The severe combined immunodeficiencies (SCID) are a set of genetic diseases in which patients are born with mutations in single genes and are unable to develop functional immune systems. While bone marrow transplantation can be curative for these diseases there remain significant limitations to this approach. Gene therapy using retroviral vectors have been used for SCID with mixed results. Tens of patients have developed functional immune systems such that they can live without having to worry about developing life-threatening infections. A handful of patients, however, have developed iatrogenic leukemia from the insertional activation of a proto-oncogene. In the next generation of viral based gene therapy trials for these diseases, the viral vectors have been engineered to decrease the probability of activating proto- oncogenes. The results of these trials, particularly the oncogenic risk, will not be known for several years. We have been working towards a third generation gene therapy approach to SCID. In contrast to using viral vectors to deliver transgenes in an uncontrolled fashion, we are working towards using homologous recombination to precisely modify the genome. We have shown that using engineered nucleases we can stimulate gene modification by homologous recombination (gene targeting) at frequencies that should be therapeutically useful (>10%) in cell lines and primary cels. The overall focus of the proposal is to translate the findings in a methodical and careful fashion to use in patient-derived umbilical cord blood derived CD34+ cells. The three specific aims are focused on translating our basic science studies into a clinical trial. Specific aim 1 is focused o developing nuclease mediated gene targeting for the IL2RG gene. Specific aim 2 is focused on developing nuclease mediated gene targeting for the ADA gene. Specific aim 3 is focused on using whole genome approaches to better understand nuclease on-target activity and off-target effects. Our goal with this proposal is to complete the IND-enabling experiments necessary to initiate a first-in-man gene therapy clinical trial using homologous recombination to cure SCID.