Unanswered AAV Biology and Genome Fate Modulation Across Viral and Synthetic Substrates

About This Grant

Project Abstract Despite over sixty years of research, many unanswered questions on Adeno-associated virus (AAV) biology exist. Following AAV delivery, a small fraction of AAV genomes persists as episomes, yet the mechanisms underlying this “Selective Persistence” remain poorly understood. Ours and others’ work has shown that AAV inverted terminal repeats (ITRs) play important roles in genome conversion, persistence, and safety. My recent work identified novel ITR design that attenuates toxicity and unique genetic “signatures” associated with long-term episomal persistence in mice, we have further shown proof-of-concept that grafting these persistence “signatures” onto synthetic substrates prolongs gene expression in mice. However, it remains to be determined whether the persistence “signatures” we observed in mice are also present in humans. To address this, we will answer three questions in this proposed work. First, what is the fate of AAV genomes across human tissues, and what molecular features the AAV episomes possess? Second, can genome fate be modulated by ITR engineering to enhance stable episome formation and reduce vector dose requirements? Third, to what extent can AAV-derived persistence mechanisms be grafted onto nonviral vectors to achieve durable expression? My lab integrates virology, genome engineering, genome–protein profiling and organ-on-chip systems with state- of-the-art techniques: long-read Sequencing, Spatial Biology, and Mass Spectrometry. Uniquely, through the human decedent H2H platform, we can directly examine AAV genome fate across human tissues—an unprecedented opportunity for the field. By combining these technologies, we may uncover how AAV episomes form, persist, and interact with host factors, expand our understanding of AAV biology, provide a foundation for safer and more efficient AAV therapies, and pioneer cross-platform strategies to enhance the durability of nonviral (including synthetic substrates) gene delivery.