Pharmaceutical composition comprising nucleic acid construct and medical use thereof

Abstract

A pharmaceutical composition comprising a nucleic acid construct and the medical use thereof. Specifically, the present invention relates to a lipid nanoparticle comprising a nucleic acid construct represented by formula I, which can achieve highly efficient delivery of a exogenous target gene, allowing the exogenous target gene to be highly efficiently and rapidly expressed in the body. The present invention has the advantages of no gene integration risk and being easy to scale up to an industrial level, is a more ideal treatment approach than naked plasmids, and can be used as a gene therapy drug for a plurality of diseases.

Description

A pharmaceutical composition containing a nucleic acid construct and its medical use Technical Field

[0001] The present disclosure relates to the field of pharmaceutical preparations, and in particular to a pharmaceutical composition containing a nucleic acid construct, and its medical use. Background Art

[0002] Gene therapy and gene vaccination can provide highly specific and personalized treatment and prevention options for a variety of diseases, including genetic diseases, autoimmune diseases, cancer or tumor-related diseases, and inflammatory diseases.

[0003] Both DNA and RNA can be used for gene therapy or gene vaccination. DNA is stable and easy to handle, but there is a risk that DNA fragments may be inserted into the patient's genome, leading to mutation events (such as loss of function of damaged genes). RNA can avoid undesirable genomic integration, but due to ubiquitous RNases, RNA is easily degraded. Therefore, it is necessary to improve the stability of RNA so that the protein products it encodes accumulate in the body to achieve treatment or prevention of diseases, as well as to maintain the integrity of RNA structure and function during storage and administration. It has been found that naturally occurring eukaryotic mRNA molecules contain stabilizing elements, for example, the untranslated regions (UTRs) at their 5' and 3' ends, as well as other structural features such as 5' cap structures or 3' poly(A) tails. 5' UTR and 3' UTR are premature mRNA elements. During the mRNA processing process, structural features unique to mature mRNA (such as 5' caps and 3' poly(A) tails) are added to the transcribed (premature) mRNA. Regarding the correlation between UTR and mRNA stability, previous studies have shown that the 3'UTR of α-globin mRNA is an important factor in α-globin mRNA stability (Nancy D Rodgers et al, RNA. 2002 Dec; 8(12): 1526-37; Z Wang et al, Mol Cell Biol. 1999 Jul; 19(7): 4552-60.).

[0004] Peripheral artery disease (PAD) refers to a noncoronary syndrome caused by structural and functional abnormalities in the arteries supplying the limbs, visceral organs, and brain. It is characterized by stenosis, occlusion, and tumor-like lesions in the noncoronary arterial circulation, affecting the aorta and its branches. Among PADs, lower limb ischemia is the most common clinically, with major etiologies including atherosclerosis (ASO), diabetic artery obliterans (DAO), and thromboangiitis obliterans (TAO). Critical limb ischemia (CLI) is a late stage of ASO, and diabetic foot ulcer (DFU) is a type of DAO. Both are peripheral vascular diseases characterized by lower limb pain, ulcers, and necrosis due to stenosis or occlusion of lower limb vessels and inadequate distal blood perfusion. Currently, effective treatments for CLI and DFU are revascularization through surgery or endovascular intervention. However, over 40% of patients, due to age, comorbidities, and other factors, do not meet the requirements for revascularization and must rely on conservative medication. Medication can only slow the progression of the disease, not cure it.

[0005] Hepatocyte growth factor (HGF) is a multifunctional mesenchymal growth factor. When it binds to the cell membrane surface receptor c-met, it causes intracellular tyrosine phosphorylation, recruits adaptor proteins, promotes kinase activity, and activates downstream signaling pathways. HGF is an important regulator of processes such as embryonic development, tissue and organ regeneration, wound healing, and angiogenesis. 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. Research results show that intramuscular injection of naked plasmid hHGF-cDNA can effectively promote blood perfusion in the ischemic hindlimbs of rats and rabbits (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. 2001 Feb; 8(3): 181-9). Clinical data show that naked plasmid injection into the calf muscle can promote wound healing in patients (S Cui et al. Clinical Safety and Preliminary Efficacy of Plasmid pUDK-HGF Expressing Human Hepatocyte Growth Factor (HGF) in Patients with Critical Limb Ischemia. Eur J Vasc Endovasc Surg. 2015 Oct; 50(4): 494-501.). However, the use of naked plasmids to deliver drugs has disadvantages such as low in vivo transfection efficiency, high dosage burden, high DNA integration risk, and high treatment costs. In addition, clinical data show that it does not improve the toe-brachial index, transcutaneous oxygen partial pressure, and amputation rate. Therefore, there is still much room for improvement in the drug.

[0006] 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 the efficiency of protein translation. The mRNA has higher stability and can be used in gene therapy and gene vaccination. In addition, the present disclosure provides mRNA capable of expressing human hepatocyte growth factor (hHGF) and its lipid nanoparticle (LNP) delivery system, which can achieve efficient and rapid conversion of exogenous hHGF protein in vivo, has the advantages of no integration risk and easy industrial-grade amplification, and is a more ideal treatment option than naked plasmids. It can be used as a gene therapy drug for various diseases such as CLI and DFU.

[0007] Summary of the Invention

[0008] The present disclosure provides a lipid nanoparticle comprising a nucleic acid construct and a lipid phase.

[0009] In some embodiments, the lipid phase comprises at least one cationic lipid.

[0010] In some embodiments, the cationic lipids described herein are selected from tertiary amine lipids.

[0011] In some embodiments, the cationic lipids described herein comprise a tertiary amine, an ester linker, and a branched tail.

[0012] In some embodiments, the cationic lipids described herein comprise a tertiary amine, an ester linker, and at least three branched tails.

[0013] In some embodiments, the cationic lipids described herein comprise a tertiary amine, an ester linker, and three branched tails.

[0014] In some embodiments, the cationic lipids described herein comprise a tertiary amine, an ester linker, and at least four branched tails.

[0015] In some embodiments, the branched tails described herein may be saturated or unsaturated hydrophobic tails containing 6 to 20 carbon atoms.

[0016] In some embodiments, the branched tails described herein may be saturated hydrophobic tails comprising 6 to 20 carbon atoms.

[0017] In some embodiments, the cationic lipids described herein comprise a free hydroxyl terminus.

[0018] In some embodiments, the cationic lipid described in the present disclosure is a compound represented by formula A,

[0019] Among them, L 1 and L 2 Each independently selected from -C(O)O-, -OC(O)-, -C(O)-, -OC(O)O-, -O-, -S(O) x -, -SS-, -C(O)S-, -SC(O)-, -NR a C(O)-、-C(O)NR a -、-NR a C(O)NR a -、-NR a C(O)O-、-OC(O)NR a - or key, R a Selected from hydrogen or C 1-6 Alkyl or C 2-6 alkenyl;

[0020] H 1 and H 2 Each independently selected from C 1-12 Heteroalkylene, C 1-12 Alkylene, C 2-12 alkenylene;

[0021] H 3 Selected from C 1-24 Alkylene, C 2-24 Alkenylene, C 3-8 Cycloalkylene or C 3-8 cycloalkenylene;

[0022] R 1 and R 2 Each independently selected from C 1-24 Alkyl or C 2-24 alkenyl;

[0023] R 3 Selected from hydrogen, -CN, -C(O)OR 4 、-OC(O)R 4 、-OR 5 or -NR 5 C(O)R 4 , R 4 Selected from C 1-6 Alkyl or C 2-6 Alkenyl, R 5 Selected from hydrogen, C 1-6 Alkyl or C 2-6 alkenyl;

[0024] x is selected from 0, 1 or 2.

[0025] In some embodiments, the cationic lipid described in the present disclosure is a compound represented by formula A,

[0026] Among them, L 1 and L 2 Each independently selected from -C(O)O-, -OC(O)-, -C(O)-, -OC(O)O-, -O-;

[0027] H 1 and H 2 Each independently selected from C 1-12 Heteroalkylene, C 1-12 Alkylene, C 2-12 alkenylene;

[0028] H 3 Selected from C 1-24 Alkylene, C 1-24 Heteroalkylene, C 2-24 alkenylene;

[0029] R 1 and R 2 Each independently selected from C 1-24 Alkyl or C 2-24 alkenyl;

[0030] R 3 Selected from hydrogen, -CN, -C(O)OR 4 、-OC(O)R 4 、-OR 5 or -NR 5 C(O)R 4 , R 4 Selected from C 1-6 Alkyl or C 2-6 Alkenyl, R 5 Selected from hydrogen, C 1-6 Alkyl or C 2-6 Alkenyl.

[0031] In some embodiments, the cationic lipid described in the present disclosure is a compound represented by formula A,

[0032] Among them, L 1 and L 2 Each independently selected from -C(O)O-, -OC(O)-;

[0033] H 1 and H 2 Each independently selected from C 1-12 Heteroalkylene, C 1-12 Alkylene, C 2-12 alkenylene;

[0034] H 3 Selected from C 1-24 Alkylene, C 1-24 Heteroalkylene, C 2-24alkenylene;

[0035] R 1 and R 2 Each independently selected from C 1-24 Alkyl or C 2-24 alkenyl;

[0036] R 3 Selected from hydrogen, -CN, -C(O)OR 4 、-OC(O)R 4 、-OR 5 or -NR 5 C(O)R 4 , R 4 Selected from C 1-6 Alkyl or C 2-6 Alkenyl, R 5 Selected from hydrogen, C 1-6 Alkyl or C 2-6 Alkenyl.

[0037] In some embodiments, the cationic lipid described in the present disclosure is a compound represented by formula A,

[0038] Among them, L 1 and L 2 Each independently selected from -C(O)O-, -OC(O)-;

[0039] H 1 and H 2 Each independently selected from C 1-12 Heteroalkylene, C 1-12 Alkylene, C 2-12 alkenylene;

[0040] H 3 Selected from C 1-24 Alkylene, C 1-24 Heteroalkylene, C 2-24 alkenylene;

[0041] R 1 and R 2 Each independently selected from C 1-24 Alkyl or C 2-24 alkenyl;

[0042] R 3 Select from -OR 5 , R 5 Selected from hydrogen.

[0043] In some embodiments, H in the compound represented by Formula A or its salt is 1 and H 2 At least one of the groups is a heteroalkylene group, wherein the heteroalkylene group contains at least one heteroatom selected from O, N and S.

[0044] In some embodiments, H in the compound represented by Formula A or its salt is 1 Selected from C 1-12 Heteroalkylene, preferably C 2-9 In some embodiments, the heteroalkylene group is a heteroalkylene group containing at least one oxygen atom. In some embodiments, the heteroalkylene group is a heteroalkylene group containing two oxygen atoms. In some embodiments, the heteroalkylene group is a heteroalkylene group containing at least one nitrogen atom. In some embodiments, the heteroalkylene group is a heteroalkylene group containing at least one oxygen atom.

[0045] In some embodiments, H in the compound represented by Formula A or its salt is 1 and H 2 At least one of them is C 1-9 Alkylene, C 2-10 Alkenylene.

[0046] In some embodiments, H in the compound represented by Formula A or its salt is 3 Selected from C 2-24 Alkenylene; or H 3 Selected from C 1-24 In other embodiments, H 3 Selected from C 1-24 Heteroalkylene.

[0047] In some embodiments, H in the compound represented by Formula A or its salt is 3 Selected from C 1-8 In some embodiments, H in the compound represented by Formula A or its salt 3 Selected from C 2-6 Alkylene.

[0048] In some embodiments, L in the compound represented by Formula A or its salt 1 and L 2 Selected from -C(O)O-.

[0049] In some embodiments, L in the compound represented by Formula A or its salt 1 and L 2 Selected from -OC(O)-.

[0050] In some embodiments, R 1 and R 2 Each independently selected from C 2-24 Alkyl (including but not limited to C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, C 10 Alkyl, C 11 Alkyl, C 12 Alkyl, C13 Alkyl, C 14 Alkyl, C 15 Alkyl, C 16 Alkyl, C 17 Alkyl, C 18 Alkyl, C 19 Alkyl, C 20 Alkyl, C 21 In other embodiments, R 1 and R 2 Each independently selected from C 4-18 alkyl.

[0051] In some embodiments, R 1 and R 2 Each independently selected from branched C 4-18 alkyl.

[0052] In some embodiments, R 1 and R 2 Each independently selected from a linear C 4-18 alkyl.

[0053] In some embodiments, R 1 Selected from straight chain C 4-18 Alkyl, R 2 Selected from branched C 4-18 alkyl.

[0054] In some embodiments, R 1 Selected from branched C 4-18 Alkyl, R 2 Selected from branched C 4-18 alkyl.

[0055] Some embodiments provide compounds represented by formula A or salts thereof wherein R 1 and R 2 Each independently selected from C 2-24 Alkenyl (including but not limited to C2 alkenyl, C3 alkenyl, C4 alkenyl, C5 alkenyl, C6 alkenyl, C7 alkenyl, C8 alkenyl, C9 alkenyl, C 10 Alkenyl, C 11 Alkenyl, C 12 Alkenyl, C 13 Alkenyl, C 14 Alkenyl, C 15 Alkenyl, C 16 Alkenyl, C 17 Alkenyl, C 18 Alkenyl, C 19 Alkenyl, C 20 Alkenyl, C21 In some other embodiments, the compound represented by formula A or its salt is R 1 and R 2 Each independently selected from C 4-18 Alkenyl.

[0056] In some embodiments, R 1 and R 2 Each independently selected from branched C 4-18 Alkenyl.

[0057] In some embodiments, R 1 and R 2 Each independently selected from a linear C 4-18 Alkenyl.

[0058] In some embodiments, R 1 Selected from straight chain C 4-18 Alkenyl, R 2 Selected from branched C 4-18 Alkenyl.

[0059] In some embodiments, R 1 Selected from branched C 4-18 Alkenyl, R 2 Selected from branched C 4-18 Alkenyl.

[0060] In some embodiments, the cationic lipids of the present disclosure may be selected from N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 1,2-dioleoyltrimethylpropane ammonium chloride (DOTAP) (also known as N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride and 1,2-dioleoyloxy-3-trimethylaminopropane chloride salt), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-γ- Linenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyl-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyl-3-morpholinylpropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyl-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyl-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazinyl)propane (DLin-MPZ) or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or its analogs, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-yl)-1,2-diol -amine, (6Z,9Z,28Z,31Z)-heptahexaenoic acid-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (C12-200), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-DMA), (6Z,9Z,28Z,31Z)-heptahexaenoic acid-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (C12-200), 31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-M-C3-DMA), 3-((6Z,9Z,28Z,31Z)-heptahexanoic acid-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropane-1-amine (MC3Ether), 4-((6Z,9Z,28Z,31Z)-heptahexanoic acid-6,9,28,31-tetraen-19 -yloxy)-N,N-dimethylbutane-1-amine (MC4Ether), ((4-hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoic acid 1-octylnonyl ester (SM-102), the compound represented by formula I, or any combination of the above cationic lipids.

[0061] In some embodiments, the cationic lipid described in the present disclosure is a compound represented by Formula I.

[0062] In some embodiments, the cationic lipid described in the present disclosure is ((4-hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315).

[0063] In some embodiments, the cationic lipid of the present disclosure is 1-octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoate (SM-102).

[0064] In some embodiments, the cationic lipids of the present disclosure comprise 10-75% by mole of the total lipid present in the lipid nanoparticles, including but not limited to 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, or any value in between.

[0065] In some embodiments, the cationic lipids of the present disclosure comprise 15-49% of the molar amount of total lipids present in the lipid nanoparticles, including but not limited to 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or any value therebetween.

[0066] In some embodiments, the cationic lipids described herein comprise 20-70% by mole of the total lipids present in the lipid nanoparticles.

[0067] In some embodiments, the cationic lipids described herein comprise 30-70% by mole of the total lipids present in the lipid nanoparticles.

[0068] In some embodiments, the cationic lipids described herein comprise 20-60% by mole of the total lipids present in the lipid nanoparticles.

[0069] In some embodiments, the cationic lipids described herein comprise 30-60% by mole of the total lipids present in the lipid nanoparticles.

[0070] In some embodiments, the cationic lipids of the present disclosure comprise 42-49% by mole of the total lipids present in the lipid nanoparticles.

[0071] In some embodiments, the cationic lipids of the present disclosure comprise 45% by mole of the total lipids present in the lipid nanoparticles.

[0072] In some embodiments, the cationic lipids of the present disclosure comprise 46% by mole of the total lipids present in the lipid nanoparticles.

[0073] In some embodiments, the cationic lipids of the present disclosure comprise 47% by mole of the total lipids present in the lipid nanoparticles.

[0074] In some embodiments, the cationic lipids of the present disclosure comprise 48% by mole of the total lipids present in the lipid nanoparticles.

[0075] In some embodiments, the cationic lipids of the present disclosure comprise 49% by mole of the total lipids present in the lipid nanoparticles.

[0076] In some embodiments, the cationic lipids of the present disclosure comprise 5-40% of the molar amount of total lipids present in the lipid nanoparticles, including but not limited to 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, or any value therebetween.

[0077] In some embodiments, the cationic lipids of the present disclosure comprise 40-70% of the molar amount of total lipids present in the lipid nanoparticles, including but not limited to 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, or any value therebetween.

[0078] In some embodiments, the lipid phase described herein comprises at least one non-cationic lipid.

[0079] In some embodiments, the non-cationic lipids of the present disclosure comprise 20-70% of the molar amount of total lipids present in the lipid nanoparticles, including but not limited to 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, or any value therebetween.

[0080] In some embodiments, the non-cationic lipids described herein comprise 50-65% by mole of the total lipids present in the lipid nanoparticles.

[0081] In some embodiments, the non-cationic lipids of the present disclosure comprise 40-55% by mole of the total lipids present in the lipid nanoparticles.

[0082] In some embodiments, the non-cationic lipids described herein comprise 55-62% of the molar amount of the total lipids present in the lipid nanoparticles.

[0083] In some embodiments, the non-cationic lipids of the present disclosure comprise 30-60% by mole of the total lipids present in the lipid nanoparticles.

[0084] In some embodiments, the non-cationic lipids described herein comprise 30-50% by mole of the total lipids present in the lipid nanoparticles.

[0085] In some embodiments, the non-cationic lipids of the present disclosure comprise 20-50% by mole of the total lipids present in the lipid nanoparticles.

[0086] In some embodiments, the non-cationic lipids of the present disclosure comprise 20-40% by mole of the total lipids present in the lipid nanoparticles.

[0087] In some embodiments, the non-cationic lipids described in the present disclosure may be selected from a mixture of phospholipids and cholesterol or its derivatives.

[0088] In some embodiments, the phospholipids described in the present disclosure can be selected from 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diondecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16Lyso PC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-bisdocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.

[0089] In some embodiments, the phospholipid described herein is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0090] In some embodiments, the phospholipid described herein is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0091] In some embodiments, the weight ratio of the phospholipids to the cationic lipids disclosed herein is 1:20 to 10:1; preferably, the weight ratio of the phospholipids to the cationic lipids is 1:20 to 5:1; preferably, the weight ratio of the phospholipids to the cationic lipids is 1:10 to 5:1; more preferably, the weight ratio of the phospholipids to the cationic lipids is 1:10 to 1:1.

[0092] In some embodiments, the phospholipids described in the present disclosure are present in an amount of 5-40% by mole of the total lipid present in the lipid nanoparticles, including but not limited to 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, or any value therebetween.

[0093] In some embodiments, the phospholipids described herein are present in an amount of 5-20% by mole of the total lipids present in the lipid nanoparticles.

[0094] In some embodiments, the phospholipids described herein are present in an amount of 15% by mole of the total lipids present in the lipid nanoparticles.

[0095] In some embodiments, the phospholipids described herein are present in an amount of 20% by mole of the total lipids present in the lipid nanoparticles.

[0096] In some embodiments, the phospholipids described herein are present in an amount of 10-20% by mole of the total lipids present in the lipid nanoparticles.

[0097] In some embodiments, the phospholipids described herein are present in an amount of 10% by mole of the total lipids present in the lipid nanoparticles.

[0098] In some embodiments, cholesterol derivatives include but are not limited to sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid.

[0099] In another aspect, the cholesterol or derivatives thereof described in the present disclosure comprise 20-60% by mole of the total lipid present in the lipid nanoparticles, including but not limited to 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, or any value therebetween.

[0100] In some embodiments, the cholesterol or its derivatives described herein are present in an amount of 30-60% by mole of the total lipids present in the lipid nanoparticles.

[0101] In some embodiments, the cholesterol or its derivatives described herein are present in an amount of 20-45% by mole of the total lipids present in the lipid nanoparticles.

[0102] In some embodiments, the cholesterol or its derivatives described herein are present in an amount of 35-45% by mole of the total lipids present in the lipid nanoparticles.

[0103] In some embodiments, the cholesterol or its derivatives described herein are present in an amount of 20-35% by mole of the total lipids present in the lipid nanoparticles.

[0104] In some embodiments, the cholesterol or derivatives thereof described herein is present in an amount of 40.5% by mole of the total lipids present in the lipid nanoparticles.

[0105] In some embodiments, the cholesterol or derivatives thereof described herein is present in an amount of 45% by mole of the total lipid present in the lipid nanoparticles.

[0106] In some embodiments, the molar ratio of cholesterol or its derivatives to phospholipids described in the present disclosure is 1.0-5.0, including but not limited to 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0 or any value therebetween.

[0107] In some embodiments, the molar ratio of cholesterol or its derivatives to phospholipids in the lipid nanoparticles described herein is 2.0 to 5.0.

[0108] On the other hand, the lipid phase described in the present disclosure further contains at least one conjugated lipid, and the conjugated lipid includes but is not limited to PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0109] In some embodiments, the conjugated lipid is selected from distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000) and methoxy polyethylene glycol ditetradecanoyl acetamide (ALC-0159).

[0110] In some embodiments, the conjugated lipid is selected from dimyristoylglycerol-3-methoxypolyethylene glycol 2000.

[0111] In some embodiments, the conjugated lipid is selected from distearoylphosphatidylethanolamine polyethylene glycol 2000.

[0112] In some embodiments, the conjugated lipid acts to inhibit lipid nanoparticle (particle) aggregation.

[0113] In some embodiments, the conjugated lipids described herein are present in an amount of 0.5-4% by mole of the total lipid present in the lipid nanoparticles, including but not limited to 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.2%, 1.4%, 1.6%, 1.8%, 2.0%, 2.2%, 2.4%, 2.6%, 2.8%, 3.0%, 3.2%, 3.4%, 3.6%, 3.8%, 4.0%, or any value therebetween.

[0114] In some embodiments, the conjugated lipids described herein are present in an amount of 0.5-1.0% by mole of the total lipids present in the lipid nanoparticles.

[0115] In some embodiments, the conjugated lipids described herein are present in an amount of 1.5-2.5% by mole of the total lipids present in the lipid nanoparticles.

[0116] In some embodiments, the conjugated lipids described herein are present in an amount of 1-2% by mole of the total lipids present in the lipid nanoparticles.

