Nucleic acid molecule for treating peripheral artery diseases of lower limbs, pharmaceutical composition and application of nucleic acid molecule

Abstract

The invention provides a nucleic acid molecule for treating peripheral artery diseases of lower limbs and encoding multiple angiogenesis factors, a pharmaceutical composition and application of the nucleic acid molecule. Compared with the prior art, the pharmaceutical composition for treating the peripheral artery diseases of the lower limbs, provided by the invention, can remarkably promote angiogenesis of the lower limbs and improve ischemia of the lower limbs.

Description

[0001] This application claims priority to the prior application filed on April 2, 2024, with the China National Intellectual Property Office, Patent Application No. 202410395803.5, entitled “Nucleic acid molecules, pharmaceutical compositions and uses thereof for treating lower extremity peripheral arterial disease”. TECHNICAL FIELD

[0002] The present application relates to the field of gene therapy, in particular to nucleic acid molecules, pharmaceutical compositions and uses thereof for treating lower extremity peripheral arterial disease. BACKGROUND

[0003] The term “critical limb ischemia” (CLI) was first used by P.R.F. Bell in 1982 to describe a group of diseases associated with leg pain at rest, trophic ulcers, and distal necrosis of the lower extremity. Critical limb ischemia is a state in which arterial blood flow to the tissues of the lower extremity is almost completely stopped. With the improvement of people's living standards, changes in eating habits and the aging of the population, the incidence of peripheral arterial disease is increasing, with an incidence of 3%-10% in the general population, and is increasing with the increasing incidence of risk factors such as diabetes and obesity. Peripheral arterial disease is mainly caused by atherosclerosis, thromboangiitis obliterans and diabetes, and is manifested as ischemia, claudication, and even severe limb ischemia, rest pain, ulceration, gangrene, and limb loss. Peripheral arterial disease (PAD) of the lower extremity is the early symptom of CLI, and is most commonly manifested as pain when walking, which is called “intermittent claudication”.

[0004] Currently, there are 8-12 million PAD patients in North America, and this number has exceeded 200 million worldwide. Even worse, due to population aging and increasing obesity, the global incidence of PAD is expected to reach 400 million by 2050. CLI is the most severe stage of PAD and is usually a general condition caused by atherosclerosis in patients with age, smoking, hypercholesterolemia, diabetes and other risk factors. A PAD patient management guideline states that the mortality rate of CLI patients within one year after diagnosis reaches 25%, and in addition, amputation is even as high as 30%. Exercise, drug therapy and smoking cessation can alleviate some symptoms. The risk of death caused by coexistence of coronary and cerebral atherosclerosis masks the risk of limb loss. The main treatment should be aimed at the systemic atherosclerotic process, and blood lipids, blood sugar and blood pressure should be controlled. In contrast, when there is pain at rest, ischemic ulcers or gangrene, the risk of limb loss becomes great.

[0005] For CLI patients, interventions such as balloon angioplasty, stent placement and surgical revascularization should be considered. The choice of intervention depends on the anatomical structure of the stenosis or occlusion; when the lesion is focal and short, percutaneous intervention is appropriate, but longer lesions must be treated by surgical revascularization to achieve acceptable long-term results. Surgical procedures have the disadvantages of large incision area, slow healing and poor prognosis. Interventional therapy is not suitable for patients with severe complications, sepsis, gangrene and other symptoms. Since the 1990s, the development of vascular gene therapy has brought new hope for lower extremity peripheral arterial disease. This treatment method, which promotes the proliferation and migration of endothelial cells by applying vascular growth factors or transgenic therapy, is called angiogenesis therapy, which can promote angiogenesis and collateral vessel formation in ischemic tissues and improve blood supply to the limbs.

[0006] Although the study of new angiogenesis therapy for lower extremity ischemia has been carried out for more than thirty years, there is still no gene or cell drug for this therapy. This is because the introduction of exogenous pro-angiogenic factor genes has the risk of causing pathological angiogenesis, such as atherosclerotic plaque growth, solid tumor angiogenesis and hemangioma. In addition, the efficiency of gene transfection, safe dose, targeting of target genes, strict regulation of gene expression, and stability of new blood vessels still need further study. Conservative drug therapy and surgical treatment are still the main treatment for lower extremity ischemia.

[0007] Therapeutic angiogenesis based on vascular endothelial growth factor (VEGF) has been studied experimentally and clinically for several decades. However, the half-life of VEGF protein is too short, less than 30 minutes, which limits its direct use in protein therapy. Long-term expression of VEGF protein using gene therapy can cause toxicity due to excessive vascular permeability. Like many paracrine factors, VEGF is not systemically active, but is secreted locally in a pulsatile manner, reaches target cells in a dose-dependent manner, and is rapidly degraded.

