Anti-aging nucleic acid molecules, compositions and uses thereof

Abstract

This invention belongs to the field of biomedical technology, specifically relating to an anti-aging nucleic acid molecule, its composition, and its applications. This invention utilizes a nucleic acid molecule encoding vascular endothelial growth factor A (VEGF). A Nucleic acid molecules that express VEGF A The nucleic acid molecules are delivered into the body, encoding VEGF, which has anti-systemic aging properties. A This protein alleviates hair thinning and neuromuscular dysfunction in the elderly, improves metabolic function of adipose tissue and liver in the elderly, treats and reduces inflammatory aging, systematically reverses the biological process of aging, and prevents and treats age-related systemic diseases with good safety. Furthermore, it expresses VEGF... A Adding a sequence expressing ferritin to a nucleic acid molecule to express VEGF A Protein and ferritin form a 24-mer fusion protein, which prolongs VEGF. A The longer the in vivo half-life, the more its anti-aging effects can be enhanced.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an anti-aging nucleic acid molecule, its composition, and its applications. Background Technology

[0002] Aging is not only defined as a shortened lifespan, but more importantly, as the systemic decline in organ function and the disruption of physiological homeostasis. With a deeper understanding of the mechanisms of aging, the focus of anti-aging interventions has shifted from simply extending lifespan to improving healthy lifespan, emphasizing the restoration of physiological function and the prevention of age-related diseases. Recently, strategies targeting molecular pathways such as mTOR, AMPK, and sirtuins have shown potential in preclinical models, primarily employing small molecule drugs, gene therapy, and immunotherapy. However, the clinical translation of these methods still faces challenges, particularly in achieving systemic functional reversal and ensuring long-term efficacy and safety. Therefore, there is a need to develop therapeutic strategies that can both target specific molecular pathways and restore the overall function of biological systems.

[0003] Vascular endothelial growth factor A (VEGF) A ) is composed of VEGF A A gene-encoded protein. Studies have shown that VEGF... A By binding to VEGFR1 and VEGFR2 receptors, VEGF plays a central regulatory role in angiogenesis, cell survival, tissue repair, and regeneration, and has been shown to stimulate endothelial cell mitosis and cell migration. Furthermore, VEGF... A It can inhibit neutrophil brain infiltration in Alzheimer's disease by targeting endothelial cell Cdk5 signaling and suppress obesity by regulating macrophages. However, regarding VEGF... A Its application in the field of anti-aging has not been fully explored, and supplementation with VEGF... A Whether proteins can intervene in aging requires further systematic research. Summary of the Invention

[0004] The purpose of this invention is to provide an anti-aging nucleic acid molecule and its composition for use in the preparation of anti-aging products, and an anti-aging mRNA-LNP nanoparticle to enhance VEGF. A It binds to receptors, prolongs the half-life of proteins in vivo, provides systemic anti-aging, reverses the biological process of aging, prevents and treats age-related systemic diseases, and is safe to use.

[0005] This invention provides the application of nucleic acid molecules encoding vascular endothelial growth factor A in the preparation of anti-aging products and / or in the preparation of drugs for the prevention and treatment of age-related systemic diseases.

[0006] Preferably, the nucleic acid molecule includes one or more of DNA, mRNA, circRNA, and saRNA.

[0007] Preferably, the nucleic acid molecule contains a nucleotide sequence encoding vascular endothelial growth factor A;

[0008] The amino acid sequence of the vascular endothelial growth factor A has at least 80% identity with the amino acid sequence shown in SEQ ID NO:1.

[0009] Preferably, the nucleic acid molecule further comprises a nucleotide sequence encoding ferritin;

[0010] The amino acid sequence of the ferritin has at least 80% identity with the amino acid sequence of the light or heavy chain of the ferritin.

[0011] Preferably, the amino acid sequence encoded by the nucleic acid molecule has at least 80% identity with the amino acid shown in SEQ ID NO:8.

[0012] This invention provides an anti-aging nucleic acid molecule, wherein the open reading frame of the nucleic acid molecule contains a nucleotide sequence encoding vascular endothelial growth factor A.

[0013] Preferably, the open reading frame of the nucleic acid molecule further includes a nucleotide sequence encoding ferritin.

[0014] Preferably, the nucleic acid molecule comprises a 5' cap structure, a 5' untranslated region, the open reading frame, a 3' untranslated region, and a 3'-poly-A tail.

[0015] The present invention provides an anti-aging composition comprising the nucleic acid molecules and excipients described in the above technical solution.

[0016] Preferably, the excipients comprise one or more of the following: injection buffer media, cryoprotectants, and nanocarriers;

[0017] The nanocarrier comprises one or more of the following: liposomes, core-shell nanoparticles, microspheres, cationic polymers, nanoemulsions, micelles, and lipid nanocarriers.

[0018] Preferably, the nanocarrier comprises cationic lipids, sterol lipids, polyethylene glycol lipids, and other auxiliary lipids;

[0019] In the nanocarrier, the molar percentage of cationic lipids is 20-65%, the molar percentage of auxiliary lipids is 12-55%, the molar percentage of sterol lipids is 15-50%, and the molar percentage of polyethylene glycol lipids is 0.5-5%.

[0020] The present invention also provides the use of the nucleic acid molecules or compositions described in the above technical solutions in the preparation of anti-aging products and / or medicaments for the prevention and treatment of aging-related systemic diseases.

[0021] Beneficial effects:

[0022] This invention provides the application of nucleic acid molecules encoding vascular endothelial growth factor A in the preparation of anti-aging products and / or drugs for the prevention and treatment of age-related systemic diseases. This invention utilizes nucleic acid molecules encoding vascular endothelial growth factor A (VEGF). A Nucleic acid molecules that express VEGF A The nucleic acid molecules are delivered into the body, encoding VEGF, which has anti-systemic aging properties. A Proteins can alleviate hair thinning and neuromuscular dysfunction in the elderly, improve metabolic function of adipose tissue and liver in the elderly, treat and reduce inflammatory aging, systematically reverse the biological process of aging, prevent and treat age-related systemic diseases, and have good safety.

[0023] Furthermore, the present invention expresses VEGF A Adding a sequence expressing ferritin to a nucleic acid molecule to express VEGF A Protein and ferritin form a 24-mer fusion protein, which prolongs VEGF. A The half-life in the body enhances its interaction with receptors, thereby enhancing its anti-aging effect. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0025] Figure 1 A schematic diagram of the mRNA structures encoding VEGF protein (A) and VEGF / Ferritin protein (B);

[0026] Figure 2 Structural predictions and molecular docking results of VEGF and VEGF / Ferritin proteins with VEGFR2;

[0027] Figure 3 The three-dimensional interaction patterns of VEGF and VEGF / Ferritin proteins with VEGFR2 are shown; the green dashed lines represent hydrogen bonds, and the red dashed lines represent salt bridge interactions.