[0117] In some embodiments, the conjugated lipids described herein are present in an amount of 1.5% by mole of the total lipids present in the lipid nanoparticles.

[0118] In some embodiments, the conjugated lipids described herein are present in an amount of 2.0% by mole of the total lipids present in the lipid nanoparticles.

[0119] In some embodiments, the weight ratio of the conjugated lipid to the cationic lipid is 1:100 to 1:1; preferably, the weight ratio of the conjugated lipid to the cationic lipid is 1:100 to 1:2; preferably, the weight ratio of the conjugated lipid to the cationic lipid is 1:50 to 1:1; preferably, the weight ratio of the conjugated lipid to the cationic lipid is 1:50 to 1:2; preferably, the weight ratio of the conjugated lipid to the cationic lipid is 1:25 to 1:2; preferably, the weight ratio of the conjugated lipid to the cationic lipid is 1:20 to 1:2.

[0120] In some embodiments, the lipid nanoparticles described herein comprise:

[0121] a) nucleic acid construct;

[0122] b) a cationic lipid, wherein the cationic lipid comprises a compound represented by formula I or a pharmaceutically acceptable salt thereof, and the cationic lipid accounts for 10 to 75% of the total lipid molar amount present in the lipid nanoparticles,

[0123] c) a non-cationic lipid selected from the group consisting of a phospholipid and cholesterol or a derivative thereof, wherein the phospholipid accounts for 5 to 40% by mole of the total lipid present in the lipid nanoparticles, and the cholesterol or a derivative thereof accounts for 15 to 60% by mole of the total lipid present in the lipid nanoparticles;

[0124] and d) a conjugated lipid, said conjugate comprising 0.5-2% by mole of the total lipid present in the lipid nanoparticle.

[0125] In some embodiments, the lipid nanoparticles described herein comprise:

[0126] a) nucleic acid construct;

[0127] b) a cationic lipid comprising a compound of formula I or a pharmaceutically acceptable salt thereof,

[0128] The ionic lipids constitute 20–70% of the total molar amount of lipids present in the lipid nanoparticles;

[0129] c) a non-cationic lipid selected from the group consisting of a phospholipid and cholesterol or a derivative thereof, wherein the phospholipid accounts for 5 to 40% by mole of the total lipid present in the lipid nanoparticles, and the cholesterol or a derivative thereof accounts for 15 to 60% by mole of the total lipid present in the lipid nanoparticles;

[0130] and d) a conjugated lipid, said conjugate comprising 0.5-2% by mole of the total lipid present in the lipid nanoparticle.

[0131] In some embodiments, the lipid nanoparticles described herein comprise:

[0132] a) nucleic acid construct;

[0133] b) a cationic lipid comprising a compound of Formula I or a pharmaceutically acceptable salt thereof, wherein the cationic lipid accounts for 10 to 50% by molar amount of the total lipid present in the lipid nanoparticles;

[0134] c) a non-cationic lipid selected from the group consisting of a phospholipid and cholesterol or a derivative thereof, wherein the phospholipid accounts for 5 to 40% by mole of the total lipid present in the lipid nanoparticles, and the cholesterol or a derivative thereof accounts for 30 to 60% by mole of the total lipid present in the lipid nanoparticles;

[0135] and d) a conjugated lipid, said conjugate comprising 0.5-2% by mole of the total lipid present in the lipid nanoparticle.

[0136] On the other hand, the nanoparticle preparation described in the present disclosure has an average particle size of 10 nm to 200 nm.

[0137] In some embodiments, the nanoparticle formulation has an average particle size of 40 to 150 nm.

[0138] In some embodiments, the nanoparticle formulation has an average particle size of 40 to 140 nm.

[0139] On the other hand, the present disclosure also provides a lyophilized composition comprising the aforementioned lipid nanoparticles.

[0140] In some embodiments, the lyophilized formulation described in the present disclosure is obtained by freeze-drying the aforementioned lipid nanoparticles or the aforementioned lyophilized composition.

[0141] In some embodiments, the lyophilized composition or formulation described in the present disclosure comprises the above-mentioned lipid nanoparticles and sodium chloride.

[0142] In some embodiments, in the lyophilized composition or formulation described in the present disclosure, the weight ratio of sodium chloride to cationic lipid is 1:10 to 10:1.

[0143] In some embodiments, in the lyophilized composition or formulation described in the present disclosure, the weight ratio of sodium chloride to cationic lipid is 1:10 to 5:1.

[0144] In some embodiments, in the lyophilized composition or formulation described in the present disclosure, the weight ratio of sodium chloride to cationic lipid is 1:5 to 10:1.

[0145] In some embodiments, in the lyophilized composition or formulation described in the present disclosure, the weight ratio of sodium chloride to cationic lipid is 1:5 to 5:1.

[0146] In some embodiments, in the lyophilized composition or formulation of the present disclosure, the weight ratio of sodium chloride to cationic lipid is 1:3 to 5:1.

[0147] In some embodiments, in the lyophilized composition or formulation of the present disclosure, the weight ratio of sodium chloride to cationic lipid is 1:2 to 5:1.

[0148] In some embodiments, in the lyophilized composition or formulation described in the present disclosure, the weight ratio of sodium chloride to cationic lipid is 1:2 to 4:1.

[0149] In some embodiments, in the lyophilized composition or formulation of the present disclosure, the weight ratio of sodium chloride to cationic lipid is 1:5 to 3:1.

[0150] In some embodiments, in the lyophilized composition or formulation described in the present disclosure, the weight ratio of sodium chloride to cationic lipid is 1:3 to 3:1.

[0151] In some embodiments, in the lyophilized composition or formulation described in the present disclosure, the weight ratio of sodium chloride to cationic lipid is 1:3 to 2:1.

[0152] In some embodiments, in the lyophilized composition or formulation described herein, the weight ratio of sodium chloride to cationic lipid is 1:2 to 2:1.

[0153] In another aspect, the lyophilized composition or formulation of the present disclosure further comprises at least one lyoprotectant.

[0154] In some embodiments, the lyoprotectant is selected from one or more of sucrose, trehalose, mannitol, and maltose; preferably, the lyoprotectant is selected from sucrose, or a combination of sucrose and trehalose.

[0155] In some embodiments, the lyoprotectant is sucrose.

[0156] In some embodiments, the lyoprotectant is a combination of trehalose and mannitol;

[0157] In some embodiments, the weight ratio of the lyoprotectant to the cationic lipid is 200:1 to 1:1; preferably, the weight ratio of the lyoprotectant to the cationic lipid is 150:1 to 1:1; preferably, the weight ratio of the lyoprotectant to the cationic lipid is 100:1 to 1:1; preferably, the weight ratio of the lyoprotectant to the cationic lipid is 100:1 to 20:1; preferably, the weight ratio of the lyoprotectant to the cationic lipid is 800:1 to 20:1; preferably, the weight ratio of the lyoprotectant to the cationic lipid is 50:1 to 20:1.

[0158] In some embodiments, the lyophilized composition or formulation of the present disclosure comprises a lyoprotectant at a concentration of 5% w / v to 20% w / v, preferably 5% w / v to 15% w / v.

[0159] In some embodiments, the lyophilized composition of the present disclosure comprises sucrose at a concentration of 5% w / v to 20% w / v, preferably 5% w / v to 15% w / v, preferably 5% w / v to 10% w / v, and preferably 8% w / v.

[0160] In some embodiments, the lyophilized composition or formulation of the present disclosure comprises trehalose at a concentration of 5% w / v to 20% w / v, preferably 5% w / v to 15% w / v.

[0161] In some embodiments, the lyophilized composition or formulation of the present disclosure comprises sucrose at a concentration of 5% w / v to 10% w / v and trehalose at a concentration of 1% w / v to 10% w / v; preferably sucrose at a concentration of 5% w / v to 10% w / v and trehalose at a concentration of 2% w / v to 8% w / v.

[0162] In another aspect, the lyophilized composition or formulation of the present disclosure further comprises a buffer.

[0163] In some embodiments, the buffer is selected from (N-morpholino)propanesulfonic acid (MOPS), 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), tris(hydroxymethylaminomethane) (TRIS), 4-morpholinoethanesulfonic acid (MES), citrate, and phosphate buffered saline (PBS).

[0164] In some embodiments, the buffer is TRIS.

[0165] In some embodiments, the concentration of the buffer is from about 10 mM to about 100 mM; preferably, the concentration of the buffer is from about 15 mM to about 75 mM; more preferably, the concentration of the buffer is from about 10 mM to about 40 mM.

[0166] In some embodiments, the lyophilized compositions or formulations described herein comprise:

[0167] i. nucleic acid construct;

[0168] ii. sodium chloride, the weight ratio of sodium chloride to cationic lipid is 1:5 to 10:1;

[0169] iii. a cationic lipid comprising a compound of formula I or a pharmaceutically acceptable salt thereof, wherein the cationic lipid accounts for 10 to 75% of the total molar amount of lipid present in the lipid nanoparticles,

[0170] iv. a non-cationic lipid selected from a phospholipid and cholesterol or a derivative thereof, wherein the phospholipid accounts for 5 to 40% by mole of the total lipid present in the lipid nanoparticles and the cholesterol or a derivative thereof accounts for 15 to 60% by mole of the total lipid present in the lipid nanoparticles; and

[0171] v. conjugated lipids, wherein the conjugates account for 0.5-2% of the total lipid molar amount present in the lipid nanoparticles. In some embodiments, the lyophilized composition or formulation of the present disclosure comprises:

[0172] i. nucleic acid construct;

[0173] ii. sodium chloride, the weight ratio of sodium chloride to cationic lipid is 1:5 to 10:1;

[0174] iii. a cationic lipid comprising a compound of Formula I or a pharmaceutically acceptable salt thereof, wherein the cationic lipid accounts for 20 to 70% by molar amount of the total lipid present in the lipid nanoparticles;

[0175] iv. a non-cationic lipid selected from a phospholipid and cholesterol or a derivative thereof, wherein the phospholipid accounts for 5 to 40% by mole of the total lipid present in the lipid nanoparticles and the cholesterol or a derivative thereof accounts for 15 to 60% by mole of the total lipid present in the lipid nanoparticles; and

[0176] v. Conjugated lipid, said conjugate comprising 0.5-2% by mole of the total lipid present in the lipid nanoparticle.

[0177] In some embodiments, the lyophilized compositions or formulations described herein comprise:

[0178] i. nucleic acid construct;

[0179] ii. sodium chloride, the weight ratio of sodium chloride to cationic lipid is 1:5 to 10:1;

[0180] iii. a cationic lipid comprising a compound of Formula I or a pharmaceutically acceptable salt thereof, wherein the cationic lipid accounts for 10 to 50% by molar amount of the total lipid present in the lipid nanoparticles;

[0181] iv. a non-cationic lipid selected from a phospholipid and cholesterol or a derivative thereof, wherein the phospholipid accounts for 5 to 40% by mole of the total lipid present in the lipid nanoparticles and the cholesterol or a derivative thereof accounts for 30 to 60% by mole of the total lipid present in the lipid nanoparticles; and

[0182] v. Conjugated lipid, said conjugate comprising 0.5-2% by mole of the total lipid present in the lipid nanoparticle.

[0183] In some embodiments, the lyophilized compositions or formulations described herein comprise:

[0184] 1) sodium chloride, the weight ratio of sodium chloride to cationic lipid is 1:5 to 10:1;

[0185] 2) Lipid nanoparticles, comprising:

[0186] nucleic acid constructs;

[0187] Cationic lipids, comprising a compound represented by formula I or a pharmaceutically acceptable salt thereof, wherein the cationic lipids account for 10 to 75% of the total lipid molar amount present in the lipid nanoparticles,

[0188] A non-cationic lipid selected from the group consisting of phospholipids and cholesterol or a derivative thereof, wherein the phospholipids account for 5 to 40% by mole of the total lipid present in the lipid nanoparticles, and the cholesterol or a derivative thereof accounts for 15 to 60% by mole of the total lipid present in the lipid nanoparticles;

[0189] and a conjugated lipid, the conjugate comprising 0.5 to 2% by mole of the total lipid present in the lipid nanoparticle.

[0190] 3) Optional lyoprotectant, wherein the concentration of the lyoprotectant is 5% w / v to 15% w / v.

[0191] In some embodiments, the lyophilized compositions or formulations described herein comprise:

[0192] 1) sodium chloride, the weight ratio of sodium chloride to cationic lipid is 1:5 to 10:1;

[0193] 2) Lipid nanoparticles, comprising:

[0194] nucleic acid constructs;

[0195] A cationic lipid comprising a compound of Formula I or a pharmaceutically acceptable salt thereof, wherein the cationic lipid accounts for 20 to 70% of the total molar amount of lipid present in the lipid nanoparticles;

[0196] A non-cationic lipid selected from the group consisting of phospholipids and cholesterol or a derivative thereof, wherein the phospholipids account for 5 to 40% by mole of the total lipid present in the lipid nanoparticles, and the cholesterol or a derivative thereof accounts for 15 to 60% by mole of the total lipid present in the lipid nanoparticles;

[0197] and a conjugated lipid, the conjugate comprising 0.5 to 2 molar percent of the total lipid present in the lipid nanoparticle;

[0198] 3) Optional lyoprotectant, wherein the concentration of the lyoprotectant is 5% w / v to 15% w / v.

[0199] In some embodiments, the lyophilized compositions or formulations described herein comprise:

[0200] 1) sodium chloride, the weight ratio of sodium chloride to cationic lipid is 1:5 to 10:1;

[0201] 2) Lipid nanoparticles, comprising:

[0202] nucleic acid constructs;

[0203] A cationic lipid comprising a compound of Formula I or a pharmaceutically acceptable salt thereof, wherein the cationic lipid accounts for 10 to 50% by molar amount of the total lipid present in the lipid nanoparticles;

[0204] A non-cationic lipid selected from the group consisting of phospholipids and cholesterol or a derivative thereof, wherein the phospholipids account for 5 to 40% by mole of the total lipid present in the lipid nanoparticles, and the cholesterol or a derivative thereof accounts for 30 to 60% by mole of the total lipid present in the lipid nanoparticles;

[0205] and a conjugated lipid, the conjugate comprising 0.5 to 2 molar percent of the total lipid present in the lipid nanoparticle;

[0206] 3) Optional lyoprotectant, wherein the concentration of the lyoprotectant is 5% w / v to 15% w / v.

[0207] In some embodiments, the amount of active agent in the lyophilized composition or formulation is about 0.01 mg / mL to about 1 mg / mL, about 0.05 mg / mL to about 0.5 mg / mL, about 0.1 mg / mL to about 0.5 mg / mL, or about 0.2 mg / mL, about 0.05 mg / mL, or about 0.02 mg / mL.

[0208] In some embodiments, the weight ratio of the active agent to the cationic lipid is 1:10 to 1:60; preferably, the weight ratio of the active agent to the cationic lipid is 1:10 to 1:50; preferably, the weight ratio of the active agent to the cationic lipid is 1:10 to 1:40.

[0209] In some embodiments, any of the aforementioned lipid nanoparticles or lyophilized compositions or formulations, wherein the nucleic acid construct comprises an ORF. In some embodiments, the ORF encodes hepatocyte growth factor (HGF), an antibody, or an antigen-binding fragment thereof. In some specific embodiments, the ORF encodes human hepatocyte growth factor (HGF). In some specific embodiments, the ORF encodes an anti-PD-1 antibody or an antigen-binding fragment thereof.

[0210] The present disclosure also provides a method for preparing a lyophilized preparation, the method comprising the following steps:

[0211] a. Providing a suspension of lipid nanoparticles in a liquid medium as described above; and

[0212] b. adjusting the liquid medium to form a suspension comprising NaCl;

[0213] c. Freeze drying.

[0214] In some embodiments, the preparation method described in the present disclosure, wherein the bulk medium comprises 5% w / v to 20% w / v sucrose, preferably 5% w / v to 15% w / v sucrose.

[0215] In some embodiments, the preparation method described herein, wherein the bulk medium comprises 5% w / v to 10% w / v sucrose and 1% w / v to 10% w / v trehalose.

[0216] In some embodiments, the preparation method described in the present disclosure, wherein the body medium comprises 10-200mM NaCl; preferably comprises 10-150mM NaCl; more preferably comprises 20-150mM NaCl; more preferably comprises 20-120mM NaCl; more preferably comprises 50-150mM NaCl; more preferably comprises 50-120mM NaCl; more preferably comprises 70-120mM NaCl.

[0217] The present disclosure also provides a method for reducing the average particle size of lipid nanoparticles in a lyophilized composition or a lyophilized formulation, comprising adding or using sodium chloride during the lyophilization process.

[0218] The present disclosure also provides a method for reducing the average particle size of lipid nanoparticles after reconstitution of a lyophilized composition or a lyophilized formulation, comprising adding or using sodium chloride during the lyophilization process.

[0219] The present disclosure also provides a use of sodium chloride in reducing the average particle size of lipid nanoparticles in a freeze-dried composition or freeze-dried preparation.

[0220] The present disclosure also provides a use of sodium chloride in reducing the average particle size of lipid nanoparticles after reconstitution of a freeze-dried composition or freeze-dried preparation, comprising adding or using sodium chloride during the freeze-drying process.

[0221] In some embodiments, after reconstitution of the lyophilized composition or lyophilized formulation of the present disclosure, the average particle size of the lipid nanoparticles does not increase by more than 100%. In some embodiments, the average particle size of the lipid nanoparticles does not increase by more than 80%. In some embodiments, the average particle size of the lipid nanoparticles does not increase by more than 60%.

[0222] In some embodiments, the lyophilized composition or lyophilized formulation of the present disclosure is tested for mRNA integrity immediately after being lyophilized, wherein the mRNA integrity is not less than 75%, or the mRNA integrity is not less than 80%, or the mRNA integrity is not less than 85%, or the mRNA integrity is not less than 86%, or the mRNA integrity is not less than 87%, or the mRNA integrity is not less than 88%, or the mRNA integrity is not less than 89%, or the mRNA integrity is not less than 90%. The immediate test is performed within 1 hour after leaving the box.

[0223] In some embodiments, the lyophilized composition or lyophilized preparation described in the present disclosure is tested for mRNA integrity after being placed at 37° C. for 72 hours after being lyophilized, wherein the integrity of the mRNA is not less than 75%, or the integrity of the mRNA is not less than 80%, or the integrity of the mRNA is not less than 85%, or the integrity of the mRNA is not less than 86%, or the integrity of the mRNA is not less than 87%, or the integrity of the mRNA is not less than 88%, or the integrity of the mRNA is not less than 89%, or the integrity of the mRNA is not less than 90%.

[0224] The present disclosure provides a reconstitution solution, wherein the reconstitution solution is prepared by reconstituted the aforementioned lyophilized composition or preparation with a reconstitution solvent. In certain embodiments, the reconstitution solution is selected from but not limited to water for injection, physiological saline, glucose solution or the following reconstitution solvents.

[0225] On the other hand, the present disclosure also provides a pharmaceutical composition comprising lipid nanoparticles and a resolubilizing agent.

[0226] On the other hand, the present disclosure also provides a pharmaceutical composition comprising a lyophilized composition and a reconstituted solution.

[0227] In another aspect, the present disclosure further provides a pharmaceutical composition comprising lipid nanoparticles, sodium chloride, and a resolubilizing agent.

[0228] The present disclosure provides a method for preparing the aforementioned pharmaceutical composition, comprising adding or using sodium chloride in the preparation of a lyophilized composition. The present disclosure provides a method for preparing the aforementioned pharmaceutical composition, comprising the step of reconstituting the lyophilized composition or preparation.

[0229] The present disclosure provides a method for preparing a reconstituted solution, which comprises preparing the aforementioned lyophilized composition, subjecting the lyophilized composition to a freeze-drying treatment to obtain the lyophilized preparation; and

[0230] The lyophilized preparation is reconstituted to obtain the reconstituted solution.

[0231] The present disclosure provides a method for preparing a lyophilized preparation, which comprises preparing the aforementioned lyophilized composition, and subjecting the lyophilized composition to a freeze-drying treatment to obtain the lyophilized preparation.

[0232] In some embodiments, the resolubilizing agents described herein comprise a dispersant.

[0233] In some embodiments, the dispersant described in the present disclosure can be selected from poloxamer, polyvinyl alcohol, hydroxyethyl cellulose, polyethylene glycol, polysorbate, polyoxyethylene fatty acid ester, sorbitan fatty acid, polyoxyethylene fatty alcohol ether, polyoxyethylene hydrogenated castor oil, and N-alkyl pyrrolidone.

[0234] In some embodiments, the dispersant described in the present disclosure can be selected from poloxamer 188, polyvinyl alcohol, hydroxyethyl cellulose, polyethylene glycol 400, polysorbate 20 (Tween 20), sorbitan laurate (Span 20), polyoxyethylene monostearate (Span 45), polyoxyethylene lauryl ether (Benzyl 35), and N-ethylpyrrolidone (NMP).

[0235] In some embodiments, the dispersing agent described in the present disclosure may be selected from Poloxamer 188.

[0236] In some embodiments, the dispersant described in the present disclosure accounts for 0.01-5.0% by weight of the resolubilizing agent.

[0237] In some embodiments, the dispersant described in the present disclosure accounts for 0.01-3.0% by weight of the resolubilizing agent.

[0238] In some embodiments, the dispersant described in the present disclosure accounts for 0.05-3.0% by weight of the resolubilizing agent.

[0239] In some embodiments, the dispersant described in the present disclosure accounts for 0.05-2.0% by weight of the resolubilizing agent.

[0240] In some embodiments, the dispersant described in the present disclosure accounts for 0.05-1.0% by weight of the resolubilizing agent.

[0241] In some embodiments, the resolubilizing agents described herein further comprise a viscosity increasing agent.

[0242] In some embodiments, the viscosity increasing agent described in the present disclosure may be selected from xanthan gum (Xc), povidone (PVP), sodium hyaluronate (HA), sodium carboxymethylcellulose (CMC-Na), and chitosan (CTS).

[0243] In some embodiments, the viscosity increasing agent described in the present disclosure may be selected from xanthan gum (Xc), povidone (PVP), sodium hyaluronate (HA), and sodium carboxymethylcellulose (CMC-Na).

[0244] In some embodiments, the povidone (PVP) described in the present disclosure is povidone K90 (PVP-K90).

[0245] In some embodiments, the viscosity-increasing agent described in the present disclosure accounts for 0.01-5.0% by weight of the resolubilizing agent.

[0246] In some embodiments, the viscosity-increasing agent described in the present disclosure accounts for 0.01-3.0% by weight of the resolubilizing agent.

[0247] In some embodiments, the viscosity-increasing agent described in the present disclosure accounts for 0.05-3.0% by weight of the resolubilizing agent.

[0248] In some embodiments, the viscosity-increasing agent described in the present disclosure accounts for 0.05-2.0% by weight of the resolubilizing agent.

