FGF18 protein coding mRNA and mRNA-LNP delivery system and application in preparation of medicine for repairing cartilage and / or treating osteoarthritis

Abstract

The invention discloses mRNA (messenger Ribonucleic Acid) for coding FGF18 (Fibroblast Growth Factor 18) protein, an mRNA-LNP delivery system and application of the mRNA to preparation of a medicine for repairing cartilage and / or treating osteoarthritis, the mRNA comprises a 5 'UTR (Untranslated Region) sequence, an FGF18 coding sequence and a 3' UTR sequence which are sequentially connected, an amino acid sequence coded by the mRNA is shown as SEQ ID NO.7, the 5 'UTR sequence is shown as SEQ ID NO.3 or SEQ ID NO.4, and the 3' UTR sequence is shown as SEQ ID NO.5 or SEQ ID NO.6. The invention further discloses a preparation method of the mRNA. According to the invention, by designing and screening the UTR sequence and the coding region sequence, a plurality of mRNAs for coding the FGF18 protein, which have high stability and high translation efficiency in cartilage cells, are constructed; according to the present invention, the mRNA-LNPs is further adopted to wrap the mRNA, and the LNPs is optimized so as to finally obtain the mRNA-LNP delivery system capable of effectively infiltrating into the deep part of the cartilage and highly expressing the FGF18 protein, such that the effective treatment on the osteoarthritis is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical biotechnology, and specifically relates to an mRNA encoding FGF18 protein, an mRNA-LNP delivery system, and applications in preparing drugs for cartilage repair and / or treating osteoarthritis. Background Art

[0002] Osteoarthritis (OA) is a common joint disease worldwide, affecting more than 250 million people and placing a significant burden on the economy and society. It is a multifactorial disease with risk factors including malalignment of the lower limbs, obesity, dyslipidemia, and age. Currently, most treatments are aimed at relieving painful symptoms in the early stages of the disease, but advanced OA leads to irreversible cartilage defects, often requiring total knee replacement surgery. Therefore, patients with OA are in need of effective drugs to reverse the progression of the disease.

[0003] In recent years, biological therapies have shown promise in the treatment of OA. For example, fibroblast growth factor 18 (FGF18) has been found to promote the proliferation and differentiation of chondrocytes, and monoclonal antibodies to tumor necrosis factor-α (TNF-α) (such as adalimumab) and interleukin-1β (IL-1β) receptor antagonists have shown the potential to inhibit inflammation and slow cartilage degradation. However, despite promising preclinical results, a series of drugs have failed in clinical development. These failures are often attributed to the challenges of intrachondral drug delivery: rapid clearance and insufficient penetration depth are the main obstacles to effective intrachondral drug delivery. Even with intra-articular injection, the half-life of many compounds is only 2-4 hours. Recombinant proteins are rapidly degraded in cartilage, and only 7.7% of the recombinant proteins remain on the surface of femoral and tibial cartilage 24 hours after injection. Similarly, IL-1β receptor antagonists failed to reach effective concentrations after a single injection, resulting in Phase II clinical failure. The short half-life shortens the duration of treatment and restricts the overall efficacy. In addition, most chondrocytes are located in the middle and deep layers of the cartilage and are covered by the negatively charged cartilage matrix, making it difficult for biological macromolecules to reach the chondrocytes.

[0004] Therefore, there is an urgent need in the art to develop a new drug that can penetrate deep into cartilage and has high bioavailability in cartilage tissue, so as to achieve the goal of highly effective treatment of osteoarthritis. Summary of the invention

[0005] In order to solve the problem that existing OA treatment drugs have poor therapeutic effects due to low effective delivery rate in cartilage and low bioavailability in cartilage tissue, the present invention provides an mRNA-LNP delivery system that can effectively penetrate deep into cartilage and continuously express FGF18 protein, thereby achieving the goal of effective treatment of osteoarthritis and providing a new option for the treatment of OA patients.

[0006] To solve the above problems, the present invention first provides an mRNA encoding FGF18 protein, wherein the mRNA comprises a 5'UTR sequence, an FGF18 coding sequence and a 3'UTR sequence connected in sequence, and the amino acid sequence encoded by the mRNA is shown in SEQ ID NO.7, wherein the 5'UTR sequence is shown in SEQ ID NO.3 or SEQ ID NO.4, and the 3'UTR sequence is shown in SEQ ID NO.5 or SEQ ID NO.6.

[0007] Preferably, the sequence of the mRNA is shown as SEQ ID NO.1 or SEQ ID NO.2.

[0008] Preferably, the sequence of the mRNA includes one or more of the following sequences:

[0009] a. A nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2;

[0010] b. a nucleotide sequence in which one or more nucleotides are added, substituted, deleted or inserted into the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2;

[0011] c. a nucleotide sequence that hybridizes to the nucleotide sequence shown in a or b or its full-length complement under stringent conditions;

[0012] d. A degenerate sequence of the nucleotide sequence shown in any one of the above ac.

[0013] Preferably, the mRNA is chemically modified mRNA, and the chemical modification comprises: using chemically modified bases instead of conventional bases.

[0014] Preferably, the chemically modified bases include at least one of 5mC and 5moU.

[0015] On the other hand, the present invention also provides an mRNA-LNP delivery system, which comprises: lipid nanoparticles encapsulating any of the above-mentioned mRNAs.