[0028] Figure 4 The 2D interaction patterns of VEGF protein (A) and VEGF / Ferritin protein (B) with VEGFR2 are shown; in the figure, the dentate amino acids exhibit hydrophobic interactions, the green dashed lines represent hydrogen bond interactions, and the red dashed lines represent salt bridge interactions.

[0029] Figure 5Preparation and performance testing of VEGF / Ferritin protein mRNA-LNP nanoparticles; where A is the preparation flowchart; B is a cryo-electron microscopy image, scale bar = 50 nm; C and D are the particle size, PDI, potential, and encapsulation efficiency of LNPs measured by Malvern particle size analyzer (n=3).

[0030] Figure 6 Biodistribution of Luc mRNA-LNPs after intravenous injection in mice; where A represents fluorescence imaging results; B represents statistical results of the distribution of Luc mRNA-LNPs in the liver and spleen (n=3), ***p< 0.001;

[0031] Figure 7 The serum hEPO level in mice was determined by intravenous injection of hEPO mRNA-LNPs; where, ***p < 0.001;

[0032] Figure 8 The results of Western blot analysis of VEGF protein expression in cell supernatant (A) and whole cell lysate (B);

[0033] Figure 9 Serum VEGF protein concentration was measured at each time point after intravenous injection of GFP, VEGF, and VEGF / Ferritin mRNA LNPs (n=3); where p < 0.001.

[0034] Figure 10 The results of detecting the effects of VEGF / Ferritin mRNA-LNPs on alleviating hair thinning and neuromuscular dysfunction in the elderly are as follows: A is the experimental flowchart; B is a schematic diagram of tissue sampling; C is a representative image of each group of mice after 5 months of treatment; D is a representative histological observation image stained with HE; E is the maximum four-paw grip strength (n=8); F is the time that mice can stay on the rotating bar (n=8); G is the latency time of descent in the grid suspension test (n=8); H is the total distance run to exhaustion on the treadmill (n=8); I is HE staining; J is the fraction of the central cell nucleus measured in three sections of each mouse (three different regions per mouse, n=3 mice / group); K is the total serum calcium level (n=4); L is the relative level of p16 mRNA expression in skeletal muscle measured by RT-qPCR (n=3); M is the p16 level in skin measured by RT-qPCR. Relative levels of mRNA expression (n=8); ****p<0.0001; ***p<0.001; **p<0.05; ***p<0.05; ns, not significant;

[0035] Figure 11The results of VEGF / Ferritin mRNA-LNPs treatment on improving metabolic dysfunction in elderly adipose tissue and liver are as follows: A-C represent HE staining of BAT, iWAT, and liver sections; D represents Oil Red O staining of liver sections; E and F represent quantitative results of the average cross-sectional area of ​​BAT and iWAT (n=4); G represents the number of large lipid droplets (n=4); H represents the percentage of Oil Red O stained area (n=4); I represents anti-UCP-1 antibody immunostaining sections; J represents the percentage of UCP-1 positive area (n=4); K represents the relative level of UCP-1 mRNA expression in BAT (n=3); L represents the Prdm16 expression level in iWAT. Relative mRNA expression levels (n=3); M represents serum alanine aminotransferase (ALT) levels (n=4); N represents serum low-density lipoprotein (LDL) levels (n=4); O represents serum total cholesterol (CHOL2) levels (n=4); P represents SA-β-gal staining of frozen liver sections, scale bar = 50 μm; Q and R represent Western blotting analysis of IgG heavy and light chains in liver and white adipose tissue; ****p<0.0001; ***p<0.001; **p<0.05; ***p<0.05; ns, not significant;

[0036] Figure 12 Results of VEGF / Ferritin mRNA-LNPs treatment in alleviating inflammatory senescence; A: HE staining of lung tissue; B: Masson staining of lung tissue; C: Flow cytometry analysis of CD11b+-Ly6G+ neutrophils in CD45+ leukocytes; D: Statistical results of C (n=4); E: Percentage of neutrophils in lung tissue; F and G: Changes in IL-6 levels in serum and lung tissue; H: Western blot analysis of IgG heavy and light chains in mouse lungs; ****p<0.0001; ***p<0.001; **p<0.05; ***p<0.05; ns, not significant;

[0037] Figure 13 Safety evaluation of VEGF / Ferritin mRNA-LNPs; where A~G represent the concentrations of urea, creatinine, lipase, creatine kinase, amylase, and PHOS in mouse serum samples; H represents HE staining of liver, kidney, heart, and spleen, scale bar = 50 μm. Detailed Implementation

[0038] This invention provides the application of nucleic acid molecules encoding vascular endothelial growth factor A in the preparation of anti-aging products and / or in the preparation of drugs for the prevention and treatment of age-related systemic diseases.

[0039] In one embodiment, the nucleic acid molecule of the present invention comprises a nucleotide sequence encoding vascular endothelial growth factor A. In another embodiment, the amino acid sequence of vascular endothelial growth factor A of the present invention has at least 80% identity with the amino acid sequence shown in SEQ ID NO:1; in yet another embodiment, the amino acid sequence of vascular endothelial growth factor A of the present invention has at least 80%, 85%, 90%, 95%, or 98% identity with the amino acid sequence shown in SEQ ID NO:1. In one embodiment, the nucleotide sequence encoding vascular endothelial growth factor A of the present invention includes a) or b): a) the nucleotide sequence shown in SEQ ID NO:2; b) has an identity ≥80% and <100% with the nucleotide sequence in a), and can encode the nucleotide sequence of vascular endothelial growth factor A. In another embodiment, b) of the present invention can have an identity of at least 80%, 85%, 90%, 95%, or 98% with the nucleotide sequence in a), and can encode the nucleotide sequence of vascular endothelial growth factor A.

[0040] In one embodiment, the nucleic acid molecule of the present invention further comprises a nucleotide sequence encoding ferritin. In one embodiment, the amino acid sequence of the ferritin of the present invention has at least 80% identity with the amino acid sequence of the ferritin light chain or heavy chain; in another embodiment, the amino acid sequence of the ferritin of the present invention has at least 85%, 90%, 95%, or 98% identity with the ferritin light chain or heavy chain.

