Once its full clinical potential has been realized, hematopoietic stem cell (HSC)-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 naivete 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 retroviral-mediated gene transfer than either the neonate or adult as a result of both the 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 HSC, we developed an approach to in utero GT in which retroviral vectors were injected intraperitoneally into fetal sheep. This approach resulted in the transfer and long-term (>5 years) expression of exogenous genes at relatively low levels within the hematopoietic system of primary and secondary recipients. The overall goal of the proposed studies is to achieve therapeutic levels of gene transfer/expression in HSC following direct injection of retroviral vectors in utero. We will also evaluate safety issues that our previous studies suggested might arise from this approach, including vector distribution, fetal germline involvement, and maternal transfer. We will employ the well characterized clinically relevant fetal sheep model to: 1) determine the most efficient method for gene transfer into fetal HSC by comparing recipient gestational age, number of vector injections, and supernatant versus producer cells; 2) evaluate whether vector tissue distribution, potential for germline alteration, and maternal transfer are influenced by the conditions described in Aim #1; 3) examine whether tolerance to the vector/transgene is induced following gene transfer during the period of pre-immunity, and whether this permits post-natal """"""""boosting"""""""" by administering vector or transduced autologous cells without eliciting an immune response. The proposed studies will provide essential information about efficiently transferring exogenous genes to fetal HSC in utero while minimizing the potential fetal/maternal risks. These studies in this unique fetal model may lay the groundwork for devising clinically relevant in itero GT approaches for treating a variety of genetic diseases in human fetuses early in gestation, prior to disease onset.
