Activation of the NF-kappaB pathway by adeno-associated virus (AAV) vectors and its implications in immune response and gene therapy.

ABSTRACT: Because our in silico analysis with a human transcription factor database demonstrated the presence of several binding sites for NF-?B, a central regulator of cellular immune and inflammatory responses, in the adeno-associated virus (AAV) genome, we investigated whether AAV uses NF-?B during its life cycle. We used small molecule modulators of NF-?B in HeLa cells transduced with recombinant AAV vectors. VP16, an NF-?B activator, augmented AAV vector-mediated transgene expression up to 25-fold. Of the two NF-?B inhibitors, Bay11, which blocks both the canonical and the alternative NF-?B pathways, totally ablated transgene expression, whereas pyrrolidone dithiocarbamate, which interferes with the classical NF-?B pathway, had no effect. Western blot analyses confirmed the abundance of the nuclear p52 protein component of the alternative NF-?B pathway in the presence of VP16, which was ablated by Bay11, suggesting that AAV transduction activates the alternative NF-?B pathway. In vivo, hepatic AAV gene transfer activated the canonical NF-?B pathway within 2 h, resulting in expression of proinflammatory cytokines and chemokines (likely reflecting the sensing of viral particles by antigen-presenting cells), whereas the alternative pathway was activated by 9 h. Bay11 effectively blocked activation of both pathways without interfering with long-term transgene expression while eliminating proinflammatory cytokine expression. These studies suggest that transient immunosuppression with NF-?B inhibitors before transduction with AAV vectors should lead to a dampened immune response, which has significant implications in the optimal use of AAV vectors in human gene therapy.

SUBMITTER: Jayandharan GR 

PROVIDER: S-EPMC3048154 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature