Polynucleotides targeting ube2m and their use in the treatment of age-related osteoarthritis

Abstract

The present application discloses a polynucleotide targeting UBE2M and its use in treating aging-related osteoarthritis, the polynucleotide comprising a nucleotide sequence as shown in SEQ ID NO: 1, or a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 1 and capable of knocking down UBE2M.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the manufacture of biological drugs, and more specifically to polynucleotides targeting UBE2M and their use in the treatment of osteoarthritis. Background Technology

[0002] Osteoarthritis is a common degenerative joint disease among middle-aged and elderly people, and is one of the main factors leading to decreased mobility and quality of life in the elderly. With the increase in average human lifespan, its incidence is rising daily. Clinically, osteoarthritis is characterized by cartilage degeneration, synovitis, and osteophyte formation.

[0003] Cellular senescence, an irreversible state of cell cycle arrest, is considered one of the key cellular mechanisms driving tissue dysfunction and overall aging. Although senescent cells cease dividing, they continuously secrete large amounts of pro-inflammatory cytokines, chemokines, growth factors, and proteases (senescence-associated secretory phenotype, SASP), driving chronic inflammation and tissue dysfunction. Current treatments for osteoarthritis primarily focus on pain relief and slowing disease progression, lacking effective strategies to reverse or cure the condition. Therefore, identifying key upstream targets regulating cellular senescence and developing novel gene therapy interventions are of significant clinical importance.

[0004] Ubiquitin-conjugating enzyme E2M (UBE2M), also known as UBC12 or hUbc12, is a member of the E2 ubiquitin-conjugating enzyme family and plays a crucial role in protein ubiquitination. UBE2M participates in a specific type of ubiquitination modification—NEDD8 modification. It links to the ubiquitin-like protein NEDD8 and acts as a carrier, covalently linking the NEDD8 molecule to the target protein in the synergy of E1 activators and E3 ligases. This process is called NEDDing of the protein. Its classic target proteins are Cullin family proteins, such as Cdc53 / culin. However, there is currently no evidence that therapies targeting ubiquitin-conjugating enzyme E2M can be used for age-related diseases such as osteoarthritis.

[0005] There is an urgent need in this field for a drug that can effectively prevent and / or treat osteoarthritis. Summary of the Invention

[0006] To address the aforementioned technical problems, this application provides a nucleic acid inhibitor targeting UBE2M and its recombinant viral vector, and provides its use in the preparation of drugs for treating osteoarthritis.

[0007] This application identified UBE2M (ubiquitin-binding enzyme E2M) as a pro-aging factor in a senescent cell model using whole-genome CRISPR / Cas9 screening technology. Targeting this target, this application designed a specific small interfering RNA (siRNA / shRNA) core sequence, 5'-GGUUUAUCAUCCCAACAUUTT-3' (SEQ ID NO:1). To broaden its application scope and improve drugability, the nucleic acid inhibitor described in this application also includes a variant sequence with at least 90% identity to SEQ ID NO:1 and retaining the targeted knockdown function, as well as nucleic acid molecules that have undergone conventional chemical modifications (such as backbone or glycosyl modifications) to improve stability.

[0008] This application further provides a recombinant adeno-associated virus type 2 (AAV2) vector (AAV-shUBE2M) carrying the aforementioned nucleic acid inhibitor. This vector can specifically knock down UBE2M expression in local joint cartilage tissue, thereby inhibiting mTORC1 signaling pathway activity and significantly improving osteoarthritis symptoms in naturally aged and surgery-induced aged mice.

[0009] In one aspect, this application provides a polynucleotide for the prevention and / or treatment of osteoarthritis or anti-aging, wherein the polynucleotide comprises a nucleotide sequence as shown in SEQ ID NO:1, or a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:1 and capable of knocking down UBE2M.

[0010] In another aspect, this application also provides a bone and joint specific recombinant expression vector, wherein the vector comprises a polynucleotide, wherein the polynucleotide comprises a nucleotide sequence as shown in SEQ ID NO:1, or a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:1 and capable of knocking down UBE2M.