[0041] In one embodiment, when the nucleic acid molecule of the present invention contains a nucleotide sequence encoding ferritin, the vascular endothelial growth factor A and ferritin are linked by flexible amino acids. In another embodiment, the amino acid sequence of the flexible amino acid (i.e., the linker) of the present invention can be any one of GGGGSGGGGS (SEQ ID NO:3), GGGGSGGGGSGGGGS (SEQ ID NO:4), GGGGSGGGGSGGGGSGGGS (SEQ ID NO:5), GGGGSGGGGSGGGGSGGGSGGGS (SEQ ID NO:6), and GGGGSGGGGSGGGGSGGGSGGGSGGGS (SEQ ID NO:7).

[0042] In one embodiment, the amino acid sequence encoded by the nucleic acid molecule of the present invention has at least 80% identity with the amino acid sequence shown in SEQ ID NO:8; in another embodiment, the amino acid sequence encoded by the nucleic acid molecule of the present invention has at least 85%, 90%, 95%, or 98% identity with the amino acid sequence shown in SEQ ID NO:8. In one embodiment, the nucleotide sequence of the nucleic acid molecule of the present invention includes c) or d): c) the nucleotide sequence shown in SEQ ID NO:9; d) has an identity ≥80% and <100% with the nucleotide sequence in c), and can encode vascular endothelial growth factor A and ferritin fusion protein. In one embodiment, d) of the present invention can be a nucleotide sequence with at least 85%, 90%, 95%, or 98% identity with the nucleotide sequence in c), and can encode vascular endothelial growth factor A and ferritin fusion protein.

[0043] The sequence information of SEQ ID NO:1 and SEQ ID NO:2 of this invention is as follows:

[0044] SEQ ID NO:1: MNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQENPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRR.

[0045] SEQ ID NO: 2: 5'-ATGAACTTCCTGCTGAGCTGGGTGCACTGGAGCCTGGCCCTGCTGCTGTACCTGCACCACGCCAAGTGGAGCCAGGCCGCCCCCATGGCCGAGGGCGGCGGCCAGAACCACCACGAGGTGGTGAAGTTCATGGACGTGTACCAGAGGAGCTACTGCCACCCCATCGAAACCCTGGTGGACATCTTCCAGGAGTACCCCGACGAGATCGAGTACATCTTCAAGCCCAGCTGCGTGCCCCTGATGAGGTGCGGCGGCTGCTGCAACGACGAGGGCCTGGAGTGCGTGCCCACCGAGGAGAGCAACATCACCATGCAGATCATGAGGATCAAGCCCCACCAGGGCCAGCACATCGGCGAGATGAGCTTCCTGCAGCACAACAAGTGCGAGTGCAGGCCCAAGAAGGACAGGGCCAGGCAGGAGAACCCCTGCGGCCCCTGCAGCGAGAGGAGGAAGCACCTGTTCGTGCAGGACCCCCAGACCTGCAAGTGCAGCTGCAAGAACACCGACAGCAGGTGCAAGGCCAGGCAGCTGGAGCTGAACGAGAGGACCTGCAGGTGCGACAAGCCCAGGAGG-3';

[0046] The sequence information of SEQ ID NO: 8 to 9 of the present invention is specifically as follows:

[0047] SEQ ID NO:8:MNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQENPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRRGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEWGKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLKHD;

[0048]

[0049] In one embodiment, the nucleic acid molecule of the present invention includes one or more of DNA, mRNA, circRNA, and saRNA; in another embodiment, the nucleic acid molecule of the present invention is mRNA. In one embodiment, the mRNA of the present invention may be encoding VEGF. A and / or VEGF A -Ferritin fusion protein mRNA. As one embodiment, the VEGF described in this invention... A Including all its subtypes, including but not limited to VEGF 206 VEGF 189 VEGF 183 VEGF 165 VEGF 148 VEGF 145 VEGF 165B VEGF 121 VEGF 111 L-VEGF 165 L-VEGF 121 L-VEGF 189 VEGF 206 Ferritin includes both heavy and light chains.

[0050] As one implementation method, the VEGF described in this invention A -Ferritin fusion protein contains VEGF A and Ferritin, the VEGF A Ferritin is linked to VEGF via a flexible amino acid pathway. As one embodiment, the VEGF described in this invention... A - The ferritin fusion protein mRNA includes VEGF 165 -Ferritin fusion protein mRNA. As one embodiment, the VEGF described in this invention... A Including all its subtypes, including but not limited to VEGF 206 VEGF 189 VEGF 183 VEGF 165 VEGF 148 VEGF 145 VEGF 165B VEGF 121 VEGF 111 L-VEGF 165 L-VEGF 121 L-VEGF 189 VEGF 206Ferritin includes both heavy and light chains. In specific embodiments of the invention, VEGF is used. A A transcriptional variant of the gene (i.e., VEGF) 165 The invention is illustrated using light chains of ) and Ferritin as examples, but should not be construed as encompassing the entire scope of protection of this invention.

[0051] The present invention also provides an anti-aging nucleic acid molecule, wherein the open reading frame of the nucleic acid molecule contains a nucleotide sequence encoding vascular endothelial growth factor A.

[0052] In one embodiment, the open reading frame of the nucleic acid molecule described in this invention further includes a nucleotide sequence encoding ferritin. The nucleotide and amino acid information of vascular endothelial growth factor A and ferritin described in this invention has been described above and will not be repeated here.

[0053] In one embodiment, the nucleic acid molecule of the present invention comprises a 5' cap structure, a 5' untranslated region, the open reading frame, a 3' untranslated region, and a 3'-poly-A tail.

[0054] The present invention also provides an anti-aging composition comprising the nucleic acid molecules and excipients described in the above technical solution.

[0055] In one embodiment, the excipients of the present invention comprise one or more of the following: injection buffers, cryoprotectants, and nanocarriers. In another embodiment, the nanocarriers of the present invention comprise one or more of the following: liposomes, core-shell nanoparticles, microspheres, cationic polymers, nanoemulsions, micelles, and lipid nanocarriers.

[0056] In one embodiment, the nanocarrier of the present invention comprises cationic lipids, sterol lipids, polyethylene glycol lipids, and other auxiliary lipids. In one embodiment, the molar percentage of cationic lipids in the nanocarrier of the present invention is 20-65%, the molar percentage of auxiliary lipids is 12-55%, the molar percentage of sterol lipids is 15-50%, and the molar percentage of polyethylene glycol lipids is 0.5-5%. In another embodiment, the molar percentage of cationic lipids in the nanocarrier of the present invention is 50%, the molar percentage of auxiliary lipids is 10%, the molar percentage of sterol lipids is 38.5%, and the molar percentage of polyethylene glycol lipids is 1.5%.

