WO2023217267A1 - 包含utr的核酸构建体及其应用 - Google Patents

包含utr的核酸构建体及其应用 Download PDF

Info

Publication number
WO2023217267A1
WO2023217267A1 PCT/CN2023/093851 CN2023093851W WO2023217267A1 WO 2023217267 A1 WO2023217267 A1 WO 2023217267A1 CN 2023093851 W CN2023093851 W CN 2023093851W WO 2023217267 A1 WO2023217267 A1 WO 2023217267A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
identity
sequence
sequence shown
utr
Prior art date
Application number
PCT/CN2023/093851
Other languages
English (en)
French (fr)
Inventor
姜军
杨林凤
陈钦俊
宋晓玉
宁威
廖成
Original Assignee
上海瑞宏迪医药有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 上海瑞宏迪医药有限公司 filed Critical 上海瑞宏迪医药有限公司
Publication of WO2023217267A1 publication Critical patent/WO2023217267A1/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus

Definitions

  • the present disclosure belongs to the field of nucleic acids and relates to nucleic acid constructs comprising UTRs and their use for preventing or treating diseases.
  • the nucleic acid construct may include a nucleic acid sequence expressing human hepatocyte growth factor (hHGF) and is used as a gene therapy drug for severe lower limb ischemia, diabetic foot and other diseases.
  • hHGF human hepatocyte growth factor
  • Gene therapy and gene vaccination can provide highly specific and personalized treatment and prevention solutions for a variety of diseases, including genetic diseases, autoimmune diseases, cancer or tumor-related diseases, and inflammatory diseases.
  • RNA can be used for gene therapy or gene vaccination.
  • DNA is stable and easy to operate, but there is a risk that DNA fragments will be inserted into the patient's genome, causing mutation events (such as loss of damaged gene function).
  • RNA can avoid unwanted genomic integration, but is susceptible to degradation due to ubiquitous RNases. Therefore, it is necessary to improve the stability of RNA so that the protein products it encodes can accumulate in the body to achieve treatment or prevention of diseases, and to maintain the integrity of RNA structure and function during storage and administration.
  • Naturally occurring eukaryotic mRNA molecules have been found to contain stabilizing elements, such as their 5' and 3' untranslated regions (UTRs), as well as other structural features such as a 5' cap structure or a 3' poly(A) tail. .
  • 5'UTR and 3'UTR are premature mRNA elements.
  • mature mRNA-specific structural features such as 5' cap and 3' poly(A) tail
  • 3'UTR of ⁇ -globin mRNA is an important factor in the stability of ⁇ -globin mRNA (Nancy D Rodgers et al, RNA. 2002Dec; 8(12) :1526-37; Z Wang et al, Mol Cell Biol. 1999Jul; 19(7):4552-60.).
  • Peripheral artery disease refers to a non-coronary system syndrome caused by a series of structural and functional abnormalities in the arteries supplying the limbs, internal organs and brain. It is characterized by stenosis and stenosis of non-coronary blood circulation. Occlusive and neoplastic lesions involving the aorta and branch arteries.
  • ischemic diseases of the lower limbs are the most common clinically, and the main causes include atherosclerosis (ASO), diabetic artery obliterans (DAO), and thromboangiitis obliterans (DAO). Thrombosis angiitis obliterans, TAO).
  • CLI Critical lower limb ischemia
  • DFU diabetic foot ulcer
  • Both are peripheral vascular disease diseases, and their onset characteristics are lower limb blood vessels. Lower limb pain, ulcers and necrosis caused by stenosis or occlusion and insufficient distal blood perfusion.
  • the current effective treatment for CLI and DFU is revascularization through surgery or endovascular intervention. However, more than 40% of patients suffer from age, medical conditions, and other factors. And complications such as those that do not meet the requirements for revascularization can only be treated conservatively with drugs. Medication can only slow down the progression of the disease but cannot cure it.
  • Hepatocyte growth factor is a multifunctional mesenchymal-derived growth factor. When it binds to the cell membrane surface receptor c-met, it causes phosphorylation of intracellular tyrosine residues and recruits adapter proteins. , promote kinase activity, thereby activating downstream signaling pathways.
  • HGF is an important regulator of processes such as embryonic development, tissue and organ regeneration, wound healing, and angiogenesis. It can promote the proliferation and migration of endothelial cells and smooth muscle cells, promote the reconstruction of microvascular networks in ischemic areas, and inhibit cell apoptosis.
  • the present disclosure provides such mRNA, which contains a new structure of 5'UTR and 3'UTR, which reduces the early degradation of mRNA or stabilizes the degradation of mRNA without losing or enhancing protein translation efficiency.
  • the mRNA has higher stability and can be used in gene therapy and gene vaccination.
  • the present disclosure provides mRNA capable of expressing human hepatocyte growth factor (Human Hepatocyte growth factor, hHGF) and its lipid nanoparticles (Lipid nanoparticles, LNP) delivery system, which can realize efficient and rapid conversion of exogenous hHGF protein in the body. , has the advantages of no integration risk and easy industrial-scale amplification. It is a more ideal treatment solution than naked plasmid and can be used as a gene therapy drug for various diseases such as CLI and DFU.
  • the present disclosure provides a nucleic acid construct, which contains at least one nucleic acid element that can regulate the expression of a gene of interest, and the nucleic acid element is a UTR. And, the nucleic acid construct may contain one or more genes of interest, such as HGF.
  • nucleic acid constructs comprising:
  • the nucleic acid construct is a DNA molecule; in some embodiments, the nucleic acid construct is an RNA molecule (eg, mRNA).
  • the ORF is a polynucleotide sequence encoding a gene of interest.
  • the gene of interest is heterologous. In other embodiments, the gene of interest is endogenous. In some embodiments, the target genes are one or more (eg, 2, 3, 4).
  • the UTR is derived from genes ACTG1, ATP6V0B, ATP6VOE1, CFL1, COX4I1, CTSB, FAM166A, NDUFB9, CHCHD10, SLC38A2, NDUFA11, NDUFV3, PRDX5, GUK1, IAH1, ABHD16A, SLC25A39, ATPIF1, ANAPC11, CCDC12 , MRPL14 or APOA1BP UTR.
  • the above-mentioned genes are human genes.
  • the UTR is a 3' untranslated region element (3' UTR) or a 5' untranslated region element (5' UTR).
  • the 3'UTR and 5'UTR are from the same or different sources, such as from the same or different genes.
  • the 3'UTR is derived from the 3'UTR of the gene ACTG1
  • the 5'UTR is derived from the 5'UTR of the gene ACTG1.
  • the 3'UTR is derived from the 3'UTR of the gene CTSB
  • the 5'UTR is derived from the 5'UTR of the gene CHCHD10.
  • the 5' UTR and 3' UTR are derived from the same species or different species.
  • 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.
  • the aforementioned nucleic acid construct includes:
  • the aforementioned nucleic acid construct includes:
  • the 3'UTR is derived from the 3'UTR of the gene ACTG1, which contains SEQ ID NO: 1 or SEQ ID NO: 58 or is consistent with Any sequence with identity;
  • the 3'UTR is derived from the 3'UTR of the gene ATP6V0B, which contains the sequence shown in SEQ ID NO:2, 3 or SEQ ID NO:59, 60 or has any identity with it;
  • the 3'UTR is derived from the 3'UTR of the gene ATP6V0E1, which contains the sequence shown in SEQ ID NO:4, 5 or SEQ ID NO:61, 62 or has any identity with it;
  • the 3'UTR is derived from the 3'UTR of the gene CFL1, which contains SEQ ID NO: 6, 7, 8 or SEQ ID Sequences shown in NO: 63, 64, 65 or identical to any of them;
  • the 3'UTR is derived from the 3'UTR of the gene COX4I1, which contains a sequence shown in SEQ ID NO: 9, 10, 11 or SEQ ID NO: 66, 67, 68 or any one thereof;
  • the 3'UTR is derived from the 3'UTR of the gene CTSB, which contains a sequence shown in SEQ ID NO: 12 or SEQ ID NO: 69 or has identity with either of them;
  • the 3'UTR is derived from the 3'UTR of the gene FAM166A, which contains a sequence shown in SEQ ID NO: 13 or SEQ ID NO: 70 or has identity with either; or
  • the 3'UTR is derived from the 3'UTR of the gene NDUFB9, which contains a sequence shown in SEQ ID NO: 14 or SEQ ID NO: 71 or has identity with either of them.
  • the 5'UTR is derived from the 5'UTR of the gene ACTG1, which contains SEQ ID NO:15 or SEQ ID NO:72 or is consistent with Any sequence with identity;
  • the 5'UTR is derived from the 5'UTR of the gene ATP6V0B, which contains a sequence shown in SEQ ID NO: 16, 17 or SEQ ID NO: 73, 74 or any one thereof;
  • the 5'UTR is derived from the 5'UTR of the gene ATP6V0E1, which contains the sequence shown in SEQ ID NO: 18, 19 or SEQ ID NO: 75, 76 or has any identity with it;
  • the 5'UTR is derived from the 5'UTR of the gene CFL1, which contains the sequence shown in SEQ ID NO: 20, 21, 22 or SEQ ID NO: 77, 78, 79 or any one thereof;
  • the 5'UTR is derived from the 5'UTR of the gene COX4I1, which contains a sequence shown in SEQ ID NO: 23, 24, 25 or SEQ ID NO: 80, 81, 82 or any one thereof;
  • the 5'UTR is derived from the 5'UTR of the gene CTSB, which contains a sequence shown in SEQ ID NO: 26 or SEQ ID NO: 83 or has identity with either of them;
  • the 5'UTR is derived from the 5'UTR of the gene FAM166A, which contains the sequence shown in SEQ ID NO:27 or SEQ ID NO:84 or has identity with either;
  • the 5'UTR is derived from the 5'UTR of the gene NDUFB9, which contains the sequence shown in SEQ ID NO:28 or SEQ ID NO:85 or has identity with either;
  • the 5'UTR is derived from the 5'UTR of the gene CHCHD10, which contains the sequence shown in SEQ ID NO:29, 30 or SEQ ID NO:86, 87 or has any identity with it;
  • the 5'UTR is derived from the 5'UTR of the gene SLC38A2, which contains a sequence shown in SEQ ID NO:31 or SEQ ID NO:88 or has identity with either;
  • the 5'UTR is derived from the 5'UTR of the gene NDUFA11, which contains the sequence shown in SEQ ID NO:32 or SEQ ID NO:89 or has identity with either of them;
  • the 5'UTR is derived from the 5'UTR of the gene NDUFV3, which contains the sequence shown in SEQ ID NO:33 or SEQ ID NO:90 or has identity with either;
  • the 5'UTR is derived from the 5'UTR of the gene PRDX5, which contains the sequence shown in SEQ ID NO:34 or SEQ ID NO:91;
  • the 5'UTR is derived from the 5'UTR of the gene GUK1, which contains the sequence shown in SEQ ID NO:35, 36, 37 or SEQ ID NO:92, 93, 94 or any one thereof;
  • the 5'UTR is derived from the 5'UTR of the gene IAH1, which contains a sequence shown in SEQ ID NO:38 or SEQ ID NO:95 or has identity with either;
  • the 5'UTR is derived from the 5'UTR of the gene ABHD16A, which contains the sequence shown in SEQ ID NO:39 or SEQ ID NO:96 or has identity with either;
  • the 5'UTR is derived from the 5'UTR of the gene SLC25A39, which contains a sequence shown in SEQ ID NO:40 or SEQ ID NO:97 or has identity with either of them;
  • the 5'UTR is derived from the 5'UTR of the gene ATPIF1, which contains a sequence shown in SEQ ID NO:41 or SEQ ID NO:98 or has identity with either;
  • the 5'UTR is derived from the 5'UTR of the gene ANAPC11, which contains the sequence shown in SEQ ID NO:42, 43 or SEQ ID NO:99, 100 or has any identity with it;
  • the 5'UTR is derived from the 5'UTR of the gene CCDC12, which contains a sequence shown in SEQ ID NO: 44 or SEQ ID NO: 101 or has identity with either of them;
  • the 5'UTR is derived from the 5'UTR of the gene MRPL14, which contains a sequence shown in SEQ ID NO:45 or SEQ ID NO:102 or has identity with either; or
  • the 5'UTR is derived from the 5'UTR of the gene APOA1BP, which contains the sequence shown in SEQ ID NO:46, 47 or SEQ ID NO:103, 104 or has any identity with it.
  • nucleic acid constructs e.g., DNA or RNA molecules
  • nucleic acid constructs comprising:
  • the 3’UTR and 5’UTR are selected from any combination of the following:
  • the 3'UTR contains SEQ ID NO:1 or SEQ ID NO:58 or a sequence identical to either of them, and the 5'UTR contains any of SEQ ID NO:15-47 or SEQ ID Sequences shown in any of NO:72-104 or identical to any of them;
  • the 3'UTR contains SEQ ID NO:2 or SEQ ID NO:59 or a sequence identical to either of them, and the 5'UTR contains any of SEQ ID NO:15-47 or SEQ ID Sequences shown in any of NO:72-104 or identical to any of them;
  • the 3'UTR contains SEQ ID NO:3 or SEQ ID NO:60 or a sequence identical to either of them, and the 5'UTR contains any of SEQ ID NO:15-47 or SEQ ID Sequences shown in any of NO:72-104 or identical to any of them;
  • the 3'UTR contains SEQ ID NO:4 or SEQ ID NO:61 or a sequence identical to either of them, and the 5'UTR contains any of SEQ ID NO:15-47 or SEQ ID Sequences shown in any of NO:72-104 or identical to any of them;
  • the 3'UTR contains SEQ ID NO:5 or SEQ ID NO:62 or is identical to either of them.
  • a specific sequence, the 5'UTR contains a sequence shown in any one of SEQ ID NO: 15-47 or any one of SEQ ID NO: 72-104 or has identity with any one of it;
  • the 3'UTR contains a sequence shown in SEQ ID NO:6 or SEQ ID NO:63 or has identity with either of them, and the 5'UTR contains any of SEQ ID NO:15-47 or SEQ ID Sequences shown in any of NO:72-104 or identical to any of them;
  • the 3'UTR contains SEQ ID NO:7 or SEQ ID NO:64 or a sequence identical to either of them, and the 5'UTR contains any of SEQ ID NO:15-47 or SEQ ID Sequences shown in any of NO:72-104 or identical to any of them;
  • the 3'UTR contains a sequence shown in SEQ ID NO:8 or SEQ ID NO:65 or has identity with either of them, and the 5'UTR contains any of SEQ ID NO:15-47 or SEQ ID Sequences shown in any of NO:72-104 or identical to any of them;
  • the 3'UTR contains SEQ ID NO:9 or SEQ ID NO:66 or a sequence identical to either of them, and the 5'UTR contains any of SEQ ID NO:15-47 or SEQ ID Sequences shown in any of NO:72-104 or identical to any of them;
  • the 3'UTR contains SEQ ID NO:10 or SEQ ID NO:67 or a sequence identical to either of them, and the 5'UTR contains any of SEQ ID NO:15-47 or SEQ ID Sequences shown in any of NO:72-104 or identical to any of them;
  • the 3'UTR contains SEQ ID NO:11 or SEQ ID NO:68 or a sequence identical to either of them, and the 5'UTR contains any of SEQ ID NO:15-47 or SEQ ID Sequences shown in any of NO:72-104 or identical to any of them;
  • the 3'UTR contains SEQ ID NO:12 or SEQ ID NO:69 or a sequence identical to any of them, and the 5'UTR contains any of SEQ ID NO:15-47 or SEQ ID Sequences shown in any of NO:72-104 or identical to any of them;
  • the 3'UTR contains SEQ ID NO:13 or SEQ ID NO:70 or a sequence identical to either of them, and the 5'UTR contains any of SEQ ID NO:15-47 or SEQ ID Sequences shown in any of NO:72-104 or identical to any of them;
  • the 3'UTR contains SEQ ID NO:14 or SEQ ID NO:71 or a sequence identical to either of them, and the 5'UTR contains any of SEQ ID NO:15-47 or SEQ ID Sequences shown in any of NO:72-104 or identical to any of them;
  • the 3'UTR contains any one of SEQ ID NO:1-14 or any one of SEQ ID NO:58-71 or a sequence having identity with any one of them, and the 5'UTR contains SEQ ID NO: 15 or the sequence shown in SEQ ID NO:72 or any sequence identical to it;
  • the 3'UTR contains any one of SEQ ID NO:1-14 or any one of SEQ ID NO:58-71 or a sequence identical to any one of SEQ ID NO:58-71, and the 5'UTR contains SEQ ID NO: 16 or the sequence shown in SEQ ID NO:73 or any sequence identical to it;
  • the 3'UTR contains any one of SEQ ID NO:1-14 or any one of SEQ ID NO:58-71 or with Any sequence with identity, the 5'UTR contains a sequence shown in SEQ ID NO: 17 or SEQ ID NO: 74 or any sequence with identity;
  • the 3'UTR contains any one of SEQ ID NO:1-14 or any one of SEQ ID NO:58-71 or a sequence having identity with any one of SEQ ID NO:58-71, and the 5'UTR contains SEQ ID NO: 18 or SEQ ID NO:75 or any sequence identical to it;
  • the 3'UTR contains any one of SEQ ID NO:1-14 or any one of SEQ ID NO:58-71 or a sequence that is identical to any one of SEQ ID NO:58-71, and the 5'UTR contains SEQ ID NO: 19 or SEQ ID NO:76 or any sequence identical to it;
  • the 3'UTR contains any one of SEQ ID NO:1-14 or any one of SEQ ID NO:58-71 or a sequence that is identical to any one of SEQ ID NO:58-71, and the 5'UTR contains SEQ ID NO: 20 or the sequence shown in SEQ ID NO:77 or any sequence identical to it;
  • the 3'UTR contains any one of SEQ ID NO:1-14 or any one of SEQ ID NO:58-71 or a sequence identical to any one of SEQ ID NO:58-71, and the 5'UTR contains SEQ ID NO: 21 or SEQ ID NO:78 or any sequence identical to it;
  • the 3'UTR contains any one of SEQ ID NO:1-14 or any one of SEQ ID NO:58-71 or a sequence that is identical to any one of SEQ ID NO:58-71, and the 5'UTR contains SEQ ID NO: 22 or SEQ ID NO:79 or any sequence identical to it;
  • the 3'UTR contains any one of SEQ ID NO:1-14 or any one of SEQ ID NO:58-71 or a sequence identical to any one of SEQ ID NO:58-71, and the 5'UTR contains SEQ ID NO: 23 or SEQ ID NO:80 or any sequence identical to it;
  • the 3'UTR contains any one of SEQ ID NO:1-14 or any one of SEQ ID NO:58-71 or a sequence identical to any one of SEQ ID NO:58-71, and the 5'UTR contains SEQ ID NO: 24 or SEQ ID NO:81 or any sequence identical to it;
  • the 3'UTR contains any one of SEQ ID NO:1-14 or any one of SEQ ID NO:58-71 or a sequence that is identical to any one of SEQ ID NO:58-71, and the 5'UTR contains SEQ ID NO: 25 or SEQ ID NO:82 or any sequence identical to it;
  • the 3'UTR contains any one of SEQ ID NO:1-14 or any one of SEQ ID NO:58-71 or a sequence having identity with any one of SEQ ID NO:58-71, and the 5'UTR contains SEQ ID NO: 26 or SEQ ID NO:83 or any sequence identical to it;
  • the 3'UTR contains any one of SEQ ID NO:1-14 or any one of SEQ ID NO:58-71 or a sequence identical to any one of SEQ ID NO:58-71, and the 5'UTR contains SEQ ID NO: 27 or SEQ ID NO:84 or any sequence identical to it;
  • the 3'UTR contains any one of SEQ ID NO:1-14 or any one of SEQ ID NO:58-71 or a sequence identical to any one of SEQ ID NO:58-71, and the 5'UTR contains SEQ ID NO: 28 or SEQ ID NO:85 or any sequence identical to it;
  • the 3'UTR contains any one of SEQ ID NO:1-14 or any one of SEQ ID NO:58-71 or is consistent with Any sequence with identity, the 5'UTR contains a sequence shown in SEQ ID NO:29 or SEQ ID NO:86 or any sequence with identity;
  • the 3'UTR contains any one of SEQ ID NO:1-14 or any one of SEQ ID NO:58-71 or a sequence that is identical to any one of SEQ ID NO:58-71, and the 5'UTR contains SEQ ID NO: 30 or the sequence shown in SEQ ID NO:87 or any sequence identical to it;
  • the 3'UTR contains any one of SEQ ID NO:1-14 or any one of SEQ ID NO:58-71 or a sequence identical to any one of SEQ ID NO:58-71, and the 5'UTR contains SEQ ID NO: 31 or SEQ ID NO:88 or any sequence identical to it;
  • the 3'UTR contains any one of SEQ ID NO:1-14 or any one of SEQ ID NO:58-71 or a sequence that is identical to any one of SEQ ID NO:58-71, and the 5'UTR contains SEQ ID NO: 32 or SEQ ID NO:89 or any sequence identical to it;
  • the 3'UTR contains any one of SEQ ID NO: 1-14 or any one of SEQ ID NO: 58-71 or a sequence identical to any one of SEQ ID NO: 58-71, and the 5'UTR contains SEQ ID NO: 33 or SEQ ID NO:90 or any sequence identical to it;
  • the 3'UTR contains any one of SEQ ID NO:1-14 or any one of SEQ ID NO:58-71 or a sequence that is identical to any one of SEQ ID NO:58-71, and the 5'UTR contains SEQ ID NO: 34 or the sequence shown in SEQ ID NO:91 or any sequence identical to it;
  • the 3'UTR contains any one of SEQ ID NO:1-14 or any one of SEQ ID NO:58-71 or a sequence that is identical to any one of SEQ ID NO:58-71, and the 5'UTR contains SEQ ID NO: 35 or SEQ ID NO:92 or any sequence identical to it;
  • the 3'UTR contains any one of SEQ ID NO:1-14 or any one of SEQ ID NO:58-71 or a sequence identical to any one of SEQ ID NO:58-71, and the 5'UTR contains SEQ ID NO: 36 or the sequence shown in SEQ ID NO:93 or any sequence identical to it;
  • the 3'UTR contains any one of SEQ ID NO:1-14 or any one of SEQ ID NO:58-71 or a sequence that is identical to any one of SEQ ID NO:58-71, and the 5'UTR contains SEQ ID NO: 37 or SEQ ID NO:94 or any sequence identical to it;
  • the 3'UTR contains any one of SEQ ID NO:1-14 or any one of SEQ ID NO:58-71 or a sequence identical to any one of SEQ ID NO:58-71, and the 5'UTR contains SEQ ID NO: 38 or SEQ ID NO:95 or any sequence identical to it;
  • the 3'UTR contains any one of SEQ ID NO:1-14 or any one of SEQ ID NO:58-71 or a sequence identical to any one of SEQ ID NO:58-71, and the 5'UTR contains SEQ ID NO: 39 or SEQ ID NO:96 or any sequence identical to it;
  • the 3'UTR contains any one of SEQ ID NO:1-14 or any one of SEQ ID NO:58-71 or a sequence identical to any one of SEQ ID NO:58-71, and the 5'UTR contains SEQ ID NO: 40 or the sequence shown in SEQ ID NO:97 or any sequence identical to it;
  • the 3'UTR contains any one of SEQ ID NO:1-14 or any one of SEQ ID NO:58-71 or with Any sequence with identity, the 5'UTR contains a sequence shown in SEQ ID NO:41 or SEQ ID NO:98 or any sequence with identity;
  • the 3'UTR contains any one of SEQ ID NO:1-14 or any one of SEQ ID NO:58-71 or a sequence identical to any one of SEQ ID NO:58-71, and the 5'UTR contains SEQ ID NO: 42 or SEQ ID NO:99 or any sequence identical to it;
  • the 3'UTR contains any one of SEQ ID NO:1-14 or any one of SEQ ID NO:58-71 or a sequence identical to any one of SEQ ID NO:58-71, and the 5'UTR contains SEQ ID NO: 43 or SEQ ID NO:100 or any sequence identical to it;
  • the 3'UTR contains any one of SEQ ID NO:1-14 or any one of SEQ ID NO:58-71 or a sequence identical to any one of SEQ ID NO:58-71, and the 5'UTR contains SEQ ID NO: 44 or SEQ ID NO:101 or any sequence identical to it;
  • the 3'UTR contains any one of SEQ ID NO:1-14 or any one of SEQ ID NO:58-71 or a sequence that is identical to any one of SEQ ID NO:58-71, and the 5'UTR contains SEQ ID NO: 45 or SEQ ID NO:102 or any sequence identical to it;
  • the 3'UTR contains any one of SEQ ID NO:1-14 or any one of SEQ ID NO:58-71 or a sequence identical to any one of SEQ ID NO:58-71, and the 5'UTR contains SEQ ID NO: 46 or a sequence identical to or shown in SEQ ID NO:103; or
  • the 3'UTR contains any one of SEQ ID NO:1-14 or any one of SEQ ID NO:58-71 or a sequence identical to any one of SEQ ID NO:58-71, and the 5'UTR contains SEQ ID NO: 47 or SEQ ID NO:104 or any sequence identical to it.
  • nucleic acid constructs e.g., DNA or RNA molecules
  • nucleic acid constructs comprising:
  • the 3'UTR is derived from the 3'UTR of the gene CTSB, FAM166A or NDUFB9, and the 5'UTR is derived from the 5'UTR of the gene ACTG1, CHCHD10 or NDUFA11;
  • the 3'UTR includes any of SEQ ID NO:12, 13, 14 or any of SEQ ID NO:69, 70, 71 or a sequence having identity with any of them
  • the 5'UTR includes SEQ ID Any one of NO: 15, 29, 30, 32 or any one of SEQ ID NO: 72, 86, 87, 89 or a sequence identical to any one of them;
  • the 3'UTR includes SEQ ID NO:12 or SEQ ID NO:69 or a sequence identical to either of them, and the 5'UTR includes SEQ ID NO:15 or SEQ ID NO:72 or a sequence identical to any of the sequences shown,
  • the 3'UTR includes a sequence shown in SEQ ID NO:13 or SEQ ID NO:70 or is identical to either sequence, and the 5'UTR includes a sequence shown in SEQ ID NO:15 or SEQ ID NO:72 or any sequence identical to it,
  • the 3'UTR includes or is identical to either of SEQ ID NO:14 or SEQ ID NO:71
  • the sequence, the 5'UTR comprises the sequence shown in SEQ ID NO:15 or SEQ ID NO:72 or has identity with either of them,
  • the 3'UTR includes a sequence shown in SEQ ID NO:12 or SEQ ID NO:69 or is identical to either sequence, and the 5'UTR includes a sequence shown in SEQ ID NO:29 or SEQ ID NO:86 or any sequence identical to it,
  • the 3'UTR includes the sequence shown in SEQ ID NO:13 or SEQ ID NO:70 or has identity with either of them, and the 5'UTR includes the sequence shown in SEQ ID NO:29 or SEQ ID NO:86 or any sequence identical to it,
  • the 3'UTR includes the sequence shown in SEQ ID NO:14 or SEQ ID NO:71 or has identity with either of them, and the 5'UTR includes the sequence shown in SEQ ID NO:29 or SEQ ID NO:86 or any sequence identical to it,
  • the 3'UTR includes a sequence shown in SEQ ID NO:12 or SEQ ID NO:69 or has identity with either of them, and the 5'UTR includes a sequence shown in SEQ ID NO:30 or SEQ ID NO:87 or any sequence identical to it,
  • the 3'UTR includes a sequence shown in SEQ ID NO:13 or SEQ ID NO:70 or is identical to either sequence, and the 5'UTR includes a sequence shown in SEQ ID NO:30 or SEQ ID NO:87 or any sequence identical to it,
  • the 3'UTR includes a sequence shown in SEQ ID NO:14 or SEQ ID NO:71 or is identical to either sequence, and the 5'UTR includes a sequence shown in SEQ ID NO:30 or SEQ ID NO:87 or any sequence identical to it,
  • the 3'UTR includes a sequence shown in SEQ ID NO:12 or SEQ ID NO:69 or is identical to either sequence, and the 5'UTR includes a sequence shown in SEQ ID NO:32 or SEQ ID NO:89 or any sequence identical to it,
  • the 3'UTR includes the sequence shown in SEQ ID NO:13 or SEQ ID NO:70 or has identity with either of them, and the 5'UTR includes the sequence shown in SEQ ID NO:32 or SEQ ID NO:89 or any sequence identical to it, or
  • the 3'UTR includes the sequence shown in SEQ ID NO:14 or SEQ ID NO:71 or has identity with either of them, and the 5'UTR includes the sequence shown in SEQ ID NO:32 or SEQ ID NO:89 or any sequence identical to it.
  • having identity encompasses having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity, and between any two of the foregoing values. range, including integers and decimals. For example “having at least 90% identity” or “having at least 95% identity”.
  • the 3'UTR and/or 5'UTR are variants of the above-mentioned 3'UTR and/or 5'UTR, and the variants are, for example, truncations and nucleotide mutants.
  • the body still maintains similar activities or functions to the aforementioned 3'UTR and/or 5'UTR of the present disclosure, for example, still maintains the function of regulating the expression protein of the target gene encoded by the ORF.
  • the aforementioned nucleic acid construct provided by the present disclosure also includes:
  • the poly-A tail in the nucleic acid construct is located downstream of the 3' UTR. In some embodiments, the poly-A tail in the nucleic acid construct is located at the 3' end of the 3' UTR. In some embodiments, the poly-A tail is at the 3' end of the nucleic acid construct. In some embodiments, the poly-A tail is at least about 50, 100, 150, 200, 300, 400, 500 nucleotides in length.
  • the poly-A tail is selected from A120, A30L70, HGH polyA, SV40 polyA, BGH polyA, rbGlob polyA or SV40late polyA.
  • A30L70 is the sequence shown in SEQ ID NO:52.
  • the expressed gene of interest ie, open reading frame ORF
  • HGF hepatocyte growth factor
  • an antibody or an antigen-binding fragment thereof are hepatocyte growth factor (HGF), an antibody or an antigen-binding fragment thereof.
  • HGF hepatocyte growth factor
  • the polypeptide or protein encoded by the ORF is a fluorescent protein or luciferase, such as the sequence shown in SEQ ID NO: 126.
  • the HGF is human hepatocyte growth factor (hHGF).
  • the coding sequence of hHGF includes any one selected from the following 1)-3):
  • nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 109 or a codon-optimized nucleic acid sequence
  • the nucleic acid construct expressing HGF as the target gene contains any one of SEQ ID NO: 115, 116, 127 or has at least 80%, 85%, 90%, 95%, 96 %, 97%, 98%, 99%, 100% identical sequences.
  • the antibody or antigen-binding fragment thereof is an anti-PD-1 antibody or antigen-binding fragment thereof.
  • the coding sequence of the anti-PD-1 antibody or antigen-binding fragment thereof includes any one selected from the following 1)-4):
  • the nucleic acid construct expressing an anti-PD-1 antibody or an antigen-binding fragment thereof includes the sequences shown in SEQ ID NO: 124 and 125, or has at least 80% similarity with SEQ ID NO: 124 and 125. , 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identical sequences.
  • the aforementioned nucleic acid construct provided by the present disclosure is DNA or RNA, for example, is mRNA.
  • nucleic acid construct (DNA or RNA) provided by the present disclosure contains any one of the following 1)-6):
  • the 5'UTR and 3'UTR can be derived from the same or different genes.
  • nucleic acid construct (DNA) provided by the present disclosure contains any one of the following 1)-4):
  • the 5'UTR and 3'UTR can be derived from the same or different genes.
  • the 5'UTR in 1), 3), and 4) includes or is a nucleotide sequence as shown in any one of SEQ ID NO: 15-47.
  • the ORF in 1)-4) includes or is a nucleotide sequence as shown in SEQ ID NO: 110 or its codon-optimized nucleotide sequence (eg, SEQ ID NO: 111-113).
  • the ORF in 1)-4) includes or is the nucleotide sequence shown in SEQ ID NO: 119 and 120.
  • the 3'UTR in 2)-4) includes or is a nucleotide sequence as shown in any one of SEQ ID NO: 1-14.
  • the aforementioned nucleic acid construct (RNA or mRNA) provided by the present disclosure also includes:
  • 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 CapO (methylation of the first nucleobase, e.g., m 7 GpppN), Capl (adjacent nucleobase of m 7 GpppN) Additional methylation of the ribose of the nucleotide, such as m 7 G(5')ppp(5')(2'OMeA)pG), Cap2(m 7 Additional methylation of the ribose of the 3rd nucleotide downstream of GpppN ), Cap3 (additional methylation of ribose at the 3rd nucleotide downstream of m 7 GpppN), Cap4 (additional methylation of
  • RNA synthesis or in vitro RNA transcription is used to form a 5' Cap structure (e.g., Cap0 or Cap1).
  • a 5'-cap structure (eg, CapO or Cap1) is formed via enzymatic capping using a capping enzyme (eg, vaccinia virus capping enzyme and/or cap-dependent 2'-O methyltransferase) .
  • a capping enzyme eg, vaccinia virus capping enzyme and/or cap-dependent 2'-O methyltransferase
  • an immobilized capping enzyme is used to add a 5' cap structure (Cap0 or Cap1).
  • the capping method and means in WO2016/193226 are introduced in full here.
  • the 5'Cap is selected from 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 .
  • the 5'Cap is 3'-O-Me-m 7 G(5')ppp(5')G or m 7 G(5')ppp(5')(2'OMeA) pG.
  • nucleic acid construct (RNA or mRNA) provided by the present disclosure contains any of the following 1)-5):
  • the 5'UTR and 3'UTR can be derived from the same or different genes.
  • the 5'UTR in 1), 3), 4), and 5) includes or is a nucleotide sequence as shown in any one of SEQ ID NO: 72-104.
  • the ORF in 1)-5) includes or is a nucleotide sequence as shown in any one of SEQ ID NO: 128-131.
  • the 3'UTR in 2)-5) includes or is a nucleotide sequence as shown in any one of SEQ ID NO: 58-71.
  • structures include, but are not limited to, Cap0, Cap1 (eg, m7G (5')ppp(5')(2'OMeA)pG), Cap2, Cap3, Cap4, ARCA.
  • the present disclosure provides a nucleic acid construct (DNA), sequentially comprising a 5'UTR, ORF, and 3'UTR from the 5' to 3' direction, and optionally further comprising a poly- A tail.
  • the 5'UTR includes or is a nucleotide sequence as shown in any one of SEQ ID NO:15-47
  • the ORF includes or is as shown in SEQ ID NO:110 or its codon optimization.
  • the nucleotide sequence (such as SEQ ID NO:111-113), the 3'UTR includes or is the nucleotide sequence shown in any one of SEQ ID NO:1-14.
  • the 5'UTR comprises or is a nucleotide sequence as shown in any one of SEQ ID NOs: 15-47
  • the ORF comprises or is a nucleoside sequence as shown in SEQ ID NOs: 119 and 120 Acid sequence
  • the 3'UTR includes or is a nucleotide sequence as shown in any one of SEQ ID NO: 1-14.
  • the present disclosure provides a nucleic acid construct (RNA or mRNA), sequentially comprising a 5'UTR, ORF, and 3'UTR from the 5' to 3' direction, and optionally further comprising a 3' UTR in the 3' direction.
  • RNA or mRNA nucleic acid construct
  • the 5'UTR comprises or is a nucleotide sequence as shown in any one of SEQ ID NO:72-104
  • the ORF comprises or is a nucleotide sequence as shown in any one of SEQ ID NO:128-131
  • the 3'UTR comprises or is a nucleotide sequence as shown in any one of SEQ ID NOs: 58-71
  • the poly-A tail comprises or is 120 consecutive adenosines or as The nucleotide sequence shown in SEQ ID NO:52.
  • the nucleic acid construct comprises a nucleotide sequence as shown in SEQ ID NO: 115, 116 or 127.
  • the 5'UTR comprises or is a nucleotide sequence as shown in any one of SEQ ID NO:72-104
  • the ORF comprises or is a nucleoside sequence as shown in SEQ ID NO:121 and 122 acid sequence
  • the 3'UTR comprises or is a nucleotide sequence as shown in any one of SEQ ID NO:58-71
  • the poly-A tail comprises or is 120 consecutive adenosine nucleotides or as SEQ ID The nucleotide sequence shown in NO:52.
  • the nucleic acid construct comprises the nucleotide sequence shown in SEQ ID NO: 124 or 125.
  • any of the aforementioned nucleic acid constructs (DNA or RNA molecules), wherein the UTR is used to increase the expression level of the ORF-expressed protein.
  • the UTR is used to increase the expression level of the ORF-expressed protein.
