Bone marrow transplantation (BMT) has been used to treat a number of diverse diseases. Reconstitution of the marrow with donor hematopoietic stem cells (HSC) provides functionally competent cells and rectifies the hematologic, immunologic or metabolic defect present in these patients. Results have been remarkable. However, there are significant problems with postnatal BMT. Since many of these problems are related to immunologic disparity between the donor and recipient, and because of the potentially devastating effects of diseases with onset during fetal life, in utero transplantation with genetically corrected hematopoietic stem cells i.e. fetal gene therapy deserves consideration. Recent work in fetal gene transfer and in utero transplantation indicates that fetal hematopoietic cells can be transduced in vitro and successfully transplanted in athe fetus, resulting in long term engraftment without significant GVHD. However further investigation is required to address issues of improved transduction, sustained engraftment, early expression,a nd the ideal source and preparation of the hematopoietic stem cells, before clinical application of fetal gene therapy is a reality. This proposal addresses the issue of improving transduction, engraftment and expression of a transferred human gene encoding a house keeping enzyme glucocerebrosidase. This will be accomplished by pre-incubation with cytokines (SCF, IL3 and il6), transduction using centrifugation during viral inoculation and in- vivo cytokine (GM-CSF) administration. Initial experiments are being conducted in C57bl/6J mice. Fetal liver cells fractionated on a ficoll- hypaque gradient are cultured with growth factors and transduced with the human glucocerebrosidase gene using the retroviral vector MFG-GC. A novel protocol of centrifugation during viral inoculation will be used for transduction. Ex-vivo studies with 3T3 cells, TF-1 (human erythroleukemia cell line), human CD34+ and murine fetal liver cells (FLC) demonstrate that the use of centrifugation greatly enhances gene transfer with the retroviral vector, MFG-GC. The transduced hematopoietic cells are then transplanted into the preimmune fetus. Transplantation will be done either by single injection or by multiple injections and engraftment efficiencies will be compared. Following transplantation, cytokines (GM-CSF) will be administered to promote prenatal expression of donor HSC progeny. The transplanted animals will be analyzed following birth and periodically thereafter for chimerism, and carriage and expression of the transferred human glucocerebrosidase gene. These initial experiments will be followed by studies of transduction and expression of a human gene in a large animals (sheep) and in CD34+ cells derived from human fetal liver at 8-14 weeks of gestation. Furthermore, the efficacy of in utero stem cell transplantation and gene transfer as a therapeutic measure will be studied in the transgenic mouse model of Gaucher disease.
