Spatial Biology Tools to Accelerate and Refine Adeno-Associated Virus Engineering and Application

The transfer of exogenous genetic material into living cells is a fundamental technique for basic research and, increasingly, for the treatment of human disease. Adeno-associated viruses (AAVs) are small, unenveloped viruses that can carry a limited DNA cargo of 4.4 kb (plus 0.3 kb inverted terminal repeats). These vectors are workhorses for in vivo gene transfer into mammalian systems, both for fundamental research and for therapeutic purposes. Natural serotypes of AAVs generally show broad tropism for easy to access tissues. Engineering of AAVs, through modification to the capsid surface and/or to the DNA genome, can enable access to otherwise privileged organs (e.g., brain) and can refine tropism to specific cell types (e.g., Purkinje cells of the cerebellum). Such engineering efforts can generate hundreds to thousands of interesting variants, but there is a dearth of high-throughput methods to characterize these variants. Furthermore, despite widespread usage, including in human patients, many questions on fundamental AAV biology remain unanswered. In this thesis, I attempt to address some of these outstanding bottlenecks and open questions. In Chapter 2, I address the lack of high-throughput methods for broadly characterizing engineered AAV vectors in vivo, by developing and applying high-throughput spatial transcriptomics for AAV transcripts. In Chapter 3, I focus on understanding the biology of AAV genome processing, illuminated by novel spatial genomics methods. Using these novel methods, I then profile and mechanistically dissect transcriptional crosstalk between codelivered AAV vectors (Chapter 4). Finally, in Chapter 5, I address the limited packaging capacity of AAV vectors by leveraging AAV transcriptional crosstalk to enable minimally invasive, all-AAV cell type-specific gene editing in wildtype animals, with enough efficiency to recapitulate known phenotypes. The work presented in this thesis will help to accelerate and refine AAV engineering and application. Furthermore, this thesis highlights potential confounds for AAV genome engineering, but also opens new avenues for AAV-powered functional genetics in mammalian systems.