected from A120 (i.e., 120 consecutive adenyl acids) or A30L70 (SEQ ID NO: 52). The 5'UTR and 3'UTR element constructs were constructed at the 5' and 3' ends of the open reading frame ORF (Fluc) with suitable enzyme cleavage sites, respectively. Examples of the constructed vectors are shown in Figures 1A to 1C, and the combination of the 5'UTR and 3'UTR can be seen in Table 3.

[0130] The control vectors used in this example included Mod.-120A (sequence number 53), in which polyA is A120, Mod.-A30L70 (sequence number 54), and BioN.-A30L70 (sequence number 55), in which polyA is A30L70, and the target gene in each case was Fluc constructed by total gene synthesis. Various UTR elements were constructed in the above vectors by enzymatic digestion, among which, for vectors V-B1 to V-B14, the construction vector was Mod.-120A, AgeI and SacII were selected as the enzymatic digestion sites, and their polyA was A120 in both cases, and the control used was Mod.-120A (SEQ ID NO: 53); for vectors V-A1 to V-A14, the construction vector was Mod.-120A (SEQ ID NO: 53), BamHI and NheI were selected as the enzymatic digestion sites, and their polyA was A120 in both cases, and the control used was also Mod.-120A (SEQ ID NO: 53); for vectors V-A15 to V-A14, the construction vector was Mod.-120A (SEQ ID NO: 53), BamHI and NheI were selected as the enzymatic digestion sites, and their polyA was A120 in both cases, and the control used was also Mod.-120A (SEQ ID NO: 53); In vector 33, the construction vector was Mod.-A30L70 (sequence number 54), BamHI and NheI were selected as the enzyme cleavage sites, and the polyA was A30L70 in both cases, and the control used was Mod.-A30L70 (sequence number 54); in vectors V-A1-B12, V-A1-B13, and V-A1-B14, the construction vector was Mod.-A30L70 (sequence number 54), BamHI and SacII were selected as the enzyme cleavage sites, and the polyA was A30L70 in both cases, and the controls used were Mod.-A30L70 (sequence number 54) and BioN.-A30L70 (sequence number 55). The inserted UTR element gene fragments were all synthesized by whole gene synthesis (Suzhou Jinweizhi Biotechnology Co., Ltd.), and the fragments were linked to the corresponding vectors by enzymatic digestion. After the vector construction was completed, it was necessary to carry out enzymatic digestion, sequencing and identification. If it was determined to be correct, it was used in the next experiment.

[0131] Table 3. Combination methods of 5'UTR and 3'UTR in vectors [Table 3-1] [Table 3-2]

[0132] The vectors (i.e., DNA templates) in Table 3 were digested by restriction endonuclease treatment to linearize the DNA templates, and then transcribed in vitro with T7-RNA polymerase. For in vitro transcription, T7 RNA polymerase (Roche), the corresponding reaction buffer, pyrophosphatase, RNase inhibitor, and NTPs were used. To effectively cap the RNA, the cap analog ARCA (3'-O-Me-m) was added to the reaction. 7 ARCA was selected for capping for the RNA production used in Figures 2 to 5, and CleanCap was selected for capping for the RNA used in Figures 6 to 9. At the same time, in order to reduce the immunogenicity of mRNA and improve translation efficiency, all of the mRNAs in the present disclosure are modified with nucleic acid to replace all of the uridine triphosphate (Uridine triphosphate, abbreviated as UTP) in the reaction system with N1-methyl-pseudouridine triphosphate (N1-methyl-pseudouridine triphosphate, abbreviated as 1m-ψUTP, purchased from ThermoFisher), and the modification method is referred to in Patent US2014 / 0194494 A1. After incubating the in vitro transcription system at 37°C for 2.5 h, RNA was purified by carboxylated magnetic beads (Invitrogen), resuspended in ribozyme-free water, and analyzed spectrophotometrically on a 5200 Bioanalyzer (Agilent) to assess RNA concentration and quality. Target mRNA was obtained by detection.

[0133] The sequences from 5'UTR to polyA of Mod. of the present disclosure are shown in SEQ ID NOs: 53 and 54, and the sequence from 5'UTR to polyA of BioN. is shown in SEQ ID NO: 55. The 5'UTR and 3'UTR sequences in Mod. are consistent with the sequences in Moderna's patents and published literature (US10849920B2, WO2013151667A1, US10730924B2, DOI:10.1016 / j.cell.2017.02.017), and the 5'UTR and 3'UTR sequences of BioN. are according to patent WO2018 / 160540.

[0134] The V-B1 linearized sequence is shown by way of example, with the sequence from 5'UTR to polyA being shown in sequence number 56, and the V-A1 linearized sequence, from 5'UTR to polyA being shown in sequence number 57. Example 3. Functional verification of 5'UTR, 3'UTR, and their combination

[0135] In this example, the function of the UTR was verified using an mRNA lipid-transfected luciferase expression system.

[0136] Experimental method: Human embryonic kidney cells (HEK293, purchased from ATCC) were cultured at 4 × 10 4 The cells were seeded in a 96-well plate at a density of 100 cells / well. The next day, transfection was performed using Lipofectamine® MessengerMAX® mRNA transfection reagent, and 100 ng of the capped mRNA prepared in Example 2 above was transfected into each well. Six hours after transfection, fresh medium was replaced. 100 μL of medium was aspirated and 50 μL of luciferase substrate was added, and the luminescence intensity was detected using a PerkinElmer multifunction plate reader. The experimental methods for human cervical cancer cells (HeLa, purchased from ATCC) and human lung cancer cells (A549, purchased from ATCC) were the same as those for HEK293.

[0137] Evaluation method: In Tables 4 to 7 and Tables 9 to 11, the expression level of fluorescein in various cell lines of the reference positive control Mod. was set to 1, and the expression levels of various mRNAs were calculated relative to the positive control Mod. In Table 8, the expression level of fluorescein in various cell lines of the reference positive control BioN. was set to 1, and the expression levels of various mRNAs were calculated relative to the positive control BioN. All values ​​in the tables are multiplications of the average detection values.

[0138] 1) Detection of the ability of 3'UTR to regulate target gene expression in various cell lines To verify the ability of different 3'UTRs to regulate target gene expression in different cell lines, mRNA expressing luciferase (Fluc) was produced by the method of Example 2, and the produced RNA was capped with ARCA, with the structure of polyA tail being A120. The RNA was transfected into human HeLa, HEK293 and A549 cells, and the luciferase level was measured 24 hours after transfection, and the results are shown in Table 4 and Figure 2.

[0139] Table 4. Expression levels of luciferase (Fluc) from mRNA with various 3'UTRs (all capped with ARCA, all target genes are Fluc, and all polyA are A120) [Table 4]

[0140] As a result, compared to the Mod. positive control, the 3'UTRs of B1 to B14 had consistent effects on protein expression levels in various cell lines, with no significant differences between the various cell lines (p>0.05), indicating that the 3'UTR elements obtained by screening in the present disclosure have a universal effect on protein expression levels and can be used to improve the protein expression levels of target genes.

[0141] 2) Detection of temporal regulation of 3'UTR target gene expression in cells To detect the ability of various 3'UTRs to regulate the time of target gene expression in cells, the mRNAs in Table 4 were transfected into HEK293 cells, and the expression levels of luciferase were measured 6 h, 24 h, 48 h and 72 h after transfection, and the results are shown in Table 5 and Figure 3.

[0142] Table 5. Expression levels of luciferase (Fluc) from mRNAs with various 3'UTRs [Table 5]

[0143] As a result, in HEK293 cells, modification of 3'UTR elements can affect the expression level of target proteins. Except for the B5-3'UTR element, the other 3'UTR elements can all increase the protein expression level by more than 1.2 times, and there are significant differences at different detection time points (p<0.05). Among them, the 3'UTR elements of B12, B13 and B14 have the best effect, which can increase the protein expression level by more than 2 times and has good temporal continuity.

[0144] 3) Detection of the regulation of 5'UTR on target gene expression in cells In order to detect the ability of various 5'UTRs to regulate the expression time of target genes in cells, mRNA expressing luciferase (Fluc) was prepared and obtained by the above method, among which the RNA used in Table 6 and Figure 4 was capped with ARCA and the structure of polyA tail was A120, and the RNA used in Table 7 and Figure 5 was capped with CleanCap and the structure of polyA tail was A30L70. The RNA produced above was transfected into HEK293 cells, and the expression level of luciferase was measured 6h, 24h, 48h and 72h after transfection, and the results are shown in Table 6, Table 7 and Figure 4 and Figure 5.

[0145] Table 6. Expression levels of luciferase (Fluc) from mRNA with various 5'UTRs (all capped with ARCA, all target genes are Fluc, and all polyA are A120) [Table 6] Table 7. Expression levels of luciferase (Fluc) from mRNA with various 5'UTRs (capped with CleanCap, all target genes are Fluc, and polyA is A30L70) [Table 7]

[0146] Figure 4 and Figure 5 show the screening of various capping methods and polyA tail structures, respectively, and the results show that the modification of 5'UTR elements in various screening vectors can affect the expression level of target proteins, and 5'UTR elements have versatility in improving the expression level of target proteins. Among them, 5'UTR elements of numbers A1, A3-A7, A9-A16, A18, A21, A24, A27, A28, A32, A33 can all improve the protein expression level by more than 1.2 times, and there are significant differences at the detection time points of 24h, 48h, and 72h (p<0.05), among which, 5'UTR elements of A1, A15, A16, and A18 have the best effect, which can improve the protein expression level by more than 2 times and has good temporal continuity.

[0147] 4) Detection of modulation of target gene expression intensity of candidate 5'UTR compared to positive control BioN. vector In order to detect the ability of various candidate 5'UTRs to regulate the expression intensity of target genes compared to the positive control BioN. vector, the above-screened preferred 5'UTR elements A1, A15, A16, and A18 were constructed into a vector containing the A30L70 polyA tail element, and mRNA expressing luciferase (Fluc) was produced by the above-mentioned method and transfected into HEK293 cells, and the expression level of luciferase was measured 6 h, 24 h, 48 h and 72 h after transfection. The results are shown in Table 8 and Figure 6.

[0148] Table 8. Expression levels of luciferase (Fluc) from mRNA with various 5'UTRs (capped with Cleancap, all target genes are Fluc, and polyA is A30L70) [Table 8]

[0149] As a result, all of the candidate 5'UTR elements were able to increase the protein expression level by at least 1.2-fold at 24 h and 48 h compared to the BioN. vector, and all of the differences were significant (p<0.05).

[0150] 5) Detection of the regulation of target gene expression by various combinations of 5'UTR and 3'UTR in cells To study the effect of various 5'-UTR and 3'UTR combinations on target protein expression in mRNA, mRNAs containing various combinations of 5'UTR and 3'-UTR elements were compared with the 5'UTR and 3'UTR selected by Moderna, and the results are shown in Table 9 and Figure 7.

[0151] Table 9. Expression levels of mRNA with various combinations of 5'UTR and 3'UTR (capped with Cleancap, all target genes are Fluc, and polyA is A30L70) [Table 9]

[0152] As a result, the combination of 5'UTR and 3'-UTR elements screened in the present disclosure can improve protein expression levels by 1.3 times or more at the detection time points of 24h, 48h and 72h compared to the 5'UTR and 3'UTR elements selected by Moderna, and all of the differences are significant (p<0.05). The target proteins in 1) to 5) above are all luciferase (Fluc), and the target protein in 6) below is a secreted protein (e.g., hHGF, anti-PD-1 antibody).

[0153] 6) Detection of the regulation of secretory target protein expression by various combinations of 5'UTR and 3'UTR To study the effect of various 5'-UTR and 3'UTR combinations on mRNA expression of secreted proteins, we constructed mRNAs whose target proteins expressed by the ORFs were human hepatocyte growth factor (hHGF) or anti-PD-1 antibody.

[0154] The amino acid sequence of the above hHGF is shown in SEQ ID NO: 109, its DNA sequence is shown in SEQ ID NO: 110, and after codon optimization, mRNA OS1, OS2, and OS3 are obtained, the optimized codon sequences of which are shown in SEQ ID NOs: 129 to 131.

[0155] The heavy and light chain amino acid sequences of the above PD-1 antibody are shown in SEQ ID NOs: 117 and 118, respectively, the heavy and light chain DNA sequences are shown in SEQ ID NOs: 119 and 120, respectively, and the heavy and light chain mRNA sequences are shown in SEQ ID NOs: 121 and 122, respectively.

[0156] First, in order to improve the translation efficiency of hHGF protein, the present disclosure performs codon optimization on the nucleotide sequence of wild-type hHGF (SEQ ID NO: 110), and the optimized sequences include hHGF-OS1 (SEQ ID NO: 111), hHGF-OS2 (SEQ ID NO: 112) and hHGF-OS3 (SEQ ID NO: 113), and the corresponding mRNA sequences are shown in SEQ ID NOs: 129 to 131, respectively. The above-mentioned natural hHGF and the hHGF sequence with the optimized codon sequence were constructed into a vector to produce mRNA. The expression level of hHGF protein was detected by ELISA, and it was proved that the hHGF codon-optimized sequence can improve the expression level of hHGF protein.

