Recombinant Adeno associated virus (AAV) is the leading gene therapy candidate for a number of inherited and acquired diseases but mechanisms that allow AAV to evade cellular barriers and deliver genetic information are not fully known. In this study we found that AAV transduction is enhanced in rat neonatal cardiomyocytes (NCM) by methyl-β- cyclodextrin (MβCD), a compound used to deplete plasma membrane cholesterol and disrupt membrane structures known as caveolae. Additionally, mouse embryonic fibroblast cell lines (MEFs) derived from mice lacking Caveolin1(Cav1), the protein responsible caveolae structure and function, demonstrated significant increases in uptake and transduction for a number AAV serotypes compared to wild type MEFs. Increased transduction in cav1-/- MEFs was significantly reduced by transfection of full length Cav1 gene or by a peptide corresponding to the “scaffolding domain” of Cav1 which contains regulatory elements inhibiting a range of signaling receptors. These results imply that disruption of caveolae regulatory functions are involved in increased AAV transduction seen in cav1-/- MEFs and MβCD treated NCM. To further investigate cav mediated regulatory function we focused on epidermal growth factor receptor (EGFR), a reputed co-receptor for AAV6, with activity that has been shown to be influenced by Cav1. We found that Cav1-/- MEFs expressed increased basal EGFR and were more responsive to EGF stimulation. AAV6 transduction was inhibited by the EGFR kinase inhibitor AG1478 and PI3K inhibitor LY29004 and AAV6 was able to induce translocation of membrane EGFR to the perinuclear area. Mechanisms that regulate receptor activity at the plasma membrane contribute to determining the permissiveness of cells toward AAV vectors. Cav1 plays a significant role in regulating receptor activity and consequently the ability of AAV to utilize these receptors to mediate uptake into the cell interior.
THE ROLE OF CAVEOLIN IN ADENO ASSOCIATED VIRUS TRANSDUCTION. JD Peterson, JE Rabinowitz. Center for Translational Medicine, Temple University, Philadelphia PA.