[0011] In another aspect, this application also provides a nucleic acid inhibitor for UBE2M, wherein the nucleic acid inhibitor comprises a polynucleotide, wherein the polynucleotide comprises a nucleotide sequence as shown in SEQ ID NO:1, or a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:1 and capable of knocking down UBE2M, and wherein the nucleic acid inhibitor is capable of binding to a nucleotide region on UBE2M mRNA complementary to SEQ ID NO:1.

[0012] In another aspect, this application also provides the use of polynucleotides, recombinant expression vectors, or nucleic acid inhibitors in the preparation of medicaments for the prevention and / or treatment of osteoarthritis, wherein the vector comprises the polynucleotide, the nucleic acid inhibitor comprises the polynucleotide, and the polynucleotide comprises a nucleotide sequence as shown in SEQ ID NO:1, or a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:1 and capable of knocking down UBE2M.

[0013] In another aspect, this application also provides the use of polynucleotides, recombinant expression vectors, or nucleic acid inhibitors in the preparation of medicaments for anti-aging, wherein the vector comprises the polynucleotide, the nucleic acid inhibitor comprises the polynucleotide, and the polynucleotide comprises a nucleotide sequence as shown in SEQ ID NO:1, or a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:1 and capable of knocking down UBE2M.

[0014] In another aspect, this application also provides a pharmaceutical composition comprising a polynucleotide, a recombinant expression vector or a nucleic acid inhibitor, and a pharmaceutically acceptable excipient or carrier, wherein the carrier comprises the polynucleotide, the nucleic acid inhibitor comprises the polynucleotide, and the polynucleotide comprises a nucleotide sequence as shown in SEQ ID NO:1, or a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:1 and capable of knocking down UBE2M.

[0015] In another aspect, this application also provides a kit comprising a polynucleotide, a recombinant expression vector, or a nucleic acid inhibitor, wherein the vector comprises the polynucleotide, the nucleic acid inhibitor comprises the polynucleotide, and the polynucleotide comprises a nucleotide sequence as shown in SEQ ID NO:1, or a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:1 and capable of knocking down UBE2M. Attached Figure Description

[0016] The present application will now be described in more detail with reference to the accompanying drawings, in which: Figure 1 The results are from a knockdown experiment of UBE2M.

[0017] Figure 2 Evaluation of Micro-CT three-dimensional reconstruction and histological staining (Safranin O-Fixed Green, H&E, immunohistochemistry) results of AAV-shUBE2M intervention in osteoarthritis in naturally aged mice.

[0018] Figure 3The results show the reversal of micro-CT and cartilage degeneration pathological indicators in a mouse model of osteoarthritis induced by AAV-shUBE2M intervention surgery. Detailed Implementation

[0019] This application relates to a polynucleotide for the prevention and / or treatment of osteoarthritis or anti-aging, wherein the polynucleotide comprises a nucleotide sequence as shown in SEQ ID NO:1, or a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:1 and capable of knocking down UBE2M. In one embodiment, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:1. In one embodiment, the polynucleotide has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:1 and is capable of knocking down UBE2M. In one embodiment, the polynucleotide is 19-30 nucleotides in length. In one embodiment, the polynucleotide is 19-23 nucleotides in length. In one embodiment, the polynucleotide is 20-22 nucleotides in length. In one embodiment, the polynucleotide is about 21 nucleotides in length. In one embodiment, the polynucleotide comprises a chemically modified polynucleotide. In one embodiment, the chemical modification includes phosphate backbone modification, ribosyl modification, and / or base modification. In one embodiment, knocking down UBE2M includes reducing UBE2M expression by at least 50%, 40%, 30%, 20%, 10%, or 5%. In one embodiment, knocking down UBE2M may include knocking out UBE2M, for example, reducing UBE2M expression by at least 70%, 80%, 90%, or 100%. In one embodiment, the polynucleotide is RNA. In one embodiment, the polynucleotide is small interfering RNA.