[0016] Preferably, the lipid nanoparticles include: SM102, DSPC, cholesterol and DMG-PEG2000.

[0017] Use of any of the above-mentioned mRNAs, or the above-mentioned delivery systems in the preparation of drugs for cartilage repair and / or treatment of osteoarthritis.

[0018] Preferably, the drug is injected into the articular cavity.

[0019] Another aspect of the present invention provides a pharmaceutical composition, which comprises: any of the above-mentioned mRNAs, or the above-mentioned delivery systems, and a pharmaceutically or physiologically acceptable carrier.

[0020] Compared with the prior art, the beneficial effects of the present invention include at least:

[0021] 1. The present invention constructs a variety of mRNAs encoding FGF18 proteins with high stability and high translation efficiency in chondrocytes by designing and screening UTR sequences and coding region sequences; further, lipid nanoparticles (LNPs) are used to encapsulate the aforementioned mRNA, and the composition and proportion of the lipid nanoparticles are optimized at the same time, and finally an mRNA-LNP delivery system that can effectively penetrate deep into the cartilage and continuously express the FGF18 protein is obtained. In a specific embodiment, the mRNA-LNP delivery system expressing the FGF18 protein of the present invention has a penetration depth of more than 50 μm in the knee joint of mice, which is 5 times more than the penetration depth of directly delivering the FGF18 protein, and the expression of the FGF18 protein in the chondrocytes is also significantly increased, and finally the effective treatment goal of osteoarthritis is achieved.

[0022] 2. The preparation method of the mRNA-LNP delivery system for expressing FGF18 protein provided by the present invention is simple, does not require complex and expensive equipment, is easy to realize industrial production, and has good application prospects in the field of arthritis treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Represents the screening and optimization process and results of the LNP delivery system, where:

[0024] (A) Flow chart of the preparation of the mRNA-LNP system; (B) Particle size of LNPs measured by dynamic light scattering; (C) Representative cryo-TEM images of LNPs, scale bar: 100 nm; (DE) Representative immunofluorescence images and quantitative analysis of the penetration depth of Cy3 dye-labeled FGF18 and Dil dye-labeled LNP (encapsulated mRNA) systems in young and old mouse cartilage (n=4), the white dotted line indicates the boundary between cartilage and subchondral bone, scale bar is 10 μm; (F) Representative TEM images of the collagen fiber network in the cartilage of mice of different ages, scale bar: 1 μm; (GH) Luminescence intensity at different time points after transfection of 293T and ATDC5 cells with Lipo3000 and LNP systems (n=4); (IJ) Intra-articular injection of Luciferase in the knee joint of mice Bioluminescence imaging and signal flux quantitative analysis at different time points after mRNA-LNP (n=3); (K) Statistical graph of fluorescence values ​​of mRNA delivered by LNPs prepared with different lipids, among which SM102 has higher delivery efficiency than Dlin-MC3.

[0025] Figure 2 The figure shows the screening and optimization process and results of the FGF18 mRNA sequence, wherein:

[0026] (AC) Detection and quantitative analysis of FGF18 mRNA expression protein with different UTR sequences (n=3); (D) After cells were transfected with FGF18 mRNA with different UTR sequences, the secretion amount of FGF18 in the cell culture supernatant was determined (n=3); (EG) Detection and quantitative analysis of FGF18 mRNA and protein after transfection with different concentrations of FGF18 mRNA-LNP (n=3); (HI) Time course and quantitative analysis of FGF18 expression after FGF18 mRNA-LNP transfection (n=3); (JK) Determination of FGF18 content in the cell culture supernatant after transfection with FGF18 mRNA-LNP of different concentrations or time (n=3).

[0027] Figure 3 It indicates that the FGF18 mRNA-LNP provided by the present invention has the effect of alleviating inflammatory degeneration of chondrocytes, wherein:

[0028] (AC) COL2A1, SOX9, MMP3, MMP13 and COL1A1 mRNA transcription and protein expression levels after treatment with inflammatory factors, FGF18 or FGF18 mRNA-LNP (n=3 or 4); (DF) Immunofluorescence and quantitative analysis of COL2A1 and SOX9 expression levels in ATDC5 chondrocytes after treatment with FGF18 or FGF18 mRNA-LNP (n=4), scale bar: 50 μm; (GJ) Alcian blue and toluidine blue staining and quantitative analysis of ATDC5 chondrocyte micromass culture at different time points after treatment with FGF18 or FGF18 mRNA-LNP (n=4).

[0029] Figure 4 It indicates that the FGF18 mRNA-LNP provided by the present invention can inhibit chondrocyte senescence induced by inflammatory stimulation, wherein:

[0030] (AB) KEGG pathway enrichment analysis and GSEA (gene set enrichment analysis) of transcriptome sequencing data of ATDC5 chondrocytes stimulated by FGF18 mRNA-LNP; (CE) mRNA and protein expression levels of aging-related genes in chondrocytes treated with FGF18 or FGF18 mRNA-LNP (n=3 or 4); (FI) Ki67 immunofluorescence staining, EdU detection and aging-related β-galactosidase staining after FGF18 or FGF18 mRNA-LNP stimulation, and quantitative analysis (n=4); Scale bars: Ki67 and EdU staining images are 50 μm, and SA-β-gal staining images are 100 μm.

