AAV Mediated–BDNF Transduced Iris Pigment Epithelial Cell Transplantation: Correlation of Photoreceptor Rescue Effect and AAV–BDNF Concentration

Abstract

Abstract: : Purpose: To examine the correlation of photoreceptor rescue effect by adeno associated virus(AAV)–mediated brain–derived neurotrophic factor (BDNF) transduced iris pigment epithelial cell (IPE) transplantation and AAV–BDNF concentration. Methods: Rat IPE was prepared as we reported previously. We also constructed AAV type2 (AAV2) with BDNF cDNA (AAV–BDNF) and purified (about 1x10¹²capsids/ml). Several concentration of AAV2–BDNF was made by using tenfold serial dilution and each AAV2–BDNF was transduced to rat IPE (AAV2–BDNF–IPE). Cell viability was examined by MTS assay. We also transplanted each AAV2–BDNF–IPE to subretinal space and examined the photoreceptor rescue effects against phototoxicity. The rescue effect was evaluated by outer nuclear layer thickness of the transplanted rats. Results:AAV2–BDNF showed no toxicity on rat IPE if they were transduced by confluent condition. No statistical significance was observed when compared to those of AAV2–LacZ–IPE and IPE only transplantation if we used less than 1x10⁵capsids/mlAAV2–BDNF concentrations. Conversely, more than 1x10⁷capsids/ml AAV2–BDNF transduction showed statistical significant photoreceptor rescue effect when compared to those of AAV2–LacZ–IPE and IPE. AAV2–BDNF around 1x10⁹capsids/ml showed best results in our experiments. Conclusions: AAV2–BDNF transduction showed no toxicity on rat IPE at least 1x10¹¹ capsids/ml. More than 1x10⁷capsids/ml of AAV2–BDNF transduction will be necessary for photoreceptor protection from phototoxicity.

Keywords: iris • transplantation • gene transfer/gene therapy