Increased in vivo transduction of AAV-9 cargo in Alport podocytes.

BACKGROUND: Alport syndrome is a rare genetic disorder characterized by progressive kidney disease, hearing loss, and eye abnormalities. It is caused by variants in the COL4A3, COL4A4 or COL4A5 genes, which disrupt the synthesis, secretion and assembly of the alpha-3, -4 and -5 chains of type IV collagen. The defective collagen IV network results in the formation of an abnormal glomerular basement membrane (GBM). Adeno-associated virus (AAV)-mediated gene therapy is a promising approach for treating Alport syndrome but has not yet been realized due to technical challenges, including effective transduction of target cells. METHODS: In this study, we evaluated the transduction efficacy of Adeno-Associated Virus-9 with a green fluorescent protein cargo (AAV9-GFP) as a gene delivery vehicle in healthy and Alport ( Col4a5 knockout) podocytes. We established a quantitative testing platform using podocytes in culture, ex vivo glomeruli, and a mouse model of Alport syndrome (male C57BL/6 (tm1b) mice). RESULTS: First we compared transduction levels of AAV9-GFP between healthy and Alport podocytes in vitro. Both immortalized human podocytes and isolated mouse primary podocytes exhibited similar transduction efficiency in culture. We then incubated ex vivo glomeruli with AAV9-GFP and found increased podocyte uptake in the Alport glomeruli compared to wild type controls. Finally in mice we found an increase in transduction of AAV9-GFP in Alport podocytes following a peripheral intravenous injection. The level of transduction was dose-dependent and increased with disease progression suggesting that the pathological environment may facilitate higher penetration of the vector. CONCLUSION: These findings underscore the potential of AAV9 for effective gene delivery in the context of Alport syndrome and show that the stage of disease at the time of injection plays a role in determining the efficiency of AAV transduction. Overall, this study provides a foundation for future therapeutic strategies aimed at correcting the underlying genetic defects.