WO2025208818A1 - Nucleic acid molecule and pharmaceutical composition for treatment of peripheral arterial disease in lower limb, and use thereof - Google Patents

Abstract

Provided are a nucleic acid molecule encoding a plurality of angiogenesis factors and a pharmaceutical composition for the treatment of peripheral arterial disease in the lower limb, and use thereof. Relative to the prior art, the pharmaceutical composition for the treatment of peripheral arterial disease in the lower limb can significantly promote lower limb angiogenesis and improve lower limb ischemia.

Description

Nucleic acid molecules, pharmaceutical compositions and applications thereof for treating lower limb peripheral arterial disease

This application claims priority to the prior application with patent application number 202410395803.5 filed with the State Intellectual Property Office of China on April 2, 2024, and entitled “Nucleic acid molecules, pharmaceutical compositions and their applications for treating peripheral arterial disease of the lower limbs”.

Technical Field

The present application relates to the field of gene therapy, and specifically to nucleic acid molecules, pharmaceutical compositions and their applications for treating lower limb peripheral arterial disease.

Background Art

The term "critical limb ischemia" (CLI) was first introduced 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 limbs. Critical limb ischemia is a state of almost complete cessation of arterial blood flow in the lower limb tissues. 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. The incidence rate in the general population is 3%-10%, and it continues to increase with the increase in the incidence of risk factors such as diabetes and obesity. Peripheral arterial disease is mainly caused by atherosclerosis, thromboangiitis obliterans, and diabetes, and manifests as limb ischemia and claudication. It can even develop into critical limb ischemia, with rest pain, ulcers, gangrene, and limb loss. Peripheral arterial disease (PAD) of the lower limbs is an early symptom of CLI, most commonly manifesting as pain when walking, which is called "intermittent claudication."

Currently, there are 8 to 12 million patients with PAD in North America, and globally, this number exceeds 200 million. Furthermore, due to an aging population and increasing obesity, the global prevalence of PAD is projected to reach 400 million by 2050. CLI, the most severe stage of PAD, is a common condition in patients with atherosclerosis, often exacerbated by the continued presence of risk factors such as aging, smoking, hypercholesterolemia, and diabetes. A PAD management guideline indicates that the mortality rate for patients with CLI is 25% within one year of diagnosis, and the risk of amputation is as high as 30%. Exercise, medication, and smoking cessation can alleviate some symptoms. The mortality risk associated with coexisting coronary and cerebral atherosclerosis overshadows the risk of limb loss. Primary treatment should target systemic atherosclerosis, controlling lipids, blood sugar, and blood pressure. In contrast, the risk of limb loss becomes significantly greater when pain at rest, ischemic ulcers, or gangrene develop.

For patients with CLI, interventions such as balloon angioplasty, stenting, and surgical revascularization should be considered. The choice of intervention depends on the anatomy of the stenotic or occlusive lesion; when the lesion is focal and short, percutaneous intervention is appropriate, but longer lesions must be treated with surgical revascularization to achieve acceptable long-term results. Surgery is invasive. Disadvantages include large size, slow healing, and a poor prognosis. Interventional therapy is not suitable for patients with severe complications, sepsis, or limb gangrene. Since the 1990s, the development of vascular gene therapy has brought new hope for treating peripheral arterial disease of the lower limbs. This treatment, which uses vascular growth factors or transgenic therapy to stimulate endothelial cell proliferation and migration, thereby promoting angiogenesis and collateral vessel formation in ischemic tissues and improving limb blood supply, is known as angiogenesis therapy.

Although research on angiogenesis therapy for lower limb ischemia has been ongoing for over three decades, no gene therapy or cell therapy has yet been developed. This is because the introduction of exogenous pro-angiogenic factor genes carries the risk of inducing pathological angiogenesis, such as plaque growth, vascular proliferation in solid tumors, and hemangiomas. Furthermore, further research is needed to determine the efficiency of gene transfection, safe dosage, targeted delivery of the target gene, tight regulation of gene expression, and the stability of the newly formed vessels. Conservative medical therapy and surgical treatment remain the mainstays of treatment for lower limb ischemia.