[0157] hHGF-OS2 expression mRNA containing various combinations of 5'UTR and 3'-UTR elements (corresponding DNA sequence is SEQ ID NO: 112) was compared with hHGF-OS2 expression mRNA Mod. (hHGF) containing various combinations of 5'UTR and 3'UTR elements from Moderna (corresponding DNA sequence is SEQ ID NO: 114). The above hHGF-encoding mRNA was transfected into HEK293 cells, and the supernatant was collected 6 h, 24 h, 48 h and 72 h after transfection to detect the expression level of hHGF protein by ELISA to evaluate the increase / prolongation of HGF expression by the candidate vector. The results are shown in Table 10 and Figure 8A.

[0158] Table 10. Target protein expression levels of mRNA with various combinations of 5'UTR and 3'UTR (capped with Cleancap, all target genes are hHGF, and polyA is A30L70) [Table 10]

[0159] Full-length anti-PD-1 antibody expression mRNAs containing various combinations of 5'UTR and 3'UTR elements (corresponding DNA sequences are SEQ ID NO: 124-125) were compared with Moderna's full-length anti-PD-1 antibody expression mRNAs containing various combinations of 5'UTR and 3'UTR elements (corresponding DNA sequence is SEQ ID NO: 123). The mRNAs encoding full-length anti-PD-1 antibody were transfected into HEK293 cells, and the supernatants were collected 6 h, 24 h, 48 h and 72 h after transfection to detect the expression level of anti-PD-1 antibody by ELISA to evaluate the increase / prolongation of anti-PD-1 antibody expression by the candidate vectors. The results are shown in Table 11 and Figure 8B.

[0160] Table 11. Target protein expression levels of mRNA with various combinations of 5'UTR and 3'UTR (capped with Cleancap, target genes are heavy and light chains of anti-PD-1 antibody, polyA is A30L70) [Table 11]

[0161] As shown by the above results, after continuous expression of secretory protein hHGF and anti-PD-1 antibody for 72 hours, the combination of 5'UTR and 3'UTR screened in the present disclosure can significantly improve the expression level of secretory protein compared with the 5'UTR and 3'UTR elements selected by Moderna (p<0.05). Example 4. Verification of hHGF protein expression in mice using hHGF-expressing mRNA

[0162] To confirm the ability of the mRNA molecule m-A16-B12(hHGF) (corresponding DNA sequence is SEQ ID NO: 127) to express hHGF in animals, the mRNA was delivered by lipid nanoparticle LNP (containing 50 mol% ionizable lipid (SM-102), 10 mol% DSPC, 38.5 mol% cholesterol, 1.5 mol% PEG-DMG), and the same lipid nanoparticle LNP was used in Examples 5 and 6 below. The positive control was Collategene plasmid (AnGes), administered as a naked plasmid. Balb / c mice (6-8 weeks, male) were randomly divided into 4 groups of 33 mice each, and various doses of m-A16-B12 (hHGF) and Collategene plasmid were intramuscularly injected into the gastrocnemius muscle of the mice. According to the scheme in Table 12, the experimental group was 1) injected with 1.0 μg / mouse m-A16-B12 (hHGF), 2) injected with 0.3 μg / mouse m-A16-B12 (hHGF), 3) injected with 0.1 μg / mouse m-A16-B12 (hHGF), and 4) injected with 200 μg / mouse Collategene naked plasmid. Mouse gastrocnemius muscle specimens were obtained at 1h, 2h, 4h, 6h, 24h, 48h, 72h, 96h, 168h, 216h and 336h, and the muscle tissue was homogenized and dissolved in RIPA lysis solution containing protease inhibitors, centrifuged to obtain the supernatant, and the hHGF protein concentration was measured using a BCA protein concentration measurement reagent kit, and the expression level of hHGF was evaluated by ELISA. The data for each group were shown as mean ± standard deviation (Mean ± SD), and plotted and statistically analyzed using the software Graphpad Prism 9.0.

[0163] Table 12. Grouping of m-A16-B12 (hHGF) and Collategene in the experiment of expressing hHGF in vivo [Table 12]

[0164] As shown in Figure 9A, the experimental results showed that m-A16-B12(hHGF) was able to express hHGF protein 1 hour after intramuscular injection, and reached its peak expression 6 hours after injection, demonstrating dose-dependence. As shown in Figure 9B, Collategene reached its peak expression 7 days after injection, m-A16-B12(hHGF) showed low protein expression 48 hours after delivery, and m-A16-B12(hHGF) at a dose of 1 μg / mouse had protein expression levels equivalent to Collategene at 72 hours. At the same time, the PK data for each group was statistically analyzed, and it was found that even the lowest dose of m-A16-B12(hHGF) at 0.1 μg had a high protein expression level. max was more than 5 times that of Collategene, and 0.1 μg of m-A16-B12 (hHGF) had an AUC inf(hr*pg / mg protein) The results are comparable to those of Collategene (Table 13). Therefore, m-A16-B12 (hHGF) can achieve highly efficient expression of hHGF in animals, and hHGF expression was not detected in plasma at each time point.

[0165] Table 13. Results of in vivo hHGF expression by m-A16-B12 (hHGF) and Collategene [Table 13] Example 5. Verification of therapeutic function of hHGF-expressing mRNA in a mouse model of hindlimb ischemia

[0166] The mouse model of hindlimb ischemia is a classical mouse model that simulates human severe hindlimb ischemia. Male Balb / c mice aged 6 to 8 weeks were used for the modeling. The method is as follows: 1) The animal was anesthetized and placed on the operating table in a supine position, the hindlimbs were completely depilated, the hindlimbs were fixed, and the skin at the surgical site was disinfected. 2) An approximately 1 cm skin incision was made from the knee toward the inner thigh, and the subcutaneous adipose tissue was incised and dissected in sequence to fully expose the femoral artery. 3) The membranous femoral sheath was carefully punctured with curved micro scissors to expose the neurovascular bundle, and the proximal end of the femoral artery, femoral vein, and nerve near the groin were separated, and after clean separation, the proximal end of the femoral artery was ligated with a 6-0 suture below the proximal end of the femoral artery. 4) The femoral artery and femoral vein were isolated at their distal ends near the knee, and the end of the femoral artery was ligated to the proximal end of the popliteal artery with a 6-0 suture below the distal end. 5) The wound was sutured.

[0167] m-A16-B12 (hHGF) was delivered using the LNP in Example 4 as a vector, and Collategene was administered in the form of a naked plasmid. Mice with hindlimb ischemia model were randomly divided into 4 groups of 5 mice each, and m-A16-B12 (hHGF) and Collategene naked plasmid were intramuscularly injected into the gastrocnemius muscle of the mice. The experiment was divided into 1) a group injected with 500 ng / mouse m-A16-B12 (hHGF), 2) a group injected with 50 ng / mouse m-A16-B12 (hHGF), 3) a group injected with 200 μg / mouse Collategene naked plasmid, and 4) a group injected with an equal volume of PBS as a control group. The day the model was constructed was counted as day 0. The condition of the legs was observed on days 0, 4, 7, 10, 12, and 14 after treatment for each experimental group. The blood flow and perfusion status of the mice's lower limbs was detected using a blood flow meter and photographed to record.

[0168] The blood perfusion ratio of each group was calculated according to the formula: Blood perfusion ratio = Lower limb blood perfusion volume of the mouse on the day / Lower limb blood perfusion volume of the mouse on day 0 × 100%, and the data of each group was shown as mean ± standard deviation (Mean ± SD) and plotted and statistically analyzed using the software Graphpad Prism 9.0.

[0169] As a result, as shown in Figures 10A and 10B, when treated with 50ng / mouse, 500ng / mouse m-A16-B12 (hHGF) and 200ng / mouse Collategene naked plasmid, the blood flow perfusion ratio of the ischemic lower limbs of each mouse was significantly improved compared to the control group, and the blood flow perfusion ratio of the ischemic lower limbs was gradually restored over time. Among them, the group injected with 50ng / mouse m-A16-B12 (hHGF) showed a blood flow recovery effect similar to the group injected with 200ng / mouse Collategene naked plasmid, and in particular, the group injected with 500ng / mouse m-A16-B12 (hHGF) showed a significantly better blood flow recovery effect than the 200ng / mouse Collategene naked plasmid group, and the blood flow perfusion status was restored to more than 90%.

[0170] To further confirm the therapeutic effect of m-A16-B12 (hHGF) on the degree of necrosis of the lower limbs in a mouse model of lower limb ischemia, the state of the mouse legs was observed, photographed, and recorded on the 14th day after administration, and the degree of necrosis of the lower limbs of the mice was scored according to the criteria shown in Figure 11A, where the criteria used in this case are as follows: 0 = spontaneous detachment of the lower limbs, 1 = necrosis of the legs, 2 = necrosis of the feet, 3 = discoloration of two or more toes, 4 = discoloration of one toe, 5 = discoloration of two or more nails, 6 = discoloration of one nail, and 7 = no necrosis. The data of each group was shown as mean ± standard deviation (Mean ± SD) and plotted and statistically analyzed using the software Graphpad Prism 9.0.

[0171] As a result, as shown in FIG. 11B, after inducing hindlimb ischemia in mice, obvious ischemic necrosis occurred in the hindlimb of the control group mice within 2 weeks after surgery, and by the 14th day, the hindlimb of some mice had been completely lost. However, in the treatment groups injected with m-A16-B12 (hHGF) and Collategene naked plasmid, the hindlimb of each mouse was able to maintain good integrity and no necrosis was observed in the hindlimb, demonstrating that m-A16-B12 (hHGF) has the same ability to improve hindlimb ischemia as the control Collategene naked plasmid.

[0172] CD31 is an important marker of angiogenesis, and to confirm the effect of m-A16-B12 (hHGF) in promoting angiogenesis in a mouse model of hindlimb ischemia, samples of the gastrocnemius muscle near the mouse ischemic tissue were taken on the 14th day after administration, paraffin sections were prepared, and CD31 in the sections was immunohistochemically stained with CD31 antibody, photographed, and analyzed with ImageJ (NIH) software to calculate the stained area of ​​CD31. The data for each group were shown as mean ± standard deviation (Mean ± SD), and plotted and statistically analyzed using the software Graphpad Prism 9.0.

[0173] As a result, as shown in FIG. 12, when 50ng / mouse or 500ng / mouse of m-A16-B12 (hHGF) and 200ng / mouse of Collategene naked plasmid were administered for treatment, angiogenesis in the ischemic lower limb muscles of each group was significantly promoted, and the number of newly formed blood vessels was statistically significantly different from the PBS group (p<0.05). The 50ng / mouse m-A16-B12 (hHGF) group showed angiogenesis promotion effect equivalent to that of 200ng / mouse of Collategene naked plasmid, and the m-A16-B12 (hHGF) administration group showed a dose-dependent angiogenesis promotion state. Example 6. Verification of therapeutic function of hHGF-expressing mRNA in a Db / Db mouse model with total cortical injury

[0174] The Db / Db mouse model is a classic diabetic mouse model, and because it is a leptin receptor gene-deficient mouse, the mouse showed similar characteristics to diabetic patients, such as hyperglycemia, hyperlipidemia, and insulin resistance, as the mice grew older. This experiment simulated the difficult-to-heal skin damage of diabetic foot patients using a full cortical injury model. The method for producing the above Db / Db mouse model was as follows: Db / Db mice were anesthetized, depilated with depilatory cream, and disinfected with 75% alcohol cotton balls. A full-thickness skin wound was created in the lumbar region using a biopunch, a skin harvester with a diameter of 8 mm.

[0175] To confirm the therapeutic effect of m-A16-B12 (hHGF) on wound healing in a mouse model of total cortical injury, m-A16-B12 (hHGF) was delivered by LNP in Example 4, and control Collategene was administered in the form of naked plasmid, with the administration method being subcutaneous injection at 4 separate points. Db / Db mice in the total cortical injury model were divided into 5 groups of 7 mice each according to body weight and blood glucose level. The experiment was divided into 1) a group injected with 500ng / mouse of m-A16-B12 (hHGF), 2) a group injected with 200ng / mouse of m-A16-B12 (hHGF), 3) a group injected with 50ng / mouse of m-A16-B12 (hHGF), 4) a group injected with 200μg / mouse of Collategene naked plasmid, and 5) a control group injected with an equal volume of PBS. The day the model was constructed was counted as day 0. After treatment for each experimental group, the wound healing status was observed on days 0, 3, 5, 7, 10, 12, and 14, respectively. Photographs of the wounds were taken and the images were analyzed using ImageJ (NIH) software to calculate the wound area.

[0176] The wound healing percentage was calculated according to the formula P = (AD-A0) / A0 x 100% (P: wound healing percentage, AD: wound area on the day of photography, A0: wound area on the day after surgery).