[0008] Recombinant vascular growth factors and recombinant cytokines are both cell active ingredients, and are prone to inactivation and degradation, with short half-life and poor stability. mRNA therapy overcomes the challenges of biological macromolecule production and degradation, as well as the difficulties of intracellular delivery. mRNA protein replacement therapy produces proteins secreted by human cells by turning the human body into a protein processing factory, which is safer than existing protein preparation methods and has no rejection reaction. Combined with the pharmacokinetics of mRNA drugs in vivo, mRNA drugs are more suitable for application in the treatment of lower extremity peripheral arterial disease.

[0009] Theoretically, all diseases treated with protein can be solved by mRNA therapy. A series of clinical trials have been started using mRNA to express vascular endothelial growth factor (VEGF) to treat heart failure and mRNA based on CRISPR-Cas9 to treat rare genetic diseases. Local regenerative therapy mRNA drugs play a role in making up for missing proteins by locally administering mRNA to express specific functional proteins, so using mRNA therapy to treat lower limb ischemia by promoting neovascularization is a more potential treatment method than traditional gene therapy.

[0010] VEGF is used to promote angiogenesis in previous reports, but mostly capillary blood vessels, which can improve local blood circulation, but for lower extremity peripheral arterial disease, arterial neogenesis is also important to restore blood supply to the lower extremities. There is an urgent need for mRNA drugs for treating lower extremity peripheral arterial disease. SUMMARY

[0011] To improve the above technical problems, the present application provides nucleic acid molecules encoding various angiogenic factors, such as vascular endothelial growth factor (VEGF-A), fibroblast growth factor (FGF), angiogenin-1 (Ang-1), heat shock protein (HSP), and morphogenetic hedgehog factor (SHH), and pharmaceutical compositions and their use in the preparation of drugs for treating lower extremity peripheral arterial disease. The present application applies a lipid nanoparticle (LNP) drug delivery system that only locally delivers mRNA, and screens suitable mRNA therapy for blood vessel regeneration in small animals. The content disclosed in the present application provides a theoretical and physical basis for clinical transformation and screening of therapeutic drugs.

[0012] In one aspect, the present application first provides nucleic acid molecules encoding angiogenic factors selected from one, two, three or more combinations of vascular endothelial growth factor (VEGF-A), fibroblast growth factor (FGF), angiogenin-1 (Ang-1), heat shock protein (HSP), and morphogenetic hedgehog factor (SHH).

[0013] According to an embodiment of the present application, the angiogenic factor is selected from any combination of the following: VEGF-A, a combination of VEGF-A and FGF-2, a combination of VEGF-A and Ang-1, a combination of VEGF-A and SHH, a combination of VEGF-A and HSP70, a combination of VEGF-A, HSP70 and FGF2, a combination of VEGF-A, HSP70 and Ang-1, a combination of VEGF-A, HSP70 and SHH, and a combination of VEGF-A, FGF-2, Ang-1, SHH and HSP70.

[0014] In some embodiments, the combination of angiogenic factors is a combination of VEGF-A, HSP70 and SHH; for example, the nucleic acid molecules of VEGF-A, HSP70 and SHH are in a mass ratio of 1 : (0.25-4) : (0.25-4); exemplarily, the mass ratio is 1 : 1 : 1, 1 : 4 : 1, 4 : 4 : 1, 4 : 1 : 1, 1 : 1 : 4, 4 : 1 : 4, 2 : 1 : 1, 1 : 2 : 1, 1 : 1 : 2, 2 : 2 : 1, 2 : 1 : 2 or 1 : 2 : 2.

[0015] According to embodiments of the present application, the nucleic acid molecule is a DNA encoding a combination of angiogenic factors or a construct thereof or an mRNA encoding a combination of angiogenic factors or a construct thereof.

[0016] According to embodiments of the present application, the mRNA is a linear mRNA or a circular mRNA.

[0017] According to embodiments of the present application, the mRNA is an unmodified or chemically modified mRNA, the chemical modification is selected from one, two, three or more of pseudouridine, 1-methylpseudouridine, 2-thiouridine, 5-methylcytosine, 4-methoxy-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, 4-thiouridine, 2-thio-1-methyl-pseudouridine, 1-ethylpseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine and 5’-CAP at the 5’ end.

[0018] According to embodiments of the present application, the 5’-CAP is selected from Cap0 (m7Gppp), Cap1 (m7GpppmN), Cap2 (m7GpppmNmN) or anti-reverse cap analog ARCA (3’-O-Me-m7G(5’)ppp(5’)G).

[0019] According to embodiments of the present application, the mRNA is a combination of independent mRNAs expressing different angiogenic factors.

[0020] According to embodiments of the present application, the mRNA is a single mRNA comprising expression regions of different angiogenic factors, wherein the expression regions of different angiogenic factors are spaced apart by, for example, a 2A peptide expression region.

[0021] According to embodiments of the present application, the mRNA is a circular mRNA comprising a region encoding an angiogenic factor and a translation initiation sequence such as an IRES.

