Combining keratinocyte growth factor transfection into the airways and tracheal occlusion in a fetal sheep model of congenital diaphragmatic hernia.
BACKGROUND
In utero tracheal occlusion (TO) has been developed to improve the lung hypoplasia associated with congenital diaphragmatic hernia (CDH). However, although TO stimulates fetal lung growth, it results in a decrease of alveolar type II cells (ATII) and surfactant production. Because keratinocyte growth factor (KGF) is a potent stimulus of ATII proliferation and maturation, we evaluated, in a fetal lamb model of CDH, a gene therapy strategy combining TO and ovine KGF transfection into the fetal airways using bisguanidinium-tren-cholesterol/dioleoyl-phosphatidylethanolamine (BGTC/DOPE) cationic liposomes.


METHODS
Three groups of sheep fetuses with CDH and a group of normal fetuses were studied. The fetuses of the three groups with CDH (KGF, Medium and Hernia groups) underwent surgery at 85 days of gestation to create a diaphragmatic hernia. The KGF and medium group fetuses underwent a second surgery step at day 125 to perform TO associated with injection of the KGF transfection mixture (KGF group) or control medium (Medium group), whereas the fetuses of the Hernia group were left untreated. Normal fetuses were used as a control (Normal group). All fetuses were euthanized at 132 days of gestation and various analytical studies [lung weight, radial alveolar count (RAC), KGF and surfactant protein B (SPB) expression, number of ATII cells] were performed to assess the efficiency of KGF transfection and its effects on fetal lung development.


RESULTS
TO was associated with lung hyperplasia and increased RAC in the Medium and KGF groups versus the Hernia group. Expression of KGF was increased in the KGF group compared to all other groups and was associated with an increased synthesis of SPB by alveolar cells and an ectopic synthesis of SPB by bronchiolar cells compared to TO treatment alone.


CONCLUSIONS
Thus, BGTC/DOPE liposomes can mediate efficient KGF transfection into the airways in a fetal sheep model of CDH. Furthermore, combining KGF transfection and TO resulted not only (as did TO alone) in the correction of the CDH-associated lung hypoplasia and decreased RAC, but also in increased SPB synthesis, suggesting a better maturation of the re-growing lung (compared to TO alone). Additional studies are required to further explore the therapeutic potential of such a combined strategy; in particular, studies evaluating the lung function of in utero-treated CDH lamb newborns.
Saada
Julien
J
EA 3102, Hôpital Robert Debré, AP-HP, Paris, France.
Oudrhiri
Noufissa
N
Bonnard
Arnaud
A
de Lagausie
Pascal
P
Aissaoui
Abderrahim
A
Hauchecorne
Michelle
M
Oury
Jean-François
JF
Aigrain
Yves
Y
Peuchmaur
Michel
M
Lehn
Jean-Marie
JM
Lehn
Pierre
P
Luton
Dominique
D
D000818:Animals; D004195:Disease Models, Animal; D005333:Fetus / Q000378:metabolism / Q000473:pathology*; D051523:Fibroblast Growth Factor 7 / Q000235:genetics* / Q000378:metabolism / Q000627:therapeutic use*; D018507:Gene Expression Regulation, Developmental; D006548:Hernia, Diaphragmatic / Q000628:therapy*; D065630:Hernias, Diaphragmatic, Congenital; D008168:Lung / Q000196:embryology / Q000473:pathology; D009929:Organ Size; D011650:Pulmonary Alveoli / Q000473:pathology; D037701:Pulmonary Surfactant-Associated Protein B / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012756:Sheep; D014132:Trachea / Q000098:blood supply*; D014162:Transfection / Q000379:methods*
D037701:Pulmonary Surfactant-Associated Protein B; D051523:Fibroblast Growth Factor 7