[0177] The percentage of wound healing for each group was expressed as the mean ± standard deviation (Mean ± SD) and plotted and statistically analyzed using the software Graphpad Prism 9.0.

[0178] As a result, as shown in FIG. 13, the wounds of the mice injected with 50 ng / mouse, 200 ng / mouse, and 500 ng / mouse of m-A16-B12 (hHGF) and 200 μg / mouse of Collategene naked plasmid were all better healed than the control group. Moreover, m-A16-B12(hHGF) showed dose-dependent promotion of wound healing in mice. On the 14th day, the wound healing rate of m-A16-B12(hHGF) at a dose of 50ng / mouse reached 66%, while m-A16-B12(hHGF) at doses of 200ng / mouse and 500ng / mouse achieved 100% wound healing. Furthermore, the wound healing promotion of m-A16-B12(hHGF) was significantly superior to that of 200μg / mouse Collategene naked plasmid (p<0.05), and even the low dose (50ng / mouse) of m-A16-B12(hHGF) showed a wound healing percentage that was significantly different from that of 200μg / mouse Collategene naked plasmid (p<0.05).

[0179] To confirm the therapeutic effect of m-A16-B12 (hHGF) on tissue reconstitution in a mouse model of total cortical injury, epithelial regeneration and tissue reconstitution were evaluated by Masson staining. On the 17th day after administration, whole mouse cortical specimens were obtained, paraffin sections were prepared, and mouse skin tissue was stained by Masson staining.

[0180] As a result, as shown in Figure 14, under the treatment of m-A16-B12 (hHGF), the wound epithelium of each group of mice achieved full epithelial coverage, and the low-dose group showed abnormally thickened epithelium and collagen proliferation at the wound site, while the medium and high-dose groups showed normal epithelial thickness recovery, regular collagen arrangement, and tissue reconstruction. On the other hand, the control group and the 200μg / animal Collategene naked plasmid group did not achieve epithelial re-coverage, and collagen abnormally proliferated and structure was disrupted at the wound site.

[0181] Sequence Listing >B1(ACTG1 3'UTR) [C1] CCGACTACCTCATGAAGATCCTCACTGAGCGAGGCTACAGCTTCACCACCACGGCCGAGCGGGAAATCGTGCGCGACATCAAGGAGAAGCTGTGCTACGTCGCCCTGGACTTCGAGCAGGAGATGGCCACCGCCGCATCCTCCTCTTCTCTGGAGAAGAGCTACGAGCTGCCCGATGGCCAGGTCATCACCATTGGCAATGAGCGGTTCCGGTGTCCGGAGGCGCTGTTCCAGCCTTCCTTCCTGGGTATGGAATCTTGCGGCATCCACGAGACCACCTTCAACTCCATCATGAAGTGTGACGTGGACATCCGCAAAGACCTGTACGCCAACACGGTGCTGTCGGGCGGCACCACCATGTACCCGGGCATTGCCGACAGGATGCAGAAGGAGATCACCGCCCTGGCGCCCAGCACCATGAAGATCAAGATCATCGCACCCCCAGAGCGCAAGTACTCGGTGTGGATCGGTGGCTCCATCCTGGCCTCACTGTCCACCTTCCAGCAGATGTGGATTAG Sequence number 1 >B2(ATP6V0B 3’UTR) [Chemical formula 2] ATGATATGTGTGGGTGGGGCCGTGCCTCACTTTTATTTATTGCTGGTTTTCCTGGGACAGCTGGAGCTGTGTCCCTTAGCCTTTCAGAGGCTTGGTGTTCAGGGCCCTCCCTGCACTCCCCTCTTGCTGCGTGTTGATTTGGAGGCACTGCAGTCCAGGCCGAGTCCTCAGTGCGGGGAGCAGGCTGCTGCTGCTGACTCTGTGCAGCTGCGCACCTGTGTCCCCCACCTCCACCCTCAACCCATCTTCCTAGTGTTTGTGAAATAAACTTGGTATTTGTCT Sequence number 2 >B3(ATP6V0B 3’UTR) [Chemical formula 3] GTTGAAGAACTTCTCTCTACCGAGCAGCCCGTAACCCCCTTTTTCTCCCTCACTGCTGCAGGAGTCTGCTACACCATTTTTGATTTGGGCTTCCGCTTTGATGTGGCATGGTTCCTGACGGAGACTTCGCCCTTCATGTGGTCCAACCTGGGCATTGGCCTAGCTATCTCCCTGTCTGTGGTTGGGGCAGCCTGGGGCATCTATATTACCGGCTCCTCCATCATTGGTGGAGGAGTGAAGGCCCCCAGGATCAAGACCAAGAACCTGGTCAGCATCATCTTCTGTGAGGCTGTGGCCATCTACGGCAT Sequence number 3 >B4(ATP6V0E1 3’UTR) [Chemical formula 4] GGAAGAAGACATGCTCTACAGTGCTCAGTCTTTGAGGTGACTATGCTTGTGACCTTTCTTATCAGTATGGTCAGGCAGTCAGAGTTTGCCTTGACTTTTTCTCACATTTTATGGCCATTTTAACACGTGGCAAGGGCAAGCTGCTGAATGCAAACACGATCTGAAGAGCAGAAATAAAATTGGAACTTAAGCCTTAGCTTACCTAAATCTCATTAAGATTCTTTTT Sequence number 4 >B5(ATP6V0E1 3’UTR) [Chemical formula 5] ATGCAAAATCACCTCCAAACCAGACCACTTTTCTTGACTTGCCTGTTTTGGCCATTAGCTGCCTTAAACGTTAACAGCACATTTGAATGCCTTATTCTACAATGCAGCGTGTTTTCCTTTGCCTTTTTTGCACTTTGGTGAATTACGTGCCTCCATAACCTGAACTGTGCCGACTCCACAAAACGATTATGTACTCTTCTGAGATAGAAGATGCTGTTCTTCTGAGAGATACGTTACTCTCTCCTTGGAATCTGTGGATTTGAAGATGGCTCCTGCCTTCTCACGTGGGAATCAGTGAAGTGTTTAGAAACTGCTGCAAGACAAACAAGACTCCAGTGGGGTGGTCAGTAGGAGAGCACGTTCAGAGGGAAGAGCCATCTCAACAGAATCGCACCAAACTATACTTTCAGGATGAATTTCTTCTTTCTGCCATCTTTTGGAATAAATATTTTCCTCCTTTC Sequence number 5 >B6(CFL1 3’UTR) [Chemical 6] GCCCCTTCTGGCCCCCTGCCTGGAGCATCTGGCAGCCCCACACCTGCCCTTGGGGGTTGCAGGCTGCCCCCTTCCTGCCAGACCGGAGGGGCTGGGGGGATCCCAGCAGGGGGAGGGCAATCCCTTCACCCCAGTTGCCAAACAGACCCCCCACCCCCTGGATTTTCCTTCTCCCTCCATCCCTTGACGGTTCTGGCCTTCCCAAACTGCTTTTGATCTTTTGATTCCTCTTGGGCTGAAGCAGACCAAGTTCCCCCCAGGCACCCCAGTTGTGGGGGAGCCTGTATTTTTTTTAACAACATCCCCATTCCCCACCTGGTCCTCCCCCTTCCCATGCTGCCAACTTCTAACCGCAATAGTGACTCTGTGCTTGTCTGTTTAGTTCTGTGTATAAATGGAATGTTGTGGAGATGACCCCTCCCTGTGCCGGCTGGTTCCTCTCCCTTTTCCCCTGGTCACGGCTACTCATGGAAGCAGGACCAGTAAGGGACCTTCGATT Sequence number 6 >B7 (CFL1 3’UTR) [Chemical formula 7] GCCCCTTCTGGCCCCCTGCCTGGAGCATCTGGCAGCCCCACACCTGCCCTTGGGGGTTGCAGGCTGCCCCCTTCCTGCCAGACCGGAGGGGCTGGGGGGATCCCAGCAGGGGGAGGGCAATCCCTTCACCCCAGTTGCCAAACAGACCCCCCACCCCCTGGATTTTCCTTCTCCCTCCATCCCTTGACGGTTCTGGCCTTCCCAAACTGCTTTTGATCTTTTGATTCCTCTTGGGCTGAAGCAGACCAAGTTCCCCCCAGGCACCCCAGTTGTGGGGGAGCC Sequence number 7 >B8 (CFL1 3’UTR) [Chemical formula 8] GCCCCTTCTGGCCCCCTGCCTGGAGCATCTGGCAGCCCCACACCTGCCCTTGGGGGTTGCAGGCTGCCCCCTTCCTGCCAGACCGGAGGGGCTGGGGGGATCCCAGCAGGGGGAGGGCAATCCCTTCACCCCAGTTGCCAAACAGACCCCCCACCCCCTGGATTTTCCTTCTCCCTCCATCCCTTGACGGTTCTGGCCTTCCCAAACTGCTTTTGATCTTTTGATTCCTCTTGGGCTGAAGCAGACCAAGTTCCCCCCAGGCACCCCAGTTGTGGGGGAGCCTGTATTTTTTTTAACAACATCCCCATTCCCCACCTGGTCCTCCCCCTTCCCATGCTGCCAACTTCTAACCGCAATAGTG Sequence number 8 >B9(COX4I1 3’UTR) [Chemical formula 9] GAGATGCTGGCCTGCGCCTGCACCTGCGCCTGGCTCTGTCACCGCCATGCAACTCCATGCCTATTTACTGGAAACCTGTTATGCCAAACAGTTGTACCACTGCTAATAAATGACCAGTTTACCTGAAA Sequence number 9 >B10(COX4I1 3’UTR) [Chemical formula 10] AGGGACCCACAGGCGCGCCCAGCAGCTCTCGGAAGCGTTGTATCGCATTAAGTTCAAGGAGAGCTTTGCTGAGATGAACAGGGGCTCGAACGAGTGGAAGACGGTTGTGGGCGGTGCCATGTTCTTCATCGGTTTCACCGCGCTCGTTATCATGTGGCAGAAGCACTATGGGCTTAGCCTCCAAGTGGGACTACGAAAAGAACGAGTGGAAGAAGTGAGAGATGCTGGCCTGCGCCTGCACCTGCGCCTGGCTCTGTCACCGCCATGCAACTCCATGCCTATTTACTGGAAACCTGTTATGCCAAACAGTTGTACCACT Sequence number 10 >B11(COX4I1 3’UTR) [Chemical 11] AGAGGACCTTCTGCTTTTAGCTTATTTTGTTGCTAACTTTTTACAAACAAAGGGCTAATTTTAAAATGTCAGTGTTGTCAGTGGTCAGAAACCGTGTGTTTGAGCGGGTGTTGAGTGGCAGGTGGCTCTGCTGACCTGGTGGCTGGTGTGTCGGGAGGATTTAACCTGTGTGAGGGATTGGCCTAGAAACAACCTGTTGAGATAGTCTTGCCCCATAACCTGTCTCACACCGTAGTGTACGGCCCCCTCCCGCAAAGCTTTGACAAAGAGTGGGTGGCCAAGCAGACCAAGAGGATGCTGGAC Sequence number 11 >B12(CTSB 3’UTR) [Chemical 12] TCTGCCGTGGGCCTGTCGTGCCAGTCCTGGGGGCGAGATCGGGAGAATCACGTCCTGGAACTGCATGTTCTTGCGACTCTTGGGACTTCATCTTAACTTCTCGCTGCCCCAGCCATGTTTTCAACCATGGCATCCCTCCCCCAATTAGTTCCCTGTCATCCTCGTCAACCTTCTCTGTAAGTGCCTGGTAAGCTTGCCCTTGCTTAAGAACTCAAAACATAGCTGTGCTCTA Sequence number 12 >B13(FAM166A 3’UTR) [Chemical 13] AAATGCTTTAATGGTGGTGTCTGTACAGCATGGATGTGGACAGGGAAAACAGCAAGTGCACACAGTGGGACAGGCCACAGGACAGGCTGGGAGTGAATAAAGAGTTCACACTGCT Sequence number 13 >B14(NDUFB9 3’UTR) [Chemical 14] TATCTAAGTGTATCAATTTAACATTTATAATCACCTTACAAGTAAGTTAAAGTGCATTCCCTGGTCGTTTTGGCTTTGTCTTTGATTTTGCTTCAGATCTCCTTCTCTTTAGTCCTTTGTCTGACTTTTTTGCCTTCTTGCTATACTTTCCCTCAACCTGCCTCTCCACTATTTTCTTAGGCATTTTTCAGTCATG Sequence number 14 >A1(ACTG1 5’UTR) [Chemical formula 15] CTCTCGCACTCTGTTCTTCCGCCGCTCCGCCGTCGCGTTTCTCTGCCGGTCGCA Sequence number 15 >A2(ATP6V0B 5’UTR) [Chemical formula 16] GGGCGGGCGGACAGACTGCGGGACGGACGGTGGACGCTGGGACGCGTTTGTAGCTCCGGCCCCGCCGTTCCGACCCCCGCCGCCGTCGCCGCCATGACGGGGCTAGCACTGCTCTACTCCGGGGTCTTCGTGGCCTTCTGGGCCTGCGCGCTGGCCGTGGGTTCCTGACGGAGACTTCGCCCTTC Sequence number 16 >A3(ATP6V0B 5’UTR) [Chemical formula 17] AGACTGCGGGACGGACGGTGGACGCTGGGACGCGTTTGTAGCTCCGGCCCCGCCGTTCCGACCCCCGCCGCCGTCGCCGCC Sequence number 17 >A4(ATP6V0E1 5’UTR) [Chemical formula 18] GGGGTAGGGGTTGGCGCTCAGGCGGCGACC Sequence number 18 >A5(ATP6V0E1 5’UTR) [Chemical formula 19] GTCACGCGGTCAGCTATTGACACTTCCTGGTGGGATCCGAGTGAGGCGACGGGGTAGGGGTTGGCGCTCAGGCGGCGACC Sequence number 19 >A6 (CFL1 5’UTR) [Chemical formula 20] GGCCGGCGGGAAGACTCCGTTACCCAGCGAGCGAGGCGGCGGCGCAGGGCCAGCGGACTCCATTTCCCGTCGGCTCGCGGTGGGAGCGCCGGAAGCCCGCCCCACCCCTCATTGTGCGGCTCCTACTAAACGGAAGGGGCCGGGAGAGGCCGCGTTCAGTCGGGTCCCGGCAGCGGCTGCAGCGCTCTCGTCTTCTGCGGCTCTCGGTGCCCTCTCCTTTTCGTTTCCGGAAAC Sequence number 20 >A7 (CFL1 5’UTR) [Chemical formula 21] CTCCTGGCTCGGGCACCCCGAAGCACGACGCAGAGTAGGAAGAGTTTAAAGACCTCGAGGCCTCTGGGCACTTGAGTTTGGCCTCCGGTGTGGCTGTCTCTGATGGTGTCATCAAGGTGTTCAACGAC Sequence number 21 >A8 (CFL1 5’UTR) [Chemical formula 22] ACGTGGCCGGCTTCTTCTGCAGTTCCGGGGAGTTTGGGGGACCAGATTTACCTTGGATTGCCCCTCCCTCTCCTGGCTCGGGCACCCCGAAGCACGACGCAGAGCCTCCGGTGTGGCTGTCTCTGATGGTGTCATCAAGGTGTTCAACGAC Sequence number 22 >A9 (COX4I1 5’UTR) [Chemical formula 23] GCGGCCTTGCTCTCTTCCGGTCGCGGGACACCGGGTGTAGAGGGCGGTCGCGGCGGGCAGTGGCGGCAGA Sequence number 23 >A10(COX4I1 5'UTR)