[0249] The present disclosure also provides a product, comprising a container containing the aforementioned lipid nanoparticles, the aforementioned lyophilized composition, the aforementioned pharmaceutical composition, or the aforementioned reconstituted solution.

[0250] The present disclosure also provides a product comprising:

[0251] 1) the aforementioned lyophilized composition or lyophilized preparation, and,

[0252] 2) Resolubilization solvent.

[0253] The present disclosure also provides a spray preparation comprising the aforementioned lyophilized composition, lyophilized preparation or reconstituted solution.

[0254] In certain embodiments, the article of manufacture comprises separately packaged lyophilized compositions or formulations and reconstitution solutions.

[0255] In certain embodiments, the container is a neutral borosilicate glass tube injection bottle or a device for nebulized drug administration. In certain embodiments, the product comprises a drug instruction sheet.

[0256] The lipid nanoparticles, lyophilized compositions, pharmaceutical compositions, or reconstituted solutions provided by the present disclosure have good stability, controllable particle size growth, and excellent mRNA expression.

[0257] The present disclosure also provides the aforementioned lipid nanoparticles, the aforementioned lyophilized composition, the aforementioned pharmaceutical composition or the aforementioned reconstituted solution, which are used as a drug for treating or alleviating a disease or condition.

[0258] In some embodiments, any of the aforementioned lipid nanoparticles comprises a nucleic acid construct. The nucleic acid construct comprises: at least one nucleic acid element capable of regulating expression of a gene of interest, wherein the nucleic acid element is a UTR. Furthermore, the nucleic acid construct may contain one or more genes of interest, such as HGF.

[0259] Nucleic acid constructs

[0260] The present disclosure provides a nucleic acid construct comprising:

[0261] (a) an open reading frame (ORF), and

[0262] (b) Untranslated region elements (UTR).

[0263] In some embodiments, the nucleic acid construct is a DNA molecule; in some embodiments, the nucleic acid construct is an RNA molecule (eg, mRNA).

[0264] In some embodiments, the ORF is a polynucleotide sequence encoding a gene of interest.

[0265] In some embodiments, the target gene is heterologous. In other embodiments, the target gene is endogenous. In some embodiments, the target gene is one or more (e.g., 2, 3, 4).

[0266] In some embodiments, the UTR is derived from the UTR of gene ACTG1, ATP6V0B, ATP6V0E1, CFL1, COX4I1, CTSB, FAM166A, NDUFB9, CHCHD10, SLC38A2, NDUFA11, NDUFV3, PRDX5, GUK1, IAH1, ABHD16A, SLC25A39, ATPIF1, ANAPC11, CCDC12, MRPL14, or APOA1BP. In some embodiments, the above genes are human genes.

[0267] In some embodiments, the UTR is a 3' untranslated region element (3'UTR) or a 5' untranslated region element (5'UTR).

[0268] In some embodiments, the 3'UTR and 5'UTR are derived from the same or different sources, for example, from the same or different genes. For example, the 3'UTR is derived from the 3'UTR of the gene ACTG1, and the 5'UTR is derived from the 5'UTR of the gene ACTG1. For another example, the 3'UTR is derived from the 3'UTR of the gene CTSB, and the 5'UTR is derived from the 5'UTR of the gene CHCHD10. In some embodiments, the 5'UTR and 3'UTR are derived from the same species or different species.

[0269] In some embodiments, the 5'UTR is located upstream of the ORF. In some embodiments, the 5'UTR in the nucleic acid construct is located at the 5' end of the ORF. In some embodiments, the 3'UTR is located downstream of the ORF. In some embodiments, the 3'UTR in the nucleic acid construct is located at the 3' end of the ORF.

[0270] In some embodiments, the aforementioned nucleic acid construct comprises:

[0271] (a) open reading frame (ORF),

[0272] (b-1) a 3′UTR derived from the 3′UTR of gene ACTG1, ATP6V0B, ATP6V0E1, CFL1, COX4I1, CTSB, FAM166A, or NDUFB9; and

[0273] (b-2) 5′UTR derived from the 5′UTR of gene ACTG1, ATP6V0B, ATP6V0E1, CFL1, COX4I1, CTSB, FAM166A, NDUFB9, CHCHD10, SLC38A2, NDUFA11, NDUFV3, PRDX5, GUK1, IAH1, ABHD16A, SLC25A39, ATPIF1, ANAPC11, CCDC12, MRPL14, or APOA1BP.

[0274] In some embodiments, the aforementioned nucleic acid construct comprises:

[0275] (a) open reading frame (ORF),

[0276] (b-1) a 3'UTR derived from the 3'UTR of gene CTSB, FAM166A, or NDUFB9; and

[0277] (b-2) 5'UTR derived from the 5'UTR of gene ACTG1, CHCHD10 or NDUFA11.

[0278] In some embodiments, in the aforementioned nucleic acid construct (eg, DNA or RNA molecule), the 3'UTR is derived from the 3'UTR of the gene ACTG1, which comprises a sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 58, or a sequence identical thereto;

[0279] The 3'UTR is derived from the 3'UTR of gene ATP6V0B, which contains a sequence shown in SEQ ID NO: 2, 3 or SEQ ID NO: 59, 60 or any sequence identical thereto;

[0280] The 3'UTR is derived from the 3'UTR of the gene ATP6V0E1, which contains a sequence shown in SEQ ID NO: 4, 5 or SEQ ID NO: 61, 62 or any sequence identical thereto;

[0281] The 3'UTR is derived from the 3'UTR of the gene CFL1, which contains a sequence shown in SEQ ID NO: 6, 7, 8 or SEQ ID NO: 63, 64, 65 or any sequence identical thereto;

[0282] The 3'UTR is derived from the 3'UTR of the COX4I1 gene, which contains a sequence shown in SEQ ID NO: 9, 10, 11 or SEQ ID NO: 66, 67, 68 or any sequence identical thereto;

[0283] 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 any sequence identical thereto;

[0284] The 3'UTR is derived from the 3'UTR of gene FAM166A, which comprises a sequence as shown in SEQ ID NO: 13 or SEQ ID NO: 70 or a sequence identical thereto; or

[0285] 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 a sequence identical thereto.

[0286] In some embodiments, in the aforementioned nucleic acid construct (eg, DNA or RNA molecule), the 5'UTR is derived from the 5'UTR of the gene ACTG1, which contains a sequence as shown in SEQ ID NO: 15 or SEQ ID NO: 72, or a sequence identical thereto;

[0287] The 5'UTR is derived from the 5'UTR of gene ATP6V0B, which contains a sequence shown in SEQ ID NO: 16, 17 or SEQ ID NO: 73, 74 or any sequence identical thereto;

[0288] The 5'UTR is derived from the 5'UTR of the gene ATP6V0E1, which contains a sequence shown in SEQ ID NO: 18, 19 or SEQ ID NO: 75, 76 or any sequence identical thereto;

[0289] The 5'UTR is derived from the 5'UTR of the gene CFL1, which contains a sequence shown in SEQ ID NO: 20, 21, 22 or SEQ ID NO: 77, 78, 79 or any sequence identical thereto;

[0290] The 5'UTR is derived from the 5'UTR of the COX4I1 gene, which contains a sequence shown in SEQ ID NO: 23, 24, 25 or SEQ ID NO: 80, 81, 82 or any sequence identical thereto;

[0291] 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 a sequence identical thereto;

[0292] The 5'UTR is derived from the 5'UTR of the gene FAM166A, which comprises a sequence as shown in SEQ ID NO: 27 or SEQ ID NO: 84 or a sequence identical thereto;

[0293] The 5'UTR is derived from the 5'UTR of the gene NDUFB9, which contains a sequence shown in SEQ ID NO: 28 or SEQ ID NO: 85 or a sequence identical thereto;

[0294] The 5'UTR is derived from the 5'UTR of gene CHCHD10, which contains a sequence shown in SEQ ID NO: 29, 30 or SEQ ID NO: 86, 87 or any sequence identical thereto;

[0295] 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 a sequence identical thereto;

[0296] The 5'UTR is derived from the 5'UTR of the gene NDUFA11, which contains a sequence as shown in SEQ ID NO: 32 or SEQ ID NO: 89 or a sequence identical thereto;

[0297] The 5'UTR is derived from the 5'UTR of the gene NDUFV3, which contains a sequence as shown in SEQ ID NO: 33 or SEQ ID NO: 90 or a sequence identical thereto;

[0298] 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;

[0299] The 5'UTR is derived from the 5'UTR of the gene GUK1, which contains a sequence shown in SEQ ID NO: 35, 36, 37 or SEQ ID NO: 92, 93, 94 or any sequence identical thereto;

[0300] The 5'UTR is derived from the 5'UTR of the gene IAH1, which contains a sequence as shown in SEQ ID NO: 38 or SEQ ID NO: 95 or a sequence identical thereto;

[0301] The 5'UTR is derived from the 5'UTR of gene ABHD16A, which comprises a sequence as shown in SEQ ID NO:39 or SEQ ID NO:96 or a sequence identical thereto;

[0302] 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 any sequence identical thereto;

[0303] 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 a sequence identical thereto;

[0304] The 5'UTR is derived from the 5'UTR of the gene ANAPC11, which contains a sequence shown in SEQ ID NO: 42, 43 or SEQ ID NO: 99, 100 or any sequence identical thereto;

[0305] The 5'UTR is derived from the 5'UTR of gene CCDC12, which contains a sequence shown in SEQ ID NO:44 or SEQ ID NO:101 or a sequence identical thereto;

[0306] The 5'UTR is derived from the 5'UTR of gene MRPL14, which comprises a sequence as shown in SEQ ID NO: 45 or SEQ ID NO: 102, or a sequence identical thereto; or

[0307] The 5'UTR is derived from the 5'UTR of the gene APOA1BP, and contains a sequence shown in SEQ ID NO: 46, 47 or SEQ ID NO: 103, 104, or any sequence identical thereto.

[0308] In some embodiments, a nucleic acid construct (e.g., a DNA or RNA molecule) is provided, comprising:

[0309] (a) open reading frame (ORF),

[0310] (b-1) 3'UTR, and

[0311] (b-2) 5′UTR;

[0312] Wherein, the 3'UTR and 5'UTR are selected from any one of the following combinations:

[0313] 1) The 3'UTR comprises a sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 58, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 15-47 or any one of SEQ ID NOs: 72-104, or a sequence identical thereto;

[0314] 2) the 3'UTR comprises a sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 59, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 15-47 or any one of SEQ ID NOs: 72-104, or a sequence identical thereto;

[0315] 3) the 3'UTR comprises a sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 60, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 15-47 or any one of SEQ ID NOs: 72-104, or a sequence identical thereto;

[0316] 4) the 3'UTR comprises a sequence as set forth in SEQ ID NO:4 or SEQ ID NO:61, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in any one of SEQ ID NOs:15-47 or any one of SEQ ID NOs:72-104, or a sequence identical thereto;

[0317] 5) the 3'UTR comprises a sequence as set forth in SEQ ID NO: 5 or SEQ ID NO: 62, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 15-47 or any one of SEQ ID NOs: 72-104, or a sequence identical thereto;

[0318] 6) the 3'UTR comprises a sequence as set forth in SEQ ID NO:6 or SEQ ID NO:63, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in any one of SEQ ID NOs:15-47 or any one of SEQ ID NOs:72-104, or a sequence identical thereto;

[0319] 7) the 3'UTR comprises a sequence as set forth in SEQ ID NO:7 or SEQ ID NO:64, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in any one of SEQ ID NOs:15-47 or any one of SEQ ID NOs:72-104, or a sequence identical thereto;

[0320] 8) the 3'UTR comprises a sequence as set forth in SEQ ID NO:8 or SEQ ID NO:65, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in any one of SEQ ID NOs:15-47 or any one of SEQ ID NOs:72-104, or a sequence identical thereto;

[0321] 9) the 3'UTR comprises a sequence as set forth in SEQ ID NO: 9 or SEQ ID NO: 66, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 15-47 or any one of SEQ ID NOs: 72-104, or a sequence identical thereto;

[0322] 10) The 3'UTR comprises a sequence as set forth in SEQ ID NO: 10 or SEQ ID NO: 67, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 15-47 or any one of SEQ ID NOs: 72-104, or a sequence identical thereto;

[0323] 11) The 3'UTR comprises a sequence as set forth in SEQ ID NO: 11 or SEQ ID NO: 68, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 15-47 or any one of SEQ ID NOs: 72-104, or a sequence identical thereto;

[0324] 12) the 3'UTR comprises a sequence as set forth in SEQ ID NO: 12 or SEQ ID NO: 69, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 15-47 or any one of SEQ ID NOs: 72-104, or a sequence identical thereto;

[0325] 13) The 3'UTR comprises a sequence as set forth in SEQ ID NO: 13 or SEQ ID NO: 70, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 15-47 or any one of SEQ ID NOs: 72-104, or a sequence identical thereto;

[0326] 14) the 3'UTR comprises a sequence as set forth in SEQ ID NO: 14 or SEQ ID NO: 71, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 15-47 or any one of SEQ ID NOs: 72-104, or a sequence identical thereto;

[0327] 15) the 3'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 1-14 or 58-71, or an identical sequence thereof, and the 5'UTR comprises a sequence as set forth in SEQ ID NO: 15 or 72, or an identical sequence thereof;

[0328] 16) the 3'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 1-14 or 58-71, or an identical sequence thereof, and the 5'UTR comprises a sequence as set forth in SEQ ID NO: 16 or 73, or an identical sequence thereof;

[0329] 17) The 3'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 1-14 or 58-71, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in SEQ ID NO: 17 or 74, or a sequence identical thereto;

[0330] 18) The 3'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 1-14 or 58-71, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in SEQ ID NO: 18 or 75, or a sequence identical thereto;

[0331] 19) The 3'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 1-14 or 58-71, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in SEQ ID NO: 19 or 76, or a sequence identical thereto;

[0332] 20) The 3'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 1-14 or 58-71, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in SEQ ID NO: 20 or 77, or a sequence identical thereto;

[0333] 21) The 3'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 1-14 or 58-71, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in SEQ ID NO: 21 or 78, or a sequence identical thereto;

[0334] 22) the 3'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 1-14 or 58-71, or an identical sequence thereof, and the 5'UTR comprises a sequence as set forth in SEQ ID NO: 22 or 79, or an identical sequence thereof;

[0335] 23) The 3'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 1-14 or 58-71, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in SEQ ID NO: 23 or 80, or a sequence identical thereto;

[0336] 24) the 3'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 1-14 or 58-71, or an identical sequence thereof, and the 5'UTR comprises a sequence as set forth in SEQ ID NO: 24 or 81, or an identical sequence thereof;

[0337] 25) the 3'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 1-14 or 58-71, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in SEQ ID NO: 25 or 82, or a sequence identical thereto;

[0338] 26) The 3'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 1-14 or 58-71, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in SEQ ID NO: 26 or 83, or a sequence identical thereto;

[0339] 27) The 3'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 1-14 or 58-71, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in SEQ ID NO: 27 or 84, or a sequence identical thereto;

[0340] 28) The 3'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 1-14 or 58-71, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in SEQ ID NO: 28 or 85, or a sequence identical thereto;

[0341] 29) The 3'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 1-14 or 58-71, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in SEQ ID NO: 29 or 86, or a sequence identical thereto;

[0342] 30) the 3'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 1-14 or 58-71, or an identical sequence thereof, and the 5'UTR comprises a sequence as set forth in SEQ ID NO: 30 or 87, or an identical sequence thereof;

[0343] 31) The 3'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 1-14 or 58-71, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in SEQ ID NO: 31 or 88, or a sequence identical thereto;

[0344] 32) The 3'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 1-14 or 58-71, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in SEQ ID NO: 32 or 89, or a sequence identical thereto;

[0345] 33) The 3'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 1-14 or 58-71, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in SEQ ID NO: 33 or 90, or a sequence identical thereto;

[0346] 34) the 3'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 1-14 or 58-71, or an identical sequence thereof, and the 5'UTR comprises a sequence as set forth in SEQ ID NO: 34 or 91, or an identical sequence thereof;

[0347] 35) the 3'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 1-14 or 58-71, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in SEQ ID NO: 35 or 92, or a sequence identical thereto;

[0348] 36) the 3'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 1-14 or 58-71, or an identical sequence thereof, and the 5'UTR comprises a sequence as set forth in SEQ ID NO: 36 or 93, or an identical sequence thereof;

[0349] 37) The 3'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 1-14 or 58-71, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in SEQ ID NO: 37 or 94, or a sequence identical thereto;

[0350] 38) The 3'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 1-14 or 58-71, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in SEQ ID NO: 38 or 95, or a sequence identical thereto;

[0351] 39) The 3'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 1-14 or 58-71, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in SEQ ID NO: 39 or 96, or a sequence identical thereto;

[0352] 40) The 3'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 1-14 or 58-71, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in SEQ ID NO: 40 or 97, or a sequence identical thereto;

[0353] 41) The 3'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 1-14 or 58-71, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in SEQ ID NO: 41 or 98, or a sequence identical thereto;

[0354] 42) The 3'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 1-14 or 58-71, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in SEQ ID NO: 42 or 99, or a sequence identical thereto;

[0355] 43) the 3'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 1-14 or 58-71, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in SEQ ID NO: 43 or 100, or a sequence identical thereto;

[0356] 44) the 3'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 1-14 or 58-71, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in SEQ ID NO: 44 or 101, or a sequence identical thereto;

[0357] 45) The 3'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 1-14 or 58-71, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in SEQ ID NO: 45 or 102, or a sequence identical thereto;

[0358] 46) the 3'UTR comprises a sequence as set forth in any one of SEQ ID NOs: 1-14 or 58-71, or a sequence identical thereto, and the 5'UTR comprises a sequence as set forth in SEQ ID NO: 46 or 103, or a sequence identical thereto; or

[0359] 47) The 3'UTR contains a sequence as shown in any one of SEQ ID NOs: 1-14 or 58-71, or a sequence identical thereto, and the 5'UTR contains a sequence as shown in SEQ ID NO: 47 or 104, or a sequence identical thereto.

[0360] In some embodiments, a nucleic acid construct (e.g., a DNA or RNA molecule) is provided, comprising:

[0361] (a) open reading frame (ORF),

[0362] (b-1) 3'UTR, and

[0363] (b-2) 5′UTR;

[0364] The 3'UTR is derived from the 3'UTR of gene CTSB, FAM166A or NDUFB9, and the 5'UTR is derived from the 5'UTR of gene ACTG1, CHCHD10 or NDUFA11;

[0365] For example, the 3'UTR comprises any one of SEQ ID NOs: 12, 13, 14 or any one of SEQ ID NOs: 69, 70, 71, or a sequence identical thereto, and the 5'UTR comprises any one of SEQ ID NOs: 15, 29, 30, 32 or any one of SEQ ID NOs: 72, 86, 87, 89, or a sequence identical thereto;

[0366] For another example, the 3'UTR comprises a sequence as shown in SEQ ID NO: 12 or SEQ ID NO: 69, or a sequence identical thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 15 or SEQ ID NO: 72, or a sequence identical thereto.

[0367] The 3'UTR comprises a sequence as shown in SEQ ID NO: 13 or SEQ ID NO: 70, or a sequence identical thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 15 or SEQ ID NO: 72, or a sequence identical thereto,

[0368] The 3'UTR comprises a sequence as shown in SEQ ID NO: 14 or SEQ ID NO: 71, or a sequence identical thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 15 or SEQ ID NO: 72, or a sequence identical thereto,

[0369] The 3'UTR comprises a sequence as shown in SEQ ID NO: 12 or SEQ ID NO: 69, or a sequence identical thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 29 or SEQ ID NO: 86, or a sequence identical thereto,

[0370] The 3'UTR comprises a sequence as shown in SEQ ID NO: 13 or SEQ ID NO: 70, or a sequence identical thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 29 or SEQ ID NO: 86, or a sequence identical thereto,

[0371] The 3'UTR comprises a sequence as shown in SEQ ID NO: 14 or SEQ ID NO: 71, or a sequence identical thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 29 or SEQ ID NO: 86, or a sequence identical thereto,

[0372] The 3'UTR comprises a sequence as shown in SEQ ID NO: 12 or SEQ ID NO: 69, or a sequence identical thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 30 or SEQ ID NO: 87, or a sequence identical thereto,

[0373] The 3'UTR comprises a sequence as shown in SEQ ID NO: 13 or SEQ ID NO: 70, or a sequence identical thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 30 or SEQ ID NO: 87, or a sequence identical thereto,

[0374] The 3'UTR comprises a sequence as shown in SEQ ID NO: 14 or SEQ ID NO: 71, or a sequence identical thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 30 or SEQ ID NO: 87, or a sequence identical thereto,

[0375] The 3'UTR comprises a sequence as shown in SEQ ID NO: 12 or SEQ ID NO: 69, or a sequence identical thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 32 or SEQ ID NO: 89, or a sequence identical thereto,

[0376] The 3'UTR comprises a sequence as shown in SEQ ID NO: 13 or SEQ ID NO: 70, or a sequence identical thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 32 or SEQ ID NO: 89, or a sequence identical thereto, or

[0377] The 3'UTR comprises a sequence as shown in SEQ ID NO: 14 or SEQ ID NO: 71, or a sequence identical thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 32 or SEQ ID NO: 89, or a sequence identical thereto.

[0378] In the above embodiments, "having identity" encompasses at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity, and ranges between any two of the aforementioned values, including integers and decimals, for example, "having at least 90% identity" or "having at least 95% identity."

[0379] In some embodiments, the 3'UTR and / or 5'UTR is a variant of the above-mentioned 3'UTR and / or 5'UTR, and the variant is, for example, a truncation or a nucleotide mutant, and the variant still maintains similar activity or function to the aforementioned 3'UTR and / or 5'UTR disclosed herein, for example, still maintains the function of regulating the expression of the protein encoded by the ORF of the target gene.

[0380] In some embodiments, the nucleic acid construct provided by the present disclosure further comprises:

[0381] (c) Polyadenylic acid (poly-A) tail.

[0382] In some embodiments, 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, 120, 150, 200, 300, 400, or 500 nucleotides in length.

[0383] In some specific embodiments, the poly-A tail is selected from A120, A30L70, HGH polyA, SV40 polyA, BGH polyA, rbGlob polyA, or SV40late polyA. For example, the A30L70 is the sequence shown in SEQ ID NO: 52; and the A120 comprises 120 adenine nucleotides.

[0384] In some embodiments, the target gene (i.e., open reading frame (ORF)) expressed in the nucleic acid constructs provided herein is hepatocyte growth factor (HGF), an antibody, or an antigen-binding fragment thereof, for example, an antibody or an antigen-binding fragment thereof that binds to a tumor antigen, an antibody or an antigen-binding fragment thereof that binds to a viral antigen, etc.

[0385] In some specific embodiments, the polypeptide or protein encoded by the ORF is a fluorescent protein or luciferase, such as the sequence shown in SEQ ID NO:126.

[0386] In some embodiments, the HGF is human hepatocyte growth factor (hHGF).

