Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel cause cystic fibrosis. The ⌬F508 mutation produces defects in channel gating and cellular processing, whereas the G551D mutation produces primarily a gating defect. To identify correctors of gating, 50,000 diverse small molecules were screened at 2.5 M (with forskolin, 20 M) by an iodide uptake assay in epithelial cells coexpressing ⌬F508-CFTR and a fluorescent halide indicator (yellow fluorescent protein-H148Q/I152L) after ⌬F508-CFTR rescue by 24-h culture at 27°C. Secondary analysis and testing of Ͼ1000 structural analogs yielded two novel classes of correctors of defective ⌬F508-CFTR gating ("potentiators") with nanomolar potency that were active in human ⌬F508 and G551D cells. The most potent compound of the phenylglycine class, 2-[(2-1H-indol-3-yl-acetyl)-methylamino]-N-(4-isopropylphenyl)-2-phenylacetamide, reversibly activated ⌬F508-
Hepatic ischemia/reperfusion (I/R) injury is characterized by the generation of reactive oxygen species (ROS), such as superoxide anions and hydrogen peroxide. The aim of this study is to investigate whether antioxidative gene delivery by our polylipid nanoparticles (PLNP) is an effective approach for prevention of the injury. Polyplexes of extracellular superoxide dismutase (EC-SOD) and/or catalase genes were injected via the portal vein 1 day prior to a warm I/R procedure in mice. The effects of the gene delivery were determined 6 hours after starting reperfusion. PLNP-mediated antioxidative gene delivery led to a marked increase in human EC-SOD and catalase gene expression in the liver. Liver superoxide dismutase (SOD) and catalase activity both increased approximately 10-fold. Increased liver superoxide anion levels caused by the I/R procedure were reduced to normal levels by EC-SOD gene delivery. The overexpression of these 2 antioxidative genes significantly suppressed the I/R-induced elevation of serum alanine aminotransferase (ALT) levels, decreased liver malondialdehyde content, restored glutathione reserve, and improved liver histology. In conclusion, EC-SOD or catalase gene delivery by PLNP resulted in high levels of the transgene activity in the liver, and markedly attenuated hepatic I/R injury. The protection is directly associated with elevated antioxidative enzyme activity as the result of the gene delivery. This novel approach may become a potential therapy to improve graft function and survival after liver transplantation.
We, and others, have observed that the structure of cationic lipids appears to have a significant effect on the transfection efficacy of optimized nucleic acid/cationic lipid complexes (lipoplexes) used for in vitro and in vivo gene delivery and expression. Although there are many in vitro comparisons of lipid reagents for gene delivery, few comparisons have been made in vivo. We previously reported the effects of changes in hydrophobic domain chain length and chain asymmetry, changes in headgroup composition, and counterion exchange. We have observed in our own work over many years the apparent superiority of asymmetric versus symmetric hydrocarbon domains for otherwise similar lipids. In this investigation we use in vivo whole animal brain imaging to evaluate the contribution of symmetric versus asymmetric hydrophobic domains on what we previously determined to be optimal chain lengths for in vitro transfections. We specifically investigated several glycerol-based lipids; however, the rare reports of asymmetric non-glycerol-based lipids also support our observations. We found that asymmetric, two-chain cationic lipids of 14 to 18 carbons perform significantly better in vivo, as analyzed by whole animal imaging, than the paired symmetric lipids.
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