UBC Theses and Dissertations
In vitro and in vivo gene transfer to respiratory epithelium in experimental models of fetal gene therapy for cystic fibrosis Huang, Louise NY
Fetal gene therapy is a novel technique for correction of monogenic disorders, such as cystic fibrosis. The aims of this study were to: i) develop small animal models for fetal gene transfer to respiratory epithelium; ii) to determine the ideal gestational timing of transfection in our models; iii) evaluate in vivo gene transfer techniques which are directly applicable to human fetal therapy; and iv) assess the durability of in vivo fetal gene transfer postnatally. Vesicular Stomatitis Virus-G (VSV-G) pseudotyped lentiviral vector was used, which contains Green Fluorescence Protein (GFP), as the reporter gene. Fetal tracheas from time-mated New Zealand White pregnant rabbits were harvested on gestational day 24, 25 and 26 (term=31d) and put in organ culture medium, then transfected with 1 x 10⁶ viral particles. Following in vitro transfection, fetal tracheas began to express marker gene as early as one day after infection, with peak expression noted by day 7 post transfection. Results were confirmed by Polymerase Chain Reaction (PCR) and Immunohistochemistry (IHC). Based on the observation that 4 days of in vitro culture was necessary to achieve substantial marker gene expression, we evaluated two in vivo injection techniques on or before day 26 of fetal gestation: i) fetal tracheal injection (TI) following hysterotomy and partial fetal delivery, and ii) amniotic injection (AI) without hysterotomy. After injection of 1 x 10⁶ virus particles, fetuses and their control littermates were delivered by caesarean section on gestational day 30. Fetal tissues (trachea, lung, gut, liver, kidney gonad) were harvested and marker gene expression was confirmed by fluorescent microscopy, PCR and IHC. By PCR, there was evidence of extensive transduction of fetal tissues by AI (trachea, lung, gut, liver, kidney, skin), yet selective transduction of trachea and lung by TI. IHC localized airway expression of GFP to tracheal surface epithelium and pulmonary alveolar cells. Control fetuses expressed marker gene following TI, presumably through blood borne transmission via placental vascular connections, while comparatively few control fetuses were positive for marker gene after AI. One doe was found to have GFP DNA in lung after TI, suggesting that maternal infection is possible with this model. Marker gene expression was also observed in mid-gestational fetal CD1 mice following AI. Transfected fetuses were survived and sacrificed at various post-natal time points to test the durability of gene expression by Quantitative real-time PCR. The marker gene was detectable as late as 21 days after birth; however there was evidence that although transgene was detectable by PCR in trachea 3 weeks after birth, there was no corresponding GFP mRNA, suggesting that transgene expression may be "switched off. Lentiviral vector mediated gene transfer using direct amniotic injection techniques exhibited transduction of respiratory epithelium in fetal rabbits and mice in vivo. This experimental system should prove useful for future fetal gene therapy studies.
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