We propose to develop Gene Therapy vectors that are targeted to particular cell membrane receptors, cell types and/or tissues. Development of these precise delivery systems will help overcome potential adverse reactions and high dosing requirements imposed by the current, non-targeted, traditional Gene Therapy vectors. The goal in Phase I is to construct a combinatorial library of chimeric Adeno-associated virus (AAV) vectors to screen for tissue-specific targeting during Phase II. Because of technical limitations, AAV-based combinatorial libraries were not previously available. We plan to use a unique new technology for building and subsequent selection of AAV vector libraries. Specifically, we will exploit molecular breeding of viruses by DNA shuffling to direct evolution in vitro of AAV capsid genes through recombination of multiple homologous parental sequences of known AAV serotypes. To construct the library, we will use a new AAV production technology based on insect cells and genome pseudotyping. Researchers at AGTC and UF Powell Gene Therapy Center, with proven track records in the AAV field, will join efforts to complete the project. During the feasibility study, we will demonstrate whether it is possible to build an AAV-based library of viable viral clones of at least 106 complexity using recombination of capsid genes of AAV1 and AAV2 serotypes. The successful completion of this step will allow us to proceed to the next stage of selection of vectors with a particular cell/tissue tropism completed during Phase II of the project. We anticipate to market AAV combinatorial libraries to users within the gene therapy community, as a product to select for delivery vectors targeted to a particular tissue or tailored to a particular application, e.g. targeted delivery of pro- apoptotic or suicidal genes to tumors. This project will represent a significant advance in the development of therapeutic gene delivery systems with precise targeting to improve patient outcomes.