Cryogenic nanoimaging of AAV infection and packaging.

Project Summary/Abstract Gene therapies based on adeno-associated virus have garnered significant clinical success, but they are limited by factors including: unidentified blocks to genome delivery from the perinuclear region to the nucleus, lack of targeting control, and limits on maximum transgene length. This first factor is the foremost bottleneck affecting particle:infectious dose ratio. Due to this ratio, large doses are required in clinical practice, which drives side effects and limits which indications AAV gene therapy can be used for. The molecular basis of each of these impediments has been unclear, but recently- developed methods of cryo-electron-microscopy-based nanoimaging finally offers our field the tools it needs to directly observe key infection/transduction steps in situ. In this project, we will directly observe perinuclear and intranuclear virions by cryo-focused ion beam micromachining and electron tomography (as well as by an orthogonal optical method). This will enable us to finally determine where and how the viral genome leaves the capsid and how the “stuck” virions differ from those that delivered genomes. Similarly, using ultrathin cells to facilitate direct cryo-electron tomography, we will determine the subnanometer-resolution structures of cell-attached virions to clarify questions surrounding receptor binding. Finally, we will chemically and genetically perturb genome packaging to elucidate determinants of packaging and, in the case of one intervention, perhaps even increase the maximum possible insert length. We expect that the expanded understanding of the structural basis for blocks to infection plus biochemical details of virion state along the infection pathway will provide a basis for AAV rational design efforts by both basic and applied/translational researchers.