[0031] Figure 5 It indicates that the FGF18 mRNA-LNP provided by the present invention can activate the autophagy pathway of chondrocytes and rescue aging and cartilage degeneration, wherein:

[0032] (AB) Protein expression and quantitative analysis of FOXO3a and autophagy-related genes in ATDC5 chondrocytes after stimulation with FGF18 or FGF18 mRNA-LNP (n=3); (C) Transmission electron microscopy immunofluorescence staining of representative images of LC3 and autophagosomes after stimulation with FGF18 or FGF18 mRNA-LNP, scale bar: LC3B staining 10 μm, TEM image 500 nm; (DE) Protein expression and quantitative analysis of FOXO3a in FOXO3a knockdown cells (n=3); (FG) IL-1β (10 ng / mL), FOXO3a knockdown, 3-MA (5 m (M) Expression and quantitative analysis of BECLIN1, LC3B, P16, SOX9, and COL2A1 in ATDC5 chondrocytes under the influence of FGF18mRNA-LNP (n=3); (HJ) Alcian blue and toluidine blue staining and quantitative analysis of chondrocyte collagen (n=4); (KL) SA-β-gal staining, LC3B immunofluorescence, transmission electron microscopy representative images and related quantitative analysis (n=4); Scale bars: SA-β-gal staining is 200μm, LC3B staining is 10μm, and TEM image is 500nm.

[0033] Figure 6 The repair effect of FGF18 mRNA-LNP provided by the present invention on osteoarthritis caused by DMM is shown, wherein:

[0034] (A) Flow chart of animal experiments; (BC) Quantitative analysis of Von Frey and hotplate experiments at different time points (n=15 or 18); (DE) Representative gait trajectories of mice in different groups and quantitative analysis of maximum contact area, footstep area, average strength, and duty cycle; (F) Representative images of X-ray, micro-CT reconstruction, subchondral bone reconstruction, synovial HE staining, cartilage Safranine O / Fast Green staining, and immunohistochemical staining of Ki67, LC3, and FGF18, scale: 100 μm; (G) Quantitative analysis of subchondral bone BV / TV, trabecular number, thickness, and separation (n=6 or 7); (H) Quantitative analysis of osteophyte number (n=6 or 7); (IL) Quantitative analysis of synovial score, OARSI score, cartilage thickness and area (n=6 or 7); (MO) Quantitative analysis of FGF18 (M), Ki67 (n), and LC3 (O) positive cells (n=6 or 7).

[0035] Figure 7 The results show that the FGF18 mRNA-LNP provided by the present invention has a restorative effect on osteoarthritis symptoms in aged mice, wherein:

[0036] (AB) Representative gait trajectories of mice in different groups and quantitative analysis of maximum contact area, print area, average intensity, and duty cycle (n=15 or 18); (C) Representative images of X-ray, synovial HE staining, cartilage Safranin O / Fast Green staining, and immunohistochemical staining of Ki67, LC3, and FGF18, scale bar: 100 μm; (DG) Quantitative analysis of synovial score (D), OARSI score (E), cartilage thickness (F), and area (G); (HJ) Quantitative analysis of FGF18 (H), Ki67 (I), and LC3 (J) positive cells (n=4 or 6). DETAILED DESCRIPTION

[0037] The technical solution of the present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0038] The processes, conditions, experimental methods, etc. for implementing the present invention, except for those specifically mentioned below, are common knowledge and common common sense in the art and are not particularly limited by the present invention.

[0039] Terminology

[0040] ECM: extracellular matrixc, refers to extracellular matrix;

[0041] UTR: Untranslated Regions, which are non-coding fragments at both ends of messenger RNA (mRNA) molecules.

[0042] Delivery: refers to the process of entering cells from outside the tissue.

[0043] Previous research results show that FGF18 protein plays an important role in the occurrence and development of osteoarthritis. The FGF family and its receptors, called FGF receptors (FGFRs), contribute to the differentiation of chondrocytes and protect articular cartilage from degeneration during bone and cartilage development. However, how to construct a drug for FGF18 that can penetrate deep into cartilage is an urgent problem to be solved in this field.

[0044] mRNA therapy is a new type of treatment method developed in recent years. It has not only achieved great success in the field of vaccines, but has also been applied to protein replacement therapy to rescue protein deficiency. mRNA will not integrate into the genome, and once it enters the cytoplasm, it can be directly translated into protein. Therefore, mRNA-based therapy has shown good safety. However, the negative charge and instability of mRNA, coupled with its susceptibility to nuclease degradation and immunogenicity, require the selection of a suitable delivery system to improve its stability and therapeutic effect.

[0045] Lipid nanoparticles (LNPs) can capture mRNA and neutralize negative charges, remain neutral under normal physiological conditions, and do not interact with negatively charged red blood cells, thereby extending the life of mRNA in the patient's body. After LNPs are absorbed by cells, they are positively charged in the acidic in vivo environment. This positive charge helps mRNA-LNP escape from endosomes. LNPs are usually composed of four lipids, namely ionizable cationic lipids, phospholipids, PEG lipids and cholesterol. Although LNPs have been widely studied in soft tissues, drug delivery to cartilage remains a challenging task due to its unique inorganic and organic components.

