The results show that the articular index decreased from day 2 to day 21 post-intra-articular injection (Fig. 7a). A similar pattern was observed for the mobility index (Fig. 7b) and ankle circumference (Fig. 7c). In comparison to the rats which received HAdV5-PUMA, the mobility of the BVCARHAdV5-PUMA-treated animals improved progressively from day 8 to day 21 and their body weight improved rapidly from day 3, remaining significantly higher until the end of the study (Fig. 7d). All the parameters measured confirmed the therapeutic benefits of BVCARHAdV5-PUMA treatment with improvement of the joint function, and absence or minimal joint and bone damage, as observed in histopathology and micro-computed tomography. Control animals administered with HAdV5-PUMA alone showed no detectable beneficial effect.Fig. 7Long term in vivo and in vitro analysis of PUMA gene transfer. Rat ankle joints were injected with HAdV5-PUMA alone, or BVCARHAdV5-PUMA at a vector dose of 109 HAdV5-PUMA per joint, after the onset of arthritis at day 14. The parameters monitored over a period of 21 days were the a ankle articular index, b loss of function index, c ankle circumferences and d body weights. Histology was performed on undecalcified samples with various staining. Tartrate-resistant acid phosphatase (TRAP) staining provided red colour in multinucleated osteoclasts on the bone counter-stained with Anilin blue e. Compared to HAdV5-PUMA group, TRAP+ osteoclast staining with the TRAP was reduced at several sites of the ankle area, including distal tibia, talus, calcaneus and navicular bones in the HAdV5-PUMA group. Arrows showed important TRAP+ osteoclast concentration in the bones. In the same sites, the bone matrix was stained with Goldner trichrome staining f. Mineralized matrix was stained in green, while non-mineralized matrix was stained in red and the marrow in yellow. Arrows showed loss of bone mineral. More mineralized trabeculae and less non-mineralized areas were observed in the bones of the BVCARHAdV5-PUMA group in comparison with the HAdV5-PUMA group f. Then, Safranin O–Fast green staining was performed with bone tissue staining in turquoise and cartilage staining in red g. Compared to HAdV5-PUMA, cartilage thickness and its regularity were preserved in the BVCARHAdV5-PUMA group. Arrows indicated the loss of mineral in the bones. Inflammatory infiltrate (indicated by the ‘i’) was importantly diminished in the HAdV5-PUMA group and the bone cavity conserved its histological integrity. Scale bar, 500 µm