AAV vectors trigger DNA damage responses and STING-dependent inflammation in human CNS cells

Adeno-associated viral (AAV) vector-based gene therapy is gaining foothold as a treatment option for a variety of genetic neurodegenerative diseases. Nonetheless, dose-dependent toxicities and severe adverse events are emerging in recent clinical trials through mechanisms that remain unclear. We have modelled here AAV-mediated neurotoxicity in the context of the human brain taking advantage of human induced pluripotent stem cell-based technologies. Our work uncovers vector-induced cell-intrinsic innate immune mechanisms at the single-cell level that contribute to neurotoxicity in 2D and 3D models of the human central nervous system (CNS). The AAV genome triggered p53-dependent DNA damage responses across CNS cell types followed by induction of IL-1R- and STING-dependent inflammatory responses. In addition, transgene-expression led to MAVS-dependent RNA sensing and activation of type I interferon (IFN) signalling. Cell-intrinsic and paracrine neurotoxicity could be prevented by inhibiting p53 or acting downstream on STING- and IL-1R-mediated activation of inflammatory responses. Activation of DNA damage, type I IFN and CNS inflammation were confirmed in vivo. Together, our work sheds significant light on the cell-autonomous innate immune mechanisms of vector sensing that can contribute to AAV-associated neurotoxicity.