[0046] Based on the above research, the present invention has conducted a lot of research in order to construct a drug that can penetrate deep into the cartilage and effectively treat OA: through the design and screening of UTR sequences and coding region sequences, a variety of mRNAs encoding FGF18 proteins with high stability and high translation efficiency in chondrocytes were constructed; further, lipid nanoparticles (LNPs) were used to encapsulate the aforementioned mRNA, and the composition and proportion of the lipid nanoparticles were optimized, ultimately obtaining an mRNA-LNP delivery system that can effectively penetrate deep into the cartilage and continuously express the FGF18 protein, thereby achieving effective treatment of osteoarthritis.

[0047] Therefore, the present invention first provides an mRNA encoding FGF18 protein, wherein the mRNA comprises a 5'UTR sequence, a FGF18 coding sequence and a 3'UTR sequence connected in sequence.

[0048] Wherein, the 5'UTR sequence is shown as SEQ ID NO.3 or SEQ ID NO.4, the 3'UTR sequence is shown as SEQ ID NO.5 or SEQ ID NO.6, and the FGF18 coding sequence is shown as SEQ ID NO.8.

[0049] SEQ ID NO.8:

[0050] 5’-AUGUAUUCAGCGCCCUCCGCCUGCACUUGCCUGUGUUUACAC UUCCUGCUGCUGUGCUUCCAGGUACAGGUGCUGGUUGCCGAGGAGAACGUGGACUUCCGCAUCCACGUGGAGAACCAGACGCGGGCUCGGGACGAUGUGAGCCGUAAGCAGCUGCGGCUGUACCAGCUCUACAGCCGGACCAGUGGGAAACACAUCCAGGUCCUGGGCCGCAGGAUCAGUGCCCGCGGCGAGGAUGGGGACAAGUAUGCCCAGCUCCUAGUGGAGACAGACACCUUCGGUAGUCAAGUCCGGAUCAAGGGCAAGGAGACGGAAUUCUACCUGUGCAUGAACCGCAAAGGCAAGCUCGUGGGGAAGCCCGAUGGCACCAGCAAGGAGUGUGUGUUCAUCGAGAAGGUUCUGGAGAACAACUACACGGCCCUGAUGUCGGCUAAGUACUCCGGCUGGUACGUGGGCUUCACCAAGAAGGGGCGGCCGCGGAAGGGUCCCAAGACCCGGGAGAACCAGCAGGACGUGCAUUUCAUGAAGCGCUACCCCAAGGGGCAGCCGGAGCUUCAGAAGCCCUUCAAGUACACGACGGUGACCAAGUGA-3’。

[0051] In some embodiments, the sequence of the mRNA is as shown in SEQ ID NO.1 or SEQ ID NO.2, and the amino acid sequence encoded by the sequence shown in SEQ ID NO.1 or SEQ ID NO.2 is as shown in SEQ ID NO.7.

[0052] SEQ ID NO.1:

[0053] 5’-GAGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAG AGAACCCGCCACCAUGUAUUCAGCGCCCUCCGCCUGCACUUGCCUGUGUUUACACUUCCUGCUGCUGUGCUUCCAGGUACAGGUGCUGGUUGCCGAGGAGAACGUGGACUUCCGCAUCCACGUGGAGAACCAGACGCGGGCUCGGGACGAUGUGAGCCGUAAGCAGCUGCGGCUGUACCAGCUCUACAGCCGGACCAGUGGGAAACACAUCCAGGUCCUGGGCCGCAGGAUCAGUGCCCGCGGCGAGGAUGGGGACAAGUAUGCCCAGCUCCUAGUGGAGACAGACACCUUCGGUAGUCAAGUCCGGAUCAAGGGCAAGGAGACGGAAUUCUACCUGUGCAUGAACCGCAAAGGCAAGCUCGUGGGGAAGCCCGAUGGCACCAGCAAGGAGUGUGUGUUCAUCGAGAAGGUUCUGGAGAACAACUACACGGCCCUGAUGUCGGCUAAGUACUCCGGCUGGUACGUGGGCUUCACCAAGAAGGGGCGGCCGCGGAAGGGUCCCAAGACCCGGGAGAACCAGCAGGACGUGCAUUUCAUGAAGCGCUACCCCAAGGGGCAGCCGGAGCUUCAGAAGCCCUUCAAGUACACGACGGUGACCAAGUGACUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCC-3’。

[0054] SEQ ID NO.2:

[0055]

[0056] SEQ ID NO.7: 5'-MYSAPSACTCLCLHFLLLCFQVQVLVAEENVDFRIHVENQTRARDDVSRKQLRLYQLYSRTSGKHIQVLGRRISARGEDGDKYAQLLVETDTFGSQ VRIKGKETEFYLCMNRKGKLVGKPDGTSKECVFIEKVLENNYTALMSAKYSGWYVGFTKKGRPRKGPKTRENQQDVHFMKRYPKGQPELQKPFKYTTVTK-3'.

[0057] In some other embodiments, the mRNA sequence includes one or more of the following sequences:

[0058] a. A nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2;

[0059] b. a nucleotide sequence in which one or more nucleotides are added, substituted, deleted or inserted into the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2;

[0060] c. a nucleotide sequence that hybridizes to the nucleotide sequence shown in a or b or its full-length complement under stringent conditions;

[0061] d. A degenerate sequence of the nucleotide sequence shown in any one of the above ac.

[0062] In some embodiments, in order to further improve the stability and translation efficiency of the mRNA and reduce its immunogenicity, chemically modified bases are used instead of conventional bases when the mRNA is transcribed. The chemically modified bases include at least one of 5mC and 5moU, and the number of modifications is one or more.

