Development and in vitro validation of a targeted delivery vehicle for DNA vaccines - DNA

Development and in vitro validation of a targeted delivery vehicle for DNA vaccines
15 May 2006
Silke S. Talsmaa, Julia E. Babenseea, , Niren Murthya and Ifor R. Williams
Journal of Controlled Release
Abstract
Usage of DNA vaccination has been limited by inefficient cellular expression of plasmid constructs used in DNA vaccines. We describe a novel system for enhancing delivery of DNA vaccine plasmids into cells and their nuclei. This delivery system uses recombinant reovirus type 3 s1 attachment protein genetically modified with a nuclear localization sequence (s1-NLS) as a targeting ligand. Purified s1-NLS was covalently conjugated to the polycation polyethyleneimine (PEI) using a carboxyl-reactive cross-linking agent and complexed with plasmid DNA. The benefit of the NLS in enhancement of protein delivery into the nucleus was demonstrated by liposome-mediated loading of cells with s1 or s1-NLS. In L929 fibroblasts loaded with s1-NLS, 69% of the internalized protein was recovered in the nuclear fraction after 6 h compared to just 10% when using unmodified s1. Transfection of L929 cells with s1-NLS-conjugated PEI complexed with a luciferase expression plasmid resulted in a mean 16-fold increase in luciferase activity over complexes made with unmodified PEI, compared to a mean 3-fold boost obtained using s1-conjugated PEI. These results suggest that s1-NLS is a useful bifunctional targeting ligand suitable for enhancing DNA delivery and subsequent gene expression for both DNA vaccine applications and nonviral gene therapy.
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