746. Localization of Structural Determinants in AAV Capsid for Efficient Liver Transduction by Domain Swapping between AAV-2 and AAV-8
Authors: Xuan Shen, Hui Xu, Theresa Storm, Mark A. Kay
Journal: Molecular Therapy (2006)

Compared with the AAV-2 serotype, AAV-8 pseudotyped vectors have become another promising candidate for liver-targeted gene delivery due to its ability to transduce almost all hepatocytes in vivo. It has been proposed that the faster uncoating rate of AAV-8 compared to AAV-2 is the key to its more robust transduction efficiencies in liver. However, given the high degree of amino acid conservation, the actual mechanism or relevant structural determinants responsible for these differences are still unsolved. To locate the structural basis for the rapid uncoating rate of AAV-8, or other properties contributing to its high transduction efficiency in vivo, we adopted a domain swapping strategy to make recombinant AAV vectors packaged in chimeric capsids based on AAV-2 and AAV-8. A series of over 26 different chimeric AAV packaging helpers were generated by replacing certain regions of the AAV-8 cap gene with homologous regions from AAV-2, or vice versa. To date, 15 of these helpers were used to make liver-specific human factor IX (hFIX)-expressing vectors with chimeric capsids and infused into mice. Liver transduction efficiencies from these chimeric AAV vectors were determined by monitoring hFIX expression in C57BL/ 6 mice. Replacing the wild type AAV-8 capsid with any large portion of the AAV-2 cap gene, namely its N-terminal VP1 and VP2-unique sequence, and the interstrand loop domains I, II, III and V, resulted in similar levels of transduction to that achieved with AAV-8. We next hypothesized that the structural determinants for high transduction efficiency were most likely located within the big interstrand loop IV domain (∼420-640 a.a.). However, swapping this whole domain of either AAV-8 or AAV-2 had a deleterious effect on the vector infectivity, resulting in dramatically lowered hFIX expression levels. This observation suggested the importance and possible multiple roles of this domain in the AAV infection pathway. In an attempt to more specifically study the structure–function relations within the loop IV domain, we further divided it into four smaller subloops and made chimeric capsids by replacing each of them with the corresponding part from the other serotype. We found that some of these subloops seemed to have great influence on the liver transduction efficiency. The non-identical residues between AAV-2 and AAV-8 in these subloops are therefore plausible candidates and are currently undergoing further examination. The specific roles for most of these residues remain to be discovered, although a few of them are known for their involvement in binding to cell surface receptors or interacting with antibodies. We predict future studies will elucidate their roles in vector uncoating or other important biological processes that may contribute to the determination of AAV transduction efficiencies. These types of techniques can then be used to make AAV vectors with varied properties as required for different gene therapy approaches.