Therapeutic gene targeting in human CD34+ cells.

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The ultimate goal in the treatment of inherited disorders is the permanent correction of the mutation that causes the disease. This project aims at establishing a novel therapy for the treatment of inherited hematological disorders in a mouse model for severe combined immunodeficiency (SCID). The therapy is based on homologous recombination (HR) and corrects the underlying mutation directly in the chromosome. Because of the low frequency of HR, such an approach has only become feasible with the availability of artificial nucleases. The creation of site-specific cuts in the DNA by such nuclease stimulates HR significantly by activating the cellular DNA repair pathways. In order to correct the underlying point mutation for SCID, we will generate repair vectors that serve as a template for HR-based DNA repair as well as custom nucleases that cleave the DNA at the site of the mutation to stimulate HR. Ex vivo gene repair in hematopoietic stem cells HSCs will be achieved by transferring the gene repair components with either integrase-deficient lentiviral vectors or vectors based on different adeno-associated virus serotypes. The extent of gene repair as well as the frequency of genotoxic side effects will be determined in dose escalation studies to evaluate the risk/benefit ratio of this system for putative clinical applications.