title: Hoechst increases adeno-associated virus-mediated transgene expression in airway epithelia by inducing the cytomegalovirus promoter.
abstract: BACKGROUND
  In airway epithelia, the kinetics of recombinant adeno-associated virus (AAV) transgene expression is slow. This has negative practical implications for research, as well as for translation into therapy. The DNA minor groove-binding agent Hoechst-33342 has been shown to enhance AAV transgene expression. In the present study, we investigated the mechanism of Hoechst-related augmentation of AAV-mediated transgene expression.
  
  
  METHODS
  We investigated the effect of Hoechst-33342 on HT1080, COS-7, mouse and human airway epithelia transduced with different AAV serotypes encoding enhanced green fluorescent protein (eGFP). We exposed cells to increasing concentrations of Hoechst-33342 at different time points. We evaluated the effect on second-strand DNA synthesis using AAV with a self-complementary genome. We also investigated the effect on expression from transfected plasmids with and without AAV2 inverted terminal repeats (ITRs).
  
  
  RESULTS
  We found that Hoechst-33342 significantly accelerated AAV transgene expression for all serotypes tested. Hoechst-33342 only had an effect when the treatment was given during or after transduction, even 120 days post-transduction, suggesting an effect on transgene expression regulation. Hoechst-33342 increased transgene expression when cells were transduced with a self-complementary AAV with the cytomegalovirus promoter, although there was no effect on cells transduced with conventional single-stranded AAV encoding the Rous sarcoma virus promoter. Finally, Hoechst-33342 increases gene expression from transfected plasmids regardless of the presence of AAV2 ITRs.
  
  
  CONCLUSIONS
  Hoechst dramatically augments and accelerates AAV-mediated transgene expression in airway epithelia without altering AAV-mediated gene transfer. Hoechst activation of the cytomegalovirus promoter is seen in plasmids, although it is drastically enhanced in the context of AAV.
authors.affiliation: Department of Internal Medicine and Molecular and Cellular Biology Program, Roy J, Lucille A. Carver College of Medicine, University of Iowa, IA, USA.
authors.forename: David D
authors.initials: DD
authors.lastname: Dickey
authors.forename: Katherine J D A
authors.initials: KJ
authors.lastname: Excoffon
authors.forename: Krista R
authors.initials: KR
authors.lastname: Young
authors.forename: Kalpaj R
authors.initials: KR
authors.lastname: Parekh
authors.forename: Joseph
authors.initials: J
authors.lastname: Zabner
mesh_terms: D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology*; D019556:COS Cells; D002453:Cell Cycle / Q000187:drug effects; D002460:Cell Line; D002522:Chlorocebus aethiops; D003587:Cytomegalovirus / Q000187:drug effects / Q000235:genetics*; D004247:DNA / Q000187:drug effects; D000229:Dependovirus / Q000187:drug effects / Q000235:genetics*; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D051379:Mice; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D020545:Respiratory Mucosa / Q000187:drug effects*; D052736:Rous sarcoma virus / Q000235:genetics; D014161:Transduction, Genetic; D019076:Transgenes
chemical_list: D001562:Benzimidazoles; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004247:DNA; C017807:bisbenzimide ethoxide trihydrochloride