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Investigation of the distribution, transgene expression and immunogenicity of intramuscular non-viral DNA vaccine complexes

thesis
posted on 22.02.2017, 01:59 by Ho, Joan Kha Tu
The advancement of non-viral DNA vaccines has been hindered due to poor immunological responses in vivo, attributed to low transfection efficiency. The use of cationic materials to form complexes with plasmid DNA and promote endocytosis into cells has been a common strategy in this field. However, the highly positive surface charge of the complexes can lead to interaction with extracellular proteins upon injection, resulting in aggregation or immobilization of complexes in vivo. This is thought to hinder the distribution of the complexes to cells and thus, reduce the number of cells made available for uptake of the complexes. A strategy to reduce these electrostatic interactions has been to shield the surface charge of complexes with poly(ethylene glycol) (PEG), thereby increasing the complexes’ stability and distribution in vivo. In our study, we developed two novel DNA complexes – a cationic lipopeptide/DNA (LP/DNA) complex and a PEGylated LP/DNA complex – for intramuscular DNA vaccine delivery. To assist in understanding the effect of shielding the cationic surface charge of complexes in DNA vaccine delivery, the relationships between the distribution, transfection and immunogenicity of the two DNA complexes were investigated. Quantitative polymerase chain reaction assays indicated that PEGylation did not affect the distribution/retention of LP/DNA complexes to the calf muscle, lymph node, spleen and liver – with similar amounts of plasmid present in these tissues after administration of both DNA complexes. Luciferase reporter assays, however, demonstrated that mice injected with the PEGylated complexes had > 200-fold higher gene expression levels in the muscle than LP/DNA treated mice at 1 month. Confocal imaging of the muscle after administration of fluorescently-labelled plasmid DNA complexes indicated that the PEGylated complexes were more disperse throughout the muscle than their non-PEGylated counterparts. This suggests that shielding the surface charge of LP/DNA complexes with PEG had increased the transfection efficiency of the complexes, likely the result of increased distribution of the complexes in the muscle to a higher number of cells for uptake. To evaluate the immunogenicity of the DNA complexes, antibody response assays and in vivo epitope-specific cytotoxic T lymphocyte elimination assays were carried out in mice using ovalbumin-encoded plasmid. Results showed that immunisation with the PEGylated LP/DNA complexes in a DNA prime-DNA boost regimen, yielded a higher number of mice positive for anti-OVA activity than immunisation with the LP/DNA complexes. Flow cytometry showed that the PEGylated complexes stimulated a significant cytotoxic T cell response, eliminating a higher number of target cells (26.7 ± 2.2%) than the LP/DNA treated mice (14.3 ± 1.6%). Overall, these results demonstrated that PEGylation was able to significantly increase the transgene expression and cytotoxic T cell activity of cationic LP/DNA complexes in vivo. Thus, the PEGylation of cationic DNA delivery systems may be a promising strategy for the development of future non-viral DNA vaccines. Future efforts should explore the optimisation of the gene carrier to contain mechanisms that can overcome key hurdles in non-viral DNA delivery such as cellular uptake, complex disassembly and nuclear uptake of the complexes.

History

Campus location

Australia

Principal supervisor

Paul White

Year of Award

2015

Department, School or Centre

Drug Discovery Biology

Course

Doctor of Philosophy

Degree Type

DOCTORATE

Faculty

Faculty of Pharmacy and Pharmaceutical Sciences

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