504. Gene Transfection into Fetal Sheep Airway Epithelium Using Cationic BGTC/DOPE Liposomes

Abstract

Over the last years, we have developed a particular class of nonviral vectors, cationic cholesterol derivatives characterized by polar headgroups with guanidinium functions. Indeed, guanidinium groups have highly favorable features for DNA binding. Liposome formulations of bis(guanidinium)-tren-cholesterol (BGTC) with dioleoylphosphatidylethanolamine (DOPE) were found to be efficient for gene transfection into primary human airway epithelial cells as well as into the mouse airway epithelium in vivo by intratracheal administration (Proc. Natl. Acad. Sci. USA 1997, 94, 1651-1656). We have also shown that colloidal stabilization (via lipophilic polyethyleneglycol derivatives) of concentrated solutions of BGTC/DOPE lipoplexes allowed enhanced transfection of the mouse airways by intranasal instillation (J. Gene Med. 2001, 3, 478-487). Thus, as prenatal gene therapy may be necessary for the treatment of a variety of congenital lung diseases, we recently undertook to explore the feasibility of BGTC-mediated gene transfection into the respiratory tract of fetal sheep in utero. Practically, pCIK-CAT plasmids expressing the E. coli chloramphenicol acetyltransferase (CAT) reporter gene (kindly provided by D. Gill, Oxford, UK) were complexed with BGTC/DOPE liposomes and the resulting lipoplexes were administered to sheep fetuses at 70 days of gestation via surgical replacement of the fetal airway fluid by the transfection mixture followed by tracheal ligature. The fetal tracheas and lungs were harvested at 72 hours and examined for CAT expression and evidence of toxicity. Using an ELISA assay, we detected significant CAT levels in lung and trachea homogenates, no CAT expression being observed in control fetuses receiving naked pCIK-CAT plasmid. Immunohistochemistry analysis showed that airway epithelial cells (at both the tracheal and bronchial levels) as well as some mesenchymal cells were transfected. Pulmonary histopathology of varied severity was however observed and manifested as focal epithelial and mesenchymal lesions. These results demonstrated that BGTC/DOPE liposomes can mediate gene transfection into the fetal sheep airway epithelium in utero and they also invited the development of optimized BGTC-based formulations with a view to future experiments with therapeutic genes. Thus, we are at present investigating the utility of BGTC-based lipoplexes for transferring genes encoding pulmonary growth factors in the fetal lamb model of congenital diaphragmatic hernia (CDH). In these ongoing experiments, a first surgical step allows the creation of the diaphragmatic hernia, whereas the second step consists of the substitution of the fetal airway fluid by the lipoplex medium followed by tracheal occlusion via a balloon. Nonviral gene delivery approaches may be particularly attractive here as long-term transgene expression is probably not required for the treatment of CDH before birth.