AAV mediated VEGF gene therapy to control mandibular condylar growth

Cell and gene therapy could attenuate or correct genetic disorders in bone and cartilage. However, correcting facial deformities caused by impaired growth through gene therapy is still uncharted territories. Objective: to establish a suitable in vivo gene delivery system to enhance condylar growth. Methods: We constructed a rAAV vector encoding VEGF164 cDNA amplified using RT-PCR from rat liver. The construct was then packaged in HEK293 cells to generate rAAV-VEGF164 virus particles. The cultured medium of transduced HEK293 cells was collected to quantify the VEGF secretion level by ELISA. Ninety 35 days female Sprague-Dawley rats were randomly divided into one experimental group, which was injected with rAAV-VEGF164 virus particles into both mandibular condyles; one control group which received a control vector encoding green fluorescent protein and the other control group which received PBS injection. Each group of rats was sacrificed on the following experimental days: 7, 14, 21, 30 &60. By means of in situ hybridization, immunohistochemistry staining, PAS staining, RT-PCR and morphological measurement, the delivered gene expression and new bone formation were investigated. Results: The level of VEGF164 protein in culture medium reached a peak after cultivation of HEK293 cells transfected with rAAV-VEGF164 virus 48 hours later. The in vivo results showed that exogenous gene expression was clearly detected in the deeper layer of mandibular condyle as early as 7 days postinjection by in situ hybridization. The thickness of hypertrophic layer significantly decreased and new bone formation increased in the experimental groups reaching a maximum at 30 days. In other organs (liver, kidney, spleen and heart), expression of the delivered transgene was not observed. Conclusions: This study first sought to confirm that foreign genes can be transferred to mandibular condyle in vivo. These exciting results strongly suggest that VEGF gene delivery is a promising approach to induce mandibular condylar growth.