GTMB 7 - Gene Therapy & Molecular Biology

Gene Therapy and Molecular Biology Vol 7, page 193Amniocentesis is mainly used clinically for prenataldiagnosis. Although it is one of the safest intrauterineprocedures, intra-amniotic application of vectors may beonly of limited use in fetal gene therapy because of vectordilution by the large volume of amniotic fluid, although itwould be the ideal application route for in utero genetherapy of skin diseases.Accessing the systemic circulation has greaterpotential. In fetal medicine, fetal blood can be obtained inthe second trimester under ultrasound guidance either fromthe placental cord insertion, the fetal heart or more safelyfrom the intrahepatic umbilical vein (Chinnaiya et al,1998). The procedure has a good success rate clinically, islow risk and is used commonly for rapid karyotyping orfetal blood transfusion (Nicolini et al, 1990). From 12weeks of gestation ultrasound-guided intracardiacpuncture for fetal blood sampling has been performed onpatients undergoing surgical termination of pregnancy(Jauniaux et al, 1999). Similarly, radiolabelled fetal livercells were successfully injected into the heart of 13 weekold fetuses under ultrasound guidance (Westgren et al,1997) prior to prostaglandin termination of pregnancy. Nofetal heart rate abnormalities were detected and all fetuseswere alive at least 6 hours after the procedure.Intraperitoneal injection has been applied for in utero stemcell transplantation in humans from 14 weeks of gestation(Touraine 1999; Muench et al, 2001) and is an alternativeroute for blood transfusion before 18 weeks of gestation(Rodeck and Deans 1999). Ultrasound guidedintramuscular injection has been used to delivercorticosteroid therapy for maturation of preterm infantlungs and vitamin K to the fetus (Larsen et al, 1978;Ljubic et al, 1999).C. Direct targeting of the fetal circulationDelivery of vectors to the systemic fetal circulationappears to be a highly effective route for targeting genetherapy to a range of fetal tissues and particularly to theliver for treatment of diseases such as the haemophiliasand the metabolic and storage disorders. This can beaccomplished in small animals such as the mouse byintracardiac injection (Christensen et al, 2000; Wang et al,1998) or by injection into the yolk sac vessels (Schachtneret al, 1996). Indeed, yolk sac vessel injection of adenoviralvectors containing the hFIX gene into fetal mice resultedin therapeutic levels of hFIX expression (Waddington etal, 2002). Long-term transgene expression was observed inthe liver, heart, brain and muscle up to a year afterdelivery of lentiviral vectors containing the β-galactosidase gene into yolk sac vessels of fetal mice(Waddington et al, 2003) and was then used to achievecorrection of the haemophilic phenotype in factor IXdeffcient mice (Waddington, submitted).In larger animals such as in the sheep, intravasculardelivery can be achieved by injection via the umbilicalvein (Yang et al, 1999). Adenoviral vectors containing thelacZ or hFIX genes were delivered into the umbilical veinof late gestation fetal sheep using ultrasound-guidedpercutaneous injection from 102 days gestation (term =145 days) (Themis et al, 1999). Positive lacZ expressionwas seen in about 30% of fetal hepatocytes, and hFIXexpression in fetal and neonatal plasma by ELISA analysisreached therapeutic levels within a week of delivery in twoanimals.In early gestation, delivery of adenoviral vectors intothe umbilical vein of fetal sheep at 60 days of gestation viahysterotomy resulted in widespread transduction of fetaltissues (Yang et al, 1999). Our group has attemptedultrasound-guided umbilical vein injection of adenoviralvectors in fetal sheep at the earlier time of 53 days ofgestation but this was unsuccessful due to procedurerelatedmortality (David et al, 2003a).Ultrasound-guided intracardiac injection has beenused to deliver adenoviral vectors to the late gestation fetalrabbit (Wang et al, 1998). Transgene expression wasobserved in up to 40% of fetal hepatocytes and wastransient as expected. A fetal immune response to thevector and transgene was detected. Unfortunately theprocedure also had a 25-40% mortality rate, comparable toother studies on fetal blood sampling in rabbits (Moise etal, 1992). Although technically straightforward,ultrasound-guided intracardiac delivery of adenoviralvectors to fetal sheep in early gestation resulted in 100%mortality due to haemorrhage (David et al, 2003a).D. Alternative routes for targeting thefetal circulation and liverDue to the peculiarities of the fetal anatomy, vectordelivery via the umbilical vein or yolk sac vessels willpreferentially target the liver, which is an important organfor treatment of many genetic diseases. However in earlypregnancy this not been technically possible andalternative approaches to reach the liver and thecirculation have been tried.1. Intrahepatic injectionFetal intrahepatic injection has been performed inmice using adenoviral vectors (Lipshutz et al, 1999a, b,2000; Mitchell et al, 2000), adeno-associated vectors(Mitchell et al, 2000; Sabatino et al, 2002) and lentiviralvectors (MacKenzie et al, 2002). In these studies, highlevels of transgene expression in fetal hepatocytes wereobserved as well as gene transfer to other organs such asthe heart, spleen, lung, intestine and brain suggestinghaematogenic spread.Ultrasound guided intrahepatic injection has beenperformed in a few large animal models. In the lategestation fetal rabbit, X-gal staining of the fetalhepatocytes was seen 2 days after ultrasound guidedintrahepatic injection of adenoviral vectors containing theβ-galactosidase gene in late-gestation fetal rabbits(Baumgartner et al, 1999). Similarly, strong expression oftransgenic enhanced green fluorescent protein wasobserved in hepatocytes one month after ultrasoundguidedintrahepatic delivery of adeno-associated viralvectors to the late-gestation rhesus monkey (Lai et al,2002). Ultrasound guided intrahepatic injection in earlygestation sheep fetuses has also been performed with fetalsurvival rates of 81% (David et al, 2003a). Only low level193