[0063] The amino acid sequence encoded by the mRNA provided by the present invention comprises one or more of the following sequences:

[0064] (1) the amino acid sequence shown in SEQ ID NO.7;

[0065] (2) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO.7, and which retains the function of the amino acid sequence shown in SEQ ID NO.7;

[0066] (3) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO.7, and which retains the function of the amino acid sequence shown in SEQ ID NO.7.

[0067] The present invention also provides an mRNA-LNP delivery system, which comprises lipid nanoparticles encapsulating the mRNA. The lipid nanoparticles comprise SM102 (ionizable cationic lipid), DSPC, cholesterol and DMG-PEG2000, with a molar ratio of 50:10:38.5:1.5. The mRNA and lipid nanoparticles are combined into a complex through physical action, and the complex can be decomposed in the cell to release the mRNA into the cell.

[0068] Another aspect of the present invention provides a use of the aforementioned mRNA or the aforementioned delivery system in the preparation of a drug for cartilage repair and / or treatment of osteoarthritis.

[0069] In some embodiments, the drug is injected into the joint cavity.

[0070] Another aspect of the present invention provides a pharmaceutical composition, which comprises: the aforementioned mRNA, or the aforementioned delivery system, and a pharmaceutically or physiologically acceptable carrier.

[0071] Suitable pharmaceutically acceptable carriers are well known to those of ordinary skill in the art. A full description of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences. Pharmaceutically acceptable carriers in the composition may contain liquids such as water, phosphate buffer, Ringer's solution, physiological saline, balanced salt solution, glycerol or sorbitol, etc. In addition, auxiliary substances such as lubricants, glidants, wetting agents or emulsifiers, pH buffer substances and stabilizers such as albumin, etc. may also be present in these carriers.

[0072] The present invention has been verified through a series of in vitro and in vivo experiments and found that the mRNA-LNP delivery system (FGF18-mRNA-LNP) expressing FGF18 protein provided by the present invention can effectively penetrate deep into the cartilage and continuously express FGF18 protein, thereby achieving effective treatment of osteoarthritis.

[0073] The experimental process of the present invention is described in detail below in conjunction with specific embodiments and drawings.

[0074] (I) Design and screening of mRNA sequences and construction of mRNA-LNP delivery system of the present invention

[0075] The preparation process of mRNA-LNP delivery system is as follows Figure 1 As shown in A.

[0076] 1. Optimization of LNP delivery system

[0077] The present invention compares the effects of different components on lipid nanoparticles (LNPs), confirms the preparation quality of LNPs by detecting the particle size of LNPs and the size under an electron microscope; and injects FGF18 protein and LNPs encapsulating mRNA containing a luciferase gene into young (3 months) and old (18 months) mice, respectively, to compare the infiltration depths of FGF18 protein and LNPs; and simultaneously selects the optimal LNP formula in combination with the delivery efficiency of LNPs.

[0078] Finally, the components of the LNP provided by the present invention include SM-102, 1,2-DSPC, cholesterol and 1,2-dimyristol-decyl-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG2000), and the molar ratios are 50:10:38.5:1.5 respectively.

[0079] In this embodiment, SM-102, 1,2-DSPC, cholesterol and MG-PEG2000 were purchased from Cayman Chemical (LNP-102, USA) and dissolved in anhydrous ethanol in a molar ratio to form an ethanol lipid mixture; Luciferase mRNA (purchased from Apexbio, R1018) was dissolved in 50mM sodium acetate solution, pH 5.0, and the mixed solution was mixed with the previously prepared ethanol lipid mixture in a volume ratio of 3:1 to obtain an mRNA-LNP solution. Among them, the mixing process is carried out using a microfluidic device with a Y-shaped channel design. After mixing, the mRNA-LNP was dialyzed overnight in a 1000-fold neutral buffer. After testing, the encapsulation efficiency of the mRNA-LNP of the present invention is greater than 90%, and the following tests are also performed:

[0080] The particle size and zeta potential of LNPs were evaluated using a dynamic light scattering (DLS) instrument using a Bettersize instrument (BeNano 90). Each sample was measured three times and the average value was calculated. Figure 1 As shown in BC.

[0081] Cell luciferase expression test: One day before transfection, HEK293T cells or ATDC5 cells were cultured at 1×10 4 The cells were seeded in a black 96-well plate at a density of 100 μL DMEM per well and incubated at 37°C, 5% CO2. 12h, 24h, 48h and 72h after transfection, 100 μL Bio-Lumi TM Firefly Luciferase Reporter Gene Assay Substrate, after 10 minutes, the luciferase activity was detected by luminometer plate reader. Figure 1 As shown in GH.

