The aim of this study was to establish a nonviral method for gene delivery to the rat kidney. To this purpose, a panel of reagents was tested, including a monocationic lipid, DOTAP, a polycationic lipid, DOGS (or Transfectam), and three different forms of the cationic polymer polyethylenimine (PEI). A comparison among these compounds was performed in vivo, using luciferase as reporter gene. Complexes containing 10 μg of DNA were injected into the left renal artery of rats and allowed to remain in contact with the kidney for 10 min. Forty-eight hours later, luciferase expression levels in kidney extracts were measured. Kidneys injected with DNA complexed to the branched, 25-kD PEI polymer (PEI25k) yielded activity levels significantly higher than control, sham-operated kidneys (2.7 x 104 vs. 5.2 x 103 RLU/kidney, respectively), whereas the other transfecting agents did not yield significant activity over controls. PEI25k was therefore chosen for further optimization of transfection conditions. Dose-dependent luciferase expression was shown for 10, 50, and 100 μg of PEI-complexed DNA, reaching maximal levels of 2.4 x 105 RLU/kidney at 100 μg DNA. The optimal PEI nitrogen/DNA phosphate molar ratio was 10 equivalents. Luciferase activity peaked at 2 days, was still significantly higher than controls at 7 days, and was undetectable at 14 days post-injection. Using β-galactosidase (β-Gal) as a reporter, transgene expression was localized almost exclusively in proximal tubular cells.
|Number of pages||9|
|Journal||Human Gene Therapy|
|Publication status||Published - Jul 1 1997|
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