GTMB 7 - Gene Therapy & Molecular Biology

Gene Therapy and Molecular Biology Vol 7, page 195Immunohistochemistry for β-galactosidase showed strongstaining of the hindlimb musculature and occasionalpositively stained hepatocytes after injection of adenoviruslacZ vector. PCR analysis of vector presence in fetaltissues confirmed that broad haematogenic spread ofvector had occurred (David et al, 2003a).Figure 4A-C. Expression of β-galactosidase byimmunohistochemistry 2 days after intraperitoneal or intraamnioticdelivery of an adenoviral vector containing the β-galactosidase gene to early gestation fetal sheep. Originalmagnifications are as indicated. Intraperitoneal injection at 52days of gestation, positive staining is seen in (A) fetal smallbowel serosa, (B) surface of umbilical cord and (C) fetalsubcapsular hepatocytes.F. Targeting the fetal airways1. Intra-amniotic injectionIntra-amniotic application has been investigatedextensively in small animal models. Adenoviral vectorsexpressing the lacZ gene have been delivered to the fetalrat (Sekhon and Larson, 1995), mouse (Holzinger et al,1995; Sekhon and Larson, 1995; Douar et al, 1997; Larsonet al, 1997; Larson et al, 2000a; Mitchell et al, 2000) andguinea pig (Senoo et al, 2000) while adeno-associatedviral vectors have been applied to the fetal mouse(Mitchell et al, 2000). In general, transgene expression ismaximal in those tissues in contact with the amniotic fluid,namely the amniotic membranes and the fetal skin withless transduction of the gut and the mucosae. Indeed,therapeutic plasma concentrations of hFIX were achievedin fetal mice after intra-amniotic injection of adenoviralvectors carrying the hFIX gene (Schneider et al, 1999) andthe transgenic protein remained detectable after birth.Intra-amniotic delivery of retroviral producer cells to thefetal mouse resulted in only low level transduction of theamniotic membranes and fetal skin and no airways or guttransduction (Douar et al, 1997).In larger animals such as the fetal sheep, ultrasoundguided intra-amniotic injection of an amphotropicretroviral producer cell line encoding the lacZ generesulted in inefficient tissue transduction (Galan et al,2002). Amniotic fluid was found to have an inhibitoryeffect on retroviral mediated tissue transduction, and thiseffect increased as gestational age progressed (Bennett etal, 2001). Better results have been obtained withadenoviral vectors. Low level transgene expression wasseen in the fetal oesophagus and trachea after injection ofadenoviral lacZ vectors at laparotomy in late gestationfetal sheep (Holzinger et al, 1995). Attempts to deliveradenoviral vectors into the amniotic cavity of fetal sheepusing catheters placed at laparotomy had high mortality(Iwamoto et al, 1999). Ultrasound-guided intra-amnioticdelivery of adenoviral vectors containing the lacZ or hFIXgenes has been achieved in the early gestation fetal sheep(33 - 39 days of gestation, term = 145 days) equivalent to8 – 10 weeks gestation in humans with 86% fetal survival(David et al, 2003a). Therapeutic plasma concentrations ofhFIX were detectable up to 11 days after injection (Figure3) and immunohistochemical analysis showed positiveexpression of β-galactosidase in the fetal skin and nasalcavities (Figure 4 D-F). This suggests that transduction ofkeratinocytes in utero may be able to deliver proteins tothe circulation as well as to treat hereditary skin diseasesuch as epidermolysis bullosa.Gene transfer to the fetal airways is important for inutero treatment of cystic fibrosis.However, no significant airway or gastrointestinaltissue transduction was seen after ultrasound-guided intra-195