[0057] In one embodiment, the auxiliary lipids of the present invention comprise distearate phosphatidylcholine (DSPC) and / or dioleoylphosphatidylethanolamine (DOPE). In one embodiment, the sterol lipids of the present invention comprise cholesterol. In one embodiment, the polyethylene glycol lipids of the present invention comprise one or more of DMG-PEG1000, DMG-PEG2000, and DMG-PEG3000. In one embodiment, the cationic lipids of the present invention comprise one or more of DOTAP, DOTMA, DOTIM, DDA, DC-Chol, CCS, diCl4-amidine, DOTPA, DOSPA, DTAB, TTAB, CTAB, DORI, DORIE and its derivatives, DPRIE, DSRIE, DMRIE, DOGS, DOSC, LPLL, DODMA, DDAB, Dlin-MC3-DMA, SM102, ALC-0315, CKK-E12, C12-200, phosphatidylethanolamine, and lipids of formula I. The structural formula of the lipids of formula I of the present invention is shown below.

[0058] Formula I

[0059] Among them, X1 and X2 are each independently C4-C 12 Alkylene; X3 is a C2-C8 alkylene; R1 and R2 each independently have the structure shown in Formula III:

[0060] Formula III;

[0061] Alternatively, R1 and R2 may each independently have at least one of the following structures:

[0062]

[0063] Where n is 1 or 2, and R7 and R8 are each independently optional C4-C substitutions. 18 Alkyl, optionally substituted C4-C 18 Heteroalkyl, optionally substituted C4-C 18 alkenyl or optionally substituted C4-C 18 Heterene group;

[0064] R3, R4, R5 and R6 are each independently H, halogen or optionally substituted C1-C3 alkyl;

[0065] The substituted groups are each independently selected from one or more halogens, -OH, -SH, -NH2, -NO2, cyano or C1-C3 alkyl groups;

[0066] X1 is a C4 alkylene, X2 is a C4 alkylene, and X3 is a C2 alkylene, but they do not coexist.

[0067] In another embodiment, the lipid of formula I described in this invention can be at least one of the following structures:

[0068] , ,

[0069] , ,

[0070] , ,

[0071] , ,

[0072] , ,

[0073] , ,

[0074] , ,

[0075] , ,

[0076] , ,

[0077] , .

[0078] As one embodiment, the present invention also provides a method for preparing anti-aging nucleic acid nanoparticles, comprising the following steps: dissolving cationic lipids, sterol lipids, polyethylene glycol lipids and other auxiliary lipids in ethanol to obtain an organic phase; mixing the nucleic acid molecules described in any one of the above technical solutions with citrate buffer to obtain an aqueous phase; and removing the ethanol after mixing the organic phase and the aqueous phase to obtain anti-aging nanoparticles.

[0079] In one embodiment, the molar ratio of cationic lipids, auxiliary lipids, polyethylene glycol lipids, and sterol lipids in this invention is 20~65:12~55:0.5~5:15~50; in another embodiment, the molar ratio of cationic lipids, sterol lipids, polyethylene glycol lipids, and other auxiliary lipids in this invention is 50:38.5:1.5:10. The specific types of cationic lipids, sterol lipids, polyethylene glycol lipids, and other auxiliary lipids in this invention have been described above and will not be repeated here.

[0080] In one embodiment, the mass ratio of cationic lipids to nucleic acid molecules in this invention is 100~1:5~0.1; in another embodiment, the mass ratio of cationic lipids to nucleic acid molecules in this invention is 15:1. In one embodiment, the volume ratio of the organic phase to the aqueous phase in this invention is 1~10:1~100; in another embodiment, the volume ratio of the organic phase to the aqueous phase in this invention is 1:3. In one embodiment, the mixing flow rate in this invention is 5~500 mL / min; in another embodiment, the mixing flow rate in this invention is 12 mL / min. This invention limits the mixing ratio and flow rate, resulting in nanoparticles with more uniform particle size and higher encapsulation efficiency for nucleic acid molecules.

[0081] The present invention also provides the use of the compositions described in the above technical solutions or the nucleic acid nanoparticles described in the above technical solutions in the preparation of anti-aging drugs and / or drugs for the prevention and treatment of aging-related systemic diseases.

[0082] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes an anti-aging nucleic acid molecule, its composition, and its applications, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0083] All experiments in this invention used the C57BL / 6 mouse strain, which was housed in an SPF environment. Naturally aged mice (male, 23 months old) were purchased from Biomax Biotechnology Co., Ltd. (Jiangsu), while young mice (male, 3 months old) and middle-aged / older mice (male, 10 months old) were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing). Behavioral experiments were all conducted within the photoperiod.

[0084] All behavioral experiments followed standard methods in the field: Grasp test: The maximum gripping force of the four paws was measured using a grip dynamometer, and the best value was recorded after 3 repetitions; Suspension test: The suspension time of the mouse on an inverted grid was recorded (more than 10 seconds is valid); Rotary bar test: The mouse's dwell time was recorded while rotating the bar at an accelerated speed (4 to 40 rpm / 5 minutes); Treadmill endurance test: The speed was increased in stages, and the total distance was recorded until exhaustion.

[0085] Histological, immunohistochemical, and immunofluorescence analysis: Tissue processing: After taking tissue, fix with 4% paraformaldehyde, embed in paraffin, and section; Oil Red O staining: stain unfixed liver OCT embedded sections to observe lipid deposition; SA-β-gal activity detection: detect aging-related β-galactosidase activity in frozen sections using a kit (Servicebio); Immunohistochemistry: after antigen retrieval, paraffin sections are stained with anti-UCP-1 antibody (Abcam) and developed with DAB; Immunofluorescence: neutrophils are labeled with anti-Ly6G antibody (Servicebio), developed with Alexa Fluor 488 secondary antibody, and counterstained with DAPI on the nuclei.

[0086] Serum biochemical analysis: Whole blood was allowed to stand for 2 hours, then centrifuged to collect serum, and calcium ions (Ca) were detected. 2+ )content.

[0087] Example 1

[0088] 1. The VEGF sequence is shown. To enhance protein bioactivity and further optimize the mRNA structure, specifically: a 5' untranslated region (5'-UTR) is inserted at the N-terminus of the VEGF signal peptide, a 3' untranslated region (3'-UTR) is inserted at the C-terminus of the VEGF amino acid sequence, a 5' cap structure is inserted at the 5' end of the 5' untranslated region, and a 3'-poly-A tail is inserted at the 3' end of the 3' untranslated region, forming a VEGF protein mRNA transcription element, as illustrated in the diagram. Figure 1 As shown in Figure A, (5' cap structure (Cap), 5' untranslated region (UTR), coding region (CDS), 3' untranslated region (UTR) and poly(A) tail).