Therapeutic angiogenesis based on vascular endothelial growth factor (VEGF) has been studied experimentally and clinically for decades. However, the half-life of VEGF protein is too short, less than 30 minutes, which limits its value for direct protein therapy. Long-term expression of VEGF protein using gene therapy can lead to toxic effects such as excessive vascular permeability. Like many paracrine factors, VEGF does not act systemically, but is secreted locally in a pulsed manner, reaches target cells in a dose-dependent manner, and is then rapidly degraded.

Recombinant vascular growth factors and recombinant cytokines are both cellular active ingredients that are easily inactivated and degraded, have short half-lives, and are relatively unstable. mRNA therapy overcomes the challenges of biomacromolecule production and degradation, as well as the difficulties of intracellular delivery. mRNA protein replacement therapy transforms the human body into its own protein processing factory, producing proteins secreted by its own cells. Compared to existing protein production methods, it is safer and has no rejection reactions. Combined with the pharmacokinetics of mRNA drugs in vivo, mRNA drugs are more suitable for the treatment of lower extremity peripheral arterial disease.

In theory, all diseases treated with proteins can be addressed with mRNA therapy. A series of clinical trials have begun using mRNA to express vascular endothelial growth factor (VEGF) to treat heart failure, and using CRISPR-Cas9 mRNA to treat rare genetic diseases. Local regenerative therapy mRNA drugs express specific functional proteins through local administration of mRNA, thus compensating for missing proteins. Therefore, using mRNA therapy to treat lower limb ischemia by promoting angiogenesis is a more promising treatment approach than traditional gene therapy.

Previous reports have shown that VEGF promotes angiogenesis, but this is mostly in capillaries, improving local blood circulation. However, for lower extremity peripheral arterial disease, arterial regeneration and restoration of lower extremity blood flow are equally important. Currently, there is an urgent need for mRNA drugs to treat lower extremity peripheral arterial disease.

Summary of the Invention

In order to improve the above technical problems, the present application provides nucleic acid molecules encoding multiple angiogenesis factors, such as vascular endothelial growth factor (VEGF-A), fibroblast growth factor (FGF), angiopoietin-1 (Ang-1), heat shock protein (HSP) and morphogenetic protein (VEGF). Nucleic acid molecules and pharmaceutical compositions encoding the morphogen hedgehog (SHH), and their use in the preparation of medicaments for treating lower extremity peripheral arterial disease. This application utilizes a lipid nanoparticle (LNP) drug delivery system that delivers mRNA only locally to screen for suitable angiogenesis mRNA therapies in small animals. The disclosure provides a theoretical and practical foundation for clinical translation and drug screening.

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

According to an embodiment of the present invention, 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.

In some embodiments, the combination of angiogenic factors is a combination of VEGF-A, HSP70 and SHH; for example, the mass ratio of the nucleic acid molecules of VEGF-A, HSP70 and SHH is 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.

According to an embodiment of the present invention, 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.

According to an embodiment of the present invention, the mRNA is a linear mRNA or a circular mRNA.

According to an embodiment of the present invention, the mRNA is unmodified or chemically modified mRNA, and the chemical modification is selected from one, two, three or more of pseudouridine, 1-methylpseudouridine, 2-thiouridine, 5-methylcytosine, 4-methoxy-pseudouridine, 2-thiol-1-methyl-1-deaza-pseudouridine, 4-thiolidine, 2-thiol-1-methyl-pseudouridine, 1-ethylpseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine and 5'-CAP at the 5' end.

According to an embodiment of the present invention, 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).

According to an embodiment of the present invention, the mRNA is a combination of independent mRNAs expressing different angiogenic factors.

According to an embodiment of the present invention, the mRNA is a single mRNA comprising different angiogenic factor expression regions, wherein the expression regions of the different angiogenic factors are separated by, for example, 2A peptide expression regions.