[0020] This application also relates to a bone and joint-specific recombinant expression vector, wherein the vector comprises the polynucleotides described in any of the foregoing embodiments. In one embodiment, the recombinant expression vector comprises a bone and joint-specific targeting vector. In one embodiment, the recombinant expression vector comprises a viral vector. In one embodiment, the recombinant expression vector comprises an adeno-associated virus vector. In one embodiment, the recombinant expression vector comprises a recombinant adeno-associated virus type 2 (AAV2) vector.

[0021] This application also relates to a nucleic acid inhibitor of UBE2M, wherein the nucleic acid inhibitor comprises the polynucleotide described in any of the foregoing embodiments and is capable of binding to a nucleotide region on UBE2M mRNA complementary to SEQ ID NO:1. In one embodiment, the nucleotide region is partially complementary to SEQ ID NO:1. In one embodiment, the nucleotide region is at least 80%, 85%, 90%, 95%, or 99% complementary to SEQ ID NO:1. In one embodiment, the nucleotide region is completely complementary to SEQ ID NO:1.

[0022] This application also relates to the use of the polynucleotides, recombinant expression vectors, or nucleic acid inhibitors described in any of the foregoing embodiments in the preparation of medicaments for the prevention and / or treatment of osteoarthritis. In one embodiment, osteoarthritis includes age-related osteoarthritis or degenerative osteoarthritis. In one embodiment, osteoarthritis includes traumatic osteoarthritis. In one embodiment, prevention and / or treatment of osteoarthritis includes increasing the thickness of the articular cartilage layer, reducing bone microstructure damage, reducing osteophyte formation, or delaying the loss of proteoglycans in the cartilage matrix. This application also relates to the use of the polynucleotides, recombinant expression vectors, or nucleic acid inhibitors described in any of the foregoing embodiments in the preparation of medicaments for anti-aging. In one embodiment, anti-aging includes increased cell proliferation capacity. In one embodiment, the medicament is administered via parenteral injection, including intravenous, subcutaneous, intraperitoneal, intramuscular, intra-articular, or infusion. In a preferred embodiment, the medicament is administered via intra-articular injection. In one embodiment, the medicament is administered to mammals. In one embodiment, the subject includes rodents. In one embodiment, the subject includes mice. In one embodiment, the subject includes humans.

[0023] This application also relates to a pharmaceutical composition comprising the polynucleotide described in any of the foregoing embodiments, the recombinant expression vector described in any of the foregoing embodiments, or the nucleic acid inhibitor described in any of the foregoing embodiments, and pharmaceutically acceptable excipients or carriers.

[0024] This application also relates to a kit comprising the polynucleotide described in any of the foregoing embodiments, the recombinant expression vector described in any of the foregoing embodiments, or the nucleic acid inhibitor described in any of the foregoing embodiments.

[0025] In this application, the terms "drug" or "pharmaceutical composition" refer to formulations of the compounds of this application with media generally accepted in the art for delivering bioactive compounds to mammals (e.g., humans). Such media include pharmaceutically acceptable carriers or excipients. The purpose of a drug or pharmaceutical composition is to facilitate administration to an organism, thereby facilitating the absorption of the active ingredient and the exertion of its bioactivity. As used in this application, the term "pharmaceuticalally acceptable" refers to a substance (such as an excipient, carrier, or diluent) that does not affect the bioactivity or properties of the compounds of this invention and is relatively non-toxic, i.e., the substance can be administered to an individual without causing an adverse biological response or interacting adversely with any component contained in the composition. In this application, the term "pharmaceuticalally acceptable excipient or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, flow aid, sweetener, diluent, preservative, dye / coloring agent, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier permitted by the relevant governmental regulatory authority for acceptable use in humans or livestock.

[0026] Optionally, this application may also relate to a method for preventing and / or treating osteoarthritis, the method comprising administering to a subject a preventatively or therapeutically effective amount of the polynucleotide described in any of the foregoing embodiments, the recombinant expression vector described in any of the foregoing embodiments, or the nucleic acid inhibitor described in any of the foregoing embodiments. This application may also relate to an anti-aging method, the method comprising administering to a subject a therapeutically effective amount of the polynucleotide described in any of the foregoing embodiments, the recombinant expression vector described in any of the foregoing embodiments, or the nucleic acid inhibitor described in any of the foregoing embodiments.

