Nonviral Gene Delivery to the Rat Kidney with Polyethylenimine

Abstract

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 (PEI 25k) yielded activity levels significantly higher than control, sham-operated kidneys (2.7 × 10⁴ vs. 5.2 × 10³ RLU/kidney, respectively), whereas the other transfecting agents did not yield significant activity over controls. PEI 25k 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 × 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. We have established a nonviral method for gene delivery to the rat kidney, as an alternative to the existing approaches based on viral vectors or on DNA-containing liposomes including viral components. Among different cationic lipids and polycationic polymers, the branched, 25-kD cationic polymer polyethylenimine (PEI 25k) was chosen as the only compound yielding significantly high luciferase expression. Optimal transfection conditions were obtained with 100 μg of DNA complexed to PEI 25k at a ratio of 10 PEI nitrogen/DNA phosphate. Transgene expression was transient, peaking at 2 days after transfection. Expression of β-galactosidase was mainly localized in proximal tubular cells.