[0387] In some specific embodiments, the coding sequence of hHGF comprises any one selected from the following 1)-3):

[0388] 1) a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 109 or a codon-optimized nucleic acid sequence;

[0389] 2) a DNA sequence as shown in SEQ ID NO: 110-113 or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto;

[0390] 3) An RNA sequence as shown in any one of SEQ ID NOs: 128-131 or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto.

[0391] In some specific embodiments, the nucleic acid construct expressing HGF as the target gene comprises a sequence shown in any one of SEQ ID NOs: 115, 116, and 127, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.

[0392] In some specific embodiments, the antibody or antigen-binding fragment thereof is an anti-PD-1 antibody or antigen-binding fragment thereof.

[0393] In some specific embodiments, the coding sequence of the anti-PD-1 antibody or antigen-binding fragment thereof comprises any one selected from the following 1)-4):

[0394] 1) a nucleic acid sequence encoding the heavy chain amino acid sequence of SEQ ID NO: 117 or a codon-optimized nucleic acid sequence, and a nucleic acid sequence encoding the light chain amino acid sequence of SEQ ID NO: 118 or a codon-optimized nucleic acid sequence;

[0395] 2) a nucleic acid sequence encoding HCDR1, HCDR2, and HCDR3 in the heavy chain amino acid sequence of SEQ ID NO: 117, or a codon-optimized nucleic acid sequence thereof, and a nucleic acid sequence encoding LCDR1, LCDR2, and LCDR3 in the light chain amino acid sequence of SEQ ID NO: 118, or a codon-optimized nucleic acid sequence thereof, wherein the CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems, for example, according to the Kabat numbering system;

[0396] 3) a DNA sequence as set forth in SEQ ID NO: 119 or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and a DNA sequence as set forth in SEQ ID NO: 120 or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto;

[0397] 4) a DNA sequence as set forth in SEQ ID NO: 121 or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identical thereto, and a DNA sequence as set forth in SEQ ID NO: 122 or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identical thereto.

[0398] In some specific embodiments, the nucleic acid construct expressing the anti-PD-1 antibody or antigen-binding fragment thereof comprises the sequences shown in SEQ ID NOs: 124 and 125, or sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs: 124 and 125.

[0399] In some embodiments, the nucleic acid construct provided by the present disclosure is DNA or RNA, for example, mRNA.

[0400] In some embodiments, from the 5' to 3' direction, the nucleic acid construct (DNA or RNA) provided by the present disclosure contains any one of the following 1)-6):

[0401] 1) 5'UTR and ORF;

[0402] 2) ORF, and 3'UTR;

[0403] 3) 5'UTR, ORF, and 3'UTR;

[0404] 4) 5'UTR, ORF, 3'UTR, and poly-A tail;

[0405] 5) 5'UTR, ORF, and poly-A tail;

[0406] 6) ORF, 3'UTR, and poly-A tail;

[0407] The 5'UTR and 3'UTR may be derived from the same or different genes.

[0408] In some embodiments, from the 5' to 3' direction, the nucleic acid construct (DNA) provided by the present disclosure contains any one of the following 1)-4):

[0409] 1) 5'UTR and ORF;

[0410] 2) ORF, and 3'UTR;

[0411] 3) 5'UTR, ORF, and 3'UTR;

[0412] 4) 5'UTR, ORF, 3'UTR, and poly-A tail;

[0413] The 5'UTR and 3'UTR may be derived from the same or different genes.

[0414] In some specific embodiments, the 5'UTR in 1), 3), and 4) comprises or is a nucleotide sequence as shown in any one of SEQ ID NOs: 15-47. In some specific embodiments, the ORF in 1)-4) comprises or is a nucleotide sequence as shown in SEQ ID NO: 110 or a codon-optimized nucleotide sequence thereof (e.g., SEQ ID NOs: 111-113). In some specific embodiments, the ORF in 1)-4) comprises or is a nucleotide sequence as shown in SEQ ID NOs: 119 and 120. In some specific embodiments, the 3'UTR in 2)-4) comprises or is a nucleotide sequence as shown in any one of SEQ ID NOs: 1-14.

[0415] In some embodiments, the nucleic acid construct (RNA or mRNA) provided by the present disclosure further comprises:

[0416] (d) 5' cap structure (5'Cap).

[0417] In some specific embodiments, the 5'cap structure in the RNA molecule is located upstream of the 5'UTR. In some embodiments, the 5'cap structure in the RNA molecule is located at the 5' end of the 5'UTR. In some embodiments, the 5'cap structure is a cap structure known to those skilled in the art, such as Cap0 (methylation of the first nucleobase, e.g., m 7 GpppN)、Cap1(m 7 Additional methylation of the ribose sugars of the adjacent nucleotides of GpppN, such as m 7 G(5')ppp(5')(2'OMeA)pG), Cap2(m 7 Additional methylation of the ribose of the third nucleotide downstream of GpppN), Cap3 (m 7 Additional methylation of the ribose of the third nucleotide downstream of GpppN), Cap4 (m 7 Additional methylation of the ribose of the fourth nucleotide downstream of GpppN), ARCA (anti-reverse cap analog), modified ARCA (e.g., phosphorothioate-modified ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0418] In some embodiments, chemical RNA synthesis or RNA in vitro transcription (co-transcriptional capping) is used to form the 5' Cap structure (eg, Cap0 or Cap1).

[0419] In some embodiments, a capping enzyme (e.g., vaccinia virus capping enzyme and / or cap-dependent 2'-O methyltransferase) is used to form a 5'-cap structure (e.g., Cap0 or Cap1) via enzymatic capping. In some embodiments, an immobilized capping enzyme is used to add the 5' cap structure (Cap0 or Cap1). The capping methods and means described in WO2016 / 193226 are incorporated herein in their entirety.

[0420] In some specific embodiments, 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 7G(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. In some embodiments, the 5'Cap is 3'-O-Me-m 7 G(5')ppp(5')G or m 7 G(5')ppp(5')(2'OMeA)pG.

[0421] In some embodiments, from the 5' to 3' direction, the nucleic acid construct (RNA or mRNA) provided by the present disclosure contains any one of the following 1)-5):

[0422] 1) 5'UTR and ORF;

[0423] 2) ORF, and 3'UTR;

[0424] 3) 5'UTR, ORF, and 3'UTR;

[0425] 4) 5'UTR, ORF, 3'UTR, and poly-A tail;

[0426] 5) 5' Cap, 5' UTR, ORF, 3' UTR, and poly-A tail;

[0427] The 5'UTR and 3'UTR may be derived from the same or different genes.

[0428] In some specific embodiments, the 5'UTR in 1), 3), 4), and 5) comprises or is a nucleotide sequence as shown in any one of SEQ ID NOs: 72-104. In some specific embodiments, the ORF in 1)-5) comprises or is a nucleotide sequence as shown in any one of SEQ ID NOs: 128-131. In some specific embodiments, the 3'UTR in 2)-5) comprises or is a nucleotide sequence as shown in any one of SEQ ID NOs: 58-71. In some specific embodiments, the structure includes but is not limited to Cap0, Cap1 (e.g., m 7 G(5')ppp(5')(2'OMeA)pG), Cap2, Cap3, Cap4, ARCA.

[0429] In some embodiments, the present disclosure provides a nucleic acid construct (DNA) comprising, from 5' to 3' direction, a 5'UTR, an ORF, and a 3'UTR, optionally further comprising a poly-A tail in the 3' direction. In some embodiments, 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 nucleotide sequence as shown in SEQ ID NO: 110 or a codon-optimized nucleotide sequence thereof (e.g., SEQ ID NOs: 111-113), and the 3'UTR comprises or is a nucleotide sequence as shown in any one of SEQ ID NOs: 1-14. In some embodiments, 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 nucleotide sequence as shown in SEQ ID NOs: 119 and 120, and the 3'UTR comprises or is a nucleotide sequence as shown in any one of SEQ ID NOs: 1-14. In some specific embodiments, the nucleic acid construct comprises a nucleotide sequence as shown in SEQ ID NOs: 115, 116, or 127.

[0430] In some embodiments, the present disclosure provides a nucleic acid construct (RNA or mRNA) comprising, from 5' to 3' direction, a 5'UTR, an ORF, and a 3'UTR, optionally, further comprising a poly-A tail in the 3' direction. In some embodiments, the 5'UTR comprises or is a nucleotide sequence as shown in any one of SEQ ID NOs: 72-104, the ORF comprises or is a nucleotide sequence as shown in any one of SEQ ID NOs: 128-131, the 3'UTR comprises or is a nucleotide sequence as shown in any one of SEQ ID NOs: 58-71, and the poly-A tail comprises or is 120 consecutive adenine nucleotides or a nucleotide sequence as shown in SEQ ID NO: 52. In some specific embodiments, the nucleic acid construct comprises the nucleotide sequence as shown in SEQ ID NO: 132.

[0431] In some embodiments, the 5'UTR comprises or is the nucleotide sequence shown in any one of SEQ ID NOs: 72-104, the ORF comprises or is the nucleotide sequence shown in SEQ ID NOs: 121 and 122, the 3'UTR comprises or is the nucleotide sequence shown in any one of SEQ ID NOs: 58-71, and the poly-A tail comprises or is 120 consecutive adenine nucleotides or the nucleotide sequence shown in SEQ ID NO: 52. In some specific embodiments, the nucleic acid construct comprises the nucleotide sequence shown in SEQ ID NOs: 124 or 125.

[0432] In some embodiments, any of the aforementioned nucleic acid constructs (DNA or RNA molecules), wherein the UTR is used to increase the expression level of the ORF expression protein. Exemplarily, in some specific embodiments, the 5'UTR shown in any of the sequences of SEQ ID NOs: 15-47, 72-104 in the present disclosure has an increased expression level of the target protein for regulating ORF expression compared to the 5'UTR shown in SEQ ID NO: 48 or 50. In some specific embodiments, the 3'UTR shown in any of the sequences of SEQ ID NOs: 1-14, 58-71 in the present disclosure has an increased expression level of the target protein for regulating ORF expression compared to the 5'UTR shown in SEQ ID NO: 49 or 51. In some specific embodiments, the combination of the 5'UTR set forth in any one of SEQ ID NOs: 15-47, 72-104 in the present disclosure and the 3'UTR set forth in any one of SEQ ID NOs: 1-14, 58-71 has an increased expression level of the target protein that regulates ORF expression, compared to the combination of the 5'UTR and 3'UTR set forth in SEQ ID NOs: 48 and 49. In some specific embodiments, the combination of the 5'UTR set forth in any one of SEQ ID NOs: 15-47, 72-104 in the present disclosure and the 3'UTR set forth in any one of SEQ ID NOs: 1-14, 58-71 has an increased expression level of the target protein that regulates ORF expression, compared to the combination of the 5'UTR and 3'UTR set forth in SEQ ID NOs: 50 and 51.

[0433] In some embodiments, the nucleic acid constructs of the present disclosure express a target protein at an expression level of about 1 to about 20 times that of the BioN vector, for example, about 1 time, about 1.1 times, about 1.2 times, about 1.3 times, about 1.4 times, about 1.5 times, about 1.6 times, about 1.7 times, about 1.8 times, about 1.9 times, about 2 times, about 2.1 times, about 2.3 times, about 2.5 times, about 2.8 times, about 3 times, about 3.2 times, about 3.4 times, about 3.8 times, about 4 times, about 4.5 times, about 5 times, about 5.2 times, about 5.5 times, about 5.8 times, about 6 times, about 7 times, about 8 times, about 10 times, about 12 times, about 15 times, etc. In some specific embodiments, the nucleic acid constructs of the present disclosure are mRNA molecules that express a target protein, and the expression level of the target protein expressed by the mRNA molecule is about 1 to about 20 times that of the BioN vector. In some specific embodiments, the BioN vector comprises the 5'UTR shown in SEQ ID NO: 50 or 107; and / or, comprises the 3'UTR shown in SEQ ID NO: 51 or 108.

[0434] In some embodiments, the nucleic acid construct of the present disclosure expresses a target protein at an expression level of about 1 to about 20 times that of a Mod vector, for example, about 1 time, about 1.1 times, about 1.2 times, about 1.3 times, about 1.4 times, about 1.5 times, about 1.6 times, about 1.7 times, about 1.8 times, about 1.9 times, about 2 times, about 2.1 times, about 2.3 times, about 2.5 times, about 2.8 times, about 3 times, about 3.2 times, about 3.4 times, about 3.8 times, about 4 times, about 4.5 times, about 5 times, about 5.2 times, about 5.5 times, about 5.8 times, about 6 times, about 7 times, about 8 times, about 10 times, about 12 times, about 15 times, etc. In some specific embodiments, the nucleic acid construct of the present disclosure is an mRNA molecule expressing a target protein, and the expression level of the target protein expressed by the mRNA molecule is about 1 to about 20 times that of a BioN vector. In some specific embodiments, the Mod vector comprises the 5'UTR shown in SEQ ID NO: 48 or 105; and / or, comprises the 3'UTR shown in SEQ ID NO: 49 or 106.

[0435] In some embodiments, the nucleic acid constructs of the present disclosure are delivered to a subject and express a target protein (e.g., hHGF protein) after about 0.5-1.5 hours. In some embodiments, the nucleic acid constructs of the present disclosure are delivered to a subject and reach peak expression at about 2 hours to 10 hours (e.g., about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 10 hours, etc.). In some specific embodiments, the nucleic acid constructs of the present disclosure are mRNA molecules that express a target protein, for example, the nucleic acid constructs are mRNA molecules that express hHGF protein.

[0436] In some embodiments, the nucleic acid constructs of the present disclosure are delivered to a subject and have a pharmacokinetic property that is higher than that of the Collategene plasmid. In some specific embodiments, the nucleic acid constructs are mRNA molecules that have improved pharmacokinetic properties (e.g., Cmax, AUC, etc.) compared to the Collategene plasmid. 0-inf , MRT 0-inf ).

[0437] In some embodiments, the nucleic acid constructs disclosed herein (e.g., m-A16-B12 (hHGF) expressing hHGF protein) can improve the blood perfusion rate of the lower limb of a subject with ischemia. For example, mice with lower limb ischemia injected with 50 ng or 500 ng of m-A16-B12 (hHGF) can achieve improved blood perfusion rates. In particular, mice with lower limb ischemia injected with approximately 500 ng of m-A16-B12 (hHGF) can achieve recovery of blood perfusion to approximately 90% or greater.

[0438] In some embodiments, the nucleic acid constructs disclosed herein (e.g., m-A16-B12 (hHGF) expressing the hHGF protein) can improve ischemic necrosis in subjects undergoing hind limb ischemia. For example, the hind limbs of mice with hind limb ischemia injected with 50 ng or 500 ng of m-A16-B12 (hHGF) remained intact and did not develop hind limb necrosis.

[0439] In some embodiments, the nucleic acid constructs disclosed herein (e.g., m-A16-B12 (hHGF) expressing hHGF protein) can promote angiogenesis in ischemic lower limb muscles in various groups. For example, administration of 50 ng / animal and 500 ng / animal of m-A16-B12 (hHGF) significantly promoted angiogenesis in ischemic lower limb muscles in each group, with the number of newly formed blood vessels showing statistically significant differences compared to the PBS group (p < 0.05).

[0440] In some embodiments, the nucleic acid constructs disclosed herein (e.g., m-A16-B12 (hHGF) expressing the hHGF protein) can improve wound healing in diabetic subjects. For example, in a Db / Db diabetic mouse model, wounds in mice injected with 50 ng / mouse, 200 ng / mouse, and 500 ng / mouse of m-A16-B12 (hHGF) all healed well. On day 14, the wound healing rate reached 66% with a 50 ng / mouse dose of m-A16-B12 (hHGF), while 100% wound healing was achieved with 200 ng / mouse and 500 ng / mouse doses of m-A16-B12 (hHGF).

[0441] The mRNA provided by the present disclosure contains a 5'UTR and a 3'UTR with a new structure, which reduces or stabilizes the early degradation of the mRNA without losing or enhancing the efficiency of protein translation. The mRNA has higher stability and can be used in gene therapy and gene vaccination. In addition, the present disclosure provides an mRNA capable of expressing human hepatocyte growth factor (hHGF) and a lipid nanoparticle (LNP) delivery system thereof, which can achieve efficient and rapid conversion of exogenous hHGF protein in vivo, has the advantages of no integration risk and easy industrial-grade amplification, and is a more ideal treatment option than naked plasmids. It can be used as a gene therapy drug for various diseases such as CLI and DFU.

[0442] polynucleotides

[0443] The present disclosure also provides an isolated polynucleotide comprising (a) an open reading frame (ORF). Exemplarily, the open reading frame (ORF) encodes a hepatocyte growth factor (HGF), such as human hepatocyte growth factor (hHGF).

[0444] In some embodiments, the coding sequence of hHGF comprises any one selected from the following 1)-3):

[0445] 1) a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 109 or a codon-optimized sequence thereof;

[0446] 2) a DNA sequence as shown in any one of SEQ ID NOs: 110-113, or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto;

[0447] 3) An RNA sequence as shown in any one of SEQ ID NOs: 128-131 or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto.

[0448] In some specific embodiments, the 5' end of the polynucleotide may comprise any 5'UTR provided herein, and / or the 3' end of the polynucleotide may comprise any 3'UTR provided herein.

[0449] In some specific embodiments, the 5' end of the polynucleotide may comprise any 5' Cap provided herein, and / or the 3' end of the polynucleotide may comprise any poly-A tail provided herein.

[0450] In some embodiments, the polynucleotide is RNA, such as mRNA.

[0451] Modification

[0452] To further improve the stability of the RNA or polynucleotides of the present disclosure with respect to protein expression, the RNA or polynucleotides may further comprise one or more modifications (including chemical modifications), such as backbone modifications, sugar modifications, base modifications, and / or lipid modifications. In some embodiments, the RNA or polynucleotides are uniformly modified to a particular modification (e.g., completely modified throughout the sequence). For example, the RNA can be uniformly modified with pseudouridine (e.g., N1-methylpseudouridine) such that every U in the sequence is a pseudouridine.

[0453] The backbone modification relevant to the present disclosure refers to the chemical modification of the phosphate of the backbone of the nucleotide included in the RNA or polynucleotide of the present disclosure. In some embodiments, the backbone modification includes but is not limited to completely replacing the unmodified phosphate part in the backbone with the modified phosphate, for example, the phosphate group of the backbone can be modified by replacing one or more oxygen atoms with different substituents. In some embodiments, the modified phosphate includes but is not limited to phosphorothioate, phosphite selenate, borane phosphate, borane phosphate, hydrogen phosphonate, phosphoramidate, alkyl or aryl phosphonate and phosphotriester.

[0454] Sugar modifications related to the present disclosure refer to chemical modifications of the sugars of the nucleotides contained in the RNA or polynucleotides of the present disclosure. In some embodiments, the sugar modifications include but are not limited to modifying or replacing the 2' hydroxyl (OH) of the RNA molecule with many different "oxy" or "deoxy" substituents. In some embodiments, the "oxy" modification includes but is not limited to substitution modifications of alkoxy, aryloxy, polyethylene glycol (PEG) and the like. In some embodiments, "deoxy" modifications include but are not limited to hydrogen, amino (e.g., NH2, alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino or amino acid) modifications.

[0455] Base modification relevant to the present disclosure refers to the chemical modification of the base portion of the nucleotide contained in the RNA or polynucleotide of the present disclosure. In some embodiments, the base modification includes the modification of adenine, guanine, cytosine and uracil in the nucleotide. For example, the nucleosides and nucleotides described herein can be chemically modified on the main groove surface. In some embodiments, the main groove chemical modification can include amino, thiol, alkyl or halogen groups. In some embodiments, the base modification includes but is not limited to modification with pseudouridine, 1-methyl-pseudouridine, 5-azacytidine, 5-methylcytosine-5'-triphosphate or 2-methoxyadenine. In some embodiments, the base modification is pseudouridine modification. For example, RNA can be uniformly modified with pseudouridine so that each U in the sequence is a pseudouridine.

[0456] Lipid modification in connection with the present disclosure refers to the inclusion of lipid modification in the RNA or polynucleotide of the present disclosure. In some embodiments, the lipid modification includes but is not limited to the covalent attachment of the RNA or polynucleotide of the present disclosure to at least one linker, and the covalent attachment of the corresponding linker to at least one lipid. In some embodiments, the lipid modification includes but is not limited to the covalent attachment of the RNA or polynucleotide of the present disclosure to at least one lipid (without a linker).

[0457] UTRs

[0458] UTRs (5'UTR and / or 3'UTR) can be provided as flanking regions to nucleic acid constructs, RNA or polynucleotide molecules of the present disclosure. UTRs can be homologous or heterologous to the coding regions in nucleic acid constructs, RNA or polynucleotide molecules of the present disclosure. Flanking regions can comprise one or more 5'UTRs and / or 3'UTRs, and the UTRs can be identical or different sequences. Any portion of the flanking regions can be codon optimized. Before and / or after codon optimization, any portion of the flanking regions can independently comprise one or more different structures or chemical modifications.

[0459] In order to change one or more characteristics of the nucleic acid construct, RNA or polynucleotide of the present invention, UTRs heterologous to the ORF of the present invention are introduced or engineered into the nucleic acid construct, RNA or polynucleotide of the present invention. The recombinant nucleic acid construct, RNA or polynucleotide is then applied to cells, tissues or organisms, and the results, such as protein levels, localization and / or half-life, are measured to evaluate the beneficial effects of the heterologous UTR on the RNA or polynucleotide of the present invention. In some embodiments, the UTR includes a wild-type UTR or a variant thereof, and the UTR variant includes adding or removing one or more nucleotides at the end, including A, T, C or G. In some embodiments, the UTR variant also includes codon optimization or modification performed in any manner. In some embodiments, the UTR variant also includes a derivative sequence of any embodiment of the present invention, for example, based on the natural UTR sequence, some nucleotides are subjected to point mutation, and the expression amount and stability of the target gene of the variant after mutation are maintained unchanged or improved. The detection method for the expression amount and stability of the target gene is conventional in the art, such as the detection method in Examples 3 and 4 of the present invention.

[0460] carrier

[0461] The present disclosure also provides a vector, which comprises the nucleic acid construct, RNA or polynucleotide described in any of the foregoing. Wherein the nucleic acid construct, RNA or polynucleotide may be present in a vector and / or may be a part of a vector, such as a plasmid, a cosmid, a YAC or a viral vector. The vector may be an expression vector, i.e., a vector for the expression of a nucleic acid construct, RNA or polynucleotide encoded polypeptide. The expression vector generally comprises at least one nucleic acid of the present disclosure, which may be operably linked to one or more suitable expression control elements (such as promoters, terminators, etc.). It is common sense for those skilled in the art to select the elements and their sequences for expression in a specific host. The regulatory elements and other elements useful or necessary for the expression of the coded polypeptide of the present disclosure are, for example, promoters, terminators, selection markers, leader sequences, reporter genes, etc.

