Successful expression of β-galactosidase and factor IX transgenes in fetal and neonatal sheep after ultrasound-guided percutaneous adenovirus vector administration into the umbilical vein

Abstract

In utero somatic gene therapy in the later stages of pregmaternal liver. Expression of the ␤-galactosidase transnancy may allow targeting of organ systems which are difgene was detected in many fetal tissues by RT-PCR. High ficult to reach later in life and to prevent the development ␤-galactosidase production was shown by immuno-histoof tissue damage otherwise caused by the early onset of chemistry predominantly in the liver, where about 30% of inherited diseases. We report here on the percutaneous the hepatocytes stained positive, and in the adrenal cortex. delivery of two adenoviral vectors, containing the ␤-Production of factor IX was determined by ELISA in the galactosidase reporter gene and the human Factor IX gene plasma of treated fetuses and newborn lambs and reached respectively, to the fetal liver and circulation by ultrasoundat birth up to 80% of the normal human plasma concenguided umbilical vein puncture similar to procedures used tration. This demonstrates a very hopeful proof of principle in human pregnancy. Vector spread, as detected by PCR for the development of prenatal treatment of many genetic analysis for the ␤-galactosidase encoding vector, was diseases but also requires more detailed investigations found in almost all fetal and neonatal organs and in the with respect to the observed systemic spread of the vector.

Possible mechanism of gene transfer into early to mid-gestational mouse fetuses by tail vein injection

“…12 Ultrasoundguided transcutaneous intra-umbilical injection has been applied to gene transfer to the circulation of fetal sheep. 13 Unfortunately, with these methods, gene delivery is laborious since each embryo has to be treated individually, or the abdomen of each pregnant female requires incision. Additionally, transduction is found in very limited tissues, such as the regions peripheral to the site of injection in the case of direct injection into the embryo, or surface tissues in the case of intra-amniotic injection.…”

“…They include intrauterine injection into the fetus, 7 placenta, [8][9][10] amniotic cavity, 1,11 yolk sac vessel 12 and umbilical cord. 13 For example, efficient in utero gene delivery to the circulation of mid-gestational murine fetuses has been achieved by direct injection into the yolk sac vessel. 12 Ultrasoundguided transcutaneous intra-umbilical injection has been applied to gene transfer to the circulation of fetal sheep.…”

Long-term transgene expression by administration of a lentivirus-based vector to the fetal circulation of immuno-competent mice

“…28 Since the efficacy of vector delivery to the fetal circulation cannot be evaluated in murine models via this procedure, alternative injection routes directly into the liver 6 and placenta 29 have been investigated. Liver injection results in limited gene delivery to other tissues 30 and placental injection carries the disadvantage of vector loss within the placenta as well as the risk of vector spread into the maternal circulation.…”

On the scientific and ethical issues of fetal somatic gene therapy

“…[7][8][9] In the sheep fetus we are using minimally invasive ultrasound-guided procedures, which are applied in human fetal medicine, to deliver the vector to several sites. We have successfully accessed the fetal circulation via the umbilical vein on day 102 of gestation (0.7), 10 while attempts at early gestation (0.3) were not successful. Successful i.p., i.a.…”

“…With regards to expression, we have achieved therapeutic levels of human factor IX by adenovirus-mediated gene delivery to the yolk sack vessels (Waddington et al, submitted) and the amniotic cavity and musculature in the mouse fetus 8,9 and by intra-umbilical injection in the late gestation of sheep, 10 although only for the relatively short period of about 3 weeks. Significantly lower levels were observed in the early gestation of sheep by i.p., i.a.…”