[0022] According to an embodiment of the present application, the mRNA comprises a nucleotide sequence encoding an amino acid sequence as set forth in any one of SEQ ID NOs: 1-5.

[0023] In one aspect, the present application provides a pharmaceutical composition for treating peripheral arterial disease of lower extremities, the pharmaceutical composition comprising the above-mentioned nucleic acid molecule, or further comprising a pharmaceutically acceptable carrier, for example, the pharmaceutically acceptable carrier is a lipid nanoparticle (abbreviated as LNP).

[0024] According to an embodiment of the present application, the pharmaceutical composition comprises the above-mentioned nucleic acid molecule and a lipid nanoparticle.

[0025] According to an embodiment of the present application, the nucleic acid molecule is a combination of nucleic acid molecules expressing different angiogenic factors. In one embodiment, the nucleic acid molecules expressing different angiogenic factors are present in separate lipid nanoparticle formulations. Further, the nucleic acid molecules expressing different angiogenic factors in the nucleic acid combination are single lipid nanoparticle formulations.

[0026] According to an exemplary embodiment of the present application, the pharmaceutical composition is selected from one, two or more than three of the following compositions: Ang-1 mRNA-LNP composition, VEGF-A mRNA-LNP composition, FGF2 mRNA-LNP composition, HSP70 mRNA-LNP composition, SHH mRNA-LNP composition, or a composition in which VEGF-A, HSP70 and SHH are present in LNP at the same time.

[0027] According to an embodiment of the present application, the lipid nanoparticle comprises an ionizable cationic lipid, a structural lipid, a helper lipid, and a polyethylene glycol lipid.

[0028] According to an embodiment of the present application, the lipid nanoparticle comprises, in mole percent (mol%), 20-60 mol% ionizable cationic lipid, 25-55 mol% structural lipid, 5-25 mol% helper lipid, and 0.5-15 mol% polyethylene glycol lipid.

[0029] According to an embodiment of the present application, the structural lipid is selected from one or both of cholesterol and cholesterol derivatives, preferably cholesterol.

[0030] According to an embodiment of the present application, the cationic lipid is selected from one or more than two of SM-102, ALC-0315, ALC-0519, Dlin-MC3-DMA, DODMA, DLin-KC2-DMA, and DlinDMA, preferably SM-102.

[0031] According to embodiments of the application, the helper lipid is selected from DSPC, DOPE, DOPC, DOPG or DOPS, preferably DOPE.

[0032] According to embodiments of the application, the polyethylene glycol lipid is selected from PEG1000-DMG, PEG2000-DMG, PEG-DSPE or DTDA-PEG2000, preferably PEG1000-DMG.

[0033] In one aspect, the present application provides a method for treating a disease, comprising administering to a subject in need thereof a therapeutically effective amount of the aforementioned nucleic acid molecule or pharmaceutical composition; the disease is a disease that can be treated by repair and / or regeneration, for example, lower extremity peripheral arterial disease, and preferably lower extremity critical ischemia.

[0034] The present application also provides a method for repairing or regenerating a tissue or organ, comprising administering to a subject in need thereof a therapeutically effective amount of the aforementioned nucleic acid molecule or pharmaceutical composition.

[0035] In one aspect, the present application also provides use of the aforementioned nucleic acid molecule or pharmaceutical composition in the preparation of a medicament for treating a disease that can be treated by repair and / or regeneration, for example, lower extremity peripheral arterial disease, and preferably lower extremity critical ischemia.

[0036] In one embodiment, the present application provides use of the aforementioned nucleic acid molecule or pharmaceutical composition in the preparation of a medicament for treating lower extremity peripheral arterial disease suffered by a subject in need thereof.

[0037] According to embodiments of the application, the lower extremity peripheral arterial disease includes leg artery narrowing, limb ischemia (for example, lower extremity critical ischemia), claudication, and chronic ischemic rest pain, ulceration or gangrene and limb loss of the lower extremity due to arterial occlusion.

[0038] In preferred embodiments of the present application, the pharmaceutical composition treats lower extremity peripheral arterial disease, especially lower extremity critical ischemia, by vascular regeneration and / or bone regeneration.

[0039] In one aspect, the present application also provides use of the aforementioned nucleic acid molecule or pharmaceutical composition in the preparation of a medicament for repairing or regenerating a tissue or organ.

[0040] In the present application, the repair or regeneration includes, but is not limited to, repair or regeneration of cells, tissues and / or organs, for example, including but not limited to repair or regeneration of cells, blood vessels, bones, cartilages, bone tissues, ligaments, nerves, skins, myocardiums, islets, pancreases, livers, kidneys, retinas, tendons, etc. Among them, the blood vessels include arterial blood vessels and / or capillary blood vessels; the cells include muscle cells, fibroblasts, myofibroblasts, neurons, dorsal root ganglion cells, neuronal structures such as axons, neural precursor cells, neural stem cells, glial cells, endogenous stem cells, neutrophils, mesenchymal stem cells, satellite cells, myoblasts, myotubes, muscle progenitor cells, adipocytes, preadipocytes, chondrocytes, osteoblasts, osteoclasts, preosteoblasts, tendon progenitor cells, tendon cells, hair follicle cells, stem cells (hematopoietic stem cells), and / or endothelial cells, etc.

