In utero gene transfer into fetal sheep: A large animal model for in utero gene therapy

We investigated the feasibility of long-term gene transfer/expression in vivo by direct injection of retroviral vectors into pre-immune fetal sheep. Sixteen pre-immune sheep fetuses were injected intraperitoneally with the GlnBgSvNa8.1 retroviral vector supernatant (titre: 1 x 107 cfu/m1). Over the four-year time course of these studies, the presence and expression of the NeoR and LacZ genes were demonstrated in 12 of the 14 animals evaluated by several immunological and biochemical methods ranging between 1.7--9.0% in the peripheral blood. Vector distribution was widespread without any detectable pathology. Humoral and cellular immune responses against viral-encoded protein were absent in the experimental animals suggesting that the in utero transduced sheep are tolerized to the foreign protein. Importantly, PCR analyses and breeding experiments demonstrated that the germ line was not altered.;We also used the in utero human/sheep transplantation model to determine whether retroviral-mediated transduction of long-term engrafting human hematopoietic stem cells (LTE-HSC) can occur. In experiment 1, human bone marrow (BM) CD34+ cells transduced ex vivo (GINaSvAd.24) were transplanted into fifteen 1° preimmune fetal sheep and examined for evidence of human cell and transgene activity at intervals post-transplant. Human cell activity was present until 18 months post-transplant and was transferable into 2° recipients. However, human progenitors exhibiting neomycin phosphotransferase (NPT) were detected only in 1° animals until 6 months posttransplant and were not present in 2° recipients at any time. Thus the ex vivo procedure did not result in the transduction of LTE-HSC. In experiment II, 11 preimmune sheep fetuses were transplanted with BM CD34+ cells on day 56 of gestation. On day 80 of gestation, each fetus was injected with retroviral vectors (GlNaSvAd.24, 4ml/fetus). Of the three animals that were evaluated for one year, high frequency of transduced human progenitors (21%) was detected. Human CD45+ cells were isolated from BM of 1° recipients at 1 year post-transplanted and transplanted into 2° fetal sheep. Evaluation of human BM from 2° recipients at six months post-transplant revealed the presence of NPT in human progenitors (11%). These studies confirmed that direct injection of an engineered retrovirus is a feasible means of safely delivering foreign genes into a developing fetus and thus achieving long-term expression of the transgenes within the recipients' hematopoietic stem cells.