Gene transfection into fetal sheep airways in utero using guanidinium-cholesterol cationic lipids.
BACKGROUND
Over the last several years, we have developed a novel class of cationic lipids, cholesterol derivatives characterized by polar head groups with guanidinium functions. We have in particular shown that bis(guanidinium)-tren-cholesterol/dioleoylphosphatidylethanolamine (BGTC/DOPE) cationic liposomes can mediate efficient gene transfection into the mouse airways in vivo via direct intratracheal administration or intranasal instillation. As prenatal gene therapy may be necessary for the treatment of a variety of congenital lung diseases, we have explored in the present work the feasibility of BGTC-mediated gene transfection into the respiratory tract of fetal sheep in utero.


METHODS
Thus, BGTC/DOPE liposomes were complexed with plasmids expressing the Escherichia coli chloramphenicol acetyltransferase (CAT) reporter gene and the resulting lipoplexes were administered to fetal sheep at 70 days of gestation via surgical replacement of the airway fluid by the transfection mixture followed by tracheal occlusion. The fetal lungs and tracheas were harvested at 72 h and examined for CAT expression and evidence of toxicity.


RESULTS
CAT expression was detected in both lung and trachea homogenates, no CAT expression being observed in control fetuses receiving naked plasmid DNA. Immunohistochemical analysis showed that airway epithelial cells and some mesenchymal cells were transfected. Pulmonary histopathology of varied severity was however observed under our transfection conditions and manifested as focal epithelial and mesenchymal lesions.


CONCLUSIONS
These results show that BGTC/DOPE liposomes can mediate gene transfection into the fetal sheep airway epithelium. They also invite the development of optimized BGTC-based formulations and administration conditions with a view to future prenatal gene transfer experiments involving therapeutic genes.
Luton
Dominique
D
Maternité, Hôpital Robert Debré, AP-HP, 48 Boulevard Sérurier, 75019 Paris, France. plehn@infobiogen.fr
Oudrhiri
Noufissa
N
de Lagausie
Pascal
P
Aissaoui
Abderrahim
A
Hauchecorne
Michelle
M
Julia
Sébastien
S
Oury
Jean-François
JF
Aigrain
Yves
Y
Peuchmaur
Michel
M
Vigneron
Jean-Pierre
JP
Lehn
Jean-Marie
JM
Lehn
Pierre
P
D000818:Animals; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis / Q000235:genetics; D002784:Cholesterol / Q000031:analogs & derivatives*; D005260:Female; D005333:Fetus / Q000378:metabolism*; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors; D006146:Guanidines; D007120:Immunochemistry; D008081:Liposomes / Q000737:chemistry; D008168:Lung / Q000196:embryology / Q000473:pathology; D010714:Phosphatidylethanolamines / Q000737:chemistry; D010957:Plasmids / Q000008:administration & dosage; D011247:Pregnancy; D020545:Respiratory Mucosa / Q000196:embryology / Q000378:metabolism*; D012756:Sheep / Q000196:embryology; D014132:Trachea / Q000473:pathology; D014162:Transfection / Q000379:methods*
C101389:3-(N(4)-(N(1),N(8)-diguanidinospermidine)carbamoyl)cholesterol; D006146:Guanidines; D008081:Liposomes; D010714:Phosphatidylethanolamines; C094877:dioleoyl phosphatidylethanolamine; D002784:Cholesterol; D015500:Chloramphenicol O-Acetyltransferase