Wednesday, February 5, 2025
2/5/2025
PROJECT SUMMARY Recombinant adeno-associated virus (rAAV) vector has emerged as one of the preferred gene delivery vehicles in clinical gene therapy. To date, three rAAV-based drugs, Luxturna, Zolgensma, and Roctavian have been approved by the US FDA for treatment of an inherited blindness (RPE65 deficient), spinal muscular atrophy, and severe hemophilia A, respectively. In addition, over 250 clinical trials of human gene therapy using rAAV vectors have been carried out or are ongoing and have yielded inspiring outcomes. However, there remain many barriers from vector binding, entry, intracellular trafficking, nuclear import, to gene expression to be resolved, in particular, the mechanism of the receptor-mediated entry and intracellular trafficking. Different rAAV vectors use different glycans as the primary attachment receptors for vector binding and a proteinaceous receptor for vector entry of cells. KIAA0319L, a type I transmembrane protein, has been identified as a multi- serotype AAV receptor involved in transduction of rAAV1-3, 5, 6, 8, and 9. It directly binds to rAAV vector in vitro, and an anti-KIAA0319L antibody or purified recombinant extracellular domains of KIAA0319L efficiently block rAAV transduction. However, the physiological function of KIAA0319L remains elusive. We identified KIAA0319L is both N- and O-glycosylated and is the bona fide 150-kDa AAV2 binding protein that has been previously identified. Its glycosylation does not contribute to the binding and transduction of rAAV vectors. Biochemically, unlike its closely related protein KIAA0319, KIAA0319L per se is not homodimerized, but likely heterodimerized. Swapping of the KIAA0319L PDK2 domain to KIAA0319 confers KIAA0319 to mediate rAAV2 transduction at the same efficiency as KIAA0319L. Notably, KIAA0319 is endocytosed through a clathrin- dependent endocytosis pathway in a homodimer format; however, KIAA0319L is dynamically recycled from the plasma membrane to the trans-Golgi network (TGN). Therefore, we hypothesize that KIAA0319L interacts with cellular membrane proteins to form a heterodimer that directs the endocytosis and intracellular trafficking of rAAV towards the TGN retrograde transport pathway. This proposal is aimed at identifying the KIAA0319L- heterodimerized partner(s), and the endocytic vesicles to the TGN pathway mediated by the heterodimer. 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. The basic characterization of the KIAA0319L protein is the first step on the road towards understanding the role that KIAA0319L plays as a multi-serotype AAV receptor, and also to determine the role of KIAA039L-interatcing protein(s) which may function as a co-receptor and mediate unique tropism of different AAV capsids to cells and tissues.
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