Minicircle DNA Provides Enhanced and Prolonged Transgene Expression Following Airway Gene Transfer.

Mustafa M Munye,Steven J Howe,Stephen L Hart,Aristides D Tagalakis,Robin J Mcanulty,Rachel E Brown,Josephine L Barnes

doi:10.1038/srep23125

Abstract

Gene therapy for cystic fibrosis using non-viral, plasmid-based formulations has been the subject of intensive research for over two decades but a clinically viable product has yet to materialise in large part due to inefficient transgene expression. Minicircle DNA give enhanced and more persistent transgene expression compared to plasmid DNA in a number of organ systems but has not been assessed in the lung. In this study we compared minicircle DNA with plasmid DNA in transfections of airway epithelial cells. In vitro, luciferase gene expression from minicircles was 5–10-fold higher than with plasmid DNA. In eGFP transfections in vitro both the mean fluorescence intensity and percentage of cells transfected was 2–4-fold higher with minicircle DNA. Administration of equimolar amounts of DNA to mouse lungs resulted in a reduced inflammatory response and more persistent transgene expression, with luciferase activity persisting for 2 weeks from minicircle DNA compared to plasmid formulations. Transfection of equal mass amounts of DNA in mouse lungs resulted in a 6-fold increase in transgene expression in addition to more persistent transgene expression. Our findings have clear implications for gene therapy of airway disorders where plasmid DNA transfections have so far proven inefficient in clinical trials.

Highlights

Lung diseases are a major cause of morbidity including genetic diseases, such as cystic fibrosis (CF), primary ciliary dyskinesia (PCD), surfactant protein B (SPB)-deficiency and alpha 1-antitrypsin (A1AT) –deficiency[1]
The mean fluorescent intensity of cells transfected with enhanced green fluorescent protein (eGFP) minicircle DNA was 2.5 to 3-fold higher than cells transfected with plasmid DNA (Fig. 2c; p < 0.001with all doses tested)
Gene delivery to the airway epithelium has been investigated for a number of diseases with most progress being made in the context of CF lung disease

Summary

Introduction

Lung diseases are a major cause of morbidity including genetic diseases, such as cystic fibrosis (CF), primary ciliary dyskinesia (PCD), surfactant protein B (SPB)-deficiency and alpha 1-antitrypsin (A1AT) –deficiency[1]. A number of human clinical trials have been conducted[4,5,6,7,8,9,10,11] but clinically viable gene therapy treatments for CF or other genetic lung disorders have yet to materialise. This lack of clinical efficacy has been in large part due to inefficient transgene expression resulting from the mucociliary barriers that the respiratory system has evolved to clear inhaled foreign particles. Towards developing optimal formulations and protocols for airway epithelial gene transfer, we optimised the methods of nebulised delivery to the conducting airways in mice[21] and pigs[22]

Methods

Results

Conclusion