Innate immune response to AAV-based gene therapy vectors: Mechanisms of complement activation and cytokine release

Recombinant adeno-associated viruses (rAAV) have emerged as a preferred strategy for <i>in vivo</i> gene delivery. However, the immune response to rAAV presents a major limitation, leading to serious adverse events in clinical trials. This study investigates the interaction between rAAV and the innate immune system. A whole blood assay (WBA) was used to assess complement activation and cytokine release upon stimulation with rAAV in 20 healthy blood donors. Results demonstrate that AAV2 and AAV8 capsids can activate the complement system, primarily through the antibody-dependent classical pathway. Complement activation also occurred in some of the seronegative donors showing the contribution of the alternative pathway. In this WBA setting, there were significant increases in the release of various cytokines and chemokines, with monocytes, natural killer cells, T cells, and B cells identified as the responding cell types using transcriptomics. Cytokine and/or chemokine release was more prominently observed with AAV2 compared with AAV8 and was enhanced by the presence of pre-existing anti-capsid antibodies. Interferon-α release appeared directly dependent on the cytosine-phosphate-guanine (CpG) content of the vector genome. These findings underscore the effects of innate and adaptive immunity to rAAV capsid and genome on the activation of complement pathways and release of inflammatory mediators.