LAUSR

Significance Adeno-associated viral (AAV) gene therapy is becoming an important therapeutic modality, especially for ocular diseases, due to its efficiency of gene delivery and relative lack of pathogenicity. However, AAV sometimes can cause inflammation and toxicity. We explored such effects using injections into the mouse eye. We found a strong correlation of toxicity and inflammation with the use of promoters that were broadly active, or specifically active in the retinal pigment epithelium. AAVs with photoreceptor-specific promoters were found to be nontoxic at all doses tested. These studies reveal that safer vectors can be designed if assays for relevant and specific cell types are developed and tested with a range of vectors with different genomic elements. Adeno-associated viral vectors (AAVs) have become popular for gene therapy, given their many advantages, including their reduced inflammatory profile compared with that of other viruses. However, even in areas of immune privilege such as the eye, AAV vectors are capable of eliciting host-cell responses. To investigate the effects of such responses on several ocular cell types, we tested multiple AAV genome structures and capsid types using subretinal injections in mice. Assays of morphology, inflammation, and physiology were performed. Pathological effects on photoreceptors and the retinal pigment epithelium (RPE) were observed. Müller glia and microglia were activated, and the proinflammatory cytokines TNF-α and IL-1β were up-regulated. There was a strong correlation between cis-regulatory sequences and toxicity. AAVs with any one of three broadly active promoters, or an RPE-specific promoter, were toxic, while AAVs with four different photoreceptor-specific promoters were not toxic at the highest doses tested. There was little correlation between toxicity and transgene, capsid type, preparation method, or cellular contaminants within a preparation. The toxic effect was dose-dependent, with the RPE being more sensitive than photoreceptors. Our results suggest that ocular AAV toxicity is associated with certain AAV cis-regulatory sequences and/or their activity and that retinal damage occurs due to responses by the RPE and/or microglia. By applying multiple, sensitive assays of toxicity, AAV vectors can be designed so that they can be used safely at high dose, potentially providing greater therapeutic efficacy.