  • the 5' UTR shown in SEQ ID NO: 48 or 50 compared with the 5' UTR shown in any sequence of SEQ ID NO: 15-47, 72-104 in the present disclosure UTR has the ability to increase the expression of target proteins that regulate ORF expression.
  • the 3'UTR shown in any sequence of SEQ ID NO:1-14, 58-71 in the present disclosure has an improved Regulate the expression level of ORF expression target protein.
  • the sequence shown in any one of SEQ ID NO:15-47 and 72-104 in the present disclosure is The combination of 5'UTR and the 3'UTR shown in any of SEQ ID NO: 1-14 and 58-71 has the ability to increase the expression of the target protein that regulates ORF expression.
  • the sequence shown in any one of SEQ ID NO:15-47 and 72-104 in the present disclosure is The combination of 5'UTR and the 3'UTR shown in any of SEQ ID NO: 1-14 and 58-71 has the ability to increase the expression of the target protein that regulates ORF expression.
  • the nucleic acid construct in the present disclosure expresses the target protein at an expression level that is about 1 to about 20 times that of the BioN vector, such as about 1 time, about 1.1 times, about 1.2 times, about 1.3 times, about 1.4 times. 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.
  • the nucleic acid construct in the present disclosure is an mRNA molecule that expresses the target protein, and the expression amount of the mRNA molecule expressing the target protein is about 1 to about 20 times that of the BioN vector.
  • the BioN vector includes the 5'UTR shown in SEQ ID NO:50 or 107; and/or, includes the 3'UTR shown in SEQ ID NO:51 or 108.
  • the nucleic acid construct in the present disclosure expresses the target protein at an expression level that is about 1 to about 20 times that of the Mod vector, such as about 1 time, about 1.1 times, about 1.2 times, about 1.3 times, about 1.4 times. 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.
  • the nucleic acid construct in the present disclosure is an mRNA molecule that expresses the target protein, and the expression amount of the mRNA molecule expressing the target protein is about 1 to about 20 times that of the BioN vector.
  • the Mod vector contains the 5'UTR shown in SEQ ID NO:48 or 105; and/or, contains the 3'UTR shown in SEQ ID NO:49 or 106.
  • the nucleic acid constructs of the present disclosure are delivered to a subject and the protein of interest (eg, hHGF protein) is expressed after about 0.5-1.5 hours. In some embodiments, the nucleic acid constructs of the present disclosure are delivered to a subject within about 2h-10h (e.g., about 2h, about 3h, about 4h, about 5h, about 6h, about 7h, about 8h, about 10h, etc.) to reach the peak expression. In some specific embodiments, the nucleic acid construct in the present disclosure is an mRNA molecule that expresses the target protein. For example, the nucleotide construct is an mRNA molecule that expresses hHGF protein.
  • the nucleic acid constructs of the present disclosure are delivered into a subject with higher pharmacokinetic properties than Collategene plasmids.
  • the nucleic acid construct is an mRNA molecule that has improved pharmacokinetic properties (eg, Cmax, AUC 0-inf , MRT 0-inf ) compared to the Collategene plasmid.
  • the nucleic acid constructs of the present disclosure can improve the perfusion ratio of the lower limbs in ischemic subjects.
  • m-A16-B12 (hHGF) expressing hHGF protein can improve the perfusion ratio of the lower limbs in ischemic subjects.
  • hHGF m-A16-B12
  • the nucleic acid constructs of the present disclosure can improve the perfusion ratio of the lower limbs in ischemic subjects.
  • hHGF m-A16-B12 expressing hHGF protein
  • hHGF m-A16-B12 expressing hHGF protein
  • mice with lower limb ischemia injected with about 500ng/mox of m-A16-B12 (hHGF) can recover to about 90% or more of blood perfusion.
  • the nucleic acid constructs of the present disclosure are capable of ameliorating ischemic necrosis in subjects with induced lower limb ischemia.
  • the lower limbs of mice with lower limb ischemia injected with 50 ng/mouse or 500 ng/mouse of m-A16-B12 (hHGF) maintained good integrity and no necrosis of the lower limbs occurred.
  • the nucleic acid constructs of the present disclosure can promote angiogenesis in various groups of ischemic lower limb muscles.
  • m-A16-B12 (hHGF) expressing hHGF protein can promote angiogenesis in various groups of ischemic lower limb muscles.
  • hHGF m-A16-B12
  • the number of new blood vessels has a significant statistical difference compared to the PBS group ( p ⁇ 0.05).
  • the nucleic acid constructs of the present disclosure can improve wound healing in diabetic subjects.
  • m-A16-B12 (hHGF) expressing hHGF protein can improve wound healing in diabetic subjects.
  • the wounds of the mice were all healed well.
  • the wound healing rate of m-A16-B12 (hHGF) at the dose of 50ng/bird reached 66%
  • the wound healing rate of m-A16-B12 (hHGF) at the dose of 200ng/bird and 500ng/bird could achieve 100%. heal.
  • the mRNA provided by the present disclosure contains a new structure of 5'UTR and 3'UTR, which reduces the early degradation or stabilizes the degradation of the mRNA without losing or enhancing the protein translation efficiency.
  • the mRNA has higher stability and can be used in gene therapy and gene vaccination.
  • the present disclosure provides mRNA capable of expressing human hepatocyte growth factor (Human Hepatocyte growth factor, hHGF) and its lipid nanoparticles (Lipid nanoparticles, LNP) delivery system, which can realize efficient and rapid conversion of exogenous hHGF protein in the body. , has the advantages of no integration risk and easy industrial-scale amplification. It is a more ideal treatment solution than naked plasmid and can be used as a gene therapy drug for various diseases such as CLI and DFU.
  • the present disclosure also provides isolated polynucleotides comprising (a) an open reading frame (ORF).
  • ORF open reading frame
  • the open reading frame (ORF) encodes a hepatocyte growth factor (HGF), such as human hepatocyte growth factor (hHGF).
  • HGF hepatocyte growth factor
  • hHGF human hepatocyte growth factor
  • the coding sequence of hHGF comprises any one selected from the following 1)-3):
  • the 5' end of the polynucleotide may include any 5'UTR provided by the disclosure, and/or the 3' end of the polynucleotide may include any 3'UTR provided by the disclosure. .
  • the 5' end of the polynucleotide can include any 5'Cap provided by the disclosure, and/or the 3' end of the polynucleotide can include any poly-A provided by the disclosure. tail.
  • the polynucleotide is RNA, such as mRNA.
  • the RNA or polynucleotide may further comprise one or more modifications (including chemical modifications), such as backbone modifications, sugar modifications, base modifications, etc. modification and/or lipid modification, etc.
  • the RNA or polynucleotide is uniformly modified to a particular modification (eg, completely modified throughout the sequence).
  • RNA can be uniformly modified with pseudouridine (eg, N1-methylpseudouridine) such that every U in the sequence is a pseudouridine.
  • Backbone modifications in connection with the present disclosure refer to chemical modifications of the phosphates of the backbone of the nucleotides contained in the RNA or polynucleotides of the present disclosure.
  • the backbone modification includes, but is not limited to, completely replacing the unmodified phosphate portion of the backbone with a modified phosphate ester.
  • the phosphate of the backbone can be modified by replacing one or more oxygen atoms with different substituents. group.
  • the modified phosphates include, but are not limited to, phosphorothioates, phosphoselenates, borane phosphates, borane phosphates, hydrogen phosphonates, phosphoramidates, alkyl or aryl esters. Phosphonates and phosphate triesters.
  • sugar modifications in connection with the present disclosure refer to chemical modifications of the sugars of the nucleotides contained in the RNA or polynucleotides of the present disclosure.
  • the sugar modifications include, but are not limited to, modifying or replacing the 2' hydroxyl (OH) of the RNA molecule with a number of different "oxy" or “deoxy” substituents.
  • the "oxygen” modification includes, but is not limited to, substitution modifications of alkoxy, aryloxy, polyethylene glycol (PEG), and the like.
  • deoxy modifications include, but are not limited to, hydrogen, amino (e.g., NH2, alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroaryl amino acid or amino acid) modification.
  • amino e.g., NH2, alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroaryl amino acid or amino acid
  • Base modifications in connection with the present disclosure refer to chemical modifications of the base portion of the nucleotides contained in the RNA or polynucleotides of the present disclosure.
  • the base modifications include modifications to adenine, guanine, cytosine, and uracil in nucleotides.
  • the nucleosides and nucleotides described herein can be chemically modified on the primary groove surface.
  • the primary groove chemical modification may include amino, thiol, alkyl, or halogen groups.
  • the base modification includes, but is not limited to, pseudouridine, 1-methyl-pseudouridine, 5-azacytidine, 5-methylcytosine-5'-triphosphate or 2- Methoxyadenine modification.
  • the base modification is a pseudouridine modification.
  • RNA can be modified uniformly with pseudouridines so that every U in the sequence is a pseudouridine.
  • Lipid modification in relation to the present disclosure refers to a lipid modification included in the RNA or polynucleotide of the present disclosure.
  • the lipid modification includes, but is not limited to, the RNA or polynucleotide of the present disclosure covalently linked to at least one linker, and the corresponding linker covalently linked to at least one lipid.
  • the lipid modification includes, but is not limited to, an RNA or polynucleotide of the disclosure covalently attached to at least one lipid (no linker).
  • UTRs (5'UTR and/or 3'UTR) can be provided as flanking regions to the nucleic acid constructs, RNA or polynucleotide molecules of the present disclosure. UTRs may be homologous or heterologous to the coding region in the nucleic acid construct, RNA, or polynucleotide molecule of the present disclosure.
  • the flanking region may contain one or more 5'UTRs and/or 3'UTRs, which may be the same or different sequences. Any portion of the flanking region can be codon optimized. Any portion of the flanking regions may independently contain one or more different structural or chemical modifications before and/or after codon optimization.
  • RNA or polynucleotide of the present disclosure In order 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. middle. The recombinant nucleic acid construct, RNA or polynucleotide is then administered to a cell, tissue or organism and the results, such as protein levels, localization and/or half-life, are measured to assess the effect of the heterologous UTR on the present disclosure, Beneficial effects produced by RNA or polynucleotides.
  • the UTR includes a wild UTR or a variant thereof that includes the addition or removal of one or more nucleotides at the terminus, including A, T, C, or G.
  • the UTR variants also include codon optimization or modification in any manner.
  • the UTR variant also includes a derivative sequence of any embodiment of the present disclosure. For example, based on the natural UTR sequence, partial nucleotides are subjected to point mutations. The mutated variant has a higher expression level of the target gene. , stability remains unchanged or improved.
  • the detection methods for the expression level and stability of the target gene are routine in the art, such as the detection methods in Examples 3 and 4 of this disclosure.
  • the present disclosure also provides vectors comprising the nucleic acid construct, RNA or polynucleotide of any one of the preceding.
  • the nucleic acid construct, RNA or polynucleotide may be present in and/or may be part of a vector, such as a plasmid, cohesive plasmid, YAC or viral vector.
  • the vector may be an expression vector, ie, a vector that provides expression of a nucleic acid construct, RNA, or polypeptide encoded by a polynucleotide.
  • the expression vector typically contains at least one nucleic acid of the present disclosure operably linked to one or more suitable expression control elements (eg, promoter, terminator, etc.).
  • Regulatory elements and other elements useful or necessary for the expression of the encoded polypeptide of the present disclosure are, for example, promoters, terminators, selection markers, leader sequences, reporter genes, etc.
  • the vector is a therapeutic vector capable of expressing a gene of interest of the present disclosure (eg, HGF), such as a plasmid (eg, a naked plasmid), an adenoviral vector, an adeno-associated virus vector, and a lentiviral vector.
  • a gene of interest of the present disclosure eg, HGF
  • a plasmid eg, a naked plasmid
  • an adenoviral vector eg, an adenoviral vector
  • an adeno-associated virus vector e.g, lentiviral vector.
  • the nucleic acid constructs of the present disclosure may be prepared or obtained by known means (eg, by automated DNA synthesis and/or recombinant DNA technology) based on the information of the nucleotide sequences of the present disclosure, and/or may be isolated from suitable natural sources. .
  • the vector of the present disclosure also contains a promoter, for example, the promoter is at the 5' end of the 5'UTR of the nucleic acid construct, for example, the promoter is a T7 promoter, a T7lac promoter, a Tac promoter , Lac promoter, Trp promoter.
  • the promoter is at the 5' end of the 5'UTR of the nucleic acid construct, for example, the promoter is a T7 promoter, a T7lac promoter, a Tac promoter , Lac promoter, Trp promoter.
  • the present disclosure also provides a host cell comprising the nucleic acid construct, RNA or polynucleotide of any one of the preceding.
  • the cells are capable of expressing polypeptides encoded by one or more nucleic acid constructs, RNAs, or polynucleotides of the present disclosure.
  • the host cell is a bacterial cell, a fungal cell, or a mammalian cell.
  • Bacterial cells include, for example, Gram-negative bacterial strains (such as Escherichia coli strains, Proteus strains, and Pseudomonas strains) and Gram-positive bacterial strains (such as Bacillus spp. (Bacillus) strain, Streptomyces strain, Staphylococcus strain and Lactococcus strain) cells.
  • Gram-negative bacterial strains such as Escherichia coli strains, Proteus strains, and Pseudomonas strains
  • Gram-positive bacterial strains such as Bacillus spp. (Bacillus) strain, Streptomyces strain, Staphylococcus strain and Lactococcus strain
  • Fungal cells include, for example, cells of species of the genus Trichoderma, Neurospora, and Aspergillus; or include cells of the genus Saccharomyces (such as Saccharomyces cerevisiae), fission yeast Schizosaccharomyces (e.g. Schizosaccharomyces pombe (Schizosaccharomyces pombe), cells of Pichia species (such as Pichia pastoris and Pichia methanolica) and Hansenula species.
  • Saccharomyces such as Saccharomyces cerevisiae
  • fission yeast Schizosaccharomyces e.g. Schizosaccharomyces pombe (Schizosaccharomyces pombe)
  • Pichia species such as Pichia pastoris and Pichia methanolica
  • Mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.
  • the present disclosure may also use amphibian cells, insect cells, plant cells, and any other cells in the art for expressing heterologous proteins.
  • the present disclosure provides a method of preparing a nucleic acid construct, RNA, or polynucleotide of the present disclosure, as well as a method of preparing the polypeptide encoded therein.
  • nucleic acid constructs RNA or polynucleotides, and polypeptides encoded by them, such as specifically suitable vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, culture conditions, etc. in this field is known.
  • protein isolation and purification techniques suitable for use in methods of making the encoded polypeptides of the present disclosure are well known to those skilled in the art.
  • the method of preparing a nucleic acid construct or polynucleotide includes culturing the aforementioned host cell, and recovering the produced nucleic acid construct or polynucleotide from the culture.
  • the nucleic acid constructs or polynucleotides of the present disclosure, and the polypeptides they encode, can also be obtained by other production methods known in the art, such as chemical synthesis, including solid phase or liquid phase synthesis.
  • the method of preparing RNA molecules includes: preparing a nucleic acid construct or vector, and then using the nucleic acid construct or vector to perform reverse transcription 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.
  • the RNA may further comprise one or more modifications (including chemical modifications), such as backbone modifications, sugar modifications, base modifications and/or lipid modifications, etc.
  • the RNA or polynucleotide is uniformly modified to a particular modification (eg, completely modified throughout the sequence).
  • RNA can be uniformly modified with pseudouridine (eg, N1-methylpseudouridine) such that every U in the sequence is a pseudouridine (eg, N1-methylpseudouridine).
  • the present disclosure also provides a delivery vehicle comprising the nucleic acid construct of any of the foregoing, any of the RNA molecules, polynucleotides or vectors of the foregoing, wherein the delivery vehicle is a cationic lipid delivery particle.
  • the particles are nanoparticles.
  • the delivery vehicle is a lipid nanoparticle.
  • the nucleic acid constructs, RNA molecule polynucleotides or vectors in the present disclosure can be delivered into cells and/or in vivo using any type of nanolipid particles in the art.
  • nanolipid particles include but are not limited to WO2017075531 , WO2018081480A1, WO2017049245A2, WO2017099823A1, WO2022245888Al, WO2022150717A1, CN101291653A, CN102119217A, WO2011000107A1, CN107028886A, US 9868692B2, the above patent is incorporated by reference in its entirety.
  • the delivery vehicle includes nanolipid particles as shown in US9868692B2, for example, the ionizable lipid of Formula B (also known as SM-102).
  • the delivery vehicle includes nanolipid particles, phospholipids, structural lipids, and/or PEG lipids.
  • the delivery vehicle includes a lipid component including about 50 mol% of the ionizable lipid (eg, SM-102), about 10 mol% of the phospholipid (eg, DSPC), and about 38.5 mol% of the structural lipid. (e.g., cholesterol) and about 1.5 mol% PEG lipid (e.g., PEG-DMG).
  • the present disclosure also provides a pharmaceutical composition, which includes any of the aforementioned nucleic acid constructs, any of the aforementioned RNA molecules, any of the aforementioned polynucleotides, any of the aforementioned vectors, and/or any of the aforementioned delivery vehicles, and a pharmaceutical Acceptable carrier, diluent or excipient, specifically, the pharmaceutical composition is a solid preparation, injection, external preparation, spray, liquid preparation, or compound preparation.
  • kits which include the nucleic acid construct described in any one of the aforementioned, the RNA molecule described in any of the aforementioned, the polynucleotide described in any of the aforementioned, the vaccine described in any of the aforementioned, any of the aforementioned.
  • the kit can be used to provide relevant detection or diagnostic purposes.
  • the present disclosure also provides methods for treating or preventing diseases, including using a therapeutically and/or preventively effective amount of any of the aforementioned nucleic acid constructs, any of the aforementioned RNA molecules, any of the aforementioned polynucleotides, any of the aforementioned, to a subject in need. carrier, any of the aforementioned delivery vehicles, any of the aforementioned pharmaceutical compositions, and/or any of the aforementioned products or kits.
  • the present disclosure also provides any of the aforementioned nucleic acid constructs, any of the aforementioned RNA molecules, any of the aforementioned polynucleotides, any of the aforementioned vectors, any of the aforementioned delivery vehicles, any of the aforementioned pharmaceutical compositions, and/or any of the aforementioned products. or the use of the kit for the preparation of medicines for the treatment and/or prevention of diseases.
  • the disease is one in which the subject may benefit from the activity of native hHGF.
  • the disease is selected from ischemic disease, metabolic syndrome, diabetes and its complications, restenosis, and nerve damage.
  • the disease is an ischemic disease, such as coronary artery disease (CAD) or peripheral artery disease (PAD), such as myocardial infarction or lower extremity arterial ischemia.
  • CAD coronary artery disease
  • PAD peripheral artery disease
  • the disease is diabetes or a complication thereof, such as diabetic peripheral neuropathy.
  • the disease is restenosis, such as post-operative restenosis and post-perfusion restenosis.
  • the disease is nerve damage, such as neurodegenerative disease (e.g., amyotrophic lateral sclerosis (ALS), Parkinson's disease, dementia), traumatic nerve injury, peripheral neuropathy (e.g., diabetes mellitus) Peripheral neuropathy).
  • neurodegenerative disease e.g., amyotrophic lateral sclerosis (ALS), Parkinson's disease, dementia
  • peripheral neuropathy e.g., diabetes mellitus
  • Peripheral neuropathy Peripheral neuropathy
  • the disease is selected from the group consisting of lower extremity arterial ischemia, myocardial infarction, and diabetic peripheral neuropathy.
  • the disease is selected from the group consisting of peripheral arterial disease (PAD), atherosclerosis (ASO), diabetic arterial disease obliterans (DAO), Thrombosis angiitis obliterans (TAO).
  • PID peripheral arterial disease
  • ASO atherosclerosis
  • DAO diabetic arterial disease obliterans
  • TAO Thrombosis angiitis obliterans
  • the disease is selected from limb ischemia (eg, lower limb ischemia, critical limb ischemia (CLI)), diabetic foot ulcer (DFU).
  • limb ischemia eg, lower limb ischemia, critical limb ischemia (CLI)
  • DFU diabetic foot ulcer
  • a method for promoting the growth and/or migration of endothelial cells comprising administering to the endothelial cells or subject in need thereof an effective amount of any of the aforementioned polynucleotides of the present disclosure, the aforementioned Any carrier, any of the aforementioned delivery vehicles, any of the aforementioned pharmaceutical compositions, and/or any of the aforementioned products or kits.
  • relevant pharmaceutical uses for preparing drugs that promote endothelial cell growth and/or migration are provided.
  • a method for promoting angiogenesis comprising administering an effective amount of any of the foregoing polynucleotides of the present disclosure, any of the foregoing vectors, any of the foregoing delivery vehicles to endothelial cells or subjects in need thereof, the foregoing Any pharmaceutical composition, and/or any of the aforementioned products or kits.
  • the vasculogenesis is microvasculogenesis.
  • relevant pharmaceutical uses for preparing drugs that promote angiogenesis are provided.
  • the nucleic acid constructs, vectors, delivery vehicles, pharmaceutical compositions, products or kits whose target gene is HGF provided by this disclosure can be advantageously used in one or more of the following aspects: promoting endothelial cell growth and/or migration; promoting blood vessels (e.g., tiny blood vessels); treatment of ischemic diseases, such as coronary artery disease (CAD) or peripheral artery disease (PAD), such as limb ischemia (e.g., lower limb ischemia, severe lower limb ischemia); treatment of metabolic syndrome and diabetes and its complications (e.g., diabetic peripheral neuropathy, diabetic foot); inhibit restenosis; and promote repair of nerve damage (e.g., neurodegenerative diseases, traumatic nerve injury, peripheral neuropathy).
  • CAD coronary artery disease
  • PED peripheral artery disease
  • limb ischemia e.g., lower limb ischemia, severe lower limb ischemia
  • metabolic syndrome and diabetes and its complications e.g., diabetic peripheral neuropathy, diabetic foot
  • Figure 1A to Figure 1C are schematic diagrams of vector construction.
  • Figure 1A is a schematic diagram of the construction of the 5'UTR element screening vector.
  • the control is the mRNA sequence containing the 5'UTR and 3'UTR elements of Moderna Company, namely 5'UTR-Fluc- ⁇ globulin 3'UTR-120A (abbreviation is Mod.), its 5'-UTR is an artificial nucleic acid sequence, and the 3'UTR is derived from human ⁇ -globulin mRNA.
  • PmeI is a linearized enzyme cutting site.
  • Figure 1B is a schematic diagram of the construction of a 3’UTR element screening vector.
  • the control is Mod.. When constructing a 3’UTR screening vector, replace the 3’UTR region with an appropriate restriction site.
  • Figure 1C is a schematic diagram of the construction of a 5' and 3' UTR element combination screening vector.
  • the control is Mod.. When constructing a 5' UTR and 3' UTR element combination screening vector, replace the 5' and 3' UTR with appropriate restriction sites. Regional or whole gene synthesis.
  • Figure 2 shows the results of evaluating the effects of different 3'UTR elements of the present disclosure in different cell lines.
  • mRNA containing different 3'UTR elements was transfected into HEK293, HeLa and A549 cells, and luciferase expression was detected 24 hours after transfection to evaluate the impact of 3'UTR sequences on protein expression.
  • Use Mod. as a control and set the expression level of Mod. to 1. The results showed that the 3'UTR element had a consistent impact on protein expression in different cell lines.
  • Figure 3 shows the results of the impact of different 3'UTR elements of the present disclosure on mRNA expression efficiency.
  • the mRNA containing different 3'UTR elements was transduced into HEK293 cells by lipofection, and the expression level of luciferase was detected 6h, 24h, 48h and 72h after transfection.
  • Use Mod. as a control and set its expression level to 1.
  • the results show that the 3’UTR elements numbered B9, B10, B12, B13, and B14 can significantly increase the expression of the protein.
  • Figure 4 shows the results of the impact of different 5'UTR elements of the present disclosure on mRNA expression efficiency.
  • the mRNA containing different 5'UTR elements was transduced into HEK293 cells by lipofection, and the expression level of luciferase was detected 6h, 24h, 48h and 72h after transfection.
  • Use Mod. as a control and set its expression level to 1.
  • the results show that the 5’UTR elements numbered A1, A3-A7, and A9-A14 can significantly increase the expression of the protein.
  • Figure 5 shows the results of the impact of different 5'UTR elements of the present disclosure on mRNA expression efficiency.
  • the mRNA containing different 5'UTR elements was transduced into HEK293 cells by lipofection, and the expression level of luciferase was detected 6h, 24h, 48h and 72h after transfection.
  • Use Mod. as a control and set its expression level to 1.
  • the results showed that the 5’UTR elements numbered A15, A16, A18-A19, A21, A24, A27, A28, and A30-A33 can significantly increase the expression of the protein.
  • Figure 6 shows the results of the impact of different 5'UTR elements of the present disclosure on mRNA expression efficiency.
  • the mRNA containing different 5'UTR elements was transduced into HEK293 cells by lipofection, and the expression level of luciferase was detected 6h, 24h and 48h 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 could significantly increase the protein expression compared with the control nucleic acid molecules.
  • Figure 7 shows the effect of UTR element combinations of the present disclosure on mRNA expression efficiency.
  • the mRNA containing different 5'UTR and 3'UTR elements was transduced into HEK293 cells by lipofection, and the expression level of luciferase was detected 6h, 24h, 48h and 72h after transfection.
  • Use Mod. as a control and set its expression level to 1.
  • the results show that the combination of the 5’UTR elements numbered A1, A15, A16, and A18 and the 3’UTR elements numbered B12, B13, and B14 can significantly increase the expression of the protein.
  • Figures 8A to 8B show the results of the impact of UTR element combinations of the present disclosure on the expression efficiency of different target proteins.
  • Figure 8A shows the regulation of hHGF expression by mRNA containing the 5'UTR and 3'UTR elements screened out in this disclosure in HEK293 cells. The results show that compared with the control Mod., the 5' of A1A15, A16, and A18 Combining UTR elements with 3'UTR elements numbered B12, B13, and B14 can significantly increase the expression of hHGF.
  • Figure 8B shows the regulation of anti-PD-1 antibody expression by mRNA containing the 5'UTR and 3'UTR elements screened in this disclosure in HEK293 cells. The results show that compared to the control Mod., A1-B12 and A15-B12 The combination of UTRs can significantly increase the expression of anti-PD-1 antibodies.
  • FIG. 9 is a schematic diagram of experimental results showing changes in the hHGF protein expression levels of m-A16-B12 (hHGF) and control plasmids of the present disclosure over time in mouse muscle tissue.
  • the results show that injection of m-A16-B12 (hHGF) and Collategene plasmid can effectively express hHGF protein.
  • m-A16-B12 (hHGF) can express hHGF 1 hour after intramuscular injection. The peak expression of hHGF appears 6 hours after injection. It is dose-dependent, and a low dose of m-A16-B12 (hHGF) can reach a level comparable to Collategene expression AUC inf (hr*pg/mg protein) .
  • Figures 10A to 10B show the results of the therapeutic effect of m-A16-B12 (hHGF) and the control plasmid of the present disclosure on the lower limb ischemia mouse model.
  • Figure 10A is a schematic diagram of the research plan and photos of the experimental results showing the effects of m-A16-B12 (hHGF) and Collategene plasmid on blood perfusion in the lower limb ischemia mouse model.
  • Figure 10B shows the statistical results showing the effects of m-A16-B12 (hHGF) and Collategene plasmid on the blood perfusion ratio of the lower limb ischemia mouse model.
  • the 50ng/animal m-A16-B12 (hHGF) group showed a similar blood flow recovery effect to the 200ng/animal Collategene naked plasmid group;
  • the blood flow recovery effect of the 500ng/animal m-A16-B12 (hHGF) group was significantly better than that of the 200ng/animal Collategene naked plasmid group, and the blood flow perfusion could be restored to more than 90%.
  • Figures 11A to 11B show the results of the therapeutic effects of m-A16-B12 (hHGF) and control plasmids of the present disclosure on lower limb ischemia mouse models.
  • Figure 11A shows the scoring standards and representative photos of different degrees of lower limb necrosis in the lower limb ischemia mouse model.
  • Figure 11B is a statistical result showing the effects of m-A16-B12 (hHGF) and Collategene plasmid on lower limb necrosis in a mouse model of lower limb ischemia.
  • Experimental results showed that in the treatment groups injected with m-A16-B12 (hHGF) and Collategene naked plasmid, the lower limbs of each mouse maintained good integrity and no necrosis of the lower limbs occurred.
  • Figure 12 shows representative photos and statistical results of the effects of m-A16-B12 (hHGF) and control plasmids of the present disclosure on angiogenesis in a mouse model of lower limb ischemia.
  • the results showed that treatment with 50ng/animal, 500ng/animal m-A16-B12 (hHGF) and 200ng/animal Collategene naked plasmid could significantly promote angiogenesis in ischemic lower limb muscles.
  • Figure 13 shows the treatment plan, representative photos and statistical results of the effects of m-A16-B12 (hHGF) and control plasmids of the present disclosure on the wounds of the Db/Db mouse model with full-cortex injury.
  • the results showed that under the treatment of 50ng/mouse, 200ng/mouse and 500ng/mouse m-A16-B12 (hHGF) and 200 ⁇ g/mouse Collategene naked plasmid, compared with the control group, the wounds of mice were healed well, and m-A16-B12 (hHGF) promotes wound healing significantly better than 200 ⁇ g/collategene naked plasmid.
  • Figure 14 is a representative photo showing the effects of m-A16-B12 (hHGF) and control plasmids of the present disclosure on tissue reconstruction in the Db/Db mouse model with full-cortex injury.
  • hHGF m-A16-B12
  • the results showed that under the treatment of m-A16-B12 (hHGF), the wound epithelium of mice in each group completed full epithelial coverage and completed tissue reconstruction.
  • the control group and the 200 ⁇ g/animal Collategene naked plasmid group did not complete epithelial re-covering, and the collagen at the wound was abnormally proliferated and the structure was disordered.
