This study will investigate proteasome inhibition as a novel mechanism to augment the expression of nonviral gene delivery systems in vivo. The gene delivery system under investigation is a multi-component peptide condensed targeted gene delivery system. The key components consist of plasmid DNA condensed by a sulfhydryl cross-linking polymer composed of polyethylene glycol (PEG)-peptide, targeting glycopeptide, and fusogenic peptide. Specific targeting is achieved using glycopeptides that bind to either the asialoglycoprotein receptor on hepatocytes or the mannose receptor on Kupffer cells. Following internalization via receptor mediated endocytosis, sulfhydryl cross-linked DNA condensates depolymerize in the reducing environment of the endosome. Released fusogenic peptides facilitate endosomal lysis and DNA escape into the cytosol. Following condensate un-coating, DNA targets the nucleus. The efficiency of gene transfer is limited by the ability of DNA condensates to escape endosomes and avoid degradation in the cytosol. Preliminary studies indicate the proteasome metabolizes peptide-DNA condensates in the cytosol, resulting in premature degradation of plasmid DNA by cytosolic DNAse. The present proposal will investigate the use of proteasome inhibitors to block DNA condensate metabolism in the cytosol and enhance gene transfer efficiency. The central hypothesis to be tested is that proteasome inhibition will enhance the level and duration of transient gene expression in vivo by stabilizing plasmid DNA from metabolism. The proposed studies aim to incorporate intrinsic peptide-based proteasome inhibitors into sulfhydryl cross-linked non-viral gene delivery systems that target either hepatocytes or Kupffer cells. The overall objective of the proposed study is to increase the efficiency of non-viral gene delivery using a novel mechanism of proteasome inhibition to block DNA condensate metabolism. The successful outcome of these studies should provide a rational approach to develop more efficient gene delivery systems that can be used to treat a variety of human diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
3R01DK066212-02S1
Application #
7279038
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Mckeon, Catherine T
Project Start
2005-04-01
Project End
2008-03-31
Budget Start
2006-04-01
Budget End
2007-03-31
Support Year
2
Fiscal Year
2006
Total Cost
$45,514
Indirect Cost
Name
University of Iowa
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
062761671
City
Iowa City
State
IA
Country
United States
Zip Code
52242
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Anderson, Kevin; Fernandez, Christian; Rice, Kevin G (2010) N-glycan targeted gene delivery to the dendritic cell SIGN receptor. Bioconjug Chem 21:1479-85
Baumhover, Nicholas J; Anderson, Kevin; Fernandez, Christian A et al. (2010) Synthesis and in vitro testing of new potent polyacridine-melittin gene delivery peptides. Bioconjug Chem 21:74-83
Fernandez, Christian A; Baumhover, Nicholas J; Anderson, Kevin et al. (2010) Discovery of metabolically stabilized electronegative polyacridine-PEG peptide DNA open polyplexes. Bioconjug Chem 21:723-30
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McAnuff, Marie A; Rettig, Garrett R; Rice, Kevin G (2007) Potency of siRNA versus shRNA mediated knockdown in vivo. J Pharm Sci 96:2922-30
Rettig, Garrett R; McAnuff, Marie; Liu, Dijie et al. (2006) Quantitative bioluminescence imaging of transgene expression in vivo. Anal Biochem 355:90-4
Kim, J; Chen, C-P; Rice, K G (2005) The proteasome metabolizes peptide-mediated nonviral gene delivery systems. Gene Ther 12:1581-90