Focusing on the treatment of monogenic immunodeficiencies with curative intent, this project will develop and evaluate new methods for genetic stem cell modification, which have wide implications for many other disorders. A central theme of the proposal is an unbiased efficiency and safety evaluation of the key technology used in the genetic modification of hematopoietic stem cells and their progeny: novel lentiviral vector-mediated transgenesis with reduced risks from insertion mutagenesis. Tailored lentiviral vectors have been developed for two target disorders (i.e, X-linked agammaglobulinemia and RAG- severe combined immunodeficiency, both characterized by a differentiation block of immune competent cells) and will be evaluated for efficacy and safety in disease-specific preclinical models. Designed with a translational aim, relevant basic studies in hematopoietic stem cell biology by clonality and integration site analyses will complement this research. This will also create a paradigmatic data-mining activity to obtain insights into crucial issues of clonal kinetics of gene-modified cells in vivo. Depending on the outcome of the preclinical evaluation, one disease entity will be brought to clinical implementation under this project. The project is intended to create the basis for gene therapy technology development and simultaneously promote patient safety. Translational dissemination of know-how will generate the expertise to establish future clinical gene therapy for other inherited monogenic diseases with improved predictability, efficacy and safety. Gene therapy is within reach for a variety of monogenic inherited diseases. At the current state-of-the-art, the rapid clinical implementation of gene therapy needs selection of diseases in which: -A specific animal model is available for efficacy and safety evaluation Major tasks: 1.Production/improvement of high titer RD114 pseudotyped lentiviral vectors for BTK, RAG1 and RAG2 Gene therapy of hematopoietic stem cells eventually will provide a cure for many inherited diseases, but in the past twenty years its development has met considerable scientific hurdles. Clinical gene therapy has been successfully applied to two inherited disease: X-linked severe combined immunodeficiency (X-linked SCID) and ADA-SCID. This X-linked SCID trial was the first to demonstrate proof of efficacy with 9/11 patients successfully cured. However, two of the nine patients cured developed a lymphoproliferative syndrome resembling acute lymphoblastic leukemia, as a serious adverse effect (SAE) of therapy. As both have been successfully treated, the SCID-X1 gene therapy trial's current follow-up shows a 2-year survival rate of 100% and a disease free survival rate of 80%. Although the numbers are still too small to confidentially estimate the frequency of this complication, the rate of serious complications (including treatment related mortality) is probably significantly less than that after the alternative treatment of haploidentical family or matched unrelated donor stem cell transplantation available for this category of patients. Also, the outcome seems superior to allogeneic transplantation of bone marrow from HLA matched family donors in terms of restoration of immune functions. Improvement in vector design, stem cell purification and stem cell dose reduction may further reduce the oncogenic risk several orders of magnitude, potentially resulting in a progressively favorable benefit/risk balance. The actual genotoxicity risk deserves intensive attention. In principle, every viral insertion results in a mutation. In this proposal, it is envisaged to use self-inactivating (SIN) lentiviral vectors, which contrary to the more conventional gammaretrovirus vectors do not integrate with preference for the promoter region of active genes. This will considerably reduce the risk to an extent that will be determined specifically in the project.