No abstract

The combination of induced pluripotent stem (iPS) cell technology and targeted gene modification by homologous recombination (HR) represents a promising new approach to generate genetically corrected, patient-derived cells that could be used for autologous transplantation therapies. This strategy has several potential advantages over conventional gene therapy including eliminating the need for immunosuppression, avoiding the risk of insertional mutagenesis by therapeutic vectors, and maintaining expression of the corrected gene by endogenous control elements rather than a constitutive promoter. However, gene targeting in human pluripotent cells has remained challenging and inefficient. Recently, engineered zinc finger nucleases (ZFNs) have been shown to substantially increase HR frequencies in human iPS cells, raising the prospect of employing this technology to correct disease-causing mutations. Here we describe the generation of iPS cell lines from sickle cell anemia patients and in situ correction of the disease-causing mutation using three ZFN pairs made by the publicly available Oligomerized Pool Engineering (OPEN) method. Gene-corrected cells retained full pluripotency and a normal karyotype following removal of reprogramming factor and drug-resistance genes. By testing various conditions, we also demonstrated that HR events in human iPS cells can occur as far as 82 bps from a ZFN-induced break. Our approach delineates a roadmap for using ZFNs made by an open-source method to achieve efficient, transgene-free correction of monogenic disease mutations in patient-derived iPS cells. Our results provide an important proof of principle that ZFNs can be used to produce gene-corrected human iPS cells that could be used for therapeutic applications.