[0082] Infiltration depth test: DIL dye (MedChemExpress, HY-D0083) solution was added to the mRNA-LNP solution to a final concentration of 100 μM. After sufficient mixing, it was incubated at 37°C in the dark for 8 hours to obtain Dil-LNPs. In addition, Cy3 NHSEster (MedChemExpress, HY-112498) and FGF18 protein (MedChemExpress, HY-P9908) were mixed in 0.1 M sodium bicarbonate buffer at a pH of 8.5. After mixing, they were incubated at room temperature in the dark for 8 hours to obtain Cy3-ADA. Cy3-ADA and Dil-LNPs were purified using a molecular sieve chromatography column (Thermo Fisher Scientific, 89882) to remove unlabeled fluorescent molecules. Then, Cy3-ADA and Dil-LNPs were injected into the joints of 12-week-old male C57BL / 6J mice, and frozen sections of the knee joints were taken 24 hours later to detect the infiltration depth of the fluorescent markers, so as to compare the differences in protein delivery depth and efficiency between direct injection of FGF18 protein and delivery of mRNA using LNPs. Figure 1 As shown in D and E, the penetration depth of the mRNA-LNP delivery system in the mouse knee joint exceeds 50 μm, which is 5 times deeper than the penetration depth of direct delivery of FGF18 protein. The delivery of mRNA via LNP not only has a higher delivery effect and a deeper penetration depth, but can also enter deeper levels and a wider distribution range within the cartilage, and can successfully express the target protein.

[0083] 2. Design and screening of mRNA sequences

[0084] The mRNA encoding FGF18 protein of the present invention comprises a 5'UTR sequence, an FGF18 coding sequence and a 3'UTR sequence connected in sequence, wherein the 5'UTR mainly affects the translation efficiency of the mRNA, and the 3'UTR mainly regulates the stability of the mRNA. In the human body, excessive expression of FGF18 can cause abnormal proliferation of cells, which may induce cancer, so it is necessary to express the FGF18 protein in a short and controllable manner.

[0085] In order to screen and obtain suitable mRNA, the present invention designs and compares the effects of different UTRs on mRNA expression of FGF18 protein, including: mUTR derived from mice, synUTR designed and constructed by the present invention, and natural hUTR of humans. The specific sequences are shown in Table 1, and the FGF18 coding sequence designed and screened is shown in SEQ ID NO.8. Plasmids containing the above three different UTRs were constructed, and these plasmids were used as templates to perform in vitro transcription using an in vitro mRNA transcription system (T7 RNA Polymerase in vitro transcription (IVT) kit, Yeasen, 10673ES). During the mRNA transcription process, uracil was replaced by N1-methyl pseudouracil (m1ψ). Transcription, mRNA capping and tailing, and subsequent mRNA purification were performed according to the instructions of the kit (Yeasen, 10673ES). The concentration of mRNA was measured using a Nanodrop spectrophotometer (Thermo Fisher Scientific, USA).

[0086] Table 1 Specific sequences of mUTR, synUTR and hUTR

[0087]

[0088]

[0089] Furthermore, FGF18 mRNA containing different UTRs was encapsulated using the optimized LNPs of the present invention. One day before transfection, cells were plated at 1×10 4 The cells were inoculated in a black 96-well plate at a density of 100 μL of DMEM per well, and incubated at 37°C, 5% CO 2 . After 12h, 24h, 48h and 72h of transfection, the cells were collected for western blot detection of protein expression.

[0090] The results are as follows Figure 2 It was shown that compared with hUTR, when the untranslated region sequence was mUTR or synUTR, the expression efficiency of recombinant FGF18 mRNA was better, among which the recombinant FGF18 mRNA containing synUTR had the highest expression level, and the expression level was significantly improved ( Figure 2BD).

[0091] More importantly, according to Figure 2 As can be seen from the EK diagram, the expression level of FGF18 is positively correlated with the mRNA content, and decreases with the decrease of the mRNA content. Therefore, the expression level of FGF18 can be controlled by controlling the mRNA content. In addition, with the passage of time, the expression of FGF18 protein gradually decreases. Therefore, the FGF18 mRNA-LNP provided by the present invention can express FGF18 protein transiently and controllably, and will not overexpress FGF18 protein, thereby reducing the risk of carcinogenesis.

[0092] (II) FGF18 mRNA-LNP reduces chondrocyte inflammatory degeneration

[0093] The present invention compares the effects of recombinant FGF18 protein and FGF18 mRNA-LNP system on cell culture under inflammatory (10 ng / mL IL-1β) and non-inflammatory conditions.

[0094] The high-density culture experiment was performed according to the following steps: after digestion, centrifugation, and suspension, ATDC5 chondrocyte suspension was prepared at a concentration of 1.5×10 7 Cells / mL, 10 μL of cell suspension was added to each well of a 24-well culture plate, and the cells were incubated in a cell culture incubator for two hours to allow the cells to attach; then, 1 mL of cell culture medium containing 1% insulin-transferrin-selenium (ITS) (41400045, Thermo Fisher) and 100 ng FGF18 protein / 3 μg FGF18 mRNA-LNP was added to each well, and the cell culture medium was replaced every two days. After different time periods, the cell clusters were fixed and stained with alcian blue or toluidine blue for 30 minutes. The integrated optical density (IOD) value of each micromass was calculated using Image J software, and the cells were collected for qPCR and Western blot detection.

