Elucidating Host Responses to AAV Ocular Gene Therapy Vectors - Elucidating Host Responses to AAV Ocular Gene Therapy Vectors Vectors derived from adeno-associated virus (AAV) have been clinically validated for gene therapy to treat ocular diseases. This was showcased in 2017 by the FDA approval of Luxturna, an AAV2-based gene therapy to treat an inherited form of blindness. Luxturna made history by becoming the first FDA-approved therapy to treat a genetic disease. Despite its clinical success, intraocular inflammation and toxicity has been reported for Luxturna and other AAV-based therapies for retinal disease. Gene expression data from the literature has demonstrated that double-stranded (dsRNA)-responsive pathways, such as the RIG-I and MDA5 signaling pathways, are upregulated upon subretinal and systemic administration of AAV gene therapy vectors in mice and non-human primates. AAV vectors have also been previously documented to exhibit intrinsic promoter activity within the inverted terminal repeats (ITRs), which can produce sense and antisense transcripts in transduced cells. Unpublished data from our lab has shown a slight (but not statistically significant) alleviation of toxicity upon subretinal injection of a toxic AAV into mice lacking functional MAVS (a dsRNA signaling adaptor) relative to wild-type controls. Taken together, these data suggest that there may be a role for dsRNA sensing pathways in mediating AAV ocular toxicity, which we hope to test directly using the experiments in Aim 1. We hypothesize that ITR-directed sense and antisense transcripts in the retinal pigment epithelium (RPE) hybridize to produce dsRNA, which activates cellular dsRNA sensors like RIG-I, MDA5, or others leading to ocular toxicity. Because MAVS KO data did not produce full rescue of cone toxicity, nor did any other single innate or adaptive KO strain tested, we hypothesize there are multiple pathways, immune or non-immune related, contributing to toxicity. We will therefore perform an unbiased search for other pathways that could be mediating toxicity in Aim 2 using a spatial transcriptomics approach developed in our lab, Light-Seq. Light-Seq enables us to read out gene expression in user-defined regions of the same sample (e.g., AAV-transduced vs. untransduced regions) without requiring cellular dissociation. I will use immunofluorescence (IF) and RNA fluorescent in situ hybridization (FISH, e.g., SABER-FISH or HCR-FISH) coupled with confocal imaging throughout my proposal to confirm AAV tissue transduction, validate pathway hits, and quantify ocular toxicity. Additionally, I will use genetic and pharmacologic perturbations to validate pathway candidates. Completion of this proposal will expand our knowledge of antiviral responses to AAV vectors in the eye and may help guide development of safer, more effective AAV ocular therapeutics. Additionally, insights gained in this proposal may inform the development of safer AAV vectors delivered to other organs of therapeutic relevance.
