One of the great aspirations of gene therapy is to develop technology that will provide a feasible approach to correct genetic defects and combat infectious diseases. We are engaged in studying the molecular biology of the human parvovirus adeno-associated virus (AAV) with the intent to develop a safe and efficient viral vector for human gene therapy. AAV is a dependent parvovirus which requires co-infection with another virus (either adenovirus or certain members of the herpes virus group) to undergo a productive infection in cultured cells. In the absence of co-infection with helper virus, the wild type (wt) AAV genome integrates via its ends into a specific host chromosomal site and remains latent until helper virus infection. The interest in AAV as a vector has centered around the biology of this virus. In addition to its unique life-cycle, AAV has a broad host range for infectivity (human, mouse, monkey, dog, etc.), it is ubiquitous in humans, and is completely non-pathogenic. Our research pioneered the use of recombinant AAV (rAAV) as a gene delivery system for central nervous system, and muscle cells, with vector expression for over 1.5 years without immune consequences or vector toxicity. With respect to the airway system, the subject of this proposal, we initiated studies that uncovered a rate limiting steps involved in vector transduction. This rate limiting step, which involved second strand synthesis of the single-stranded viral genome, could be augmented by genetic (Ad open reading frame (ORF) 6) Physical (heat shock, UV, X-ray irradiation) and chemical steps (hydroxyl urea, butyrate, etc.). This has lead to a better understanding for the initial event involved in AAV infection and more practically, has resulted in a new approach for generating Ad free rAAV preps. Our continued efforts to dissect the primary steps involved in AAV infection has recently led to the identification of the AAV receptor. We are now poised to take advantage of this information in determining in vivo which cells are true targets for AAV infection, mapping the epitopes on the virus capsid involved in binding, and utilizing this information for purifying AAV vectors. Identification of the AAV receptor, rate limiting for vector transduction and ability to efficiently transduce primary brain and muscle cells, but not airway cells in vivo, has provided a unique paradigm for studying the molecular steps involved in efficient vector transduction. The overall objective of the proposed work is to study the primary steps involved in rAAV transduction and persistence in airway cells using highly defined rAAV preps, a unique in vitro airway model, and hybrid targeting vectors capable of carrying genes larger than 5.0 kb. The long range objective is to better understand these molecular steps in primary airway cells with the ultimate goal of developing specific viral vectors with efficient targeted transducing capability of CFTR.