  • HGF in this disclosure encompasses native or wild-type HGF and its homologues derived from different species, including Biologically active, naturally occurring human hepatocyte growth factor (hHGF) and its variants.
  • the amino acid sequence of native or wild-type hHGF is readily available from various public databases (eg, the GenBank database).
  • the amino acid sequence of native hHGF can be found in the GenBank database accession number: NP_000592.3.
  • the present disclosure provides hHGF having an amino acid sequence such as SEQ ID NO: 109, a DNA sequence such as SEQ ID NO: 110, and a codon-optimized sequence as shown in SEQ ID NO: 111-113.
  • HGF has a variety of biological activities, including but not limited to one or more of the following activities: (1) Promoting endothelial cell growth and/or migration; (2) Promoting vasculogenesis (e.g., microvessels); and/or, ( 3) Promote the repair of nerve damage (such as peripheral neuropathy, such as diabetic peripheral neuropathy).
  • HGF can have application prospects in many aspects, including but not limited to: (1) promoting endothelial cell growth and/or migration; (2) promoting vasculogenesis (such as microvessels); (3) treating ischemic diseases, For example, coronary artery disease (CAD) or peripheral arterial disease (PAD), such as lower extremity arterial ischemia; (4) treatment of metabolic syndrome and diabetes and their complications (e.g., diabetic peripheral neuropathy); (5) inhibition of restenosis; and (6) promote repair of nerve damage (e.g., neurodegenerative diseases, traumatic nerve injury, peripheral neuropathy).
  • CAD coronary artery disease
  • PAD peripheral arterial disease
  • nerve damage e.g., neurodegenerative diseases, traumatic nerve injury, peripheral neuropathy.
  • examples of the term “diseases that may benefit from the activity of native hHGF” include, but are not limited to, the above-mentioned diseases, for example, ischemic disease, metabolic syndrome, diabetes and its complications, restenosis, nerve damage, etc.
  • Nucleic acid or “nucleotide” includes RNA, DNA and cDNA molecules. It will be appreciated that due to the degeneracy of the genetic code, a large number of nucleotide sequences encoding a given protein can be generated.
  • nucleic acid is used interchangeably with the term “polynucleotide.”
  • Olionucleotides are short-chain nucleic acid molecules.
  • a “primer” is an oligonucleotide, whether naturally occurring in a purified restriction digest or produced synthetically, that when placed under conditions inducing the synthesis of a primer extension product complementary to a nucleic acid strand (i.e., between the nucleotide and the inducer For example, in the presence of DNA polymerase and at appropriate temperature and pH) it can serve as a starting point for synthesis.
  • primers are preferably single-stranded but may alternatively be double-stranded. If double-stranded, the primer is first treated to separate its strands before being used to prepare extension products.
  • the primers are deoxyribonucleotides. The primer must be long enough to initiate the synthesis of the extension product in the presence of the inducer. The exact length of the primer will depend on many factors, including temperature, primer source, and method used.
  • Vector refers to a replicon, such as a plasmid, bacmid, phage, virus, virion or cosmid, to which another DNA segment, i.e., an "insert” is ligated to achieve ligation of the segments in a cell of copy.
  • a vector may be a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells.
  • the vector may be viral or non-viral in origin and/or final form, such as the PUC57 DNA vector as used herein.
  • the term “vector” encompasses any genetic element capable of replicating and transferring a genetic sequence to a cell when combined with appropriate control elements.
  • the vector may be an expression vector or a recombinant vector.
  • Promoter refers to any nucleic acid sequence that regulates the expression of another nucleic acid sequence, which may be a heterologous target gene encoding a protein or RNA, by driving the transcription of the nucleic acid sequence. Promoters can be constitutive, inducible, repressive, tissue-specific, or any combination thereof. A promoter is the control region of a nucleic acid sequence where the initiation and rate of transcription of the remainder of the nucleic acid sequence is controlled.
  • Gene refers to a DNA segment involved in the production of a polypeptide chain, which may or may not include preceding and following coding regions, e.g., a 5' untranslated (5'UTR) or “leader” sequence and a 3'UTR or “untranslated” sequence. "Transcript tail” sequence, as well as the intervening sequences (introns) between the respective coding segments (exons).
  • Recombinant means that a polynucleotide is the product of various combinations of cloning, restriction, or ligation steps, as well as other steps that result in a construct that is different and/or different from the naturally occurring polynucleotide.
  • “Introduction” in the context of inserting a nucleic acid sequence into a cell, means “transfection”, “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid sequence into a eukaryotic or prokaryotic cell in which the nucleic acid sequence may be incorporated into the cell
  • the genome e.g., chromosome, plasmid, plasmid, or mitochondrial DNA
  • transient expression e.g., transfected mRNA
  • nucleic acid construct refers to a single- or double-stranded nucleic acid molecule.
  • nucleic acids are constructed as DNA molecules that have been modified or synthesized to contain nucleic acid segments in a manner that does not otherwise occur in nature, the nucleic acid molecules containing one or more control sequences or regulatory elements.
  • the nucleic acid is constructed as an RNA molecule formed after DNA is transcribed.
  • a nucleic acid construct contains a recombinant nucleotide sequence consisting essentially of optionally one, two, three or more isolated nucleotide sequences: including 5' UTR, open reading frame (ORF), 3'UTR.
  • ORF open reading frame
  • the sequences are operably linked to each other in the construct.
  • “Derived sequence” refers to a sequence that is highly homologous to the UTR sequence of the disclosure (e.g., at least 80%, 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98%, 99% to the UTR sequence of the disclosure) %, 100% identity) and still retain a nucleotide sequence with similar functional activity as the UTR sequence of the present disclosure.
  • the derived sequence is a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotides based on the natural UTR sequence.
  • the derived sequence is a nucleotide sequence obtained by truncating the natural UTR sequence.
  • operably linked is defined herein as a structure in which a control sequence, namely a promoter sequence and/or a 5'UTR sequence, is suitably positioned relative to the coding DNA sequence such that the control sequence directs the movement of the coding sequence. Transcription and translation of the mRNA into the polypeptide sequence encoded by the coding DNA.
  • ORF Open reading frame
  • the ORF consists of contiguous non-overlapping in-frame codons, starting with a start codon and ending with a stop codon in the mRNA sequence, as translated by the ribosome.
  • Endogenous refers to any substance from or produced within an organism, cell, tissue or system.
  • Exogenous refers to any substance introduced or produced from outside an organism, cell, tissue or system.
  • sequence identity refers to sequence identity between genes or proteins at the nucleotide or amino acid level, respectively.
  • sequence identity or “sequence identity” is a measure of identity between proteins at the amino acid level and between nucleic acids at the nucleotide level. Protein sequence identity can be determined by comparing the amino acid sequences at a given position in each sequence when the sequences are aligned. Similarly, nucleic acid sequence identity can be determined by comparing the nucleotide sequence at a given position in each sequence when the sequences are aligned. Methods for aligning sequences for comparison are well known in the art. Such methods Including GAP, BESTFIT, BLAST, FASTA and TFASTA.
  • the method for comparing sequences is BLAST, and the BLAST algorithm calculates percent sequence identity and performs statistical analysis on the similarity between two sequences.
  • Software for performing BLAST analyzes is publicly available through the National Center for Biotechnology Information (NCBI) website.
  • “Homology” or “homology” is defined as a nucleotide that is identical to a nucleotide residue in the corresponding sequence on the target chromosome after aligning the sequences and introducing gaps if necessary to achieve maximum percent sequence identity. Percentage of residues. Alignments for the purpose of determining percent nucleotide sequence homology can be accomplished in various ways within the skill of the art, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN, ClustalW2 or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximal alignment over the entire length of the sequences being compared.
  • the nucleic acid sequence (eg, DNA sequence) of the homology arm 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, the said Sequences are considered "homologous".
  • substitution is defined as a change in an amino acid or nucleotide sequence resulting from the replacement of one or more amino acids or nucleotides, respectively, by different amino acids or nucleotides, as compared to the amino acid sequence or nucleotide sequence of the reference polypeptide. If the substitution is conservative, the amino acid substituted into the polypeptide has similar structural or chemical properties (eg, charge, polarity, hydrophobicity, etc.) to the amino acid it replaces. In some embodiments, polypeptide variants may have "non-conservative" changes, where the substituted amino acids differ in structure and/or chemical properties.
  • deletion is defined as a change in an amino acid or nucleotide sequence that is missing one or more amino acids or nucleotide residues, respectively, compared to the amino acid sequence or nucleotide sequence of a reference polypeptide.
  • deletions may involve deletions of 2, 5, 10, up to 20, up to 30 or up to 50 or more amino acid or nucleotide residues.
  • “Insertion” or “addition” refers to a change in an 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 sequence or nucleotide sequence of a reference polypeptide. base. "Insertion” generally refers to the addition of one or more amino acid residues (or nucleotide residues within a polynucleotide) within the amino acid sequence of a polypeptide, while “addition” can be insertion or refers to the addition of one or more amino acid residues within the amino acid sequence of a polypeptide.
  • insertion or addition may be up to 10, up to 20, up to 30, up to 50 or more amino acids (or nucleotide residues).
  • Codon optimization refers to replacing the codons present in the target sequence that are generally rare in highly expressed genes of a given species with codons that are generally common in highly expressed genes of such species, and the codons before and after the replacement The codons code for the same amino acid.
  • Various species show specific preferences for certain codons for specific amino acids. Codon preference (differences in codon usage between organisms) is often associated with messenger RNA (mRNA)
  • mRNA messenger RNA
  • tRNA transfer RNA
  • the dominance of a selected tRNA in a cell is often a reflection of the codons most commonly used in peptide synthesis. Therefore, based on codon optimization, genes can be modified for optimal gene expression in a given organism. Therefore, the choice of optimal codons depends on the codon usage preference of the host genome.
  • Cell or “host cell” includes any cell type susceptible to transformation, transfection, transduction, etc., by the nucleic acid constructs or vectors of the present disclosure.
  • the host cell can be an isolated primary cell, a pluripotent stem cell, a CD34+ cell, an induced pluripotent stem cell, or any of a number of immortalized cell lines (eg, HepG2 cells).
  • the host cell may be an in situ or in vivo cell in a tissue, organ or organism.
  • Treat means administering an internal or external therapeutic agent, such as a composition comprising any of the nucleic acid constructs of the present disclosure, to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to affect Has therapeutic properties.
  • a therapeutic agent is administered to a subject or population in an amount effective to alleviate one or more symptoms of a disease, to induce regression of such symptoms, or to inhibit the progression of such symptoms to any clinically measurable extent.
  • the amount of therapeutic agent effective to alleviate the symptoms of any particular disease can vary depending on a variety of factors, such as the patient's disease state, age and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether disease symptoms have been alleviated may be assessed by any of the clinical tests commonly used by physicians or other health care professionals to assess the severity or progression of symptoms.
  • an “effective amount” or “pharmaceutically effective amount” includes an amount sufficient to ameliorate or prevent symptoms or conditions of a medical disease.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular patient or veterinary subject may vary depending on factors such as, for example, the condition being treated, the patient's general health, the method, route and dosage of administration, and the severity of the side effects.
  • An effective amount may be the maximum dosage or dosage regimen that avoids significant side effects or toxic effects.
  • an "effective amount” is an effective dose of RNA that produces an antigen-specific immune response.
  • “Pharmaceutically acceptable” means those therapeutic agents, materials, compositions and/or dosage forms that, within the scope of reasonable medical judgment, are suitable for contact with patient tissue without undue toxicity, irritation, allergic reactions or other problems or complications, have a reasonable benefit/risk ratio, and be effective for the intended use.
  • Polypeptide and “protein” have the same meaning and are used interchangeably.
  • Subject refers to mammals, including but not limited to humans, rodents (mouse, rat, guinea pig), dog, horse, cow, cat, pig, monkey, chimpanzee), etc. Preferably, the subject is human.
  • this example screened UTR sequences in housekeeping gene mRNA.
  • 301 candidate housekeeping genes including Abhd16a were identified through the database (https://esbl.nhlbi.nih.gov/helixweb/Database/NephronRNAseq/Housekeeping_Genes.html). Then, bioinformatics methods were used to analyze the expression levels and expression levels of the above genes, and retrieve the transcripts and UTR sequence information of the genes. The UTR elements were further screened based on the expression level of the gene and the length of the UTR sequence, and multiple candidate UTR sequences were obtained, from which 33 5'UTRs numbered A1 to A33, and 14 3'UTRs numbered B1 to B14 were obtained. 'UTR, gene source and sequence number are shown in Table 1 and Table 2.
  • the UTR obtained by screening in Example 1 was constructed into a DNA vector used for in vitro transcription to obtain mRNA that stably expresses the 5'UTR and 3'UTR elements.
  • the vector contains a T7 promoter, a sequence encoding firefly luciferase (Fluc) as an open reading frame (ORF) (SEQ ID NO: 126), and a polyadenylic acid (polyA) sequence, a polyA sequence Followed by restriction sites for linearizing the vector.
  • the polyA is selected from A120 (i.e., 120 consecutive adenosines) or A30L70 (SEQ ID NO: 52).
  • the 5'UTR and 3'UTR elements were constructed into the 5' end and 3' end of the open reading frame ORF (Fluc) respectively through appropriate restriction sites. Examples of constructed vectors are shown in Figure 1A to Figure 1C. The combination of 5’UTR and 3’UTR is shown in Table 3.
  • control vectors used in this example include polyA A120Mod.-120A (SEQ ID NO:53), polyA A30L70 Mod.-A30L70 (SEQ ID NO:54) and BioN.-A30L70 (SEQ ID NO:55 ), the target genes are all Fluc, constructed through total gene synthesis. Different UTR elements were constructed into the above-mentioned vectors through enzyme digestion.
  • the construction vector of vectors V-B1 to V-B14 was Mod.-120A
  • the selected enzyme cutting sites are AgeI and SacII, their polyA is A120, and the control used is Mod.-120A (SEQ ID NO: 53);
  • the construction vector of vectors V-A1 to V-A14 is Mod.-120A ( SEQ ID NO:53), the restriction sites used are BamHI and NheI, their polyA is both A120, and the control used is Mod.-120A (SEQ ID NO:53);
  • Construction of V-A15 to V-A33 The vector is Mod.-A30L70 (SEQ ID NO:54), the restriction sites used are BamHI and NheI, the polyA is A30L70, and the control used is Mod.-A30L70 (SEQ ID NO:54);
  • the vector V- The construction vector of A1-B12, V-A1-B13 and V-A1-B14 is Mod.-A30L70 (SEQ ID NO:54), the selected enzyme
  • the used Controls are Mod.-A30L70 (SEQ ID NO:54) and BioN.-A30L70 (SEQ ID NO:55).
  • the inserted UTR element gene fragments are all fully gene synthesized (Suzhou Jinweizhi Biotechnology Co., Ltd.), and the fragments are constructed through enzyme digestion and ligation to the corresponding vector. After the vector construction is completed, enzyme digestion and sequencing identification are required. If the identification is correct, it will be used in the next step. experiment.
  • the vector (i.e., DNA template) in Table 3 was digested by restriction endonuclease treatment to linearize the DNA template, and then transcribed in vitro using T7-RNA polymerase.
  • T7 RNA polymerase (Roche), corresponding reaction buffer, pyrophosphatase, RNase inhibitor and NTP were used.
  • RNA For efficient capping of RNA, add excess capping compound ARCA (3'-O-Me-m 7 G(5')ppp(5')G, Trilink, N-7003-1) or CleanCap (m7G) to the reaction (5')ppp(5')(2'OMeA)pG, Trilink, N-7113), in which the RNA used in Figures 2 to 5 was produced using ARCA for capping, and the RNA used in Figures 6 to 9 was capped. CleanCap performs capping.
  • ARCA 3'-O-Me-m 7 G(5')ppp(5')G, Trilink, N-7003-1
  • CleanCap m7G
  • the mRNA in this disclosure has undergone nucleic acid modification, and all uridine triphosphate (UTP) in the reaction system has been replaced with N1-methyl -Pseudouridine triphosphate (N1-methyl-pseudouridine triphosphate, abbreviated as 1m- ⁇ UTP, purchased from ThermoFisher), this modification method refers to patent US 2014/0194494 A1.
  • the RNA was purified by carboxylated magnetic beads (Invitrogen) and resuspended in ribozyme-free water. The RNA was evaluated spectrophotometrically and analyzed on a 5200 bioanalyzer (Agilent). concentration and quality. After detection, the target mRNA is obtained.
  • the sequences from the 5'UTR to polyA of Mod. of the present disclosure are shown in SEQ ID NO: 53 and 54, and the sequence from the 5'UTR to polyA of BioN. is shown in SEQ ID NO: 55.
  • the 5'UTR and 3'UTR sequences in Mod. are the same as The sequences in Moderna's patents and published documents are consistent (US10849920B2, WO2013151667A1, US10730924B2, DOI: 10.1016/j.cell.2017.02.017), and BioN.'s 5'UTR and 3'UTR sequences are derived from patent WO 2018/160540 .
  • the linearized sequence of V-B1, the sequence from 5'UTR to polyA is shown in SEQ ID NO:56; the linearized sequence of V-A1, the sequence from 5'UTR to polyA is shown in SEQ ID NO: 57 shown.
  • This example uses the luciferase expression system of mRNA lipofection to verify the function of UTR.
  • HEK293 Human embryonic kidney cells (HEK293, purchased from ATCC) were seeded in a 96-well plate at a density of 4 ⁇ 10 4 cells/well. The next day, transfection was performed using Lipofectamine TM MessengerMAX TM mRNA transfection reagent. Each well was transfected with 100 ng of the capped mRNA prepared in Example 2. After 6 hours of transfection, fresh medium was replaced. Aspirate 100 ⁇ L of culture medium and add 50 ⁇ L of luciferase substrate, and detect the luminescence intensity using a PerkinElmer multifunctional microplate 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 for HEK293.
  • HeLa human cervical cancer cells
  • A549 purchased from ATCC
  • Evaluation method In Tables 4 to 7 and 9 to 11, set the fluorescein expression level of the reference positive control Mod. in different cell lines to 1, and calculate the expression multiples of different mRNAs relative to the positive control Mod. .
  • the fluorescein expression level of the reference positive control BioN. in different cell lines is set to 1, and the expression multiples of different mRNAs relative to the positive control BioN. are calculated.
  • the values in the table are multiples of the detection average.
  • Example 2 In order to verify the ability of different 3'UTRs to regulate the expression of target genes in different cell lines, the method of Example 2 was used to produce mRNA expressing luciferase (Fluc), and the produced RNA was ARCA-capped, and the polyA tail structure was A120. RNA was transfected into human HeLa, HEK293 and A549 cells, and luciferase levels were measured 24 hours after transfection. The results are shown in Table 4 and Figure 2.
  • the mRNA in Table 4 was transfected into HEK293 cells, and the luciferase expression levels were measured at 6h, 24h, 48h and 72h after transfection. The results See Table 5 and Figure 3.
  • RNA used in Table 6 and Figure 4 is ARCA-capped and the polyA tail structure is A120.
  • the RNA used in Table 7 and Figure 5 is CleanCap-capped and the polyA tail is The structure is A30L70.
  • the RNA produced above was transfected into HEK293 cells respectively, and the luciferase expression levels were measured at 6h, 24h, 48h and 72h after transfection. The results are shown in Table 6, Table 7 and Figure 4 and Figure 5.
  • Figure 4 and Figure 5 were screened through different capping methods and polyA tail structures respectively.
  • the results show that in different screening vectors, changes in the 5'UTR element can affect the expression of the target protein.
  • the 5'UTR element has a negative impact on the target protein.
  • the improvement of protein expression levels is universal.
  • the 5'UTR elements numbered A1, A3 to A7, A9 to A16, A18, A21, A24, A27, A28, A32, and A33 can increase the protein expression by more than 1.2 times, and at 24h, 48h, and 72h There are significant differences at all detection time points (p ⁇ 0.05).
  • the 5'UTR elements of A1, A15, A16, and A18 have the best effect, which can increase protein expression by more than 2 times and have good time continuity.
  • the better 5'UTR elements of A1, A15, A16, and A18 selected above were constructed into the 5'UTR elements containing the A30L70polyA tail element.
  • mRNA expressing luciferase Fluc was produced using the aforementioned method and transfected into HEK293 cells. Luciferase expression levels were measured at 6h, 24h, 48h and 72h after transfection. The results are shown in Table 8 and Figure 6.
  • the target proteins in 1)-5) above are all luciferase (Fluc), and the target proteins in 6) below are secreted proteins (such as hHGF, anti-PD-1 antibody).
  • the mRNA is constructed as an ORF and the target protein expressed is human hepatocyte growth factor (Hepatocyte growth factor, hHGF) or anti-PD-1 antibody.
  • Hepatocyte growth factor human hepatocyte growth factor, hHGF
  • anti-PD-1 antibody anti-PD-1 antibody
  • hHGF The amino acid sequence of hHGF is shown in SEQ ID NO: 109, and its DNA sequence is shown in SEQ ID NO: 110. After codon optimization, mRNA OS1, OS2, and OS3 were obtained, and the optimized codon sequence is SEQ ID NO. :129-131.
  • the amino acid sequences of the heavy chain and light chain of the PD-1 antibody are shown in SEQ ID NO: 117 and 118 respectively.
  • the DNA sequences of the heavy chain and light chain are shown in SEQ ID NO: 119 and 120 respectively.
  • the heavy chain and The mRNA sequences of the light chain are shown in SEQ ID NO: 121 and 122 respectively.
  • the present disclosure has carried out codon optimization on the nucleotide sequence of wild-type hHGF (SEQ ID NO: 110).
  • the optimized sequences include hHGF-OS1 (SEQ ID NO: 111), hHGF-OS2 (SEQ ID NO: 112) and hHGF-OS3 (SEQ ID NO: 113), the corresponding mRNA sequences are shown in SEQ ID NO: 129-131 respectively.
  • the expression level of hHGF protein was detected by ELISA, which proved that the hHGF codon optimized sequence can increase the expression level of hHGF protein.
  • hHGF-OS2 The mRNA expressing hHGF-OS2 containing different combinations of 5'UTR and 3'-UTR elements (the corresponding DNA sequence is SEQ ID NO: 112) was compared with the expression of hHGF containing a combination of Moderna's 5'UTR and 3'UTR elements.
  • -OS2 mRNA Mod.(hHGF) (corresponding DNA sequence is SEQ ID NO:114).
  • HEK293 cells were transfected with the hHGF-encoding mRNA. The supernatant was collected at 6h, 24h, 48h and 72h after transfection and the expression of hHGF protein was detected by ELISA to evaluate the increase/prolongation of HGF expression by the candidate vector. Results are shown in Table 10 and Figure 8A.
  • Target protein expression of mRNA carrying different 5'UTR and 3'UTR combinations (Cleancap capped, target genes are all hHGF, polyA is A30L70)
  • the full-length mRNA expressing the anti-PD-1 antibody containing different combinations of 5'UTR and 3'UTR elements was compared with the 5'UTR and 3'UTR of Moderna.
  • the element combination is compared with the mRNA expressing anti-PD-1 antibody (the corresponding DNA sequence is SEQ ID NO: 123).
  • HEK293 cells were transfected with mRNA encoding the full length of anti-PD-1 antibody. The supernatant was collected at 6h, 24h, 48h and 72h after transfection and the expression of anti-PD-1 antibody was detected by ELISA to evaluate the expression of anti-PD-1 by candidate vectors. 1 antibody increase/prolongation, results are shown in Table 11 and Figure 8B.
  • Target protein expression of mRNA carrying different 5'UTR and 3'UTR combinations (Cleancap capped, target genes are both heavy and light chains of anti-PD-1 antibodies, polyA is A30L70)
  • lipid nanoparticles LNP containing 50 mol% ionizable lipid (SM-102) were used ), 10 mol% DSPC, 38.5 mol% cholesterol, 1.5 mol% PEG-DMG) for delivery of mRNA, and the same lipid nanoparticle LNP was also used in the following Examples 5 and 6.
  • the positive control was Collategene plasmid (AnGes Company), administered via naked plasmid.
  • Balb/c mice (6-8w, male) were randomly divided into 4 groups, with 33 mice in each group.
  • m-A16-B12 hHGF
  • Collategene plasmid Different doses of m-A16-B12 (hHGF) and Collategene plasmid were injected intramuscularly into the gastrocnemius muscle of the mice, and then the arrangements were as follows according to Table 12.
  • 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 using RIPA lysis solution containing protease inhibitors. The supernatant was centrifuged and used The BCA protein concentration assay kit was used to measure the hHGF protein concentration, and the ELISA method was used to evaluate the hHGF expression level. The data of each group are expressed as mean ⁇ standard deviation (Mean ⁇ SD), and Graphpad Prism 9.0 software is used for drawing and statistics.
  • m-A16-B12 can express hHGF protein 1 hour after intramuscular injection, and the expression peaks at 6 hours after injection, in a dose-dependent manner.
  • Collategene reached its peak expression 7 days after injection; the protein expression decreased after 48 hours of m-A16-B12 (hHGF) delivery, and the 1 ⁇ g/mouse dose of m-A16-B12 (hHGF) at 72 hours The protein expression level is comparable to Collategene.
  • the lower limb ischemia mouse model is a classic mouse model that simulates severe lower limb ischemia in humans. Male Balb/c mice of 6-8 weeks are used for modeling. The method is as follows: 1) Anesthetize the animal and place it on the operating table in a supine position.
  • the LNP in Example 4 was used as a carrier to deliver m-A16-B12 (hHGF), and Collategene was administered as a naked plasmid.
  • hHGF m-A16-B12
  • Collategene naked plasmid were intramuscularly injected into the gastrocnemius muscles of the mice respectively.
  • the experimental groups were as follows: 1) Inject 500ng/mouse of m -A16-B12(hHGF); 2) Inject 50ng/animal of m-A16-B12(hHGF); 3) Inject 200 ⁇ g/animal Collategene naked plasmid; 4) Inject an equal volume of PBS as a control group.
  • the day of modeling was counted as day 0.
  • the condition of the legs was observed on the 0th day, the 4th day, the 7th day, the 10th day, the 12th day and the 14th day after the treatment of each experimental group, and a blood flow meter was used to detect The blood perfusion of the lower limbs of mice was photographed and recorded.
  • Blood perfusion ratio the blood perfusion volume of the lower limbs of the mouse on that day/the blood perfusion volume of the lower limbs of the mouse on day 0 ⁇ 100%; the data of each group are average ⁇ standard Difference (Mean ⁇ SD) is expressed, using Graphpad Prism 9.0 software for drawing and statistics.
  • the group injected with 50ng/animal m-A16-B12 (hHGF) showed a similar blood flow recovery effect to the group injected with 200ng/animal Collategene naked plasmid; in particular, the group injected with 500ng/animal m-A16-B12 (hHGF)
  • the blood flow recovery effect of the 200ng/collategene naked plasmid group was significantly better than that of the 200ng/animal Collategene naked plasmid group, and the blood flow perfusion could be restored to more than 90%.
  • CD31 is an important marker of angiogenesis.
  • m-A16-B12 hHGF
  • hHGF m-A16-B12
  • samples from the vicinity of the ischemic tissue of the mice were taken.
  • Gastrocnemius muscle samples were prepared into paraffin sections, and CD31 in the sectioned samples was immunohistochemically stained with CD31 antibodies.
  • Photographs were taken and analyzed by ImageJ (NIH) software to calculate the CD31 staining area. The data of each group are expressed as mean ⁇ standard deviation (Mean ⁇ SD), and Graphpad Prism 9.0 software is used for drawing and statistics.
  • the Db/Db mouse model is a classic diabetic mouse model. Because mice are deficient in the leptin receptor gene, the mice exhibit characteristics such as hyperglycemia, hyperlipidemia, and insulin resistance similar to those of diabetic patients as they age.
  • This experiment uses a full-thickness injury model to simulate skin injuries that are difficult to heal in diabetic foot patients.
  • the manufacturing method of the above-mentioned Db/Db mouse model is as follows: the small Db/Db mice are anesthetized, depilated with hair removal cream, and sterilized with 75% alcohol cotton balls. A full-thickness skin wound was made on the lower back using the biopunch, a skin harvesting device with a diameter of 8 mm.
  • Example 4 In order to determine the therapeutic effect of m-A16-B12 (hHGF) on wound healing in a mouse model of full-thickness injury, the LNP in Example 4 was used to deliver m-A16-B12 (hHGF), and Collategene was administered as a naked plasmid.
  • the administration method is subcutaneous injection at 4 points.
  • the Db/Db mice in the full cortex injury model were divided into 5 groups according to body weight and blood glucose level, with 7 mice in each group.
  • the experimental groups are as follows: 1) Inject 500ng/animal m-A16-B12 (hHGF); 2) Inject 200ng/animal m-A16-B12 (hHGF); 3) Inject 50ng/animal m-A16-B12 (hHGF) ); 4) Inject 200 ⁇ g/animal of Collategene naked plasmid; 5) Inject an equal volume of PBS into the control group.
  • the day of modeling was counted as day 0. After treatment in each experimental group, the recovery status of the sores was observed on day 0, day 3, day 5, day 7, day 10, day 12 and day 14 respectively. , take photos of the trauma site, and the images are analyzed by ImageJ (NIH) software to calculate the wound area.
  • NIR ImageJ
  • the percentage of wound healing in each group was expressed as mean ⁇ SD, and Graphpad Prism9.0 software was used for drawing and statistics.
  • m-A16-B12 hHGF
  • m-A16-B12 hHGF
  • hHGF promotes wound healing significantly better than 200 ⁇ g/animal Collategene naked plasmid (p ⁇ 0.05)
  • even a lower dose (50ng/animal) of m-A16-B12 (hHGF) showed a significantly different wound healing percentage than 200 ⁇ g/animal Collategene naked plasmid (p ⁇ 0.05).
  • m-A16-B12 hHGF
  • epithelial regeneration and tissue remodeling were evaluated by Masson staining.
  • whole cortex specimens of mice were obtained, paraffin sections were prepared, and Masson staining was used to stain the mouse skin tissues.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Communicable Diseases (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Dispersion Chemistry (AREA)
  • Pulmonology (AREA)
  • Genetics & Genomics (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Oncology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

