Induction of immune tolerance to FIX by intramuscular AAV gene transfer is independent of the activation status of dendritic cells.

The nature of viral vectors is suggested to be a significant contributor to undesirable immune responses subsequent to gene transfer. Such viral vectors, recognized as danger signals by the host immune system, activate dendritic cells (DCs), causing unwanted antivector and/or transgene product immunity. We recently reported efficient induction of immune tolerance to coagulation factor IX (FIX) by direct intramuscular injection of adeno-associated virus (AAV)-FIX. AAV vectors are nonpathogenic and elicit minimal inflammatory response. We hypothesized that the nonpathogenic nature of AAV plays a critical role in induction of tolerance after AAV gene transfer. We observed inefficient recruitment and activation of DCs subsequent to intramuscular injection of AAV. To further validate our hypothesis, we examined immune responses to FIX after intramuscular injection of AAV with simultaneous activation of DCs. We were able to achieve phenotypic and functional activation of DCs after administration of lipopolysaccharide and anti-CD40 antibody. However, we observed efficient induction of FIX tolerance irrespective of DC activation in mice with different genetic and major histocompatibility complex backgrounds. Furthermore, activation of DCs did not exaggerate the immune response induced after intramuscular injection of AAV serotype 2 vector. Our results demonstrate that induction of FIX tolerance after AAV gene transfer is independent of DC activation status.