[24] GGGACACCGGGTGTAGAGGGCGGTCGCGGCGGGCAGTGGCGGCAGA SEQ ID NO:24 >A11(COX4I1 5'UTR)

[25] CGCGGCCTTGCTCTCTTCCGGTCGCGGGACACCGGGTGTAGAGGGCGGTCGCGGCGGGCAGTGGCGGCAGA SEQ ID NO:25 >A12(CTSB 5'UTR)

[26] GGCCGGGGCTGGCCCAGGCTACGGCGGCTGCAGGGCTCCGGCAACCGCTCGGCAACGCCAACCGCTCCGCTGCGCGCAGGCTGGGCTGCAGGCTCTCGGCTGCAGCGCTGGGTGGATCTAGGATCCGGCTTCCAAC SEQ ID NO:26 >A13(FAM166A 5'UTR)

[27] AAGGGAGCTGGATGCCGGGAGGGACTGGAGCCAGCAAGGCCAGAGTGAAAGCAAA SEQ ID NO:27 >A14(NDUFB9 5'UTR)

[28] GCAGCAGGCGTGCAGTTTCCCGGCTCTCCGCCGGCCGGGGAAGGTCAGCGCCGTA SEQ ID NO:28 >A15(CHCHD10 5'UTR) [C29] GGCATTTGTCCCCGCGACAGCACCGCTGCCGCCGTCTCTAAGGTCGCCCGGGTCCCACCGCCGCCACC SEQ ID NO:29 >A16(CHCHD10 5'UTR)

[30] CCGCTGCCGCCGTCTCTAAGGTCGCCCGGGTCCCACCGCCGCCACC SEQ ID NO:30 >A17(SLC38A2 5'UTR)

[31] ATGTCTTTTTTGTGTGTTTGTTTTCATGGTATTCCTATGAA SEQ ID NO:31 >A18(NDUFA11 5'UTR)

[32] GCTTCCCGAGCTGGCGGGGTCCGTGGTGCGGGATCGAGATTGCGGGCT SEQ ID NO:32 >A19(NDUFV3 5'UTR)

[33] GGCGGCTGTTCAGGCGCGGGTGCGCGCGCAGCTGCTGTGGCCCTGCTTGGTGCGCCCGCTGTCACCGCC SEQ ID NO:33 >A20(PRDX5 5'UTR)

[34] CGCGCCTGCGCAGTGGAGGCGGCCCAGGCCCGCCTTCCGCAGGGTGTCGCCGCTGTGCCGCTAGCGGTGCCCCGCCTGCTGCGGTGGCACCAGCCAGGAGGCGGAGTGGAAGTGGCCGTGGGGCGGGT SEQ ID NO:34 >A21(GUK1 5'UTR)

[35] CAGCAGATGGGGACTAGAGGCCGCACTGCTATCCACAGCCTCTCTTCTCACCCCCAGGC SEQ ID NO:35 >A22(GUK1 5'UTR)

[36] GCTGGCCGGGCTGGCTGCGGCCGCCCTGGGCCGGGCCCCACCGGACGCCTCTCTTCTCACCCCCAGGC SEQ ID NO:36 >A23(GUK1 5'UTR)

[37] AGAGGTGGCCCCGGATGCTGCGGCGCCCGCTGGCCGGGCTGGCTGCGGCCGCCCTGGGCCGGGCCCCACCGGACGCCTCTCTTCTCACCCCCAGGC SEQ ID NO:37 >A24(IAH1 5'UTR)

[38] TGGCTGGCGGCCCCGCCCCGCCCCGCCCGGCTGCTCC SEQ ID NO:38 >A25(ABHD16A 5'UTR)

[39] GGAGGGCGGGGCCGGCAGGGGGACCTGCTGCTGGAAGAGCAGCGGCCCGAGCCGGGGCC SEQ ID NO:39 >A26(SLC25A39 5'UTR)

[40] TTTCGGCGTCGGCCTAGGTGCGCTGCGAGCGCGCGGACCGCGCACAGGCGGCGGAGCCGGTATGGGCCCGCCTGACCCTGGGCGCCGCGCCGCACGAGCACCAGCCTAGAGCCAGGACTGAAGCTTCAAG SEQ ID NO:40 >A27(ATPIF1 5'UTR)

[41] CGAGAGACTGCTTGCTGCGGCAGAGACGCCAGAGGTGCAGCTCCAGCAGCA SEQ ID NO:41 >A28(ANAPC11 5'UTR)

[42] CGGAGTTTCGTCATGTTGGCCAGGCCCATTTGAGATCTTTGAAGATATCCTCAACGTGAGGGCTCTGCTGCC SEQ ID NO:42 >A29(ANAPC11 5'UTR)

[43] GGGCGCGGCTTCGGCGGGCGGCAGCCGCTGCAGACGAGCTGCGGGCTCTGCTGCC SEQ ID NO:43 >A30(CCDC12 5'UTR)

[44] GCCTGCGCGATGCAAGACGGGAGAAAAGGAGGGGCGTACGCGGGCAAG SEQ ID NO:44 >A31(MRPL14 5'UTR)

[45] ATCGCGTCCGCCGGGCTGGGCCTGGCGCGCAGGCGCTAGGAAGAGGCCGCGTGGGGCGAAGGCGGCGCTTGGCTGGTGGGGCCCGCGGCGGGATTTTCCCGGGCGGCGAGAGCGGATCTATCTTGGGATCCC SEQ ID NO:45 >A32(APOA1BP 5'UTR)

[46] GCCGGGGGCGCGCGCTCTGCGAGCTGG SEQ ID NO:46 >A33(APOA1BP 5'UTR)

[47] GCCGGGGCCGGGCCGGGCCGGGGGCGCGCTCTGCGAGCTGG SEQ ID NO:47 >Mod.5'UTR

[48] GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC SEQ ID NO:48 > α-globin 3'UTR

[49] TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCC TCCCCCAGCCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTT TGAATAAAGTCTGAGTGGGCGGC SEQ ID NO:49 >α-1 globin 5'UTR [Chemical 50] AGACGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC Sequence number 50 >haplogroup U8b1b1 mitochondrion 3’UTR [Chemical 51] CTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTGCTATACTAATCGCTAGCCGCGTCGCTCCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCGAGACCTGGTCCAGAGCCCAAGCACGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAACAGCAATGCA GCTCAAAACGCTTAGCCTAG Sequence number 51 >A30L70 PolyA [Chemical 52] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Sequence number 52 >5’UTR-Fluc-α globin 3’UTR-120A (Mod.-120A) [Chemical 53] Allocation number 53 >5'UTR-Fluc-α グロビン3'UTR-A30L70 (Mod.-A30L70) [Chemistry 54] Allocation number 54 >α-1-グロビン5'UTR-Fluc-haplogroup U8b1b1 mitochondrion 3'UTR-A30L70 (BioN.) [Chemistry 55] SEQ ID NO:55 >5'UTR-Fluc-ACTG1 3'UTR-120A(B1)