涉及包含UTR的核酸构建体及其应用，具体地，UTR能够显著提高核酸构建体中目的基因的表达效率。所述核酸构建体可包含表达例如人肝细胞生长因子(hHGF)的核酸序列，用于严重下肢缺血、糖尿病足等疾病的基因治疗药物。

Description

包含UTR的核酸构建体及其应用
本公开要求如下专利申请的优先权：于2022年05月13日提交，申请号为CN202210522056.8，发明名称为“包含UTR的核酸构建体及其应用”的中国专利申请；上述专利申请的全部内容通过引用结合在本公开中。
技术领域
本公开属于核酸领域，涉及包含UTR的核酸构建体及其用于预防或治疗疾病的用途。所述核酸构建体可包含表达人肝细胞生长因子(hHGF)的核酸序列，用于严重下肢缺血、糖尿病足等疾病的基因治疗药物。
背景技术
基因治疗和基因接种疫苗可以为多种疾病提供高度特异性和个性化治疗、预防方案，包括遗传病、自身免疫疾病、癌或肿瘤相关疾病以及炎性疾病等。
DNA、RNA均可用作基因治疗或基因接种疫苗。DNA稳定、易操作，但存在DNA片段插入患者基因组导致突变事件(如受损基因功能丧失)等的风险。RNA能避免不希望的基因组整合，但因为遍在的RNA酶，RNA易被降解。因此，需要提高RNA的稳定性，使其编码的蛋白产物在体内积累，以实现对疾病的治疗或预防，以及，在储存、施用的过程中保持RNA结构和功能的完整性。已经发现天然存在的真核mRNA分子中含有稳定化元件，例如，其5’端、3’端的非翻译区(UTR)，以及如5’帽结构或3’聚腺苷酸尾的其它结构特征。5’UTR和3’UTR是成熟前(premature)mRNA元件，在mRNA加工过程中，成熟mRNA特有的结构特征(如5’帽和3’聚腺苷酸尾)被添加于转录的(成熟前)mRNA。关于UTR和mRNA稳定性的相关性，已有研究显示，α-球蛋白mRNA的3’UTR是α-球蛋白mRNA稳定性的重要因素(Nancy D Rodgers et al,RNA.2002Dec；8(12):1526-37；Z Wang et al,Mol Cell Biol.1999Jul；19(7):4552-60.)。
外周动脉疾病(Peripheral artery disease,PAD)是指由一系列由供应肢体动脉、内脏器官及脑部的结构和功能异常导致的非冠状动脉系统综合征，其特征在于非冠状动脉血液循环发生狭窄、闭塞和瘤样病变，累及主动脉及分支动脉。在PAD中，下肢缺血性疾病在临床上最为常见，主要病因包括动脉粥样硬化(Atherosclerosis obliterans,ASO)、糖尿病性动脉硬化闭塞症(Diabetic artery obliterans,DAO)和血栓闭塞性脉管炎(Thrombosis angiitis obliterans,TAO)。严重下肢缺血(Critical limb ischemia,CLI)属于ASO发展的晚期阶段，糖尿病足(Diabetic foot ulcer,DFU)属于DAO的一种，两者均属于外周血管病变性疾病，其发病特征均为下肢血管狭窄或闭塞、远端血液灌注不足导致的下肢疼痛、溃疡及坏死。目前治疗CLI和DFU的有效措施是通过手术或者腔内介入进行血运重建，但超40％的患者因为年龄、合 并并发症等不满足血运重建要求，只能通过药物保守治疗。药物治疗仅能延缓疾病进程，无法治愈。
肝细胞生长因子(Hepatocyte growth factor,HGF)是一种多功能间质源性生长因子，当其与细胞膜表面受体c-met结合后，引起胞内酪氨酸残基磷酸化，招募衔接蛋白，促进激酶活性，进而激活下游信号通路。HGF是胚胎发育、组织器官再生、伤口愈合和血管发生等过程的重要调节因子，能促进内皮细胞和平滑肌细胞增殖和迁移，促进缺血部位微血管网络重建，抑制细胞凋亡。研究结果显示，裸质粒肌肉注射递送hHGF-cDNA能有效促进鼠和家兔缺血下肢的血流灌注(Y Taniyama et al.Therapeutic angiogenesis induced by human hepatocyte growth factor gene in rat and rabbit hindlimb ischemia models:preclinical study for treatment of peripheral arterial disease.Gene Ther.2001Feb；8(3):181-9)。临床数据显示，小腿肌肉注射裸质粒可促进患者创面愈(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.2015Oct；50(4):494-501.)。但是，使用裸质粒递送药物存在体内转染效率低、给药剂量负担大、DNA整合风险高及治疗费用成本高等不足，且临床数据显示并未改善趾肱指数、经皮氧分压及截肢率，因此药物仍有较大提升空间。
本公开提供这样的mRNA，其含有新结构的5’UTR、3’UTR，降低mRNA的早期降解或稳定mRNA的降解，但不损失更或增强蛋白翻译效率。所述mRNA稳定性更高，可应用于基因治疗、基因接种疫苗。以及，本公开提供能够表达人肝细胞生长因子(Human Hepatocyte growth factor,hHGF)的mRNA，及其脂质纳米颗粒(Lipid nanoparticles,LNP)递送系统，可以实现外源hHGF蛋白在体内高效、快速转化，具有无整合风险、易于工业级放大的优点，是较裸质粒更为理想的治疗方案，可用作CLI、DFU等多种疾病的基因治疗药物。
发明内容
本公开提供一种核酸构建体，其包含至少一个可调节目的基因表达的核酸元件，所述核酸元件为UTR。以及，所述核酸构建体可含有一个或多个目的基因，所述目的基因例如为HGF。
核酸构建体
本公开提供核酸构建体，其包含:
(a)开放阅读框(ORF)，和
(b)非翻译区元件(UTR)。
一些实施方案中，核酸构建体为DNA分子；一些实施方案中，核酸构建体为RNA分子(例如，mRNA)。
一些实施方案中，所述ORF为编码目的基因的多核苷酸序列。
一些实施方案中，所述目的基因为异源的。另一些实施方案中，所述目的基因为内源的。一些实施方案中，所述目的基因为一个或多个(例如，2、3、4个)。
一些实施方案中，所述UTR源自基因ACTG1、ATP6V0B、ATP6V0E1、CFL1、COX4I1、CTSB、FAM166A、NDUFB9、CHCHD10、SLC38A2、NDUFA11、NDUFV3、PRDX5、GUK1、IAH1、ABHD16A、SLC25A39、ATPIF1、ANAPC11、CCDC12、MRPL14或APOA1BP的UTR。一些实施方案中，上述基因是人基因。
一些实施方案中，所述UTR为3’非翻译区元件(3’UTR)或5’非翻译区元件(5’UTR)。
一些实施方案中，所述3’UTR和5’UTR是相同或不同来源的，例如源自相同或不同的基因。例如，3’UTR源自基因ACTG1的3’UTR，5’UTR源自基因ACTG1的5’UTR。又例如，3’UTR源自基因CTSB的3’UTR，5’UTR源自基因CHCHD10的5’UTR。一些实施方案中，所述5’UTR和3’UTR源自相同物种或不同物种。
一些实施方案中，所述5’UTR位于ORF的上游。一些实施方案中，所述核酸构建体中的5’UTR位于ORF的5’末端。一些实施方案中，所述3’UTR位于ORF的下游。一些实施方案中，所述核酸构建体中的3’UTR位于ORF的3’末端。
一些实施方案中，前述核酸构建体中，其包含:
(a)开放阅读框(ORF)，
(b-1)3’UTR，所述3’UTR源自基因ACTG1、ATP6V0B、ATP6V0E1、CFL1、COX4I1、CTSB、FAM166A或NDUFB9的3’UTR；和
(b-2)5’UTR，所述5’UTR源自基因ACTG1、ATP6V0B、ATP6V0E1、CFL1、COX4I1、CTSB、FAM166A、NDUFB9、CHCHD10、SLC38A2、NDUFA11、NDUFV3、PRDX5、GUK1、IAH1、ABHD16A、SLC25A39、ATPIF1、ANAPC11、CCDC12、MRPL14或APOA1BP的5’UTR。
一些实施方案中，前述核酸构建体中，其包含:
(a)开放阅读框(ORF)，
(b-1)3’UTR，所述3’UTR源自基因CTSB、FAM166A或NDUFB9的3’UTR；和
(b-2)5’UTR，所述5’UTR源自基因ACTG1、CHCHD10或NDUFA11的5’UTR。
一些实施方案中，前述核酸构建体(例如，DNA或RNA分子)中，所述3’UTR源自基因ACTG1的3’UTR，其含有SEQ ID NO:1或SEQ ID NO:58所示或与之任一具有同一性的序列；
所述3’UTR源自基因ATP6V0B的3’UTR，其含有SEQ ID NO:2、3或SEQ ID NO:59、60所示或与之任一具有同一性的序列；
所述3’UTR源自基因ATP6V0E1的3’UTR，其含有SEQ ID NO:4、5或SEQ ID NO:61、62所示或与之任一具有同一性的序列；
所述3’UTR源自基因CFL1的3’UTR，其含有SEQ ID NO:6、7、8或SEQ ID  NO:63、64、65所示或与之任一具有同一性的序列；
所述3’UTR源自基因COX4I1的3’UTR，其含有SEQ ID NO:9、10、11或SEQ ID NO:66、67、68所示或与之任一具有同一性的序列；
所述3’UTR源自基因CTSB的3’UTR，其含有SEQ ID NO:12或SEQ ID NO:69所示或与之任一具有同一性的序列；
所述3’UTR源自基因FAM166A的3’UTR，其含有SEQ ID NO:13或SEQ ID NO:70所示或与之任一具有同一性的序列；或
所述3’UTR源自基因NDUFB9的3’UTR，其含有SEQ ID NO:14或SEQ ID NO:71所示或与之任一具有同一性的序列。
一些实施方案中，前述核酸构建体(例如，DNA或RNA分子)中，所述5’UTR源自基因ACTG1的5’UTR，其含有SEQ ID NO:15或SEQ ID NO:72所示或与之任一具有同一性的序列；
所述5’UTR源自基因ATP6V0B的5’UTR，其含有SEQ ID NO:16、17或SEQ ID NO:73、74所示或与之任一具有同一性的序列；
所述5’UTR源自基因ATP6V0E1的5’UTR，其含有SEQ ID NO:18、19或SEQ ID NO:75、76所示或与之任一具有同一性的序列；
所述5’UTR源自基因CFL1的5’UTR，其含有SEQ ID NO:20、21、22或SEQ ID NO:77、78、79所示或与之任一具有同一性的序列；
所述5’UTR源自基因COX4I1的5’UTR，其含有SEQ ID NO:23、24、25或SEQ ID NO:80、81、82所示或与之任一具有同一性的序列；
所述5’UTR源自基因CTSB的5’UTR，其含有SEQ ID NO:26或SEQ ID NO:83所示或与之任一具有同一性的序列；
所述5’UTR源自基因FAM166A的5’UTR，其含有SEQ ID NO:27或SEQ ID NO:84所示或与之任一具有同一性的序列；
所述5’UTR源自基因NDUFB9的5’UTR，其含有SEQ ID NO:28或SEQ ID NO:85所示或与之任一具有同一性的序列；
所述5’UTR源自基因CHCHD10的5’UTR，其含有SEQ ID NO:29、30或SEQ ID NO:86、87所示或与之任一具有同一性的序列；
所述5’UTR源自基因SLC38A2的5’UTR，其含有SEQ ID NO:31或SEQ ID NO:88所示或与之任一具有同一性的序列；
所述5’UTR源自基因NDUFA11的5’UTR，其含有SEQ ID NO:32或SEQ ID NO:89所示或与之任一具有同一性的序列；
所述5’UTR源自基因NDUFV3的5’UTR，其含有SEQ ID NO:33或SEQ ID NO:90所示或与之任一具有同一性的序列；
所述5’UTR源自基因PRDX5的5’UTR，其含有SEQ ID NO:34或SEQ ID NO:91所示的序列；
所述5’UTR源自基因GUK1的5’UTR，其含有SEQ ID NO:35、36、37或SEQ ID NO:92、93、94所示或与之任一具有同一性的序列；
所述5’UTR源自基因IAH1的5’UTR，其含有SEQ ID NO:38或SEQ ID NO:95所示或与之任一具有同一性的序列；
所述5’UTR源自基因ABHD16A的5’UTR，其含有SEQ ID NO:39或SEQ ID NO:96所示或与之任一具有同一性的序列；
所述5’UTR源自基因SLC25A39的5’UTR，其含有SEQ ID NO:40或SEQ ID NO:97所示或与之任一具有同一性的序列；
所述5’UTR源自基因ATPIF1的5’UTR，其含有SEQ ID NO:41或SEQ ID NO:98所示或与之任一具有同一性的序列；
所述5’UTR源自基因ANAPC11的5’UTR，其含有SEQ ID NO:42、43或SEQ ID NO:99、100所示或与之任一具有同一性的序列；
所述5’UTR源自基因CCDC12的5’UTR，其含有SEQ ID NO:44或SEQ ID NO:101所示或与之任一具有同一性的序列；
所述5’UTR源自基因MRPL14的5’UTR，其含有SEQ ID NO:45或SEQ ID NO:102所示或与之任一具有同一性的序列；或
所述5’UTR源自基因APOA1BP的5’UTR，其含有SEQ ID NO:46、47或SEQ ID NO:103、104所示或与之任一具有同一性的序列。
一些实施方案中，提供核酸构建体(例如，DNA或RNA分子)，其包含:
(a)开放阅读框(ORF)，
(b-1)3’UTR，和
(b-2)5’UTR；
其中，所述3’UTR和5’UTR选自如下任一组合:
1)所述3’UTR含有SEQ ID NO:1或SEQ ID NO:58所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:15-47任一或SEQ ID NO:72-104任一所示或与之任一具有同一性的序列；
2)所述3’UTR含有SEQ ID NO:2或SEQ ID NO:59所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:15-47任一或SEQ ID NO:72-104任一所示或与之任一具有同一性的序列；
3)所述3’UTR含有SEQ ID NO:3或SEQ ID NO:60所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:15-47任一或SEQ ID NO:72-104任一所示或与之任一具有同一性的序列；
4)所述3’UTR含有SEQ ID NO:4或SEQ ID NO:61所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:15-47任一或SEQ ID NO:72-104任一所示或与之任一具有同一性的序列；
5)所述3’UTR含有SEQ ID NO:5或SEQ ID NO:62所示或与之任一具有同一 性的序列，所述5’UTR含有SEQ ID NO:15-47任一或SEQ ID NO:72-104任一所示或与之任一具有同一性的序列；
6)所述3’UTR含有SEQ ID NO:6或SEQ ID NO:63所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:15-47任一或SEQ ID NO:72-104任一所示或与之任一具有同一性的序列；
7)所述3’UTR含有SEQ ID NO:7或SEQ ID NO:64所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:15-47任一或SEQ ID NO:72-104任一所示或与之任一具有同一性的序列；
8)所述3’UTR含有SEQ ID NO:8或SEQ ID NO:65所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:15-47任一或SEQ ID NO:72-104任一所示或与之任一具有同一性的序列；
9)所述3’UTR含有SEQ ID NO:9或SEQ ID NO:66所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:15-47任一或SEQ ID NO:72-104任一所示或与之任一具有同一性的序列；
10)所述3’UTR含有SEQ ID NO:10或SEQ ID NO:67所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:15-47任一或SEQ ID NO:72-104任一所示或与之任一具有同一性的序列；
11)所述3’UTR含有SEQ ID NO:11或SEQ ID NO:68所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:15-47任一或SEQ ID NO:72-104任一所示或与之任一具有同一性的序列；
12)所述3’UTR含有SEQ ID NO:12或SEQ ID NO:69所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:15-47任一或SEQ ID NO:72-104任一所示或与之任一具有同一性的序列；
13)所述3’UTR含有SEQ ID NO:13或SEQ ID NO:70所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:15-47任一或SEQ ID NO:72-104任一所示或与之任一具有同一性的序列；
14)所述3’UTR含有SEQ ID NO:14或SEQ ID NO:71所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:15-47任一或SEQ ID NO:72-104任一所示或与之任一具有同一性的序列；
15)所述3’UTR含有SEQ ID NO:1-14任一或SEQ ID NO:58-71任一所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:15或SEQ ID NO:72所示或与之任一具有同一性的序列；
16)所述3’UTR含有SEQ ID NO:1-14任一或SEQ ID NO:58-71任一所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:16或SEQ ID NO:73所示或与之任一具有同一性的序列；
17)所述3’UTR含有SEQ ID NO:1-14任一或SEQ ID NO:58-71任一所示或与 之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:17或SEQ ID NO:74所示或与之任一具有同一性的序列；
18)所述3’UTR含有SEQ ID NO:1-14任一或SEQ ID NO:58-71任一所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:18或SEQ ID NO:75所示或与之任一具有同一性的序列；
19)所述3’UTR含有SEQ ID NO:1-14任一或SEQ ID NO:58-71任一所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:19或SEQ ID NO:76所示或与之任一具有同一性的序列；
20)所述3’UTR含有SEQ ID NO:1-14任一或SEQ ID NO:58-71任一所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:20或SEQ ID NO:77所示或与之任一具有同一性的序列；
21)所述3’UTR含有SEQ ID NO:1-14任一或SEQ ID NO:58-71任一所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:21或SEQ ID NO:78所示或与之任一具有同一性的序列；
22)所述3’UTR含有SEQ ID NO:1-14任一或SEQ ID NO:58-71任一所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:22或SEQ ID NO:79所示或与之任一具有同一性的序列；
23)所述3’UTR含有SEQ ID NO:1-14任一或SEQ ID NO:58-71任一所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:23或SEQ ID NO:80所示或与之任一具有同一性的序列；
24)所述3’UTR含有SEQ ID NO:1-14任一或SEQ ID NO:58-71任一所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:24或SEQ ID NO:81所示或与之任一具有同一性的序列；
25)所述3’UTR含有SEQ ID NO:1-14任一或SEQ ID NO:58-71任一所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:25或SEQ ID NO:82所示或与之任一具有同一性的序列；
26)所述3’UTR含有SEQ ID NO:1-14任一或SEQ ID NO:58-71任一所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:26或SEQ ID NO:83所示或与之任一具有同一性的序列；
27)所述3’UTR含有SEQ ID NO:1-14任一或SEQ ID NO:58-71任一所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:27或SEQ ID NO:84所示或与之任一具有同一性的序列；
28)所述3’UTR含有SEQ ID NO:1-14任一或SEQ ID NO:58-71任一所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:28或SEQ ID NO:85所示或与之任一具有同一性的序列；
29)所述3’UTR含有SEQ ID NO:1-14任一或SEQ ID NO:58-71任一所示或与 之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:29或SEQ ID NO:86所示或与之任一具有同一性的序列；
30)所述3’UTR含有SEQ ID NO:1-14任一或SEQ ID NO:58-71任一所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:30或SEQ ID NO:87所示或与之任一具有同一性的序列；
31)所述3’UTR含有SEQ ID NO:1-14任一或SEQ ID NO:58-71任一所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:31或SEQ ID NO:88所示或与之任一具有同一性的序列；
32)所述3’UTR含有SEQ ID NO:1-14任一或SEQ ID NO:58-71任一所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:32或SEQ ID NO:89所示或与之任一具有同一性的序列；
33)所述3’UTR含有SEQ ID NO:1-14任一或SEQ ID NO:58-71任一所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:33或SEQ ID NO:90所示或与之任一具有同一性的序列；
34)所述3’UTR含有SEQ ID NO:1-14任一或SEQ ID NO:58-71任一所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:34或SEQ ID NO:91所示或与之任一具有同一性的序列；
35)所述3’UTR含有SEQ ID NO:1-14任一或SEQ ID NO:58-71任一所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:35或SEQ ID NO:92所示或与之任一具有同一性的序列；
36)所述3’UTR含有SEQ ID NO:1-14任一或SEQ ID NO:58-71任一所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:36或SEQ ID NO:93所示或与之任一具有同一性的序列；
37)所述3’UTR含有SEQ ID NO:1-14任一或SEQ ID NO:58-71任一所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:37或SEQ ID NO:94所示或与之任一具有同一性的序列；
38)所述3’UTR含有SEQ ID NO:1-14任一或SEQ ID NO:58-71任一所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:38或SEQ ID NO:95所示或与之任一具有同一性的序列；
39)所述3’UTR含有SEQ ID NO:1-14任一或SEQ ID NO:58-71任一所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:39或SEQ ID NO:96所示或与之任一具有同一性的序列；
40)所述3’UTR含有SEQ ID NO:1-14任一或SEQ ID NO:58-71任一所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:40或SEQ ID NO:97所示或与之任一具有同一性的序列；
41)所述3’UTR含有SEQ ID NO:1-14任一或SEQ ID NO:58-71任一所示或与 之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:41或SEQ ID NO:98所示或与之任一具有同一性的序列；
42)所述3’UTR含有SEQ ID NO:1-14任一或SEQ ID NO:58-71任一所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:42或SEQ ID NO:99所示或与之任一具有同一性的序列；
43)所述3’UTR含有SEQ ID NO:1-14任一或SEQ ID NO:58-71任一所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:43或SEQ ID NO:100所示或与之任一具有同一性的序列；
44)所述3’UTR含有SEQ ID NO:1-14任一或SEQ ID NO:58-71任一所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:44或SEQ ID NO:101所示或与之任一具有同一性的序列；
45)所述3’UTR含有SEQ ID NO:1-14任一或SEQ ID NO:58-71任一所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:45或SEQ ID NO:102所示或与之任一具有同一性的序列；
46)所述3’UTR含有SEQ ID NO:1-14任一或SEQ ID NO:58-71任一所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:46或SEQ ID NO:103所示或与之任一具有同一性的序列；或
47)所述3’UTR含有SEQ ID NO:1-14任一或SEQ ID NO:58-71任一所示或与之任一具有同一性的序列，所述5’UTR含有SEQ ID NO:47或SEQ ID NO:104所示或与之任一具有同一性的序列。
一些实施方案中，提供核酸构建体(例如，DNA或RNA分子)，其包含:
(a)开放阅读框(ORF)，
(b-1)3’UTR，和
(b-2)5’UTR；
所述3’UTR源自基因CTSB、FAM166A或NDUFB9的3’UTR，所述5’UTR源自基因ACTG1、CHCHD10或NDUFA11的5’UTR；
例如，所述3’UTR包含SEQ ID NO:12、13、14任一或SEQ ID NO:69、70、71任一或与之任一具有同一性的序列，所述5’UTR包含SEQ ID NO:15、29、30、32任一或SEQ ID NO:72、86、87、89任一或与之任一具有同一性的序列；
又例如，所述3’UTR包含SEQ ID NO:12或SEQ ID NO:69所示或与之任一具有同一性的序列，所述5’UTR包含SEQ ID NO:15或SEQ ID NO:72所示或与之任一具有同一性的序列，
所述3’UTR包含SEQ ID NO:13或SEQ ID NO:70所示或与之任一具有同一性的序列，所述5’UTR包含SEQ ID NO:15或SEQ ID NO:72所示或与之任一具有同一性的序列，
所述3’UTR包含SEQ ID NO:14或SEQ ID NO:71所示或与之任一具有同一性 的序列，所述5’UTR包含SEQ ID NO:15或SEQ ID NO:72所示或与之任一具有同一性的序列，
所述3’UTR包含SEQ ID NO:12或SEQ ID NO:69所示或与之任一具有同一性的序列，所述5’UTR包含SEQ ID NO:29或SEQ ID NO:86所示或与之任一具有同一性的序列，
所述3’UTR包含SEQ ID NO:13或SEQ ID NO:70所示或与之任一具有同一性的序列，所述5’UTR包含SEQ ID NO:29或SEQ ID NO:86所示或与之任一具有同一性的序列，
所述3’UTR包含SEQ ID NO:14或SEQ ID NO:71所示或与之任一具有同一性的序列，所述5’UTR包含SEQ ID NO:29或SEQ ID NO:86所示或与之任一具有同一性的序列，
所述3’UTR包含SEQ ID NO:12或SEQ ID NO:69所示或与之任一具有同一性的序列，所述5’UTR包含SEQ ID NO:30或SEQ ID NO:87所示或与之任一具有同一性的序列，
所述3’UTR包含SEQ ID NO:13或SEQ ID NO:70所示或与之任一具有同一性的序列，所述5’UTR包含SEQ ID NO:30或SEQ ID NO:87所示或与之任一具有同一性的序列，
所述3’UTR包含SEQ ID NO:14或SEQ ID NO:71所示或与之任一具有同一性的序列，所述5’UTR包含SEQ ID NO:30或SEQ ID NO:87所示或与之任一具有同一性的序列，
所述3’UTR包含SEQ ID NO:12或SEQ ID NO:69所示或与之任一具有同一性的序列，所述5’UTR包含SEQ ID NO:32或SEQ ID NO:89所示或与之任一具有同一性的序列，
所述3’UTR包含SEQ ID NO:13或SEQ ID NO:70所示或与之任一具有同一性的序列，所述5’UTR包含SEQ ID NO:32或SEQ ID NO:89所示或与之任一具有同一性的序列，或
所述3’UTR包含SEQ ID NO:14或SEQ ID NO:71所示或与之任一具有同一性的序列，所述5’UTR包含SEQ ID NO:32或SEQ ID NO:89所示或与之任一具有同一性的序列。
以上实施方案中，“具有同一性”涵盖具有至少80％、85％、90％、95％、96％、97％、98％、99％、100％同一性、以及前述任意两个数值之间的范围，包括整数和小数。例如“具有至少90％同一性”或“具有至少95％同一性”。
一些实施方案中，所述3’UTR和/或5’UTR为上述3’UTR和/或5’UTR的变体，所述变体例如为截短体、核苷酸突变体，所述变体仍然保持与前述本公开的3’UTR和/或5’UTR相似的活性或功能，例如仍然保持调控ORF编码目的基因表达蛋白的功能。
一些实施方案中，前述本公开提供的核酸构建体中，还包含:
(c)多聚腺苷酸(poly-A)尾。
一些具体实施方案中，所述核酸构建体中的poly-A尾位于3’UTR的下游。一些具体实施方案中，所述核酸构建体中的poly-A尾位于3’UTR的3’末端。一些具体实施方案中，所述poly-A尾在所述核酸构建体的3’末端。一些具体实施方案中，poly-A尾的长度为至少约50、100、150、200、300、400、500个核苷酸。
一些具体实施方案中，所述poly-A尾选自A120、A30L70、HGH polyA、SV40polyA、BGH polyA、rbGlob polyA或SV40late polyA。例如，所述A30L70为SEQ ID NO:52所示的序列。
一些实施方案中，前述本公开提供的核酸构建体中，表达的目的基因(即，开放阅读框ORF)为肝细胞生长因子(HGF)、抗体或其抗原结合片段。例如，与肿瘤抗原结合的抗体或其抗原结合片段、与病毒抗原结合的抗体或其抗原结合片段等。
一些具体实施方案中，所述ORF编码的多肽或蛋白是荧光蛋白或荧光素酶(luciferase)，例如SEQ ID NO:126所示的序列。
一些具体实施方案中，所述HGF为人肝细胞生长因子(hHGF)。
一些具体实施方案中，hHGF的编码序列包含选自如下1)-3)的任一项:
1)编码如SEQ ID NO:109所示氨基酸序列的核酸序列或经密码子优化的核酸序列；
2)如SEQ ID NO:110-113所示或与之具有至少80％、85％、90％、95％、96％、97％、98％、99％、100％同一性的DNA序列；
3)如SEQ ID NO:128-131任一所示或与之具有至少80％、85％、90％、95％、96％、97％、98％、99％、100％同一性的RNA序列。
一些具体实施方案中，所述表达HGF作为目的基因的核酸构建体中包含SEQ ID NO:115、116、127任一所示或与之具有至少80％、85％、90％、95％、96％、97％、98％、99％、100％同一性的序列。
一些具体实施方案中，所述抗体或其抗原结合片段为抗PD-1抗体或其抗原结合片段。
一些具体实施方案中，所述抗PD-1抗体或其抗原结合片段的编码序列包含选自如下1)-4)的任一项:
1)编码如SEQ ID NO:117所示重链氨基酸序列的核酸序列或经密码子优化的核酸序列，和编码如SEQ ID NO:118所示轻链氨基酸序列的核酸序列或经密码子优化的核酸序列；
2)编码如SEQ ID NO:117所示重链氨基酸序列中HCDR1、HCDR2和HCDR3的核酸序列或经密码子优化的核酸序列，和编码如SEQ ID NO:118所示轻链氨基酸序列中LCDR1、LCDR2和LCDR3的核酸序列或经密码子优化的核酸序列，所述CDR是根据Kabat、IMGT、Chothia、AbM或Contact编号系统定义的，例如， 是根据Kabat编号系统定义的；
3)如SEQ ID NO:119所示或与之具有至少80％、85％、90％、95％、96％、97％、98％、99％、100％同一性的DNA序列，和如SEQ ID NO:120所示或与之具有至少80％、85％、90％、95％、96％、97％、98％、99％、100％同一性的DNA序列；
4)如SEQ ID NO:121所示或与之具有至少80％、85％、90％、95％、96％、97％、98％、99％、100％同一性的DNA序列，和如SEQ ID NO:122所示或与之具有至少80％、85％、90％、95％、96％、97％、98％、99％、100％同一性的DNA序列。
一些具体实施方案中，所述表达抗PD-1抗体或其抗原结合片段的核酸构建体中包含SEQ ID NO:124和125所示的序列，或与SEQ ID NO:124和125具有至少80％、85％、90％、95％、96％、97％、98％、99％、100％同一性的序列。
一些实施方案中，前述本公开提供的核酸构建体为DNA或RNA，例如，为mRNA。
一些实施方案中，从5’至3’方向上，前述本公开提供的核酸构建体(DNA或RNA)含有如下1)-6)中任一:
1)5’UTR，和ORF；
2)ORF，和3’UTR；
3)5’UTR，ORF，和3’UTR；
4)5’UTR，ORF，3’UTR，和poly-A尾巴；
5)5’UTR，ORF，和poly-A尾巴；
6)ORF，3’UTR，和poly-A尾巴；
所述5’UTR和3’UTR可以源自相同或不同的基因。
一些实施方案中，从5’至3’方向上，前述本公开提供的核酸构建体(DNA)含有如下1)-4)中任一:
1)5’UTR，和ORF；
2)ORF，和3’UTR；
3)5’UTR，ORF，和3’UTR；
4)5’UTR，ORF，3’UTR，和poly-A尾巴；
所述5’UTR和3’UTR可以源自相同或不同的基因。
一些具体实施方案中，所述1)、3)、4)中的5’UTR包含或为如SEQ ID NO:15-47任一所示的核苷酸序列。一些具体实施方案中，所述1)-4)中的ORF包含或为如SEQ ID NO:110所示或其密码子优化的核苷酸序列(例如SEQ ID NO:111-113)。一些具体实施方案中，所述1)-4)中的ORF包含或为如SEQ ID NO:119和120所示的核苷酸序列。一些具体实施方案中，所述2)-4)中的3’UTR包含或为如SEQ ID NO:1-14任一所示的核苷酸序列。
一些实施方案中，前述本公开提供的核酸构建体(RNA或mRNA)还包含:
(d)5’帽结构(5’Cap)。
一些具体实施方案中，所述RNA分子中的5’Cap结构位于5’UTR的上游。一些实施方案中，所述RNA分子中的5’Cap结构位于5’UTR的5’末端。在一些实施方案中，5’帽结构是本领域技术人员已知的帽结构，如Cap0(第一个核碱基的甲基化，例如m7GpppN)、Cap1(m7GpppN的相邻核苷酸的核糖的额外甲基化，例如m7G(5’)ppp(5’)(2’OMeA)pG)、Cap2(m7GpppN下游第3个核苷酸的核糖的额外甲基化)、Cap3(m7GpppN下游第3个核苷酸的核糖的额外甲基化)、Cap4(m7GpppN下游第4个核苷酸的核糖的额外甲基化)、ARCA(抗反向帽类似物)、修饰的ARCA(例如，硫代磷酸酯修饰的ARCA)、肌苷、N1-甲基-鸟苷、2’-氟代-鸟苷、7-脱氮-鸟苷、8-氧代-鸟苷、2-氨基-鸟苷、LNA-鸟苷和2-叠氮基-鸟苷。
一些具体实施方案中，使用化学RNA合成或RNA体外转录(共转录加帽)形成5’Cap结构(如Cap0或Cap1)。
一些具体实施方案中，使用加帽酶(例如牛痘病毒加帽酶和/或帽依赖性2’-O甲基转移酶)经由酶促加帽来形成5’-帽结构(如Cap0或Cap1)。一些实施方案中，使用固定化加帽酶，添加5’帽结构(Cap0或Cap1)。此处全文引入WO2016/193226中的加帽方法和手段。
一些具体实施方案中，所述5’Cap选自ARCA、3’-O-Me-m7G(5’)ppp(5’)G、m7G(5’)ppp(5’)(2’OMeA)pU、m7Gppp(A2’O-MOE)pG、m7G(5’)ppp(5’)(2’OMeA)pG、m7G(5’)ppp(5’)(2’OMeG)pG、m7(3’OMeG)(5’)ppp(5’)(2’OMeG)pG或m7(3’OMeG)(5’)ppp(5’)(2’OMeA)pG。一些具体实施方案中，所述5’Cap为3’-O-Me-m7G(5’)ppp(5’)G或m7G(5’)ppp(5’)(2’OMeA)pG。
一些实施方案中，从5’至3’方向上，前述本公开提供的核酸构建体(RNA或mRNA)含有如下1)-5)中任一:
1)5’UTR，和ORF；
2)ORF，和3’UTR；
3)5’UTR，ORF，和3’UTR；
4)5’UTR，ORF，3’UTR，和poly-A尾巴；
5)5’Cap，5’UTR，ORF，3’UTR，和poly-A尾；
所述5’UTR和3’UTR可以源自相同或不同的基因。
一些具体实施方案中，所述1)、3)、4)、5)中的5’UTR包含或为如SEQ ID NO:72-104任一所示的核苷酸序列。一些具体实施方案中，所述1)-5)中的ORF包含或为如SEQ ID NO:128-131任一所示的核苷酸序列。一些具体实施方案中，所述2)-5)中的3’UTR包含或为如SEQ ID NO:58-71任一所示的核苷酸序列。一些具体实施方案中，结构包括但不限于Cap0、Cap1(例如m7G(5’)ppp(5’)(2’OMeA)pG)、Cap2、Cap3、Cap4、ARCA。
