The first project area aims to characterize the interactions that define viral tropism, (e.g. the interactions that define viral tropism) and define the different functional domains on the particles. We have pioneered the understanding of AAV transcytosis or ability to pass through barrier epithelia and determined for some serotypes this activity represents a distinct cell entry pathway from the transduction pathway. Using biochemical and glycan microarray approaches we have identified the first transcytosis receptor for one serotype, BAAV. Similarly we have developed microarray technology to identify and characterize protein receptors for AAV6 as well as several proteins involved in Ebola virus entry. As a complementary approach we have developed a forward genetics based screening technology and demonstrated its utility in identifying genes associated with AAV5 transduction. Both of these approaches not only have the potential to provide new insights into vector biology and AAV natural tropism, but also to provide a guide for the optimal use of vectors in gene therapy applications. As our understanding of the interactions necessary for AAV transduction has improved we have worked to map these sites to the surface of the particle using crystallographic based difference electron density analysis and identified the sialic acid binding site on AAV5. Interestingly, mutagensis of this region can improve AAV5 transduction, suggesting further study of this domain could be used to enhance vector transduction. The second area focuses on the development, and application of new AAV vectors with particular interest on those vectors that target neuronal or epithelial cells, two cell types directly related to the mission of the NIDCR. We have demonstrated that AAV12, a non-heparin, non-sialic acid binding AAV which is highly resistant to neutralization by circulating human antibodies has a unique tropism for nasal epithelia and can by used as part of a nasal vaccination strategy against influenza. In several publications we describe the use of AAV vectors for the treatment of several diseases;including hereditary hearing loss, cystic fibrosis, diabetes, radiation induced xerostomia, as well as highly active gene transfer vectors that display distinct tropism for the epithelial or mesenchymal cell populations in the developing salivary gland. The third project area focuses on applying this technology to better understand the etiology of Sjogrens syndrome. By combining the gene transfer ability of AAV vectors with our microarray data on key population of Sjogrens patients or healthy volunteers we have defined the transcriptome of the normal human salivary gland, identified key regulatory changes in the male population of Sjogrens patients, and identified epithelial gene changes related to the xerostomia associated with the disease. In addition we have explored immunomodulatory protein therapy for this disease and again identified key molecules associated with immune activation. Our future research will build on our current work with the goals of improving the transduction activity of AAV vectors and developing a better understanding of parvovirus biology. Just as our work with AAV has led to a proposed trial for the treatment of radiation induced xerostomia, we anticipate our continued studies of the basic biology of AAV will also translate into new therapies. We will continue our ongoing efforts to map regions important for transduction on AAV particles and explore the tropism and biodistribution of novel AAV isolates. In addition to applying our bioinformatics- and new genetics based analysis of AAV transduction to the characterization of AAVs to better target their application in gene therapy, we will apply this technology to another complex interaction important in AAVs lifecycle: vector production. Similarly we will continue to combine our AAV expertise with our Sjogrens syndrome microarray databases and clinical and physiology expertise in the Branch to identify and characterize gene changes associated with this disease and utilize this information to develop novel therapies.
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