According to an embodiment of the present invention, the mRNA is a circular mRNA, which comprises a region encoding an angiogenic factor and a translation initiation sequence such as IRES.

According to an embodiment of the present invention, the mRNA contains a nucleotide sequence encoding an amino acid sequence as shown in any one of SEQ ID NO:1-5.

In one aspect, the present application provides a pharmaceutical composition for treating lower extremity peripheral arterial disease, which comprises the above-mentioned nucleic acid molecule, or further comprises a pharmaceutically acceptable carrier, for example, the pharmaceutically acceptable carrier is lipid nanoparticles (abbreviated as LNP).

According to an embodiment of the present invention, the pharmaceutical composition comprises the above-mentioned nucleic acid molecules and lipid nanoparticles.

According to an embodiment of the present invention, 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. Furthermore, the nucleic acid molecules expressing different angiogenic factors in the nucleic acid combination are present in a single lipid nanoparticle formulation.

According to an exemplary embodiment of the present invention, the pharmaceutical composition is selected from one, two or more 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 simultaneously present in LNP.

According to an embodiment of the present invention, the lipid nanoparticles comprise ionizable cationic lipids, structural lipids, helper lipids and polyethylene glycol lipids.

According to an embodiment of the present invention, the lipid nanoparticles comprise, in terms of molar percentage (mol%), 20-60 mol% ionizable cationic lipids, 25-55 mol% structural lipids, 5-25 mol% helper lipids and 0.5-15 mol% polyethylene glycol lipids.

According to an embodiment of the present invention, the structured lipid is selected from one or two of cholesterol and cholesterol derivatives, preferably cholesterol.

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

According to an embodiment of the present invention, the helper lipid is selected from DSPC, DOPE, DOPC, DOPG or DOPS, preferably DOPE.

According to an embodiment of the present invention, the polyethylene glycol lipid is selected from PEG1000-DMG, PEG2000-DMG, PEG-DSPE or DTDA-PEG2000, preferably PEG1000-DMG.

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

The present application also provides a method for repairing or regenerating a tissue or organ, which comprises administering a therapeutic agent to a subject in need thereof. An effective amount of the aforementioned nucleic acid molecule or pharmaceutical composition.

In one aspect, the present application also provides the use of the aforementioned nucleic acid molecules or pharmaceutical compositions in the preparation of drugs for treating diseases that are treated by repair and/or regeneration, for example, the disease is peripheral arterial disease of the lower limbs, and preferably severe ischemia of the lower limbs.

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 limb peripheral arterial disease in a subject in need thereof.

According to an embodiment of the present invention, the lower limb peripheral arterial disease includes leg artery stenosis, limb ischemia (such as severe lower limb ischemia), claudication and chronic ischemic rest pain of the lower limbs due to arterial occlusion, ulcer or gangrene and limb loss.

In a preferred embodiment of the present application, the pharmaceutical composition treats lower limb peripheral arterial disease, especially severe lower limb ischemia, through angiogenesis and/or bone regeneration.

In one aspect, the present application also provides the use of the aforementioned nucleic acid molecules or pharmaceutical compositions in the preparation of drugs for repairing or regenerating tissues or organs.

In the present application, the repair or regeneration includes, but is not limited to, the repair or regeneration of cells, tissues, and/or organs, such as, but not limited to, the repair or regeneration of cells, blood vessels, bones, cartilage, bone tissue, ligaments, nerves, skin, myocardium, islets of Langerhans, pancreas, liver, kidney, retina, tendon, etc. Wherein, the blood vessels include arteries and/or capillaries; 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, tenocytes, hair follicle cells, stem cells (hematopoietic stem cells), and/or endothelial cells, etc.

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), angiogenesis can promote bone regeneration, so the above-mentioned bone regeneration is preferably bone regeneration promoted by angiogenesis.

definition

As used herein, "angiogenic factor" refers to a molecule that stimulates vascular development, such as promoting angiogenesis, endothelial cell growth, vascular stability and/or vasculogenesis. In one embodiment, 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), CTGF and its family members, FGF2, Ang-1, SHH, HSP70, and TGF-α and TGF-β. See, for example, 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): 1359-1361. 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).

