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Once its full clinical potential has been realized, hematopoietic stem cell based gene therapy (GT) promises to cure a wide array of both inborn and acquired diseases. For many genetic disorders, early onset and irreparable tissue and organ damage necessitate innovative methods that allow therapeutic intervention early in development, if a full cure is to be realized. Performing GT in utero would allow early correction prior to disease onset and is thus one of the few therapeutic modalities that could promise the birth of a healthy infant. Several features of the developing fetus may circumvent obstacles that have thus far been observed in GT trials. For example, the immune naïveté of the early gestational fetus may evade immune reactions to the vector and transgene product. Furthermore, fetal exposure to foreign antigens can result in sustained tolerance, suggesting that induction of tolerance to the vector/transgene product could allow postnatal treatment to be performed successfully. In addition to these immunologic advantages, the fetal hematopoietic system promises to be more amenable to retrovirus-mediated gene transfer than either the neonate or adult as a result of both proliferation and expansion of the stem/progenitor cell pool that take place during fetal development. To investigate whether these characteristics of the developing fetus could be used to advantage to efficiently transduce hematopoietic stem cells, we developed an approach to in utero GT, in which retroviral vectors were directly injected into the peritoneal cavity of preimmune fetal sheep. This approach resulted in the transfer and long-term (>5 years) expression of exogenous genes within the hematopoietic system of primary and secondary recipients, albeit at relatively low levels that would not likely be therapeutic in most diseases. These studies also demonstrated that the direct injection of retroviral vectors into preimmune fetal sheep results not only in the successful transduction of long-term engrafting hematopoietic stem cells, but also in the widespread distribution of vector to all other tissues examined, including the reproductive organs. In an effort to increase the hematopoietic cell transduction to clinically relevant levels, we repeated our initial studies with 1,000-fold higher titer vectors. This led to only a modest (two- to fourfold) increase in the transduction levels, suggesting that factors other than absolute vector dosage were responsible for the low levels of gene transfer. For this reason, we have more recently begun evaluating the effect of recipient gestational age on the efficiency of gene transfer to both hematopoietic and nonhematopoietic tissues. Thus far, we have observed an inverse relation between the gestational age at the time of vector administration and the level of transduction and expression of the transgene within the hematopoietic system, such that fetuses injected earlier in gestation have higher levels of hematopoietic cell transduction. These elevated levels have persisted for at least 1 year after injection, suggesting that the enhancement is at the level of primitive stem/progenitor cells. When analyzing the liver sections from animals that had received the vector at different gestational ages, we also observed an inverse correlation between recipient age and efficiency of gene transfer to the hepatocytes, such that a high efficiency of gene transfer occurred at early ages, while very little occurred at later stages of gestation. In contrast to the findings in the hematopoietic system and in the liver, analysis of the lungs of these same animals revealed that the efficiency of transduction of nonhematopoietic lung tissue increased with increasing gestational age. These results demonstrate that both hematopoietic cells and nonhematopoietic cells within liver and lung are transduced following direct injection of murine retroviral vector supernatants into the peritoneal cavity of preimmune fetal sheep and suggest that the developmental stage of each organ at the time of injection may determine its etermine its susceptibility to in utero gene transfer.