[56] Allocation number 56 >ACTG1 5'UTR-Fluc-α グロビン3'UTR-120A (A1) [Chemistry 57] Accession No. 57 >B1 mRNA sequence (ACTG1 3’UTR) [Chemical 58] CCGACUACCUCAUGAAGAUCCUCACUGAGCGAGGCUACAGCUUCACCACCACGGCCGAGCGGGAAAUCGUGCGCGACAUCAAGGAGAAGCUGUGCUACGUCGCCCUGGACUUCGAGCAGGAGAUGGCCACCGCCGCAUCCUCCUCUUCUCUGGAGAAGAGCUACGAGCUGCCCGAUGGCCAGGUCAUCACCAUUGGCAAUGAGCGGUUCCGGUGUCCGGAGGCGCUGUUCCAGCCUUCCUUCCUGGGUAUGGAAUCUUGCGGCAUCCACGAGACCACCUUCAACUCCAUCAUGAAGUGUGACGUGGACAUCCGCAAAGACCUGUACGCCAACACGGUGCUGUCGGGCGGCACCACCAUGUACCCGGGCAUUGCCGACAGGAUGCAGAAGGAGAUCACCGCCCUGGCGCCCAGCACCAUGAAGAUCAAGAUCAUCGCACCCCCAGAGCGCAAGUACUCGGUGUGGAUCGGUGGCUCCAUCCUGGCCUCACUGUCCACCUUCCAGCAGAUGUGGAUUAG Accession No. 58 >B2 mRNA sequence (ATP6V0B 3’UTR) [Chemical 59] AUGAUAUGUGUGGGUGGGGCCGUGCCUCACUUUUAUUUAUUGCUGGUUUUCCUGGGACAGCUGGAGCUGUGUCCCUUAGCCUUUCAGAGGCUUGGUGUUCAGGGCCCUCCCUGCACUCCCCUCUUGCUGCGUGUUGAUUUGGAGGCACUGCAGUCCAGGCCGAGUCCUCAGUGCGGGGAGCAGGCUGCUGCUGCUGACUCUGUGCAGCUGCGCACCUGUGUCCCCCACCUCCACCCUCAACCCAUCUUCCUAGUGUUUGUGAAAUAAACUUGGUAUUUGUCU Accession No. 59 >B3 mRNA sequence (ATP6V0B 3’UTR) [Chemical formula 60] GUUGAAGAACUUCUCUCUACCGAGCAGCCCGUAACCCCCUUUUUCUCCCUCACUGCUGCAGGAGUCUGCUACACCAUUUUUGAUUUGGGCUUCCGCUUUGAUGUGGCAUGGUUCCUGACGGAGACUUCGCCCUUCAUGUGGUCCAACCUGGGCAUUGGCCUAGCUAUCUCCCUGUCUGUGGUUGGGGCAGCCUGGGGCAUCUAUAUUACCGGCUCCUCCAUCAUUGGUGGAGGAGUGAAGGCCCCCAGGAUCAAGACCAAGAACCUGGUCAGCAUCAUCUUCUGUGAGGCUGUGGCCAUCUACGGCAU Sequence number 60 >B4 mRNA sequence (ATP6V0E1 3’UTR) [Chemical formula 61] GGAAGAAGACAUGCUCUACAGUGCUCAGUCUUUGAGGUGACUAUGCUUGUGACCUUUCUUAUCAGUAUGGUCAGGCAGUCAGAGUUUGCCUUGACUUUUUCUCACAUUUUAUGGCCAUUUUAACACGUGGCAAGGGCAAGCUGCUGAAUGCAAACACGAUCUGAAGAGCAGAAAUAAAAUUGGAACUUAAGCCUUAGCUUACCUAAAUCUCAUUAAGAUUCUUUUU Sequence number 61 >B5 mRNA sequence (ATP6V0E1 3’UTR) [Chemical formula 62] AUGCAAAAUCACCUCCAAACCAGACCACUUUUCUUGACUUGCCUGUUUUGGCCAUUAGCUGCCUUAAACGUUAACAGCACAUUUGAAUGCCUUAUUCUACAAUGCAGCGUGUUUUCCUUUGCCUUUUUUGCACUUUGGUGAAUUACGUGCCUCCAUAACCUGAACUGUGCCGACUCCACAAAACGAUUAUGUACUCUUCUGAGAUAGAAGAUGCUGUUCUUCUGAGAGAUACGUUACUCUCUCCUUGGAAUCUGUGGAUUUGAAGAUGGCUCCUGCCUUCUCACGUGGGAAUCAGUGAAGUGUUUAGAAACUGCUGCAAGACAAACAAGACUCCAGUGGGGUGGUCAGUAGGAGAGCACGUUCAGAGGGAAGAGCCAUCUCAACAGAAUCGCACCAAACUAUACUUUCAGGAUGAAUUUCUUCUUUCUGCCAUCUUUUGGAAUAAAUAUUUUCCUCCUUUC Accession No. 62 >B6 mRNA sequence (CFL1 3’UTR) [Chemical 63] GCCCCUUCUGGCCCCCUGCCUGGAGCAUCUGGCAGCCCCACACCUGCCCUUGGGGGUUGCAGGCUGCCCCCUUCCUGCCAGACCGGAGGGGCUGGGGGGAUCCCAGCAGGGGGAGGGCAAUCCCUUCACCCCAGUUGCCAAACAGACCCCCCACCCCCUGGAUUUUCCUUCUCCCUCCAUCCCUUGACGGUUCUGGCCUUCCCAAACUGCUUUUGAUCUUUUGAUUCCUCUUGGGCUGAAGCAGACCAAGUUCCCCCCAGGCACCCCAGUUGUGGGGGAGCCUGUAUUUUUUUUAACAACAUCCCCAUUCCCCACCUGGUCCUCCCCCUUCCCAUGCUGCCAACUUCUAACCGCAAUAGUGACUCUGUGCUUGUCUGUUUAGUUCUGUGUAUAAAUGGAAUGUUGUGGAGAUGACCCCUCCCUGUGCCGGCUGGUUCCUCUCCCUUUUCCCCUGGUCACGGCUACUCAUGGAAGCAGGACCAGUAAGGGACCUUCGAUU Sequence number 63 >B7 mRNA sequence (CFL1 3’UTR) [Chemical formula 64] GCCCCUUCUGGCCCCCUGCCUGGAGCAUCUGGCAGCCCCACACCUGCCCUUGGGGGUUGCAGGCUGCCCCCUUCCUGCCAGACCGGAGGGGCUGGGGGGAUCCCAGCAGGGGGAGGGCAAUCCCUUCACCCCAGUUGCCAAACAGACCCCCCACCCCCUGGAUUUUCCUUCUCCCUCCAUCCCUUGACGGUUCUGGCCUUCCCAAACUGCUUUUGAUCUUUUGAUUCCUCUUGGGCUGAAGCAGACCAAGUUCCCCCCAGGCACCCCAGUUGUGGGGGAGCC Sequence number 64 >B8 mRNA sequence (CFL1 3’UTR) [Chemical formula 65] GCCCCUUCUGGCCCCCUGCCUGGAGCAUCUGGCAGCCCCACACCUGCCCUUGGGGGUUGCAGGCUGCCCCCUUCCUGCCAGACCGGAGGGGCUGGGGGGAUCCCAGCAGGGGGAGGGCAAUCCCUUCACCCCAGUUGCCAAACAGACCCCCCACCCCCUGGAUUUUCCUUCUCCCUCCAUCCCUUGACGGUUCUGGCCUUCCCAAACUGCUUUUGAUCUUUUGAUUCCUCUUGGGCUGAAGCAGACCAAGUUCCCCCCAGGCACCCCAGUUGUGGGGGAGCCUGUAUUUUUUUUAACAACAUCCCCAUUCCCCACCUGGUCCUCCCCCUUCCCAUGCUGCCAACUUCUAACCGCAAUAGUG Sequence number 65 >B9 mRNA sequence (COX4I1 3’UTR) [Chemical formula 66] GAGAUGCUGGCCUGCGCCUGCACCUGCGCCUGGCUCUGUCACCGCCAUGCAACUCCAUGCCUAUUUACUGGAAACCUGUUAUGCCAAACAGUUGUACCACUGCUAAUAAAUGACCAGUUUACCUGAAA Sequence number 66 >B10 mRNA sequence (COX4I1 3’UTR) [Chemical formula 67] AGGGACCCACAGGCGCGCCCAGCAGCUCUCGGAAGCGUUGUAUCGCAUUAAGUUCAAGGAGAGCUUUGCUGAGAUGAACAGGGGCUCGAACGAGUGGAAGACGGUUGUGGGCGGUGCCAUGUUCUUCAUCGGUUUCACCGCGCUCGUUAUCAUGUGGCAGAAGCACUAUGGGCUUAGCCUCCAAGUGGGACUACGAAAAGAACGAGUGGAAGAAGUGAGAGAUGCUGGCCUGCGCCUGCACCUGCGCCUGGCUCUGUCACCGCCAUGCAACUCCAUGCCUAUUUACUGGAAACCUGUUAUGCCAAACAGUUGUACCACU SEQ ID NO:67 >B11 mRNA sequence (COX4I1 3'UTR)

[68] AGAGGACCUUCUGCUUUUAGCUUAUUUUGUUGCUAACUUUUUACAAACAAAGGGCUAAUUUUAAAAUGUCAGUGUUGUCAGUGGUUCAGAAACCGUGUGUUUGAGCGGGUGUUGAGUGGCAGGUGGCUCUGCUGACCUGGUGGCUGGUGUGU CGGGAGGAUUUAACCUGUGAGGGAUUGGCCUAGAAACAACCUGUUGAGAUAGUCUUGCCCCAUAACCUGUCUCACACCGUAGUGUACGGCCCCCUCCCGCAAAGCUUUGACAAAGAGUGGUGGCCAAGCAGACCAAGAGGAUGCUGGAC SEQ ID NO:68 >B12 mRNA sequence (CTSB 3'UTR)

[69] UCUGCCGUGGGCCUGUCGUGCCAGUCCUGGGGGCGAGAUCGGGAGAAUCACGUCCUGGAACUGCAUGUUCUUGCGACUCUUGGGACUUCAUCUUAACUUCUCGCUGCCCCAGCCAU GUUUUCAACCAUGGCAUCCCUCCCCCAAUUAGUUCCCUGUCAUCCUCGUCAACCUUCUCUGUAAGUGCCUGGUAAGCUUGCCCUUGCUUAAGAACUCAAAACAUAGCUGUGCUCUA SEQ ID NO:69 >B13 mRNA sequence (FAM166A 3'UTR)

[70] AAAUGCUUUAAUGGUGGUGUCUGUACAGCAUGGAUGUGGACAGGGAAAACAGCAAGUGCACACAGUGGGACAGGCCACAGGACAGGCUGGGAGUGAAUAAAGAGUUCACACUGCU SEQ ID NO:70 >B14 mRNA sequence (NDUFB9 3'UTR)

[71] UAUCUAAGUGUAUCAAUUUAACAUUUAUAAUCACCUUACAAGUAAGUUAAAGUGCAUUCCCUGGUCGUUUUGGCUUUGUCUUUGAUUUUGCUUCAGAUCUCCUUCUCUUUAGUCCUUUGUCUGACUUUUUUGCCUUCUUGCUAUACUUUCCCUCAACCUGCCUCUCCACUAUUUUCUUAGGCAUUUUUCAGUCAUG SEQ ID NO:71 >A1 mRNA sequence (ACTG1 5'UTR)

[72] CUCUCGCACUCUGUUCUUCCGCCGCUCCGCCGUCGCGUUUCUCUGCCGGUGGCA SEQ ID NO:72 >A2 mRNA sequence (ATP6V0B 5'UTR)

[73] GGGCGGGCGGACAGACUGCGGGACGGACGGUGGACGCUGGGACGCGUUUGUAGCUCCGGCCCCGCCGUUCCGACCCCCGCCGCCGUCGCCGCCAUGACGGGGCUAGCACUGCUCUACUCCGGGGUCUUGUGGCCUUCUGGGCCUGCGCGCUGGCCGUGGGUUCCUGACGGAGACUUCGCCCUUC SEQ ID NO:73 >A3 mRNA sequence (ATP6V0B 5'UTR)

[74] AGACUGCGGGACGGACGGUGGACGCUGGGACGCGUUUGUAGCUCCGGCCCCGCCGUUCCGACCCCCGCCGCCGUCGCCGCC SEQ ID NO:74 >A4 mRNA sequence (ATP6V0E1 5'UTR)

[75] GGGGUAGGGGUUGGCGCUCAGGCGGCGACC SEQ ID NO:75 >A5 mRNA sequence (ATP6V0E1 5'UTR)

[76] GUCACGCGGUCAGCUAUUGACACUUCCUGGUGGGAUCCGAGUGAGGCGACGGGGUAGGGGUUGGCGCUCAGGCGGCGACC Sequence number 76 >A6 mRNA sequence (CFL1 5’UTR) [Chemical formula 77] GGCCGGCGGGAAGACUCCGUUACCCAGCGAGCGAGGCGGCGGCGCAGGGCCAGCGGACUCCAUUUCCCGUCGGCUCGCGGUGGGAGCGCCGGAAGCCCGCCCCACCCCUCAUUGUGCGGCUCCUACUAAACGGAAGGGGCCGGGAGAGGCCGCGUUCAGUCGGGUCCCGGCAGCGGCUGCAGCGCUCUCGUCUUCUGCGGCUCUCGGUGCCCUCUCCUUUUCGUUUCCGGAAAC Sequence number 77 >A7 mRNA sequence (CFL1 5’UTR) [Chemical formula 78] CUCCUGGCUCGGGCACCCCGAAGCACGACGCAGAGUAGGAAGAGUUUAAAGACCUCGAGGCCUCUGGGCACUUGAGUUUGGCCUCCGGUGUGGCUGUCUCUGAUGGUGUCAUCAAGGUGUUCAACGAC Sequence number 78 >A8 mRNA sequence (CFL1 5’UTR) [Chemical formula 79] ACGUGGCCGGCUUCUUCUGCAGUUCCGGGGAGUUUGGGGGACCAGAUUUACCUUGGAUUGCCCCUCCCUCUCCUGGCUCGGGCACCCCGAAGCACGACGCAGAGCCUCCGGUGUGGCUGUCUCUGAUGGUGUCAUCAAGGUGUUCAACGAC Sequence number 79 >A9 mRNA sequence (COX4I1 5’UTR) [Chemical formula 80] GCGGCCUUGCUCUCUUCCGGUCGCGGGACACCGGGUGUAGAGGGCGGUCGCGGCGGGCAGUGGCGGCAGA SEQ ID NO:80 >A10 mRNA sequence (COX4I1 5'UTR)

[81] GGGACACCGGGUGUAGAGGGCGGUCGCGGCGGGCAGUGGCGGCAGA SEQ ID NO:81 >A11 mRNA sequence (COX4I1 5'UTR)

[82] CGCGGCCUUGCUCUCUUCCGGUCGCGGGACACCGGGUGUAGAGGGCGGUCGCGGCGGGCAGUGGCGGCAGA SEQ ID NO:82 >A12 mRNA sequence (CUSB 5'UTR)

[83] GGCCGGGGCUGGCCCAGGCUACGGCGGCUGCAGGGCUCCGGCAACCGCUCCGGCAACGCCAACCGCUCCGCUGCGCGCAGGCUGGGCUGCAGGCUCUCGGCUGCAGCGCUGGGUGGAUCUAGGAUCCGGCUUCCAAC SEQ ID NO:83 >A13 mRNA sequence (FAM166A 5'UTR)

[84] AAGGGAGCUGGAUGCCGGGAGGGACUGGAGCCAGCAAGGCCAGAGUGAAAGCAAA SEQ ID NO:84 >A14 mRNA sequence (NDUFB9 5'UTR)

[85] GCAGCAGGCUGCAGUUUCCCGGCUCUCCGCCGGCCGGGGAAGGUCAGCGCCGUA SEQ ID NO:85 >A15 mRNA sequence (CHCHD10 5'UTR)

