AAV capsid assembly, viral entry, and viral tropism

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Work from a number of laboratories, including those involved in this program project, has demonstrated that Adeno-associated virus (AAV) holds significant promise for the correction of human disease. AAV2, the most commonly used serotype to data, shows a broad host range and broad tropism. Although a broad host range is useful, it is clearly time to begin exploring ways of developing AAV vectors that have a more restricted or specific tropism, or vectors that have special properties. In particular, it would be extremely helpful if methods could be found to target AAV vectors to specific tissues. Vector targeting is still in its infancy with all vectors and is particularly hampered in the case of AAV by the relative lack of information about capsid assembly, particle entry and intracellular trafficking. To address these problems, this proposal focused in part (during the previous funding period) on developing a comprehensive series of mutations in the AAV2 capsid genes. This provided a resource that can be used to study these problems. More importantly, it will provide valuable information about how and where to insert foreign ligands into the AAV capsid for the purpose of changing the viral tropism. The specific aims are: Specific aim 1: To characterize mutants that are defective in binding the viral cell surface receptors or in intracellular trafficking. In particular, surface residues of the capsid gene involved in heparin binding will be identified, as well as residues involved in endosome escape or nuclear entry. Both molecular biology and confocal and cryoelectron microscopy techniques will be used. Specific aim 2: To characterize mutants defective in assembly of AAV capsids. In particular, attempts will be made to identify the intermediates of capsid assembly and regions of the capsid that are involved in viral assembly. Specific aim 3: Finally,, attempts will be made to change the tropism of the AAV2 capsid by inserting foreign ligands to the capsid coding regions identified as non-essential in the genetic screens. This will include testing of appropriate foreign ligands in vivo to see if the biodistribution of AAV can be significantly changed. It is anticipated that these studies will produce valuable information that will impact on the use of AAV vectors for virtually all gene therapy studies.