title: 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-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.
authors.affiliation: Division of Cellular and Molecular Therapy, Department of Pediatrics, Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville, FL 32611, USA.
authors.forename: Giridhara R
authors.initials: GR
authors.lastname: Jayandharan
authors.forename: George
authors.initials: G
authors.lastname: Aslanidi
authors.forename: Ashley T
authors.initials: AT
authors.lastname: Martino
authors.forename: Stephan C
authors.initials: SC
authors.lastname: Jahn
authors.forename: George Q
authors.initials: GQ
authors.lastname: Perrin
authors.forename: Roland W
authors.initials: RW
authors.lastname: Herzog
authors.forename: Arun
authors.initials: A
authors.lastname: Srivastava
mesh_terms: D000818:Animals; D000938:Antigen-Presenting Cells / Q000187:drug effects / Q000378:metabolism / Q000821:virology; D001665:Binding Sites; D016207:Cytokines / Q000378:metabolism; D000229:Dependovirus / Q000187:drug effects / Q000235:genetics*; D015870:Gene Expression / Q000187:drug effects; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D007109:Immunity / Q000187:drug effects / Q000235:genetics*; D018836:Inflammation Mediators / Q000378:metabolism; D008099:Liver / Q000187:drug effects / Q000378:metabolism / Q000473:pathology / Q000821:virology; D051379:Mice; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009570:Nitriles / Q000494:pharmacology; D010322:Parvoviridae Infections / Q000235:genetics / Q000378:metabolism / Q000821:virology; D011485:Protein Binding / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects; D013450:Sulfones / Q000494:pharmacology; D020079:Terminal Repeat Sequences / Q000235:genetics; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D019076:Transgenes
chemical_list: C434003:3-(4-methylphenylsulfonyl)-2-propenenitrile; D016207:Cytokines; D018836:Inflammation Mediators; D016328:NF-kappa B; D009570:Nitriles; D013450:Sulfones; D014157:Transcription Factors