Site specific integration and gene correction

Gene transfer with integrating vectors has an excellent therapeutic potential as demonstrated in recent clinical trials. A concurrent risk, however, is that random integration of the vector in the host cellular genome may alter the function of cellular genes found at or near the insertion site, with deleterious consequences. The recent development of a new powerful technology for gene targeting has brought the possibility of inserting the therapeutic transgene in a predetermined safe site of the genome, or directly correcting the disease gene. This approach is based on engineered Zinc Finger Nucleases (ZFNs), which are custom-designed proteins that recognize a specific DNA sequences in the genome in which the exogenous corrective gene is to be inserted. Within this project, we will capitalize on our expertise in the ZFN technology to establish the rules that make a genomic site suitable for integration of the therapeutic transgenes, with the goal to devise a low-impact modality of gene transfer. By establishing the criteria for "sustainable" gene transfer, we will identify novel "safe harbor" sites in the genome that should overcome the risks associated with the use of randomly integrating vectors. Furthermore, we will attempt to correct endogenous mutations in human primary lymphocytes and hematopoietic stem/progenitor cells (HSPCs) derived from Immunodysregulation Polyendocrinopathy Enteropathy X-linked (IPEX) and X-linked Severe Combined Immunodeficiency (SCID-X1) patients, two lethal inherited diseases. To these aims, we will increase both the specificity of ZFNs and their efficiency in HSPCs. In particular, we will optimize the stem cell culture conditions and evaluate several strategies to deliver ZFN into the cells. Finally, we will combine gene correction with strategies for safe genetic reprogramming of patient-derived fibroblasts to induced pluripotent stem cells (iPSC), in order to provide an alternative source of curative stem cells. If successful, these results may set a new gold standard for regenerative medicine and will make it possible to extend the benefits of site-specific gene transfer to an increasing number of future applications.