In Situ Genetic Correction of the Sickle Cell Anemia Mutation in Human Induced Pluripotent Stem Cells Using Engineered Zinc Finger Nucleases

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.

High-efficiency motor neuron differentiation from human pluripotent stem cells and the function of Islet-1

“…We used three previously established hiPSC lines-MMW2, RND5 and ID28. MMW2 and RND5 lines were derived by the use of lentivirus-mediated gene delivery 10,35 , while the ID28 hiPSC line was generated using an integration-free reprogramming approach 36 . When cultured under self-renewal conditions, these hiPSC lines all exhibited typical pluripotent stem cell morphologies and expressed pluripotent markers OCT-4 and NANOG ( Supplementary Fig.…”

“…When cultured under self-renewal conditions, these hiPSC lines all exhibited typical pluripotent stem cell morphologies and expressed pluripotent markers OCT-4 and NANOG ( Supplementary Fig. 3a) 10,35,36 . When subjected to the motoneuron differentiation method developed for hESCs, hiPSCs exhibited a range of efficiencies.…”

“…The ID28 hiPSCs, kindly provided by Dr Marius Wernig (Stanford University), were derived from human dermal fibroblasts using a polycistronic lentiviral vector containing the reprogramming factors, which were subsequently removed by the use of Cre recombinase 36 . The hiPSCs were cultured under the same conditions as hESCs.…”

Promise and challenges of human iPSC-based hematologic disease modeling and treatment

“…Despite these potential advantages of iPSC-based modeling, there have only been very limited numbers of iPSCbased blood disease models that have shown either the disease features or the feasibility of gene therapy (Table 2) [14,[26][27][28][29][30]. The first comprehensive proof-of-principle study to demonstrate the potential of iPSCs in any disease modeling and treatment was conducted in the mouse model N/D not determined of sickle cell disease (SCD) [31].…”

“…Inherited diseases with well-defined genetic lesion(s) like SCD are the most straightforward to develop iPSC models. Human SCD iPSCs have been derived for developing disease models and treatment [10,20,27,29,32,33]. Besides SCD, X-linked chronic granulomatous disease (X-CGD) is another example of such recessive genetic diseases.…”