Hematopoietic stem cell (HSC) gene therapy for genetic diseases remains an attractive alternative to allogeneic transplantation. Nonhuman primates have been invaluable in studying HSC gene transfer strategies. In contrast to mouse studies, nonhuman primate studies more closely reflect conditions applicable to humans, e.g., the use of the same HSC enrichment methods and cytokines, the same culture conditions, and similar engraftment issues. We have used nonhuman primates to study different retrovirus vectors, transduction conditions, conditioning regimens and in vivo selection of gene-modified repopulating cells. We have developed transduction and transplantation conditions in the nonhuman primate model resulting in high- level in vivo marking in hematopoietic repopulating cells. Using these optimized conditions and in vivo selection we have achieved marking levels sufficient to cure not only immunodeficiencies, but also genetic diseases which require relatively high marking levels including hemoglobinopathies. These studies, however, required high-dose conditioning with total body irradiation (TBI), which would not be an appropriate conditioning regimen for such genetic diseases. We have also used the nonhuman primate model to perform extensive insertion site analyses and found that lentivirus vectors and foamy virus vectors maybe less prone for insertional mutagenesis than gammaretroviral vectors. Based on these findings, we propose here to develop safer and more clinically applicable HSC gene therapy protocols for genetic diseases, especially for hemoglobinopathies. In aim 1, we propose to study an inducible promoter system to more safely select gene- corrected cells in vivo. We will also test and compare the use of lentivirus and foamy virus vectors in this setting and analyze integration sites before and after in vivo selection using whole genome-adapted pyrosequencing. In aim 2, we will determine whether we can expand HSC and select gene-corrected cells before infusion into the recipient. In aim 3, we will evaluate engraftment in a nonmyeloablative transplant setting using a combination of busulfan and stem cell mobilization.. In aim 4, we will evaluate our optimum conditions with a clinical globin vector to determine the efficacy and safety of our strategies to treat hemoglobinopathies. Protocols developed in the nonhuman primate model should be readily translatable to the clinical setting.