Background: Osteoarthritis (OA) is the most prevalent joint disease worldwide, imposing a substantial burden on healthcare systems. Despite this burden, there are no approved disease-modifying OA drugs (DMOADs), and current guidelines largely aim at pain relief rather than altering structural progression. While numerous compounds have been evaluated in clinical trials, reproducible structure-modifying efficacy has not been achieved. A key contributor to these failures is late treatment initiation, which has redirected attention to early intervention—the “window of opportunity”—assuming that therapy begun before irreversible tissue injury accumulates improves long-term results. However, translating this to practice raises concerns because aggressive pharmacologic intervention is not easily justified in patients with minimal symptoms and merely Kellgren–Lawrence (K–L) grade 1 findings. Accordingly, treatment is generally deferred until structural progression is present and patients report clear pain or stiffness. In this context, strategies leveraging endogenous cartilage regeneration have become a central therapeutic target in OA. Alongside tissue-engineering approaches, a growing body of work is exploring gene therapy for OA. Of the candidate payloads, SRY-box transcription factor 9 (SOX9) is particularly attractive as the master regulator driving mesenchymal progenitors toward chondrogenesis. Identified as an HMG-box transcription factor within the SRY family, SOX9 is fundamental to chondrogenesis; skeletal malformations seen with haploinsufficiency affirm its central role. Mechanistically, SOX9 cooperates with SOX5 and SOX6 to form the SOX trio, which commits mesenchymal progenitors to the chondrogenic lineage and binds enhancers of key ECM genes such as COL2A1 and ACAN, driving hyaline matrix synthesis and stabilizing the articular chondrocyte phenotype. In parallel, SOX9 restrains hypertrophic drift by counteracting RUNX2/Ihh signaling and integrates upstream cues from pathways such as TGF-β/SMAD. These properties make SOX9 an appealing therapeutic node for promoting in situ endogenous cartilage repair. Building on these fundamentals, multiple groups have tested SOX9-based gene therapy. Recombinant AAV (rAAV) vectors delivering SOX9 have enhanced repair of focal osteochondral lesions in animal models and remodeled human OA cartilage ex vivo; polymer-assisted formulations further improved persistence. Adenoviral SOX9 transfer has likewise promoted chondrogenesis and matrix deposition. More recently, combinatorial AAV strategies (e.g., co-delivery with IL-1 receptor antagonist) have shown additive benefits—dampening inflammation while reinforcing chondrogenic transcriptional programs. Collectively, these studies support SOX9 as a credible payload for attempts to regenerate cartilage and slow structural deterioration. In the present work, we leverage these insights to evaluate SOX9-directed gene therapy as a strategy to re-establish chondrocyte homeostasis and ECM integrity within osteoarthritic cartilage. We hypothesize that targeted augmentation of SOX9 signaling in situ can shift the transcriptional landscape toward a hyaline, non-hypertrophic state, thereby creating a regenerative niche in joints where conventional pharmacotherapy has failed to modify the disease course.

Objectives: SRY-box transcription factor 9 (Sox9) is a master regulator essential for chondrogenesis and cartilage homeostasis. However, effective delivery of Sox9 for therapeutic purposes remains a challenge in osteoarthritis (OA), and the precise molecular mechanisms underlying its therapeutic effects are not fully understood. This study aimed to investigate the therapeutic efficacy and underlying molecular mechanisms of intra-articular delivery of Sox9 mRNA encapsulated in lipid nanoparticles (LNPs) in a rat surgical OA model.

Methods: Sox9 mRNA was synthesized and encapsulated into LNPs using a microfluidic system. The physicochemical properties of the LNPs were characterized by dynamic light scattering, scanning electron microscopy, and Fourier transform-infrared spectroscopy. In vitro validation was performed using IL-1β-stimulated chondrocytes. For in vivo evaluation, a rat anterior cruciate ligament transection with partial medial meniscectomy (ACLT+pMMx) model was established. Rats received intra-articular injections of Sox9 mRNA-LNPs, Lorecivivint (positive control), or PBS. Therapeutic outcomes were assessed via weight-bearing index, micro-CT, and histological scoring. Additionally, bulk RNA sequencing analyses of articular cartilages were conducted to elucidate transcriptomic alterations and chondrocyte subpopulation shifts.

Results: Sox9 mRNA-LNPs exhibited stable physicochemical properties and robustly induced Sox9 protein expression in vitro . In vivo , intra-articular administration of Sox9 mRNA-LNPs significantly attenuated pain behavior and preserved articular cartilage structure compared to the PBS-treated group, demonstrating therapeutic efficacy comparable to that of Lorecivivint. Immunofluorescence confirmed enhanced Sox9 expression and suppressed MMP13 expression within the cartilage. Transcriptomic profiling revealed that Sox9 treatment enriched pathways associated with cilium movement and cytosolic calcium regulation, while concurrently suppressing Wnt signaling. Furthermore, deconvolution analysis demonstrated that Sox9 therapy restored the cellular landscape by increasing the fractions of regulatory and proliferative chondrocytes while reducing the effector chondrocyte subpopulation compared to OA controls.

Conclusions: Intra-articular delivery of Sox9 mRNA-LNPs effectively retards OA progression and provides pain relief by enhancing cilium movement and suppressing pathological Wnt signaling, which in turn restores the anabolic balance and modulates chondrocyte subpopulations.

REFERENCES: NIL.

Acknowledgments: NIL.

Disclosure of Interests: None declared.