[0462] In some embodiments, the vector is a therapeutic vector capable of expressing the gene of interest (eg, HGF) of the present disclosure, such as a plasmid (eg, naked plasmid), an adenoviral vector, an adeno-associated viral vector, and a lentiviral vector.

[0463] The nucleic acid constructs disclosed herein can 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 disclosed herein, and / or can be isolated from suitable natural sources.

[0464] In some embodiments, the vector of the present disclosure further comprises 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 T7 promoter, T7lac promoter, Tac promoter, Lac promoter, Trp promoter.

[0465] host cells

[0466] The present disclosure also provides a host cell comprising any of the aforementioned nucleic acid constructs, RNA, or polynucleotides. In some embodiments, the cell is capable of expressing one or more polypeptides encoded by the nucleic acid constructs, RNA, or polynucleotides of the present disclosure. In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0467] Examples of bacterial cells include cells of gram-negative bacterial strains (e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains) and gram-positive bacterial strains (e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains).

[0468] Exemplary fungal cells include cells of species of the genera Trichoderma, Neurospora, and Aspergillus; or cells of species of the genera Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0469] Examples of mammalian cells include HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0470] However, the present disclosure may also be used with amphibian cells, insect cells, plant cells, and any other cells known in the art for expressing heterologous proteins.

[0471] Production or preparation method

[0472] The present disclosure provides a method for preparing the nucleic acid construct, RNA or polynucleotide of the present disclosure, and a method for preparing the polypeptide encoded therein.

[0473] Methods and reagents for preparing nucleic acid constructs, RNA or polynucleotides, and their encoded polypeptides, such as specific suitable vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, culture conditions, etc. are known in the art. Similarly, protein isolation and purification techniques suitable for use in the methods of producing the encoded polypeptides of the present disclosure are well known to those skilled in the art.

[0474] In some embodiments, the method for preparing the nucleic acid construct or polynucleotide comprises culturing the aforementioned host cell and recovering the produced nucleic acid construct or polynucleotide from the culture. The nucleic acid construct or polynucleotide of the present disclosure, and its encoded polypeptide can also be obtained by other production methods known in the art, such as chemical synthesis, including solid phase or liquid phase synthesis.

[0475] In some embodiments, the method for preparing an RNA molecule comprises: preparing a nucleic acid construct or vector, and then performing reverse transcription using the nucleic acid construct or vector to obtain an RNA molecule. In some specific embodiments, the method further comprises adding a 5' Cap to the 5' end of the RNA molecule.

[0476] In some embodiments, the RNA may further comprise one or more modifications (including chemical modifications), such as backbone modifications, sugar modifications, base modifications, and / or lipid modifications. In some embodiments, the RNA or polynucleotide is uniformly modified to a particular modification (e.g., completely modified throughout the sequence). For example, the RNA can be uniformly modified with pseudouridine (e.g., N1-methylpseudouridine) such that every U in the sequence is a pseudouridine (e.g., N1-methylpseudouridine).

[0477] Methods for treating diseases and pharmaceutical uses

[0478] The present disclosure provides pharmaceutical uses of the aforementioned lipid nanoparticles, lyophilized compositions, lyophilized preparations, reconstituted solutions, spray preparations or pharmaceutical compositions for preparing drugs for preventing, treating or alleviating the aforementioned diseases or symptoms.

[0479] The present disclosure provides uses and methods of the aforementioned lipid nanoparticles, lyophilized compositions, lyophilized preparations, reconstituted solutions, spray preparations or pharmaceutical compositions in preventing, treating or alleviating diseases or symptoms.

[0480] The present disclosure provides a method for preventing, treating, or alleviating a disease or symptom, comprising administering to a patient or subject a preventively and / or therapeutically effective amount of the aforementioned lipid nanoparticles, lyophilized composition, lyophilized formulation, reconstituted solution, spray formulation, or pharmaceutical composition.

[0481] In some embodiments, the present invention provides a method for preventing, treating, or alleviating a disease or symptom, comprising administering an effective amount of the aforementioned lipid nanoparticles, lyophilized composition, lyophilized formulation, reconstituted solution, spray formulation, or pharmaceutical composition to a subject in need thereof.

[0482] In some embodiments, the disease or condition is selected from ischemic disease, metabolic syndrome, diabetes and its complications, restenosis, and nerve damage;

[0483] Preferably, the ischemic disease is selected from coronary artery disease (CAD), peripheral arterial disease (PAD), myocardial infarction, limb ischemia, thromboangiitis obliterans (TAO), and diabetic arteriosclerosis obliterans (DAO); more preferably, the limb ischemia is lower limb ischemia, and most preferably, the limb ischemia is critical lower limb ischemia (CLI);

[0484] Preferably, the diabetes and its complications are selected from diabetic peripheral neuropathy, diabetic foot (DFU), and diabetic arteriosclerosis obliterans (DAO);

[0485] Preferably, the restenosis is selected from post-operative restenosis and post-perfusion restenosis;

[0486] Preferably, the nerve damage is selected from neurodegenerative diseases, traumatic nerve injury, and peripheral neuropathy; more preferably, the neurodegenerative disease is selected from amyotrophic lateral sclerosis (ALS), Parkinson's disease, and dementia, and the peripheral neuropathy is diabetic peripheral neuropathy.

[0487] The pharmaceutical composition of the present disclosure can be used to treat patients in need of such treatment by parenteral administration. The parenteral administration route can be selected from subcutaneous injection, intramuscular injection or intravenous injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0488] Figures 1A to 1C are schematic diagrams of vector construction. Among them, Figure 1A is a schematic diagram of the construction of a 5'UTR element screening vector, and the control is an mRNA sequence containing Moderna's 5'UTR and 3'UTR elements, i.e., 5'UTR-Fluc-α globulin 3'UTR-120A (abbreviated as Mod.), whose 5'-UTR is an artificial nucleic acid sequence, and the 3'UTR is derived from the mRNA of human α globulin. When constructing a 5'UTR screening vector, the 5'UTR region is replaced by a suitable restriction enzyme site. PmeI is a linearization restriction enzyme site. Figure 1B is a schematic diagram of the construction of a 3'UTR element screening vector, and the control is Mod. When constructing a 3'UTR screening vector, the 3'UTR region is replaced by a suitable restriction enzyme site. Figure 1C is a schematic diagram of the construction of a 5' and 3'UTR element combination screening vector, and the control is Mod. When constructing a 5'UTR and 3'UTR element combination screening vector, the 5' and 3'UTR regions or full gene synthesis are replaced by suitable restriction enzyme sites.

[0489] Figure 2 shows the results of evaluating the effects of different 3'UTR elements disclosed herein 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 effect of the 3'UTR sequence on protein expression. Mod. was used as a control, and the expression level of Mod. was set to 1. The results showed that the effect of the 3'UTR element on protein expression was consistent across different cell lines.

[0490] Figure 3 shows the effects of different 3'UTR elements on mRNA expression efficiency. mRNA containing different 3'UTR elements was transduced into HEK293 cells via lipofection, and luciferase expression levels were measured 6, 24, 48, and 72 hours after transfection. Mod. was used as a control, and its expression level was set to 1. The results showed that 3'UTR elements numbered B9, B10, B12, B13, and B14 significantly increased protein expression.

[0491] Figure 4 shows the effects of different 5'UTR elements on mRNA expression efficiency. mRNA containing different 5'UTR elements was transduced into HEK293 cells via lipofection, and luciferase expression levels were measured 6, 24, 48, and 72 hours after transfection. Mod. was used as a control, and its expression level was set to 1. The results showed that 5'UTR elements numbered A1, A3-A7, and A9-A14 significantly increased protein expression.

[0492] Figure 5 shows the effects of different 5'UTR elements on mRNA expression efficiency. mRNA containing different 5'UTR elements was transduced into HEK293 cells by lipofection, and the expression level of luciferase was measured 6h, 24h, 48h, and 72h after transfection. Mod. was used as a control, and its expression level was set to 1. The results showed that 5'UTR elements numbered A15, A16, A18-A19, A21, A24, A27, A28, and A30-A33 significantly increased protein expression.

[0493] Figure 6 shows the effects of different 5'UTR elements on mRNA expression efficiency. mRNAs containing different 5'UTR elements were transduced into HEK293 cells via lipofection, and luciferase expression levels were measured 6 hours, 24 hours, and 48 hours after transfection. BioN. was used as a control, and its expression level was set to 1. The results showed that 5'UTR elements numbered A1, A15, A16, and A18 significantly increased protein expression compared to the control nucleic acid molecules.

[0494] Figure 7 shows the effects of the disclosed UTR element combinations on mRNA expression efficiency. 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. Mod. was used as a control, and its expression level was set to 1. The results showed that the combination of 5'UTR elements numbered A1, A15, A16, and A18 with 3'UTR elements numbered B12, B13, and B14 significantly increased protein expression.

[0495] Figures 8A to 8B show the effects of the UTR element combinations of the present disclosure on the expression efficiency of different target proteins. Figure 8A shows the regulation of hHGF expression in HEK293 cells by mRNA containing the 5'UTR and 3'UTR elements screened by the present disclosure. The results show that compared with the control Mod., the combination of 5'UTR elements numbered A1A15, A16, and A18 and 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 in HEK293 cells by mRNA containing the 5'UTR and 3'UTR elements screened by the present disclosure. The results show that compared with the control Mod., the UTR combination of A1-B12 and A15-B12 can significantly increase the expression of anti-PD-1 antibody.

[0496] Figure 9 is a schematic diagram of the experimental results of the present disclosure, showing the changes in hHGF protein expression levels over time in mouse muscle tissue using m-A16-B12 (hHGF) and a control plasmid. The results show that both m-A16-B12 (hHGF) and Collategene plasmids effectively express hHGF protein. m-A16-B12 (hHGF) can express hHGF as early as one hour after intramuscular injection, with peak hHGF expression occurring six hours after injection. This is dose-dependent, and even low doses of m-A16-B12 (hHGF) can achieve comparable expression levels (AUCinf) (hr*pg / mg protein) as Collategene.

[0497] Figures 10A and 10B illustrate the therapeutic effects of m-A16-B12 (hHGF) and a control plasmid in a mouse model of lower limb ischemia. Figure 10A is a schematic diagram of the research plan and photographs of experimental results demonstrating the effects of m-A16-B12 (hHGF) and the Collategene plasmid on blood perfusion in a mouse model of lower limb ischemia. Figure 10B is a statistical analysis of the effects of m-A16-B12 (hHGF) and the Collategene plasmid on blood perfusion ratios in a mouse model of lower limb ischemia. The results showed that when treated with 50ng / mouse, 500ng / mouse m-A16-B12 (hHGF) and 200ng / mouse Collategene naked plasmid, the blood perfusion ratio of the ischemic lower limbs of each mouse was significantly improved compared with the control group, and the blood perfusion ratio of the ischemic lower limbs was gradually restored over time; the 50ng / mouse m-A16-B12 (hHGF) group showed a blood flow recovery effect similar to that of the 200ng / mouse Collategene naked plasmid group; the 500ng / mouse m-A16-B12 (hHGF) group had a blood flow recovery effect significantly better than that of the 200ng / mouse Collategene naked plasmid group, and could achieve a blood perfusion recovery of more than 90%.

[0498] Figures 11A and 11B show the therapeutic effects of the disclosed m-A16-B12 (hHGF) and control plasmids on a mouse model of lower limb ischemia. Figure 11A shows the scoring criteria and representative photographs for varying degrees of lower limb necrosis in a mouse model of lower limb ischemia. Figure 11B shows statistical results demonstrating the effects of m-A16-B12 (hHGF) and Collategene plasmids on lower limb necrosis in a mouse model of lower limb ischemia. The experimental results showed that in the groups treated with m-A16-B12 (hHGF) and Collategene naked plasmids, the lower limbs of all mice remained intact and no lower limb necrosis occurred.

[0499] Figure 12 shows representative photographs and statistical results of the effects of m-A16-B12 (hHGF) and a control plasmid on angiogenesis in a mouse model of lower limb ischemia. The results show that treatment with 50 ng and 500 ng of m-A16-B12 (hHGF) per mouse, as well as 200 ng of Collategene naked plasmid per mouse, significantly promoted angiogenesis in ischemic lower limb muscles.

[0500] Figure 13 shows the treatment regimen, representative photos, and statistical results of the effects of m-A16-B12 (hHGF) and a control plasmid on wounds in a Db / Db mouse model with full-thickness cortical injury. The results showed that compared to the control group, the wounds of mice treated with 50 ng / mouse, 200 ng / mouse, and 500 ng / mouse of m-A16-B12 (hHGF) and 200 μg / mouse of Collategene naked plasmid healed well, and m-A16-B12 (hHGF) significantly promoted wound healing compared to 200 μg / mouse of Collategene naked plasmid.

[0501] Figure 14 shows representative photographs demonstrating the effects of m-A16-B12 (hHGF) and a control plasmid on tissue remodeling in a full-thickness cortical injury Db / Db mouse model. The results show that m-A16-B12 (hHGF) treatment achieved complete epithelial coverage and tissue remodeling in all groups of mice. However, neither the control group nor the 200 μg / mouse Collategene naked plasmid group achieved complete epithelial remodeling, with abnormal collagen proliferation and structural disorganization at the wound site.

[0502] Figure 15: Particle size of formulations 1-4 in different buffers before lyophilization.

[0503] Figure 16: Particle size of formulations 1-4 after lyophilization and reconstitution.

[0504] Figure 17: Particle size change rate of formulations 1-4 before and after freeze-drying.

[0505] Figure 18: mRNA integrity of formulations 1-4 after lyophilization.

[0506] Figure 19: mRNA integrity of formulations 7-9 after lyophilization.

[0507] Figure 20: mRNA integrity of Formulations 1-4 at 37°C.

[0508] Figure 21: mRNA integrity of formulations 7-9 at 37°C.

[0509] Figure 22: Fluorescence intensity of cell transfection expression of freeze-dried products of formulations 7 and 9. DETAILED DESCRIPTION

[0510] 1. Terminology

[0511] To facilitate understanding of the present disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present disclosure pertains. The three-letter and one-letter amino acid codes used herein are as described in J. Biol. Chem, 243, p3558 (1968).

[0512] "Sequence" should generally be understood to include both the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding the sequence, unless the present disclosure requires further limited explanation.

[0513] "Homology" or "identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if every position in two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, if 6 out of 10 positions in the two sequences match or are homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. Generally, a comparison is made when the two sequences are aligned to achieve the maximum percent homology.

[0514] "About" and "approximately" refer to values ​​that are within an acceptable error range for the specific value as determined by one of ordinary skill in the art, which value depends in part on how it is measured or determined (i.e., the limits of the measurement system). For example, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" or "substantially comprising" can mean a range of up to 20%, such as between 1% and 15%, between 1% and 10%, between 1% and 5%, between 0.5% and 5%, between 0.5% and 1%, and in this disclosure, each instance of a number or numerical range preceded by the term "about" also includes embodiments of the given number. Unless otherwise stated, when a specific value appears in the present disclosure and claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that specific value.

[0515] "Buffer" refers to a buffer that tolerates changes in pH through the action of its acid-base conjugate components. Examples of buffers that control pH within an appropriate range include tris (Tris), acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0516] A "histidine salt buffer" is a buffer containing histidine ions. Examples of histidine salt buffers include histidine hydrochloride, histidine acetate, histidine phosphate, histidine sulfate, and the like. Preferred are histidine hydrochloride buffers or histidine acetate buffers. Histidine acetate buffers are prepared from histidine and acetic acid, and histidine hydrochloride buffers are prepared from histidine and histidine hydrochloride, or histidine and hydrochloric acid.

[0517] "Phosphate buffer" is a buffer containing phosphate ions. Examples of phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-citric acid, and the like. Preferably, the phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.

[0518] A "pharmaceutical composition" refers to a mixture containing one or more antibodies described herein with other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to maintain the stability of the active ingredient, facilitate administration to an organism, and promote absorption of the active ingredient to exert its biological activity.

[0519] In the present disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0520] Unless otherwise specified, the solvent in the solution form of the pharmaceutical composition described in the present disclosure is water.

[0521] "Lyophilized formulation" refers to a pharmaceutical composition in liquid or solution form or a formulation or pharmaceutical composition obtained after a liquid or solution formulation has been subjected to a vacuum freeze-drying step.

[0522] "Administer," "give," and "treat," as they apply to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administer," "give," and "treat" can refer to, for example, therapeutic, pharmacokinetics, diagnostics, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administer," "give," and "treat" also mean the in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic, a binding composition, or by another cell. "Treatment," as it applies to humans, veterinary medicine, or research subjects, refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0523] "Treatment" means administering an internal or external therapeutic agent, such as any of the antibodies disclosed herein or pharmaceutical compositions thereof, to a subject who has, is suspected of having, or is predisposed to having one or more proliferative diseases or symptoms thereof, and for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to the treated subject or population in an amount effective to alleviate one or more symptoms of the disease, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable extent. The amount of the therapeutic agent that effectively alleviates any specific disease symptom (also referred to as a "therapeutically effective amount") can vary according to a variety of factors, such as the disease state, age, and weight of the subject, and the ability of the drug to produce the desired therapeutic effect in the subject. Whether the symptoms of the disease have been alleviated can be evaluated by any clinical detection method commonly used by doctors or other health care professionals to evaluate the severity or progression of the symptoms. Although an embodiment of the present disclosure (e.g., a method of treatment or article of manufacture) may not be effective in alleviating the symptoms of the target disease in a certain subject, it should alleviate the symptoms of the target disease in a statistically significant number of subjects as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0524] An "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also refers to an amount sufficient to permit or facilitate diagnosis. The effective amount for a subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects. The subject of the present disclosure may be an animal or a human subject.

[0525] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0526] "Subject" or "patient" refers to a mammal, particularly a primate, and especially a human.

[0527] Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "having," "including," etc. should be construed to have an inclusive sense rather than an exclusive or exhaustive sense; that is, in the sense of "including but not limited to."

[0528] The term "lipid phase" refers to the general term for all lipids in lipid nanoparticles, including but not limited to cationic lipids, non-cationic lipids, phospholipids, cholesterol or its derivatives, conjugated lipids, and other lipids that are well known to those skilled in the art and can be used to prepare lipid nanoparticles.

[0529] The term "lipid" refers to a class of organic compounds that include, but are not limited to, esters of fatty acids and are characterized by being insoluble in water but soluble in many organic solvents. They are generally divided into at least three categories: (1) "simple lipids," which include fats and oils as well as waxes; (2) "compound lipids," which include phospholipids and glycolipids; and (3) "derivative lipids" such as steroids.

[0530] The term "cationic lipid" refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH (eg, pH about 7.0).

[0531] The term "anionic lipid" refers to any lipid that is negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and other anionic modifying groups attached to neutral lipids.

[0532] The term "neutral lipid" refers to any of a number of lipid species that exist as uncharged or neutral zwitterionic forms at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerol.

[0533] The term "amphiphilic lipid" refers, in part, to any suitable material, wherein the hydrophobic portion of the lipid material is oriented to the hydrophobic phase, while the hydrophilic portion is oriented to the aqueous phase. The hydrophilic nature results from the presence of polar or charged groups such as carbohydrates, phosphates, carboxyls, sulfates, aminos, sulfhydryls, nitros, hydroxyls, and other similar groups. Hydrophobicity can be imparted by including non-polar groups, including but not limited to long-chain saturated and unsaturated aliphatic hydrocarbons and such groups substituted by one or more aromatic, alicyclic, or heterocyclic groups. Examples of amphiphilic compounds include but are not limited to phospholipids, amino lipids, and sphingolipids.

[0534] The term "non-cationic lipid" refers to any amphipathic lipid as well as any other neutral lipid or anionic lipid.

[0535] The term "conjugated lipid" refers to a conjugated lipid that inhibits aggregation of lipid particles. The conjugated lipid includes, but is not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0536] The term "mammal" refers to any mammalian species such as humans, mice, rats, dogs, cats, hamsters, guinea pigs, rabbits, livestock, and the like.

[0537] The "average particle size" (Z-average Size) described herein, for example, "average particle size less than 2000 nm," is the average light intensity, calculated from the light intensity contributed by different types of particles. The "average particle size" can be measured using conventional particle size measurement techniques familiar to those skilled in the art. Such techniques include, for example, sedimentation field flow fractionation, photon correlation spectroscopy, and light scattering.

[0538] The polydispersity index (PDI) of the present disclosure reflects the uniformity of particle size and is an important index for characterizing particle size.

[0539] The terms "mixed" and "mixed" mean that the order of adding the components is not limited. For example, mixing A into B can mean A is added to B, or B is added to A. Mixing A and B can mean A is added to B and the mixture can also mean B is added to A and the mixture can also mean B is added to A and the mixture can also mean.

[0540] The numerical values ​​in this disclosure are instrumental measurements and are subject to a certain degree of error. Generally speaking, within a reasonable error range of plus or minus 10%. The context in which the numerical value is used must be considered. For example, the particle size of an active ingredient, where the error after measurement does not exceed plus or minus 10%, may be plus or minus 9%, plus or minus 8%, plus or minus 7%, plus or minus 6%, plus or minus 5%, plus or minus 4%, plus or minus 3%, plus or minus 2%, or plus or minus 1%, preferably plus or minus 5%.

[0541] The compound of formula I disclosed in the present invention is prepared by referring to the method in WO2023125738A, and the relevant content is disclosed herein for illustration.

[0542] The equipment and methods used in the testing process are as follows:

[0543] Total content and encapsulation efficiency:

[0544] mRNA-LNPs, when exposed to 2% Triton X-100, release mRNA encapsulated in lipids. The mRNA binds to RiboGreen and can be read on a microplate reader in fluorescence mode, selecting excitation at 485 nm and emission at 535 nm. Fluorescent dye assays are used to measure mRNA levels in samples before and after release from Triton X-100 treatment. Free RNA and total RNA levels are measured, respectively. The difference between total RNA and free RNA represents the ratio of total RNA to total RNA, representing the sample encapsulation efficiency.

[0545] Total content and encapsulation efficiency detection instrument: Molecular Devices-multi-function microplate reader.

[0546] dsRNA detection:

[0547] The double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) method is used to detect the dsRNA content in the original solution of in vitro transcribed RNA products. A microplate is coated with a capture antibody to form a solid-phase antibody. The dsRNA standard and the sample to be tested are added to the solid-phase antibody microplate, followed by the detection antibody. Finally, a horseradish peroxidase-labeled enzyme-labeled secondary antibody is added to form a "coated antibody-antigen-enzyme-labeled detection antibody" complex. After washing, a color developing solution is added for color development. The color developing solution turns blue under the catalysis of HRP and eventually yellow under the action of acid. The color depth is positively correlated with the amount of dsRNA in the sample.