[0027] Example

[0028] This application will be described in detail through the following exemplary embodiments. These embodiments are only intended to help those skilled in the art better understand the invention of this application. It should be noted that the spirit and scope of protection of this application are not limited to the following specific embodiments.

[0029] It should be understood that in the actual industrial drug development process, those skilled in the art can make conventional length adjustments (such as 19~30 nt) or chemical modifications based on the core sequence (SEQ ID NO:1) of this invention to improve nucleic acid stability, all of which fall within the scope of protection of this invention.

[0030] Example 1: Validation of the in vitro anti-cellular senescence effect of a nucleic acid inhibitor targeting UBE2M

[0031] 1. Sequence design and transfection: A small interfering RNA sequence, 5'-GGUUUAUCAUCCCAACAUUTT-3' (siUBE2M) (synthesized by Gemma Biotechnology), was designed for the mouse UBE2M gene and transfected into mouse fibroblast L929 (Pronosai, CL-0137).

[0032] 2. Anti-aging phenotype detection: L929 cells were stimulated for 24 hours with medium containing 4 μM etoposide (MCE, HY-13629), then replaced with fresh medium and cultured for another 3 days to complete the induction of cell senescence. Results showed that knocking down UBE2M significantly reduced the proportion of SA-β-Gal staining (Cell Signaling Technology, #9860) positive cells (P < 0.001).

[0033] 3. SASP and inhibition of aging marker proteins: qPCR results (reagents purchased from Novizan, CAT#RC102, CAT#711, CAT#R333) showed that the mRNA expression levels of aging-related inflammatory factors IL-6 (P < 0.001) and IL-1β (P = 0.003) were significantly inhibited; Western blot (reagents purchased from GenScript, CAT#M01159, CAT#M00680; CST, FGFR1-CAT#9740, cGAS-CAT#31659, p-IRF3-CAT#4947, p-STAT1-CAT#9167) confirmed that the expression levels of aging marker proteins p16 and p53 were significantly downregulated.

[0034] 4. Recovery of cell proliferation capacity: After knocking down UBE2M, the cell colony formation capacity was significantly enhanced, and the proportion of EdU-stained (Raybot Biotechnology, C10310-1) positive cells increased significantly from 1.2% in the control group to 6.6% (P < 0.0001).

[0035] To verify the function of UBE2M in cellular senescence, we performed a knockdown experiment on UBE2M at the L929 cell level using RNA interference. The results showed that in an etoposide-induced cellular senescence model, knockdown of UBE2M significantly reduced the proportion of SA-β-Gal staining-positive cells (a marker of senescence). Figure 1 A) indicates a reduction in senescent cells. Simultaneously, qPCR detection revealed that the mRNA expression levels of key inflammatory factors IL6 and IL1β in the senescence-associated secretory phenotype (SASP) were also significantly suppressed. Figure 1B). Further Western blot analysis revealed that knocking down UBE2M significantly downregulated the expression levels of aging marker proteins SA-β-Gal (Proteintech, 15518-1-AP), p16 (Cell Signaling Technology, #23200), and p53 (Cell Signaling Technology, #2524). Figure 1 B), suggesting that UBE2M deficiency can partially reverse the cellular senescence process.

[0036] Cellular senescence is often accompanied by a loss of cell proliferation capacity. To further investigate the effect of UBE2M on cell proliferation, we conducted colony formation assays and EdU incorporation assays. The results showed that knocking down UBE2M significantly enhanced the colony formation capacity of cells (Beyotime, C1021). Figure 1 D), the proportion of EdU-positive cells also increased significantly ( Figure 1 E), indicating that UBE2M deficiency can promote cell proliferation and help cells escape aging-related proliferative arrest.

