Background/purpose: Advancements in gene transfer technology and prenatal diagnosis have allowed investigators to consider an in utero gene therapy approach for fatal genetic diseases. The authors sought to develop fetoscopic techniques for gene delivery and investigate the efficacy and safety of recombinant adenoviral vectors in the fetus.
Methods: Fetal sheep between 60 and 130 days' gestation (dGA) underwent either fetoscopic intratracheal injection or umbilical vein (UV) injection of recombinant adenovirus, AdCMVlacZ. At death, fetal organs were examined for beta-galactosidase expression, histopathology, and CD45 immunostaining. Fetal serum was compared with preimmune serum for transaminase levels and the presence of antiadenoviral neutralizing antibodies.
Results: Fetoscopic intratracheal delivery of AdCMVlacZ in late-gestation sheep fetuses resulted in efficient alveolar gene transfer, but, antiadenoviral immunologic reactions limited the longevity of transgene expression to 14 days. This prompted an examination of whether early gestational exposure could induce tolerance in the fetus to adenoviral and transgene antigens. AdCMVlacZ (1 x 10(11) particles) was injected via UV into fetuses at 60 dGA. Within 3 days, beta-galactosidase expression was localized to the fetal liver, adrenal glands, kidneys, and endocardium. Although adrenal expression was nearly constant over 28 days, expression in fetal liver disappeared within 14 to 28 days. Loss of hepatic expression did not appear to be immune mediated because there was no evidence of hepatic inflammation or appearance of antiadenoviral neutralizing antibodies. Fetuses injected with AdCMVlacZ at 60 dGA were reinjected with 1 x 10(13) particles at 125 dGA and antiadenoviral humoral immune responses were recorded. Despite early-gestation adenovirus injection, fetuses still responded to the late-gestation adenoviral exposure, developing antiadenoviral neutralizing antibodies similar to control fetuses.
Conclusions: The authors developed fetoscopic access for pulmonary adenovirus delivery in late-gestation sheep. Although initial alveolar transduction was highly efficient, antiadenoviral immune responses limited the duration of transgene expression. In contrast, early-gestation adenoviral delivery did not elicit antiadenoviral immune responses despite achieving efficient transduction of many fetal tissues. Furthermore, early-gestation adenovirus delivery did not affect late-gestation antiadenoviral immune responses. These findings suggest that the early-gestation sheep fetus is not amenable to adenoviral tolerance induction by UV injection and that it is incompetent of immunologic response to adenovirus. For the purposes of in utero gene therapy, recombinant adenovirus may be applied optimally to genetic diseases requiring transient in utero expression.