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

Proc Natl Acad Sci U S A. 2011 Mar 1;108(9):3743-8. doi: 10.1073/pnas.1012753108. Epub 2011 Feb 14.

Abstract

Because our in silico analysis with a human transcription factor database demonstrated the presence of several binding sites for NF-κB, a central regulator of cellular immune and inflammatory responses, in the adeno-associated virus (AAV) genome, we investigated whether AAV uses NF-κB during its life cycle. We used small molecule modulators of NF-κB in HeLa cells transduced with recombinant AAV vectors. VP16, an NF-κB activator, augmented AAV vector-mediated transgene expression up to 25-fold. Of the two NF-κB inhibitors, Bay11, which blocks both the canonical and the alternative NF-κB pathways, totally ablated transgene expression, whereas pyrrolidone dithiocarbamate, which interferes with the classical NF-κB pathway, had no effect. Western blot analyses confirmed the abundance of the nuclear p52 protein component of the alternative NF-κB pathway in the presence of VP16, which was ablated by Bay11, suggesting that AAV transduction activates the alternative NF-κB pathway. In vivo, hepatic AAV gene transfer activated the canonical NF-κB 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-κB 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Retracted Publication

MeSH terms

  • Animals
  • Antigen-Presenting Cells / drug effects
  • Antigen-Presenting Cells / metabolism
  • Antigen-Presenting Cells / virology
  • Binding Sites
  • Cytokines / metabolism
  • Dependovirus / drug effects
  • Dependovirus / genetics*
  • Gene Expression / drug effects
  • Genetic Therapy*
  • Genetic Vectors / genetics*
  • HeLa Cells
  • Humans
  • Immunity / drug effects
  • Immunity / genetics*
  • Inflammation Mediators / metabolism
  • Liver / drug effects
  • Liver / metabolism
  • Liver / pathology
  • Liver / virology
  • Mice
  • NF-kappa B / antagonists & inhibitors
  • NF-kappa B / metabolism*
  • Nitriles / pharmacology
  • Parvoviridae Infections / genetics
  • Parvoviridae Infections / metabolism
  • Parvoviridae Infections / virology
  • Protein Binding / drug effects
  • Signal Transduction* / drug effects
  • Sulfones / pharmacology
  • Terminal Repeat Sequences / genetics
  • Time Factors
  • Transcription Factors / metabolism
  • Transgenes

Substances

  • 3-(4-methylphenylsulfonyl)-2-propenenitrile
  • Cytokines
  • Inflammation Mediators
  • NF-kappa B
  • Nitriles
  • Sulfones
  • Transcription Factors