[86] GGCAUUUGUCCCCGCGACAGCACCGCUGCCGCCGUCUCUAAGGUCGCCCGGGUCCCACCGCCGCCACC SEQ ID NO:86 >A16 mRNA sequence (CHCHD10 5'UTR)

[87] CCGCUGCCGCCGUCUCUAAGGUCGCCCGGGUCCCACCGCCGCCACC SEQ ID NO:87 >A17 mRNA sequence (SLC38A2 5'UTR)

[88] AUGUCUUUUUUUGUGUGUUUGUUUUCAUGGUAUUCCUAUGAA SEQ ID NO:88 >A18 mRNA sequence (NDUFA11 5'UTR)

[89] GCUUCCCGAGCUGGCGGGGUCCGUGGUGCGGGAUCGAGAUUGCGGGCU SEQ ID NO:89 >A19 mRNA sequence (NDUFV3 5'UTR)

[90] GGCGGCUGUUCAGGCGCGGGUGCGCGCGCAGCUGCUGUGGCCCUGCUUGGUGCGCCCGCUGUCACCGCC SEQ ID NO:90 >A20 mRNA sequence (PRDX5 5'UTR)

[91] CGCGCCUGCGCAGUGGAGGCGGCCCAGGCCCGCCUUCCGCAGGGUGUCGCCGCUGUGCCGCUAGCGGUGCCCCGCCUGCUGCGGUGGCACCAGCCAGGAGGCGGAGUGGAAGUGGCCGUGGGGCGGGU SEQ ID NO:91 >A21 mRNA sequence (GUK1 5'UTR)

[92] CAGCAGAUGGGGACUAGAGGCCGCACUGCUAUCCACAGCCUCUCUUCUCACCCCCAGGC SEQ ID NO:92 >A22 mRNA sequence (GUK1 5'UTR)

[93] GCUGGCCGGGCUGGCUGCGGCCGCCCUGGGCCGGGCCCCACCGGACGCCUCUCUUCUCACCCCCAGGC SEQ ID NO:93 >A23 mRNA sequence (GUK1 5'UTR)

[94] AGAGGUGGCCCCGGAUGCUGCGGCGCCCGCUGGCCGGGCUGGCUGCGGCCGCCCUGGGCCGGGCCCCACCGGACGCCUCUCUUCUCACCCCCAGGC SEQ ID NO:94 >A24 mRNA sequence (IAH1 5'UTR)

[95] UGGCUGGCGGCCCCGCCCCGCCCCGCCCGGCUGCUCC SEQ ID NO:95 >A25 mRNA sequence (ABHD16A 5'UTR)

[96] GGAGGGCGGGGCCGGCAGGGGGACCUGCUGCUGGAAGAGCAGCGGCCCGAGCCGGGGCC SEQ ID NO:96 >A26 mRNA sequence (SLC25A39 5'UTR)

[97] UUUCGGCGUCGGCCUAGGUGCGCUGCGAGCGCGCGGACCGCGCACAGGCGGCGGAGCCGGUAUGGGCCCGCCUGACCCUGGGCGCCGCGCCGCACGAGCACCAGCCUAGAGCCAGGACUGAAGCUUCAAG SEQ ID NO:97 >A27 mRNA sequence (AUPIF1 5'UTR)

[98] CGAGAGACUGCUUGCUGCGGCAGAGACGCCAGAGGUGCAGCUCCAGCAGCAGCA SEQ ID NO:98 >A28 mRNA sequence (ANAPC11 5'UTR)

[99] CGGAGUUUCGUCAUGUUGGCCAGGCCCAUUUGAGAUCUUUGAAGAUAUCCUCAACGUGAGGGCUCUGCUGCC SEQ ID NO:99 >A29 mRNA sequence (ANAPC11 5'UTR)

[100] GGGCGCGGCUUCGGCGGGCGGCAGCCGCUGGCAGACGAGCUGCGGGCUCUGCUGCC SEQ ID NO:100 >A30 mRNA sequence (CCDC12 5'UTR) [C101] GCCUGCGCGAUGCAAGACGGGAGAAAAGGAGGGGCGUACGCGGGCAAG SEQ ID NO:101 >A31 mRNA sequence (MRPL14 5'UTR) [C102] AUCGCGUCCGCCGGGCUGGGCCUGGCGCGCAGGCGCUAGGAAGAGGCCGCGUGGGGCGAAGGCGGCGCUUGGCUGUGGGGCCCGCGGCGGGAUUUUCCCGGGCGGCGAGAGCGGAUCUAUCUUGGGAUCCC SEQ ID NO:102 >A32 mRNA sequence (APOA1BP 5'UTR) [C103] GCCGGGGGCGCGCGCUCUGCGAGCUGG SEQ ID NO:103 >A33 mRNA sequence (APOA1BP 5'UTR) [C104] GCCGGGGCCGGGCCGGGCCGGGGGCGCGCGCUCUGCGAGCUGG SEQ ID NO:104 > Mod.5'UTR mRNA sequence [C105] GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC SEQ ID NO:105 > α-globin 3'UTR mRNA sequence [C106] UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCC UCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUU UGAAUAAAGUCUGAGUGGGCGGC SEQ ID NO:106 >α-1 globin 5'UTR mRNA sequence [C107] AGACGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC SEQ ID NO:107 > Haplogroup U8b1b1 mitochondrion 3'UTR mRNA sequence [C108] CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUGCUAUACU AAUCGCUAGCCGCGUCGCUCCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCGAGACCUGGUCCAGAGCCCAAGCACGCCACACCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAACAGCAAUGCA GCUCAAAACGCUUAGCCUAG SEQ ID NO:108 >hHGF amino acid sequence [C109] MWVTKLLPALLLQHVLLHLLLLPIAIPYAEGQRKRRNTIHEFKKSAKTTLIKIDPALKIKTKKVNTADQCANRCTRNKGLPFTCKAFVFDKARKQCLWFPFNSMSSGVKKEFGHEFDLYENKDYIRNCIIGKGRSYKGTVSITKSGIKCQPWSSMIPHEHSFLPSSYRGKDLQENYCRNPRG EEGGPWCFTSNPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTPHRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPLETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGESESPW CFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNMEDLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTPTIVNLDHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRDLKDYEA WLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLPNYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAGAEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKIILTYKVPQS SEQ ID NO:109 >hHGF nucleotide sequence [C110] SEQ ID NO:110 >hHGF codon-optimized sequence 1 (hHGF-OS1) [C111] SEQ ID NO:111 >hHGF codon-optimized sequence 2 (hHGF-OS2) [C112] SEQ ID NO:112 >hHGF codon-optimized sequence 3 (hHGF-OS3) [C113] SEQ ID NO:113 >5'UTR-hHGF-α Globin 3'UTR-A30L70 (Mod.-hHGF-OS2) [C114] SEQ ID NO:114 >ACTG1 5'UTR-hHGF-CTSB 3'UTR-A30L70(A1B12-hHGF-OS2) [C115] SEQ ID NO:115 >CHCHD10 5'UTR-hHGF-CTSB 3'UTR-A30L70(A15B12-hHGF-OS2) [C116] SEQ ID NO:116 >PD-1 antibody heavy chain amino acid sequence [C117] MEWSWVFLFFLSVTTGVHSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWVATISGGGANTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC ARQLYYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDK RVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO:117 >PD-1 antibody light chain amino acid sequence [C118] MSVPTQVLGLLLLWLTDARCDIQMTQSPSSLSASVGDRVTITCLASQTIGTWLTWYQQKPGKAPKLLIYTATSLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYSIPWT FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:118 >PD-1 antibody heavy chain DNA sequence [C119] Accession No. 119 >PD-1 antibody light chain DNA sequence [Chemical 120] ATGTCCGTGCCTACCCAGGTGCTGGGACTGCTGCTGCTGTGGCTGACCGACGCCAGATGCGACATCCAGATGACCCAGTCCCCCTCCAGCCTGTCTGCCTCTGTGGGCGACAGAGTGACCATCACCTGTCTGGCCTCCCAGACCATCGGCACCTGGCTGACCTGGTATCAGCAGAAGCCTGGCAAGGCCCCCAAGCTGCTGATCTACACCGCCACCAGTCTGGCCGATGGCGTGCCCTCTAGATTCTCCGGCTCTGGCTCTGGCACCGACTTTACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGTGTACTCCATCCCCTGGACCTTTGGCGGAGGCACCAAGGTGGAAATCAAGCGGACCGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGAACCGCCAGTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGC Accession No. 120 >PD-1 antibody heavy chain mRNA sequence [Chemical 121] Accession No. 121 >PD-1 antibody light chain mRNA sequence [Chemical 122] AUGUCCGUGCCUACCCAGGUGCUGGGACUGCUGCUGCUGUGGCUGACCGACGCCAGAUGCGACAUCCAGAUGACCCAGUCCCCCUCCAGCCUGUCUGCCUCUGUGGGCGACAGAGUGACCAUCACCUGUCUGGCCUCCCAGACCAUCGGCACCUGGCUGACCUGGUAUCAGCAGAAGCCUGGCAAGGCCCCCAAGCUGCUGAUCUACACCGCCACCAGUCUGGCCGAUGGCGUGCCCUCUAGAUUCUCCGGCUCUGGCUCUGGCACCGACUUUACCCUGACCAUCAGCUCCCUGCAGCCCGAGGACUUCGCCACCUACUACUGCCAGCAGGUGUACUCCAUCCCCUGGACCUUUGGCGGAGGCACCAAGGUGGAAAUCAAGCGGACCGUGGCCGCUCCCUCCGUGUUCAUCUUCCCACCUUCCGACGAGCAGCUGAAGUCCGGAACCGCCAGUGUCGUGUGCCUGCUGAACAACUUCUACCCCCGCGAGGCCAAGGUGCAGUGGAAGGUGGACAACGCCCUGCAGUCCGGCAACUCCCAGGAAUCCGUGACCGAGCAGGACUCCAAGGACAGCACCUACUCCCUGUCCUCCACCCUGACCCUGUCCAAGGCCGACUACGAGAAGCACAAGGUGUACGCCUGCGAAGUGACCCACCAGGGCCUGUCUAGCCCCGUGACCAAGUCUUUCAACCGGGGCGAGUGC Accession No. 122 >5’UTR-anti PD-1-α globin 3’UTR-A30L70 (Mod.- anti PD-1) [Chemical 123] SEQ ID NO:123 >ACTG1 5'UTR-anti PD-1-CTSB 3'UTR-A30L70(A1B12-anti PD-1)

[124] SEQ ID NO:124 >CHCHD10 5'UTR-anti PD-1-CTSB 3'UTR-A30L70(A15B12-anti PD-1) [C125] SEQ ID NO:125 >Firefly Luciferase Nucleotide Sequence (Fluc) [C126] SEQ ID NO:126 >HGF-OS2 expression vector CHCHD10 5'UTR- hHGF-CTSB 3'UTR-A30L70(A16B12-HGF-OS2)

[127] SEQ ID NO:127 > hHGF protein mRNA sequence

[128] SEQ ID NO:128 >hHGF codon-optimized mRNA sequence 1 (hHGF-OS1) [C129] SEQ ID NO:129 >hHGF codon-optimized mRNA sequence 2 (hHGF-OS2) [C130] SEQ ID NO:130 >hHGF codon-optimized mRNA sequence 3 (hHGF-OS3) [C131] SEQ ID NO:131

Claims

1. A nucleic acid construct comprising: (a) an open reading frame (ORF); (b) Any one of SEQ ID NOs: 12, 26 or a sequence having at least 90% identity thereto; Any one of SEQ ID NOs: 1, 15 or a sequence having at least 90% identity thereto; Any one of SEQ ID NOs: 4-5, 18-19 or a sequence having at least 90% identity thereto; Any one of SEQ ID NOs: 6 to 8, 20 to 22 or a sequence having at least 90% identity thereto; Any one of SEQ ID NOs: 9 to 11, 23 to 25 or a sequence having at least 90% identity thereto; Any one of SEQ ID NOs: 13, 27 or a sequence having at least 90% identity thereto; Any one of SEQ ID NOs: 14, 28 or a sequence having at least 90% identity thereto; Any one of SEQ ID NOs: 29, 30 or a sequence having at least 90% identity thereto; Any one of SEQ ID NOs: 35 to 37 or a sequence having at least 90% identity thereto; Any one of SEQ ID NOs: 42 and 43 or a sequence having at least 90% identity thereto; Any one of SEQ ID NOs: 46, 47 or a sequence having at least 90% identity thereto; or and an untranslated region (UTR) comprising any one of SEQ ID NOs: 31-34, 38-41, 44-45, or a sequence having at least 90% identity thereto; Nucleic acid constructs.