[0089] 2. Considering that ferritin can self-assemble into a 24-mer structure, the N-terminus of the ferritin sequence is linked to the C-terminus of VEGF via a linker to form a VEGF-linker-ferritin fusion protein (VEGF / Ferritin or VEGF / Fer). A 5' untranslated region (5'-UTR) is inserted at the 5' end of the VEGF / Ferritin nucleotide sequence, and a 3' untranslated region (3'-UTR) is inserted at the 3' end of the VEGF / Ferritin nucleotide sequence. A 5' cap structure is inserted at the 5' end of the 5' untranslated region, and a 3'-poly-A tail is inserted at the 3' end of the 3' untranslated region, forming a VEGF / Ferritin protein mRNA transcription element, as shown in the diagram. Figure 1 As shown in B.

[0090] 3. To verify that the VEGF / Ferritin protein self-assembles into a 24-mer in vivo, enhancing its biological activity, three-dimensional structures of VEGF and VEGFR2 were constructed using Alphafold2. VEGF / Ferritin protein monomers and their 24-mers were constructed using SWISS-MODEL and Alphafold2, and three-dimensional models of VEGFR2 and the VEGF / Ferritin fusion protein were built to simulate the binding of the VEGF / Ferritin 24-mer to its receptor. Figure 2 Since many age-related histopathologies are primarily associated with insufficient VEGFR2 signaling, VEGFR2 was chosen as a model to investigate whether the VEGF / Ferritin fusion protein could enhance the interaction between VEGF and its receptor. Protein-protein / nucleic acid docking simulations using HADDOCK revealed that the binding energy of the VEGFR2-VEGF monomer was van der Waals force -79.0±16.0 kcal / mol and electrostatic energy -302.8±90.7 kcal / mol, while the binding energy of the VEGFR2-VEGF / Ferritin 24-mer was significantly enhanced (van der Waals force -84.9±3.6 kcal / mol, electrostatic energy -404.5±25.9 kcal / mol). Figure 2 This indicates that the 24-mer design can improve the binding affinity of VEGF to VEGFR2.

[0091] 4. Further analysis of the binding sites revealed that VEGFR2 forms 15 hydrogen bonds and 2 salt bridges with VEGF monomers, while it forms 18 hydrogen bonds and 2 salt bridges with VEGF / Ferritin 24 polymers. Figure 3 This involves additional residues in both the receptor and ligand. Numerous hydrophobic interactions between the two further stabilize the receptor-ligand complex (…). Figure 4 ).

[0092] Through de novo modeling, homology modeling, and protein-protein docking, it was demonstrated that VEGFR2 has a higher binding affinity for the VEGF / Ferritin 24-mer than for the VEGF monomer, providing a structural basis for mRNA-encoded protein therapies designed based on the 24-mer.

[0093] Example 2

[0094] mRNA in vitro transcription

[0095] 1. The transcribed template DNA was inserted into the pUC57 vector, followed by fermentation amplification, plasmid extraction, and purification to prepare plasmid DNA. Then, referring to *Molecular Cloning: A Laboratory Manual (4th Edition)* and the instructions for commercially available restriction endonucleases and DNA purification kits, the plasmid DNA was digested to obtain linearized plasmid DNA template, which was then purified. The concentration and purity of the plasmid DNA and linearized DNA template were detected by spectrophotometry and gel electrophoresis, and complete linearization was confirmed by electrophoresis.

[0096] 2. RNA polymerase, NTPs, and cap analogs were used to synthesize and prepare mRNA. Precursor mRNA was transcribed in vitro according to the kit (Novizan TR101-02). Specifically: Add the relevant reactants to 1.5 mL centrifuge tubes according to the dosage and order. Mix by pipetting three times or gently tapping the bottom of the tube with a finger. Centrifuge briefly to collect the reaction solution at the bottom of the tube. Incubate at 37°C for 4 h. Add 1 μL of DNase and incubate at 37°C for 15 min. Then add 179 μL of nuclease-free water, followed by 200 μL of phenol / chloroform / isoamyl alcohol solution for mRNA extraction. After mixing, centrifuge at 15000 rpm for 10 min and collect the aqueous phase. Add 200 μL of 5 M ammonium acetate solution and incubate overnight at 4°C for mRNA precipitation. Then centrifuge at 15000 rpm for 10 min at 4°C and carefully remove the supernatant. Wash the precipitate with 1 mL of 70% ethanol, centrifuge at 15000 rpm for 10 min at 4°C, carefully remove the supernatant, and air-dry the precipitate. Then reconstitute with 20 μL of nuclease-free water to obtain the mRNA solution.

[0097] In this embodiment, five template DNAs were transcribed according to steps 1 and 2, specifically the VEGF protein mRNA transcription element obtained in Example 1, the VEGF / Ferritin protein mRNA transcription element obtained in Example 1, the Luc mRNA transcription element, the GFP mRNA transcription element, and human erythropoietin (hEPO) mRNA, and VEGF protein mRNA solution, VEGF / Ferritin protein mRNA solution, Luc mRNA solution, GFP mRNA solution, and hEPO-mRNA solution were obtained sequentially.

[0098] Example 3

[0099] Preparation of VEGF / Ferritin protein mRNA-LNP nanoparticles

[0100] according to Figure 5 The specific steps for preparing VEGF / Ferritin protein mRNA-LNP nanoparticles using the procedure described in section A are as follows:

[0101] (1) The lipids, DSPC, cholesterol and DMG-PEG2000 of Formula I were dissolved in ethanol in a molar ratio of 50:10:38.5:1.5 to form the organic phase.

[0102] (2) The VEGF / Ferritin protein mRNA obtained in Example 2 was dissolved in 50 mM citrate buffer (pH 4.0) as the aqueous phase.

[0103] (3) According to Formula I, the mass ratio of lipid to mRNA is 15:1, the volume ratio of organic phase in step (1) to aqueous phase in step (2) is 1:3, and the organic phase and aqueous phase are mixed at a flow rate of 12 mL / min using a microfluidic device to prepare undiluted lipid nanoparticles (LNPs), and then ethanol is removed by ultrafiltration.

[0104] (4) Morphological observation: The morphology of LNPs was observed using a transmission electron microscope (Hitachi H-600). Transmission electron microscopy (TEM) showed that they were uniform spherical nanoparticles. Figure 5 (B)

[0105] (5) Particle size and potential analysis: The particle size and zeta potential of LNPs were determined using a Zetasizer Nano ZS90 (Malvin). The mRNA encapsulation efficiency was calculated using the formula (W total - W free) / W total × 100%, where W total is the total mRNA content, determined by Stunner (Unchained Labs), and W free is the free mRNA content, quantified using the Quant-iT RiboGreen RNA kit (Invitrogen).

