title: Topoisomerase inhibition accelerates gene expression after adeno-associated virus-mediated gene transfer to the mammalian heart.
abstract: Utility of adeno-associated virus 2 (AAV2) vectors for cardiac gene therapy is limited by the prolonged lag phase before maximal gene expression. Topoisomerase inhibition can induce AAV2-mediated gene expression in vivo, but with variable success in different tissues. In this study, we demonstrate that topoisomerase inhibition can accelerate AAV2-mediated gene expression in the mouse heart. We used an AAV2 vector expressing firefly luciferase and monitored expression kinetics using non-invasive bioluminescence imaging. In the group receiving vector alone, cardiac luciferase activity was evident from week 2 onward and increased progressively to reach a steady plateau by 9 weeks postinjection. In the group receiving vector and camptothecine (CPT), luciferase expression was evident from days 2 to 4 onward and increased rapidly to reach a steady plateau by 3-4 weeks postinjection, nearly three times faster than in the absence of CPT (P<0.05). Southern blot analysis of AAV2 genomes in cardiac tissue showed rapid conversion of the AAV2 genome from its single-stranded to double-stranded form in CPT-treated mice. Non-invasive determinations of luciferase expression correlated well with in vitro luciferase assays. Direct injection of the AAV2 vector and long-term luciferase gene expression had no detectable effects on normal cardiac function as assessed by magnetic resonance imaging.
authors.affiliation: Department of Biomedical Engineering, University of Virginia, Charlottesville, Virginia 22903, USA.
authors.forename: Konkal-Matt R
authors.initials: KM
authors.lastname: Prasad
authors.forename: Yaqin
authors.initials: Y
authors.lastname: Xu
authors.forename: Zequan
authors.initials: Z
authors.lastname: Yang
authors.forename: Marie-Claire
authors.initials: MC
authors.lastname: Toufektsian
authors.forename: Stuart S
authors.initials: SS
authors.lastname: Berr
authors.forename: Brent A
authors.initials: BA
authors.lastname: French
mesh_terms: D000818:Animals; D001483:Base Sequence; D002166:Camptothecin / Q000494:pharmacology; D003327:Coronary Disease / Q000628:therapy; D017931:DNA Primers / Q000235:genetics; D000229:Dependovirus / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D066298:In Vitro Techniques; D049409:Luciferases, Firefly / Q000235:genetics; D008163:Luminescent Measurements; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009206:Myocardium / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics; D059004:Topoisomerase I Inhibitors; D014161:Transduction, Genetic
chemical_list: D017931:DNA Primers; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D059004:Topoisomerase I Inhibitors; D049409:Luciferases, Firefly; D002166:Camptothecin