As used herein, "nucleic acid molecule" refers to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), typically linked together by a phosphodiester bond. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded and can be cDNA.

As used herein, "pharmaceutical composition" refers to a variety of preparations. The preparation containing a therapeutically effective amount of the nucleic acid molecules provided herein is in the form of a sterile liquid solution, liquid suspension or lyophilized form, optionally containing a stabilizer or excipient.

It will be understood that the aforementioned nucleic acid molecules will be administered with suitable pharmaceutically acceptable carriers, excipients, and other agents incorporated into formulations to provide improved transfer, delivery, tolerability, etc. A large number of suitable formulations can be found in the pharmacopoeia known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th ed., Mack Publishing Company, Easton, Pa. (1975)), in particular Chapter 87 of Blaug and Seymour therein. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid-containing (cationic or anionic) carriers (e.g., Lipofectin, TMSM102, DOPE, cholesterol, and PEG 1000-DMG), DNA conjugates, anhydrous slurries, oil-in-water and water-in-oil emulsions, polyethylene glycol emulsions (polyethylene glycol of various molecular weights), semisolid gels, and semisolid mixtures containing polyethylene glycol. Any of the foregoing mixtures may be suitable for use in the treatment or therapy according to the present application, provided that the active ingredient in the formulation is not inactivated by the formulation and that the formulation is physiologically compatible and tolerated for the route of administration.

As used herein, "peripheral arterial disease of the lower extremities" is understood to mean diseases related to arteries other than those of the heart or brain, including narrowing of the arteries in the legs, limb ischemia, claudication, and chronic ischemic rest pain in the lower extremities due to arterial occlusion, ulcers or gangrene, and limb loss. Peripheral arterial disease typically affects the legs, but other arteries may be involved.

As used herein, "critical lower extremity ischemia" is understood to be a subdivision of peripheral arterial disease in which the condition is characterized by chronic ischemic rest pain, ulcers, or gangrene in one or both legs due to objectively demonstrated arterial occlusive disease.

As used herein, "treating" an individual suffering from a disease or condition means that the individual's symptoms are partially or completely alleviated, or remain unchanged after treatment. Thus, treatment includes prevention, treatment, and/or cure. Prevention refers to preventing a potential disease and/or preventing symptoms from worsening or disease progression. Treatment also includes any pharmaceutical use of any nucleic acid molecule provided and the compositions provided herein.

As used herein, a "therapeutically effective amount" refers to an amount of a substance, nucleic acid molecule, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is the amount necessary to prevent, cure, ameliorate, arrest, or partially arrest the symptoms of a disease or condition.

As used herein, a "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 desired prophylactic effect, e.g., preventing or delaying the onset or recurrence of a disease or symptom, or reducing the likelihood of the onset or recurrence of a disease or symptom. A completely prophylactically effective dose need not occur by administering one dose, and may occur only after administering a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations.

As used herein, the term "subject" refers to mammals, such as humans, cows, and dogs.

As used herein and unless otherwise specified, the terms "comprises," "includes," "has," "contains," and their grammatical equivalents should generally be understood as open-ended and non-limiting, e.g., not excluding other unlisted elements or steps.

Beneficial effects

The present application provides a pharmaceutical composition that can significantly promote cell, tissue, or organ regeneration. For example, a pharmaceutical composition that promotes angiogenesis and reduces inflammatory responses in the lower limbs can effectively restore blood flow and improve necrosis in ischemic lower limbs.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of the screening of angiogenesis factor combinations. Among the five angiogenesis factors, Ang-1 (numbered 3), VEGF-A (numbered 1), FGF2 (numbered 2), HSP70 (numbered 5), and SHH (numbered 4), VEGF-A was used as the basis for screening for multiple combinations: A is SAM immunohistochemistry, where SAM characterizes neovascularization in the lower limbs; B is CD31 immunohistochemistry, where CD31 characterizes neovascularization in the lower limbs; C is HE staining, which characterizes the pathological morphology of lower limb muscles and observes muscle morphology and inflammation. Combining the above indicators, the VEGF-A + SHH + HSP70 combination can significantly promote angiogenesis and improve the physiological environment of ischemic lower limbs.