[0041] As disclosed in the document Zhang, M., Fukushima, Y., Nozaki, K. et al. Enhancement of bone regeneration by coadministration of angiogenic and osteogenic factors using messenger RNA. Inflamm Regener 43, 32 (2023), bone regeneration can be promoted by vascular regeneration, so the above-mentioned bone regeneration is preferably bone regeneration promoted by vascular regeneration.

[0042] Definitions

[0043] As used herein, "angiogenic factor" refers to a molecule that stimulates blood vessel development, e.g., promotes angiogenesis, endothelial cell growth, vascular stability, and / or vasculogenesis, etc. In one embodiment, an angiogenic factor refers to a factor that accelerates wound healing, including but not limited to one or more growth hormone, insulin-like growth factor-I (IGF-I), VIGF, epidermal growth factor (EGF), members of the CTGF family, FGF2, Ang-1, SHH, HSP70, and TGF-α and TGF-β. See, e.g., Klagsbrun and D'Amore, Annu. Rev. Physiol., 53:217-39 (1991); Streit and Detmar, Oncogene, 22:3172-3179 (2003); Ferrara & Alitalo, Nature Medicine 5(12): 1359-1364 (1999); Tonini et al., Oncogene, 22:6549-6556 (2003) (e.g., Table 1 listing known angiogenic factors); Sato Int. J. Clin. Oncol., 8:200-206 (2003).

[0044] As used herein, "nucleic acid molecule" refers to an oligomer or polymer of at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) that are typically linked together by phosphodiester bonds. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. A nucleic acid molecule can be single-stranded or double-stranded, and can be cDNA.

[0045] As used herein, "pharmaceutical composition" refers to a preparation of a plurality of agents. A preparation containing a therapeutically effective amount of a nucleic acid molecule provided herein is a sterile liquid solution, liquid suspension, or lyophilized form, optionally including a stabilizer or excipient.

[0046] It will be appreciated that the aforementioned nucleic acid molecules will be administered with a suitable pharmaceutically acceptable carrier, excipient, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, etc. A wide variety of appropriate formulations can be found in Remington's Pharmaceutical Sciences (15th Ed., Mack Publishing Company, Easton, Pa. (1975)), particularly Chapter 87 by Blaug, Seymour. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing (cationic or anionic) carriers (e.g., Lipofectin, TMS M102, DOPE, cholesterol, and PEG 1000-DMG), DNA conjugates, anhydrous abs, oil-in-water and water-in-oil emulsions, emulsions in polyethyleneglycol (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing polyethylene glycol. Any of the aforementioned mixtures can be suitable for use in the treatment or therapy according to the present application, provided that the active ingredients in the formulation are not inactivated by the formulation and the formulation is physiologically compatible and tolerable to the route of administration.

[0047] As used herein, "lower extremity peripheral arterial disease" is understood to be a disease associated with arteries other than those of the heart or brain, including narrowing of leg arteries, ischemia of a limb, claudication, and chronic ischemic rest pain, ulceration or gangrene of the lower extremities due to arterial occlusion. Peripheral arterial disease typically affects the legs, but can involve other arteries.

[0048] As used herein, "critical limb ischemia" is understood to be a subdivision of peripheral arterial disease, wherein the condition is characterized by chronic ischemic rest pain, ulceration or gangrene in one or both legs due to objectively documented arterial occlusive disease.

[0049] As used herein, "treatment" of an individual having a disease or condition means partial or total alleviation of symptoms of the individual, or remaining unchanged after treatment. Thus, treatment includes prevention, therapy and / or cure. Prevention refers to preventing the underlying disease and / or preventing the symptoms from worsening or the disease from developing. Treatment also includes any pharmaceutical use of any of the nucleic acid molecules provided herein and compositions provided herein.

[0050] As used herein, "therapeutically effective amount" means an amount of a substance, nucleic acid molecule, compound, material, or composition comprising a compound, administered to a subject which is at least sufficient to produce a therapeutic effect. Thus, it is the amount necessary to prevent, cure, ameliorate, retard or partially retard symptoms of a disease or disorder.

[0051] As used herein, "prophylactically effective amount" refers to an amount of a substance, nucleic acid molecule, compound, material, or composition comprising a compound that, when administered to a subject, will have the intended prophylactic effect, e.g., prevent or delay the onset of a disease or symptom, reduce the likelihood of the onset of a disease or symptom. A fully prophylactically effective dose need not occur by administration of one dose, and can only occur after administration of a series of doses. Thus, a prophylactically effective amount can be administered in one or more administrations.

