The research objective of this project is to investigate the interactions of adeno-associated virus (AAV) with its host cell. The underlying hypothesis is that by understanding these interactions as they apply to the biology of the virus we can contribute to the use of AAV vectors for gene therapy. Staff members focus on two types of interactions: those involved in viral transduction of the target cell, and those between the Rep proteins of the wild type AAV and their cellular partners. Current projects study the tropism and transduction pathways of AAV serotypes, the lifecycle of these viral isolates, and the identification and functional characterization of novel cellular proteins that interact with the AAV Rep proteins. In addition, the Unit has continued to identify new AAV serotypes. These new viral isolates are being studied both as natural mutations of other serotypes for understanding the biology of this genus of virus and because of their unique cell tropism, as novel vectors for gene transfer. As a result of our work, AAV4 and AAV5 are now accepted as useful vectors for gene transfer and are actively being evaluated in several gene therapy applications including gene transfer to the lung, CNS, eye, and salivary gland. Our recent publication describing the development of AAV5 based vectors for the treatment of cystic fibrosis and AAV4 based vectors for the treatment of lysosomal storage diseases are representative of this acceptance (Ostegaard 2005, Liu 2005). While current AAV2 vectors can deliver genes to the ductal cells of salivary glands, we have begun to explore the utility of AAV4 and AAV5 in the salivary gland. Our initial results demonstrate that improved gene transfer activity is possible by using AAV4 or AAV5 based systems. Like AAV2 based vectors, these vectors exclusively target ductal cells (Katano In Press). In collaboration with NIDCR researchers we have continued to assess the gene transfer activity of other AAV vectors to deliver genes to cells for the treatment of both salivary gland and systemic disease. Salivary glands are natural secretory organs able to secrete large amounts of protein either in the saliva or into the blood for systemic circulation. We have demonstrated that salivary glands treated with AAV2 based vectors can express erythropoeitin at therapeutic levels for the treatment of chronic anemia associated with kidney disease. Furthermore, the activity is long lived and expression is stable for at least 1 year (Voutetakis 2004, 2005). We believe that just as our characterizations of AAV4 and AAV5 have advanced the field of gene therapy, the development of new vectors also will have an impact on other gene therapy applications as well as our understanding of parvovirus biology. As a first step in this endeavor, we have developed a PCR based method for rapidly identifying and cloning divergent AAV isolates (Katano 2004). We have recently utilized this system to clone a new AAV serotype from a bovine adenovirus sample termed bovine AAV (BAAV) (Schmidt 2004). In collaboration with other intramural researchers, we have begun to identify unique applications for this vector. We have recently demonstrated that BAAV can efficiently deliver genes to hair cells in the inner ear, an important target cell for the treatment of deafness and balance disorders (Di Pasquale 2005). Our knowledge of the interactions necessary for cellular transduction with AAV4 and AAV5 clearly has been helpful in defining their biology and likely will identify novel applications for these vectors. Furthermore, the tools we have developed while examining AAV4 and AAV5 tropism can be applied to other novel vectors we have isolated. We expect this information will aid in identifying optimal target cells for the different AAV vectors and also aid in the development of a new generation of vectors for gene transfer with very defined cell tropism. In the future, we plan to further refine our tools for studying interactions necessary for cellular transduction, identify critical interactions in the transduction pathway, identify the domains critical for these interaction on the virus surface, as well as to identify new AAV isolates that maybe useful for gene therapy applications.
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