Notch1 Augments Intracellular Trafficking of Adeno-Associated Virus Type 2

ABSTRACT We report here the significance of the Notch1 receptor in intracellular trafficking of recombinant adeno-associated virus type 2 (rAAV2). RNA profiling of human prostate cancer cell lines with various degrees of AAV transduction indicated a correlation of the amount of Notch1 with rAAV transgene expression. A definitive role of Notch1 in enhancing AAV transduction was confirmed by developing clonal derivatives of DU145 cells overexpressing either full-length or intracellular Notch1. To discern stages of AAV2 transduction influenced by Notch1, competitive binding with soluble heparin and Notch1 antibody, intracellular trafficking using Cy3-labeled rAAV2, and blocking assays for proteasome and dynamin pathways were performed. Results indicated that in the absence or low-level expression of Notch1, only binding of virus was found on the cell surface and internalization was impaired. However, increased Notch1 expression in these cells allowed efficient perinuclear accumulation of labeled capsids. Nuclear transport of the vector was evident by transgene expression and real-time PCR analyses. Dynamin levels were not found to be different among these cell lines, but blocking dynamin function abrogated AAV2 transduction in DU145 clones overexpressing full-length Notch1 but not in clones overexpressing intracellular Notch1. These studies provide evidence for the role of activated Notch1 in intracellular trafficking of AAV2, which may have implications in the optimal use of AAV2 in human gene therapy.