2. The nucleic acid construct of claim 1, wherein (b) comprises a combination of any one of the following 3' untranslated region elements (3'UTR) and 5' untranslated region elements (5'UTR): 1) the 3'UTR comprises a sequence represented by SEQ ID NO: 1 or a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by any one of SEQ ID NOs: 15 to 47 or a sequence having at least 90% identity thereto; 2) the 3'UTR comprises a sequence represented by SEQ ID NO: 2 or a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by any one of SEQ ID NOs: 15 to 47 or a sequence having at least 90% identity thereto; 3) the 3'UTR comprises a sequence represented by SEQ ID NO: 3 or a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by any one of SEQ ID NOs: 15 to 47 or a sequence having at least 90% identity thereto; 4) the 3'UTR comprises a sequence represented by SEQ ID NO: 4 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by any one of SEQ ID NOs: 15 to 47 or having at least 90% identity thereto; 5) the 3'UTR comprises a sequence represented by SEQ ID NO: 5 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by any one of SEQ ID NOs: 15 to 47 or having at least 90% identity thereto; 6) The 3'UTR comprises a sequence represented by SEQ ID NO: 6 or a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by any one of SEQ ID NOs: 15 to 47 or a sequence having at least 90% identity thereto; 7) The 3'UTR comprises a sequence represented by SEQ ID NO: 7 or a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by any one of SEQ ID NOs: 15 to 47 or a sequence having at least 90% identity thereto; 8) The 3'UTR comprises a sequence represented by SEQ ID NO: 8 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by any one of SEQ ID NOs: 15 to 47 or having at least 90% identity thereto; 9) The 3'UTR comprises a sequence represented by SEQ ID NO: 9 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by any one of SEQ ID NOs: 15 to 47 or having at least 90% identity thereto; 10) The 3'UTR comprises a sequence represented by SEQ ID NO: 10 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by any one of SEQ ID NOs: 15 to 47 or having at least 90% identity thereto; 11) The 3'UTR comprises a sequence represented by SEQ ID NO: 11 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by any one of SEQ ID NOs: 15 to 47 or having at least 90% identity thereto; 12) The 3'UTR comprises a sequence represented by SEQ ID NO: 12 or a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by any one of SEQ ID NOs: 15 to 47 or a sequence having at least 90% identity thereto; 13) The 3'UTR comprises a sequence represented by SEQ ID NO: 13 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by any one of SEQ ID NOs: 15 to 47 or having at least 90% identity thereto; 14) The 3'UTR comprises a sequence represented by SEQ ID NO: 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by any one of SEQ ID NOs: 15 to 47 or having at least 90% identity thereto; 15) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 15 or having at least 90% identity thereto; 16) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 16 or having at least 90% identity thereto; 17) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 17 or having at least 90% identity thereto; 18) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 18 or having at least 90% identity thereto; 19) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 19 or having at least 90% identity thereto; 20) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 20 or having at least 90% identity thereto; 21) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 21 or having at least 90% identity thereto; 22) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 22 or having at least 90% identity thereto; 23) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 23 or having at least 90% identity thereto; 24) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 24 or having at least 90% identity thereto; 25) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 25 or having at least 90% identity thereto; 26) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 26 or having at least 90% identity thereto; 27) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 27 or having at least 90% identity thereto; 28) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 28 or having at least 90% identity thereto; 29) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 29 or having at least 90% identity thereto; 30) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 30 or having at least 90% identity thereto; 31) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 31 or having at least 90% identity thereto; 32) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 32 or having at least 90% identity thereto; 33) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 33 or having at least 90% identity thereto; 34) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 34 or having at least 90%, 95% identity thereto; 35) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 35 or having at least 90% identity thereto; 36) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 36 or having at least 90% identity thereto; 37) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 37 or having at least 90% identity thereto; 38) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 38 or having at least 90% identity thereto; 39) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 39 or having at least 90% identity thereto; 40) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 40 or having at least 90% identity thereto; 41) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 41 or having at least 90% identity thereto; 42) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 42 or having at least 90% identity thereto; 43) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 43 or having at least 90% identity thereto; 44) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 1 to 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 44 or having at least 90% identity thereto; 45) The 3'UTR comprises a sequence having at least 90% identity with any one of SEQ ID NOs: 1 to 14, and the 5'UTR comprises a sequence having at least 90% identity with any one of SEQ ID NOs: 45, 46) The 3'UTR comprises a sequence having at least 90% identity to any one of SEQ ID NOs: 1 to 14, and the 5'UTR comprises a sequence having at least 90% identity to any one of SEQ ID NOs: 46, or 47) The 3'UTR comprises a sequence set forth in any one of SEQ ID NOs: 1 to 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence set forth in SEQ ID NO: 47 or having at least 90% identity thereto.

3. The 3'UTR comprises a sequence having at least 90% identity with any one of SEQ ID NOs: 12 to 14, and the 5'UTR comprises a sequence having at least 90% identity with any one of SEQ ID NOs: 15, 29, 30, and 32, Preferably, The 3'UTR comprises a sequence set forth in SEQ ID NO: 12 or a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence set forth in any one of SEQ ID NOs: 15, 29, 30, and 32 or a sequence having at least 90% identity thereto; The 3'UTR comprises a sequence set forth in SEQ ID NO: 13 or a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence set forth in any one of SEQ ID NOs: 15, 29, 30, and 32 or a sequence having at least 90% identity thereto; The 3'UTR comprises a sequence set forth in SEQ ID NO: 14 or a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence set forth in any one of SEQ ID NOs: 15, 29, 30, and 32 or a sequence having at least 90% identity thereto; The 3'UTR comprises a sequence set forth in any one of SEQ ID NOs: 12 to 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence set forth in SEQ ID NO: 15 or having at least 90% identity thereto; The 3'UTR comprises a sequence set forth in any one of SEQ ID NOs: 12 to 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence set forth in SEQ ID NO: 29 or having at least 90% identity thereto; the 3'UTR comprises a sequence set forth in any one of SEQ ID NOs: 12-14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence set forth in SEQ ID NO: 30 or having at least 90% identity thereto; or The 3'UTR comprises a sequence set forth in any one of SEQ ID NOs: 12 to 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence set forth in SEQ ID NO: 32 or having at least 90% identity thereto; The nucleic acid construct of claim 1.

4. (c) further comprising a polyadenylic acid (poly-A) tail; Preferably, the poly-A tail is selected from A120, A30L70, HGH polyA, SV40 polyA, BGH polyA, rbGlob polyA or SV40late polyA; Preferably, the poly-A tail is selected from A120 or A30L70, wherein A120 comprises 120 adenine nucleotides, and A30L70 comprises a sequence as set forth in SEQ ID NO:52 or having at least 90% identity thereto; A nucleic acid construct according to any one of claims 1 to 3.

5. The ORF encodes hepatocyte growth factor (HGF), an antibody or an antigen-binding fragment thereof, preferably human hepatocyte growth factor (hHGF), an anti-PD-1 antibody or an antigen-binding fragment thereof; A nucleic acid construct according to any one of claims 1 to 4.

6. The ORF is the following 1) to 2), that is, 1) a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 109; 2) A polynucleotide sequence having at least 90% identity to any one of SEQ ID NOs: 110 to 113; Alternatively, the ORF may be any one of the following 1) to 3), i.e. 1) a polynucleotide sequence encoding the heavy chain amino acid sequence shown in SEQ ID NO: 117, and / or a polynucleotide sequence encoding the light chain amino acid sequence shown in SEQ ID NO: 118; 2) a polynucleotide sequence encoding HCDR1, HCDR2 and HCDR3 in the heavy chain amino acid sequence shown in SEQ ID NO: 117, and a polynucleotide sequence encoding LCDR1, LCDR2 and LCDR3 in the light chain amino acid sequence shown in SEQ ID NO: 118, wherein the CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system, preferably according to the Kabat numbering system; 3) A polynucleotide sequence represented by SEQ ID NO: 119 or having at least 90% identity thereto, and / or a polynucleotide sequence represented by SEQ ID NO: 120 or having at least 90% identity thereto, The nucleic acid construct of claim 5.

7. It comprises a sequence represented by any one of SEQ ID NOs: 115, 116, 127 or a sequence having at least 90% identity thereto, or a sequence represented by SEQ ID NO: 124 or a sequence having at least 90% identity thereto and / or a sequence represented by SEQ ID NO: 125 or a sequence having at least 90% identity thereto; A nucleic acid construct according to any one of claims 5 to 6.

8. 1. An RNA molecule comprising: (a) an open reading frame (ORF); (b) Any one of SEQ ID NOs: 69, 83 or a sequence having at least 90% identity thereto; Any one of SEQ ID NOs: 58, 72 or a sequence having at least 90% identity thereto; Any one of SEQ ID NOs: 59-60, 73-74 or a sequence having at least 90% identity thereto; Any one of SEQ ID NOs: 61-62, 75-76 or a sequence having at least 90% identity thereto; Any one of SEQ ID NOs: 63 to 65, 77 to 79 or a sequence having at least 90% identity thereto; Any one of SEQ ID NOs: 66 to 68, 80 to 82 or a sequence having at least 90% identity thereto; Any one of SEQ ID NOs: 70, 84 or a sequence having at least 90% identity thereto; Any one of SEQ ID NOs: 71, 85 or a sequence having at least 90% identity thereto; Any one of SEQ ID NOs: 86, 87 or a sequence having at least 90% identity thereto; Any one of SEQ ID NOs: 92 to 94 or a sequence having at least 90% identity thereto; Any one of SEQ ID NOs: 99, 100 or a sequence having at least 90% identity thereto; Any one of SEQ ID NOs: 103, 104 or a sequence having at least 90% identity thereto; or and an untranslated region (UTR) comprising any one of SEQ ID NOs: 88-91, 95-98, 101-102, or a sequence having at least 90% identity thereto; RNA molecule.

9. The RNA molecule of claim 8, wherein (b) comprises a combination of any one of the following 3' untranslated region elements (3'UTR) and 5' untranslated region elements (5'UTR): 1) the 3'UTR comprises a sequence represented by SEQ ID NO: 58 or a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 72 to 104 or a sequence having at least 90% identity thereto; 2) the 3'UTR comprises a sequence represented by SEQ ID NO: 59 or a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 72 to 104 or a sequence having at least 90% identity thereto; 3) the 3'UTR comprises a sequence represented by SEQ ID NO: 60 or a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 72 to 104 or a sequence having at least 90% identity thereto; 4) the 3'UTR comprises a sequence represented by SEQ ID NO: 61 or a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 72 to 104 or a sequence having at least 90% identity thereto; 5) the 3'UTR comprises a sequence represented by SEQ ID NO: 62 or a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 72 to 104 or a sequence having at least 90% identity thereto; 6) The 3'UTR comprises a sequence represented by SEQ ID NO: 63 or a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 72 to 104 or a sequence having at least 90% identity thereto; 7) The 3'UTR comprises a sequence represented by SEQ ID NO: 64 or a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 72 to 104 or a sequence having at least 90% identity thereto; 8) The 3'UTR comprises a sequence represented by SEQ ID NO: 65 or a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 72 to 104 or a sequence having at least 90% identity thereto; 9) The 3'UTR comprises a sequence represented by SEQ ID NO: 66 or a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 72 to 104 or a sequence having at least 90% identity thereto; 10) The 3'UTR comprises a sequence represented by SEQ ID NO: 67 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by any one of SEQ ID NOs: 72 to 104 or having at least 90% identity thereto; 11) The 3'UTR comprises a sequence represented by SEQ ID NO: 68 or a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 72 to 104 or a sequence having at least 90% identity thereto; 12) The 3'UTR comprises a sequence represented by SEQ ID NO: 69 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by any one of SEQ ID NOs: 72 to 104 or having at least 90% identity thereto; 13) The 3'UTR comprises a sequence represented by SEQ ID NO: 70 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by any one of SEQ ID NOs: 72 to 104 or having at least 90% identity thereto; 14) The 3'UTR comprises a sequence represented by SEQ ID NO: 71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by any one of SEQ ID NOs: 72 to 104 or having at least 90% identity thereto; 15) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 58 to 71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 72 or having at least 90% identity thereto; 16) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 58 to 71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 73 or having at least 90% identity thereto; 17) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 58 to 71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 74 or having at least 90% identity thereto; 18) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 58 to 71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 75 or having at least 90% identity thereto; 19) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 58 to 71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 76 or having at least 90% identity thereto; 20) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 58 to 71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 77 or having at least 90% identity thereto; 21) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 58 to 71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 78 or having at least 90% identity thereto; 22) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 58 to 71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 79 or having at least 90% identity thereto; 23) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 58 to 71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 80 or having at least 90% identity thereto; 24) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 58 to 71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 81 or having at least 90% identity thereto; 25) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 58 to 71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 82 or having at least 90% identity thereto; 26) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 58 to 71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 83 or having at least 90% identity thereto; 27) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 58 to 71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 84 or having at least 90% identity thereto; 28) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 58 to 71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 85 or having at least 90% identity thereto; 29) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 58 to 71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 86 or having at least 90% identity thereto; 30) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 58 to 71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 87 or having at least 90% identity thereto; 31) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 58 to 71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 88 or having at least 90% identity thereto; 32) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 58 to 71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 89 or having at least 90% identity thereto; 33) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 58 to 71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 90 or having at least 90% identity thereto; 34) The 3'UTR comprises a sequence having at least 90% identity with any one of SEQ ID NOs: 58 to 71, and the 5'UTR comprises a sequence having at least 90%, 95% identity with any one of SEQ ID NOs: 91, 35) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 58 to 71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 92 or having at least 90% identity thereto; 36) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 58 to 71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 93 or having at least 90% identity thereto; 37) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 58 to 71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 94 or having at least 90% identity thereto; 38) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 58 to 71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 95 or having at least 90% identity thereto; 39) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 58 to 71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 96 or having at least 90% identity thereto; 40) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 58 to 71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 97 or having at least 90% identity thereto; 41) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 58 to 71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 98 or having at least 90% identity thereto; 42) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 58 to 71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 99 or having at least 90% identity thereto; 43) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 58 to 71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 100 or having at least 90% identity thereto; 44) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 58 to 71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 101 or having at least 90% identity thereto; 45) The 3'UTR comprises a sequence represented by any one of SEQ ID NOs: 58 to 71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence represented by SEQ ID NO: 102 or having at least 90% identity thereto; 46) The 3'UTR comprises a sequence set forth in any one of SEQ ID NOs: 58-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence set forth in SEQ ID NO: 103 or having at least 90% identity thereto; or 47) The 3'UTR comprises a sequence set forth in any one of SEQ ID NOs: 58-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence set forth in SEQ ID NO: 104 or having at least 90% identity thereto.