一些实施方案中，本公开提供核酸构建体(DNA)，从5’至3’方向上，依次包含5’UTR、ORF、3’UTR，可选地，可在3’方向上进一步包含poly-A尾。一些实施方案中，所述5’UTR包含或为如SEQ ID NO:15-47任一所示的核苷酸序列，所述ORF包含或为如SEQ ID NO:110所示或其密码子优化的核苷酸序列(例如SEQ ID NO:111-113)，所述3’UTR包含或为如SEQ ID NO:1-14任一所示的核苷酸序列。一些实施方案中，所述5’UTR包含或为如SEQ ID NO:15-47任一所示的核苷酸序列，所述ORF包含或为如SEQ ID NO:119和120所示的核苷酸序列，所述3’UTR包含或为如SEQ ID NO:1-14任一所示的核苷酸序列。
一些实施方案中，本公开提供核酸构建体(RNA或mRNA)，从5’至3’方向上，依次包含5’UTR、ORF、3’UTR，可选地，可在3’方向上进一步包含poly-A尾。一些实施方案中，所述5’UTR包含或为如SEQ ID NO:72-104任一所示的核苷酸序列，所述ORF包含或为如SEQ ID NO:128-131任一所示的核苷酸序列，所述3’UTR包含或为如SEQ ID NO:58-71任一所示的核苷酸序列，和所述poly-A尾巴包含或为120个连续的腺苷酸或如SEQ ID NO:52所示的核苷酸序列。一些具体实施方案中，所述核酸构建体包含如SEQ ID NO:115、116或127所示的核苷酸序列。
一些实施方案中，所述5’UTR包含或为如SEQ ID NO:72-104任一所示的核苷酸序列，所述ORF包含或为如SEQ ID NO:121和122所示的核苷酸序列，所述3’UTR包含或为如SEQ ID NO:58-71任一所示的核苷酸序列，和所述poly-A尾巴包含或为120个连续的腺苷酸或如SEQ ID NO:52所示的核苷酸序列。一些具体实施方案中，所述核酸构建体包含如SEQ ID NO:124或125所示的核苷酸序列。
一些实施方案中，前述任一种核酸构建体(DNA或RNA分子)，其中，所述UTR用于提高所述ORF表达蛋白的表达水平。示例性地，一些具体的实施方案中，与SEQ ID NO:48或50所示的5’UTR相比，本公开中SEQ ID NO:15-47、72-104任一序列所示的5’UTR具有提高的调控ORF表达目标蛋白的表达量。一些具体的实施方案中，与SEQ ID NO:49或51所示的5’UTR相比，本公开中SEQ ID NO:1-14、58-71任一序列所示的3’UTR具有提高的调控ORF表达目标蛋白的表达量。一些具体的实施方案中，与SEQ ID NO:48和49所示的5’UTR和3’UTR的组合相比，本公开中SEQ ID NO:15-47、72-104任一序列所示的5’UTR，与SEQ ID NO:1-14、58-71任一序列所示的3’UTR的组合，具有提高的调控ORF表达目标蛋白的表达量。一些具体的实施方案中，与SEQ ID NO:50和51所示的5’UTR和3’UTR的组合相比，本公开中SEQ ID NO:15-47、72-104任一序列所示的5’UTR，与SEQ ID NO:1-14、58-71任一序列所示的3’UTR的组合，具有提高的调控ORF表达目标蛋白的表达量。
一些实施方案中，本公开中的核酸构建体，其表达目标蛋白的表达量是BioN载体的约1-约20倍，例如约1倍、约1.1倍、约1.2倍、约1.3倍、约1.4倍、约1.5倍、约1.6倍、约1.7倍、约1.8倍、约1.9倍、约2倍、约2.1倍、约2.3倍、 约2.5倍、约2.8倍、约3倍、约3.2倍、约3.4倍、约3.8倍、约4倍、约4.5倍、约5倍、约5.2倍、约5.5倍、约5.8倍、约6倍、约7倍、约8倍、约10倍、约12倍、约15倍等。一些具体的实施方案中，本公开中的核酸构建体为表达目标蛋白的mRNA分子，所述mRNA分子表达目标蛋白的表达量是BioN载体的约1-约20倍。一些具体的实施方案中，所述BioN载体包含SEQ ID NO:50或107所示的5’UTR；和/或，包含SEQ ID NO:51或108所示的3’UTR。
一些实施方案中，本公开中的核酸构建体，其表达目标蛋白的表达量是Mod载体的约1-约20倍，例如约1倍、约1.1倍、约1.2倍、约1.3倍、约1.4倍、约1.5倍、约1.6倍、约1.7倍、约1.8倍、约1.9倍、约2倍、约2.1倍、约2.3倍、约2.5倍、约2.8倍、约3倍、约3.2倍、约3.4倍、约3.8倍、约4倍、约4.5倍、约5倍、约5.2倍、约5.5倍、约5.8倍、约6倍、约7倍、约8倍、约10倍、约12倍、约15倍等。一些具体的实施方案中，本公开中的核酸构建体为表达目标蛋白的mRNA分子，所述mRNA分子表达目标蛋白的表达量是BioN载体的约1-约20倍。一些具体的实施方案中，所述Mod载体包含SEQ ID NO:48或105所示的5’UTR；和/或，包含SEQ ID NO:49或106所示的3’UTR。
一些实施方案中，本公开中的核酸构建体被递送于受试者体内，在约0.5-1.5h后表达目标蛋白(例如，hHGF蛋白)。一些实施方案中，本公开中的核酸构建体被递送于受试者体内，在约2h-10h(例如，约2h、约3h、约4h、约5h、约6h、约7h、约8h、约10h等)达到表达峰值。一些具体的实施方案中，本公开中的核酸构建体为表达目标蛋白的mRNA分子，例如，所述核苷酸构建体为表达hHGF蛋白的mRNA分子。
一些实施方案中，本公开中的核酸构建体被递送于受试者体内，具有高于Collategene质粒的药代动力学性能。一些具体的实施方案中，核酸构建体为mRNA分子，所述mRNA分子与Collategene质粒相比，具有提高的药代动力学性能(例如，Cmax、AUC0-inf、MRT0-inf)。
一些实施方案中，本公开中的核酸构建体(例如，表达hHGF蛋白的m-A16-B12(hHGF))，能够改善缺受试者血下肢的血流灌注比率。示例性地，注射50ng/只、500ng/只的m-A16-B12(hHGF)的下肢缺血小鼠可实现改善的血流灌注比率。其中，注射约500ng/只m-A16-B12(hHGF)的下肢缺血小鼠可实现恢复至约90％以上的血流灌注。
一些实施方案中，本公开中的核酸构建体(例如，表达hHGF蛋白的m-A16-B12(hHGF))，能够改善诱导下肢缺血的受试者的缺血性坏死。示例性地，注射50ng/只、500ng/只的m-A16-B12(hHGF)的下肢缺血小鼠的下肢均能保持良好的完整性且并未出现下肢坏死情况。
一些实施实施方案中，本公开中的核酸构建体(例如，表达hHGF蛋白的m-A16-B12(hHGF))，可促进各组缺血下肢肌肉中的血管新生。示例性地，在给予 50ng/只、500ng/只的m-A16-B12(hHGF)治疗的情况下，均可显著促进各组缺血下肢肌肉中的血管新生，且新生血管数量相对PBS组具有明显的统计学差异(p<0.05)。
一些实施方案中，本公开中的核酸构建体(例如，表达hHGF蛋白的m-A16-B12(hHGF))，能够改善糖尿病受试者的创面愈合。示例性地，在注射50ng/只、200ng/只和500ng/只m-A16-B12(hHGF)的Db/Db糖尿病小鼠模型，小鼠的创面均得到良好的愈合。其中，在第14天，50ng/只剂量的m-A16-B12(hHGF)创面愈合率达66％，200ng/只和500ng/只剂量的m-A16-B12(hHGF)能实现100％的伤口愈合。
本公开提供的mRNA，其含有新结构的5’UTR、3’UTR，使得mRNA的早期降解降低或稳定降解，但不损失更或增强蛋白翻译效率。所述mRNA稳定性更高，可应用于基因治疗、基因接种疫苗。以及，本公开提供能够表达人肝细胞生长因子(Human Hepatocyte growth factor,hHGF)的mRNA，及其脂质纳米颗粒(Lipid nanoparticles,LNP)递送系统，可以实现外源hHGF蛋白在体内高效、快速转化，具有无整合风险、易于工业级放大的优点，是较裸质粒更为理想的治疗方案，可用作CLI、DFU等多种疾病的基因治疗药物。
多核苷酸
本公开还提供经分离的多核苷酸，其包含(a)开放阅读框(ORF)。示例性地，开放阅读框(ORF)编码肝细胞生长因子(HGF)，例如人肝细胞生长因子(hHGF)。
一些实施方案中，所述hHGF的编码序列包含选自如下1)-3)的任一项:
1)编码如SEQ ID NO:109所示氨基酸序列的核酸序列或其密码子优化的序列；
2)如SEQ ID NO:110-113任一所示或与之具有至少80％、85％、90％、95％、96％、97％、98％、99％、100％同一性的DNA序列；
3)如SEQ ID NO:128-131任一所示或与之具有至少80％、85％、90％、95％、96％、97％、98％、99％、100％同一性的RNA序列。
一些具体实施方案中，所述多核苷酸的5’端可以包含前述本公开提供的任意5’UTR，和/或所述多核苷酸的3’端可以包含前述本公开提供的任意3’UTR。
一些具体实施方案中，所述多核苷酸的5’端可以包含前述本公开提供的任意5’Cap，和/或所述多核苷酸的3’端可以包含前述本公开提供的任意poly-A尾。
一些实施方案中，所述多核苷酸是RNA，例如mRNA。
修饰
为了进一步改善本公开的RNA或多核苷酸的关于蛋白质表达的稳定性，所述RNA或多核苷酸可进一步包含一种或多种修饰(包括化学修饰)，例如骨架修饰、糖修饰、碱基修饰和/或脂质修饰等。在一些实施方案中，所述RNA或多核苷酸被均匀地修饰成某个特定修饰(例如，在整个序列中完全修饰)。例如，可以用假尿苷(例如，N1-甲基假尿苷)均匀地修饰RNA，使得序列中的每个U是假尿苷。
与本公开有关的骨架修饰是指本公开RNA或多核苷酸中包含的核苷酸的骨架的磷酸酯的化学修饰。一些实施方案中，所述骨架修饰包括但不限于用修饰的磷酸酯完全取代骨架中未修饰的磷酸酯部分，例如可以通过用不同的取代基取代一个或多个氧原子来修饰主链的磷酸基团。一些实施方案中，所述修饰的磷酸酯包括但不限于硫代磷酸酯、亚磷酸硒酸酯、硼烷磷酸酯、硼烷磷酸酯、膦酸氢酯、氨基磷酸酯、烷基或芳基膦酸酯和磷酸三酯。
与本公开有关的糖修饰是指本公开RNA或多核苷酸中包含的核苷酸的糖的化学修饰。一些实施方案中，所述糖修饰包括但不限于将RNA分子的2’羟基(OH)修饰或替换为许多不同的”氧基”或”脱氧”取代基。一些实施方案中，所述”氧基”修饰包括但不限于烷氧基、芳氧基、聚乙二醇(PEG)等的取代修饰。一些实施方案中，”脱氧”修饰包括但不限于氢、氨基(例如NH2、烷基氨基、二烷基氨基、杂环基、芳基氨基、二芳基氨基、杂芳基氨基、二杂芳基氨基或氨基酸)修饰。
与本公开有关的碱基修饰是指本公开RNA或多核苷酸中包含的核苷酸的碱基部分的化学修饰。一些实施方案中，所述碱基修饰包括对核苷酸中腺嘌呤、鸟嘌呤、胞嘧啶和尿嘧啶的修饰。例如，本文所述的核苷和核苷酸可在主凹槽表面上被化学修饰。一些实施方案中，主要的凹槽化学修饰可包括氨基、硫醇基、烷基或卤素基团。一些实施方案中，所述碱基修饰包括但不限于用假尿苷、1-甲基-伪尿苷、5-氮杂胞苷、5-甲基胞嘧啶-5’-三磷酸或2-甲氧基腺嘌呤修饰。一些实施方案中，所述碱基修饰为假尿苷修饰。例如，可以用假尿苷均匀地修饰RNA，使得序列中的每个U是假尿苷。
与本公开有关的脂质修饰是指本公开RNA或多核苷酸中包含脂质修饰。一些实施方案中，所述脂质修饰包括但不限于本公开的RNA或多核苷酸共价连接至少一个接头，以及相应的接头与至少一个脂质共价连接。一些实施方案中，所述脂质修饰包括但不限于本公开的RNA或多核苷酸与至少一个脂质共价连接(无接头)。
UTRs
UTRs(5’UTR和/或3’UTR)可以作为侧翼区域提供给本公开的核酸构建体、RNA或多核苷酸分子。UTRs可以与在本公开的核酸构建体、RNA或多核苷酸分子中的编码区同源或异源。侧翼区域可以包含一个或多个5’UTR和/或3’UTR，所述UTRs可以是相同的或不同的序列。侧翼区域的任何部分可以进行密码子优化。在密码子优化之前和/或之后，侧翼区域的任何部分可以独立地包含一个或多个不同的结构或化学修饰。
为了改变本公开的核酸构建体、RNA或多核苷酸的一种或多种特性，将与本公开的ORF异源的UTRs引入或工程化合成到本公开的核酸构建体、RNA或多核苷酸中。然后将所述重组核酸构建体、RNA或多核苷酸施用于细胞、组织或生物体，并测量结果，如蛋白质水平、定位和/或半衰期，以评估异源UTR对本公开的、 RNA或多核苷酸产生的有益影响。一些实施方案中，所述UTR包括野生UTR或其变体，所述UTR变体包括在末端添加或去除一个或多个核苷酸，包括A、T、C或G。一些实施方案中，所述UTR变体也包括任何方式进行的密码子优化或修改。一些实施方案中，所述UTR变体也包括本公开任何实施方式的衍生序列，例如在天然UTR序列的基础上，将部分核苷酸进行点突变，突变后的变体的对目的基因表达量、稳定性维持不变或得到提高。所述对目的基因表达量、稳定性的检测方法是本领域常规的，例如本公开实施例3、4中的检测方法。
载体
本公开还提供载体，其包含前述任一项所述的核酸构建体、RNA或多核苷酸。其中核酸构建体、RNA或多核苷酸可存在于载体中和/或可为载体的一部分，该载体例如质粒、粘端质粒、YAC或病毒载体。载体可为表达载体，即可提供核酸构建体、RNA或多核苷酸编码多肽表达的载体。该表达载体通常包含至少一种本公开的核酸，其可操作地连接至一个或多个适合的表达调控元件(例如启动子、终止子等)。针对在特定宿主中的表达对所述元件及其序列进行选择为本领域技术人员的常识。对本公开的编码多肽的表达有用或必需的调控元件及其他元件例如为启动子、终止子、选择标记物、前导序列、报告基因等。
一些实施方案中，所述载体是能表达本公开目的基因(例如HGF)的治疗载体，例如质粒(例如裸质粒)，腺病毒载体，腺相关病毒载体，和慢病毒载体。
本公开的核酸构建体可基于本公开的核苷酸序列的信息通过已知的方式(例如通过自动DNA合成和/或重组DNA技术)制备或获得，和/或可从适合的天然来源加以分离。
一些实施方案中，本公开的载体还包含启动子，例如所述启动子在所述核酸构建体5’UTR的5’末端，例如所述启动子为T7启动子、T7lac启动子、Tac启动子、Lac启动子、Trp启动子。
宿主细胞
本公开还提供一种宿主细胞，其包含前述任一项所述的核酸构建体、RNA或多核苷酸。一些实施方案中，所述细胞能够表达一种或多种本公开核酸构建体、RNA或多核苷酸编码的多肽。一些实施方案中，所述宿主细胞为细菌细胞、真菌细胞或哺乳动物细胞。
细菌细胞例如包括革兰氏阴性细菌菌株(例如大肠杆菌(Escherichia coli)菌株、变形杆菌属(Proteus)菌株及假单胞菌属(Pseudomonas)菌株)及革兰氏阳性细菌菌株(例如芽孢杆菌属(Bacillus)菌株、链霉菌属(Streptomyces)菌株、葡萄球菌属(Staphylococcus)菌株及乳球菌属(Lactococcus)菌株)的细胞。
真菌细胞例如包括木霉属(Trichoderma)、脉孢菌属(Neurospora)及曲菌属(Aspergillus)的物种的细胞；或者包括酵母属(Saccharomyces)(例如酿酒酵母(Saccharomyces cerevisiae))、裂殖酵母属(Schizosaccharomyces)(例如粟酒裂殖酵母 (Schizosaccharomyces pombe))、毕赤酵母属(Pichia)(例如巴斯德毕赤酵母(Pichia pastoris)及嗜甲醇毕赤酵母(Pichia methanolica))及汉森酵母属(HansenuLa)的物种的细胞。
哺乳动物细胞例如包括例如HEK293细胞、CHO细胞、BHK细胞、HeLa细胞、COS细胞等。
然而，本公开也可使用两栖类细胞、昆虫细胞、植物细胞及本领域中用于表达异源蛋白的任何其他细胞。
生产或制备方法
本公开提供一种制备本公开核酸构建体、RNA或多核苷酸的方法，以及制备其编码多肽的方法。
用于制备产生核酸构建体、RNA或多核苷酸，及其编码多肽的方法及试剂，例如特定适合载体、转化或转染方法、选择标记物、诱导蛋白表达的方法、培养条件等在本领域中是已知的。类似地，适用于制造本公开的编码多肽的方法中的蛋白分离及纯化技术为本领域技术人员所公知。
一些实施方案中，所述制备核酸构建体或多核苷酸的方法包括培养前述的宿主细胞，并从培养物中回收产生的核酸构建体或多核苷酸。本公开的核酸构建体或多核苷酸，及其编码多肽也可以通过本领域已知的其它产生方法获得，例如化学合成，包括固相或液相合成。
一些实施方案中，制备RNA分子的方法包括：制备核酸构建体或载体，然后利用所述核酸构建体或载体进行逆转录，得到RNA分子。一些具体的实施方案中，所述方法还包括对所述RNA分子的5’端添加5’Cap。
一些实施方案中，RNA可进一步包含一种或多种修饰(包括化学修饰)，例如骨架修饰、糖修饰、碱基修饰和/或脂质修饰等。在一些实施方案中，所述RNA或多核苷酸被均匀地修饰成某个特定修饰(例如，在整个序列中完全修饰)。例如，可以用假尿苷(例如，N1-甲基假尿苷)均匀地修饰RNA，使得序列中的每个U是假尿苷(例如，N1-甲基假尿苷)。
递送媒介物
本公开还提供递送媒介物，其包含前述任一项所述的核酸构建体、前述任意的RNA分子、多核苷酸或载体，其中所述递送媒介物是阳离子脂质递送颗粒。一些实施方案中，其中所述颗粒是纳米颗粒。一些实施方案中，所述递送媒介物是纳米脂质颗粒。本公开中的核酸构建体、RNA分子多核苷酸或载体可以使用本领域任意类型的纳米脂质颗粒实现向细胞内和/或体内的递送，示例性地，纳米脂质颗粒包括但不限于WO2017075531、WO2018081480A1、WO2017049245A2、WO2017099823A1、WO2022245888Al、WO2022150717A1、CN101291653A、CN102119217A、WO2011000107A1、CN107028886A、US9868692B2中所公开的脂质颗粒，上述专利通过引用全文并入本公开。
一些实施方案中，递送媒介物包含US9868692B2中示出的纳米脂质颗粒，例如，式B所示的可离子化脂质(又称，SM-102)。一些实施方案中，递送媒介物包含纳米脂质颗粒、磷脂、结构脂质、和/或PEG脂质。示例性地，递送媒介物包含脂质组分包含约50mol％的所述可电离脂质(例如，SM-102)、约10mol％的磷脂(例如，DSPC)、约38.5mol％的结构脂质(例如，胆固醇)和约1.5mol％的PEG脂质(例如，PEG-DMG)。
药物组合物
本公开还提供药物组合物，其包含本公开前述任意的核酸构建体、前述任意的RNA分子、前述任意的多核苷酸、前述任意的载体，和/或前述任意的递送媒介物，以及药学上可接受的载体、稀释剂或赋形剂，具体地，所述药物组合物为固体制剂、注射剂、外用制剂、喷剂、液体制剂、或复方制剂。
产品或试剂盒
本公开提供产品或试剂盒，其包含前述任一项所述的核酸构建体、前述任意的RNA分子、前述任一项所述的多核苷酸、前述任一项所述的疫苗、前述任一项所述的载体、前述任一项所述的递送媒介物，和/或前述任一项所述的药物组合物。所述试剂盒可用于提供相关检测或诊断用途。
治疗和/或预防疾病的方法和制药用途
本公开还提供治疗或预防疾病的方法，包括向有需要的受试者使用治疗和/或预防有效量的前述任意的核酸构建体、前述任意的RNA分子、前述任意的多核苷酸、前述任意的载体、前述任意的递送媒介物，前述任意的药物组合物，和/或前述任意的产品或试剂盒。
本公开同时提供前述任意的核酸构建体、前述任意的RNA分子、前述任意的多核苷酸、前述任意的载体、前述任意的递送媒介物，前述任意的药物组合物，和/或前述任意的产品或试剂盒用于制备治疗和/或预防疾病的药物的用途。
一些实施方案中，所述疾病为受试者可受益于天然hHGF的活性的疾病。
一些实施方案中，所述疾病选自缺血性疾病、代谢综合征、糖尿病及其并发症、再狭窄，以及神经损伤。一些具体实施方案中，所述疾病为缺血性疾病，例如冠状动脉疾病(CAD)或外周动脉疾病(Peripheral artery disease,PAD)，例如心肌梗死或下肢动脉缺血。一些具体实施方案中，所述疾病为糖尿病或其并发症，例如糖尿病周围神经病变。一些具体实施方案中，所述疾病为再狭窄，例如手术后再狭窄和灌注后再狭窄。一些具体实施方案中，所述疾病为神经损伤，例如神经退行性疾病(例如肌萎缩性侧索硬化(ALS)，帕金森氏病，痴呆病)、创伤性神经损伤、周围神经病变(例如糖尿病周围神经病变)。一些具体实施方案中，所述疾病选自下肢动脉缺血、心肌梗死和糖尿病周围神经病变。
一些实施方案中，所述疾病选自外周动脉疾病(PAD)、动脉粥样硬化(Atherosclerosis obliterans,ASO)、糖尿病性动脉硬化闭塞症(Diabetic artery  obliterans,DAO)、血栓闭塞性脉管炎(Thrombosis angiitis obliterans,TAO)。一些实施方案中，所述疾病选自肢体缺血(例如，下肢缺血、严重下肢缺血(Critical limb ischemia,CLI))、糖尿病足(Diabetic foot ulcer,DFU)。
一些实施方案中，提供了促进内皮细胞(例如脐静脉内皮细胞)生长和/或迁移的方法，包括向有需要的内皮细胞或受试者施用有效量的本公开前述任意的多核苷酸、前述任意的载体、前述任意的递送媒介物，前述任意的药物组合物，和/或前述任意的产品或试剂盒。同时提供相关制备促进内皮细胞生长和/或迁移药物的制药用途。
一些实施方案中，提供了促进血管发生的方法，包括向有需要的内皮细胞或受试者施用有效量的本公开前述任意的多核苷酸、前述任意的载体、前述任意的递送媒介物，前述任意的药物组合物，和/或前述任意的产品或试剂盒。一些实施方案中，所述血管发生为微小血管发生。同时提供相关制备促进血管发生(例如微小血管发生)药物的制药用途。
有益效果
本公开提供的目的基因为HGF的核酸构建体、载体、递送媒介物、药物组合物、产品或试剂盒可有利地应用于以下一个或多个方面:促进内皮细胞生长和/或迁移；促进血管(例如微小血管)发生；治疗缺血性疾病，例如冠状动脉疾病(CAD)或外周动脉疾病(PAD)，例如肢体缺血(例如下肢缺血、严重下肢缺血)；治疗代谢综合征和糖尿病及其并发症(例如，糖尿病周围神经病变、糖尿病足)；抑制再狭窄；和促进神经损伤(例如，神经退行性疾病，创伤性神经损伤，周围神经病变)修复。
附图说明
图1A至图1C为载体构建示意图。其中，图1A为5’UTR元件筛选载体构建的示意图，对照为含有Moderna公司的5’UTR及3’UTR元件的mRNA序列，即5’UTR-Fluc-α球蛋白3’UTR-120A(简称为Mod.)，其5’-UTR为人工核酸序列，3’UTR源自人α球蛋白的mRNA。构建5’UTR筛选载体时，通过合适的酶切位点替换5’UTR区域。PmeI为线性化酶切位点。图1B为3’UTR元件筛选载体构建的示意图，对照为Mod.，构建3’UTR筛选载体时，通过合适的酶切位点替换3’UTR区域。图1C为5’及3’UTR元件组合筛选载体构建的示意图，对照为Mod.，构建5’UTR及3’UTR元件组合筛选载体时，通过合适的酶切位点替换5’及3’UTR区域或全基因合成。
图2为本公开不同的3’UTR元件在不同细胞系中的效果评估结果。将含有不同3’UTR元件的mRNA转染至HEK293、HeLa和A549细胞中，在转染后24h检测荧光素酶表达，评估3’UTR序列对蛋白表达量的影响。使用Mod.作为对照，并将Mod.的表达水平设为1。结果显示，在不同细胞系中，3’UTR元件对蛋白表达量的影响具有一致性。
图3为本公开不同3’UTR元件对mRNA表达效率的影响结果。将含有不同3’UTR元件的mRNA通过脂转染转导至HEK293细胞中，在转染6h、24h、48h和72h后检测荧光素酶的表达水平。使用Mod.作为对照，将其表达水平设为1。结果显示，编号为B9、B10、B12、B13、B14的3’UTR元件可显著提高蛋白的表达量。
图4为本公开不同5’UTR元件对mRNA表达效率的影响结果。将含有不同5’UTR元件的mRNA通过脂转染转导至HEK293细胞中，在转染6h、24h、48h和72h后检测荧光素酶的表达水平。使用Mod.作为对照，将其表达水平设为1。结果显示，编号为A1、A3-A7、A9-A14的5’UTR元件可显著提高蛋白的表达量。
图5为本公开不同5’UTR元件对mRNA表达效率的影响结果。将含有不同5’UTR元件的mRNA通过脂转染转导至HEK293细胞中，在转染6h、24h、48h和72h后检测荧光素酶的表达水平。使用Mod.作为对照，将其表达水平设为1。结果显示，编号为A15、A16、A18-A19、A21、A24、A27、A28、A30-A33的5’UTR元件可显著提高蛋白的表达量。
图6为本公开不同5’UTR元件对mRNA表达效率的影响结果。将含有不同5’UTR元件的mRNA通过脂转染转导至HEK293细胞中，在转染6h、24h和48h后检测荧光素酶的表达水平。使用BioN.作为对照，将其表达水平设为1。结果显示，编号为A1、A15、A16和A18的5’UTR元件相较于对照的核酸分子均可显著提高蛋白的表达量。
图7为本公开UTR元件组合对mRNA表达效率的影响结果。将含有不同5’UTR和3’UTR元件的mRNA通过脂转染转导至HEK293细胞中，在转染6h、24h、48h和72h后检测荧光素酶的表达水平。使用Mod.作为对照，将其表达水平设为1。结果显示，编号为A1、A15、A16、A18的5’UTR元件与编号为B12、B13、B14的3’UTR元件进行组合均可显著提高蛋白的表达量。
图8A至图8B为本公开UTR元件组合对不同目的蛋白的表达效率的影响结果。图8A为HEK293细胞中，含有本公开筛选出的5’UTR及3’UTR元件的mRNA对hHGF表达量的调控，结果显示，相较于对照Mod.，编号为A1A15、A16、A18的5’UTR元件与编号为B12、B13、B14的3’UTR元件进行组合可显著提高hHGF的表达量。图8B为HEK293细胞中，含有本公开筛选出的5’UTR及3’UTR元件的mRNA对抗PD-1抗体表达量的调控，结果显示，相较于对照Mod.，A1-B12、A15-B12的UTR组合可显著提高抗PD-1抗体的表达量。
图9为本公开m-A16-B12(hHGF)及对照质粒在小鼠肌肉组织中hHGF蛋白表达水平随时间变化的实验结果示意图。结果显示，注射m-A16-B12(hHGF)及Collategene质粒均可有效表达hHGF蛋白，m-A16-B12(hHGF)于肌肉注射1h后即可表达hHGF，注射后6h，出现hHGF的表达峰值，呈剂量依赖，且低剂量的m-A16-B12(hHGF)即可达到与Collategene表达AUCinf(hr*pg/mg protein)相当的水平。
图10A至图10B为本公开m-A16-B12(hHGF)及对照质粒对下肢缺血小鼠模型治疗效果的结果。图10A为表明m-A16-B12(hHGF)及Collategene质粒对下肢缺血小鼠模型血流灌注影响的研究方案示意图及实验结果照片。图10B为表明m-A16-B12(hHGF)及Collategene质粒对下肢缺血小鼠模型血流灌注比率的统计结果。结果显示，在50ng/只、500ng/只的m-A16-B12(hHGF)和200ng/只的Collategene裸质粒进行治疗的情况下，各小鼠缺血下肢的血流灌注比率相对于对照组有明显的改善，且缺血下肢的血流灌注比率随时间的进展逐渐得到恢复；50ng/只m-A16-B12(hHGF)组表现出与200ng/只Collategene裸质粒组相近的血流恢复效果；500ng/只m-A16-B12(hHGF)组显著优于200ng/只Collategene裸质粒组的血流恢复效果，可实现恢复至90％以上的血流灌注情况。
图11A至图11B为本公开m-A16-B12(hHGF)及对照质粒对下肢缺血小鼠模型治疗效果的结果。图11A为下肢缺血小鼠模型下肢坏死不同程度的评分标准及代表性照片。图11B为表明m-A16-B12(hHGF)及Collategene质粒对下肢缺血小鼠模型下肢坏死影响的统计结果。实验结果显示，在注射m-A16-B12(hHGF)和Collategene裸质粒治疗组中，各小鼠的下肢均能保持良好的完整性且并未出现下肢坏死情况。
图12为本公开m-A16-B12(hHGF)及对照质粒对下肢缺血小鼠模型血管新生影响的代表性照片及统计结果。结果显示，给予50ng/只、500ng/只的m-A16-B12(hHGF)和200ng/只的Collategene裸质粒治疗的情况下，均可显著促进缺血下肢肌肉中的血管新生。
图13为本公开m-A16-B12(hHGF)及对照质粒对全皮层损伤的Db/Db小鼠模型创口影响的治疗方案、代表性照片及统计结果。结果显示，在50ng/只、200ng/只和500ng/只m-A16-B12(hHGF)和200μg/只Collategene裸质粒的处理下，相对于对照组，小鼠的创面均得到良好的愈合，且m-A16-B12(hHGF)的促创面愈合情况显著优于200μg/只Collategene裸质粒。
图14为本公开m-A16-B12(hHGF)及对照质粒对全皮层损伤的Db/Db小鼠模型组织重构影响的代表性照片。结果显示，在m-A16-B12(hHGF)的治疗下，各组小鼠创面上皮均完成上皮全覆盖，完成组织重构。而对照组及200μg/只Collategene裸质粒组均未完成上皮再覆盖，创口处胶原蛋白异常增生，结构紊乱。
具体实施方式
术语
为了更容易理解本公开，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。
本公开的“HGF”涵盖天然或野生型HGF及其不同物种来源的同源物，包括 具有生物学活性的、天然存在的人肝细胞生长因子(human hepatocyte growth factor，hHGF)及其变体。可方便地从各种公共数据库(例如，GenBank数据库)获得天然或野生型hHGF的氨基酸序列。例如，天然hHGF的氨基酸序列可见于GenBank数据库登录号:NP_000592.3。又例如，本公开提供氨基酸序列如SEQ ID NO:109、DNA序列如SEQ ID NO:110、密码子优化序列如SEQ ID NO:111-113所示的hHGF。HGF具有多种生物学活性，包括但不限于以下的一种或多种活性:(1)促进内皮细胞生长和/或迁移；(2)促进血管(例如微小血管)发生；和/或，(3)促进神经损伤(例如周围神经病变，例如糖尿病周围神经病变)修复。因此，HGF可在多个方面具有应用前景，包括但不限于:(1)促进内皮细胞生长和/或迁移；(2)促进血管(例如微小血管)发生；(3)治疗缺血性疾病，例如冠状动脉疾病(CAD)或外周动脉疾病(PAD)，例如下肢动脉缺血；(4)治疗代谢综合征和糖尿病及其并发症(例如，糖尿病周围神经病变)；(5)抑制再狭窄；和(6)促进神经损伤(例如，神经退行性疾病，创伤性神经损伤，周围神经病变)修复。因此，术语“可受益于天然hHGF的活性的疾病”的实例包括但不限于上述疾病，例如，缺血性疾病，代谢综合征，糖尿病及其并发症，再狭窄，神经损伤等。
“核酸”或“核苷酸”包括RNA、DNA和cDNA分子。应当理解，由于遗传密码的简并性，可以产生编码给定蛋白质的大量核苷酸序列。术语核酸可以与术语“多核苷酸”互换使用。“寡核苷酸”是短链核酸分子。“引物”是寡核苷酸，无论是天然存在于纯化的限制酶切消化中还是合成产生，当置于诱导与核酸链互补的引物延伸产物的合成的条件(即在核苷酸和诱导剂例如DNA聚合酶的存在下，并在合适的温度和pH下)下能够充当合成起始点。为了最大化扩增效率，引物优选是单链的，但另外可选地可以是双链的。如果是双链的，则在用于制备延伸产物之前，首先对引物进行处理以分离其链。优选地，引物是脱氧核糖核苷酸。引物必须足够长以在诱导剂的存在下引发延伸产物的合成。引物的确切长度将取决于许多因素，包括温度、引物来源和使用的方法。
“载体”或“表达载体”是指复制子，例如质粒、杆粒、噬菌体、病毒、病毒体或粘粒，可连接另一个DNA区段，即“插入物”以实现连接区段在细胞中的复制。载体可以是设计用于递送至宿主细胞或用于在不同宿主细胞之间转移的核酸构建体。如本文所用，载体在起源和/或最终形式上可以是病毒或非病毒的，如本文所用的PUC57DNA载体。术语“载体”涵盖与适当的控制元件结合时能够复制并且可以将基因序列转移至细胞的任何遗传元件。在一些实施方案中，载体可以是表达载体或重组载体。
“启动子”是指通过驱动核酸序列的转录来调节另一核酸序列表达的任何核酸序列，其可以是编码蛋白质或RNA的异源靶基因。启动子可以是组成型的、诱导型的、阻遏型的、组织特异性的或其任何组合。启动子是核酸序列的控制区域，在此核酸序列的其余部分的启动和转录速率是受控的。
“基因”是指涉及产生多肽链的DNA片段，可以包括或不包括在前和在后的编码区域，例如，5'非翻译(5'UTR)或“先导”序列和3'UTR或“非转录尾区”序列，以及在各自编码片段(外显子)之间的插入序列(内含子)。
“重组”是指多核苷酸是克隆、限制或连接步骤的各种组合的产物，以及导致与天然存在的多核苷酸差异和/或不同的构建体的其他步骤的产物。
“引入”，在将核酸序列插入细胞的内容中，意思为“转染”、“转化”或“转导”和包括参考核酸序列并入真核或原核细胞中，其中核酸序列可以并入细胞的基因组(例如，染色体、质粒、质体，或线粒体DNA)，转入自主复制，或暂时表达(例如，转染mRNA)。
“核酸构建体”是指单链或双链的核酸分子。一些实施方案中，核酸构建为DNA分子，其被修饰或合成为以天然本来不存在的方式包含核酸区段，所述核酸分子包含一个或多个控制序列或调控元件。一些实施方案中，核酸构建为DNA转录后形成的RNA分子。在本公开的上下文中，核酸构建体含有重组核苷酸序列，该重组核苷酸序列基本上由任选地一个、两个、三个或多个分离的核苷酸序列组成:包括5'UTR、开放阅读框(ORF)、3'UTR。在涉及包括两个或更多个序列的构建体的实施方式中，序列在构建体中彼此可操作地连接。
“衍生序列”是指与本公开UTR序列高度同源(例如与本公开UTR序列至少80％、85％、88％、90％、93％、95％、96％、97％、98％、99％、100％同一性)且仍保留与本公开UTR序列具有相似功能活性的核苷酸序列。一些实施方案中，所述衍生序列为在天然UTR序列的基础上经过一个或多个核苷酸的取代、缺失或添加得到的核苷酸序列。一些实施方案中，所述衍生序列为在天然UTR序列的基础上经过截短的得到的核苷酸序列。
“可操作地连接”在本文中定义为如下结构:其中控制序列即启动子序列和/或5'UTR序列，被适当地置于相对于编码DNA序列的位置处，使得控制序列指导编码序列的转录和mRNA翻译成由编码DNA编码的多肽序列。
“开放阅读框架”缩写为“ORF”，是指编码多肽的核酸序列的片段或区域。ORF包括连续的非重叠的框架内密码子，在mRNA序列中从起始密码子开始并用终止密码子结束，由核糖体翻译。
“内源的”是指来自或在生物体、细胞、组织或系统内部产生的任何物质。
“外源的”是指从生物体、细胞、组织或系统外部引入或产生的任何物质。
“同一性的序列”或“序列同一性”是指基因或蛋白质之间分别在核苷酸或氨基酸水平上的序列同一性。“同一性的序列”或“序列同一性”是蛋白质之间在氨基酸水平上的同一性量度以及核酸之间在核苷酸水平上的同一性量度。蛋白质序列同一性可以通过在比对序列时比较每个序列中给定位置的氨基酸序列来确定。类似地，核酸序列同一性可以通过在比对序列时比较每个序列中给定位置的核苷酸序列来确定。用于比对序列以供比较的方法是本领域所熟知的，此类方法 包括GAP、BESTFIT、BLAST、FASTA和TFASTA。一些实施方案中，比对序列的方法为BLAST，BLAST算法计算序列同一性百分比并对两个序列之间的相似性进行统计学分析。用于进行BLAST分析的软件可通过国家生物技术信息中心(National Center for Biotechnology Information，NCBI)网站公开获得。
“同源性”或“同源”定义为，在对准序列且必要时引入空位以实现最大序列一致性百分比之后，与靶染色体上的相应序列中的核苷酸残基一致的核苷酸残基的百分比。为了确定核苷酸序列同源性百分比的比对可以用本领域技术范围内的各种方式来实现，例如使用公开可用的计算机软件，例如BLAST、BLAST-2、ALIGN、ClustalW2或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的合适参数，包括在所比较的序列的全长上实现最大比对所需的任何算法。在一些实施方案中，当同源臂的例如核酸序列(例如DNA序列)与宿主细胞的相应原生或未经编辑的核酸序列(例如基因组序列)至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或更多一致时，所述序列被视为“同源”。
“替换”定义为氨基酸或核苷酸序列的如下变化:与参比多肽的氨基酸序列或核苷酸序列相比，分别由不同氨基酸或核苷酸替换一个或多个氨基酸或核苷酸产生。如果替换是保守的，则替换成多肽的氨基酸具有与其替换的氨基酸相似的结构或化学性质(例如，电荷、极性、疏水性等)。在一些实施方案中，多肽变体可具有“非保守”变化，其中替换的氨基酸在结构和/或化学性质上不同。
“缺失”定义为氨基酸或核苷酸序列的如下变化:与参比多肽的氨基酸序列或核苷酸序列相比，分别缺少一个或多个氨基酸或核苷酸残基。在多肽或多核苷酸序列的情况下，考虑到被修饰的多肽或多核苷酸序列的长度，缺失可涉及缺失2个、5个、10个、高至20个、高至30个或高至50个或更多个氨基酸或核苷酸残基。
“插入”或“添加”是指氨基酸或核苷酸序列的如下变化:与参比多肽的氨基酸序列或核苷酸序列相比，该变化导致分别加入了一个或多个氨基酸或核苷酸残基。”插入”通常是指在多肽的氨基酸序列内添加一个或多个氨基酸残基(或多核苷酸内的核苷酸残基)，而”添加”可以是插入或指在多肽的N-或C-末端添加的氨基酸残基(或在多核苷酸的5'或3'末端添加的核苷酸残基)。在多肽或多核苷酸序列的情况下，插入或添加可以是高至10个、高至20个、高至30个、高至50个或更多个氨基酸(或核苷酸残基)。
“密码子优化”是指将目标序列中存在的在给定物种的高度表达的基因中一般罕见的密码子替换为在这类物种的高度表达的基因中一般常见的密码子，而替换前后的密码子编码相同的氨基酸。各种物种对特定氨基酸的某些密码子表现出特定的偏好。密码子偏好(生物体之间密码子使用的差异)通常与信使RNA(mRNA) 的翻译效率相关，继而认为所述信使RNA尤其取决于翻译的密码子的特性和具体的转运RNA(tRNA)分子的利用度。选择的tRNA在细胞中的优势通常是肽合成中最常使用的密码子的反映。因此，基于密码子优化，基因可以针对给定生物体中的最佳基因表达进行修改。因此，最佳密码子的选择取决于宿主基因组的密码子使用偏好。
“细胞”或“宿主细胞”包括易于被本公开内容的核酸构建体或载体转化、转染、转导等的任何细胞类型。作为非限制性实例，宿主细胞可以是分离的原代细胞、多能干细胞、CD34+细胞、诱导的多能干细胞或许多永生化细胞系(例如HepG2细胞)中的任何一种。或者，宿主细胞可以是组织、器官或生物体中的原位或体内细胞。
“治疗”意指给予患者内用或外用治疗剂，例如包含本公开的任一种核酸构建体的组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，以诱导这类症状退化或抑制这类症状发展到任何临床可测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。
“有效量”或“药物学上的有效量”包含足以改善或预防医学疾病的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化:例如，待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。一些实施方案中，“有效量”是一种有效的RNA的剂量，可以产生抗原特异性免疫应答。
“药学上可接受的”是指这些治疗剂、材料、组合物和/或剂型，在合理的医学判断范围内，适用于与患者组织接触而没有过度毒性、刺激性、过敏反应或其他问题或并发症，具有合理的获益/风险比，并且对预期的用途是有效。
“多肽”和“蛋白质”具有相同的含义，可互换使用。
“受试者”是指哺乳动物，包括但不限于，人、啮齿类动物(小鼠、大鼠、豚鼠)、狗、马、牛、猫、猪、猴、黑猩猩)等。优选地，受试者是人。
实施例
以下结合实施例用于进一步描述本公开，但这些实施例并非限制本公开的范围。本公开实施例中未注明具体条件的实验方法，通常按照常规条件，如冷泉港的抗体技术实验手册，分子克隆手册；或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1. 5’UTR和3’UTR的筛选
为了筛选到可提高蛋白表达效率的3’-非翻译区元件(3’UTR元件)、5’-非翻译区元件(5’UTR元件)，本实施例筛选了管家基因mRNA中的UTR序列。
首先，通过数据库(https://esbl.nhlbi.nih.gov/helixweb/Database/NephronRNAseq/Housekeeping_Genes.htmL)确定了包括Abhd16a等301个候选人管家基因。然后，通过生物信息学方法对上述基因的表达水平进行分析及表达量排序，并调取基因的转录本及UTR序列信息。进一步根据基因的表达水平及UTR序列长短对UTR元件进行筛选，获得了多个候选UTR序列，从中筛选获得了编号为A1至A33的33个5’UTR，和编号为B1至B14的14个3’UTR，基因来源和序列号如表1和表2所示。
表1.筛选获得的新3’UTR
表2.筛选获得的新5’UTR

