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Unpublished data from our lab has shown a slight (but not statistically significant) alleviation of toxicity upon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.