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Hoechst increases adeno‐associated virus‐mediated transgene expression in airway epithelia by inducing the cytomegalovirus promoter
Journal article   Open access

Hoechst increases adeno‐associated virus‐mediated transgene expression in airway epithelia by inducing the cytomegalovirus promoter

David D Dickey, Katherine J. D. A Excoffon, Krista R Young, Kalpaj R Parekh and Joseph Zabner
The journal of gene medicine, Vol.14(6), pp.366-373
06/2012
DOI: 10.1002/jgm.2632
PMID: 22610695
url
http://doi.org/10.1002/jgm.2632View
Open Access

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. Copyright © 2012 John Wiley & Sons, Ltd.
 expression transgene CMV promoter Hoechst AAV

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