Substantial evidence supports the therapeutic potential of ex vivo gene therapy based on Hematopoietic Stem Cell (HSC) or T lymphocytes to treat inherited diseases or cancer. Yet, the intrinsic limitations of current gene replacement approaches based on semi-randomly integrating vectors, and the occurrence in some clinical trials of severe adverse events related to gene transfer, prevent safe deployment and broad application of gene therapy. This project aims to overcome these limits by exploiting the results of an earlier highly successful integrated project (PERSIST), which demonstrated the potential of gene targeting technologies based on engineered nucleases to provide radical new solutions to these hurdles. By homology-driven repair of a nuclease-targeted dysfunctional gene, we will insert a functional copy downstream its own endogenous promoter in HSC, thus restoring both function and physiological expression control. For adoptive T-cell therapy, we will combine nuclease-mediated disruption of the endogenous TCR genes with transfer of tumor-specific TCR, thus editing T-cell specificity at the genetic level. We will develop these innovative approaches into robust, scalable and clinically ready processes for safe ex vivo genetic modification of hematopoietic cells, and apply them to the treatment of paradigmatic diseases that provide a favourable risk-benefit ratio for clinical testing. Because both HSC and T-cell based gene therapy strategies share methodological aspects and technological challenges, we will address them systematically by involving a SME which holds unique know-how and top-level expertise in vector manufacturing and HSC and T-cell processing, as already established for our previous and ongoing gene therapy trials. Our findings will lead to the design of new clinical trials to provide durable benefits or even a cure to patients suffering from severe and otherwise fatal diseases, and representing a template for broaden application in medicine.