GTMB 7 - Gene Therapy & Molecular Biology

David et al: Current status and future direction of fetal gene therapycould prolong the pregnancy until fetal maturity wasattained and reduce the likelihood of long-termcomplications in the mother and fetus.Intrauterine growth restriction (IUGR) affects up to8% of all pregnancies. It commonly occurs in pregnanciescomplicated by pre-eclampsia but can also arise innormotensive pregnancy. As well as leading to neonatalproblems, the long-term consequences are serious sinceIUGR infants exhibit higher rates of coronary heartdisease, type 2-diabetes, hypertension and stroke as adults(Barker et al, 1993). Abnormalities in placentaldevelopment are believed to adversely affect placentalfunction and deprive the fetus of the nutrients required foroptimal growth. Transport of amino acids and essentialfatty acids across the placenta is altered in IUGR fetusesand impaired oxygenation and acid base balance may beseen in severe cases (Pardi et al, 2002). Prenatal genetherapy could target placental transport mechanisms andincrease the availability of essential nutrients to the fetus.III. Vectors for in utero gene deliveryThe development of efficient vector systems iscrucial for the success of gene therapy. The ideal vectorfor fetal somatic gene therapy would introduce atranscriptionally regulated therapeutic gene into all organsrelevant to the genetic disorder by a single safeapplication. Although none of the present vector systemsmeet all these criteria, many of them have characteristicsthat may be beneficial to the fetal approach.A. Non-viral vectorsCationic liposome/DNA complexes have theadvantage of being relatively non-toxic and nonimmunogenicbut are still very inefficient in vivo. Anotherdrawback with these vehicles is that the DNA introducedas plasmid molecules remains episomal and will be lostover time following cell division. This is a particulardisadvantage in the fetus where cell populations arerapidly dividing. However, short term transgeneexpression has been shown to be a promising approach tomaintain a patent ductus arteriosus prior to surgery forcongenital heart defects in neonates (Mason et al, 1999).Liposomes containing plasmid expressing a decoy RNAdesigned to sequester fibronectin mRNA binding proteinwere delivered to the ductus arteriorus in fetal sheep at 90days of gestation, prior to the onset of intimal cushionformation at 100 days of gestation. Fibronectin synthesiswas inhibited resulting in a 60% reduction in intimalthickness and increased ductal patency at term.More recently, non-viral systems have beendeveloped that integrate into the host genome and couldthus in principle provide long term gene expression, butthese vectors are still at an early stage of experimentaldesign (Olivares et al, 2002).B. Viral vectorsStudies of in utero gene therapy have thereforeconcentrated on viral vectors, many of which have beendesigned to deliver reporter genes such as the β-galactosidase gene (lacZ). These allow tracking of thetransduced cells and to define tissue expression bybiochemical staining assays. Alternatively, use of vectorscarrying therapeutic genes allows the assessment ofpotentially curative levels of the expressed protein and, inanimal models of disease, even the observation ofphenotype correction. The hFIX gene for instance, can beused both as a marker gene, allowing the analysis of bloodlevels of the hFIX protein over time in non-haemophiliacanimals, and to study the correction of the blood clottingparameters in animal models of haemophilia. Postnatalreadministration of hFIX protein or the hFIX vector tofetally treated animals can be used to examine whetherimmune tolerance has been achieved.1. RetrovirusVectors that are able to integrate into the hostgenome such as retroviruses, lentiviruses and to a lesserextent adeno-associated viruses, may offer the possibilityof permanent gene delivery. Although only fairly lowvirus titres can be produced, virus gene transfer may beimproved by complexing vectors with cationic agents,(Themis et al, 1998) or by the administration of retrovirusproducer cells in vivo to allow localised gene deliveryclose to the site of cell transfer (Douar et al, 1997; Russelet al, 1995).Retroviruses require dividing cells for gene transfer(Miller DG et al, 1990) which suggests that they may bebetter suited for use in fetal tissues where cells are rapidlydividing rather than in adult applications. Other problemsinclude reports of premature promoter shutdown (Palmeret al, 1991; Challita and Kohn 1994) leading totranscriptional shutoff. Human serum can almostcompletely inactivate some retroviral particles (Welsh etal, 1975) which limits their use in vivo although increasedresistance to serum inactivation can be achieved bygenerating retroviruses from particular human packagingcells (Cosset et al, 1995) or by pseudotyping, whichreplaces the natural envelope of the retrovirus with aheterologous envelope (Engelstädter et al, 2001). Aparticular problem with in utero application is thatamniotic fluid has also been shown in vitro to have a mildinhibitory effect on retrovirus infection (Douar et al,1996). A further difficulty is the relatively short half-lifeof the retroviral particles in vivo which may hindertransduction because fetal cell division is nonsynchronizedand only those cells undergoing cell divisionat the time of infection will become transduced.Retroviruses were used in the first successful genetherapy trial, where bone marrow stem cells transduced exvivo with retroviral vectors expressing the correct cDNAwere delivered to infants suffering from an X-linked formof severe combined immunodeficiency (SCID)(Cavazzana-Calvo et al, 2000). The infants were able toleave protective isolation, discontinue treatment andappear to be developing normally (Hacein-Bey-Abina etal, 2002). However two of the fifteen patients treated forX-linked SCID have developed leukemia which has beenshown to involve insertional mutagenesis. An expandedclonal population of T-cells was demonstrated to be188