Inefficient transduction of sheep in utero after intra-amniotic injection of retroviral producer cells

OBJECTIVE: Our purpose was to test the hypothesis that the intra-amniotic injection of a retroviral vector producer cell line into pregnant sheep will result in retrovirus-mediated transduction and stable gene transfer to the ovine fetus. STUDY DESIGN: Thirteen pregnant ewes at various gestational ages underwent amniocentesis and injection of cells producing the retrovirus vector LIRESgeo, which is derived from Maloney murine leukemia virus and encodes Escherichia coli LacZ (beta-galactosidase) as a marker gene. Pregnant ewes and fetuses were killed, and amniotic fluid, placenta, and fetal tissues were collected and assayed for transgene expression 7 to 77 days after intraamniotic injection. In addition, serum was collected and analyzed for evidence of specific immune responses against the producer cells, and amniotic fluid was collected and analyzed for deleterious effects on producer cell viability, vector production, and vector transduction. RESULTS: Only 1 of 10 fetuses exposed to the retroviral producer cells demonstrated beta-galactosidase activity that correlated with positive immunohistochemistry for LacZ in lung, trachea, pancreas, and small intestine. However, the presence of the LacZ gene could not be confirmed by polymerase chain reaction. Thus, we could not confirm transduction after any of the injections. The retroviral producer cells survived well in amniotic fluid and continued to produce high levels of retroviral vector after intra-amniotic injection, although amniotic fluid inhibited the transducing activity of the vector in a manner dependent on gestational age. CONCLUSIONS: Intra-amniotic retroviral gene transfer with the use of these amphotropic producer cells does not result in reproducible gene transfer in the ovine fetus although amniotic fluid sustains producer cell viability and vector production. Possible reasons for the inefficient transduction are discussed.