Lentiviral-mediated fetal gene therapy for monogenic disorders: development of an in vitro rabbit model.
OBJECTIVE
Fetal gene replacement is a novel, potential therapy for monogenic disorders which are diagnosed prenatally. The purpose of this study was to develop in vitro, respiratory-epithelium targeted, lentiviral (LV)-mediated gene transfer in fetal rabbit tracheas.


METHODS
Via triple plasmid transfection, vesicular stomatitis virus-G (VSV-G)-pseudotyped LV vector containing green fluorescent protein (GFP) marker gene, under the control of a cytomegalovirus promoter was constructed. LV bioavailability in rabbit amniotic fluid (AF) was evaluated by infectivity assays of 293T cell monolayers in variable concentrations of AF. Fetal tracheas from time-mated rabbits (term gestation, G = 31 days) were collected on G 23-25 days, and placed in tissue culture (substrate-enriched DMEM, 37 degrees C, 5% CO(2)/room air). The tracheal cultures were transfected with 1 x 10(5) LV particles, and analyzed daily for: reporter gene by polymerase chain reaction, and reporter gene product (GFP) by whole-mount fluoroscopy and immunohistochemistry.


RESULTS
293T cell infectivity assays confirmed bioavailability of LV in rabbit AF. Following in vitro transfection, GFP DNA and GFP were detectable in fetal rabbit tracheas by 4 and 5 days, respectively. Immunocytochemistry localized GFP to the luminal aspect of tracheal epithelium.


CONCLUSIONS
In vitro, LV-mediated GFP gene transfer to fetal rabbit tracheas occurs within 4 days, and gene expression is evident by 5 days post-transfection. This observation, and the bioavailability of LV through AF, suggests the appropriateness of this model for the future evaluation of in vivo, transamniotic gene delivery strategies.
Huang
Louis
L
Division of Pediatric General Surgery, British Columbia Children's Hospital, Vancouver, Canada.
Reebye
Stacy C
SC
Yeung
Adam Y
AY
Jia
William W
WW
Skarsgard
Erik D
ED
D000653:Amniotic Fluid; D000818:Animals; D005315:Fetal Diseases / Q000628:therapy*; D030342:Genetic Diseases, Inborn / Q000628:therapy*; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D005865:Gestational Age; D049452:Green Fluorescent Proteins / Q000235:genetics; D016086:Lentivirus / Q000235:genetics*; D023421:Models, Animal; D011817:Rabbits; D046509:Tissue Culture Techniques; D014132:Trachea / Q000196:embryology; D014162:Transfection
D049452:Green Fluorescent Proteins