实施例2. mRNA的制备
将实施例1筛选获得的UTR构建至用于体外转录的DNA载体中，以获得稳定表达5’UTR及3’UTR元件的mRNA。所述载体含有T7启动子，作为开放阅读框(ORF)的编码萤火虫荧光素酶(Firefly Luciferase，Fluc)的序列(SEQ ID NO:126)，和多聚腺苷酸(polyA)序列，polyA序列后接用于线性化载体的限制性位点。所述polyA选自A120(即，120个连续的腺苷酸)或A30L70(SEQ ID NO:52)。通过合适的酶切位点将5’UTR及3’UTR元件构建分别构建至开放阅读框ORF(Fluc)的5’端及3’端。构建好的载体示例如图1A至图1C，5’UTR和3’UTR的组合方式参见表3。
本实施例中所使用的对照载体包括polyA为A120Mod.-120A(SEQ ID NO:53)、polyA为A30L70的Mod.-A30L70(SEQ ID NO:54)和BioN.-A30L70(SEQ ID NO:55)，目的基因均为Fluc，通过全基因合成构建。不同UTR元件分别通过酶切的方法构建至上述载体，其中，载体V-B1至V-B14的构建载体为Mod.-120A， 选用酶切位点为AgeI和SacII，其polyA均为A120，其使用的对照为Mod.-120A(SEQ ID NO:53)；载体V-A1至V-A14的构建载体为Mod.-120A(SEQ ID NO:53)，选用酶切位点为BamHI和NheI，其polyA均为A120，其使用的对照也为Mod.-120A(SEQ ID NO:53)；V-A15至V-A33的构建载体为Mod.-A30L70(SEQ ID NO:54)，选用酶切位点为BamHI和NheI，其polyA均为A30L70，其使用的对照为Mod.-A30L70(SEQ ID NO:54)；载体V-A1-B12、V-A1-B13和V-A1-B14的构建载体为Mod.-A30L70(SEQ ID NO:54)，选用酶切位点为BamHI和SacII，其polyA均为A30L70,其使用的对照为Mod.-A30L70(SEQ ID NO:54)和BioN.-A30L70(SEQ ID NO:55)。插入的UTR元件基因片段均为全基因合成(苏州金唯智生物科技有限公司)，片段通过酶切连接构建至相应载体，载体构建完成后需进行酶切及测序鉴定，鉴定正确后用于下一步实验。
表3.载体中5’UTR和3’UTR的组合方式

