Recombinant Adeno-associated virus (rAAV) is an attractive viral vector for gene therapy to the heart and lung. Although clinical trials for cystic fibrosis (CF) lung disease with rAAV have thus far not been successful, considerable interest remains in using this virus to treat CF and other NHLBI focus diseases. In part, the lack of clinical success with rAAV vectors has been due to a minimal understanding of mechanisms that control viral transduction (i.e., expression of an encoded transgene). This proposal seeks to delineate receptor/co-receptor-dependent endocytic mechanisms that control highly efficient transduction by rAAV vectors, with a focus on the type-2 serotype (rAAV2). Although receptors and co-receptors for various rAAV serotypes have been identified, the signaling mechanisms that control endocytosis and the intracellular fate of rAAV are largely unknown. Our laboratory has determined that the mechanism of rAAV uptake significantly influences how efficiently the virus is processed through the endosomal compartment. Preliminary data from this project suggest that 1521-integrin-dependent endocytosis of rAAV2 leads to more efficient infection than do competing pathways of entry. However, the signaling events that regulate integrin-dependent endocytosis following rAAV2/integrin docking remain unclear. A large body of literature has linked integrin activation to the recruitment and activation of numerous intracellular signaling proteins, such as the non-receptor tyrosine kinases, focal adhesion kinase (FAK) and c-Src. These integrin effectors are known to bind and regulate Rac1 and phosphoinositide 3-kinase (PI3K), both of which have been shown to regulate rAAV2 endocytosis and vesicular trafficking. Our long-term goal is to identify approaches that will enhance the efficiency of viral transduction by rAAV2 and other integrin-dependent AAV serotypes. The proposed research will aid in this by identifying the events surrounding integrin-dependent endocytosis of rAAV2, as well as the downstream signaling pathways that lead to efficient nuclear transport and transduction of the virus. In addition, clarifying how cell signaling is activated by rAAV infection may aid the field in understanding the genesis of innate immune responses to this virus-a second barrier to the development of rAAV vectors for gene therapy applications.