Fetal gene therapy may treat genetic diseases before significant organ damage, target stem cell populations and avoid immune sensitisation. Candidate diseases include cystic fibrosis, haemophilia and lysosomal storage disorders. This thesis developed ultrasound-guided delivery of viral vectors to the sheep fetus for treatment of these diseases. For haemophilia B treatment we delivered adenovirus vectors containing the p-galactosidase reporter gene (adlacZ) or the human factor IX gene (adhFIX) by ultrasound guidance to the early gestation sheep fetus, when it is considered to be pre-immune. Intraperitoneal injection allowed the earliest time point for gene delivery, achieved the highest hFIX levels and the most localised p-galactosidase expression. Therapeutic hFIX levels were detected after intramuscular and intra-amniotic delivery suggesting that these are potentially alternative sites for therapeutic gene expression. For each route examined, no humoral immune response was observed to the transgene, although antibodies to the adenovirus vector were identified. We achieved intravascular delivery via umbilical vein injection therapeutic hFIX levels were detected. We developed ultrasound-guided transthoracic injection of the mid-gestation fetal trachea for cystic fibrosis treatment, p-galactosidase expression, measured by ELISA, was low after delivery of adlacZ vector alone, but increased 10 fold when the vector was complexed with DEAE dextran. Pretreatment of the fetal airways with sodium caprate increased expression by 90 fold the effect of the two agents was synergistic. Perflubron instillation following vector injection redistributed transgene expression from the large to the small airways. We developed ultrasound-guided fetal intragastric injection and achieved widespread transgene expression throughout the gastrointestinal epithelia after adlacZ vector delivery. For brain manifestation of lysosomal storage disorders we injected adlacZ vectors to the fetal ventricles under ultrasound guidance. Transduction of the choroid plexus was seen. Future application of integrating vectors such as lentivirus may allow for long term therapeutic correction and induce immune tolerance to the transgene
