GTMB 7 - Gene Therapy & Molecular Biology

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David et al: Current status and future direction of fetal gene therapyFigure 6: Na-caprate stimulation of DEAE dextran complexed adenovirus mediated airway transduction. Panel 1: Examples of stainingin the peripheral lungs after virus alone (a) and DEAE complexed virus (b) and of the trachea after Na-caprate pre-treatment anduncomplexed virus administration (c) in fetal sheep injected between 102 and 109 days of gestation. Panel 2a: Na-caprate pre-treatmentfollowed by DEAE dextran complexed virus in a 108 day sheep. Widespread gene expression was seen in the small (a), medium (b) andlarge (c) airways and also the main bronchi (d) and trachea (e). Panel 2b: Similar results were observed in a fetus injected at 81 days ofgestation. Expression was seen in the airways (a & b) and trachea (c) Panel 3: Na-caprate pre-treatment followed by DEAE dextrancomplexed virus followed by perflubron. Staining of the peripheral airways in transverse sections (a & b) and longitudinal sectionshowing gene expression was limited to the terminal branches of the bronchial tree (c). Some staining of the bronchioles (d) and trachea(e) was also observed, although less than in the absence of perflubron. Scale bar = 5mm in all cases. (Peebles et al 2003).198
Gene Therapy and Molecular Biology Vol 7, page 199Although they are not capable of producing a definitivecytotoxic response until 18 weeks of gestation (Mackenzieand Maclean, 1980) natural killer cells and some T celllines may provide a limited immune response earlier ingestation (Miyagawa et al, 1992; Phillips et al, 1992). Thefetal lamb is able to produce detectable circulatingantibodies in response to some antigenic stimuli from 66days of gestation (Silverstein et al, 1963) and to reject skingrafts after 77 days of gestation (Silverstein et al, 1964).This would suggest a 'window of opportunity' in the firstthird to half of pregnancy during which time introductionof foreign genetic material may not produce an immuneresponse. No humoral immune response to the transgenewas observed in early gestation fetal sheep, althoughantibodies to the adenoviral vector were detected for eachroute of injection (David et al, 2003a). Similarly, umbilicalvein injection of adenoviral vectors into fetal sheep at 60days of gestation via hysterotomy resulted in widespreadtransduction of fetal tissues with no humoral immuneresponse to the adenoviral vector (Yang et al, 1999).Expression of a foreign antigen during early fetaldevelopment may also result in its recognition as “self”where exposure of the fetus to foreign antigen ismaintained (Billingham et al, 1956; Binns, 1967) thusallowing development of tolerance. Evidence to supportinduced tolerance has been reported after in uterointraperitoneal delivery of retroviral vectors in fetal sheep(Tran et al, 2001).Induction of tolerance to transgene in adults althoughpossible, is expensive, therefore, prenatal induction oftolerance may provide an excellent alternative. Forexample, a single injection of adenovirus expressing thefactor IX gene into the fetal mouse was shown to providelong term, albeit diminishing expression over five months.Furthermore, 56% of these adult mice remained tolerant torepeated challenges with hFIX protein (Figure 8). Incontrast, a group of mice which received adenovirus forthe first time as adults developed high levels of anti-hFIXantibodies (Waddington et al, 2002). This provides proofof principle that gene therapy applicaton in utero mayallow induction of immune tolerance.However the paradigm of self/non-self immunetolerance and sensitisation has been recently challenged bythe hypothesis of Matzinger (2002). This suggests thatimmunity arises as a consequence of cellular alarm signalsfrom distressed or injured cells stimulating antigenpresenting cells. A recent study examined the idea that thefetus is particularly susceptible to induction of tolerance;the study concluded that, rather than being due toignorance, timing-based tolerance or properties of naïve Tcells in early life, tolerance induction in fetus may arisefrom differences in fetal antigen presentation; this remainsto be identified (Anderson, et al, 2001).VI. Ethical and safety issuesThere are various ethical issues in relation to in uterogene therapy that need to be addressed before such therapycould be applied clinically (Fletcher and Richter, 1996;Recombinant DNA Advisory Committee 2000). Onemajor concern is that fetal gene therapy has potentialadverse effects such as injury, infection, severe immunereactions or preterm labour on the fetus as well as on themother. Furthermore, many parents decide to terminate anaffected pregnancy, and therefore the option of in uterotreatment must be at least as safe for the mother, andshould also reliably treat the disease (Coutelle andRodeck, 2002).There is a theoretical risk that the therapeutic geneproduct or vector that is required at a certain stage duringfetal development could cause oncogenesis. In addition,insertion of vector sequences may cause developmentalaberrations to occur.While one of the aims of prenatal gene therapy is toachieve immune tolerance to the transgene and deliverysystem, vectors must be designed to be sufficientlydifferent to the wild type so that the immune systemremains able to mount an effective immune responseagainst wild-type virus infection.The problem of insertional mutagenesis as apotential risk of retroviral gene therapy has been debatedfor some years. This serious adverse event has now beenidentified in a trial of gene therapy for X-linked severecombined immunodeficiency syndrome in which CD34 +haemopoietic stem cells were transduced ex vivo with theγc gene using retroviral vectors. Two patients out offifteen treated developed acute lymphoblastic leukemia(ALL) three years after successful gene therapy treatment.Analysis of the lymphocytes showed that the transgenehad been inserted adjacent to an oncogene, LMO2, theproduct of which has been implicated in the pathogenesisof ALL (Juengst, 2003). Further work is needed to addressthis issue and to devise strategies to determine andpossibly direct integration sites.Germline transmission is another risk that raisesethical concerns. Fetal somatic gene therapy does not aimto modify the genetic content of the germ-line butinadvertent gene transfer to the germ-line could occur.Compartmentalisation of the primordial germ cells in thegonads is complete by 7 weeks of gestation in humans andit is unlikely therefore that any therapy applied after thistime would result in germ-line transduction. Examinationof germ cells after delivery of retroviral vectors (Porada etal, 1998; Tran et al, 2000) or adenoviral vectors to earlygestation fetal sheep has not shown any detectabletransmission (David et al, 2003a). Following intravascularadministration of adenoviral vectors to late gestation fetalsheep, vector DNA was detectable by PCR in the gonads,but extensive investigation by RT-PCR could not detectany gene expression. A similar risk of germlinetransduction occurs with AAV that can integrate into thegenome. No AAV sequences were detectable in thegermline tissues of fetal mice receiving injection of AAVvectors via the intraperitoneal route nor the tissues of theirprogeny (Lipshutz et al, 2001). Many of these issues arenot confined to in utero or even adult gene therapy andconcerns regarding germ-line transmission can be raised inparticular for chemotherapy and infertility treatment(Schneider and Coutelle 1999).Finally there is the concernthat fetal gene therapy research poses special challenges toinformed consent (Burger and Wilfond 2000).199
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