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.
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, Suite 2127, Atlanta, GA 30332-0535, USA.
Silke S
SS
Talsma
Julia E
JE
Babensee
Niren
N
Murthy
Ifor R
IR
Williams
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