Directing Integrin-linked Endocytosis of Recombinant AAV Enhances Productive FAK-dependent Transduction

Abstract

Recombinant adeno-associated virus (rAAV) is a widely used gene therapy vector. Although a wide range of rAAV serotypes can effectively enter most cell types, their transduction efficiencies (i.e., transgene expression) can vary widely depending on the target cell type. Integrins play important roles as coreceptors for rAAV infection, however, it remains unclear how integrin-dependent and -independent mechanisms of rAAV endocytosis influence the efficiency of intracellular virus processing and ultimately transgene expression. In this study, we examined the contribution of integrin-mediated endocytosis to transduction of fibroblasts by rAAV2. Mn(++)-induced integrin activation significantly enhanced (~17-fold) the efficiency of rAAV2 transduction, without altering viral binding or endocytosis. rAAV2 subcellular localization studies demonstrated that Mn(++) promotes increased clustering of rAAV2 on integrins and recruitment of intracellular vinculin (an integrin effector) to sites of rAAV2 binding at the cell surface. Focal adhesion kinase (FAK), a downstream effector of integrin signals, was essential for rAAV2/integrin complex internalization and transduction. These findings support a model whereby integrin activation at the cell surface can redirect rAAV2 toward a FAK-dependent entry pathway that is more productive for cellular transduction. This pathway appears to be conserved for other rAAV serotypes that contain a capsid integrin-binding domain (AAV1 and AAV6).

Gene delivery to rat and human Schwann cells and nerve segments: a comparison of AAV 1–9 and lentiviral vectors

“…These processes may differ in different cells and for different AAV capsids. [71][72][73] The differential transduction rates between rat and human SCs may be due to inherent biological difference between SCs from these two species; however, we cannot exclude the possibility that the different procedures used to obtain rat and human SCs have contributed to this result. Although both rat and human SCs are obtained directly from nerve segments by a dissociation procedure, rat SCs are isolated from nerve segments kept in culture medium for 6 weeks to allow fibroblast to migrate out of the segments, and human SCs are obtained directly from nerve segments kept in medium for a couple of hours (for details, see the Materials and methods section).…”

Hepatitis B virus infection enhances susceptibility toward adeno-associated viral vector transductionin vitroandin vivo

“…Both FAK and its effector, PI3K, which remodulate the actin cytoskeleton and enable trafficking and nuclear import of AAV, can be targets of HBx . Therefore, we measured PI3K activity using protein kinase B (Akt) phosphorylation as an indicator.…”

“…Cell attachment is followed by binding to coreceptors, which either support the initial contact or—as with αvβ5 and α5β1 integrins—initiate vector internalization through clathrin‐dependent endocytosis . In addition, integrin‐binding activates phosphatidylinositol 3‐kinase (PI3K) through focal adhesion kinase (FAK), a step required for trafficking of AAV‐containing endosomes along the cytoskeleton to the nucleus, where transgene expression takes place …”

Novel Roles of Focal Adhesion Kinase in Cytoplasmic Entry and Replication of Influenza A Viruses

“…(16,18,27) (and presumably other signaling components), the mechanism that initiates these signaling events during early IAV binding is still elusive. PI3K signaling initiated by integrins and growth factor receptors (i.e., platelet-derived growth factor [PDGF] and EGF receptor tyrosine kinases) is regulated by FAK, which also regulates FA complex disassembly and subsequent endocytic receptor internalization (38,39,42). Moreover, several recent studies have reported that FAK activation is a postbinding step during Kaposi's sarcoma-associated herpesvirus or herpes simplex virus infection (30)(31)(32)43).…”