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Nonviral gene transfer by chitosan polymer-based nanotechnology

University of British Columbia

Gene therapy provides a potential alternative to conventional protein drug administration, allowing the body to produce its own drugs based on the exogenously introduced genetic information. Increasing efforts have been devoted to developing nonviral-based gene transfer systems owing to its attractive safety features. In this thesis, a nonviral chitosan/ФC31 gene transfer technology was evaluated for the delivery of two transgenes, SEAP and leptin. While SEAP reporter gene transfer allowed evaluation of delivery methods, leptin gene transfer permitted the assessment of therapeutic efficacy. Chitosan nanoparticles encapsulating 2 µg SEAP or leptin transgene were able to transfect HEK 293T cells, which secreted SEAP and human leptin proteins that achieved as much as 9414 ± 169 and 74 ± 5 ng/ml, respectively. In culture, the human leptin protein expressed by the leptin constructs effectively activated the leptin signalling pathway, represented by the phosphorylation of STAT3, demonstrating the protein’s biological activity. In vivo, SEAP gene transfer, in C57BL6/j mice, was achieved via intrasplenic and i.p. chitosan nanoparticle delivery. For the i.p. route, it was revealed that, at Day 1 post gene transfer, a 60% decrease in the DNA dose from 625 to 250 µg led to an at least ~7-fold increase in circulating SEAP, detected at 371 ng/ml. Furthermore, a similar level of SEAP (353 ng/ml) was obtainable with an even lower dose of DNA (50 µg). Mice receiving a single i.p. injection of chitosan nanoparticles containing 50 µg gWIZTM-SEAP plasmid showed transgene expression kinetics characteristic of those observed with a CMV promoter, with circulating SEAP measurements of 342 ± 60, 108 ± 18, 30 ± 3, and 7 ± 1 ng/ml at Day 1, 3, 5, and 7 post-treatment. Furthermore, a rare case of successful oral SEAP gene transfer was observed following a single oral feeding of chitosan nanoparticles. These data demonstrate that the use of the chitosan/ФC31 gene transfer system can lead to successful SEAP gene transfer via the i.p. route of administration, although in vivo data did not support effective leptin gene transfer. Future studies on improving transgene vector design are warranted for further development of this gene transfer system.

Thesis/Dissertation

application/pdf

2009-08-26

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/12571

Physiology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/