[0052] As used herein, the term "subject" refers to a mammal, such as a human, a cow, and a dog.

[0053] As used herein and unless otherwise indicated, the terms "comprising," "including," "having," "containing," and their grammatical variants, are generally understood to be open-ended and non-limiting, e.g., not excluding additional unrecited elements or steps.

[0054] Beneficial effects

[0055] The present application provides pharmaceutical compositions that can significantly promote the regeneration of cells, tissues, or organs. For example, pharmaceutical compositions that promote angiogenesis and reduce inflammation in the lower limbs can effectively reestablish blood circulation and improve the necrosis of ischemic lower limbs. BRIEF DESCRIPTION OF DRAWINGS

[0056] Figure 1 A schematic diagram for screening combinations of angiogenic factors. Among the five angiogenic factors, Ang-1 (labeled as No. 3), VEGF-A (labeled as No. 1), FGF2 (labeled as No. 2), HSP70 (labeled as No. 5), and SHH (labeled as No. 4), VEGF-A was used to screen multiple combinations: A is SAM immunohistochemistry, in which SAM represents the newly formed arteries in the lower limbs; B is CD31 immunohistochemistry, in which CD31 represents the newly formed capillaries in the lower limbs; C is HE staining, which represents the pathological morphology of the muscles in the lower limbs, and the morphology and inflammation of the muscles are observed. In combination with the above indicators, the combination of VEGF-A + SHH + HSP70 can significantly promote angiogenesis and improve the physiological environment of ischemic lower limbs.

[0057] Figure 2The schematic diagram for screening the proportion of each factor in the VEGF-A+SHH+HSP70 angiogenesis factor combination. Based on the combination of the three angiogenesis factors VEGF-A, SHH and HSP70, the proportion of the three factors is screened: A is SAM immunohistochemistry, SAM represents the newly formed arteries in the lower limbs; B is CD31 immunohistochemistry, CD31 represents the newly formed capillaries in the lower limbs; C is HE staining and inflammation evaluation, HE staining represents the pathological morphology of the lower limb muscles, and the muscle morphology and inflammation are observed, and the inflammation is verified and evaluated by detecting IL-1β; D is the functional score of the lower limbs, and the recovery of the lower limbs is evaluated. In combination with the above indexes, when the proportion of VEGF-A, SHH and HSP70 is equal, the inflammation is the lowest, the blood supply is the best, and the morphology of the lower limb necrosis is significantly improved. DETAILED DESCRIPTION

[0058] The technical solutions of the present application will be further described in detail below in combination with specific examples. It should be understood that the following examples are only illustratively described and explained, and should not be interpreted as limiting the scope of protection of the present application. Any technology realized based on the above description of the present application is covered within the scope of protection intended by the present application.

[0059] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0060] Example 1: Preparation of mRNA for lower limb peripheral arterial disease

[0061] 1.1 Amino acid sequence of protein for treating lower limb peripheral arterial disease

[0062] The amino acid sequences of Ang-1, VEGF-A, FGF2, HSP70 and SHH proteins were obtained through the Uniprot protein database.

[0063] 1.1.1 Amino acid sequence of Ang-1 protein:

[0064] MTVFLSFAFLAAILTHIGCSNQRRSPENSGRRYNRIQHGQCAYTFILPEHDGNCRESTTDQYNTNALQRDAPHVEPDFSSQKLQHLEHVMENYTQWLQKLENYIVENMKSEMAQIQQNAVQNHTATMLEIGTSLLSQTAEQTRKLTDVETQVLNQTSRLEIQLLENSLSTYKLEKQLLQQTNEILKIHEKNSLLEHKILEMEGKHKEELDTLKEEKENLQGLVTRQTYIIQELEKQLNRATTNNSVLQKQQLELMDTVHNLVNLCTKEGVLLKGGKREEEKPFRDCADVYQAGFNKSGIYTIYINNMPEPKKVFCNMDVNGGGWTVIQHREDGSLDFQRGWKEYKMGFGNPSGEYWLGNEFIFAITSQRQYMLRIELMDWEGNRAYSQYDRFHIGNEKQNYRLYLKGHTGTAGKQSSLILHGADFSTKDADNDNCMCKCALMLTGGWWFDACGPSNLNGMFYTAGQNHGKLNGIKWHYFKGPSYSLRSTTMMIRPLDF*(SEQ ID NO: 1)

[0065] 1.1.2 Amino acid sequence of VEGF-A protein:

[0066] MNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQENPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRR*(SEQ ID NO: 2)

[0067] 1.1.3 Amino acid sequence of FGF2 protein:

[0068] MAAGSITTLPALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDP HIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSR KYTSWYVALKRTGQYKLGSKTGPGQKAILFLPMSAKS*(SEQ ID NO:3)

[0069] 1.1.4HSP70蛋白的氨基酸序列:

