AAV-associated toxicity caused by transgene expression in the retinal pigment epithelium

Abstract

The field of AAV gene therapy has recently experienced multiple clinical successes, and AAV continues to be a relatively safe viral vector. Nevertheless, there is a long history of toxic side effects at higher doses, culminating in the first cases of AAV-associated fatalities in 2020. As gene therapy moves forward, it is critical to understand and account for potential factors driving AAV-associated toxicity. Here we use the murine eye as a model system to study AAV-associated toxicity from transgene expression. In our model, high expression of EGFP induces degeneration of the retinal pigment epithelium (RPE), which leads to deterioration of photoreceptors. By evaluating the impact of various AAV vectors on the eye, we show that expression-based toxicity can occur independently from the dose of AAV capsid or DNA. As expected, AAV-associated toxicity due to EGFP increases with increasing expression. More surprisingly, toxicity can remain unaffected across multiple immune knockout lines, including knockouts of the Toll-like receptors (TLRs), cytotoxic T cells, and the viral RNA sensor MAVS. We show that EGFP RNA is not sufficient for inducing toxicity, suggesting that it is being caused by off-target activity of the expressed protein. Along that line, as different proteins have different activities, we observed that the severity of toxicity varied by transgene. In addition, we do not see a clear correlation between toxicity and self vs. non-self transgenes. Taken as a whole, our results illustrate the risks of aiming to maximize transgene expression. Although optimizing for increased expression has been attractive in the effort to lower the doses for AAV gene therapy, it is worthwhile to distinguish the effective level of expression per organism from the effective level per cell. Identifying the maximum level of beneficial transgene expression at the cellular level may be an important factor in mitigating AAV-associated toxicity.