[0095] The results are as follows Figure 3 As shown in the results, the mRNA expression and protein translation of genes related to cartilage matrix synthesis were enhanced after treatment with recombinant FGF18 protein (FGF18 group) or FGF18 mRNA-LNP (LNP group), among which COL2A1 and SOX9 increased by 2 times, while the mRNA expression related to matrix degradation and fibrosis (such as MMP3, MMP13 and COL1A1) was inhibited compared with the IL-1β group alone ( Figure 3 Meanwhile, immunofluorescence results showed that after treatment with recombinant FGF18 protein or FGF18mRNA-LNP, the proportion of SOX9-positive cells and the fluorescence intensity of COL2A1 increased by 30% ( Figure 3High-density micromass culture experiments showed that both FGF18 mRNA-LNP and recombinant FGF18 protein could effectively increase chondrocyte matrix secretion under inflammatory conditions and restore matrix consumption after 4, 6, and 8 days ( Figure 3 In addition, after FGF18 or FGF18 mRNA-LNP treatment, the IOD value of toluidine blue staining increased by 20% compared with the control group or IL-1β alone group ( Figure 3 G and H), the IOD values ​​of alician blue also increased by 20% to 50% compared with the control group or the IL-1β group alone, depending on the duration ( Figure 3 I and J).

[0096] The above results indicate that the mRNA-LNP delivery system expressing FGF18 protein provided by the present invention has the effect of alleviating inflammatory degeneration of chondrocytes.

[0097] (III) FGF18 mRNA-LNP inhibits chondrocyte senescence induced by inflammatory stimulation

[0098] The present invention uses RNA sequencing to perform enrichment analysis on genes related to cell cycle and aging, such as E2F1 and CCND1, in ATDC5 chondrocytes treated with FGF18 mRNA-LNP for 24 hours. RNA sequencing experimental steps: Chondrocytes were cultured at 4×10 6 The cells were seeded at a density of 1.54 μg / mL in six-well plates and incubated with 10 ng / mL TNF-α (C600052, Sangon) or 3 μg / mL FGF18 mRNA-LNP for 24 h. Total RNA was extracted using a total RNA extraction kit (R6812-01HP, Omega, USA). RNA sequencing was performed by Wuhan BGI Genomics Co., Ltd., China, and gene ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and gene set enrichment analysis (GSEA) were performed using the Mybgi platform (Wuhan BGI Genomics, https: / / mybgi.bgi.com / tech / login). The results are shown in Figure 4 As shown in Figure AC.

[0099] Further aging-related studies were conducted on primary mouse chondrocytes. Figure 4 As shown in Figure 2, RT-qPCR and western blot experiments showed that compared with the IL-1β group alone, the expression of cell senescence-related genes P16INK4a, P21, and P53 could be downregulated by 40%-60% after treatment with recombinant FGF18 protein (FGF18 group) or FGF18 mRNA-LNP (LNP group). Figure 4DF). Consistent with these results, SA-β-gal staining experiments showed that under inflammatory conditions, treatment with FGF18 mRNA-LNP or recombinant FGF18 protein reduced the number of aged primary chondrocytes in mice by 50%. In addition, EdU detection and Ki67 immunofluorescence experiments ultimately showed that both FGF18 mRNA-LNP and recombinant FGF18 protein could enhance the proliferation potential of primary chondrocytes, and the proportion of EdU-positive and Ki67-positive cells increased by about 100% compared with the IL-1β group alone ( Figure 4 of GJ).

[0100] The above results indicate that the mRNA-LNP expressing FGF18 protein provided by the present invention can inhibit chondrocyte senescence induced by inflammatory stimulation.

[0101] (IV) FGF18 mRNA-LNP activates chondrocyte autophagy pathway and rescues aging and cartilage degeneration

[0102] In order to clarify the relationship between autophagy and chondrocyte aging and degeneration, the present invention constructed a FOXO3a knockdown cell line ( Figure 5 DE), and 3-MA was used as an inhibitor to inhibit autophagy. The effects of FGF18 mRNA-LNP and FGF18 protein treatment on chondrocyte autophagy were examined by using a series of techniques, including western blot, high-density micromass culture, LC3B immunofluorescence, SA-β-gal staining, and transmission electron microscopy.

[0103] The results showed that the FGF18 mRNA-LNP provided by the present invention can protect chondrocytes from degeneration and aging by enhancing autophagy. Specifically, it was first found that the addition of FGF18 mRNA-LNP led to increased expression of FOXO3a and BECLIN1, thereby increasing the LC3B II / I ratio ( Figure 5 AB); immunofluorescence and transmission electron microscopy revealed a decrease in the number of autolysosomes in an inflammatory setting ( Figure 5 C). But importantly, the FGF18 mRNA-LNP provided by the present invention can significantly improve these changes: the expression of cartilage matrix synthesis-related genes COL2A1 and SOX9 increased by 100%; the expression of BECLIN1 and LC3B II / I ratio was restored, while the expression of the aging gene P16 decreased by 50%. In addition, the proportion of senescent cells decreased by 60%, and the matrix secretion function and autophagosome number of chondrocytes were significantly enhanced. At the same time, when these cells were subjected to FOXO3a knockout and 3-MA treatment, it can be seen by SA-β-gal staining and micromass culture that the protective effect of FGF18 mRNA-LNP was reversed under inflammatory conditions ( Figure 5 of FJ).

[0104] The above results indicate that the mRNA-LNP expressing FGF18 protein provided by the present invention can protect chondrocytes from degeneration and aging by enhancing autophagy.

[0105] (V) Effect of FGF18 mRNA-LNP on the treatment of OA in vivo

[0106] In order to further evaluate the effect of FGF18 mRNA-LNP in treating OA in vivo, the present invention established the models of DMM surgery-induced and elderly-induced osteoarthritis, respectively. The motor ability and reaction sensitivity of mice were quantitatively analyzed by Von Frey and hot plate stimulation experiments at different time points. The walking movement of mice was quantitatively analyzed by analyzing the maximum contact area, footprint area, average intensity, and duty cycle in the gait trajectory. The bones of mice were subjected to X-ray, micro-CT reconstruction, and subchondral bone reconstruction to analyze the changes in the microstructure of the bones of mice. The histological changes of mice were analyzed by synovial HE staining, cartilage Safranine O / Fast Green staining, immunohistochemical staining, etc.

