Prenatal Orogastric Gene Delivery Results in Transduction of the Small Bowel in the Fetal Rabbit

Abstract

Background: Gene therapy for meconium ileus, or other genetic diseases involving the gastrointestinal epithelium, may be possible with prenatal delivery of the CFTR gene to the gastrointestinal tract. Although minimally invasive techniques will probably be used for any future therapy of gastrointestinal disease, it is important to first test this strategy with a reliable animal model. Methods: The technique of orogastric fetal gene delivery was assessed using 7 pregnant rabbits (22 days’ gestation n = 1, 25 days’ gestation n = 4, 28 days’ gestation n = 2). Four fetuses from each litter were given an adenoviral vector carrying a marker gene by instilling it into the posterior pharynx with an animal feeding needle (1 × 10¹⁰ particles of ADV/RSV/LacZ suspended in 0.3 ml of saline), with the untreated fetuses serving as control animals. Results: There were no recoverable fetuses from the does that had surgery at 22 and 28 days (n = 3) due to maternal death (n = 2) and premature delivery (n = 1). Among the 4 does that underwent hysterotomy at 25 days of gestation, 1 underwent cesarean section 2 days after fetal gene delivery and 3 delivered at term, 5 (n = 1) or 6 (n = 2) days following gene delivery. Eleven of the 16 experimental pups and 7 untreated control animals were collected alive, and were sacrificed at delivery for study. Nine of the 11 experimental pups (82%) showed positive blue (LacZ+) nuclei in the small intestine by X-gal staining. No positive cells were found in 7/7 control animals. The presence of the reporter gene LacZ was confirmed in 8/11 (73%) virus-treated pups by PCR with 5/5 control animals negative for LacZ by PCR. Conclusions: There was significant maternal and fetal loss related to anesthetic and husbandry issues when surgery was performed at 22 or 28 days of gestation. Based on these preliminary results, we conclude that orogastric gene delivery in the rabbit fetus at 25 days’ gestation is an encouraging animal model to study fetal delivery to the gastrointestinal tract.

Feasibility of Retroviral Vector-Mediated in utero Gene Transfer to the Fetal Rabbit

“…Desirable features of this animal model include a short gestational period, an optimal size for fetal intervention (28-day gestation fetal rabbits are similar in size to an adult mouse), and the possibility of treating simultaneously several fetuses located within individual sacks in the bicornic uterus of a single doe. Extensive experience has endowed the rabbit as a well-recognized model for fetal therapy, allowing accurate access to different organs with acceptable levels of fetal survival [12,[21][22][23] . Furthermore, embryological and maturational evolution of several organ systems and tissues proceed through similar stages as the human fetus, with the exception of neural growth [24,25] .…”

“…The rabbit is a middle-sized animal model allowing accurate organ-directed manipulation and delivery of gene transfer vectors. Previously reported gene transfer studies in fetal rabbits have focused on recombinant adenovirus [10][11][12] and adenoassociated virus [13] vectors, but there is no available experimental data on the use of retroviral vectors for in utero gene transfer in the pregnant rabbit model.…”

Stem Cell Transplantation and Gene Therapy in utero

“…Furthermore, recent data from experiments using stem cell transplantation into mouse blastocysts show that gene expression of donor cells largely depends on the developmental stage of the host microenvironment [83], which could mean that gene expression is enhanced after prenatal gene transfer compared to postnatal gene therapy. Different in utero gene transfer models targeted to various cells and organs have been developed, including pulmonary epithelial cells [84][85][86][87], hepatocytes [88], skin [89], intestine [90], heart [91], and ductus arteriosus cells [92]. The models used intraamniotic, intratracheal, intraperitoneal, intrahepatic, intravascular, intraplacental, or in situ (ductus arteriosus region) administration of the vector gene construct.…”