*3.1. E*ffi*cacy of rAAV-Mediated sox9 Overexpression in Conditions of Inflammation upon Vector Delivery via Polymeric Micelles*

We first evaluated whether the presence of pro-inflammatory cytokines may alter the overexpression of SOX9 in human OA chondrocytes monolayer cultures.

In agreement with our previous observations [23], effective SOX9 overexpression was noted in the cells via rAAV-FLAG-hsox9 transduction (up to a 1.4-fold increase relative to the negative control in the absence of cytokines, *p* = 0.021) (Figure 1A,B). Similarly, supply of rAAV in polymeric micelles led to the most intense SOX9 immunoreactivity (up to a 1.8-fold increase when compared with the negative control in the absence of cytokines on day 1, *p* = 0.015), leading to more sustained levels of expression over time (up to a 1.4-fold difference with respect to the cell control in the absence of cytokines on day 10, *p* = 0.009) (Figure 1A,B).

Treatment with IL-1β did not alter the levels of SOX9 expression early on (*p* = 0.450 compared with the negative control in the absence of cytokines on day 1) (Figure 1A,B versus Figure 1C,D) while a reduction was noted after 10 days (up to a 1.1-fold difference with respect to the negative control in the absence of cytokines, *p* = 0.106) (Figure 1A,B versus Figure 1C,D). Interestingly, the treatment with rAAV-FLAG-hsox9 promoted a significant enhancement in SOX9 expression levels following IL-1β treatment (up to a 1.5-fold difference with respect to the cell control in the presence of IL-1β, *p* = 0.024), especially upon vector delivery via micellar systems (up to a 1.7-fold difference when compared with the cell control in the presence of IL-1β on day 1, *p* = 0.040) (Figure 1C,D). Such effects were also maintained over the time of evaluation (up to a 1.5-fold increase with respect to the cell control in the presence of IL-1β on day 10, *p* = 0.045) (Figure 1C,D).

Administration of TNF-α did not affect the levels of SOX9 expression, regardless of the time points evaluated (*p* = 0.090 compared with the cell control in the absence of cytokines) (Figure 1A,B versus Figure 1E,F). Overexpression of sox9 via rAAV led to increased levels of SOX9 expression (up to a 1.4-fold increase with respect to the cell control in the presence of TNF-α on day 10, *p* = 0.048) (Figure 1E,F). Notably, delivery of the vector via micellar carriers resulted in the highest levels of SOX9

expression (up to a 1.7-fold difference with respect to the cell control in the presence of TNF-α on day 1, *p* = 0.021) (Figure 1E,F).

Concomitant IL-1β/TNF-α application led to a decrease in the levels of SOX9 expression (up to a 1.2-fold difference with respect to the negative control in the absence of cytokines on day 10, *p* = 0.150) (Figure 1A,B versus Figure 1G,H). Significantly increased SOX9 levels were noted either using free rAAV-FLAG-hsox9 form (up to a 1.5-fold difference relative to the cell control in the presence of IL-1β/TNF-α on day 10, *p* = 0.019) (Figure 1G,H), or via delivery in PF68 or T908-based micelles (up to a 1.6-fold increase with respect to the cell control in the presence of IL-1β/TNF-α on day 1, *p* = 0.020) (Figure 1G,H).

**Figure 1.** Transgene expression in rAAV-FLAG-h*sox9*-transduced human OA chondrocytes using polymeric micelles. Cells in mnolayer culture were directly transduced with the rAAV/polymeric micelles (**A**,**B**) or after pre-incubation for 4 h with IL-1β (10 ng/mL) (**C**,**D**), TNF-α (100 ng/mL) (**E**,**F**), or IL-1β/TNF-α (10/100 ng/mL) (**G**,**H**), as described in the Materials and Methods. The cultures were then processed after 1 and 10 days to detect SOX9 expression by immunocytochemistry (magnification x4, scale bar 500 μm; all representative data) (**A**,**C**,**E**,**G**) with corresponding histomorphometric analyses (**B**,**D**,**F**,**H**), as described in the Materials and Methods. Control conditions included the absence of copolymer or vector treatment (negative control) and the application of free rAAV vector (positive control). \* Statistically significant compared with the negative control at similar time points.