[0106] The results showed that dynamic light scattering (DLS) analysis of VEGF / Ferritin protein mRNA-LNP nanoparticles indicated an average particle size of 97.67 nm and a polydispersity index (PDI) of 0.12, confirming their high monodispersity. Figure 5 (C and D); the zeta potential was -0.36 mV, and the encapsulation efficiency reached 95%, verifying the effective loading of VEGF / Ferritin protein mRNA ( Figure 5 (D).

[0107] Comparative Example 1

[0108] The preparation method of VEGF protein mRNA-LNP nanoparticles is the same as in Example 3, except that the VEGF / Ferritin protein mRNA is replaced with the VEGF protein mRNA obtained in Example 2.

[0109] Comparative Example 2

[0110] The preparation method of Luc mRNA-LNPs nanoparticles is the same as in Example 3, except that the VEGF / Ferritin protein mRNA is replaced with the Luc mRNA obtained in Example 2.

[0111] Comparative Example 3

[0112] The preparation method of VEGF / Ferritin protein mRNA-LNPs nanoparticles is the same as in Example 3, except that the lipid of Formula I is replaced with the ionizable lipid SM-102.

[0113] Comparative Example 4

[0114] The preparation method of VEGF protein mRNA-LNP nanoparticles is the same as that of Comparative Example 1, except that the lipid in Formula I is replaced with an ionizable lipid SM-102.

[0115] Comparative Example 5

[0116] The preparation method of Luc mRNA-LNPs nanoparticles is the same as that of Comparative Example 2, except that the lipid in Formula I is replaced with the ionizable lipid SM-102.

[0117] Comparative Example 6

[0118] The preparation method of GFP mRNA-LNP nanoparticles is the same as in Example 3, except that the VEGF / Ferritin protein mRNA is replaced with the GFP mRNA obtained in Example 2.

[0119] Comparative Example 7

[0120] The preparation method of GFP mRNA-LNP nanoparticles is the same as that of Comparative Example 6, except that the lipid in Formula I is replaced with the ionizable lipid SM-102.

[0121] Comparative Example 8

[0122] The preparation method of hEPO mRNA-LNPs nanoparticles is the same as in Example 3, except that the VEGF / Ferritin protein mRNA is replaced with the hEPO-mRNA obtained in Example 2.

[0123] Comparative Example 9

[0124] Preparation of hEPO mRNA-LNPs nanoparticles, specifically the preparation method described in Comparative Example 8, with the only difference being that the lipid in Formula I was replaced with the ionizable lipid SM-102.

[0125] Comparative Example 10

[0126] The preparation method for empty LNP nanoparticles is the same as in Example 3, except that VEGF / Ferritin protein mRNA is not added, and 50 mM citrate buffer (pH 4.0) is used as the aqueous phase.

[0127] Example 4

[0128] (1) Young mice (male, 3 months old) were randomly divided into two treatment groups and treated as follows:

[0129] Treatment group 1 (Formula I lipid): C57BL / 6J mice were intravenously injected with Luc mRNA-LNP nanoparticles obtained in Comparative Example 2 containing 20 µg Luc mRNA;

[0130] Treatment group 2 (SM-102): C57BL / 6J mice were intravenously injected with Luc mRNA-LNP nanoparticles obtained in Comparative Example 5 containing 20 µg Luc mRNA.

[0131] Six hours after injection, mice in each treatment group were intraperitoneally injected with 150 mg / kg D-fluorescein potassium salt. Ten minutes later, the heart, liver, spleen, lungs, and kidneys were dissected and harvested. Bioluminescence imaging was performed using the IVIS Lumina system, and fluorescence intensity was calculated. The results showed that, compared to the clinically approved ionizable lipid (SM-102), treatment group 1 (LNPs prepared from formula I lipids) exhibited significantly improved in vivo delivery efficiency. Figure 6 In the middle A, from left to right, three repeats are represented. In young C57BL / 6 mice, intravenous injection of mRNA-LNPs encoding luciferinase (Luc) resulted in stronger specific fluorescent signals in the liver, spleen, and lungs of the formula I liposome. Figure 6 (B)

[0132] (2) Young mice (male, 3 months old) were randomly divided into two treatment groups and treated as follows:

[0133] Treatment group 1 (Formula I lipid): C57BL / 6J mice were intravenously injected with hEPO mRNA-LNPs nanoparticles obtained in Comparative Example 8 containing 20 µg hEPO mRNA.

[0134] Treatment group 2 (SM-102): C57BL / 6J mice were intravenously injected with hEPO mRNA-LNPs nanoparticles obtained in Comparative Example 9 containing 20 µg hEPO mRNA.

[0135] Six hours after injection, serum hEPO levels were measured in mice in each treatment group. The results showed that the hEPO levels in the Formula I lipid group were twice that in the SM-102 group, further highlighting the superiority of Formula I lipid for the delivery of secretory proteins. Figure 7 ).

[0136] Example 5

[0137] 293T cells were used at a rate of 2×10 5 The cells were seeded at a density of cells / well in 24-well plates. After 24 hours of incubation, the cells were randomly divided into three treatment groups and treated as follows:

[0138] Treatment Group 1 (GFP): The GFP mRNA obtained in Example 2 was mixed with Lipofectamine 2000 transfection reagent to obtain the transfection solution; 293T cells were transfected at a dose of 1 µg GFP mRNA / well;

[0139] Treatment Group 2 (VEGF): The VEGF protein mRNA obtained in Example 2 was mixed with Lipofectamine 2000 transfection reagent to obtain the transfection solution; 293T cells were transfected at a dose of 1 µg VEGF protein mRNA / well;

[0140] Treatment group 3 (VEGF / Ferritin): The VEGF / Ferritin protein mRNA obtained in Example 2 was mixed with Lipofectamine 2000 transfection reagent to obtain the transfection solution; 293T cells were transfected at a dose of 1 µg VEGF / Ferritin protein mRNA / well;

[0141] Twelve hours after transfection, the expression levels of VEGF protein in cell supernatant and whole-cell lysate (WCL) were detected by Western blot (under reducing and non-reducing conditions). The results showed that VEGF and VEGF / Ferritin were partially secreted extracellularly and partially retained intracellularly; PAGE gel electrophoresis under non-reducing conditions (without loading buffer) confirmed the aggregate structure of the VEGF / Ferritin complex. Figure 8 ).