Figure 2 is a schematic diagram of the selection of the ratios of the various factors within the VEGF-A+SHH+HSP70 angiogenesis factor combination. Based on the combination of VEGF-A, SHH, and HSP70, the ratios of the three factors were screened: A is SAM immunohistochemistry, which characterizes arterial neoplasia in the lower limbs; B is CD31 immunohistochemistry, which characterizes capillary neoplasia in the lower limbs; C is HE staining and inflammation assessment, which characterizes lower limb muscle pathology and inflammation, and verifies the assessment by quantitatively measuring IL-1β; D is the lower limb functional score, which assesses lower limb recovery. Combining these indicators, the combination of VEGF-A, SHH, and HSP70 showed the lowest inflammation, the best blood circulation recovery, and significant improvement in lower limb necrosis morphology when the three factors, VEGF-A, SHH, and HSP70, were in equal ratios.

DETAILED DESCRIPTION

The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

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

Example 1: Preparation of mRNA for lower extremity peripheral arterial disease

1.1 Amino acid sequences of proteins related to the treatment of lower limb peripheral arterial disease

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

1.1.1 Amino acid sequence of Ang-1 protein:

1.1.2 Amino acid sequence of VEGF-A protein:

1.1.3 Amino acid sequence of FGF2 protein:

1.1.4 Amino acid sequence of HSP70 protein:

1.1.5 Amino acid sequence of SHH protein:

1.2 Obtaining the mRNA sequence encoding the corresponding protein

Plasmids containing nucleotide sequences encoding five angiogenic factors, Ang-1, VEGF-A, FGF2, HSP70, and SHH proteins, as shown in SEQ ID NOs: 1-5, were linearized with the restriction endonuclease BspQ1. Transcription was performed using a T7 in vitro transcription kit and cap analogs (Zhaowei Cat#ON-040; Cat#ON-134) to obtain capped mRNA. The transcription templates were digested with DNase I and purified using an mRNA purification kit (Novozymes). The capped mRNA was purified using the CellTissue Total RNA Isolation Kit V2 #RC112).

Example 2: Preparation of mRNA-LNP composition

mRNA encoding Ang-1, VEGF-A, FGF2, HSP70, and SHH proteins was dissolved in 50 mM citrate solution (pH 4) at a concentration of 170 ng/μl to obtain an aqueous phase. Lipids containing SM102, DOPE, cholesterol, and PEG 1000-DMG were dissolved in anhydrous ethanol at a molar ratio of 47.2:15.1:36.3:1.4, using a nitrogen-to-phosphorus ratio of 6:1.

The aqueous phase and the organic phase were rapidly mixed and encapsulated in a fishbone microfluidic chip at a volume ratio of 3:1, and the mixture was dialyzed in PBS at 4°C overnight to restore the pH to neutral to obtain mRNA-LNP. The encapsulation efficiency/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.

Example 3: Construction of mouse lower limb ischemia model

The construction process of the mouse model of acute lower limb ischemia induced by femoral artery ligation is as follows: make an approximately 1 cm long incision from the knee to the inner thigh of the lower limb of C57BL/6 mice to expose the muscle; cut the subcutaneous fat tissue transversely to expose the nerves and arteriovenous vessels; after isolating the femoral artery, separate the femoral artery from the femoral vein at the distal position near the knee; pass an 8-0 suture under the distal femoral artery, pass an 8-0 suture under the proximal femoral artery, block the proximal femoral artery with a double knot, and ligate it. Finally, the incision was closed with 5-0 sutures.

