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. Recent publications describing the development of AAV5 based vectors for the transduction of salivary glands, AAV4 based vectors for the treatment of lysosomal storage diseases in the CNS and BAAV vectors for gene delivery to hair cells in the inner ear for treating deafness and balance disorders, and the growth of new bone to repair spinal injury are representative of this acceptance (Liu et al. 2005a, Liu et al. 2005b, Katano et al. 2006, Li et al. 2006, DiPasquale et al. 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 et al. 2004). We have recently utilized this system to clone a new AAV serotype from a bovine adenovirus sample termed bovine AAV (BAAV) as well several new isolates in stocks of adenovirus stored at the American Type Culture Collection (Schmidt et al. 2004, Schmidt et al. 2006). In collaboration with other intramural researchers we have begun to identify unique applications for this vector as well as characterize their mechanism of entry. ? 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. ? While natural isolates have served as a rich source of vectors for gene transfer, an alternative approach would be to develop vectors with defined tropism. The need to manipulate AAV capsids for specific tissue delivery has generated interest in understanding their capsid structures. Previously we have reported the structure of the AAV5 capsid (Walters et al. 2004) and more recently we have solved the structure for one of the most antigenically distinct serotypes, AAV4, to 13-? resolution by cryo-electron microscopy and image reconstruction (Pardon et al. 2005). A pseudoatomic model was built for the AAV4 capsid by use of a structure-based sequence alignment of its major capsid protein, VP3, with that of AAV2, to which AAV4 is 58% identical and constrained by its reconstructed density envelope. The model showed variations in the surface loops that may account for the differences in receptor binding and antigenicity between AAV2 and AAV4. The AAV4 capsid surface topology also shows an unpredicted structural similarity to that of Aleutian mink disease virus and human parvovirus B19, autonomous members of the genus, despite their limited sequence homology. In the future we plan to further refine these models and develop models of viral capsids complexed with their cellular receptors to define any structural changes that occur in the viral particles as a result of receptor binding. We have also continued our work with AAV5 and recently developed crystals for high resolution study (DiMattia et al. 2005).? In addition to our structural studies we have continued to study the transduction pathway of different AAV vectors with the goal of better targeting them to specific applications. To better understand the entry pathway and cell tropism of BAAV, we have characterized the initial cell surface interactions that are required for transduction with BAAV vectors. Our results show that, like other AAVs, BAAV requires cell surface sialic acid for transduction. However, in contrast to other AAVs, terminal sialic acid groups are not required for cell attachment and BAAV does not utilize proteins for binding and entry. Instead, like SV40 and polyomavirus , BAAV requires gangliosides for internalization and infection (Schmidt et al. 2006).? In summary, the future directions for the AAVBU will be to 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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