[0037] In conclusion, UBE2M plays a positive regulatory role in cellular senescence. Knocking down UBE2M can effectively alleviate senescence phenotypes and restore cell proliferation capacity, further supporting its functional positioning as a pro-senescence gene.

[0038] Example 2: Therapeutic effect of recombinant AAV vector on osteoarthritis in naturally aging mice

[0039] 1. Dosing regimen: 18-month-old naturally senescent C57BL / 6 female mice (purchased from Shanghai Southern Model Animal Co., Ltd.) were selected. The experimental group received a single intra-articular injection of 10 μl of adeno-associated virus vector (AAV2-shUBE2M, titer 5×10¹) carrying the aforementioned interfering sequence into the right knee joint cavity. 0 vg), the control group was injected with an equal volume of AAV-NC viral fluid. Knee joint tissue was harvested 6 weeks after injection (e.g., Figure 2 (As shown in A).

[0040] The recombinant adeno-associated virus type 2 interference vector (AAV2-shUBE2M) and negative control vector (AAV2-NC) targeting the Ube2m gene were designed, constructed, and prepared by Jiangsu Saisofe Biotechnology Co., Ltd. (Jiangsu, China). In short, the shRNA interference sequence targeting the Ube2m gene was cloned into the pAAV-shRNA expression plasmid. Subsequently, using a standard three-plasmid co-transfection system, the recombinant expression plasmid (pAAV-shUBE2M or pAAV-NC), the helper packaging plasmid pAAV-RC2, and the adenovirus helper plasmid pHelper were co-transfected into HEK293 cells for viral packaging. 72 hours after transfection, cells were collected and lysed, and AAV virus particles were isolated and purified using iodixanol density gradient ultracentrifugation. Viral titer was determined by quantitative real-time fluorescence specific PCR (qPCR) targeting the inverted terminal repeat (ITR) region. Finally, the titers of purified AAV2-shUBE2M and AAV2-NC viruses used for intra-articular injection in mice were uniformly diluted with sterile PBS and calibrated to 5 × 10¹. 0 vg.

[0041] 2. Micro-CT Quantitative Analysis (Ping Sheng Medical Technology, VNC-102): Imaging reconstruction showed a significant reduction in knee osteophyte formation in mice in the AAV-shUBE2M group. Quantitative analysis indicated that knockdown of UBE2M significantly increased bone volume fraction (BV / TV) (P = 0.0027), increased trabecular bone number (Tb.N) (P = 0.0001), and decreased trabecular bone separation (Tb.Sp) (P = 0.0146). Figure 2 (As shown in B and 2C).

[0042] 3. Cartilage Protection and Anti-inflammatory Assessment: Safranin O-Fixed Green (SO&FG) staining (Saiwell Biotech, #G1053) showed significant thickening of the knee cartilage layer and a substantial reduction in proteoglycan loss in the treatment group; H&E staining (Saiwell Biotech, #G1005) showed reduced synovial hyperplasia. Immunohistochemistry confirmed that the expression of inflammatory factors TNF-α (Saiwell Biotech, #GB15188), IL-6 (Saiwell Biotech, GB11117), and matrix metalloproteinase MMP13 (Saiwell Biotech, GB11247) was significantly downregulated in the treatment group, while the expression of chondroitin proteoglycan (Aggrecan) (Saiwell Biotech, GB155687) was upregulated (e.g., ...). Figure 2 As shown in DH).

[0043] Example 3: Therapeutic effect of recombinant AAV vector on traumatic osteoarthritis (OA)

[0044] 1. Drug administration and model establishment: Ten-week-old C57BL / 6 mice were used, and each mouse was pre-injected into the right knee joint cavity with 10 μl of AAV-shUBE2M or AAV-NC virus solution (titer of 5×10¹). 0 vg). One week after injection, anterior cruciate ligament transection (ACLT) was performed to induce osteoarthritis. Patients were monitored for 6 weeks post-surgery (e.g., ...). Figure 3 (As shown in A).

