PROJECT SUMMARY Recombinant adeno-associated virus (rAAV) vectors have emerged as one of the preferred gene delivery agents for clinical gene therapy. To date, two rAAV-based drugs, Luxturna and Zolgensma, have been approved by the US FDA, and hundreds of clinical trials of human gene therapy using rAAV vectors have been carried out or are ongoing, and some have yielded positive outcomes. However, various barriers still remain to be resolved, in particular the tissue tropism of AAV vectors controlled by the primary attachment glycan receptor and the proteinaceous entry receptor. A type I transmembrane protein, KIAA0319L, denoted hereafter as AAV receptor (AAVR), has been identified as a multiple serotype AAV receptor involved in transduction of rAAV1, 2, 3, 5, 6, 8, and 9 (Clades A-F and H AAVs). These AAV capsids directly interact with AAVR on virus overlay assays. However, Clade G AAVs (AAV4 and AAVrh32.33) do not use the AAVR for vector binding and transduction, rather through a so called AAVR-independent entry. We have identified two AAV4-binding proteins (AAV4-BPs) at ~80-kDa and ~35-kDa, respectively, on the virus overlay assays using the rAAV4 or rAAVrh32.33 vectors. Importantly, Clade A-F and G AAVs, including AAV4 and AAVrh32.33, but not the Clade H AAV5, utilize the trans-Golgi network (TGN)-localized GPR108 (G protein-coupled receptor-like protein 108) for TGN vascular escaping during intracellular trafficking. Therefore, we hypothesize that Clade G AAVs bind to and use the AAV4- BP(s) as a proteinaceous receptor for vector cell entry and intracellular trafficking towards the TGN retrograde transport pathway where the vector escapes and enters into the cytoplasm for nuclear entry. The subject of this proposal aims to identify the AAV4-BPs by using a proteomics approach and to elucidate the AAVR-independent AAV entry and GPR108-depedent intracellular trafficking pathway for productive transduction of Clade G AAVs. Our long-term goal is to identify steps that limit rAAV transduction and that can be altered, and, therefore, can be used to enhance rAAV transduction in various cells and tissue in human gene therapy applications.