[0070] MAKAAAIGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSYVAFTDTERLIGDAAKNQVALNPQNTVFDAKRLIGRKFGDPVVQSDMKHWPFQVINDGDKPKVQVSYKGETKAFYPEEISSMVLTKMKEIAEAYLGYPVTNAVITVPAYFNDSQRQATKDAGVIAGLNVLRIINEPTAA AIAYGLDRTGKGERNVLIFDLGGGTFDVSILTIDDGIFEVKATAGDTHLGGEDFDNRLVNHFVEEFKRKHKKDISQNKRAVRRLRTACERAKRTLSSSTQASLEIDSLFEGIDFYTSITRARFEELCSDLFRSTLEPVEKALRDAKLDKAQIHDLVLVGGSTRIPKVQKLLQDFFNGRDLNKSINPDEAVAYGAAVQAAILMGDKSENVQDLLLLDVAPLSLGLETAGGVMTALIKRNSTIPTKQTQIFTTYSDNQPGVLIQVYEGERAMTKDNNLLGRFELSGIPPAPRGVPQIEVTFDIDANGILNVTATDKSTGKANKITITNDKGRLSKEEIERMVQEAEKYKAEDEVQRERVSAKNALESYAFNMKSAVEDEGLKGKISEADKKKVLDKCQEVISWLDANTLAEKDEFEHKRKELEQVCNPIISGLYQGAGGPGPGGFGAQGPKGGSGSGPTIEEVD*(SEQ ID NO:4)

[0071] 1.1.5SHH蛋白的氨基酸序列:

[0072] MLLLARCLLLVLVSSLLVCSGLACGPGRGFGKRRHPKKLTPLAYKQFIPNVAEKTLGASGRYEGKISRNSERFKELTPNYNPDIIFKDEENTGADRLMTQRCKDKLNALAISVMNQWPGVKLRVTEGWDEDGHHSEESLHYEGRAVDITTSDRDRSKYGMLARLAVEAGFDWVYYESKAHIHCSVKAENSVAAKSGGCFPGSATVHLEQGGTKLVKDLSPGDRVLAADDQGRLLYSDFLTFLDRDDGAKKVFYVIETREPRERLLLTAAHLLFVAPHNDSATGEPEASSGSGPPSGGALGPRALFASRVRPGQRVYVVAERDGDRRLLPAAVHSVTLSEEAAGAYAPLTAQGTILINRVLASCYAVIEEHSWAHRAFAPFRLAHALLAALAPARTDRGGDSGGGDRGGGGGRVALTAPGAADAPGAGATAGIHWYSQLLYQIGTWLLDSEALHPLGMAVKSS*(SEQ ID NO: 5)

[0073] 1.2 Obtaining mRNA sequences encoding the respective proteins

[0074] Plasmids containing nucleotide sequences encoding the five angiogenic factors, Ang-1, VEGF-A, FGF2, HSP70 and SHH proteins, whose amino acid sequences are shown in SEQ ID NOs: 1-5, respectively, were linearized with the restriction endonuclease BspQl. Transcription was performed with the T7 in vitro transcription kit and cap analog (Megabio Cat# ON-040; Cat# ON-134), obtaining capped mRNA. The transcription templates were digested with DNase I, and the capped mRNA was purified with the mRNA purification kit (Norgen Biotec CellTissue Total RNA Isolation Kit V2 #RC112), respectively.

[0075] Example 2: Preparation of mRNA-LNP compositions

[0076] The water phase was obtained by dissolving mRNA encoding Ang-1, VEGF-A, FGF2, HSP70 and SHH proteins in 50 mM citrate solution (pH = 4) at a concentration of 170 ng / μl. The organic phase was obtained by dissolving lipids in anhydrous ethanol according to the nitrogen-phosphorus ratio of 6 / 1, wherein the lipids comprise SM102, DOPE, cholesterol and PEG 1000-DMG at a ratio of SM102:DOPE:cholesterol:PEG 1000-DMG = 47.2:15.1:36.3:1.4, according to the molar ratio.

[0077] The water phase and the organic phase were mixed at a volume ratio of 3:1 by fishbone microfluidic chip rapid mixing encapsulation, respectively. The mixture of the water phase and the organic phase was dialyzed in PBS at 4°C overnight to restore the pH to neutral to obtain mRNA-LNP, and the encapsulation rate / particle size and other characterization parameters were detected to prepare Ang-1 mRNA-LNP composition, VEGF-A mRNA-LNP composition, FGF2 mRNA-LNP composition, HSP70 mRNA-LNP composition and SHH mRNA-LNP composition, respectively.

[0078] Example 3: Construction of mouse lower limb ischemia model

[0079] The construction process of the acute lower limb ischemia mouse model by ligating the femoral artery is as follows: make an incision of about 1 cm long from the knee to the medial thigh of the lower limb of C57BL / 6 mouse, expose the muscle; cut the subcutaneous adipose tissue transversely to expose the nerve and arteriovenous vessels; separate the femoral artery, and separate the femoral artery from the femoral vein at the distal position close to the knee; pass an 8-0 suture thread under the distal femoral artery, pass an 8-0 suture thread under the proximal femoral artery, block the proximal femoral artery with double knots, and ligate, and finally close the incision with a 5-0 suture thread.