[0548] dsRNA detection instrument: Molecular Devices-Multi-function Microplate Reader

[0549] HGF detection:

[0550] In this experiment, supernatants from HEK293 cells infected with mRNA (liposome-formulated) for three days were collected as test samples. Using a double-antibody sandwich ELISA, anti-human HGF antibodies were coated onto microplates. Human HGF in the samples and standards bound to the antibodies immobilized on the plate, and free components were washed away. Biotinylated anti-human HGF detection antibodies were then added and incubated. Unbound material was washed away, and streptavidin-labeled horseradish peroxidase (Streptavidin-HRP) was added and incubated. After washing away unbound reagents, TMB substrate solution (developer) was added. The color of the solution was proportional to the bound target protein. Stop solution was added, and the absorbance was measured using a microplate reader.

[0551] HGF detection instrument: Molecular Devices-Multi-function Microplate Reader

[0552] Examples and test cases

[0553] The present disclosure is further described in detail by the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present disclosure.

[0554] Experimental methods in the examples disclosed herein, where specific conditions are not specified, generally followed conventional conditions or the conditions recommended by the raw material or commercial manufacturer. See Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory; and Current Methods in Molecular Biology, Ausubel et al., Greene Publishing Associates, Wiley Interscience, NY. Reagents whose sources are not specified were commercially available.

[0555] Example 1. Preparation of mRNA

[0556] 1.1 Screening of 5'UTR and 3'UTR

[0557] In order to screen for 3'-untranslated region elements (3'UTR elements) and 5'-untranslated region elements (5'UTR elements) that can improve protein expression efficiency, this example screened the UTR sequences in housekeeping gene mRNAs.

[0558] First, 301 candidate housekeeping genes, including Abhd16a, were identified using the database (https: / / esbl.nhlbi.nih.gov / helixweb / Database / NephronRNAseq / Housekeeping_Genes.htmL). Then, bioinformatics methods were used to analyze and rank the expression levels of these genes, and to retrieve gene transcript and UTR sequence information. UTR elements were further screened based on gene expression levels and UTR sequence lengths, resulting in multiple candidate UTR sequences. Thirty-three 5'UTRs, numbered A1 to A33, and 14 3'UTRs, numbered B1 to B14, were identified. The gene sources and sequence numbers are shown in Tables 1 and 2.

[0559] Table 1. New 3'UTRs obtained through screening

[0560] Table 2. New 5'UTRs obtained by screening

[0561] 1.2 mRNA preparation

[0562] The UTR obtained by screening in 1.1 is constructed into a DNA vector for in vitro transcription to obtain stably expressed mRNA of 5'UTR and 3'UTR elements. The vector contains a T7 promoter, a sequence (SEQ ID NO: 126) encoding firefly luciferase (Firefly Luciferase, Fluc) as an open reading frame (ORF), and a polyadenylic acid (polyA) sequence, which is followed by a restriction site for linearization of the vector. The polyA is selected from A120 (i.e., 120 continuous adenylic acids) or A30L70 (SEQ ID NO: 52). By suitable restriction enzyme sites, 5'UTR and 3'UTR element constructions are respectively constructed to the 5' end and 3' end of the open reading frame ORF (Fluc). The constructed vector example is shown in Figure 1A to Figure 1C, and the combination of 5'UTR and 3'UTR is shown in Table 3.

[0563] The control vectors used in this example include Mod.-120A (SEQ ID NO: 53), Mod.-A30L70 (SEQ ID NO: 54), and BioN.-A30L70 (SEQ ID NO: 55), each containing a polyA of A120 (SEQ ID NO: 53), and the target gene is Fluc, which was constructed by whole-gene synthesis. Different UTR elements were constructed into the above vectors by enzyme digestion. Among them, the construction vectors of vectors V-B1 to V-B14 were Mod.-120A, and the restriction sites were AgeI and SacII. The poly A of each was A120, and the control used was Mod.-120A (SEQ ID NO: 53); the construction vectors of vectors V-A1 to V-A14 were Mod.-120A (SEQ ID NO: 53), and the restriction sites were BamHI and NheI. The poly A of each was A120, and the control used was also Mod.-120A (SEQ ID NO: 53); the construction vectors of V-A15 to V-A33 were Mod.-A30L70 (SEQ ID NO: 54), and the restriction sites were BamHI and NheI. The poly A of each was A30L70, and the control used was Mod.-A30L70 (SEQ ID NO: 55). NO:54); the construction vector of vectors V-A1-B12, V-A1-B13, and V-A1-B14 is Mod.-A30L70 (SEQ ID NO:54), and the restriction sites are BamHI and SacII. The poly A of each is A30L70. The controls used 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 into the corresponding vectors by restriction enzyme digestion and ligation. After the vector construction is completed, it needs to be digested and sequenced for identification. After correct identification, it is used in the next experiment.

[0564] Table 3. Combinations of 5'UTR and 3'UTR in vectors

[0565] The vectors (i.e., DNA templates) in Table 3 were digested by restriction endonuclease treatment to linearize the DNA templates and then transcribed in vitro using T7-RNA polymerase. For in vitro transcription, T7 RNA polymerase (Roche), the corresponding reaction buffer, pyrophosphatase, RNase inhibitor, and NTP were used. In order to effectively cap RNA, an excess of the cap-like compound ARCA (3'-O-Me-m 7G(5')ppp(5')G, Trilink, N-7003-1) or CleanCap (m7G(5')ppp(5')(2'OMeA)pG, Trilink, N-7113), wherein the RNA used in Figures 2 to 5 was capped with ARCA, and the RNA used in Figures 6 to 9 was capped with CleanCap. At the same time, in order to reduce the immunogenicity of mRNA and improve translation efficiency, the mRNA in the present disclosure was nucleic acid modified, and all uridine triphosphate (UTP) in the reaction system was replaced with N1-methyl-pseudouridine triphosphate (1m-ψUTP, purchased from ThermoFisher). The modification method refers to patent US2014 / 0194494A1. After incubation at 37°C for 2.5 hours, the in vitro transcription system was purified using carboxylated magnetic beads (Invitrogen) and resuspended in nuclease-free water. RNA concentration and quality were assessed by spectrophotometry and analysis on an Agilent 5200 Bioanalyzer. The target mRNA was obtained.

[0566] The sequences from the 5'UTR to the polyA region of Mod. of the present disclosure are shown in SEQ ID NOs: 53 and 54, and the sequences from the 5'UTR to the polyA region of BioN. are shown in SEQ ID NO: 55. The 5'UTR and 3'UTR sequences in Mod. are consistent with those in Moderna's patents and publications (US10849920B2, WO2013151667A1, US10730924B2, DOI: 10.1016 / j.cell.2017.02.017). The 5'UTR and 3'UTR sequences of BioN. are derived from patent WO 2018 / 160540.

[0567] As an example, 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.

[0568] 1.3 Functional validation of 5'UTR, 3'UTR, and their combination

[0569] In this example, the luciferase expression system using mRNA lipid transfection was used to verify the function of UTR.

[0570] Experimental method: Human embryonic kidney cells (HEK293, purchased from ATCC) were cultured at 4×10 4The cells were seeded at a density of 10 cells / well in a 96-well plate. TM MessengerMAX TM Transfection was performed using an mRNA transfection reagent, with 100 ng of the capped mRNA prepared in Example 2 being transfected per well. Six hours after transfection, fresh medium was replaced. 100 μL of medium was aspirated and 50 μL of luciferase substrate was added. Luminescence intensity was measured using a PerkinElmer multi-function microplate reader. Human cervical cancer cells (HeLa, purchased from ATCC) and human lung cancer cells (A549, purchased from ATCC) were assayed using the same protocol as HEK293.

[0571] Evaluation Method: In Tables 4 to 7 and 9 to 11, the fluorescein expression level of the reference positive control Mod. in different cell lines was set to 1, and the expression levels of different mRNAs relative to the positive control Mod. were calculated. In Table 8, the fluorescein expression level of the reference positive control BioN. in different cell lines was set to 1, and the expression levels of different mRNAs relative to the positive control BioN. were calculated. The values ​​in the table are all multiples of the average detection value.

[0572] 1) Detection of the ability of 3'UTR to regulate target gene expression in different cell lines

[0573] To verify the ability of different 3'UTRs to regulate target gene expression in different cell lines, mRNA expressing luciferase (Fluc) was produced using the method of Example 2. The produced RNA was ARCA-capped with an A120 polyA tail. The 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.

[0574] Table 4. Expression of luciferase (Fluc) expressed in mRNAs carrying different 3'UTRs (ARCA capping, target genes are all Fluc, poly A is all A120)

[0575] The results showed that in different cell lines, compared with the Mod. positive control, the effects of the 3'UTRs of B1 to B14 on protein expression were consistent, and there were no significant differences between different cell lines (p>0.05), indicating that the effects of the 3'UTR elements obtained by the present disclosure on protein expression are universal and can be used to increase the protein expression level of the target gene.

[0576] 2) Detection of the duration of target gene expression regulated by 3'UTR in cells

[0577] To test the ability of different 3'UTRs to regulate the expression time of target genes in cells, the mRNAs listed in Table 4 were transfected into HEK293 cells, and the luciferase expression levels were measured 6 h, 24 h, 48 h, and 72 h after transfection. The results are shown in Table 5 and Figure 3.

[0578] Table 5. Expression levels of luciferase (Fluc) expressed by mRNAs carrying different 3'UTRs

[0579] The results showed that in HEK293 cells, changes in 3'UTR elements could affect the expression level of the target protein. Except for the B5-3'UTR element, other 3'UTR elements could increase protein expression by more than 1.2 times, and there were significant differences at different detection time points (p<0.05). Among them, the 3'UTR elements of B12, B13, and B14 had the best effect, which could increase protein expression by more than 2 times and had good time continuity.

[0580] 3) Detection of 5'UTR regulation of target gene expression in cells

[0581] To test the ability of different 5'UTRs to regulate the expression time of target genes in cells, mRNA expressing luciferase (Fluc) was prepared using the aforementioned method. The RNAs used in Table 6 and Figure 4 were ARCA-capped with an A120 polyA tail, while the RNAs used in Table 7 and Figure 5 were CleanCap-capped with an A30L70 polyA tail. The RNAs produced above were transfected into HEK293 cells, and luciferase expression levels were measured 6, 24, 48, and 72 hours after transfection. The results are shown in Tables 6 and 7 and Figures 4 and 5.

[0582] Table 6. Expression of luciferase (Fluc) expressed in mRNAs carrying different 5'UTRs (ARCA capping, target genes are all Fluc, poly A is all A120)

[0583] Table 7. Expression of luciferase (Fluc) expressed in mRNAs carrying different 5'UTRs (CleanCap capping, target genes are all Fluc, polyA is all A30L70)

[0584] Figures 4 and 5 were screened using different capping methods and polyA tail structures, respectively. The results showed that in different screening vectors, changes in the 5'UTR element can affect the expression level of the target protein, and the 5'UTR element has universality in improving the expression level of the target protein. Among them, the 5'UTR elements numbered A1, A3 to A7, A9 to A16, A18, A21, A24, A27, A28, A32, and A33 can all increase protein expression by more than 1.2 times, and there are significant differences at the detection time points of 24h, 48h, and 72h (p<0.05). Among them, 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.

[0585] 4) Detection of the expression intensity of target gene regulated by candidate 5'UTR compared with the positive control BioN. vector

[0586] To test the ability of different candidate 5'UTRs to regulate the expression intensity of the target gene compared to the positive control BioN. vector, the preferred 5'UTR elements A1, A15, A16, and A18 screened above were constructed into a vector containing an A30L70 polyA tail element. Luciferase (Fluc) mRNA was produced using the aforementioned method and transfected into HEK293 cells. Luciferase expression levels were measured 6 h, 24 h, 48 h, and 72 h after transfection. The results are shown in Table 8 and Figure 6.

[0587] Table 8. Expression of luciferase (Fluc) expressed in mRNAs carrying different 5'UTRs (Cleancap capping, target genes are all Fluc, poly A is all A30L70)

[0588] The results showed that the candidate 5'UTR elements could increase protein expression by at least 1.2 times compared with the BioN. vector at 24h and 48h, and the differences were significant (p<0.05).

[0589] 5) Detection of the regulation of target gene expression by different 5'UTR and 3'UTR combinations in cells

[0590] To investigate the effects of different 5'-UTR and 3'UTR combinations on target protein expression in mRNA, mRNAs containing different combinations of 5'UTR and 3'UTR elements were compared with the 5'UTR and 3'UTR selected by Moderna. The results are shown in Table 9 and Figure 7 .

[0591] Table 9. Expression levels of mRNAs carrying different 5'UTR and 3'UTR combinations (Cleancap capping, target genes are all Fluc, polyA is all A30L70)

[0592] The results showed that the 5'UTR and 3'UTR element combinations screened in this disclosure increased protein expression by more than 1.3-fold compared to the 5'UTR and 3'UTR elements selected by Moderna at the 24h, 48h, and 72h detection time points, with significant differences (p < 0.05). The target proteins in 1)-5) above are all luciferase (Fluc), and the target protein in 6) below is a secreted protein (e.g., hHGF, anti-PD-1 antibody).

[0593] 6) Detection of different 5'UTR and 3'UTR combinations regulating the expression of secreted target proteins

[0594] In order to study the effects of different 5'-UTR and 3'UTR combinations on the expression of secreted proteins by mRNA, the target protein expressed by mRNA was constructed as ORF, which was human hepatocyte growth factor (hHGF) or anti-PD-1 antibody.

[0595] 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 their optimized codon sequences are shown in SEQ ID NOs: 129-131.

[0596] The amino acid sequences of the heavy chain and light chain of the PD-1 antibody are shown in SEQ ID NOs: 117 and 118, respectively, the DNA sequences of the heavy chain and light chain are shown in SEQ ID NOs: 119 and 120, respectively, and the mRNA sequences of the heavy chain and light chain are shown in SEQ ID NOs: 121 and 122, respectively.

[0597] First, to improve the translation efficiency of hHGF protein, the present disclosure codon-optimized the nucleotide sequence of wild-type hHGF (SEQ ID NO: 110). The optimized sequences are hHGF-OS1 (SEQ ID NO: 111), hHGF-OS2 (SEQ ID NO: 112), and hHGF-OS3 (SEQ ID NO: 113), and the corresponding mRNA sequences are shown in SEQ ID NOs: 129-131, respectively. The native hHGF and codon-optimized hHGF sequences were constructed into vectors to produce mRNA. The expression level of hHGF protein was measured by ELISA, demonstrating that the codon-optimized hHGF sequences can increase hHGF protein expression.

[0598] hHGF-OS2 mRNAs containing different combinations of 5'UTR and 3'UTR elements (DNA sequence: SEQ ID NO: 112) were compared with the hHGF-OS2 mRNA Mod.(hHGF) (DNA sequence: SEQ ID NO: 114) containing different combinations of 5'UTR and 3'UTR elements from Moderna. HEK293 cells were transfected with the hHGF mRNAs. Supernatants were collected 6, 24, 48, and 72 hours after transfection and assayed for hHGF protein expression by ELISA to assess the ability of the candidate vectors to increase / prolong HGF expression. Results are shown in Table 10 and Figure 8A.

[0599] Table 10. Target protein expression levels of mRNAs carrying different 5'UTR and 3'UTR combinations (Cleancap capping, target genes are all hHGF, poly A is all A30L70)

[0600] mRNAs expressing full-length anti-PD-1 antibodies containing different combinations of 5'UTR and 3'UTR elements (corresponding DNA sequences are SEQ ID NOs: 124-125) were compared with mRNA expressing anti-PD-1 antibodies containing combinations of 5'UTR and 3'UTR elements from Moderna (corresponding DNA sequence is SEQ ID NO: 123). HEK293 cells were transfected with mRNA encoding full-length anti-PD-1 antibodies. Supernatants were collected 6, 24, 48, and 72 hours after transfection and assayed for anti-PD-1 antibody expression by ELISA to assess the increased / extended expression of anti-PD-1 antibodies by candidate vectors. The results are shown in Table 11 and Figure 8B.

[0601] Table 11. Target protein expression levels of mRNAs carrying different 5'UTR and 3'UTR combinations (Cleancap capping, target genes are all anti-PD-1 antibody heavy and light chains, poly A is A30L70)

[0602] The above results show that after 72 hours of continuous expression of secretory protein hHGF and anti-PD-1 antibody, the 5'UTR and 3'UTR combination screened by the present disclosure can significantly increase the expression level of secretory protein compared with the 5'UTR and 3'UTR elements selected by Moderna (p<0.05).

[0603] 1.4 Verification of hHGF protein expression in mice by hHGF mRNA

[0604] To determine the ability of the mRNA molecule m-A16-B12 (hHGF) (corresponding DNA sequence is SEQ ID NO: 127) to express hHGF in animals, lipid nanoparticles LNP (comprising 50 mol% ionizable lipid (SM-102), 10 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% PEG-DMG) were used for mRNA delivery. The same lipid nanoparticles LNP were also used in Examples 5 and 6 below. The positive control was Collategene plasmid (AnGes), which was administered via naked plasmid administration. Balb / c mice (6-8 weeks, male) were randomly divided into 4 groups, with 33 mice in each group. Different doses of m-A16-B12 (hHGF) and Collategene plasmid were injected into the gastrocnemius muscle of the mice. Then, the experimental groups were arranged as follows according to the scheme in Table 12: 1) injection of 1.0 μg / mouse m-A16-B12 (hHGF); 2) injection of 0.3 μg / mouse m-A16-B12 (hHGF); 3) injection of 0.1 μg / mouse m-A16-B12 (hHGF); 4) injection of 200 μg / mouse naked Collategene plasmid. Gastrocnemius muscle samples were obtained from mice at 1, 2, 4, 6, 24, 48, 72, 96, 168, 216, and 336 hours post-incubation. Muscle tissue was homogenized using RIPA lysis buffer containing protease inhibitors, and the supernatant was collected by centrifugation. hHGF protein concentration was determined using a BCA protein concentration assay kit, and hHGF expression was assessed using ELISA. Data are presented as mean ± standard deviation (SD) for each group, and graphs and statistics were generated using Graphpad Prism 9.0 software.

[0605] Table 12. Experimental groups of m-A16-B12 (hHGF) and Collategene in vivo expression of hHGF

[0606] The experimental results are shown in Figure 9A. m-A16-B12 (hHGF) can express hHGF protein 1 hour after intramuscular injection, and reaches the peak expression 6 hours after injection, showing a dose-dependent manner. As shown in Figure 9B, Collategene reaches the peak expression 7 days after injection; the protein expression level of m-A16-B12 (hHGF) decreases 48 hours after delivery. The protein expression level of m-A16-B12 (hHGF) at a dose of 1μg / mouse is comparable to that of Collategene at 72 hours. At the same time, statistics of the PK data of each group found that even the lowest dose of 0.1μg of m-A16-B12 (hHGF) had a C max It is also more than 5 times that of Collategene, and the AUC of 0.1μg m-A16-B12 (hHGF) is inf(hr*pg / mg protein) The expression of hHGF in the plasma was comparable to that of Collategene (Table 13).

[0607] Table 13. Results of in vivo expression of hHGF by m-A16-B12 (hHGF) and Collategene

[0608] 1.5 Verification of the therapeutic function of hHGF mRNA expression in a mouse model of lower limb ischemia

[0609] The lower limb ischemia mouse model is a classic mouse model that simulates severe lower limb ischemia in humans. 6-8 week old male Balb / c mice were used for modeling. The method is as follows: 1) Anesthetize the animal and place it in the supine position on the operating table. The hind limbs are thoroughly depilated, the hind limbs are fixed, and the skin at the surgical site is disinfected. 2) A skin incision approximately 1 cm long is made from the knee to the inner thigh. The subcutaneous fat tissue is sequentially incised and dissected to fully expose the femoral artery. 3) Using micro-curved forceps, gently pierce the membranous femoral sheath to expose the neurovascular package. The femoral artery is separated from the femoral vein and nerve at the proximal position near the groin. After clean separation, the proximal femoral artery is ligated with a 6-0 suture below the proximal femoral artery. 4) The femoral artery and femoral vein are separated distally near the knee. The distal end of the femoral artery is ligated with a 6-0 suture below the proximal popliteal artery. 5) Suture the wound.

[0610] m-A16-B12 (hHGF) was delivered using the LNP described in Example 1.4, and Collategene was administered as a naked plasmid. Mice modeling lower limb ischemia were randomly divided into four groups of five. Both m-A16-B12 (hHGF) and naked Collategene plasmid were injected intramuscularly into the gastrocnemius muscle. The experimental groups were as follows: 1) 500 ng / mouse m-A16-B12 (hHGF); 2) 50 ng / mouse m-A16-B12 (hHGF); 3) 200 μg / mouse naked Collategene plasmid; and 4) an equal volume of PBS was injected as a control group. The day of modeling was designated as day 0. Legs were observed on days 0, 4, 7, 10, 12, and 14 after treatment in each experimental group. Lower limb blood perfusion was measured using a blood flowmeter and photographed.

[0611] The blood perfusion ratio of each group was calculated according to the following formula: blood perfusion ratio = blood perfusion volume of the lower limb of the mouse on that day / blood perfusion volume of the lower limb of the mouse on day 0 × 100%; the data of each group were expressed as mean ± standard deviation (Mean ± SD), and graphs and statistics were prepared using Graphpad Prism 9.0 software.

[0612] The results, as shown in Figures 10A and 10B, show that when treated with 50 ng / mouse, 500 ng / mouse m-A16-B12 (hHGF), and 200 ng / mouse Collategene naked plasmid, the blood perfusion ratio of the ischemic lower limb of each mouse significantly improved compared to the control group, and the blood perfusion ratio of the ischemic lower limb gradually recovered over time. The group receiving 50 ng / mouse m-A16-B12 (hHGF) demonstrated a blood flow recovery effect similar to that of the group receiving 200 ng / mouse Collategene naked plasmid. In particular, the group receiving 500 ng / mouse m-A16-B12 (hHGF) significantly improved blood flow recovery compared to the group receiving 200 ng / mouse Collategene naked plasmid, achieving a blood perfusion recovery of over 90%.

[0613] To further determine the therapeutic effect of m-A16-B12 (hHGF) on the extent of limb necrosis in a mouse model of limb ischemia, the legs of the mice were observed and photographed on day 14 after administration. The severity of limb necrosis was scored using the criteria shown in Figure 11A : 0 = limb self-detachment; 1 = leg necrosis; 2 = foot necrosis; 3 = >2 toe discoloration; 4 = 1 toe discoloration; 5 = >2 nail discoloration; 6 = 1 nail discoloration; 7 = no necrosis. Data are expressed as mean ± standard deviation (mean ± SD) for each group, and graphs and statistics were generated using Graphpad Prism 9.0 software.

[0614] The results are shown in Figure 11B. After inducing lower limb ischemia in mice, the mice in the control group developed obvious ischemic necrosis in the lower limbs within 2 weeks after surgery. By the 14th day, the lower limbs of some mice were completely detached. In the m-A16-B12 (hHGF) and Collategene naked plasmid injection treatment groups, the lower limbs of each mouse maintained good integrity and no lower limb necrosis occurred, indicating that m-A16-B12 (hHGF) has the same ability to improve lower limb ischemia as the control Collategene naked plasmid.