[0142] Example 6

[0143] Drug-time curve monitoring

[0144] To evaluate the in vivo circulation kinetics of mRNA-encoded proteins, young male BALB / c mice (SPF grade, provided by Beijing Biotechnology Co., Ltd.), aged 3 months and weighing 20–22 g, were randomly divided into 6 treatment groups and subjected to the following treatments:

[0145] Treatment group 1 (Formula I lipid-GFP): 10 μg of GFP mRNA-LNPs nanoparticles obtained in Comparative Example 6 were administered via intramuscular injection; the mass of the GFP mRNA-LNPs nanoparticles was based on the mass of the mRNA.

[0146] Treatment group 2 (SM-102-GFP): 10 μg of GFP mRNA-LNPs nanoparticles obtained in Comparative Example 7 were administered via intramuscular injection; the mass of the GFP mRNA-LNPs nanoparticles was based on the mass of the mRNA.

[0147] Three treatment groups (Formula I lipid-VEGF): 10 μg of VEGF protein mRNA-LNPs nanoparticles obtained in Comparative Example 1 were administered via intramuscular injection; the mass of the VEGF protein mRNA-LNPs nanoparticles was based on the mass of the mRNA.

[0148] Treatment group 4 (SM-102-VEGF): 10 μg of VEGF protein mRNA-LNPs nanoparticles obtained in Comparative Example 4 were administered via intramuscular injection; the mass of VEGF protein mRNA-LNPs nanoparticles was based on the mass of mRNA.

[0149] Five groups were treated (Formula I lipid-VEGF / Ferritin): 10 μg of VEGF / Ferritin protein mRNA-LNPs nanoparticles obtained in Example 3 were administered via intramuscular injection; the mass of the VEGF / Ferritin protein mRNA-LNPs nanoparticles was based on the mass of the mRNA.

[0150] Six groups (SM-102-VEGF / Ferritin) were treated: 10 μg of VEGF / Ferritin protein mRNA-LNP nanoparticles obtained in Comparative Example 3 were administered via intramuscular injection. The mass of the VEGF / Ferritin protein mRNA-LNP nanoparticles was calculated based on the mass of mRNA.

[0151] Blood was collected from the orbital sinus of mice at 0, 1.5, 3, 6, 12, 24, 48, and 72 hours after injection in each treatment group. Centrifugation conditions: Serum was separated by centrifugation at 12000g. Special treatment: Serum from the SM-102-VEGF / Ferritin and Formula I lipid-VEGF / Ferritin groups required pre-incubation at 37°C for 30 minutes before testing. Detection reagent: VEGF was used. A ELISA kits were used to measure serum VEGF levels. Results showed that the serum VEGF levels of VEGF or VEGF / Ferritin mRNA encapsulated in LNPs prepared with Formula I lipids were more than 6 times higher than those in the SM-102 group after 3 hours. Serum VEGF levels in the Formula I lipid-VEGF / Ferritin group peaked at 1.5 hours and remained elevated for at least 72 hours, significantly prolonging the protein half-life. Figure 9 ).

[0152] Example 7

[0153] VEGF / Ferritin mRNA-LNPs alleviate age-related hair thinning and neuromuscular dysfunction.

[0154] Because intervention in old age is more clinically feasible than lifelong treatment, according to Figure 10 In the A-treatment procedure, young male mice (3 months old) served as the normal control group, while naturally aging mice (male, 23 months old, equivalent to approximately 60 years old in humans) were randomly divided into three treatment groups and subjected to the following treatments:

[0155] Normal control group (Y-Ctrl): Young mice were injected with PBS 12 times via tail vein, with a 5-day interval between two consecutive injections;

[0156] Treatment Group 1 (O-Ctrl): Naturally aged mice were injected with PBS 12 times via tail vein, with a 5-day interval between two consecutive injections;

[0157] Treatment Group 2 (O-LNP): Naturally aged mice were injected with empty LNP nanoparticles via tail vein 12 times as control Example 10, with a 5-day interval between two consecutive injections and a dose of 10 µg / mouse per injection.

[0158] Treatment Group 3 (O-VEGF / Ferritin): Naturally aged mice were injected 12 times via tail vein with VEGF / Ferritin protein mRNA-LNP nanoparticles obtained in Example 3, with a 5-day interval between two consecutive injections and a dose of 10 µg per mouse per injection.

[0159] Behavioral parameters of mice in each treatment group were monitored at 1 and 3 months after treatment, and the mice were sacrificed at 5 months for histological analysis. Figure 10 (B) Closely monitor the overall health of the mice during the experiment.

[0160] The results showed that the overall health status of aged mice in the third treatment group (O-VEGF / Ferritin) was significantly improved compared with the first and second treatment groups. Hair loss and graying of hair are typical phenotypes of aging. Young mice had black and shiny skin on their backs, while aged mice showed white hair and alopecia areata. Notably, only the O-VEGF / Ferritin group significantly inhibited hair loss in aged mice. Figure 10 (C) Histological analysis showed that hair follicles degenerated in the control group of aged mice, while the density of dorsal hair follicles in the O-VEGF / Ferritin group was significantly restored. Figure 10 (D). The control groups (O-Ctrl and O-LNP groups) all showed significant decline in grip strength, twirling bar time, grid suspension time, and treadmill endurance test. Figure 10In the control group, the cross-sectional area of ​​skeletal muscle (CSA) was decreased and nuclear centrifugation was increased, while in the O-VEGF / Ferritin group, all indicators were significantly improved, suggesting that it can alleviate neuromuscular dysfunction. Histological analysis showed that the CSA of skeletal muscle in aged mice was decreased and nuclear centrifugation was increased, while the CSA of the O-VEGF / Ferritin group was significantly increased. Figure 10 (I), nuclear anomalies decreased ( Figure 10 (J). Furthermore, serum calcium levels (a marker of muscle loss) and the expression of p16, a marker of aging, in muscle and skin were significantly decreased in the O-VEGF / Ferritin group. Figure 10 The presence of KM (in the middle KM) indicates that it delays cell senescence.

[0161] Example 8

[0162] VEGF / Ferritin mRNA-LNPs treatment improves metabolic dysfunction in elderly patients with adipose tissue and liver.

[0163] Mice were treated as described in Example 7, and sacrificed at 5 months. The health status of brown adipose tissue (BAT), white adipose tissue (iWAT), and liver in aged mice was analyzed. Results showed increased lipid accumulation in BAT and iWAT in aged mice, suggesting decreased thermogenesis. Compared to the young control group, the lipid contents of BAT and iWAT were significantly increased in the O-Ctrl and O-LNP groups, while significantly decreased in the O-VEGF / Ferritin group. Figure 11 (A and B). Quantitative analysis further showed that the adipocytes in the O-VEGF / Ferritin group were smaller ( Figure 11 The presence of E and F in the middle indicates that it inhibits adipose tissue albinism.