[0080] Example 4: Combination formula screening

[0081] 4.1 Combination screening of mRNA-LNP compositions of angiogenic factors

[0082] The following mRNA-LNP compositions of angiogenic factors were used for combination screening:

[0083] 1. mRNA-LNP composition of vascular endothelial growth factor (VEGF-A);

[0084] 2. mRNA-LNP composition of fibroblast growth factor (FGF2);

[0085] 3. mRNA-LNP composition of angiopoietin-1 (Ang-1);

[0086] 4. mRNA-LNP composition of morphogen Sonic Hedgehog (SHH);

[0087] 5. mRNA-LNP composition of heat shock protein (HSP70).

[0088] The angiogenic factor combination screening procedure is as follows:

[0089] On the second day after modeling, the muscle was injected with 0.1 mg / kg mRNA-LNP, and the recovery of the ischemic lower limbs of the mice was observed on days 0 / 7 / 14. After 14 days, the muscle tissue of the lower limbs of the mice was taken to make paraffin sections, and the immunohistochemistry of α-smooth muscle actin (SAM) representing arterial angiogenesis and platelet endothelial cell adhesion molecule (CD31) representing capillary angiogenesis was detected;

[0090] (1) Paraffin embedding, sectioning, dewaxing, and hydration of muscle tissue.

[0091] (2) Antigen repair: the sample was treated with an appropriate antigen repair solution to expose the target antigen.

[0092] (3) Blocking endogenous peroxidase: eliminating non-specific binding.

[0093] (4) Serum blocking: a histology circle was drawn around the section with a histology pen, and 3% BSA was added dropwise to evenly cover the tissue in the histology circle, and the section was blocked at room temperature for 30 min.

[0094] (5) Addition of primary antibody: the sample was incubated with SAM primary antibody (proteintech 55135-1-AP) / CD31 primary antibody (bcamab182981) to bind to the target antigen.

[0095] (6) Addition of secondary antibody: the sample was incubated with the secondary antibody to bind to the primary antibody.

[0096] (7) DAB color development; counterstaining of cell nuclei

[0097] (8) Dehydration and mounting; counting the number of blood vessels of mouse angiogenesis.

[0098] Based on VEGF-A(1), multiple combinations were screened for administration, and the lower limb pathological morphology and the number of angiogenesis were screened, among which VEGF-A+FGF2(1+2), VEGF-A+SHH(1+4), VEGF-A+HSP70(1+5), and VEGF-A+HSP70+SHH(1+5+4) all significantly promoted the angiogenesis of the lower limbs of the mice, and VEGF-A+HSP70+SHH(1+5+4) had good pathological morphology without inflammatory infiltration, so the mRNA-LNP composition of VEGF-A+SHH+HSP70 was used as the optimal combination for further research (seeFigure 1 ).

[0099] 4.2 Angiogenic factor ratio screening

[0100] The ratio of each factor in the VEGF-A+SHH+HSP70 combination was screened, and eight groups of dose ratios were set. The following lower limb pathological morphology, IL-1β inflammation, angiogenesis number and functional score were further screened.

[0101] The steps of muscle inflammation detection are as follows:

[0102] 1. Take 0.1 g of mouse muscle tissue, grind with 100 μl PBS, and centrifuge at 5000 rpm to take the supernatant.

[0103] 2. Detect the IL-1β content in the supernatant by mouse interleukin 1β enzyme-linked immunosorbent assay kit.

[0104] According to the proportion of 2.5 μg, 5 μg or 10 μg mRNA per mouse (wherein 10 μg is converted to about 0.5 mg / kg according to body weight), the administration ratio combination is optimized. When the administration ratio of VEGF-A, SHH and HSP70 is 10 μg: 10 μg: 10 μg, more blood vessels are increased, the number of arterial angiogenesis is 470% of that of VEGF-A alone, the number of capillary angiogenesis is 270% of that of VEGF-A alone, the inflammation is lower, the functional score is good, and it is explained that the administration of VEGF-A, SHH and HSP70 has a positive effect on the treatment of lower limb severe ischemia by revascularization, improvement of inflammatory response, etc. (see Figure 2 ).

[0105] The above describes the embodiments of the present application. However, the present application is not limited to the above-described embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A nucleic acid molecule encoding an angiogenic factor, wherein the angiogenic factor is selected from one, two, three or more of vascular endothelial growth factor (VEGF-A), fibroblast growth factor (FGF), angiopoietin-1 (Ang-1), heat shock protein (HSP) and morphogen hedgehog (SHH).