[0615] CD31 is a key marker of angiogenesis. To determine the role of m-A16-B12 (hHGF) in promoting angiogenesis in a mouse model of lower limb ischemia, paraffin sections were prepared from gastrocnemius muscle samples near the ischemic tissue of the mice 14 days after administration. Immunohistochemical staining of CD31 in the sections was performed using a CD31 antibody. Images were taken and analyzed using ImageJ (NIH) software to calculate the CD31 staining area. Data for each group are expressed as mean ± standard deviation (mean ± SD) and graphed and statistically analyzed using Graphpad Prism 9.0 software.

[0616] The results are shown in Figure 12. When treated with 50 ng / mouse, 500 ng / mouse m-A16-B12 (hHGF) and 200 ng / mouse Collategene naked plasmid, angiogenesis in the ischemic lower limb muscles of each group was significantly promoted, and the number of new blood vessels was significantly different from that of the PBS group (p<0.05). The 50 ng / mouse m-A16-B12 (hHGF) group showed an angiogenesis-promoting effect comparable to that of the 200 ng / mouse Collategene naked plasmid, and the m-A16-B12 (hHGF)-administered group showed a dose-dependent angiogenesis-promoting effect.

[0617] 1.6 Verification of the therapeutic function of hHGF mRNA expression in the Db / Db mouse model of full-thickness cortical injury

[0618] The Db / Db mouse model is a classic diabetic mouse model. Due to its Leptin receptor gene deficiency, the mice develop features similar to those of diabetic patients, including hyperglycemia, hyperlipidemia, and insulin resistance, over age. This experiment employed a full-thickness skin injury model to simulate the difficult-to-heal skin lesions seen in patients with diabetic foot. The Db / Db mouse model was created as follows: After anesthesia, small Db / Db mice were depilated with a depilatory cream and disinfected with 75% alcohol cotton balls. A full-thickness skin wound was created on the lower back using an 8mm diameter biopunch.

[0619] To determine the therapeutic effect of m-A16-B12 (hHGF) on wound healing in a full-thickness cortical injury mouse model, m-A16-B12 (hHGF) was delivered using the LNPs described in Example 1.4. A control, naked Collategene plasmid, was administered subcutaneously at four different sites. Db / Db mice undergoing a full-thickness cortical injury model were divided into five groups of seven mice based on body weight and blood glucose levels. The experimental groups were as follows: 1) 500 ng / mouse m-A16-B12 (hHGF); 2) 200 ng / mouse m-A16-B12 (hHGF); 3) 50 ng / mouse m-A16-B12 (hHGF); 4) 200 μg / mouse naked Collategene plasmid; and 5) a control group injected with an equal volume of PBS. The day of modeling was counted as day 0. After treatment in each experimental group, the wound recovery was observed on day 0, 3, 5, 7, 10, 12, and 14. The wound site was photographed, and the images were analyzed using ImageJ (NIH) software to calculate the wound area.

[0620] The wound healing percentage was calculated according to the following formula: P = (AD-A0) / A0×100% (P: wound healing percentage; AD: wound area on the day of photography; A0: wound area on the day of surgery).

[0621] The wound healing percentage of each group was expressed as mean ± standard deviation (Mean ± SD), and Graphpad Prism 9.0 software was used for graphing and statistics.

[0622] The results are shown in FIG13 . When injected with 50 ng / mouse, 200 ng / mouse, and 500 ng / mouse m-A16-B12 (hHGF) and 200 μg / mouse Collategene naked plasmid, the wounds of the mice healed well compared with the control group. Moreover, m-A16-B12 (hHGF) promoted wound healing in mice in a dose-dependent manner. On day 14, the wound healing rate of m-A16-B12 (hHGF) at a dose of 50 ng / mouse reached 66%, while m-A16-B12 (hHGF) at doses of 200 ng / mouse and 500 ng / mouse achieved 100% wound healing. At the same time, m-A16-B12 (hHGF) promoted wound healing significantly better than 200 μg / mouse Collategene naked plasmid (p < 0.05). Even the lower dose (50 ng / mouse) of m-A16-B12 (hHGF) showed a significantly different wound healing percentage from 200 μg / mouse Collategene naked plasmid (p < 0.05).

[0623] To determine the therapeutic efficacy of m-A16-B12 (hHGF) on tissue remodeling in a full-thickness cortical injury mouse model, Masson's staining was used to evaluate epithelial regeneration and tissue remodeling. On day 17 after administration, full-thickness cortical specimens were obtained from the mice, paraffin sections were prepared, and the mouse skin tissue was stained using Masson's stain.

[0624] The results, as shown in Figure 14, show that under m-A16-B12 (hHGF) treatment, the wounds of all mice in each group were fully covered with epithelial cells. In the low-dose group, the epithelium at the wound site was abnormally thickened, with collagen proliferation. In the medium- and high-dose groups, the epithelium returned to normal thickness, with collagen arranged in an orderly manner, completing tissue reconstruction. In contrast, in the control group and the 200 μg / mouse Collategene naked plasmid group, epithelial re-covery was not completed, with abnormal collagen proliferation and structural disorder at the wound site.

[0625] Example 2:

[0626] Preparation of LNP lipid particles

[0627] Compound 1 (Formula I compound prepared with reference to WO2023125738A), DSPC, cholesterol, and DMG-PEG 2000 solution were mixed in a certain molar percentage (mol%), that is, the molar amount of each component accounted for the percentage of the molar amount of the total lipids present in the lipid nanoparticles, and an ethanol lipid solution was prepared. The molar ratios of the four components were prepared as shown in Table 14. The mRNA encoding the luciferase protein was dissolved in a pH 4 acetate buffer to prepare an mRNA aqueous solution. The ethanol lipid solution and the mRNA aqueous solution were mixed in a 1:3 volume ratio by microfluidics, and the weight ratio of total lipids to mRNA was about 20:1 to prepare liposomes. Ethanol was removed by dialyzing in a 20mM Tris solution to obtain a liposome nanoparticle composition encapsulating mRNA.

[0628] Lipid particle composition characterization

[0629] Characterization methods

[0630] The particle size and polydispersity index (PDI) of the liposome nanoparticles were determined by dynamic light scattering (DLS) using a Malvern Zetasizer Pro in backscattering mode at 173°.

[0631] The liposome encapsulation efficiency was determined using the Quant-iT RiboGreen RNA Assay Kit.

[0632] Table 14

[0633] Example 3:

[0634] Lyophilization of lipid particle compositions

[0635] Prepare lipid nanoparticles according to Example 2, and replace the prepared lipid nanoparticles into a freeze-dried preparation buffer (containing 20mM Tris, pH 7.5, 8% sucrose, 5% trehalose and sodium chloride solutions of different concentrations corresponding to the table below) by ultrafiltration. Fill the freeze-dried preparation solution of lipid nanoparticles into 2mL cillin bottles, 0.6mL / bottle, and the cationic lipid concentration in each bottle is 1.78mg / mL. Place in a freeze dryer. Pre-freeze at -45°C for 3 hours, dry at -40°C for 30 hours, and dry at 30°C for 10 hours, with a vacuum degree of 0.05mmbar. The final freeze-dried product is out of the box.

[0636] The lyophilized product was reconstituted with 0.6 mL of water for injection and the lipid nanoparticles were then measured using the aforementioned method. The particle sizes of the lipid nanoparticles before and after lyophilization were compared. The results are shown in Table 15 and Figures 15-17.

[0637] Table 15

[0638] The lipid nanoparticle stock solutions from the above experiments were diluted to 1 / 2 and 1 / 3 concentrations using a lyophilization formulation buffer. The resulting lipid nanoparticles were then bottled and lyophilized according to the above experimental procedures. The particle size of the lyophilized and reconstituted lipid nanoparticles was measured. The results are shown in Table 16.

[0639] Table 16

[0640] The prepared lipid nanoparticles were ultrafiltrated into a separate lyophilization buffer (containing 20 mM Tris, pH 7.5, 8% sucrose, and various sodium chloride concentrations as shown in the table below). The lyophilized lipid nanoparticle solution was then filled into 2 mL vials at a rate of 0.6 mL per vial, with a cationic lipid concentration of 2.38 mg / mL per vial. The vials were placed in a lyophilizer. Pre-freeze at -45°C for 3 hours, dry at -40°C for 30 hours, and dry at 30°C for 10 hours, all under a vacuum of 0.05 mmbar. The final lyophilized product was then removed from the freezer.

[0641] The lyophilized product was reconstituted with 0.6 mL of water for injection and the lipid nanoparticles were then tested for particle size according to the aforementioned method. The particle sizes of the lipid nanoparticles before and after lyophilization were compared, and the test results are shown in Table 17.

[0642] Table 17

[0643] Example 4:

[0644] Assessing mRNA integrity in lipid nanoparticle compositions

[0645] Freeze-dried lipid nanoparticles were obtained according to Example 3, and the integrity of mRNA in each group of samples was detected using a capillary electrophoresis instrument (Agilent 5200 fragment analyzer).

[0646] mRNA is easily degraded and affected by temperature. As the temperature rises, mRNA stability decreases. With prolonged storage time, degradation occurs under high-temperature conditions, reducing its integrity. During the freeze-drying process, the temperature rises from primary to secondary drying, with the secondary drying temperature being higher. Therefore, the mRNA integrity of sodium chloride in different formulations was evaluated during the freeze-drying process and under high-temperature storage conditions. As shown in Table 18 and Figures 18-21, mRNA integrity improves after freeze-drying with increasing sodium chloride concentration. Similarly, under high-temperature storage conditions at 37°C, mRNA integrity improves with increasing sodium chloride concentration.

[0647] Table 18

[0648] Example 5:

[0649] Evaluating the mRNA delivery efficiency of lipid nanoparticles in cells

[0650] HEK 293 cells were seeded into 96-well plates and cultured overnight. When the cell density reached more than 80%, the lipid nanoparticle solution encapsulating luciferase mRNA (lyophilized product of prescription 7 and lyophilized product of prescription 9) was added to the cell plate well culture medium, and the mRNA dose was 100 ng / well. After 24 hours, the fluorescence intensity of the expressed luciferase protein was detected using a luciferase reporter gene assay kit (Promega) and a microplate reader. The fluorescence intensity value is the fluorescence value detected by the microplate reader, which represents the expression level of the luciferase protein. The higher the intensity of the strong light, the higher the protein expression level. The average fluorescence intensity was calculated for at least 3 groups of lipid nanoparticles corresponding to each compound.

[0651] Table 19

[0652] Conclusion: As shown in Table 19 and Figure 22, the fluorescence intensity of the lipid nanoparticle freeze-dried product corresponding to prescription 9 was significantly higher than that of prescription 7. In Figure 22, *** represents a statistically significant difference, P < 0.001.

[0653] Example 6 Preparation process of HGF-mRNA-LNP spray formulation:

[0654] Preparation of HGF-mRNA-LNP lyophilized powder. HGF-mRNA-LNP (0.09 mg m-A16-B12, 1.07 mg cationic lipid of Formula I, with the molar amounts of the cationic lipid, DSPC, cholesterol, and DMG-PEG 2000 accounting for 48 mol%, 10 mol%, 40.5 mol%, and 1.5 mol% of the total molar amount of the lipid nanoparticles, respectively) was prepared according to the method described in Example 2. HGF-mRNA-LNP lyophilized powder (Formulation 10) was prepared according to Formula 4 in Example 3. The lyophilized product was reconstituted in 0.6 mL of water for injection, and the particle size of the lipid nanoparticles was measured according to the aforementioned method. The particle size of the lipid nanoparticles before and after lyophilization was compared. The results are shown in Table 20.

[0655] Table 20

[0656] A reconstitution solvent was prepared, sterilized and filtered, and used to reconstitute the above-mentioned HGF-mRNA-LNP lyophilized powder to prepare a spray preparation.

[0657] Example 7. Screening of dispersant components

[0658] When mRNA-LNP is in solution (after reconstitution of lyophilized powder), its particles exhibit significant stability, manifested by a tendency to aggregate and increase in size. When LNP is atomized via a nebulizer, mechanical compression or shear forces further increase the likelihood of aggregation. To reduce the risk of LNP particle aggregation during atomization, a dispersant is added to the reconstitution solvent. The reconstitution solvent was prepared (by weight) as follows: WFI, 0.2% PF68 (Poloxamer 188), 0.2% PF127 (Poloxamer 407), 0.25% PVA (Polyvinyl Alcohol), 0.1% HEC (Hydroxyethylcellulose), and 1% PEG400 (Polyethylene Glycol 400). Nebulization was performed using a disposable nasal delivery device. The atomized solution was collected 3 cm from the atomizer nozzle. Particle size, PDI, total mRNA content, and encapsulation efficiency were measured in the collected atomized solution and the reconstituted solution before atomization as evaluation indicators.

[0659] The results are shown in Table 21. Reconstitution of the lyophilized LNP powder with different resolvents significantly decreased its particle size (81.3 nm vs. 96.7 nm) compared to reconstitution with water for injection (WFI). This suggests that reconstitution with 0.2% PF127 may have disrupted the LNP structure. The LNP particle size after reconstitution with the other resolvents was not significantly different from that after reconstitution with WFI. Furthermore, the total mRNA content and encapsulation efficiency of the solutions reconstituted with the other resolvents were comparable to those after reconstitution with WFI. In terms of changes in particle size, PDI, total mRNA content, and encapsulation efficiency before and after spraying, no significant differences were observed between the solutions reconstituted with the other resolvents. This suggests that the risk of LNP particle damage and drug adsorption during spraying using a disposable nasal delivery device is low.

[0660] In summary, 0.2% PF68, 0.25% PVA, 0.1% HEC, and 1% PEG400 can all be used as the complex solvent component-dispersant.

[0661] Table 21. Screening results of resolvent components-dispersants

[0662] Example 8. Screening of Resolvent Components - Viscosity Enhancers

[0663] As a topical spray preparation, this product needs to have a certain viscosity to increase the retention time of the drug solution at the administration site. Xanthan gum (Xc), povidone K90 (PVP-K90), sodium hyaluronate (HA), sodium carboxymethyl cellulose (CMC-Na), and chitosan (CTS) were selected as viscosity enhancers. The specific formula is as follows (weight ratio):

[0664] 1) 0.24% xanthan gum (Xc);

[0665] 2) 0.5% povidone K90 (PVP-K90);

[0666] 3) 0.1% sodium hyaluronate (HA);

[0667] 4) 0.5% sodium carboxymethylcellulose (CMC-Na);

[0668] 5) 0.2% chitosan (CTS).

[0669] Each of the above reconstitution solutions was prepared, filtered, filled, stoppered, and capped for later use. Each of the above reconstitution solutions was pipetted into a disposable nasal delivery device and then reconstituted with the lyophilized powder. The sprayed samples were collected. The particle size, PDI, encapsulation efficiency, and total mRNA content of the samples before and after spraying were measured. The results are shown in Table 22 below.

[0670] Table 22. Screening of Resolvent Components - Viscosity Enhancers

[0671] Based on the above results, the particle size and PDI of the reconstituted sample after spraying with 0.2% chitosan (CTS) increased compared to those before spraying (107.5 nm vs. 99.2 nm), suggesting that the sample may have aggregated after spraying. Several other reconstituted solvents (0.24% Xc, 0.5% PVPK90, 0.1% HA, and 0.5% CMC-Na) were able to maintain the stability of the LNP particle structure before and after spraying.

[0672] At the same time, the above-mentioned complex solvents were screened using the viscosity of the sprayed liquid and the spray particle size as evaluation indicators. The structures are shown in Table 23 below:

[0673] Table 23. Screening of Resolvent Components - Viscosifiers (Spray Pattern and Mode)

[0674] The above results indicate that compared with spraying with water for injection (WFI), spraying with 0.5% PVPK90 and 0.2% CTS resulted in a higher viscosity, smaller spray area, and higher ellipticity. This suggests that reconstitution with 0.5% PVPK90 and 0.2% CTS results in a thicker solution, making it more difficult to achieve a better spray pattern (spray area and ellipticity). The spraying effects of the other reconstitution solvents (0.24% Xc, 0.1% HA, and 0.2% CTS) were not significantly different from those with water for injection (WFI).

[0675] Example 9. Determination of Resolvent Components

[0676] The resolvent components must be carefully considered, considering factors such as the viscosity of the spray solution after spraying, the stability of the LNP structure, and the spray morphology and pattern. The resolvent components were prepared according to Table 24 below. The spray was then prepared using a disposable nasal delivery device. Samples were collected after spraying to measure the viscosity of the spray solution, particle size, PDI, total mRNA content, and encapsulation efficiency. These were used as evaluation indicators for screening resolvent components. The results are shown in Table 25 below.

[0677] Table 24 Design of screening test for resolvent components

[0678] Table 25. Results of the screening of resolvent components

[0679] The experimental results show that the resolvent containing 1% PVP-K90 has the highest viscosity, followed by 0.5% PVP-K90, and the resolvent containing 0.25% PVP-K900 has the lowest viscosity. Changes in particle size and PDI before and after spraying, as well as changes in total mRNA content and encapsulation efficiency, and compared to WFI, show that the resolvent containing 1% PEG-400 has a lower encapsulation efficiency and total mRNA content after spraying compared to before spraying. Therefore, 0.2% PF68 + 1% PVP-K90, 0.2% PF68 + 0.5% PVP-K90, and 0.2% PF68 + 0.25% PVP-K90 are preferred as resolvent components.

[0680] Example 10. Determination of Reconstitution Components - HGF Expression

[0681] Furthermore, it is necessary to introduce the spray morphology and pattern as well as the biological activity of the main component of the spray solution (LNP) as key indicators to further screen and evaluate the resolvent components. The resolvent components are as follows:

[0682] 1) 1% PVP-K90 + 1% PEG-400

[0683] 2) 1% PVP-K90 + 0.2% PF68

[0684] 3) 0.5% PVP-K90 + 1% PEG-400

[0685] 4) 0.5% PVP-K90 + 0.2% PF68

[0686] 5)0.25% PVP-K90+1% PEG-400

[0687] 6) 0.25% PVP-K90 + 0.2% PF68

[0688] 7) 0.2% PF68

[0689] 8) 1.5% PK90

[0690] 9) 1% PEG-400

[0691] 10) 0.1% HEC

[0692] 11) 0.25% PVA

[0693] 12) 0.2% PF127

[0694] 13)WFI

[0695] Each reconstituted solution was prepared according to the above formula, and then filtered, filled, stoppered, and capped for use. A disposable nasal delivery device was used to transfer each reconstituted solution to the lyophilized powder and sprayed, and the sprayed sample was collected. The viscosity, osmotic pressure, particle size, PDI, encapsulation efficiency, total mRNA content, and spray pattern of the sprayed sample were tested. The collected samples were also tested for HGF expression in HEK293 cells (CCTCC). The results are shown in Table 26 below.

[0696] Table 26. Screening of Resolvent Components (Biological Activity)

[0697] As shown in Table 26, after reconstitution and spraying of the sample with Formulation 12 (0.2% PF127), HGF expression in the sample was almost zero, indicating that this component reconstitution solvent may have a significant destructive effect on the LNP, causing it to lose its biological activity after reconstitution and spraying. The reconstituted samples of Formulations 1 (1% PVP-K90 + 1% PEG-400), 2 (1% PVP-K90 + 0.2% PF68), 5 (0.25% PVP-K90 + 1% PEG-400), and 8 (1.5% PVP-K90) exhibited larger spray particle size, smaller spray area, and higher liquid viscosity after spraying. The reconstitution solvents of the remaining formulations all exhibited higher HGF expression levels, moderate viscosity, larger spray area, and smaller spray particle size.

[0698] Sequence Listing

Claims

1. A lipid nanoparticle comprising a nucleic acid construct and a lipid phase, The nucleic acid construct comprises: (a) an open reading frame (ORF), and (b) an untranslated region element (UTR) derived from the UTR of gene ACTG1, ATP6V0B, ATP6V0E1, CFL1, COX4I1, CTSB, FAM166A, NDUFB9, CHCHD10, SLC38A2, NDUFA11, NDUFV3, PRDX5, GUK1, IAH1, ABHD16A, SLC25A39, ATPIF1, ANAPC11, CCDC12, MRPL14, or APOA1BP; The lipid phase comprises a cationic lipid of formula I, 2. According to the lipid nanoparticles described in claim 1, the cationic lipids account for 20 to 70% of the molar amount of the total lipids present in the lipid nanoparticles; preferably, the cationic lipids account for 30 to 70% of the molar amount of the total lipids present in the lipid nanoparticles; more preferably, the cationic lipids account for 40 to 70% of the molar amount of the total lipids present in the lipid nanoparticles.

3. The lipid nanoparticle according to claim 1, wherein the lipid nanoparticle comprises at least one non-cationic lipid, preferably the non-cationic lipid is selected from a mixture of phospholipids and cholesterol or its derivatives.

4. The lipid nanoparticles according to claim 3, wherein the non-cationic lipids account for 20 to 70% of the molar amount of the total lipids present in the lipid nanoparticles; preferably, the non-cationic lipids account for 30 to 60% of the molar amount of the total lipids present in the lipid nanoparticles; more preferably, the non-cationic lipids account for 20 to 50% of the molar amount of the total lipids present in the lipid nanoparticles.

5. The lipid nanoparticle according to claim 3, wherein the content of phospholipids accounts for 5 to 30% of the molar amount of total lipids present in the lipid nanoparticle.

6. The lipid nanoparticles according to claim 3, wherein cholesterol or its derivatives account for 20 to 60% of the molar amount of total lipids present in the lipid nanoparticles, preferably, the cholesterol or its derivatives content accounts for 30 to 60% of the molar amount of total lipids present in the lipid nanoparticles; more preferably, the cholesterol or its derivatives content accounts for 20 to 45% of the molar amount of total lipids present in the lipid nanoparticles.

7. The lipid nanoparticle according to claim 1, wherein the lipid nanoparticle further contains at least one conjugated lipid; preferably, the content of the conjugated lipid in the lyophilized composition accounts for 0.5 to 5% of the molar amount of the total lipids present in the lipid nanoparticle.

8. The lipid nanoparticle according to claim 1, comprising a) nucleic acid construct; b) a cationic lipid, wherein the cationic lipid comprises a compound of formula I or a pharmaceutically acceptable salt thereof, and the cationic lipid accounts for 10 to 75% of the total lipid molar amount present in the lipid nanoparticles, c) a non-cationic lipid selected from the group consisting of phospholipids and cholesterol or a derivative thereof, wherein the phospholipids account for 5 to 40% by molar amount of the total lipids present in the lipid nanoparticles, and the cholesterol or a derivative thereof accounts for 15 to 60% by molar amount of the total lipids present in the lipid nanoparticles; and d) a conjugated lipid, said conjugate comprising 0.5-2% by mole of the total lipid present in the lipid nanoparticle.

