GTMB 7 - Gene Therapy & Molecular Biology

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David et al: Current status and future direction of fetal gene therapyFigure 1. Upper panel. Representative sections of fetal livers harvested at 72h, 7, 14, 28, 79, 168 days and 1 year after yolk sacinjection of high titre titre EAIV SMART2Z (equine infectious anaemia virus vector expressing the β-galactosidase gene driven by theCMV promoter) lentiviral vector (n=1, 1, 3, 1 and 1, respectively). Uniform hepatocyte staining is observed after 72 h followed by theemergence of clusters of β-galactosidase-stained hepatocytes to day 79. Macroscopic appearance of liver sections (top row, x 10).Microscopic analyses (bottom row, x 400). Age matched noninfected control livers of 3 day old and 1-year-old animals are shown in thelower panel. Lower panel. Representative sections of fetal tissues harvested at 72 h, 7, 14, 79 days and 1 year after yolk sac injection ofhigh titre EAIV SMART2Z lentiviral vector (n…1, 1, 3 and 1, respectively). High-level staining is observed after 72 h and 79 days inbrain, 7, 14 and 79 days in heart and 14 and 79 days in skeletal muscle. Low-level expression is shown in lung and kidney at 79 dayspostinjection. Macroscopic appearance of tissues (left columns, x 10). Microscopic analysis (right column, x 400). (Waddington et al2003). Republished with permission from Nature Publishing Group.190
Gene Therapy and Molecular Biology Vol 7, page 191Because adenoviruses provide highly efficient genetransfer yet transient expression, novel hybrid vectors havebeen developed to take advantage of adenovirus infectivityand the permanent nature of integrative vectors such asretroviruses and lentiviruses (Murphy et al, 2002; Kuboand Mitani, 2003). Hybrid vectors may offer efficient geneexpression to fetal organs such as the lung in which it hasso far proved difficult to achieve high level gene transferwith integrating vectors.5. Sendai virusRecently, the negative strand RNA cytoplasmicallyreplicating Sendai virus, a member of the paramyxovirusfamily was developed as a gene transfer vector. Earlyvectors still capable of self-propagation, were found toprovide very high levels of marker gene expression in awide range of tissues including bronchial epithelium(Yonemitsu et al, 2000), skeletal muscle (Shiotani et al,2001) and vascular endothelium (Masaki et al, 2001).Second generation vectors, although still capable of intracytoplasmicreplication of the RNA genome, are incapableof intercellular propagation. In these vectors, genesencoding surface glycoproteins including thehaemaglutinin-neuraminidase (HN) protein or the fusion(F) protein, which are responsible for cell binding andinfection, have been deleted from the viral genome (Inoueet al, 2003). Injection of F-deficient Sendai virus vectorinto the fetal mouse via various routes including intravascular,intra-amniotic, intra-muscular, intra-peritonealand intra-spinal resulted in expression of marker gene ingut wall, lung, muscle, peritoneal mesothelia and dorsalroute ganglia respectively. Further optimisation will beneeded to develop these first generation constructs intoclinically applicable vectors (Waddington et al,submitted).IV. Fetal gene therapy studiesSince the initial attempts in the early 1990s, in uterogene therapy has been investigated in a range of differentanimals using a variety of techniques. The possible routesof administration are illustrated in Figure 2.Figure 2. Routes of administration of gene therapy to the fetus. Routes in italics have not yet been applied in a fetal animal model usingultrasound guided injection.191
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