In situ genetic correction of the sickle cell anemia mutation in human induced pluripotent stem cells using engineered zinc finger nucleases

Vittorio Sebastiano, Morgan L. Maeder, James F. Angstman, Bahareh Haddad, Cyd Khayter, Dana T. Yeo, Mathew J. Goodwin, John S. Hawkins, Cherie L. Ramirez, Luis F.Z. Batista, Steven E. Artandi, Marius Wernig, J. Keith Joung

Research output: Contribution to journalArticlepeer-review

273 Scopus citations


The combination of induced pluripotent stem cell (iPSC) 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 iPSCs, raising the prospect of using this technology to correct disease causing mutations. Here, we describe the generation of iPSC 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 method (OPEN). 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 iPSCs 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 patientderived iPSCs. Our results provide an important proof of principle that ZFNs can be used to produce gene-corrected human iPSCs that could be used for therapeutic applications.

Original languageEnglish
Pages (from-to)1717-1726
Number of pages10
Issue number11
StatePublished - Nov 2011


  • Anemia
  • Gene targeting
  • Gene therapy
  • Induced pluripotency
  • Zinc finger nucleases


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