GTMB 7 - Gene Therapy & Molecular Biology

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David et al: Current status and future direction of fetal gene therapyamniotic delivery of adenoviral vectors to early gestationfetal sheep (David et al, 2003a). Similarly ultrasoundguidedintra-amniotic injection of adenoviral vectors inmid-trimester rhesus macaque fetuses resulted insignificant transgene spread to tissues coming into contactwith amniotic fluid but low level transgene expression inthe fetal airways and intestine (Larson et al, 2000b).Similar findings were observed in fetal rabbits(Boyle et al, 2001). Low levels of airway transduction areprobably due to dilution of the vector by the relativelylarger volume of the amniotic fluid as well as the lack offetal breathing movements or fetal swallowing at this earlygestation. It may be possible to enhance fetal breathingmovements in later gestation using agents such astheophylline (Moss and Scarpelli, 1981) that lead to anintake of amniotic fluid to the lungs against the continuousoutflow of tracheal fluid (Badalian et al, 1993; Kalache etal, 2000). Indeed increased intake of marker dye and someenhancement of adenovirus mediated marker geneexpression was observed in mouse fetuses aftertheophylline administration. However other still unknownfactors appear to influence the level of gene transfer to thefetal airways more effectively (Buckley, in preparation).Recent work in our laboratory aimed to reproduce theiconoclastic report by Larson et al, (1997) that the CFphenotypein CFTR-knockout mice can be cured by shorttermprenatal expression of CFTR from an adenovirusvector, could not substantiate this claim (Buckley et al,2003). We are, therefore, constructing integratingexpression vector systems under tissue specific promotercontrol to achieve long-term postnatal CFTR-geneexpression after in utero gene delivery.2. Direct lung parenchymal injectionDirect injection of the lung parenchyma has beenattempted to access the fetal airways but with poor results.In mid-gestation fetal primates, ultrasound guidedinjection of lentiviral vectors into the lung resulted in lowlevel transgene expression in the fetal airways (Tarantal etal, 2001a). However, in the mid-gestation sheep fetus,ultrasound-guided delivery of an adenoviral vector to thelung parenchyma elicited only localized gene transfer andno spread within the airways could be detected(unpublished results).Figure 4 D-F. Intra-amniotic injection at 33 days of gestation,positive staining is seen in (D) surface of umbilical cord, (E)fetal nasal cavity and (F) fetal skin (David AL et al 2003a).Republished with permission from Mary Ann Liebert Inc,Publishers3. Tracheal injectionDirect instillation of vector into the trachea has beenmore successful. Placement of catheters in the tracheae offetal sheep can be performed by highly invasivetechniques at laparotomy (McCray et al, 1995; Pitt et al,1995; Vincent et al, 1995) or fetoscopically (Sylvester etal, 1997; Yang et al, 1999). Low level transduction of theproximal airways can be achieved using adenoviral orretroviral vectors, and occlusion of the trachea with aballoon improves distal airway transduction. Thesetechniques however, carry a significant morbidity andmortality.Recently a percutaneous transthoracic route ofinjection of the fetal trachea has been developed in midgestationsheep using ultrasound guidance to target the196
Gene Therapy and Molecular Biology Vol 7, page 197fetal airways as illustrated in Figure 5 (David et al,2003b). Using this technique we achieved good transgeneexpression in the fetal trachea and airways followingintratracheal delivery of an adenovirus containing the β-galactosidase gene (Peebles et al, 2003). Transgeneexpression was enhanced by pretreatment of the fetalairways with sodium caprate, a fatty acid that opens thetight junctions between airways epithelial cells. Thisallows the vector to reach the basolateral surface where thecoxsackie-adenovirus receptor (CAR receptor) responsiblefor binding adenovirus is located. Further enhancement oftransgene expression was achieved by complexing theadenoviral vector with DEAE dextran, a polycation thatneutralizes the negative charge on the vector, improvingvector binding to the CAR receptor (Figure 6 and Figure7).Instillation of perflubron, an inert fluorocarbon, resulted ina redistribution of expression from the upper to theperipheral airways and is most likely due to flushing of thevector solution further down the airways by the waterimmiscible perflubron (Weiss et al, 1999b). These resultsshow proof of principle for the relatively safe andminimally invasive in utero delivery of a gene therapyvector to the fetal airways that resulted in levels oftransgene expression in the airway epithelia that may berelevant to a therapeutic application in cystic fibrosis genetherapy.G. Targeting the fetal gutIntrapharyngeal delivery has been attempted once infetal rabbits at laparotomy to target the fetalgastrointestinal system as a model for the treatment ofmeconium ileus due to cystic fibrosis (Wu et al, 1999).Gene transfer to the small bowel enterocytes was achievedbut there was significant maternal and fetal loss related toanaesthesia and the invasive surgery used. Ultrasoundguidedinjection of barium into the fetal stomach of rabbitshas been performed successfully (Brandt et al, 1997) andthis technique could be extended to deliver gene to thefetal gut. Gene delivery to the gut of fetal mice has beenobserved after intra-amniotic vector application and wasmost likely a result of fetal swallowing (Douar et al,1997).H. Delivery to the placentaTargeting the placenta could be used in the treatmentof placental disorders such as pre-eclampsia or intrauterinegrowth restriction. Low level gene transfer to theplacenta has been achieved using angiographically guidedinjection of non-viral vectors into the uterine artery(Heikkilä et al, 2001). The intraplacental route has beenattempted in mice, rats, guinea pigs and rabbits. Somaticgene transfer to the fetal heart and liver was achieved insome studies using mice (Woo et al, 1997; Türkay et al,1999), but others have found little or no fetal gene transferin mice and guinea pigs (Senoo et al, 2000) or rats (Xinget al, 2000). Commonly, the placenta showed the mosttransfection, but maternal tissues also demonstratedtransgene expression, which although not unexpected, isundesirable in therapy aimed at the fetus.Figure 5: (A) Ultrasonogram and (B) diagram of sheep fetus at114 days of gestation in longitudinal section. A 20 Gauge spinalneedle is inserted into the fetal thorax between the 3rd and 4thrib, penetrates the lung parenchyma and enters the fetal tracheajust proximal to the carina (David et al 2003b). Republished withpermission from S Karger AG, Basel.V. Development of the fetal immunesystemA major restriction in adult gene therapy is theimmune response to vector and/or transgene. In uteroapplication, on the other hand, aims to circumvent this bytreatment before maturity of the functional immune systemand this depends critically on the time at which fetaltolerance might be induced. The human immune systemdevelops progressively through the first trimester and isnot fully functional until 1-2 years after birth (Riley,1998). Lymphoid cells appear first in the fetal liver from 8weeks of gestation, with B lymphocytes and natural killercells predominating over T cells (Pahal et al, 2000). Tlymphocytes increase in number in the fetal liver andcirculation from 12 weeks of gestation.197
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