GTMB 7 - Gene Therapy & Molecular Biology

Gene Therapy and Molecular Biology Vol 7, page 187returned in the form of a skin graft (Uitto and Pulkkinen,2000). Preliminary studies show keratinocytes andfibroblasts from patients with DEB can be successfullytransduced using lentiviral vectors containing theCOL7A1 transgene in vitro resulting in long-termexpression and synthesis of type VII collagen (Chen et al,2002). In a canine animal model of DEB, transduction ofkeratinocytes with a retrovirus containing the collagentype VII cDNA corrected the observable defects in in vitroreconstructed skin (Baldeschi et al, 2003). A non-viralgene transfer approach has been used for junctionalepidermolysis bullosa (JEB) in which there is severelaminin-5 deficiency. Integration of an attB-containinglaminin 5 β3 expression plasmid using φC31 integrase intohuman keratinocytes from JEB patients produced skintissue with no histological evidence of subepidermalblistering when regenerated on SCID mice (Ortiz-Urda etal, 2003).Epidermolysis bullosa however, is a generalizeddisorder affecting the entire skin and the extracutaneoustissues. Prenatal therapy delivered into the amniotic fluidwould bathe the entire skin surface and reach thegastrointestinal system by fetal swallowing. Injection intothe amniotic cavity can be performed safely at relativelyearly gestation, but the timing of intra-amniotic deliverywill be important from developmental considerations.Even at 20 weeks gestation, the fetal epidermis isincompletely keratinized and this would aid gene tranfer.However there is a high rate of apotosis in fetalkeratinocytes and therefore the ideal strategy would be totarget stem cells (Haake and Cooklis, 1997). Prenataldiagnosis for epidermolysis bullosa can now be performedwith a 98% success rate in at risk families, paving the wayfor preliminary studies into prenatal treatment (Pfendner etal, 2003). Disorders of defective keratinisation such asharlequin ichthyosis, an autosomal recessive severe andusually fatal congenital ichthyosis (Akiyama, 1998), mayalso be amenable to prenatal gene transfer.I. Perinatal diseasePulmonary hypoplasia is another important cause ofneonatal morbidity and mortality. In this condition, thefetal lungs fail to develop resulting in respiratoryinsufficiency at birth. Current neonatal management issupportive and involves surfactant replacement, carefulmechanical ventilation avoiding barotrauma and treatmentof pulmonary hypertension. Pulmonary hypoplasia canoccur when there is reduced or no liquor surrounding thefetus (oligo or anhydramnios) prior to 22 weeks gestation,most commonly because of preterm premature rupture ofthe membranes (PPROM). Serial amnioinfusion has beenused for the prevention of pulmonary hypoplasia withsome success but has a high complication rate (Tan et al,2003). Space occupying lesions that compress the lungswithin the chest cavity also result in pulmonaryhypoplasia. Examples of such conditions include pleuraleffusion associated with congenital cardiac defects andcongenital diaphragmatic hernia (CDH) in which thebowel herniates through the diaphragmatic defect. Fetalinterventions such as drainage of pleural effusions can beused to treat the underlying cause of the pulmonaryhypoplasia. Temporary occlusion of the trachea with anexpandable balloon for treatment of CDH results inimpressive expansion of the hypoplastic lung with trachealfluid. However ‘plugging’ has yet to be shown to improveoutcome in the long term (Harrison et al, 1998). Studiessuggest that pulmonary hypoplasia in CDH begins duringembryogenesis as an abnormality in growth factorsignalling and actually precedes the development of theanatomical defect (Jesudason 2002). Prenatal gene therapycould be envisaged in the future to enhance antenatal lunggrowth and maturation by the targeted delivery of growthfactors at specific times during lung development.J. Infectious diseaseInfectious diseases with pathogens such as Group Bstreptococcus, human immunodeficiency virus, hepatitis Bvirus and herpes simplex virus are a major cause ofneonatal morbidity and mortality. Transmission of thesediseases from mother to infant often occurs shortly before,during, or after birth by early rupture of the amnioticmembranes or direct contact with infectious secretionsduring labor and delivery. Delivery by caesarean section toprevent such contact, and antibiotic and maternal antiviraltreatments have been used with some success, particularlyin the prevention of vertical HIV transmission.Immunisation of the fetus with DNA vaccines in latepregnancy has been proposed as an alternative approach toprevent neonatal infection (Gerdts et al, 2000; Sarzotti etal, 1996; Watts et al, 1999). The mucosal surfaces of theeyes, respiratory and gastrointestinal tract are the primarysite of entry for infectious agents during birth and theneonatal period. Thus intra-amniotic or intra-oral deliveryof antigen would probably provide the best diseaseprotection. Studies in the fetal mouse (Sarzotti et al, 1996),sheep (Gerdts, et al, 2000) and baboon (Watts et al, 1999)have shown that fetal immunisation can induce activeimmunity in the newborn. In particular, in the fetal sheep,intra-oral administration of hepatitis B surface antigenDNA resulted in a higher protective antibody titre than anintramuscular injection of the recombinant protein vaccine(Gerdts, et al, 2003). The timing of such an intervention iscrucial since exposure of the fetus to the antigen beforeimmune competence is reached may result in tolerance. Inaddition a single in utero injection may not be sufficient tomaintain immunity. At present there is no clinicalindication for such a prenatal immunization strategy.K. Placental disordersPre-eclampsia/eclampsia is one of the leading causesof maternal and fetal morbidity and mortality. Theunderlying defect is believed to be inadequate deepplacentation that fails to transform the spiral arteries intouteroplacental vessels and thus limits placental blood flow(Brosens et al, 2002). Secondary damage such as fibrindeposition and thrombosis then limit placental perfusionfurther and there is also widespread activation of thematernal vascular endothelium leading to decreasedformation of vasodilators such as nitric oxide (Walker,2000). Gene therapy could be used to improve uteroplacentalperfusion by for example, temporary expressionof nitric oxide synthase or placental growth factor. This187