AAV vectors based on serotype 2 have been evaluated extensively for lung-directed gene transfer in the treatment of cystic fibrosis (CF). Low levels of gene transfer, however, have limited the utility of AAV serotype 2 vectors in this application. Higher levels of transduction have indeed been achieved with AAV vectors based on the other known serotypes 1 and 5 indicating that entry may be a barrier to efficient gene transfer, in an attempt to further assess the potential of AAV for gene therapy of CF, we undertook a comprehensive analysis of human and nonhuman primate tissue for the presence of endogenous AAV. The goals of these studies were to further understand the biology of natural AAV infections as well as to identifydifferent AAV that would yield vectors with improved gene transfer to lung. This recently completed survey revealed endogenous AAV sequences in almost 20% of all tissues screened. Characterization of these sequences demonstrated substantial structural, serologic, and functional heterogeneity. Phylogenetic analysis demonstrated the presence of related groups of AAVs which we call clades. These data provide a basis for identifying key elements of AAV biology relevant to gene therapy as well as to create potentially improved vectors.
The Specific Aims are as follows. 1) Develop AAV vectors utilizing capsid sequences from the novel isolates. Preliminary studies indicate an extremely high level of gene transfer to lung utilizingmembers of the AAV8 and 9 clades. These will be further characterized with respect to in vivo gene transfer in both mice and nonhuman primates as well as for serologic properties. 2) The basis for the enhanced gene transfer with novel AAVs will be further evaluated. An attempt will be made to identify the receptors to at least two of the new AAVs and to evaluate the tissue distribution of these novel receptors. 3) A number of approaches will be undertaken to improve the utility of the novel AAV vectors. We will develop better methods of purification, evaluate the role of double copy vectors to overcome post-entry blocks, and assess pharmacologic agents to inhibit proteosome-mediated degradation of AAV.
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