**4. Discussion**

A potential means to counterbalance the disrupted cartilage homeostasis altered during OA disease is based on the correction of specific chondrocyte gene expression patterns [19]. Herein, transcription factors are critical mediators of cartilage metabolism prompting chondrogenesis in both physiologic and pathologic conditions [19]. Among them, SOX9 plays vital roles in the settlement of skeletal and cartilage formation [36] and the differentiation of chondrocytes [37]. Several studies have reported a decline in SOX9 expression in OA pathology [38,39]. Therefore, genetic adjustment of the levels of SOX9 expression may constitute a valuable strategy for re-equilibrating the disturbed balance characteristic of OA cartilage towards the synthesis of ECM compounds, affording the rescue of a native articular cartilage surface [19]. rAAV vectors are convenient carriers for efficiently and steadily targeting human OA chondrocytes [11,19] and avoiding the shortcomings and/or risks inherent to other types of vectors (short-term nonviral vectors, immunogenic adenoviral vectors, potentially tumorigenic retro-/lentiviral vectors) [40,41]. However, clinical administration of rAAV for OA treatments in patients may be hindered by the prevalence of circulating anti-AAV capsid antibodies in the subjects [42], especially in the synovial fluid from patients affected with joint disorders [20]. To overcome this hurdle, we evidenced the capability of PEO-PPO-PEO-based polymeric micelles (PF68 and T908) to efficiently and durably deliver rAAV vectors with increased stability and bioactivity to chondrocytes and mesenchymal stem cells (MSCs), affording protection against neutralizing antibodies [21,31]. Equally important, rAAV-mediated gene transfer of *sox9* via polymeric micelle delivery resulted in the remodeling of OA cartilage, with increased proteoglycan accumulation and cell proliferation in OA chondrocytes relative to free vector administration [23].

In light of these observations, the goal of the present study was to test the potentiality of these micellar nanocarriers to deliver the rAAV-FLAG-h*sox9* vector to human OA chondrocytes in an environment similar to that in OA, i.e., in the presence of pro-inflammatory IL-1β and TNF-α cytokines [4,5,24]. First, and in good concordance with our previous findings [23], the data indicate that the transfer of rAAV-FLAG-h*sox9* to human OA chondrocytes via polymeric micelles led to enhanced levels of SOX9 expression over time relative to free vector treatment. Of note, rAAV-FLAG-h*sox9* transduction of chondrocytes prompted elevated and sustained levels of SOX9 expression in cells treated with IL-1β, especially when the vectors were carried by the polymeric micelles. A similar trend was observed in the presence of TNF-α alone or combined with IL-1β (IL-β/TNF-α condition), showing that delivery of the vectors via micellar systems to the highest levels of SOX9 expression. Likewise, rAAV-FLAG-h*sox9*-mediated treatment in the presence of IL-β/TNF-α increased the levels of SOX9 expression. These results are in agreement with previous work reporting an increased rAAV-mediated modification of fibroblast-like synoviocytes in conditions of inflammation [43].

The results next indicate that rAAV *sox9* treatment led to significantly higher levels of type-II collagen deposition compared with untransduced controls, most particularly when the vectors were delivered via polymeric micelles, concordant with our previous work when providing rAAV-FLAG-h*sox9* to experimental human osteochondral defects [23] and with the pro-anabolic properties of this transcription factor [44,45]. Of further note, while administration of IL-1β to chondrocyte cultures decreased the levels of type-II collagen deposition, in agreement with previous findings [7], transduction with rAAV-FLAG-h*sox9* reversed such undesirable effects by increasing type-II collagen deposition in IL-1β-treated chondrocytes especially when providing the construct via polymeric micelles, expanding earlier work using lentiviral delivery of *sox9* [8]. In this regard, the use of rAAV provides strong advantages for clinical translation, as they do not carry the risk of insertional mutagenesis inherent to lentiviruses [9]. Similar observations were made following TNF-α treatment (alone or combined with IL-1β) and genetic modification via rAAV-FLAG-h*sox9*, with increased type-II collagen deposition especially using polymeric micelle-guided rAAV gene transfer. Also remarkably, SOX9 overexpression via rAAV was capable of reverting the inhibitory effects of the cytokines upon the deposition of proteoglycans and the proliferation index [7], especially when delivering the therapeutic construct in polymeric micelles, again expanding work with lentiviral gene delivery of *sox9* [8], and concordant with the pro-anabolic activities of the transcription factor [45]. Moreover, no detrimental effects were noted, regardless of the gene transfer method adopted, as previously described with rAAV [23], and SOX9 overexpression was again capable to counteract the cytotoxic effects of the cytokines by preserving the viability of the OA chondrocytes, in agreement with work highlighting the role of SOX9 to preserve chondrocyte survival [8].
