Comparison of efficiency and function of VEGF adenovirus vectors in endothelial cells for gene therapy of placental insufficiency

Abstract

Severe fetal growth restriction (FGR) affects 1:500 pregnancies, is untreatable and causes serious neonatal morbidity and death. Reduced uterine blood flow (UBF) and lack of bioavailable VEGF due to placental insufficiency is a major cause. Transduction of uterine arteries in normal or FGR sheep and guinea pigs using an adenovirus (Ad) encoding VEGF isoforms A (Ad.VEGF-A165) and a FLAG-tagged pre-processed short form D (DΔNΔC, Ad.VEGF-DΔNΔC-FLAG) increases endothelial nitric oxide expression, enhances relaxation and reduces constriction of the uterine arteries and their branches. UBF and angiogenesis are increased long term, improving fetal growth in utero. For clinical trial development we compared Ad.VEGF vector transduction efficiency and function in endothelial cells (ECs) derived from different species. We aimed to compare the transduction efficiency and function of the pre-clinical study Ad. constructs (Ad.VEGF-A165, Ad.VEGF-DΔNΔC-FLAG) with the intended clinical trial construct (Ad.VEGF-DΔNΔC) where the FLAG tag is removed. We infected ECs from human umbilical vein, pregnant sheep uterine artery, pregnant guinea pig aorta and non-pregnant rabbit aorta, with increasing multiplicity of infection (MOI) for 24 or 48 hours of three Ad.VEGF vectors, compared to control Ad. containing the LacZ gene (Ad.LacZ). VEGF supernatant expression was analysed by ELISA. Functional assessment used tube formation assay and Erk-Akt phosphorylation by ELISA. VEGF expression was higher after Ad.VEGF-DΔNΔC-FLAG and Ad.VEGF-DΔNΔC transduction compared to Ad.VEGF-A165 in all EC types (*p < 0.001). Tube formation was higher in ECs transduced with Ad.VEGF-DΔNΔC in all species compared to other constructs (***p < 0.001, *p < 0.05 with rabbit aortic ECs). Phospho-Erk and phospho-Akt assays displayed no differences between the three vector constructs, whose effect was, as in other experiments, higher than Ad.LacZ (***p < 0.001). In conclusion, we observed high transduction efficiency and functional effects of Ad.VEGF-DΔNΔC vector with comparability in major pathway activation to constructs used in pre-clinical studies, supporting its use in a clinical trial.