Applications and Host Responses to Adeno-Associated Virus (AAV) as a Gene Therapy Vector in the Eye

Abstract

Adeno-associated virus (AAV) vectors are state-of-the-art delivery vectors to treat genetic diseases. In 2017, the first AAV gene therapy was approved by the Food and Drug Administration. Called Luxturna, this AAV gene therapy helps restore vision in patients suffering from an inherited form of blindness called Leber’s Congenital Amaurosis Type 2 (LCA2). Beyond LCA2, there are many blinding diseases, such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD), for which it may be possible to slow vision loss via gene agnostic therapies leveraging AAV delivery vectors. Dry AMD is the most frequent cause of visual impairment in individuals over age 50 in developed countries. It is characterized by subretinal deposits of oxidized proteins and lipids and results in progressive loss of high acuity vision. One major risk factor is smoking, which causes oxidative stress in many tissues, including the eye. We previously showed that an adeno-associated viral vector expressing human NRF2 (AAV8/Best1-Nrf2), a transcription factor that regulates responses to oxidative damage, slowed degeneration in mouse models of another blinding disorder, RP, which also includes oxidative stress. Here, our AAV8/Best1-Nrf2 vector was tested in a model of oxidative stress wherein sodium iodate was injected systemically, as this is often used to model dry AMD. Sodium iodate causes acute oxidative damage to supporting cells of the retina, the retinal pigment epithelial cells, and ultimately leads to photoreceptor death. Subretinal injection of AAV8/Best1-Nrf2 led to protection of the retinal pigment epithelium and photoreceptors, as well as preservation of visual function, in rat and mouse sodium iodate models. AAV8/Best1-Nrf2 may serve as an effective gene-agnostic therapy for diseases with oxidative stress, including dry AMD. As AAVs have also been reported to elicit ocular toxicity in the clinic, we additionally sought to elucidate mechanisms of toxicity upon subretinal injection of toxic or nontoxic AAVs in wild type and immune KO mice. Several transgenes, self and non-self, were tested for toxicity, with no clear correlation for this variable. Bulk RPE RNA-sequencing revealed upregulation of translational processes, cell stress, cytokine release, antiviral responses, and leukocyte infiltration pathways. Possible toxicity-inducing pathways were explored for causality by injecting toxic AAVs into mice deficient for intrinsic, innate, or adaptive immune pathways. Knocking out the interferon receptor, IFNAR1, partially alleviated toxicity. RNA-sequencing revealed >200 interferon stimulated genes upregulated in toxic vs. non-toxic AAV-injected RPE samples. In situ hybridization of interferon pathway transcripts (IFNB1, IFNAR1) revealed that the RPE and retina can produce and potentially respond to interferon. These data suggest that transgene-induced cell stress responses and host interferon responses contribute to toxicity following subretinal injection of AAVs encoding particular transgenes. This work elucidates mechanisms of toxicity following ocular AAV administration.