9. A lyophilized composition comprising the lipid nanoparticles according to any one of claims 1 to 8.

10. The freeze-dried composition according to claim 9, comprising the lipid nanoparticles according to any one of claims 1-8 and sodium chloride, preferably the weight ratio of sodium chloride to cationic lipid is 1:10 to 10:1; more preferably the weight ratio of sodium chloride to cationic lipid is 1:5 to 10:1; more preferably the weight ratio of sodium chloride to cationic lipid is 1:5 to 5:1; more preferably the weight ratio of sodium chloride to cationic lipid is 1:3 to 5:1; more preferably the weight ratio of sodium chloride to cationic lipid is 1:2 to 5:1; more preferably the weight ratio of sodium chloride to cationic lipid is 1:2 to 4:

1.

11. The freeze-dried composition according to any one of claims 9 to 10, comprising at least one freeze-dried protective agent, preferably the freeze-dried protective agent is selected from one or more of sucrose, trehalose, mannitol, and maltose; preferably the freeze-dried protective agent is selected from sucrose, a combination of sucrose and trehalose.

12. A lyophilized preparation, which is obtained by freeze-drying the lipid nanoparticles according to any one of claims 1-8 or the lyophilized composition according to any one of claims 9-11; preferably, in the lyophilized preparation, the weight ratio of sodium chloride to cationic lipid is 1:10-10:1; more preferably, the weight ratio of sodium chloride to cationic lipid is 1:5-10:1; more preferably, the weight ratio of sodium chloride to cationic lipid is 1:5-5:1; more preferably, the weight ratio of sodium chloride to cationic lipid is 1:3-5:1; more preferably, the weight ratio of sodium chloride to cationic lipid is 1:2-5:1; more preferably, the weight ratio of sodium chloride to cationic lipid is 1:2-4:

1.

13. A reconstituted solution, which is obtained by reconstituted the lyophilized preparation according to claim 12 with a reconstituted solvent; preferably, the reconstituted solvent is water; more preferably, the reconstituted solvent is water for injection.

14. A product comprising: 1) the freeze-dried composition according to any one of claims 9 to 11 or the freeze-dried preparation according to claim 12, and, 2) resolubilizing agent; Preferably, the preparation comprises independently packaged lyophilized compositions and resolubilizing agents, preferably the resolubilizing agent is water; more preferably the resolubilizing agent is water for injection.

15. A spray preparation comprising the freeze-dried composition according to any one of claims 9 to 11, the freeze-dried preparation according to claim 12, or the reconstituted solution according to claim 13.

16. A method for preparing a reconstituted solution, comprising preparing the lyophilized composition according to any one of claims 9 to 11, wherein the lyophilized composition is freeze-dried to obtain the lyophilized preparation; and The lyophilized preparation is reconstituted to obtain the reconstituted solution; Preferably, the resolubilizing agent is water; more preferably, the resolubilizing agent is water for injection.

17. A method for preparing a lyophilized preparation, comprising preparing the lyophilized composition according to any one of claims 9 to 11, wherein the lyophilized composition is subjected to a freeze-drying treatment to obtain the lyophilized preparation.

18. The lipid nanoparticle according to any one of claims 1 to 8, wherein The nucleic acid construct comprises: (a) an open reading frame (ORF), and (b) an untranslated region element (UTR), the UTR comprising the following sequence: Any of SEQ ID NO: 12, 26, or a sequence having at least 90% identity thereto, Any of SEQ ID NO: 1, 15, or a sequence having at least 90% identity thereto, Any of SEQ ID NO: 2, 3, 16, 17, or a sequence having at least 90% identity thereto, Any of SEQ ID NOs: 4-5, 18-19, or a sequence having at least 90% identity thereto, Any of SEQ ID NOs: 6-8, 20-22, or a sequence having at least 90% identity thereto, Any of SEQ ID NOs: 9-11, 23-25, or a sequence having at least 90% identity thereto, Any of SEQ ID NO: 13, 27, or a sequence having at least 90% identity thereto, Any of SEQ ID NO: 14, 28, or a sequence having at least 90% identity thereto, Any of SEQ ID NO: 29, 30, or a sequence having at least 90% identity thereto, Any one of SEQ ID NOs: 35-37, or a sequence at least 90% identical thereto, Any of SEQ ID NO:42, 43, or a sequence having at least 90% identity thereto, Any of SEQ ID NO:46, 47, or a sequence having at least 90% identity thereto, Any of SEQ ID NOs: 31-34, 38-41, 44-45, or a sequence having at least 90% identity thereto, Any of SEQ ID NO:69, 83, or a sequence having at least 90% identity thereto, Any of SEQ ID NO:58, 72, or a sequence having at least 90% identity thereto, Any of SEQ ID NOs: 59-60, 73-74, or a sequence having at least 90% identity thereto, Any of SEQ ID NOs: 61-62, 75-76, or a sequence having at least 90% identity thereto, Any of SEQ ID NOs: 63-65, 77-79, or a sequence having at least 90% identity thereto, Any of SEQ ID NOs: 66-68, 80-82, or a sequence having at least 90% identity thereto, Any of SEQ ID NO:70, 84, or a sequence having at least 90% identity thereto, Any of SEQ ID NO:71, 85, or a sequence having at least 90% identity thereto, Any of SEQ ID NO:86, 87, or a sequence having at least 90% identity thereto, Any of SEQ ID NOs: 92-94, or a sequence at least 90% identical thereto, Any of SEQ ID NO:99, 100, or a sequence having at least 90% identity thereto, Any of SEQ ID NO: 103, 104, or a sequence having at least 90% identity thereto, or Any of SEQ ID NOs: 88-91, 95-98, 101-102, or a sequence at least 90% identical thereto.

19. The lipid nanoparticle according to claim 18, wherein (b) comprising a combination of a 3' untranslated region element (3'UTR) and a 5' untranslated region element (5'UTR) selected from any one of the following: 1) the 3'UTR comprises a sequence as shown in SEQ ID NO: 1 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in any one of SEQ ID NOs: 15-47 or having at least 90% identity thereto; 2) the 3'UTR comprises a sequence as shown in SEQ ID NO:2 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in any one of SEQ ID NOs:15-47 or having at least 90% identity thereto; 3) the 3'UTR comprises a sequence as shown in SEQ ID NO:3 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in any one of SEQ ID NOs:15-47 or having at least 90% identity thereto; 4) the 3'UTR comprises a sequence as shown in SEQ ID NO:4 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in any one of SEQ ID NOs:15-47 or having at least 90% identity thereto; 5) the 3'UTR comprises a sequence as shown in SEQ ID NO:5 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in any one of SEQ ID NOs:15-47 or having at least 90% identity thereto; 6) the 3'UTR comprises a sequence as shown in SEQ ID NO:6 or a sequence having at least 90% identity thereto, The 5'UTR comprises a sequence shown in any one of SEQ ID NOs: 15-47 or a sequence having at least 90% identity thereto; 7) the 3'UTR comprises a sequence as shown in SEQ ID NO:7 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in any one of SEQ ID NOs:15-47 or having at least 90% identity thereto; 8) the 3'UTR comprises a sequence as shown in SEQ ID NO:8 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in any one of SEQ ID NOs:15-47 or having at least 90% identity thereto; 9) the 3'UTR comprises a sequence as shown in SEQ ID NO:9 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in any one of SEQ ID NOs:15-47 or having at least 90% identity thereto; 10) the 3'UTR comprises a sequence as shown in SEQ ID NO: 10 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in any one of SEQ ID NOs: 15-47 or having at least 90% identity thereto; 11) the 3'UTR comprises a sequence as shown in SEQ ID NO: 11 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in any one of SEQ ID NOs: 15-47 or having at least 90% identity thereto; 12) the 3'UTR comprises a sequence as shown in SEQ ID NO: 12 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in any one of SEQ ID NOs: 15-47 or having at least 90% identity thereto; 13) the 3'UTR comprises a sequence as shown in SEQ ID NO: 13 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in any one of SEQ ID NOs: 15-47 or having at least 90% identity thereto; 14) the 3'UTR comprises a sequence as shown in SEQ ID NO: 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in any one of SEQ ID NOs: 15-47 or having at least 90% identity thereto; 15) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs: 1-14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 15 or having at least 90% identity thereto; 16) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs: 1-14 or a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 16 or a sequence having at least 90% identity thereto; 17) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs: 1-14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 17 or having at least 90% identity thereto; 18) The 3'UTR contains any one of SEQ ID NOs: 1-14 or any one of them having at least 90% homology thereto. The 5'UTR comprises a sequence as shown in SEQ ID NO: 18 or a sequence having at least 90% identity thereto; 19) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs: 1-14 or a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 19 or a sequence having at least 90% identity thereto; 20) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs: 1-14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 20 or having at least 90% identity thereto; 21) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs: 1-14 or a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 21 or a sequence having at least 90% identity thereto; 22) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs: 1-14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 22 or having at least 90% identity thereto; 23) the 3'UTR comprises a sequence shown in any one of SEQ ID NOs: 1-14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence shown in SEQ ID NO: 23 or having at least 90% identity thereto; 24) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs: 1-14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 24 or having at least 90% identity thereto; 25) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs: 1-14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 25 or having at least 90% identity thereto; 26) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs: 1-14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 26 or having at least 90% identity thereto; 27) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs: 1-14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 27 or having at least 90% identity thereto; 28) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs: 1-14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 28 or having at least 90% identity thereto; 29) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs: 1-14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 29 or having at least 90% identity thereto; 30) The 3'UTR contains any one of SEQ ID NOs: 1-14 or any one of which has at least 90% homology thereto. The 5'UTR comprises a sequence as shown in SEQ ID NO:30 or a sequence having at least 90% identity thereto; 31) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs: 1-14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 31 or having at least 90% identity thereto; 32) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs: 1-14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 32 or having at least 90% identity thereto; 33) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs: 1-14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 33 or having at least 90% identity thereto; 34) the 3'UTR comprises a sequence shown in any one of SEQ ID NOs: 1-14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence shown in SEQ ID NO: 34 or having at least 90%, 95% identity thereto; 35) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs: 1-14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 35 or having at least 90% identity thereto; 36) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs: 1-14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 36 or having at least 90% identity thereto; 37) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs: 1-14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 37 or having at least 90% identity thereto; 38) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs: 1-14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 38 or having at least 90% identity thereto; 39) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs: 1-14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 39 or having at least 90% identity thereto; 40) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs: 1-14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 40 or having at least 90% identity thereto; 41) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs: 1-14 or a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 41 or a sequence having at least 90% identity thereto; 42) The 3'UTR contains any one of SEQ ID NOs: 1-14 or any one of which has at least 90% homology thereto. The 5'UTR comprises a sequence as shown in SEQ ID NO:42 or a sequence having at least 90% identity thereto; 43) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs: 1-14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 43 or having at least 90% identity thereto; 44) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs: 1-14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 44 or having at least 90% identity thereto; 45) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs: 1-14 or a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 45 or a sequence having at least 90% identity thereto; 46) the 3'UTR comprises a sequence shown in any one of SEQ ID NOs: 1-14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence shown in SEQ ID NO: 46 or having at least 90% identity thereto; or 47) the 3'UTR comprises a sequence shown in any one of SEQ ID NOs: 1-14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence shown in SEQ ID NO: 47 or having at least 90% identity thereto; 48) the 3'UTR comprises a sequence as shown in SEQ ID NO:58 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:72-104 or having at least 90% identity thereto; 49) the 3'UTR comprises a sequence as shown in SEQ ID NO:59 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:72-104 or having at least 90% identity thereto; 50) the 3'UTR comprises a sequence as shown in SEQ ID NO:60 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:72-104 or having at least 90% identity thereto; 51) the 3'UTR comprises a sequence as shown in SEQ ID NO:61 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:72-104 or having at least 90% identity thereto; 52) the 3'UTR comprises a sequence as shown in SEQ ID NO:62 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:72-104 or having at least 90% identity thereto; 53) the 3'UTR comprises a sequence as shown in SEQ ID NO:63 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:72-104 or having at least 90% identity thereto; 54) the 3'UTR comprises a sequence as shown in SEQ ID NO:64 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:72-104 or having at least 90% identity thereto; 55) the 3'UTR comprises a sequence as shown in SEQ ID NO:65 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:72-104 or having at least 90% identity thereto; 56) the 3'UTR comprises a sequence as shown in SEQ ID NO:66 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:72-104 or having at least 90% identity thereto; 57) the 3'UTR comprises a sequence shown in SEQ ID NO:67 or a sequence having at least 90% identity thereto, The 5'UTR comprises a sequence shown in any one of SEQ ID NOs:72-104 or a sequence having at least 90% identity thereto; 58) the 3'UTR comprises a sequence as shown in SEQ ID NO:68 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:72-104 or having at least 90% identity thereto; 59) the 3'UTR comprises a sequence as shown in SEQ ID NO:69 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in any one of SEQ ID NOs:72-104 or having at least 90% identity thereto; 60) the 3'UTR comprises a sequence as shown in SEQ ID NO:70 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in any one of SEQ ID NOs:72-104 or having at least 90% identity thereto; 61) the 3'UTR comprises a sequence as shown in SEQ ID NO:71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in any one of SEQ ID NOs:72-104 or having at least 90% identity thereto; 62) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs:58-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:72 or having at least 90% identity thereto; 63) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs:58-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:73 or having at least 90% identity thereto; 64) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs:58-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:74 or having at least 90% identity thereto; 65) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs:58-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:75 or having at least 90% identity thereto; 66) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs:58-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:76 or having at least 90% identity thereto; 67) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs:58-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:77 or having at least 90% identity thereto; 68) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs:58-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:78 or having at least 90% identity thereto; 69) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs:58-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:79 or having at least 90% identity thereto; 70) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs:58-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:80 or having at least 90% identity thereto; 71) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs:58-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:81 or having at least 90% identity thereto; 72) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs:58-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:82 or having at least 90% identity thereto; 73) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs:58-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:83 or having at least 90% identity thereto; 74) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs:58-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:84 or having at least 90% identity thereto; 75) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs:58-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:85 or having at least 90% identity thereto; 76) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs:58-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:86 or having at least 90% identity thereto; 77) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs:58-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:87 or having at least 90% identity thereto; 78) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs:58-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:88 or having at least 90% identity thereto; 79) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs:58-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:89 or having at least 90% identity thereto; 80) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs:58-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:90 or having at least 90% identity thereto; 81) The 3'UTR contains a sequence shown in any one of SEQ ID NOs: 58-71 or a sequence having at least 90% identity thereto, and the 5'UTR contains a sequence shown in SEQ ID NO: 91 or a sequence having at least 90%, 95% identity thereto; 82) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs:58-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:92 or having at least 90% identity thereto; 83) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs:58-71 or a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:93 or a sequence having at least 90% identity thereto; 84) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs:58-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:94 or having at least 90% identity thereto; 85) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs:58-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:95 or having at least 90% identity thereto; 86) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs:58-71 or a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:96 or a sequence having at least 90% identity thereto; 87) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs:58-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:97 or having at least 90% identity thereto; 88) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs:58-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:98 or having at least 90% identity thereto; 89) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs:58-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:99 or having at least 90% identity thereto; 90) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs:58-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:100 or having at least 90% identity thereto; 91) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs:58-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:101 or having at least 90% identity thereto; 92) the 3'UTR comprises a sequence shown in any one of SEQ ID NOs:58-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence shown in SEQ ID NO:102 or having at least 90% identity thereto; 93) the 3'UTR comprises a sequence as shown in any one of SEQ ID NOs:58-71 or a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:103 or a sequence having at least 90% identity thereto; or 94) The 3'UTR comprises a sequence as shown in any one of SEQ ID NOs:58-71 or a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO:104 or a sequence having at least 90% identity thereto.

20. The lipid nanoparticle according to any one of claims 1-8, 18-19, wherein The 3'UTR comprises a sequence shown in any one of SEQ ID NOs: 12-14 or a sequence having at least 90% identity thereto, and the 5'UTR comprises a sequence shown in any one of SEQ ID NOs: 15, 29, 30, 32 or a sequence having at least 90% identity thereto; or, The 3'UTR comprises a sequence shown in any one of SEQ ID NOs: 69-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence shown in any one of SEQ ID NOs: 72, 86, 87, 89 or having at least 90% identity thereto; Preferably, The 3'UTR comprises a sequence as shown in SEQ ID NO: 12 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in any one of SEQ ID NO: 15, 29, 30, 32 or having at least 90% identity thereto, The 3'UTR comprises a sequence as shown in SEQ ID NO: 13 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in any one of SEQ ID NO: 15, 29, 30, 32 or having at least 90% identity thereto, The 3'UTR comprises a sequence as shown in SEQ ID NO: 14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in any one of SEQ ID NO: 15, 29, 30, 32 or having at least 90% identity thereto, The 3'UTR comprises a sequence as shown in any one of SEQ ID NOs: 12-14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 15 or having at least 90% identity thereto, The 3'UTR comprises a sequence as shown in any one of SEQ ID NOs: 12-14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 29 or having at least 90% identity thereto, The 3'UTR comprises a sequence as shown in any one of SEQ ID NOs: 12-14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 30 or having at least 90% identity thereto, The 3'UTR comprises a sequence as shown in any one of SEQ ID NOs: 12-14 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 32 or having at least 90% identity thereto, The 3'UTR comprises a sequence as shown in SEQ ID NO: 69 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in any one of SEQ ID NO: 72, 86, 87, 89 or having at least 90% identity thereto, The 3'UTR comprises a sequence as shown in SEQ ID NO: 70 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in any one of SEQ ID NO: 72, 86, 87, 89 or having at least 90% identity thereto, The 3'UTR comprises a sequence shown in SEQ ID NO:71 or having at least 90% identity thereto, The 5'UTR comprises a sequence shown in any one of SEQ ID NOs: 72, 86, 87, 89, or a sequence having at least 90% identity thereto, The 3'UTR comprises a sequence as shown in any one of SEQ ID NOs: 69-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 72 or having at least 90% identity thereto, The 3'UTR comprises a sequence as shown in any one of SEQ ID NOs: 69-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 86 or having at least 90% identity thereto, The 3'UTR comprises a sequence as shown in any one of SEQ ID NOs: 69-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence as shown in SEQ ID NO: 87 or having at least 90% identity thereto, or The 3'UTR comprises a sequence shown in any one of SEQ ID NOs: 69-71 or having at least 90% identity thereto, and the 5'UTR comprises a sequence shown in SEQ ID NO: 89 or having at least 90% identity thereto.

21. The lipid nanoparticle according to any one of claims 1-8, 18-20, wherein The nucleic acid construct further comprises: (c) polyadenylic acid (poly-A) tail; Preferably, the poly-A tail is selected from A120, A30L70, HGH polyA, SV40polyA, BGH polyA, rbGlob polyA or SV40late polyA; Preferably, the poly-A tail is selected from A120 or A30L70, wherein A120 comprises 120 adenine nucleotides, and wherein A30L70 comprises a sequence as shown in SEQ ID NO: 52 or a sequence having at least 90% identity thereto.

22. The lipid nanoparticle according to any one of claims 1-8, 18-21, wherein The ORF encodes hepatocyte growth factor (HGF), an antibody or an antigen-binding fragment thereof, preferably human hepatocyte growth factor (hHGF), an anti-PD-1 antibody or an antigen-binding fragment thereof.

23. The lipid nanoparticle of claim 22, wherein the ORF comprises any one of 1)-2) selected from the group consisting of: 1) a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 109; 2) a polynucleotide sequence as shown in any one of SEQ ID NOs: 110-113, 128-131, or having at least 90% identity thereto; Alternatively, the ORF comprises any one selected from the following 1)-4): 1) a polynucleotide sequence encoding the heavy chain amino acid sequence shown in SEQ ID NO: 117, and / or a polynucleotide sequence encoding the light chain amino acid sequence shown in SEQ ID NO: 118; 2) a polynucleotide sequence encoding HCDR1, HCDR2 and HCDR3 in the heavy chain amino acid sequence as shown in SEQ ID NO: 117, and a polynucleotide sequence encoding LCDR1, LCDR2 and LCDR3 in the light chain amino acid sequence as shown in SEQ ID NO: 118, wherein the CDRs are selected according to Kabat, IMGT, Chothia, AbM or Contact numbering system, preferably defined according to the Kabat numbering system; 3) a polynucleotide sequence as shown in SEQ ID NO: 119 or having at least 90% identity thereto, and / or a polynucleotide sequence as shown in SEQ ID NO: 120 or having at least 90% identity thereto; 4) a polynucleotide sequence as shown in SEQ ID NO: 121 or having at least 90% identity thereto, and / or a polynucleotide sequence as shown in SEQ ID NO: 122 or having at least 90% identity thereto.

24. A lipid nanoparticle as described in any one of claims 22-23, comprising a sequence shown in any one of SEQ ID NOs: 115, 116, 127, 132, or having at least 90% identity thereto; or, comprising a sequence shown in SEQ ID NO: 124, or having at least 90% identity thereto and / or a sequence shown in SEQ ID NO: 125, or having at least 90% identity thereto.

25. The lipid nanoparticle according to any one of claims 1-8, 18-24, wherein The nucleic acid construct is DNA or RNA, preferably RNA, and more preferably mRNA.

26. The lipid nanoparticle according to claim 25, wherein The RNA further comprises: (d) a 5' cap structure (5'Cap); Preferably, the 5'Cap is selected from Cap0, Cap1, Cap2, Cap3, Cap4, ARCA, modified ARCA, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine and 2-azido-guanosine; More preferably, the 5'Cap is 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.

27. The lipid nanoparticle according to claim 25, wherein The RNA comprises one or more modifications, preferably, the modification comprises backbone modification, sugar modification, base modification and / or lipid modification; more preferably, the base modification is uracil modification.

28. A method for treating and / or preventing a disease, comprising administering to a subject in need thereof a therapeutically effective amount of the lipid nanoparticles of any one of claims 1-8, 18-27, the lyophilized composition of any one of claims 9-11, the lyophilized formulation of claim 12, the reconstituted solution of claim 13, the preparation of claim 14, or the spray formulation of claim 15; The disease is selected from ischemic disease, metabolic syndrome, diabetes and its complications, restenosis, and nerve damage; Preferably, the ischemic disease is selected from coronary artery disease (CAD), peripheral arterial disease (PAD), myocardial infarction, limb ischemia, thromboangiitis obliterans (TAO), diabetic arteriosclerosis obliterans (DAO); more preferably, the limb ischemia is lower limb ischemia, and most preferably, the limb ischemia is severe lower limb ischemia. (CLI); Preferably, the diabetes and its complications are selected from diabetic peripheral neuropathy, diabetic foot (DFU), and diabetic arteriosclerosis obliterans (DAO); Preferably, the restenosis is selected from post-operative restenosis and post-perfusion restenosis; Preferably, the nerve damage is selected from neurodegenerative diseases, traumatic nerve injury, and peripheral neuropathy; more preferably, the neurodegenerative disease is selected from amyotrophic lateral sclerosis (ALS), Parkinson's disease, and dementia, and the peripheral neuropathy is diabetic peripheral neuropathy.

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