Example 4: Combination formula screening

4.1 Combinatorial Screening of Angiogenic Factor mRNA-LNP Combinations

Combinatorial screening was performed using the following mRNA-LNP compositions of angiogenic factors:

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

2. Fibroblast growth factor (FGF2) mRNA-LNP composition;

3. Angiopoietin-1 (Ang-1) mRNA-LNP composition;

4. mRNA-LNP composition of the morphogen hedgehog (SHH);

5. Heat shock protein (HSP70) mRNA-LNP composition.

The steps for screening angiogenesis factor combinations are as follows:

On the second day after modeling, 0.1 mg/kg mRNA-LNP was injected into the muscles at multiple points. The recovery of the ischemic lower limbs of the mice was observed on days 0, 7, and 14. After 14 days, paraffin sections were prepared from the muscle tissue of the lower limbs of the mice after modeling. Immunohistochemical staining for α-smooth muscle actin (SAM), which represents arterial angiogenesis, and platelet endothelial cell adhesion molecule (CD31), which represents capillary angiogenesis, was performed.

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

(2) Antigen retrieval: Treat the sample with an appropriate antigen retrieval solution to expose the target antigen.

(3) Block endogenous peroxidase: eliminate nonspecific binding.

(4) Serum blocking: Use a histochemical pen to draw a histochemical circle around the slice, add 3% BSA in the histochemical circle to evenly cover the tissue, and block at room temperature for 30 minutes.

(5) Add primary antibody: incubate the sample with SAM primary antibody (proteintech 55135-1-AP)/CD31 primary antibody (bcam ab182981) to allow it to bind to the target antigen.

(6) Add secondary antibody: Incubate the sample with the secondary antibody to allow it to bind to the primary antibody.

(7) DAB staining; counterstaining of cell nuclei

(8) Dehydrate and seal the slides; count the number of angiogenic blood vessels in the mice.

Based on VEGF-A (1), multiple combination administrations were screened, and the lower limb pathological morphology and the number of angiogenesis were screened. Among them, 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 angiogenesis in the lower limbs of mice. Among them, VEGF-A+HSP70+SHH(1+5+4) had good pathological morphology and no inflammatory infiltration. Therefore, the mRNA-LNP composition of VEGF-A+SHH+HSP70 was selected as the optimal combination for further study (see Figure 1).

4.2 Screening of angiogenesis factor ratios

Screening the ratio of each factor in the combination of VEGF-A+SHH+HSP70, setting up 8 groups of dosage ratios, IL-1β inflammation, angiogenesis number and functional scores were further screened.

The steps for muscle inflammation testing are as follows:

1. Take 0.1g of mouse muscle tissue, grind it with 100μl PBS, and centrifuge it at 5000rpm to obtain the supernatant.

2. Mouse interleukin-1β enzyme-linked immunosorbent assay kit was used to detect the IL-1β content in the supernatant.

The dosing ratio combination was optimized according to the ratio of 2.5μg, 5μg or 10μg mRNA/mouse (where 10μg is approximately 0.5mg/kg based on body weight). When the dosing ratio of VEGF-A, SHH and HSP70 was 10μg:10μg:10μg, more blood vessels increased, the number of arterial blood vessels generated was 470% of that of VEGF-A alone, and the number of capillary blood vessels generated was 270% of that of VEGF-A alone. Inflammation was lower and the functional score was good, indicating that VEGF-A, SHH and HSP70 have a positive effect on the treatment of severe lower limb ischemia by reconstructing blood supply and improving inflammatory response (see Figure 2).

The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims (12)

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). 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. 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. 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). 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. 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. 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. 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. 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 limb 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. 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. A method for treating a disease, comprising administering a therapeutically effective amount of the nucleic acid molecule according to any one of claims 1 to 5 or the pharmaceutical composition according to any one of claims 6 to 8 to a subject in need thereof; the disease is a disease that can be treated by repair and/or regeneration, such as lower limb peripheral artery disease, and preferably lower limb severe ischemia. A method for repairing or regenerating a tissue or organ, comprising administering a therapeutically effective amount of the nucleic acid molecule according to any one of claims 1 to 5 or the pharmaceutical composition according to any one of claims 6 to 8 to a subject in need thereof.