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Published Online: 8 February 2019

Dual-Plasmid Bionic Array-Directed Gene Electrotransfer in HEK293 Cells and Cochlear Mesenchymal Cells Probes Transgene Expression and Cell Fate

Publication: Human Gene Therapy
Volume 30, Issue Number 2

Abstract

Naked plasmid DNA electrotransfer offers advantages over viral-based gene delivery, including being regulatory permissive, but factors influencing expression efficiency and cell fate impact on translational utility. This study compared co-expression of red and green fluorescence reporter plasmids with differing promoters in HEK293 cells and in vivo in guinea pig cochlear mesenchymal cells using Bionic array-Directed Gene Electrotransfer (BaDGE®). A functional plasmid copy number of ∼64 was established in HEK293 cells by co-transfecting with separate CMV-actin-globin (CAGp) promoter-driven mCherry and green fluorescent protein (GFP) reporters, where cell division diluted plasmids toward discrete red or green channels from 100% co-expression to 10% over 24 days (∼17 cell cycles). Cross-talk between promoters was identified by interchanging a cytomegalovirus promoter (CMVp)-driven GFP plasmid for the CAGp-GFP plasmid. Here, expression of the CMVp-GFP plasmid dominated, while a dual CAGp-based reporter plasmid cocktail showed persistent co-expression beyond 2 weeks. In contrast, in vivo, cochlear mesenchymal cells co-transduced with CAGp-mCherry and CMVp-GFP plasmids showed stable co-expression at ∼50%, while the total transfectant numbers diminished over 2 weeks. This is consistent with a lack of mitosis in the cochlear mesenchymal cells and shows that cell type is a factor in plasmid interaction.

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Information & Authors

Information

Published In

cover image Human Gene Therapy
Human Gene Therapy
Volume 30Issue Number 2February 2019
Pages: 211 - 224
PubMed: 30032660

History

Published online: 8 February 2019
Published in print: February 2019
Published ahead of print: 2 October 2018
Published ahead of production: 21 July 2018
Accepted: 19 July 2018
Received: 16 March 2018

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Authors

Affiliations

Jeremy L. Pinyon
Translational Neuroscience Facility and Department of Physiology, School of Medical Sciences, and UNSW Sydney, Sydney, Australia.
Matthias Klugmann
Translational Neuroscience Facility and Department of Physiology, School of Medical Sciences, and UNSW Sydney, Sydney, Australia.
Nigel H. Lovell
Graduate School of Biomedical Engineering, UNSW Sydney, Sydney, Australia.
Gary D. Housley*
Translational Neuroscience Facility and Department of Physiology, School of Medical Sciences, and UNSW Sydney, Sydney, Australia.

Notes

*
Correspondence: Dr. Gary D. Housley, Department of Physiology, School of Medical Sciences, Faculty of Medicine, UNSW Sydney, Translational Neuroscience Facility, Wallace Wurth Building, Sydney, NSW, Australia, 2052. [email protected]

Author Disclosure

All authors of this manuscript are inventors on patent applications that relate to the development of the BaDGE technology described in this manuscript.

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