[0164] Histological analysis of the liver showed that the O-Ctrl and O-LNP groups had significant hepatic steatosis (Oil Red O staining), while the O-VEGF / Ferritin group had only mild lesions. Figure 11 (C and G). Detailed analysis of the livers of 30-month-old mice revealed a significant reduction in lipid droplets in hepatocytes in the VEGF / Ferritin group. Figure 11 The presence of D and H in the blood suggests that it may improve age-related fatty liver.

[0165] To assess thermogenic function, the expression of uncoupling protein 1 (UCP-1) in BAT was detected. The results showed that the levels of UCP-1 protein and mRNA were significantly increased in the O-VEGF / Ferritin group. Figure 11 The presence of IK in iWAT indicates enhanced BAT activity. Simultaneously, the expression of the gene PRDM16 in iWAT is activated in the VEGF / Ferritin group (…). Figure 11 The presence of L (in the middle) suggests that it promotes the browning of white adipose tissue, thereby improving metabolic function.

[0166] In addition, serum alanine aminotransferase (ALT), low-density lipoprotein (LDL), and cholesterol levels were significantly elevated in aged mice, while VEGF / Ferritin treatment significantly reduced them. Figure 11 (Middle MO). These results indicate that VEGF / Ferritin reverses age-related decline in liver function.

[0167] Example 9

[0168] VEGF / Ferritin mRNA-LNPs treatment reduces inflammation and aging

[0169] Mice were treated as described in Example 7. At 5 months, the mice were sacrificed. Lung tissue was digested with collagenase and filtered into a single-cell suspension. Peripheral blood and bone marrow cells were lysed to obtain erythrocytes. The cells were labeled with CD11b-FITC, CD45-PerCP-CY5.5, and Ly6G-PE antibodies and analyzed using FlowJo software. Other assays were performed as in Examples 8 and 9.

[0170] Aging is associated with an increased risk of progressive lung dysfunction and pulmonary fibrosis. Experimental results showed that 30-month-old mice exhibited aging markers such as alveolar dilation, structural damage, and inflammatory infiltration in their lungs. VEGF / Ferritin treatment significantly alleviated these phenotypes. Figure 12 (A). Compared with the young control group, the O-Ctrl and O-LNP groups showed increased pulmonary collagen deposition, while the O-VEGF / Ferritin group showed a significant decrease ( Figure 12 (B) indicates improved lung function and fibrosis. Aging also manifests as "inflammatory aging," i.e., chronic low-grade systemic inflammation. The proportion of neutrophils in the peripheral blood of 30-month-old mice was elevated, and VEGF / Ferritin treatment restored it to normal. Figure 12 In the lung microenvironment, neutrophil infiltration was reduced in the O-VEGF / Ferritin group (C and D). Figure 12 The presence of VEGF / Ferritin (VEGF / Ferritin) indicates that it inhibits age-related lung inflammation pathology. Serum and lung tissue levels of the pro-inflammatory cytokine IL-6 significantly increase with age and accelerate the progression of age-related diseases. VEGF / Ferritin treatment significantly reduced serum and lung IL-6 levels in aged mice (…). Figure 12 In addition, IgG deposition in lung tissue was reduced (F and G). Figure 12 The presence of H in the middle of the spectrum suggests that it alleviates chronic inflammation.

[0171] Example 10

[0172] Safety evaluation of VEGF / Ferritin mRNA-LNPs

[0173] Mice were treated as described in Example 7. At 5 months of age, the mice were sacrificed, and whole blood was collected by centrifugation after standing for 2 hours. Serum levels of urea, creatinine, lipase, CK, amylase, PHOS, and AST were measured. Liver, kidney, and heart samples were collected for HE staining.

[0174] The results showed that there were no significant differences in serum urea, creatinine, lipase, CK, amylase, PHOS, and AST levels among the groups. Figure 13 (A~G). HE staining showed no tissue necrosis in the liver, kidneys, and heart of aged mice. Figure 13 (H).

[0175] As can be seen from the above, the technical solution provided by this invention can systematically reverse the biological process of aging, prevent and treat aging-related systemic diseases, and has strong safety.

[0176] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of nucleic acid molecules encoding vascular endothelial growth factor A in the preparation of anti-aging products and / or drugs for the prevention and treatment of age-related systemic diseases.

2. The application according to claim 1, characterized in that, The nucleic acid molecules include one or more of DNA, mRNA, circRNA, and saRNA.

3. The application according to claim 2, characterized in that, The nucleic acid molecule contains a nucleotide sequence encoding vascular endothelial growth factor A; the amino acid sequence of vascular endothelial growth factor A has at least 80% identity with the amino acid sequence shown in SEQ ID NO:

1.

4. The application according to claim 3, characterized in that, The nucleic acid molecule also contains a nucleotide sequence encoding ferritin; the amino acid sequence of the ferritin has at least 80% identity with the amino acid sequence of the light or heavy chain of ferritin.

5. The application according to claim 4, characterized in that, The amino acid sequence encoded by the nucleic acid molecule has at least 80% identity with the amino acid shown in SEQ ID NO:

8.

6. An anti-aging nucleic acid molecule, characterized in that, The open reading frame of the nucleic acid molecule contains a nucleotide sequence encoding vascular endothelial growth factor A.

7. The nucleic acid molecule according to claim 6, characterized in that, The open reading frame of the nucleic acid molecule also contains a nucleotide sequence encoding ferritin.

8. The nucleic acid molecule according to claim 6 or 7, characterized in that, The nucleic acid molecule comprises a 5' cap structure, a 5' untranslated region, the open reading frame, a 3' untranslated region, and a 3'-poly-A tail.

9. An anti-aging composition, characterized in that, The composition comprises the nucleic acid molecule and excipients as described in any one of claims 6 to 8.

10. The composition according to claim 9, characterized in that, The excipients include one or more of the following: injection buffer, cryoprotectant, and nanocarrier; the nanocarriers include one or more of the following: liposomes, core-shell nanoparticles, microspheres, cationic polymers, nanoemulsions, micelles, and lipid nanocarriers.

11. The composition according to claim 9, characterized in that, The nanocarrier comprises cationic lipids, sterol lipids, polyethylene glycol lipids, and other auxiliary lipids; in the nanocarrier, the molar percentage of cationic lipids is 20-65%, the molar percentage of auxiliary lipids is 12-55%, the molar percentage of sterol lipids is 15-50%, and the molar percentage of polyethylene glycol lipids is 0.5-5%.

12. The use of the nucleic acid molecule according to any one of claims 6 to 8 or the composition according to any one of claims 9 to 11 in the preparation of anti-aging products and / or medicaments for the prevention and treatment of aging-related systemic diseases.

Images