10. The 3'UTR comprises a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence having at least 90% identity thereto, and Preferably, The 3'UTR comprises a sequence set forth in SEQ ID NO: 69 or a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence set forth in any one of SEQ ID NOs: 72, 86, 87, and 89 or a sequence having at least 90% identity thereto; The 3'UTR comprises a sequence set forth in SEQ ID NO: 70 or having at least 90% identity thereto, and the 5'UTR comprises a sequence set forth in any one of SEQ ID NOs: 72, 86, 87, and 89 or having at least 90% identity thereto; The 3'UTR comprises a sequence set forth in SEQ ID NO: 71 or a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence set forth in any one of SEQ ID NOs: 72, 86, 87, and 89 or a sequence having at least 90% identity thereto; The 3'UTR comprises a sequence set forth in any one of SEQ ID NOs: 69-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence set forth in SEQ ID NO: 72 or having at least 90% identity thereto; The 3'UTR comprises a sequence set forth in any one of SEQ ID NOs: 69-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence set forth in SEQ ID NO: 86 or having at least 90% identity thereto; the 3'UTR comprises a sequence set forth in any one of SEQ ID NOs: 69-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence set forth in SEQ ID NO: 87 or having at least 90% identity thereto; or The 3'UTR comprises a sequence set forth in any one of SEQ ID NOs: 69-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence set forth in SEQ ID NO: 89 or having at least 90% identity thereto; The RNA molecule of claim 8.

11. (c) further comprising a polyadenylic acid (poly-A) tail; Preferably, the poly-A tail is selected from A120, A30L70, HGH polyA, SV40 polyA, BGH polyA, rbGlob polyA or SV40late polyA; Preferably, the poly-A tail is selected from A120 or A30L70, wherein A120 comprises 120 adenine nucleotides, and A30L70 comprises a sequence as set forth in SEQ ID NO:52 or having at least 90%, 95% identity thereto; An RNA molecule according to any one of claims 8 to 10.

12. the ORF encodes hepatocyte growth factor (HGF), an antibody or an antigen-binding fragment thereof, preferably human hepatocyte growth factor (hHGF), an anti-PD-1 antibody or an antigen-binding fragment thereof; Preferably, the ORF is one of the following 1) to 2), i.e. 1) a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO: 109, or 2) any one of SEQ ID NOs: 128 to 131 or an RNA sequence having at least 90%, preferably at least 95%, identity thereto; or The ORF includes the following 1) to 3), that is, 1) a nucleic acid sequence encoding the heavy chain amino acid sequence shown in SEQ ID NO: 117, and / or a nucleic acid sequence encoding the light chain amino acid sequence shown in SEQ ID NO: 118; 2) A nucleic acid sequence encoding HCDR1, HCDR2, and HCDR3 in the heavy chain amino acid sequence shown in SEQ ID NO: 117, and a nucleic acid sequence encoding LCDR1, LCDR2, and LCDR3 in the light chain amino acid sequence shown in SEQ ID NO: 118, wherein the CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system, preferably according to the Kabat numbering system; 3) An RNA sequence represented by SEQ ID NO: 121 or having at least 90% identity thereto, and / or an RNA sequence represented by SEQ ID NO: 122 or having at least 90% identity thereto, An RNA molecule according to any one of claims 8 to 11.

13. (d) comprising a 5' cap structure (5'Cap); Preferably, said 5'Cap is selected from Cap0, Cap1, Cap2, Cap3, Cap4, ARCA, modified ARCA, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine and 2-azido-guanosine; More preferably, the 5'Cap is ARCA, 3'-O-Me-m ７ G(5')ppp(5')G,m ７ G(5')ppp(5')(2'OMeA)pU,m ７ Gppp(A2'O-MOE)pG,m ７ G(5')ppp(5')(2'OMeA)pG,m ７ G(5')ppp(5')(2'OMeG)pG,m ７ (3'OMeG) (5') ppp (5') (2'OMeG) pG or m ７ (3'OMeG)(5')ppp(5')(2'OMeA)pG, An RNA molecule according to any one of claims 8 to 12.

14. one or more modifications, preferably the modifications include a backbone modification, a sugar modification, a base modification and / or a lipid modification, more preferably the base modification is a uracil modification; An RNA molecule according to any one of claims 8 to 13.

15. The RNA molecule is for improving the expression level of a target protein of the ORF. An RNA molecule according to any one of claims 8 to 14.

16. (a) an isolated polynucleotide comprising an open reading frame (ORF), the ORF comprising a sequence set forth in any one of SEQ ID NOs: 111-113 and 129-131, or a sequence having at least 90% identity thereto; Preferably, the polynucleotide is any one of the following (b) to (d): (b) a 5′UTR and / or a 3′UTR; (c) poly-A tail; (d) 5′Cap, Preferably, the poly-A tail is selected from A120, A30L70, HGH polyA, SV40 polyA, BGH polyA, rbGlob polyA or SV40late polyA, more preferably, the poly-A tail is A120 or A30L70; Preferably, the 5'Cap is ARCA, 3'-O-Me-m ７ G(5')ppp(5')G,m ７ G(5')ppp(5')(2'OMeA)pU,m ７ Gppp(A2'O-MOE)pG,m ７ G(5')ppp(5')(2'OMeA)pG,m ７ G(5')ppp(5')(2'OMeG)pG,m ７ (3'OMeG) (5') ppp (5') (2'OMeG) pG or m ７ (3'OMeG)(5')ppp(5')(2'OMeA)pG; Preferably, the polynucleotide comprises one or more modifications selected from a backbone modification, a sugar modification, a base modification and / or a lipid modification, among which the base modification is preferably a pseudouridine modification; Isolated polynucleotides.

17. A nucleic acid construct comprising: (a) an open reading frame (ORF); (b) a UTR derived from CTSB, FAM166A, NDUFB9, ACTG1, CHCHD10, NDUFA11, ATP6V0B, ATP6V0E1, CFL1, COX4I1, CTSB, SLC38A2, NDUFV3, PRDX5, GUK1, IAH1, ABHD16A, SLC25A39, ATPIF1, ANAPC11, CCDC12, MRPL14, or APOA1BP, which is a 3' untranslated region element (3'UTR) or a 5' untranslated region element (5'UTR); Preferably, the UTR comprises a sequence set forth in any one of SEQ ID NOs: 1 to 47 or having at least 90% identity thereto. Nucleic acid constructs.

18. 1. An RNA molecule comprising: (a) an open reading frame (ORF); (b) a UTR derived from CTSB, FAM166A, NDUFB9, ACTG1, CHCHD10, NDUFA11, ATP6V0B, ATP6V0E1, CFL1, COX4I1, CTSB, SLC38A2, NDUFV3, PRDX5, GUK1, IAH1, ABHD16A, SLC25A39, ATPIF1, ANAPC11, CCDC12, MRPL14, or APOA1BP, which is a 3' untranslated region element (3'UTR) or a 5' untranslated region element (5'UTR); Preferably, the UTR comprises a sequence set forth in any one of SEQ ID NOs: 58 to 104 or having at least 90% identity thereto. RNA molecule.

19. A nucleic acid construct according to any one of claims 1 to 7 and 17, an RNA molecule according to any one of claims 8 to 15 and 18, or a polynucleotide according to claim 16. vector.

20. 20. The vector of claim 19, host cell.

21. A method for preparing the nucleic acid construct according to any one of claims 1 to 7 and 17, comprising culturing a host cell according to claim 20 and recovering the produced nucleic acid construct from the culture. Preparation method.

22. A method for preparing an RNA molecule according to any one of claims 8 to 15 and 18, comprising reverse transcribing a nucleic acid construct according to any one of claims 1 to 7 and 17, or a vector according to claim 19, to obtain an RNA molecule, preferably further comprising adding a 5' Cap to the 5' end of the RNA molecule. Preparation method.

23. 20. A method for the preparation of a nucleic acid construct comprising the steps of: a) administering to said subject a nucleic acid construct according to any one of claims 1 to 7 and 17; b) administering to said subject an RNA molecule according to any one of claims 8 to 15 and 18; c) administering to said subject a polynucleotide according to claim 16; or d) administering to said subject a vector according to claim 19, said vector being preferably a cationic lipid delivery particle or a nanolipid particle. Delivery vehicle.

24. A pharma- ceutically acceptable carrier, diluent or excipient; A nucleic acid construct according to any one of claims 1 to 7 and 17, an RNA molecule according to any one of claims 8 to 15 and 18, a polynucleotide according to claim 16, and a vector according to claim 19 or a delivery vehicle according to claim 23, or any combination thereof. Pharmaceutical compositions.

25. The present invention relates to a method for the preparation of a pharmaceutical composition comprising the steps of: a nucleic acid construct according to any one of claims 1 to 7 and 17; an RNA molecule according to any one of claims 8 to 15 and 18; a polynucleotide according to claim 19; a delivery vehicle according to claim 23; and a pharmaceutical composition according to claim 24, or any combination thereof. Products or reagent kits.

26. of a nucleic acid construct according to any one of claims 1 to 7 and 17 or an RNA molecule according to any one of claims 8 to 15 and 18 in the preparation of a product for improving the expression level of a protein of an ORF or for improving the expression level of a protein of an ORF, use.

27. A method for treating and / or preventing a disease, comprising administering to a subject in need thereof a therapeutically effective amount of a nucleic acid construct according to any one of claims 1 to 7 and 17, an RNA molecule according to any one of claims 8 to 15 and 18, a polynucleotide according to claim 16, a delivery vehicle according to claim 23 and / or a pharmaceutical composition according to claim 24, wherein said disease is selected from ischemic diseases, metabolic syndrome, diabetes and its complications, restenosis and nerve damage, Preferably, the ischemic disease is selected from coronary artery disease (CAD), peripheral arterial disease (PAD), myocardial infarction, limb ischemia, thromboangiitis obliterans (TAO), and diabetic arteriosclerosis obliterans (DAO); more preferably, the limb ischemia is lower limb ischemia; and most preferably, the limb ischemia is critical limb ischemia (CLI); Preferably, the diabetes and its complications are selected from diabetic peripheral neuropathy, diabetic foot (DFU), and diabetic arteriosclerosis obliterans (DAO); Preferably, the restenosis is selected from post-operative restenosis, post-perfusion restenosis; Preferably, the nerve damage is selected from a neurodegenerative disease, a traumatic nerve injury, and a peripheral neuropathy, more preferably, the neurodegenerative disease is selected from amyotrophic lateral sclerosis (ALS), Parkinson's disease, and dementia, and the peripheral neuropathy is diabetic peripheral neuropathy; method.

28. A method for promoting endothelial cell growth and / or migration, comprising administering to a subject in need thereof an effective amount of a nucleic acid construct according to any one of claims 1 to 7 and 17, an RNA molecule according to any one of claims 8 to 15 and 18, a polynucleotide according to claim 16, a delivery vehicle according to claim 23 and / or a pharmaceutical composition according to claim 24. method.

29. A method for promoting angiogenesis, comprising administering to a subject in need thereof an effective amount of a nucleic acid construct according to any one of claims 1 to 7 and 17, an RNA molecule according to any one of claims 8 to 15 and 18, a polynucleotide according to claim 16, a delivery vehicle according to claim 23 and / or a pharmaceutical composition according to claim 24. method.

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