将表3中的载体(即，DNA模板)通过限制性内切酶处理消化，使DNA模板线性化，再使用T7-RNA聚合酶体外转录。对于体外转录，使用T7RNA聚合酶(Roche)、相应的反应缓冲液、焦磷酸酶、RNase抑制剂和NTP。为了有效加帽RNA，向反应中加入过量的帽类似化合物ARCA(3’-O-Me-m7G(5’)ppp(5’)G，Trilink,N-7003-1)或CleanCap(m7G(5’)ppp(5’)(2’OMeA)pG，Trilink,N-7113)，其中图2至图5中所用的RNA生产选用ARCA进行加帽，图6至图9中所用的RNA选用CleanCap进行加帽。同时，为了降低mRNA的免疫源性并提高翻译效率，本公开中的mRNA均进行了核酸修饰，将反应体系中的尿苷-三磷酸(Uridine triphosphate，简称为UTP)全部替换为N1-甲基-假尿苷-三磷酸(N1-methyl-pseudouridine triphosphate，简称为1m-ψUTP，购于ThermoFisher)，该修饰方法参考专利US 2014/0194494 A1。体外转录体系在37℃下孵育2.5小时后，通过羧化磁珠(Invitrogen)来纯化RNA并重悬于无核酶水中，通过分光光度法和在5200生物分析仪(Agilent)上分析来评估RNA的浓度和质量。经检测，获得目的mRNA。
本公开Mod.的5’UTR至polyA的序列如SEQ ID NO:53、54所示，BioN.的5’UTR至polyA的序列如SEQ ID NO:55所示。Mod.中的5’UTR及3’UTR序列与 Moderna公司的专利及发表文献中的序列一致(US10849920B2、WO2013151667A1、US10730924B2、DOI:10.1016/j.cell.2017.02.017)，BioN.的5’UTR及3’UTR序列源自于专利WO 2018/160540。
示例性的给出V-B1线性化后序列，5’UTR至polyA的序列如SEQ ID NO:56所示；V-A1线性化后的序列，5’UTR至polyA的序列如SEQ ID NO:57所示。
实施例3. 5’UTR、3’UTR及其组合的功能验证
本实施例使用mRNA脂质转染的荧光素酶表达系统进行UTR的功能验证。
实验方法:将人胚胎肾细胞(HEK293，购自ATCC)以4×104个细胞/孔的密度接种于96孔板。次日，通过LipofectamineTM MessengerMAXTM mRNA转染试剂进行转染，每孔转染100ng前述实施例2中制备获得的加帽mRNA，转染6小时后，更换新鲜培养基。吸出100μL培养基并加入50μL荧光素酶底物，使用PerkinElmer多功能酶标仪检测发光强度。人宫颈癌细胞(HeLa，购自ATCC)和人肺癌细胞(A549，购自ATCC)的实验方法同HEK293。
评估方法:表4至表7、表9至表11中，将参比的阳性对照Mod.在不同细胞系中的荧光素表达量设置为1，计算不同mRNA相对阳性对照Mod.的表达量倍数。表8中，将参比的阳性对照BioN.在不同细胞系中的荧光素表达量设置为1，计算不同mRNA相对阳性对照BioN.的表达量倍数。表格中的数值均为检测平均值的倍数。
1)3’UTR在不同细胞系中调控目的基因表达能力的检测
为验证不同3’UTR在不同细胞系中调控目的基因的表达能力，用实施例2的方法生产表达荧光素酶(Fluc)的mRNA，将生产的RNA进行ARCA加帽，polyA尾结构为A120。将RNA转染至人HeLa、HEK293和A549细胞染，并在转染后24小时测量荧光素酶水平，结果参见表4和图2。
表4.携带不同3’UTR的mRNA表达荧光素酶(Fluc)的表达量(ARCA加帽，目的基因均为Fluc，polyA均为A120)

结果显示，在不同细胞系中，相较于Mod.阳性对照，B1至B14的3’UTR对蛋白表达量的影响具有一致性，不同细胞系间无显著性差异(p>0.05)，说明本公开筛选获得的3’UTR元件对蛋白表达量的影响具有普适性，能够用于提高目的基因的蛋白表达水平。
2)3’UTR在细胞中的调控目的基因表达时长的检测
为检测不同3’UTR在细胞中调控目的基因表达时间的能力，将表4中的mRNA转染至HEK293细胞中，并在转染后6h、24h、48h和72h测量荧光素酶表达水平，结果参见表5和图3。
表5.携带不同3’UTR的mRNA表达荧光素酶(Fluc)的表达量
结果显示，在HEK293细胞中，3’UTR元件的改变可影响目的蛋白的表达量，除B5-3’UTR元件，其他3’UTR元件均可提高蛋白表达量1.2倍以上，且在不同检测时间点均具有显著性差异(p<0.05)，其中B12、B13、B14的3’UTR元件效果最佳，可提高蛋白表达量2倍以上，且具有较好的时间延续性。
3)5’UTR在细胞中的调控目的基因表达的检测
为检测不同5’UTR在细胞中调控目的基因表达时间的能力，用前述方法制备 获得表达荧光素酶(Fluc)的mRNA，其中表6及图4中所使用的RNA为ARCA加帽，polyA尾结构为A120，表7及图5中所使用的RNA为CleanCap加帽，polyA尾结构为A30L70。将上述生产的RNA分别转染至HEK293细胞中，在转染后6h、24h、48h和72h测量荧光素酶表达水平，结果参见表6、表7和图4、图5。
表6.携带不同5’UTR的mRNA表达荧光素酶(Fluc)的表达量(ARCA加帽，目的基因均为Fluc，polyA均为A120)
表7.携带不同5’UTR的mRNA表达荧光素酶(Fluc)的表达量(CleanCap加帽，目的基因均为Fluc，polyA均为A30L70)

图4和图5分别通过不同的加帽方式和polyA尾结构进行筛选，结果显示，在不同的筛选载体中，5’UTR元件的改变均可影响目的蛋白的表达量，5’UTR元件对目标蛋白表达水平的提高具有通用性。其中，编号A1、A3至A7、A9至A16、A18、A21、A24、A27、A28、A32、A33的5’UTR元件，均可提高蛋白表达量1.2倍以上，且在24h、48h、72h的检测时间点均具有显著性差异(p<0.05)，其中A1、A15、A16、A18的5’UTR元件效果最佳，可提高蛋白表达量2倍以上，且具有较好的时间延续性。
4)候选5’UTR相较于阳性对照BioN.载体调控目的基因表达强度的检测
为检测不同候选5’UTR相较于阳性对照BioN.载体调控目的基因表达强度的能力，将前述筛选到的较佳的A1、A15、A16、A18的5’UTR元件构建至含有A30L70polyA尾元件的载体中，用前述方法生产表达荧光素酶(Fluc)的mRNA并转染至HEK293细胞中，在转染后6h、24h、48h和72h测量荧光素酶表达水平，结果参见表8和图6。
表8.携带不同5’UTR的mRNA表达荧光素酶(Fluc)的表达量(Cleancap加帽，目的基因均为Fluc，polyA均为A30L70)
结果显示，候选的5’UTR元件均相较于BioN.载体，在24h和48h时均可提高蛋白表达量至少高于1.2倍，且均具有显著性差异(p<0.05)。
5)不同5’UTR和3’UTR组合在细胞中的调控目的基因表达的检测
为了研究不同的5’-UTR和3’UTR组合对mRNA中目的蛋白表达的影响，将含有不同5’UTR和3’-UTR元件组合的mRNA与Moderna公司选用的5’UTR和3’UTR相比，结果参见表9和图7。
表9.携带不同5’UTR和3’UTR组合的mRNA的表达量 (Cleancap加帽，目的基因均为Fluc，polyA均为A30L70)
结果显示，本公开筛选的5’UTR和3’-UTR元件组合相较于Moderna公司选用的5’UTR和3’UTR元件，在24h、48h及72h检测时间点，均可提高蛋白表达量1.3倍以上，且均具有显著性差异(p<0.05)。以上1)-5)中的目的蛋白均为荧光素酶(Fluc)，以下6)中的目的蛋白为分泌型蛋白(例如hHGF，抗PD-1抗体)。
6)不同5’UTR和3’UTR组合调控分泌型目的蛋白表达的检测
为了研究不同的5’-UTR和3’UTR组合对mRNA表达分泌型蛋白的影响，这里将mRNA构建为ORF表达的目的蛋白为人肝细胞生长因子(Hepatocyte growth factor,hHGF)或抗PD-1抗体。
所述hHGF的氨基酸序列为SEQ ID NO:109所示，其DNA序列为SEQ ID NO:110所示，经密码子优化后获得mRNA OS1、OS2、OS3，其优化的密码子序列为SEQ ID NO:129-131所示。
所述PD-1抗体的重链和轻链的氨基酸序列分别为SEQ ID NO:117、118所示，重链和轻链的DNA序列分别为SEQ ID NO:119、120所示，重链和轻链的mRNA序列分别为SEQ ID NO:121、122所示。
首先，为了提高hHGF蛋白的翻译效率，本公开对野生型hHGF(SEQ ID NO:110)的核苷酸序列进行了密码子优化，优化后的序列有hHGF-OS1(SEQ ID NO:111)、hHGF-OS2(SEQ ID NO:112)和hHGF-OS3(SEQ ID NO:113)，相应的mRNA序列分别为SEQ ID NO:129-131所示。将上述天然hHGF及密码子序列优化后的hHGF序列构建至载体中，生产mRNA。通过ELISA检测hHGF蛋白的表达量，证明hHGF密码子优化序列能够提高hHGF蛋白的表达量。
将含有不同5’UTR和3’-UTR元件组合的表达hHGF-OS2的mRNA(对应的DNA序列为SEQ ID NO:112)，与含有Moderna公司的5’UTR和3’UTR元件组合的表达hHGF-OS2的mRNA Mod.(hHGF)(对应的DNA序列为SEQ ID NO:114)相比。用所述编码hHGF的mRNA转染HEK293细胞，在转染后6h、24h、48h和72h收集上清并通过ELISA检测hHGF蛋白的表达量，评估候选载体表达HGF的增加/延长。结果参见表10和图8A。
表10.携带不同5’UTR和3’UTR组合的mRNA的目的蛋白表达量(Cleancap加帽，目的基因均为hHGF，polyA均为A30L70)
将含有不同5’UTR和3’UTR元件组合的表达抗PD-1抗体全长的mRNA(对应的DNA序列为SEQ ID NO:124-125)，与含有Moderna公司的5’UTR和3’UTR元件组合的表达抗PD-1抗体的mRNA(对应的DNA序列为SEQ ID NO:123)相比。用编码抗PD-1抗体全长的mRNA转染HEK293细胞，在转染后6h、24h、48h和72h收集上清并通过ELISA检测抗PD-1抗体的表达量，评估候选载体表达抗PD-1抗体的增加/延长，结果参见表11和图8B。
表11.携带不同5’UTR和3’UTR组合的mRNA的目的蛋白表达量(Cleancap加帽，目的基因均为抗PD-1抗体的重链和轻链，polyA均为A30L70)
以上结果显示，持续表达分泌蛋白hHGF和抗PD-1抗体72小时后，本公开筛选得到的5’UTR和3’UTR组合相较于Moderna公司选用的5’UTR和3’UTR元件能够显著提高分泌蛋白的表达量(p<0.05)。
实施例4. 表达hHGF的mRNA在小鼠体内表达hHGF蛋白的验证
为了确定mRNA分子m-A16-B12(hHGF)(对应DNA序列为SEQ ID NO:127)在动物体内表达hHGF的能力，使用脂质纳米颗粒LNP(包含50mol％可离子化脂质(SM-102)，10mol％DSPC，38.5mol％胆固醇，1.5mol％PEG-DMG)进行mRNA的递送，以下实施例5和实施例6中也使用相同的脂质纳米颗粒LNP。阳性对照为Collategene质粒(AnGes公司)，通过裸质粒给药方式。Balb/c小鼠(6-8w，雄)随机分成4组，每组33只，对小鼠腓肠肌肌肉注射不同剂量的m-A16-B12(hHGF)及Collategene质粒，而后按表12方案安排如下各实验组：1)注射1.0μg/只的m-A16-B12(hHGF)；2)注射0.3μg/只的m-A16-B12(hHGF)；3)注射0.1μg/只的m-A16-B12(hHGF)；4)注射200μg/只的Collategene裸质粒。在1h、2h、4h、6h、24h、48h、72h、96h、168h、216h及336h获取小鼠腓肠肌标本，使用含蛋白酶抑制剂的RIPA裂解液将肌肉组织匀浆裂解，离心取上清，使用BCA蛋白浓度测定试剂盒测定hHGF蛋白浓度，使用ELISA方法评价hHGF的表达水平。各组数据用平均值±标准差(Mean±SD)表示，使用Graphpad Prism 9.0软件作图及统计。
表12.m-A16-B12(hHGF)及Collategene在体内表达hHGF的实验分组情况
实验结果如图9的A所示，m-A16-B12(hHGF)于肌肉注射1h后即可表达hHGF蛋白，注射后6h达到表达峰值，呈剂量依赖。如图9的B所示，Collategene于注射后7天达到表达峰值；m-A16-B12(hHGF)递送48h后蛋白表达量下降，1μg/只剂量m-A16-B12(hHGF)在72h时的蛋白表达量与Collategene相当。同时，对各组PK数据进行统计发现，即使是最低剂量0.1μg的m-A16-B12(hHGF)，其Cmax也是Collategene的5倍以上，且0.1μg的m-A16-B12(hHGF)其AUCinf(hr*pg/mg protein)也与Collategene相当(表13)。因此，m-A16-B12(hHGF)可实现hHGF在动物体内 的高效表达，且各时间点血浆中未检测到hHGF表达。
表13.m-A16-B12(hHGF)及Collategene在体内表达hHGF的结果
实施例5. 表达hHGF的mRNA对下肢缺血小鼠模型的治疗功能验证
下肢缺血小鼠模型是模拟人的严重下肢缺血的经典小鼠模型，建模使用6-8周的雄性Balb/c小鼠，方法如下：1)麻醉动物，取仰卧位放置在手术台上，后肢彻底脱毛，固定后肢，消毒手术部位皮肤；2)从膝盖向大腿内侧切开一个约1厘米长的皮肤切口，依次切开、解剖皮下脂肪组织，充分显露股动脉；3)使用显微弯镊，轻轻刺穿膜状股鞘，暴露神经血管包，分离股动脉与股静脉和神经处腹股沟附近的近端位置，分离干净后，在股动脉近端下方用6-0缝合线结扎股动脉近端；4)在靠近膝盖的远端将股动脉和股静脉分开，在远端下方用6-0缝合线结扎股动脉的末端在腘动脉的近端；5)缝合创口。
使用实施例4中的LNP作为载体递送m-A16-B12(hHGF)，Collategene以裸质粒形式给药。将下肢缺血模型小鼠随机分成4组，每组5只，分别对小鼠腓肠肌进行肌肉注射m-A16-B12(hHGF)和Collategene裸质粒，实验分组如下：1)注射500ng/只的m-A16-B12(hHGF)；2)注射50ng/只的m-A16-B12(hHGF)；3)注射200μg/只的Collategene裸质粒；4)注射等体积PBS作为对照组。造模当日计为第0天，在进行各实验组治疗后的第0天、第4天、第7天、第10天、第12天以及第14天观察腿部状况，使用血流仪检测小鼠下肢血流灌注情况，拍照记录。
按以下公式计算各组的血流灌注比率：血流灌注比率＝当日小鼠下肢血流灌注量/该小鼠第0天的下肢血流灌注量×100％；各组数据用平均值±标准差(Mean±SD)表示，使用Graphpad Prism 9.0软件作图及统计。
结果如图10A和图10B所示，在注射50ng/只、500ng/只的m-A16-B12(hHGF)和200ng/只的Collategene裸质粒进行治疗的情况下，各小鼠缺血下肢的血流灌注比率相对于对照组有明显的改善，且缺血下肢的血流灌注比率随时间的进展逐渐得到恢复。在其中，注射50ng/只m-A16-B12(hHGF)组表现出与注射200ng/只Collategene裸质粒组相近的血流恢复效果；尤其，注射500ng/只m-A16-B12(hHGF) 组显著优于200ng/只Collategene裸质粒组的血流恢复效果，可实现恢复至90％以上的血流灌注情况。
为了进一步确定m-A16-B12(hHGF)对下肢缺血小鼠模型下肢坏死程度的治疗效果，在给药之后的第14天，对小鼠的腿部状况进行观察，拍照记录，并通过如图11A的基准对小鼠的下肢坏死程度进行评分，此时使用的基准如下：0＝下肢自我脱离；1＝腿部坏死；2＝脚坏死；3＝＞2个脚趾变色；4＝1个脚趾变色；5＝＞2个指甲变色；6＝1个指甲变色；7＝无坏死。各组数据用平均值±标准差(Mean±SD)表示，使用Graphpad Prism 9.0软件作图及统计。
结果如图11B所示，在诱导小鼠下肢缺血后，对照组的小鼠在术后2周内，下肢发生明显的缺血坏死情况，至第14天，部分小鼠的下肢发生完全脱落；而在注射m-A16-B12(hHGF)和Collategene裸质粒治疗组中，各小鼠的下肢均能保持良好的完整性且并未出现下肢坏死情况，表明m-A16-B12(hHGF)具有与对照Collategene裸质粒相当的下肢缺血改善能力。
CD31是血管新生的中要标志物，为了确定m-A16-B12(hHGF)在促进下肢缺血小鼠模型血管新生中的作用，在给药后第14天，取小鼠缺血组织附近的腓肠肌样本，制备石蜡切片，通过CD31抗体对切片样本中的CD31进行免疫组化染色，拍照，经ImageJ(NIH)软件分析，计算CD31染色面积。各组数据用平均值±标准差(Mean±SD)表示，使用Graphpad Prism 9.0软件作图及统计。
结果如图12所示，在给予50ng/只、500ng/只的m-A16-B12(hHGF)和200ng/只的Collategene裸质粒治疗的情况下，均可显著促进各组缺血下肢肌肉中的血管新生，且新生血管数量相对PBS组具有明显的统计学差异(p<0.05)，50ng/只m-A16-B12(hHGF)组表现出与200ng/只的Collategene裸质粒相当的促血管新生作用，且m-A16-B12(hHGF)给药组表现出剂量依赖的促血管新生情况。
实施例6. 表达hHGF的mRNA对全皮层损伤Db/Db小鼠模型的治疗功能验证
Db/Db小鼠模型是经典的糖尿病小鼠模型，因其Leptin受体基因缺陷小鼠，致使小鼠随周龄增长表现出与糖尿病病人相似的高血糖、高血脂及胰岛素抵抗等特征。本实验通过全皮层损伤模型模拟糖尿病足患者难以愈合的皮肤损伤。上述Db/Db小鼠模型的制造方法如下:小Db/Db鼠麻醉后脱毛膏脱毛，75％酒精棉球消毒。使用直径8mm皮肤取材器biopunch在腰背部造成全层皮肤创伤。
为了确定m-A16-B12(hHGF)对全皮层损伤小鼠模型创面愈合的治疗效果，而用实施例4中的LNP递送m-A16-B12(hHGF)，对照Collategene以裸质粒形式给药，给药方式为分4点的皮下注射。将全皮层损伤模型的Db/Db小鼠按体重及血糖水平分成5组，每组7只。实验分组如下：1)注射500ng/只的m-A16-B12(hHGF)；2)注射200ng/只的m-A16-B12(hHGF)；3)注射50ng/只的m-A16-B12(hHGF)；4) 注射200μg/只的Collategene裸质粒；5)注射等体积PBS的对照组。造模当日计为第0天，在进行各实验组治疗后，分别在第0天、第3天、第5天、第7天、第10天、第12天以及第14天观察疮口恢复状况，对创伤部位进行拍照，图像经ImageJ(NIH)软件分析，计算创面面积。
按以下公式计算创面愈合百分率：P＝(AD-A0)/A0×100％(P：创口愈合百分率；AD：拍照日创口面积；A0：术后当日创口面积)。
各组创面愈合百分率用平均值±标准差(Mean±SD)表示，使用Graphpad Prism9.0软件作图及统计。
结果如图13所示，在注射50ng/只、200ng/只和500ng/只m-A16-B12(hHGF)和200μg/只Collategene裸质粒的处理下，相较于对照组，小鼠的创面均得到良好的愈合。而且，m-A16-B12(hHGF)对小鼠创面愈合情况的促进呈剂量依赖性，在第14天，50ng/只剂量的m-A16-B12(hHGF)创面愈合率达66％，200ng/只和500ng/只剂量的m-A16-B12(hHGF)能实现100％的伤口愈合情况；同时，m-A16-B12(hHGF)的促创面愈合情况显著优于200μg/只Collategene裸质粒(p<0.05)，即使是较低剂量(50ng/只)的m-A16-B12(hHGF)也表现出与200μg/只Collategene裸质粒具有显著性差异的创面愈合百分率(p<0.05)。
为了确定m-A16-B12(hHGF)对全皮层损伤小鼠模型组织重构的治疗效果，而通过马松染色评价上皮再生、组织重构情况。在给药之后第17天，获取小鼠全皮层标本，制备石蜡切片，使用马松染色对小鼠皮肤组织进行染色。
结果如图14所示，在m-A16-B12(hHGF)的治疗下，各组小鼠创面上皮均完成上皮全覆盖，且低剂量组创口处上皮异常增厚，胶原蛋白增生；中高剂量组上皮恢复至正常厚度，胶原蛋白有序排列，完成组织重构。而对照组及200μg/只Collategene裸质粒组均未完成上皮再覆盖，创口处胶原蛋白异常增生，结构紊乱。
序列表

































Claims (29)

