Gene Transfer to the Eye Utilizing the Cationic Liposomes After Intravitreal Injection in Rabbits

Abstract

Abstract: : Purpose: The purpose of this study is to investigate the transfection activity of plasmid DNA (pDNA) /cationic liposome complexes to the eye in rabbits in relation to the lipid composition of cationic liposomes and the charge ratio of pDNA/ cationic liposome complexes. Methods:N–[1–(2,3–dioleyloxy)propyl]–N,N,N–trimethylammonium chloride (DOTMA)/ dioleoylphosphatidylethanolamine (DOPE) (1:1) liposomes and DOTMA/cholesterol (Chol) (1:1) liposomes were prepared. pDNA encoding luciferase gene (pCMV–Luc) complexed with cationic liposomes was injected intravitreally in rabbits. The rabbits were sacrificed and the each ocular tissue was subjected to luciferase assay. Results: At a dose of 40 µg pDNA, the luciferase activities in the ocular tissues after injection of pDNA complexed with DOTMA/DOPE liposomes were approximately 1.1–8.0 fold higher than that of naked pDNA or pDNA complexed with DOTMA/Chol liposomes. At pDNA doses of more than 50 µg, it was difficult to prepare the pDNA complexed with DOTMA/DOPE liposomes due to causing the aggregation. After injection of naked pDNA at pDNA doses of 60 and 85 µg, their luciferase activities in the ocular tissues were almost the same compared with those of 40 µg. In contrast, the luciferase activity after injection of pDNA complexed with DOTMA/Chol liposomes was enhanced according to an increase in pDNA doses (40–85 µg). Among these transfection experiments, pDNA (85 µg) complexed with DOTMA/Chol liposomes showed the highest transfection activity. As far as the charge ratios of pDNA complexed with DOTMA/Chol liposomes (+1.5, +2.0, and +2.5) were concerned, the luciferase activity was varied and complexes at a charge ratio of +2.0 produced maximal gene expression in the eye. Results from time course showed that gene expression in the ocular tissues was transient, with a peak level at 3 days, followed by dropping to less than approximately 10% of the peak level by the 7 days. Conclusions: Efficient gene transfer could be achieved by cationic liposomes in the ocular tissues of rabbits after intravitreal injection. Optimizing the lipid composition of cationic liposomes and the charge ratio of pDNA/cationic liposome complexes should enhance the transfection efficiency to the eye.

Keywords: gene transfer/gene therapy