2. The nucleic acid molecule according to claim 1, wherein the angiogenic factor is selected from any one of the following combinations: VEGF-A, a combination of VEGF-A and FGF-2, a combination of VEGF-A and Ang-1, a combination of VEGF-A and SHH, a combination of VEGF-A and HSP70, a combination of VEGF-A, HSP70 and FGF2, a combination of VEGF-A, HSP70 and Ang-1, a combination of VEGF-A, HSP70 and SHH, and a combination of VEGF-A, FGF-2, Ang-1, SHH and HSP70; Preferably, the combination of angiogenesis factors is a combination of VEGF-A, HSP70 and SHH; Preferably, the mass ratio of the nucleic acid molecules encoding the angiogenic factors VEGF-A, HSP70 and SHH in the nucleic acid molecules is 1:(0.25-4):(0.25-4); preferably, it is 1:1:1, 1:4:1, 4:4:1, 4:1:1, 1:1:4, 4:1:4, 2:1:1, 1:2:1, 1:1:2, 2:2:1, 2:1:2 or 1:2:

2.

3. The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule is a DNA encoding a combination of angiogenic factors or a construct thereof, or an mRNA encoding a combination of multiple angiogenic factors or a construct thereof; Preferably, the mRNA is a linear mRNA or a circular mRNA.

4. The nucleic acid molecule according to claim 3, wherein the mRNA is unmodified or chemically modified, and the chemical modification is selected from one, two, three or more of pseudouridine, 1-methylpseudouridine, 2-thiouridine, 5-methylcytosine, 4-methoxy-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, 4-thiouridine, 2-thio-1-methyl-pseudouridine, 1-ethylpseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine and 5'-CAP at the 5' end; Preferably, the 5'-CAP is selected from Cap0 (m7Gppp), Cap1 (m7GpppmN), Cap2 (m7GpppmNmN) or the anti-reversal cap analog ARCA (3'-O-Me-m7G(5')ppp(5')G).

5. The nucleic acid molecule according to claim 3, wherein the mRNA is a combination of independent mRNAs expressing different angiogenic factors; Preferably, the mRNA is a single mRNA comprising different angiogenic factor expression regions, wherein the expression regions of different angiogenic factors are separated by, for example, 2A peptide expression regions; Preferably, the mRNA is a circular mRNA, comprising a region encoding an angiogenic factor and a translation initiation sequence such as IRES; Preferably, the mRNA comprises a nucleotide sequence encoding an amino acid sequence as shown in any one of SEQ ID NOs: 1-5.

6. A pharmaceutical composition comprising the nucleic acid molecule according to any one of claims 1 to 5, or further comprising a pharmaceutically acceptable carrier; for example, the pharmaceutically acceptable carrier is a lipid nanoparticle; Preferably, the pharmaceutical composition comprises the nucleic acid molecule and lipid nanoparticles according to any one of claims 1 to 5; Preferably, the nucleic acid molecule is a combination of nucleic acid molecules expressing different angiogenesis factors; Preferably, the nucleic acid molecules expressing different angiogenic factors are present in independent lipid nanoparticle preparations; Preferably, the nucleic acid molecules expressing different angiogenic factors in the nucleic acid molecule combination are present in a single lipid nanoparticle formulation.

7. The pharmaceutical composition according to claim 6, wherein the lipid nanoparticles comprise ionizable cationic lipids, structural lipids, helper lipids and polyethylene glycol lipids; Preferably, in terms of molar percentage (mol%), the lipid nanoparticles comprise 20-60 mol% ionizable cationic lipids, 25-55 mol% structural lipids, 5-25 mol% helper lipids and 0.5-15 mol% polyethylene glycol lipids.

8. The pharmaceutical composition according to claim 6 or 7, wherein the structured lipid is selected from cholesterol and cholesterol derivatives, preferably cholesterol; The cationic lipid is selected from the group consisting of: SM-102, ALC-0315, ALC-0519, Dlin-MC3-DMA, DODMA, DLin-KC2-DMA, and DlinDMA, preferably SM-102; The helper lipid is selected from DSPC, DOPE, DOPC, DOPG or DOPS, preferably DOPE; The polyethylene glycol lipid is selected from PEG1000-DMG, PEG2000-DMG, PEG-DSPE or DTDA-PEG2000, preferably PEG1000-DMG.

9. Use of the nucleic acid molecule according to any one of claims 1 to 5 and the pharmaceutical composition according to any one of claims 6 to 8 in the preparation of a medicament for treating lower extremity peripheral arterial disease in a subject in need thereof; Preferably, the lower limb peripheral arterial disease includes leg artery stenosis, limb ischemia, claudication, and chronic ischemic rest pain of the lower limbs due to arterial occlusion, ulcers or gangrene, and limb loss.

10. Use of the nucleic acid molecule according to any one of claims 1 to 5 and the pharmaceutical composition according to any one of claims 6 to 8 in the preparation of a medicament for treating a disease, or for repairing or regenerating a tissue or organ.

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