Gene therapy involving the transfer of a functioning exogenous gene into appropriate somatic cells (e.g. hematopoietic stem cells - HSC) of an organism offers a precise means of treating a number of genetic diseases. Most genetic disorders in human can now be diagnosed early in gestation. In addition, procedures for directly accessing the fetus during much of the intrauterine life have been well established. It is thus possible to treat these patients quite early in gestation before the disease process has had a chance to clinically compromise the patient. The insertion of genes into hematopoietic cells has been greatly facilitated by the use of retroviral vectors. Our preliminary studies using a in utero retroviral gene transfer / HSC transplantation protocol in sheep and monkey fetuses indicate that this approach may offer a suitable procedure for applying somatic cell gene therapy before birth. The long term objective of the proposed studies is to investigate the possibility of using an in utero approach to somatic cell gene therapy that will result in the long term transfer/expression of a normal functioning gene in large animals. Specifically, we plan to use the sheep as a large animal model to: 1) Delineate the conditions the will allow the most efficient transfer of a retroviral-mediated exogenous gene into circulating hematopoietic stem cells (HSC) of fetal sheep by examining the effects of hematopoietic growth factors, time and sequence of incubation, multiplicity of infection, etc. on HSC transduction in vitro and in vivo; 2) Evaluate the safety and efficacy of multiple gene transfer procedures in sheep fetuses by comparing the efficiency of gene transfer/expression in newborn lambs that were given either 1, 2 or 3 gene therapy treatments before birth; and 3) Determine whether efficient transfer of the NeoR gene into hematopoietic and other cells can be achieved by the direct administration of the recombinant retrovirus to fetal sheep, by assessing the transfer/expression of the NeoR in marrow, blood, liver, lungs, thymus, etc. of the fetus and the newborn at intervals following the administration of the retroviral vector to sheep fetuses intraperitoneally or via the yolk sac. It is hoped that with the techniques developed here it will become possible to treat genetic diseases including those involving hemoglobin synthesis in humans early during development.
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