Fig. 4: ivcRNA-based SOX5 therapy inhibits osteoarthritis progression.

a Schematic illustration of the mouse Sox5 transcript (ENSMUST00000038815.13) binding by MSI2 protein. Blue Bars, the putative MSI2 binding elements (MBEs: r(G/A)U_1–3AGU). CDS, coding sequence. b Coimmunoprecipitated RNAs were analyzed for the enrichment of Sox5 transcripts with anti-MSI2 antibody or a control rabbit IgG in chondrocytes. The data are presented as the box-and-whisker plots show Min to Max and all points. n = 4 biological repeats. Statistical significance was determined by two-tailed unpaired t test. c WB analysis of SOX5, HMGB2, VINCULIN and MSI2 protein levels in ATDC5 cells overexpressing Flag-tagged Msi2 and Flag-tagged Msi2^RBDmut. VINCULIN was used as a reference protein. d Immunohistochemical staining with SOX5 antibody was performed on paraffin sections of cartilage from patients with arthroplasty. Scale bars = 200 μm. e Quantitative statistics of signals from immunohistochemical staining of SOX5 in (d) using Image J software (n = 9 samples per group). The data are presented as the Mean with SEM. Statistical significance was determined by two-tailed unpaired t test. f WB analysis of mouse SOX5 expression levels in chondrocyte after 24 h of transfection with various IRES-Sox5 combinations and Sox5 mRNA. ACTIN was used as a reference protein. Quantification of SOX5 bands was performed with Image J software. g Representative image of ivc-IRES3-Sox5 is analyzed using 4% denaturing urea-PAGE. RNA circles are enriched and verified with RNase R treatment. The hollow green circles indicate RNA circles; single green curves indicate nicked RNAs. h WB analysis of mouse SOX5 expression levels in chondrocytes after transfection with ivc-IRES3-Sox5. Quantification of SOX5 bands was performed with Image J software. i Schematic illustration of intra-articular injection of ivcRNA-LNPs encoding SOX5 to treatment DMM mice. ivcRNA-based therapy treated 10-week-old mice of sham and DMM. Then, the therapeutic phenotypes of OA were evaluated in sham and DMM mice after 7-week-treatment by micro-CT, SO&FG staining and immunohistochemistry. j Micro-CT scans show calcified meniscus and extra bone after intra-articular injection of ivc-IRES3-fLuc-LNPs, and ivc-IRES3-Sox5-LNPs in DMM model mice (calcified meniscus and extra bone are marked in red) (n = 10, 10, 20 mice). k Quantification of the BV of the calcified meniscus and extra bone in (j) (n = 10, 10, 20 mice). The data are presented as the Mean with SEM. Statistical significance was determined by Ordinary one-way ANOVA. l Representative images of SO&FG staining of paraffin sections from wild-type mice injected with ivcRNA encoding SOX5 and ivcRNA encoding fLuc after DMM surgery (n = 10, 10, 19 mice). Scale bars = 100 μm. m OARSI histopathological score for SO&FG staining in (l) (n = 10, 10, 19 mice). The data are presented as the Mean with SEM. Statistical significance was determined by Ordinary one-way ANOVA. n Quantification of SBP thickness in (l) (n = 10, 10, 19 mice). The data are presented as the Mean with SEM. Statistical significance was determined by Ordinary one-way ANOVA. o Immunohistochemical staining with SOX5 antibody was performed on paraffin sections of knee joint from wild-type mice injected with ivcRNA encoding SOX5 and ivcRNA encoding fLuc after DMM surgery (n = 6 samples per group). Scale bars = 200 μm (top), scale bars = 50 μm (bottom). p Quantitative statistics of signals from immunohistochemical staining of SOX5 in (o) using Image J software (n = 6 samples per group). The data are presented as the box-and-whisker plots show Min to Max and all points. Statistical significance was determined by Ordinary one-way ANOVA. q Schematic representation of ivcRNA-based therapy compensates SOX5, a downstream target of MSI2, to alleviate the phenotypes of OA in DMM mice. MSI2 can bind to the 3’UTR of Sox5 transcripts to promote SOX5 translation, and DMM surgery resulted in a significant decrease in SOX5 protein expression. Injecting ivcRNA-LNPs into the joint cavity of mice after DMM surgery to effectively express SOX5 protein in chondrocytes. Figure 4b, p: Boxes: 25th–75th percentiles; centre line: median; whiskers: 1.5× IQR. The experiments in Fig. 4c, f, g, h was repeated three times independently and similar results were obtained. Figure 4i, q:Created in BioRender. Suo, J. (2025) https://BioRender.com/4byajfm.