[0045] 2. Treatment Efficacy: Micro-CT scans showed that compared with the NC group, which exhibited severe osteoarthritis-like changes, the AAV-shUBE2M treatment group had significantly less osteophyte formation and a significantly higher bone volume fraction (BV / TV) (P < 0.0001). Histological staining revealed a significantly increased safranin-stained area in the treatment group, successfully maintaining cartilage thickness and inhibiting proteoglycan degradation. Immunohistochemistry further validated that this targeting vector significantly reduced the expression of TNF-α, IL-6, and MMP13, demonstrating excellent efficacy in delaying and treating osteoarthritis (e.g., Figure 3 As shown in BH).

[0046] This application identifies UBE2M as a positive regulator of cellular senescence through genome-wide CRISPR / Cas9 screening. This application provides a small interfering nucleic acid sequence and its homologous functional variants that specifically target and knock down UBE2M, and constructs the corresponding recombinant AAV2 viral vector. Experiments have demonstrated that intra-articular injection of this vector effectively inhibits age-related bone microstructural damage, significantly reduces osteophyte formation, delays the loss of proteoglycans in cartilage matrix, and downregulates the expression of inflammatory and catabolic factors in joint tissues. This application provides a novel targeted gene therapy product for age-related osteoarthritis, intervening at the root cause of cellular senescence.

[0047] Compared with the prior art, the present invention has the following advantages: 1. Precise targeting and safety: Using the AAV2 vector for local injection into the joint cavity can precisely target knee joint tissues, effectively avoiding the potential toxic side effects of systemic drug administration.

[0048] 2. Significantly improves bone microstructure: Micro-CT quantitative analysis shows that the drug in this application can significantly reduce the formation of osteophytes in the knee joint, increase the bone volume fraction (BV / TV) and the number of trabeculae (Tb.N) in the subchondral bone, and reduce the trabecular separation (Tb.Sp).

[0049] 3. Complete reversal of cartilage degeneration and inflammation: Histological studies have confirmed that the drug in this application can significantly increase the thickness of the knee joint cartilage layer, reduce the loss of proteoglycans in the cartilage matrix, significantly reduce the expression levels of TNF-α, IL-6 and matrix metalloproteinase MMP13 in cartilage tissue, and upregulate the expression of chondroitin proteoglycans (Aggrecan).

Claims

1. A polynucleotide for the prevention and / or treatment of osteoarthritis or anti-aging, wherein the polynucleotide comprises a nucleotide sequence as shown in SEQ ID NO:1, or a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:1 and capable of knocking down UBE2M.

2. The polynucleotide of claim 1, wherein the polynucleotide is 19-30 nucleotides in length; and / or The polynucleotides mentioned above include chemically modified polynucleotides.

3. The polynucleotide of claim 2, wherein the chemical modification includes phosphate backbone modification, ribose glycosyl modification and / or base modification.

4. A bone and joint specific recombinant expression vector, wherein the vector comprises any one of the polynucleotides according to claims 1-3.

5. The recombinant expression vector of claim 4, wherein the recombinant expression vector comprises a recombinant adeno-associated virus type 2 (AAV2) vector.

6. A nucleic acid inhibitor for UBE2M, wherein the nucleic acid inhibitor comprises the polynucleotide of any one of claims 1-3, and is capable of binding to a nucleotide region on UBE2M mRNA complementary to SEQ ID NO:

1.

7. Use of the polynucleotide of any one of claims 1-3, the recombinant expression vector of claim 4 or 5, or the nucleic acid inhibitor of claim 6 in the preparation of a medicament for the prevention and / or treatment of osteoarthritis.

8. Use of the polynucleotide of any one of claims 1-3, the recombinant expression vector of claim 4 or 5, or the nucleic acid inhibitor of claim 6 in the preparation of a medicament for anti-aging.

9. A pharmaceutical composition comprising any one of claims 1-3, the recombinant expression vector of claim 4 or 5, or the nucleic acid inhibitor of claim 6, and a pharmaceutically acceptable excipient or carrier.

10. A kit comprising the polynucleotide of any one of claims 1-3, the recombinant expression vector of claim 4 or 5, or the nucleic acid inhibitor of claim 6.

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