[0107] The results are as follows Figure 6 and Figure 7 The results showed that 500 ng of recombinant FGF18 protein or 6 μg of FGF18 mRNA-LNP were injected into the knee joint cavity weekly. Biosafety experiments confirmed that there were no side effects on other organs after injection. Von Frey and hot plate experiments confirmed that both FGF18 mRNA-LNP and recombinant FGF18 protein could significantly reduce DMM-induced lower limb pain hypersensitivity, but the Von Frey threshold and hot plate reaction time of the LNP-mRNA group were significantly higher than those of the group directly injected with recombinant FGF18 protein ( Figure 6 BC). Gait analysis showed that mice treated with FGF18 mRNA-LNP or recombinant FGF18 protein showed 20% improvement in parameters such as maximum contact area, print area, average intensity, and duty cycle compared with the DMM group or the elderly group ( Figure 6 DE Figure 7 AB).

[0108] Radiographic results showed that intra-articular injection of FGF18 mRNA-LNP or recombinant FGF18 protein treatment reduced subchondral bone sclerosis caused by the DMM model. This treatment resulted in a decrease in BV / TV and trabecular thickness compared with the DMM group. However, the number of trabecular bones remained unchanged after FGF18 mRNA-LNP or recombinant FGF18 protein administration. In addition, the number of osteophytes decreased by 60% after FGF18 mRNA-LNP injection, which was better than the 30% after recombinant FGF18 protein injection ( Figure 6 FG Figure 7 CD).

[0109] In addition, HE staining was used to detect the severity of synovitis, erythropoietin O / Fast green staining was used to evaluate erythropoietin O-stained proteoglycans in the cartilage matrix, and Alcian blue staining was used to detect acidic polysaccharides in the cartilage. These staining results showed that after injection of FGF18 mRNA-LNP or recombinant FGF18 protein, the synovitis and cartilage wear of the treated mice were significantly reduced compared with those of the DMM and elderly model groups, and the synovitis score and OARSI score were lower than those of the DMM group and the elderly model group ( Figure 6 F. Figure 7 C).

[0110] The above results indicate that the mRNA-LNP expressing FGF18 protein provided by the present invention can repair osteoarthritis caused by DMM, and also has a significant repair effect on the symptoms of osteoarthritis in elderly mice.

[0111] In summary, the present invention constructs a variety of mRNAs encoding FGF18 proteins with high stability and high translation efficiency in chondrocytes through the design and screening of UTR sequences and coding region sequences; further, lipid nanoparticles (LNPs) are used to encapsulate the aforementioned mRNA, and the composition and proportion of the lipid nanoparticles are optimized, ultimately obtaining an mRNA-LNP delivery system that can effectively penetrate deep into the cartilage and continuously express the FGF18 protein, thereby achieving the goal of effective treatment of osteoarthritis and providing a new option for the treatment of OA patients.

[0112] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. An mRNA encoding FGF18 protein, characterized in that The mRNA comprises a 5'UTR sequence, an FGF18 coding sequence and a 3'UTR sequence connected in sequence, and the amino acid sequence encoded by the mRNA is shown in SEQ ID NO.7, wherein the 5'UTR sequence is shown in SEQ ID NO.3 or SEQ ID NO.4, and the 3'UTR sequence is shown in SEQ ID NO.5 or SEQ ID NO.

6.

2. The mRNA encoding FGF18 protein according to claim 1, characterized in that The sequence of the mRNA is shown as SEQ ID NO.1 or SEQ ID NO.

2.

3. The mRNA encoding FGF18 protein according to claim 1, characterized in that The mRNA sequence includes one or more of the following sequences: a. A nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2; b. a nucleotide sequence in which one or more nucleotides are added, substituted, deleted or inserted into the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2; c. a nucleotide sequence that hybridizes to the nucleotide sequence shown in a or b or its full-length complement under stringent conditions; d. A degenerate sequence of the nucleotide sequence shown in any one of the above ac.

4. The mRNA encoding FGF18 protein according to claim 1, characterized in that The mRNA is chemically modified mRNA, and the chemical modification includes: using chemically modified bases to replace conventional bases.

5. The mRNA encoding FGF18 protein according to claim 4, characterized in that The chemically modified bases include at least one of 5mC and 5moU.

6. An mRNA-LNP delivery system, characterized in that: The delivery system comprises: lipid nanoparticles encapsulating the mRNA according to any one of claims 1 to 5.

7. The mRNA-LNP delivery system of claim 6, wherein The lipid nanoparticles include: SM102, DSPC, cholesterol and DMG-PEG2000.

8. Use of the mRNA according to any one of claims 1 to 5, or the delivery system according to claim 6 or 7 in the preparation of a medicament for cartilage repair and / or treatment of osteoarthritis.

9. The use according to claim 8, characterized in that The drug is injected into the articular cavity.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: the mRNA according to any one of claims 1 to 5, or the delivery system according to claim 6 or 7, and a pharmaceutically or physiologically acceptable carrier.

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