Amelioration Of Osteoarthritis Severity By Compensation Of Nkx3.2 Expression

Purpose: Osteoarthritis (OA), the most common chronic form of arthritis, is an inflammatory disease. However, OA pathogenesis can be affected by a broad range of dysregulations other than enflamed inflammation. In fact, maintaining proper dynamics of chondrocyte differentiation, survival, and hypertrophic calcification would be more fundamental to prohibit the initiation of osteoarthritic degeneration. These support the reason why previous attempts solely focused on anti-inflammatory rationale have not been effective in treating OA. To date, other than symptomatic or palliative therapies, there are no effective disease-modifying treatments, and significant unmet medical needs exist for a disease-modifying OA drug (DMOAD) capable of restoring normal joint function. Nkx3.2 (also termed as Bapx1) is initially expressed in chondrocyte progenitor cells to enhance chondrogenic cell fate; once chondrogenic differentiation is induced, Nkx3.2 expression is maintained at high levels in early-stage proliferative chondrocytes, while its expression is suppressed in late-stage hypertrophic chondrocytes. Consistent with these expression patterns, it has been shown that Nkx3.2 promotes chondrogenic differentiation and supports chondrocyte survival, while it inhibits chondrocyte hypertrophy and suppresses blood-vessel invasion into cartilage. Considering diverse molecular functions of Nkx3.2 identified in association with cartilage physiology, we aimed to prove our hypothesis that Nkx3.2 plays a role in suppressing OA by preserving functional cartilage, and may serve as a DMOAD candidate. Methods: We examined levels of Nkx3.2 expression under OA conditions in multiple rodent models (e.g., STR/ORT, surgery-induced, age-driven OA models) as well as human OA patients. Furthermore, modulation of OA pathogenesis by compensation of Nkx3.2 expression was examined by using transgenic mice and intra-articular injection of AAV-Nkx3.2 (Adeno-Associated Virus encoding Nkx3.2) in both surgery-induced and age-driven OA models. Besides, various in vitro and in vivo analyses were employed to elucidate molecular mechanisms, in which, Nkx3.2 suppresses OA pathogenesis. Results: Nkx3.2 expression was markedly reduced in three different OA models in mice as well as in human OA patients. Post-natal ectopic expression of Nkx3.2 using a transgenic approach suppressed DMM-OA progression in mice. Intra-articular injection of AAV-Nkx3.2 was effective in alleviating OA severity and reducing damaged cartilage in mouse DMM-OA model. Intra-articular delivery of AAV-Nkx3.2 also ameliorated degenerative joint conditions in aged normal mice. Most notably, intra-articular injection of AAV encoding Nkx3.2 recuperated degenerated articular cartilage and restrained osteophyte maturation in aged mice. Longitudinally monitored OA progression in the same animal by employing in-life micro-CT of aged mice knee joints showed that OA-related changes were progressive in vehicle injected knee joints, whereas structural anomalies were maintained, if not, mitigated in AAV-Nkx3.2 injected knee joints. In addition, key molecular functions of Nkx3.2, which would be necessary for OA suppression (e.g., chondrocyte survival, enhancement of ECM production, inhibition of cartilage destruction), were demonstrated under various in vitro and in vivo settings including human primary articular chondrocyte isolated from OA patients. Conclusions: Our results demonstrate that decreased Nkx3.2 expression was evident in OA and Nkx3.2 plays a key role in preserving functional articular cartilage. Furthermore, compensating the loss of Nkx3.2 expression can effectively ameliorate OA severity. In particular, our results obtained by using intra-articular injection of AAV-Nkx3.2 are of great interest because these findings suggest that a Nkx3.2-based AAV gene therapy could potentially serve as a DMOAD to resolve an unmet medical need for human patients.