556. Comparative In Vitro Transduction Efficiency of AAV Vector Serotypes 1-9 in Different Cellular Models

Abstract

Recombinant viral vectors can be useful tools for expressing transgenes in neuronal cells to study disease mechanisms and to test for therapeutic effect. Recombinant adeno-associated virus (AAV), generated from a nonpathogenic parvovirus, has become the most widely used vector in gene therapy applications in human. However, lack of suitable animal models that accurately reflect the human disease phenotype often stalls the development of treatment strategies. To overcome this problem, research is focused on generating induced pluripotent stem cells (iPSCs) from affected human subjects as in vitro models to explore the potential of gene augmentation therapy. An advantage of iPSCs is that these cells can be differentiated along a retinal cell lineage. In this study, we differentiated iPSCs into Neural Progenitor Cells (NPC) and compared the in vitro transduction efficiency of AAV serotypes 1-9. Differentiated NPCs were characterized by immunocytochemistry with the expression of NPCs specific markers (Sox2, Nestin, Pax6). Experiments were carried out using different AAVs serotypes containing the same transgene cassette: an enhanced green-fluorescent protein (eGFP) reporter gene driven by the cytomegalovirus immediate-early (CMV) promoter. At 24 to 48 hours post transduction, positive GFP expressing cells are examined by laser scanning in Typhoon. Relative GFP expression was evaluated by using the software ImageJ. In addition to the laser scanning method, flow cytometry was used to evaluate GFP expression in the cell population. Live cell imaging and flow cytometry evaluation revealed that AAV2 is the most efficient vector for in vitro transduction in NPC followed by AAV1, AAV3 and AAV6 while AAV4, AAV5, AAV7, AAV8 and AAV9 showed no transduction or extremely low transduction efficiency.