GTMB 7 - Gene Therapy & Molecular Biology

David et al: Current status and future direction of fetal gene therapyA. Animal modelsSmall animals are the most commonly used becausethey offer a number of advantages. Transgenic mousemodels exist for many genetic diseases such as cysticfibrosis and haemophilia and this allows the therapeuticeffect of the gene therapy to be studied. Small animals arealso cheaper to maintain and have short breeding cycleswith large litters which permit studies over severalgenerations e.g. on germline transmission. However, theirsize precludes their use for the development of minimallyinvasive techniques for gene therapy delivery as requiredin human application.Studies in large animals have mainly used sheep, sincethey are well established as an animal model relevant tohuman fetal physiology, have a good tolerance to in uteromanipulations and a consistent gestation period of 145days, which is approximately half that of the human. Thereare some differences between ovine and human biology(Newnham and Kelly 1993). In late gestation the fetalgrowth rate in sheep is over double that in humans(Fowden, 1995) and the placental weight declines from 90days gestation while it remains static in the human(Barcroft and Barron 1946). However the major differenceis in the structure of the placenta. In sheep thesynepitheliochorial placenta consists of six tissue layers,three from the mother and three from the fetus, and it isthe most complete barrier possible (Benirschke andKaufmann 1990). The maternofetal interdigitations(placentomes) are spread throughout the uterine cavity andmay be difficult to avoid during ultrasound-guided uterineinterventions. In humans, there is only a single discoidplacenta and there is extensive invasion of theendometrium by the trophoblast that removes the threematernal tissue barriers and results in a hemomonochorialplacenta at term. Probably as a result of these structuraldifferences, γ-globulin does not pass from the mother tothe fetus in the sheep, but is able to cross the placenta inhumans.Nonhuman primates are close physiologically tohumans with menstrual cycles of similar length andhormonal control, comparable cellular and endocrineprocesses of implantation, and similar timetables ofprenatal development. The placental structure in somenonhuman primates is also the same, for example in therhesus monkey the placenta is hemomonochorial andbidiscoidal (Benirschke and Kaufmann 1990). For thisreason they are used as an animal model in studies ofteratology, developmental biology, infertility andcontraception (Hendrickx and Peterson 1997). Ultrasoundguided injection techniques as used in fetal medicine havealso been applied extensively in the fetal nonhumanprimate with comparable results (Tarantal, 1990).However nonhuman primates are more costly than sheepand are difficult to maintain.The rabbit has been studied in some prenatal genetherapy studies. Minimally-invasive percutaneousultrasound guided injection and fetoscopic procedures arealso being developed (Brandt et al, 1997; Papadopulos etal, 1999). Because of the small size of the fetus and litternumber however, technically this is only possible fromlate gestation. The guinea pig has the same placentalstructure as humans but they are not commonly used inprenatal gene therapy studies because of the small fetalsize and lack of transgenic models of disease.There are unfortunately few large animal models ofhuman genetic disease available for testing of genetherapy. Efforts to produce transgenic domestic animalsare continuing particularly in the pig, sheep and cow(Piedrahita 2000). There are however, some dog modelsincluding mucopolysaccharidosis type VII, Duchennemuscular dystrophy and haemophilia B, which are usefulfor investigating the therapeutic effect of gene therapy.The dog is also a suitable model for minimally invasivedelivery techniques and studies on prenatal gene transferhave used ultrasound guided intraperitoneal or yolk sacinjection through the exposed uterus (Lutzko et al, 1999;Meertens et al, 2002).B. Application routes in fetal medicineInvasive surgical techniques such as maternallaparotomy or hysterotomy must be performed to accessthe fetus in small animal models, but have also beenapplied in large animal studies such as in the sheep (Tranet al, 2000; Vincent et al, 1995). Surgery carries a highmorbidity from wound infection and haemorrhage and therisk of mortality is significant.Minimally invasive procedures with fibreoptictelescopes are currently in use in fetal medicine and arebeing adapted for application of gene therapy in largeanimal fetuses. Fetoscopy was developed in the late 1970sfor examination of 2 nd trimester fetuses and for fetal bloodsampling (Rodeck, 1980). The morbidity from fetoscopy issignificant however, because of the relatively largerdiameter of the puncture site in the fetal membranes whichleads to premature rupture of the membranes and pretermlabour and its associated problems. With the improvementin ultrasound technology in the 1990s, more detailedanatomical survey of the fetus could be performed andfetal blood sampling by ultrasound guided injectionbecame routine practice. Operative fetoscopy has recentlyre-emerged for use together with ultrasound in endoscopicfetal surgery for conditions such as twin reversed-arterialperfusionsequence (Quintero et al, 1994), severe feto-fetaltransfusion syndrome (Ville et al, 1997) and congenitaldiaphragmatic hernia (Harrison et al, 1998).Percutaneous ultrasound-guided injection is the leastinvasive technique for accessing the fetus and is usedfrequently in the clinical setting. Coelocentesis usesultrasound to guide a needle into the extraembryoniccoelom in the early first trimester. It has a success rate of>95% at 6-11 weeks of gestation, and has been suggestedas a possible technique for stem cell engraftment in earlygestation (Wilson and Wivel 1999). It may be of little use,however for in utero gene therapy because of the limitedtransfer from the extraembryonic coelom via the amnioticmembrane to the amniotic cavity (Jauniaux and Gulbis2000). Studies on the risk of miscarriage in ongoingpregnancies beyond the 1 st trimester followingcoelocentesis gave controversial results (Makrydimas et al,1997; Ross et al, 1997; Santolaya-Forgas et al, 1998).192