Development and in vitro validation of a targeted delivery vehicle for DNA vaccines.
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 sigma1 attachment protein genetically modified with a nuclear localization sequence (sigma1-NLS) as a targeting ligand. Purified sigma1-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 sigma1 or sigma1-NLS. In L929 fibroblasts loaded with sigma1-NLS, 69% of the internalized protein was recovered in the nuclear fraction after 6 h compared to just 10% when using unmodified sigma1. Transfection of L929 cells with sigma1-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 sigma1-conjugated PEI. These results suggest that sigma1-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.
Talsma
Silke S
SS
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, Suite 2127, Atlanta, GA 30332-0535, USA.
Babensee
Julia E
JE
Murthy
Niren
N
Williams
Ifor R
IR
D000818:Animals; D016466:CHO Cells; D036022:Capsid Proteins / Q000235:genetics*; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D003432:Cross-Linking Reagents / Q000008:administration & dosage*; D008156:Luciferases / Q000235:genetics; D012089:Mammalian orthoreovirus 3 / Q000235:genetics; D051379:Mice; D019913:Nuclear Localization Signals / Q000235:genetics*; D010957:Plasmids / Q000008:administration & dosage* / Q000235:genetics; D011094:Polyethyleneimine / Q000008:administration & dosage*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014162:Transfection; D019444:Vaccines, DNA
D036022:Capsid Proteins; D003432:Cross-Linking Reagents; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D019444:Vaccines, DNA; C031728:sigma 1 protein, reovirus; D011094:Polyethyleneimine; D008156:Luciferases