  1. 核酸构建体，其包含:
    (a)开放阅读框(ORF)，和
    (b)非翻译区元件(UTR)，所述UTR包含如下序列：
    SEQ ID NO:12、26任一或与之具有至少90％同一性的序列，
    SEQ ID NO:1、15任一或与之具有至少90％同一性的序列，
    SEQ ID NO:4-5、18-19任一或与之具有至少90％同一性的序列，
    SEQ ID NO:6-8、20-22任一或与之具有至少90％同一性的序列，
    SEQ ID NO:9-11、23-25任一或与之具有至少90％同一性的序列，
    SEQ ID NO:13、27任一或与之具有至少90％同一性的序列，
    SEQ ID NO:14、28任一或与之具有至少90％同一性的序列，
    SEQ ID NO:29、30任一或与之具有至少90％同一性的序列，
    SEQ ID NO:35-37任一或与之具有至少90％同一性的序列，
    SEQ ID NO:42、43任一或与之具有至少90％同一性的序列，
    SEQ ID NO:46、47任一或与之具有至少90％同一性的序列，或
    SEQ ID NO:31-34、38-41、44-45任一或与之具有至少90％同一性的序列。
  2. 如权利要求1所述的核酸构建体，其中，(b)包含选自如下任一的3’非翻译区元件(3’UTR)和5’非翻译区元件(5’UTR)的组合：
    1)所述3’UTR含有SEQ ID NO:1所示或与之具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:15-47任一所示或与之任一具有至少90％同一性的序列；
    2)所述3’UTR含有SEQ ID NO:2所示或与之具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:15-47任一所示或与之任一具有至少90％同一性的序列；
    3)所述3’UTR含有SEQ ID NO:3所示或与之具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:15-47任一所示或与之任一具有至少90％同一性的序列；
    4)所述3’UTR含有SEQ ID NO:4所示或与之具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:15-47任一所示或与之任一具有至少90％同一性的序列；
    5)所述3’UTR含有SEQ ID NO:5所示或与之具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:15-47任一所示或与之任一具有至少90％同一性的序列；
    6)所述3’UTR含有SEQ ID NO:6所示或与之具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:15-47任一所示或与之任一具有至少90％同一性的序列；
    7)所述3’UTR含有SEQ ID NO:7所示或与之具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:15-47任一所示或与之任一具有至少90％同一性的序列；
    8)所述3’UTR含有SEQ ID NO:8所示或与之具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:15-47任一所示或与之任一具有至少90％同一性的序列；
    9)所述3’UTR含有SEQ ID NO:9所示或与之具有至少90％同一性的序列，所 述5’UTR含有SEQ ID NO:15-47任一所示或与之任一具有至少90％同一性的序列；
    10)所述3’UTR含有SEQ ID NO:10所示或与之具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:15-47任一所示或与之任一具有至少90％同一性的序列；
    11)所述3’UTR含有SEQ ID NO:11所示或与之具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:15-47任一所示或与之任一具有至少90％同一性的序列；
    12)所述3’UTR含有SEQ ID NO:12所示或与之具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:15-47任一所示或与之任一具有至少90％同一性的序列；
    13)所述3’UTR含有SEQ ID NO:13所示或与之具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:15-47任一所示或与之任一具有至少90％同一性的序列；
    14)所述3’UTR含有SEQ ID NO:14所示或与之具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:15-47任一所示或与之任一具有至少90％同一性的序列；
    15)所述3’UTR含有SEQ ID NO:1-14任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:15所示或与之具有至少90％同一性的序列；
    16)所述3’UTR含有SEQ ID NO:1-14任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:16所示或与之具有至少90％同一性的序列；
    17)所述3’UTR含有SEQ ID NO:1-14任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:17所示或与之具有至少90％同一性的序列；
    18)所述3’UTR含有SEQ ID NO:1-14任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:18所示或与之具有至少90％同一性的序列；
    19)所述3’UTR含有SEQ ID NO:1-14任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:19所示或与之具有至少90％同一性的序列；
    20)所述3’UTR含有SEQ ID NO:1-14任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:20所示或与之具有至少90％同一性的序列；
    21)所述3’UTR含有SEQ ID NO:1-14任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:21所示或与之具有至少90％同一性的 序列；
    22)所述3’UTR含有SEQ ID NO:1-14任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:22所示或与之具有至少90％同一性的序列；
    23)所述3’UTR含有SEQ ID NO:1-14任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:23所示或与之具有至少90％同一性的序列；
    24)所述3’UTR含有SEQ ID NO:1-14任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:24所示或与之具有至少90％同一性的序列；
    25)所述3’UTR含有SEQ ID NO:1-14任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:25所示或与之具有至少90％同一性的序列；
    26)所述3’UTR含有SEQ ID NO:1-14任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:26所示或与之具有至少90％同一性的序列；
    27)所述3’UTR含有SEQ ID NO:1-14任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:27所示或与之具有至少90％同一性的序列；
    28)所述3’UTR含有SEQ ID NO:1-14任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:28所示或与之具有至少90％同一性的序列；
    29)所述3’UTR含有SEQ ID NO:1-14任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:29所示或与之具有至少90％同一性的序列；
    30)所述3’UTR含有SEQ ID NO:1-14任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:30所示或与之具有至少90％同一性的序列；
    31)所述3’UTR含有SEQ ID NO:1-14任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:31所示或与之具有至少90％同一性的序列；
    32)所述3’UTR含有SEQ ID NO:1-14任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:32所示或与之具有至少90％同一性的序列；
    33)所述3’UTR含有SEQ ID NO:1-14任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:33所示或与之具有至少90％同一性的 序列；
    34)所述3’UTR含有SEQ ID NO:1-14任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:34所示或与之具有至少90％、95％同一性的序列；
    35)所述3’UTR含有SEQ ID NO:1-14任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:35所示或与之具有至少90％同一性的序列；
    36)所述3’UTR含有SEQ ID NO:1-14任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:36所示或与之具有至少90％同一性的序列；
    37)所述3’UTR含有SEQ ID NO:1-14任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:37所示或与之具有至少90％同一性的序列；
    38)所述3’UTR含有SEQ ID NO:1-14任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:38所示或与之具有至少90％同一性的序列；
    39)所述3’UTR含有SEQ ID NO:1-14任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:39所示或与之具有至少90％同一性的序列；
    40)所述3’UTR含有SEQ ID NO:1-14任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:40所示或与之具有至少90％同一性的序列；
    41)所述3’UTR含有SEQ ID NO:1-14任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:41所示或与之具有至少90％同一性的序列；
    42)所述3’UTR含有SEQ ID NO:1-14任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:42所示或与之具有至少90％同一性的序列；
    43)所述3’UTR含有SEQ ID NO:1-14任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:43所示或与之具有至少90％同一性的序列；
    44)所述3’UTR含有SEQ ID NO:1-14任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:44所示或与之具有至少90％同一性的序列；
    45)所述3’UTR含有SEQ ID NO:1-14任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:45所示或与之任一具有至少90％同一 性的序列；
    46)所述3’UTR含有SEQ ID NO:1-14任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:46所示或与之任一具有至少90％的序列；或
    47)所述3’UTR含有SEQ ID NO:1-14任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:47所示或与之任一具有至少90％同一性的序列。
  3. 如权利要求1所述的核酸构建体，其中，
    所述3’UTR包含SEQ ID NO:12-14任一所示或与之任一具有至少90％同一性的序列，所述5’UTR包含SEQ ID NO:15、29、30、32任一所示或与之任一具有至少90％同一性的序列；
    优选地，
    所述3’UTR包含SEQ ID NO:12所示或与之具有至少90％同一性的序列，所述5’UTR包含SEQ ID NO:15、29、30、32任一所示或与之具有至少90％同一性的序列，
    所述3’UTR包含SEQ ID NO:13所示或与之具有至少90％同一性的序列，所述5’UTR包含SEQ ID NO:15、29、30、32任一所示或与之具有至少90％同一性的序列，
    所述3’UTR包含SEQ ID NO:14所示或与之具有至少90％同一性的序列，所述5’UTR包含SEQ ID NO:15、29、30、32任一所示或与之具有至少90％同一性的序列，
    所述3’UTR包含SEQ ID NO:12-14任一所示或与之具有至少90％同一性的序列，所述5’UTR包含SEQ ID NO:15所示或与之具有至少90％同一性的序列，
    所述3’UTR包含SEQ ID NO:12-14任一所示或与之具有至少90％同一性的序列，所述5’UTR包含SEQ ID NO:29所示或与之具有至少90％同一性的序列，
    所述3’UTR包含SEQ ID NO:12-14任一所示或与之具有至少90％同一性的序列，所述5’UTR包含SEQ ID NO:30所示或与之具有至少90％同一性的序列，或
    所述3’UTR包含SEQ ID NO:12-14任一所示或与之具有至少90％同一性的序列，所述5’UTR包含SEQ ID NO:32所示或与之具有至少90％同一性的序列。
  4. 如权利要求1-3任一项所述的核酸构建体，其还包含:
    (c)多聚腺苷酸(poly-A)尾；
    优选地，所述poly-A尾选自A120、A30L70、HGH polyA、SV40polyA、BGH polyA、rbGlob polyA或SV40late polyA；
    优选地，所述poly-A尾选自A120或A30L70，所述A120包含120个腺嘌呤 核苷酸，所述A30L70包含SEQ ID NO:52所示或与之具有至少90％同一性的序列。
  5. 如权利要求1-4任一项所述的核酸构建体，其中，所述ORF编码肝细胞生长因子(HGF)、抗体或其抗原结合片段，优选为人肝细胞生长因子(hHGF)、抗PD-1抗体或其抗原结合片段。
  6. 如权利要求5所述的核酸构建体，所述ORF包含选自如下1)-2)中的任一项:
    1)编码如SEQ ID NO:109所示氨基酸序列的多核苷酸序列；
    2)如SEQ ID NO:110-113中任一项所示或与之具有至少90％同一性的多核苷酸序列；
    或者，所述ORF包含选自如下1)-3)的任一项:
    1)编码如SEQ ID NO:117所示重链氨基酸序列的多核苷酸序列，和/或编码如SEQ ID NO:118所示轻链氨基酸序列的多核苷酸序列；
    2)编码如SEQ ID NO:117所示重链氨基酸序列中HCDR1、HCDR2和HCDR3的多核苷酸序列，和编码如SEQ ID NO:118所示轻链氨基酸序列中LCDR1、LCDR2和LCDR3的多核苷酸序列，所述CDR是根据Kabat、IMGT、Chothia、AbM或Contact编号系统定义的，优选根据Kabat编号系统定义的；
    3)如SEQ ID NO:119所示或与之具有至少90％同一性的多核苷酸序列，和/或如SEQ ID NO:120所示或与之具有至少90％同一性的多核苷酸序列。
  7. 如权利要求5-6任一项所述的核酸构建体，其包含SEQ ID NO:115、116、127中任一所示或与之具有至少90％同一性的序列；或，包含SEQ ID NO:124所示或与之具有至少90％同一性的序列和/或SEQ ID NO:125所示或与之具有至少90％同一性的序列。
  8. RNA分子，其包含:
    (a)开放阅读框(ORF)，和
    (b)非翻译区元件(UTR)，所述UTR包含如下序列：
    SEQ ID NO:69、83任一或与之具有至少90％同一性的序列，
    SEQ ID NO:58、72任一或与之具有至少90％同一性的序列，
    SEQ ID NO:59-60、73-74任一或与之具有至少90％同一性的序列，
    SEQ ID NO:61-62、75-76任一或与之具有至少90％同一性的序列，
    SEQ ID NO:63-65、77-79任一或与之具有至少90％同一性的序列，
    SEQ ID NO:66-68、80-82任一或与之具有至少90％同一性的序列，
    SEQ ID NO:70、84任一或与之具有至少90％同一性的序列，
    SEQ ID NO:71、85任一或与之具有至少90％同一性的序列，
    SEQ ID NO:86、87任一或与之具有至少90％同一性的序列，
    SEQ ID NO:92-94任一或与之具有至少90％同一性的序列，
    SEQ ID NO:99、100任一或与之具有至少90％同一性的序列，
    SEQ ID NO:103、104任一或与之具有至少90％同一性的序列，或
    SEQ ID NO:88-91、95-98、101-102任一或与之具有至少90％同一性的序列。
  9. 如权利要求8所述的RNA分子，其中，(b)包含选自如下任一的3’非翻译区元件(3’UTR)和5’非翻译区元件(5’UTR)的组合：
    1)所述3’UTR含有SEQ ID NO:58所示或与之具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:72-104所示或与之任一具有至少90％同一性的序列；
    2)所述3’UTR含有SEQ ID NO:59所示或与之具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:72-104所示或与之任一具有至少90％同一性的序列；
    3)所述3’UTR含有SEQ ID NO:60所示或与之具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:72-104所示或与之任一具有至少90％同一性的序列；
    4)所述3’UTR含有SEQ ID NO:61所示或与之具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:72-104所示或与之任一具有至少90％同一性的序列；
    5)所述3’UTR含有SEQ ID NO:62所示或与之具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:72-104所示或与之任一具有至少90％同一性的序列；
    6)所述3’UTR含有SEQ ID NO:63所示或与之具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:72-104所示或与之任一具有至少90％同一性的序列；
    7)所述3’UTR含有SEQ ID NO:64所示或与之具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:72-104所示或与之任一具有至少90％同一性的序列；
    8)所述3’UTR含有SEQ ID NO:65所示或与之具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:72-104所示或与之任一具有至少90％同一性的序列；
    9)所述3’UTR含有SEQ ID NO:66所示或与之具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:72-104所示或与之任一具有至少90％同一性的序列；
    10)所述3’UTR含有SEQ ID NO:67所示或与之具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:72-104任一所示或与之任一具有至少90％同一性的序列；
    11)所述3’UTR含有SEQ ID NO:68所示或与之具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:72-104所示或与之任一具有至少90％同一性的序列；
    12)所述3’UTR含有SEQ ID NO:69所示或与之具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:72-104任一所示或与之任一具有至少90％同一性的序列；
    13)所述3’UTR含有SEQ ID NO:70所示或与之具有至少90％同一性的序列， 所述5’UTR含有SEQ ID NO:72-104任一所示或与之任一具有至少90％同一性的序列；
    14)所述3’UTR含有SEQ ID NO:71所示或与之具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:72-104任一所示或与之任一具有至少90％同一性的序列；
    15)所述3’UTR含有SEQ ID NO:58-71任一所示或与之具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:72所示或与之具有至少90％同一性的序列；
    16)所述3’UTR含有SEQ ID NO:58-71任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:73所示或与之具有至少90％同一性的序列；
    17)所述3’UTR含有SEQ ID NO:58-71任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:74所示或与之具有至少90％同一性的序列；
    18)所述3’UTR含有SEQ ID NO:58-71任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:75所示或与之具有至少90％同一性的序列；
    19)所述3’UTR含有SEQ ID NO:58-71任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:76所示或与之具有至少90％同一性的序列；
    20)所述3’UTR含有SEQ ID NO:58-71任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:77所示或与之具有至少90％同一性的序列；
    21)所述3’UTR含有SEQ ID NO:58-71任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:78所示或与之具有至少90％同一性的序列；
    22)所述3’UTR含有SEQ ID NO:58-71任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:79所示或与之具有至少90％同一性的序列；
    23)所述3’UTR含有SEQ ID NO:58-71任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:80所示或与之具有至少90％同一性的序列；
    24)所述3’UTR含有SEQ ID NO:58-71任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:81所示或与之具有至少90％同一性的序列；
    25)所述3’UTR含有SEQ ID NO:58-71任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:82所示或与之具有至少90％同一性的 序列；
    26)所述3’UTR含有SEQ ID NO:58-71任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:83所示或与之具有至少90％同一性的序列；
    27)所述3’UTR含有SEQ ID NO:58-71任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:84所示或与之具有至少90％同一性的序列；
    28)所述3’UTR含有SEQ ID NO:58-71任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:85所示或与之具有至少90％同一性的序列；
    29)所述3’UTR含有SEQ ID NO:58-71任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:86所示或与之具有至少90％同一性的序列；
    30)所述3’UTR含有SEQ ID NO:58-71任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:87所示或与之具有至少90％同一性的序列；
    31)所述3’UTR含有SEQ ID NO:58-71任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:88所示或与之具有至少90％同一性的序列；
    32)所述3’UTR含有SEQ ID NO:58-71任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:89所示或与之具有至少90％同一性的序列；
    33)所述3’UTR含有SEQ ID NO:58-71任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:90所示或与之具有至少90％同一性的序列；
    34)所述3’UTR含有SEQ ID NO:58-71任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:91所示或与之任一具有至少90％、95％同一性的序列；
    35)所述3’UTR含有SEQ ID NO:58-71任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:92所示或与之具有至少90％同一性的序列；
    36)所述3’UTR含有SEQ ID NO:58-71任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:93所示或与之具有至少90％同一性的序列；
    37)所述3’UTR含有SEQ ID NO:58-71任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:94所示或与之具有至少90％同一性的 序列；
    38)所述3’UTR含有SEQ ID NO:58-71任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:95所示或与之具有至少90％同一性的序列；
    39)所述3’UTR含有SEQ ID NO:58-71任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:96所示或与之具有至少90％同一性的序列；
    40)所述3’UTR含有SEQ ID NO:58-71任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:97所示或与之具有至少90％同一性的序列；
    41)所述3’UTR含有SEQ ID NO:58-71任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:98所示或与之具有至少90％同一性的序列；
    42)所述3’UTR含有SEQ ID NO:58-71任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:99所示或与之具有至少90％同一性的序列；
    43)所述3’UTR含有SEQ ID NO:58-71任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:100所示或与之具有至少90％同一性的序列；
    44)所述3’UTR含有SEQ ID NO:58-71任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:101所示或与之具有至少90％同一性的序列；
    45)所述3’UTR含有SEQ ID NO:58-71任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:102所示或与之具有至少90％同一性的序列；
    46)所述3’UTR含有SEQ ID NO:58-71任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:103所示或与之具有至少90％同一性的序列；或
    47)所述3’UTR含有SEQ ID NO:58-71任一所示或与之任一具有至少90％同一性的序列，所述5’UTR含有SEQ ID NO:104所示或与之具有至少90％同一性的序列。
  10. 如权利要求8所述的RNA分子，其中，
    所述3’UTR包含SEQ ID NO:69-71任一所示或与之具有至少90％同一性的序列，所述5’UTR包含SEQ ID NO:72、86、87、89任一所示或与之任一具有至少90％同一性的序列；
    优选地，
    所述3’UTR包含SEQ ID NO:69所示或与之具有至少90％同一性的序列，所述5’UTR包含SEQ ID NO:72、86、87、89任一所示或与之具有至少90％同一性的序列，
    所述3’UTR包含SEQ ID NO:70所示或与之具有至少90％同一性的序列，所述5’UTR包含SEQ ID NO:72、86、87、89任一所示或与之具有至少90％同一性的序列，
    所述3’UTR包含SEQ ID NO:71所示或与之具有至少90％同一性的序列，所述5’UTR包含SEQ ID NO:72、86、87、89任一所示或与之具有至少90％同一性的序列，
    所述3’UTR包含SEQ ID NO:69-71任一所示或与之具有至少90％同一性的序列，所述5’UTR包含SEQ ID NO:72所示或与之具有至少90％同一性的序列，
    所述3’UTR包含SEQ ID NO:69-71任一所示或与之具有至少90％同一性的序列，所述5’UTR包含SEQ ID NO:86所示或与之具有至少90％同一性的序列，
    所述3’UTR包含SEQ ID NO:69-71任一所示或与之具有至少90％同一性的序列，所述5’UTR包含SEQ ID NO:87所示或与之具有至少90％同一性的序列，或
    所述3’UTR包含SEQ ID NO:69-71任一所示或与之具有至少90％同一性的序列，所述5’UTR包含SEQ ID NO:89所示或与之具有至少90％同一性的序列。
  11. 如权利要求8-10任一项所述的RNA分子，其还包含:
    (c)多聚腺苷酸(poly-A)尾；
    优选地，所述poly-A尾选自A120、A30L70、HGH polyA、SV40polyA、BGH polyA、rbGlob polyA或SV40late polyA；
    优选地，所述poly-A尾选自A120或A30L70，所述A120包含120个腺嘌呤核苷酸，所述A30L70包含SEQ ID NO:52所示或与之具有至少90％、95％同一性的序列。
  12. 如权利要求8-11任一项所述的RNA分子，其中，所述ORF编码肝细胞生长因子(HGF)、抗体或其抗原结合片段，优选为人肝细胞生长因子(hHGF)、抗PD-1抗体或其抗原结合片段；
    优选地，所述ORF包含选自如下1)-2)中的任一项:
    1)编码如SEQ ID NO:109所示氨基酸序列的核酸序列；或者，
    2)如SEQ ID NO:128-131中任一所示或与之具有至少90％，优选至少95％同一性的RNA序列；或者，
    所述ORF包含选自如下1)-3)的任一项:
    1)编码如SEQ ID NO:117所示重链氨基酸序列的核酸序列，和/或编码如SEQ  ID NO:118所示轻链氨基酸序列的核酸序列；
    2)编码如SEQ ID NO:117所示重链氨基酸序列中HCDR1、HCDR2和HCDR3的核酸序列，和编码如SEQ ID NO:118所示轻链氨基酸序列中LCDR1、LCDR2和LCDR3的核酸序列，所述CDR是根据Kabat、IMGT、Chothia、AbM或Contact编号系统定义的，优选根据Kabat编号系统定义的；
    3)如SEQ ID NO:121所示或与之具有至少90％同一性的RNA序列，和/或如SEQ ID NO:122所示或与之具有至少90％同一性的RNA序列。
  13. 如权利要求8-12任一项所述的RNA分子，其包含:
    (d)5’帽结构(5’Cap)；
    优选地，所述5’Cap选自Cap0、Cap1、Cap2、Cap3、Cap4、ARCA、修饰的ARCA、肌苷、N1-甲基-鸟苷、2’-氟代-鸟苷、7-脱氮-鸟苷、8-氧代-鸟苷、2-氨基-鸟苷、LNA-鸟苷和2-叠氮基-鸟苷；
    更优选地，所述5’Cap选自ARCA、3’-O-Me-m7G(5’)ppp(5’)G、m7G(5’)ppp(5’)(2’OMeA)pU、m7Gppp(A2’O-MOE)pG、m7G(5’)ppp(5’)(2’OMeA)pG、m7G(5’)ppp(5’)(2’OMeG)pG、m7(3’OMeG)(5’)ppp(5’)(2’OMeG)pG或m7(3’OMeG)(5’)ppp(5’)(2’OMeA)pG。
  14. 如权利要求8-13任一项所述的RNA分子，其包含一种或多种修饰，优选地，所述修饰包括骨架修饰、糖修饰、碱基修饰和/或脂质修饰；更优选地，所述碱基修饰为尿嘧啶修饰。
  15. 如权利要求8-14任一项所述的RNA分子，其中，所述RNA分子用于提高所述ORF表达目标蛋白的表达量。
  16. 经分离的多核苷酸，其包含(a)开放阅读框(ORF)，所述ORF包含SEQ ID NO:111-113和129-131中任一所示或与之具有至少90％同一性的序列；
    优选地，所述多核苷酸包含如下(b)-(d)中的任一项或其任意组合:
    (b)5’UTR和/或3’UTR，
    (c)poly-A尾，
    (d)5’Cap；
    优选地，所述poly-A尾选自A120、A30L70、HGH polyA、SV40polyA、BGH polyA、rbGlob polyA或SV40late polyA；更优选地，所述poly-A尾为A120或A30L70；
    优选地，所述5’Cap选自ARCA、3’-O-Me-m7G(5’)ppp(5’)G、m7G(5’)ppp(5’)(2’OMeA)pU、m7Gppp(A2’O-MOE)pG、m7G(5’)ppp(5’)(2’OMeA)pG、 m7G(5’)ppp(5’)(2’OMeG)pG、m7(3’OMeG)(5’)ppp(5’)(2’OMeG)pG或m7(3’OMeG)(5’)ppp(5’)(2’OMeA)pG；
    优选地，所述多核苷酸包含选自骨架修饰、糖修饰、碱基修饰和/或脂质修饰的一种或多种修饰，其中，所述碱基修饰优选为假尿苷修饰。
  17. 核酸构建体，其包含:
    (a)开放阅读框(ORF)，和
    (b)非翻译区元件(UTR)，所述UTR源自CTSB、FAM166A、NDUFB9、ACTG1、CHCHD10、NDUFA11、ATP6V0B、ATP6V0E1、CFL1、COX4I1、CTSB、SLC38A2、NDUFV3、PRDX5、GUK1、IAH1、ABHD16A、SLC25A39、ATPIF1、ANAPC11、CCDC12、MRPL14或APOA1BP的UTR，所述UTR为3’非翻译区元件(3’UTR)或5’非翻译区元件(5’UTR)；
    优选地，所述UTR包含SEQ ID NO:1-47任一所示或与之具有至少90％同一性的序列。
  18. RNA分子，其包含:
    (a)开放阅读框(ORF)，和
    (b)非翻译区元件(UTR)，所述UTR源自CTSB、FAM166A、NDUFB9、ACTG1、CHCHD10、NDUFA11、ATP6V0B、ATP6V0E1、CFL1、COX4I1、CTSB、SLC38A2、NDUFV3、PRDX5、GUK1、IAH1、ABHD16A、SLC25A39、ATPIF1、ANAPC11、CCDC12、MRPL14或APOA1BP的UTR，所述UTR为3’非翻译区元件(3’UTR)或5’非翻译区元件(5’UTR)；
    优选地，所述UTR包含SEQ ID NO:58-104任一所示或与之具有至少90％同一性的序列。
  19. 载体，其包含权利要求1-7和17中任一项所述的核酸构建体、权利要求8-15和18中任一项所述的RNA分子，或权利要求16所述的多核苷酸。
  20. 宿主细胞，其包含权利要求19所述的载体。
  21. 如权利要求1-7和17中任一项所述的核酸构建体的制备方法，包括培养权利要求20所述的宿主细胞，并从培养物中回收产生的核酸构建体。
  22. 如权利要求8-15和18中任一项所述的RNA分子的制备方法，包括将权利要求1-7和17中任一项所述的核酸构建体，或权利要求19所述的载体进行逆转录，得到RNA分子；优选地，所述方法还包含对所述RNA分子的5’端添加5’Cap。
  23. 递送媒介物，其包含权利要求1-7和17中任一项所述的核酸构建体、权利要求8-15和18中任一项所述的RNA分子、权利要求16所述的多核苷酸，或权利要求19所述的载体；所述递送媒介物优选为阳离子脂质递送颗粒或纳米脂质颗粒。
  24. 药物组合物，其包含药学上可接受的载体、稀释剂或赋形剂，和选自如下的任一项或其任意组合:
    权利要求1-7和17中任一项所述的核酸构建体、权利要求8-15和18中任一项所述的RNA分子、权利要求16所述的多核苷酸、权利要求19所述的载体或权利要求23所述的递送媒介物。
  25. 产品或试剂盒，其包含选自如下的任一项或其任意组合:
    权利要求1-7和17中任一项所述的核酸构建体、权利要求8-15和18中任一项所述的RNA分子、权利要求19所述的多核苷酸、权利要求23所述的递送媒介物、权利要求24所述的药物组合物。
  26. 权利要求1-7和17中任一项所述的核酸构建体或权利要求8-15和18中任一项所述的RNA分子在用于提高ORF表达蛋白的表达量，或制备用于提高ORF表达蛋白的表达量的产品中的用途。
  27. 治疗和/或预防疾病的方法，包括向有需要的受试者施用治疗有效量的权利要求1-7和17中任一项所述的核酸构建体、权利要求8-15和18中任一项所述的RNA分子、权利要求16所述的多核苷酸、权利要求23所述的递送媒介物和/或权利要求24所述的药物组合物，所述疾病选自缺血性疾病、代谢综合征、糖尿病及其并发症、再狭窄，以及神经损伤；
    优选地，所述缺血性疾病选自冠状动脉疾病(CAD)、外周动脉疾病(PAD)、心肌梗死、肢体缺血、血栓闭塞性脉管炎(TAO)、糖尿病性动脉硬化闭塞症(DAO)；更优选地，所述肢体缺血为下肢缺血，最优选地，所述肢体缺血为严重下肢缺血(CLI)；
    优选地，所述糖尿病及其并发症选自糖尿病周围神经病变、糖尿病足(DFU)、糖尿病性动脉硬化闭塞症(DAO)；
    优选地，所述再狭窄选自手术后再狭窄、灌注后再狭窄；
    优选地，所述神经损伤选自神经退行性疾病、创伤性神经损伤、周围神经病变；更优选地，所述神经退行性疾病选自肌萎缩性侧索硬化(ALS)、帕金森氏病、痴呆病，所述周围神经病变为糖尿病周围神经病变。
  28. 促进内皮细胞生长和/或迁移的方法，包括向有需要的受试者施用有效量 的权利要求1-7和17中任一项所述的核酸构建体、权利要求8-15和18中任一项所述的RNA分子、权利要求16所述的多核苷酸、权利要求23所述的递送媒介物和/或权利要求24所述的药物组合物。
  29. 促血管发生的方法，包括向有需要的受试者施用有效量的权利要求1-7和17中任一项所述的核酸构建体、权利要求8-15和18中任一项所述的RNA分子、权利要求16所述的多核苷酸、权利要求23所述的递送媒介物和/或权利要求24所述的药物组合物。
PCT/CN2023/093851 2022-05-13 2023-05-12 包含utr的核酸构建体及其应用 WO2023217267A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210522056.8 2022-05-13
CN202210522056 2022-05-13

Publications (1)

Publication Number Publication Date
WO2023217267A1 true WO2023217267A1 (zh) 2023-11-16

Family

ID=88729768

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/093851 WO2023217267A1 (zh) 2022-05-13 2023-05-12 包含utr的核酸构建体及其应用

Country Status (2)

Country Link
TW (1) TW202400784A (zh)
WO (1) WO2023217267A1 (zh)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013151666A2 (en) * 2012-04-02 2013-10-10 modeRNA Therapeutics Modified polynucleotides for the production of biologics and proteins associated with human disease
US20170029847A1 (en) * 2013-12-30 2017-02-02 Curevac Ag Artificial nucleic acid molecules
US20180353618A1 (en) * 2015-12-09 2018-12-13 Modernatx, Inc. Heterologous UTR Sequences for Enhanced mRNA Expression
CN109535243A (zh) * 2019-01-07 2019-03-29 北京诺思兰德生物技术股份有限公司 人肝细胞生长因子突变体及其应用
CN112119162A (zh) * 2018-04-27 2020-12-22 加图立大学校产学协力团 插入表达调控序列的核酸分子、包含核酸分子的表达载体及其药物用途
CN113186203A (zh) * 2020-02-13 2021-07-30 斯微(上海)生物科技有限公司 治疗或者预防冠状病毒病的疫苗试剂
WO2022066757A1 (en) * 2020-09-23 2022-03-31 Myeloid Therapeutics, Inc. Improved methods and compositions for expression of nucleic acids in cells

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013151666A2 (en) * 2012-04-02 2013-10-10 modeRNA Therapeutics Modified polynucleotides for the production of biologics and proteins associated with human disease
US20170029847A1 (en) * 2013-12-30 2017-02-02 Curevac Ag Artificial nucleic acid molecules
US20180353618A1 (en) * 2015-12-09 2018-12-13 Modernatx, Inc. Heterologous UTR Sequences for Enhanced mRNA Expression
CN112119162A (zh) * 2018-04-27 2020-12-22 加图立大学校产学协力团 插入表达调控序列的核酸分子、包含核酸分子的表达载体及其药物用途
CN109535243A (zh) * 2019-01-07 2019-03-29 北京诺思兰德生物技术股份有限公司 人肝细胞生长因子突变体及其应用
CN113186203A (zh) * 2020-02-13 2021-07-30 斯微(上海)生物科技有限公司 治疗或者预防冠状病毒病的疫苗试剂
WO2022066757A1 (en) * 2020-09-23 2022-03-31 Myeloid Therapeutics, Inc. Improved methods and compositions for expression of nucleic acids in cells

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WANG LI, YANG GUODONG, FU HAIYAN, LU XIAOZHAO, WEI MENGYING, LUZFAN: "Construction of cyclooxygenase 2 mRNA 3'UTR luciferase reporter gene vector", CHINESE JOURNAL OF CELLULAR AND MOLECULAR IMMUNOLOGY., vol. 28, no. 6, 1 January 2012 (2012-01-01), pages 656 - 659, XP093106766, DOI: 10.13423/j.cnki.cjcmi.006454 *

Also Published As

Publication number Publication date
TW202400784A (zh) 2024-01-01

Similar Documents

Publication Publication Date Title
KR101710026B1 (ko) Cas9 단백질 및 가이드 RNA의 혼성체를 함유하는 나노 리포좀 전달체 조성물
RU2749882C2 (ru) Модулирующие полинуклеотиды
CN109640962A (zh) 编码松弛素的多核苷酸
KR102146815B1 (ko) Hmgb1 단편을 이용한 신규 심근경색의 치료법
JP2016539127A (ja) Ｃ／ｅｂｐアルファ組成物及び使用方法
EP3718561A1 (en) Therapeutic agent for inflammatory bowel disease
JP2020511470A (ja) リソソーム障害を治療する方法
KR20150102957A (ko) Hmgb1 단편을 이용한 척수 손상에 대한 신규 치료법
US20140051630A1 (en) Periostin-induced pancreatic regeneration
AU2003234840A1 (en) Decoy composition for treating and preventing inflammatory disease
JP2004099471A (ja) 心筋梗塞および心不全の治療薬
CN110734972B (zh) miR-181c-3p作为肾纤维化标志物的应用
WO2023217267A1 (zh) 包含utr的核酸构建体及其应用
JP7413629B2 (ja) Ｉｇｆ－１異型体を発現させるｄｎａコンストラクトを用いた神経病症の治療
CN109234381B (zh) miR-2682-5p作为肾纤维化标志物的应用
WO2022247740A1 (zh) 一种多肽及其在制备免疫调节药物中的应用
CN116019935A (zh) Ago2在制备治疗心衰或糖尿病性心肌病的药物方面的用途及其蛋白、基因、转化体
WO2022105903A1 (zh) 一种治疗肝纤维化的siRNA及其递送制剂
CN114432332A (zh) circUTRN在制备治疗心力衰竭药物中的应用、重组载体和治疗心力衰竭的药物
US20220362359A1 (en) Dna vaccine capable of effectively treating and/or preventing type 1 diabetes and use thereof
EP4188392A1 (en) Long non-coding rna as therapeutic target in cardiac disorders and cardiac regeneration
US20220133849A1 (en) Compositions and methods for the treatment of smooth muscle dysfunction
US10040837B2 (en) Nucleotide sequence and pharmaceutical composition based thereon with prolonged VEGF transgene expression
CN111808186A (zh) 一种人源性分泌型fndc5蛋白及其制备方法和用途
CN112813063A (zh) 脂代谢紊乱动物模型构建以及利用aav-crispr/cas9的修复

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23803035

Country of ref document: EP

Kind code of ref document: A1