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Poly (D, L-lactide-co-glycolide)/DNA microspheres to facilitate prolonged transgene expression in airway epithelium in vitro, ex vivo and in vivo

Gene Therapy volume 10, pages 1282–1288 (2003)Cite this article

Abstract

Repeat administration of gene therapy for cystic fibrosis is likely to be essential for long-term clinical efficacy. This may be minimized by the use of slow-release gene transfer preparations with more prolonged expression and longer dosing intervals for the patient. Poly(D-L-lactide-co-glycolide) (PLG) is a biodegradable and biocompatible polymer that has been used to encapsulate plasmid DNA. PLG-DNA microspheres were generated and characterized with respect to morphology, size (80% of particles <5.2 μm), and encapsulation efficiency (50.7±2.3%, n=6). Gel electrophoresis of DNA re-extracted from the microspheres confirmed that despite a decrease in the proportion of supercoiled conformation, it had not been degraded by the preparation process. Gene transfer efficiency was tested using microspheres encapsulating the reporter gene β-galactosidase in vitro on Cos 7 cells and a CF airway epithelial line (CFTEo∼) and ex vivo in a sheep tracheal (s.t.) model. In both cases, transgene exression was significantly (P<0.01) lower at the first time point tested (24 h in vitro, 48 h ex vivo) compared to lipid-#67-mediated gene transfer. However, PLG-mediated expression in vitro was sustained at 48 h, while lipid #67-mediated expression levels had dropped significantly (P<0.05) to 50.3±13.7 and 38.2±2.7% (Cos 7 and CFTEo∼ cells, respectively) of the 24-h level. This pattern was also seen in the s.t. model where at 72 h, PLG-mediated expression was 125.4±7.2% of the 48-h level demonstrating significantly (P<0.05) better retention of transfection efficiency than lipid #67, where levels had fallen to approximately half the 48 h level. By 96 h, expression was still retained in the PLG-transfected group (87.3±12.5% of 48 h expression) but was undetectable in the lipid -#67-transfected s.t. Finally, PLG microspheres, encapsulating the reporter gene chloramphenicol transferase (CAT, 80 μg) were instilled intranasally into Balb/C mice. Compared to lipid-#67-mediated delivery, where whole lung CAT expression was highest at 48 h (13.7 × 103±0.05 CAT U/μg protein, n=6) and then not detectable at further time points, CAT expression was not detectable in PLG-transfected mice at 48 h, but was detectable at 7, 14 and 21 days after transfection. These data demonstrate that PLG-mediated gene transfer can produce prolonged gene expression in airway epithelia. However, gene transfer efficiency still requires significant improvement.

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Acknowledgements

We thank Ann Dewar (Electron Microscopy, Imperial College at the National Heart and Lung Institute) for her help with the electron microscopy studies and Professor C Marriott and Mr Steve Ingham (Department of Pharmacy, King College of London) for their assistance with sizing analysis of the PLG polymers. These studies were funded by the Wellcome Trust, the Cystic Fibrosis Research Trust and by a Wellcome Trust Senior Clinical Fellowship (EWFWA).

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  1. Department of Gene Therapy, Imperial College Faculty of Medicine at the National Heart and Lung Institute, London, UK

    M Stern, K Ulrich, D M Geddes & E W F W Alton

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Stern, M., Ulrich, K., Geddes, D. et al. Poly (D, L-lactide-co-glycolide)/DNA microspheres to facilitate prolonged transgene expression in airway epithelium in vitro, ex vivo and in vivo. Gene Ther 10, 1282–1288 (2003). https://doi.org/10.1038/sj.gt.3301994

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