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

In Situ Genetic Correction of the Sickle Cell Anemia Mutation in Human Induced Pluripotent Stem... 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 patient‐derived 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. STEM CELLS 2011;29:1717–1726 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Stem Cells Wiley

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

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Publisher
Wiley
Copyright
Copyright © 2011 AlphaMed Press
ISSN
1066-5099
eISSN
1549-4918
DOI
10.1002/stem.718
Publisher site
See Article on Publisher Site

Abstract

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 patient‐derived 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. STEM CELLS 2011;29:1717–1726

Journal

Stem CellsWiley

Published: Nov 1, 2011

References

  • Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc‐finger nucleases
    Hockemeyer, Hockemeyer; Söldner, Söldner; Beard, Beard
  • Heritable targeted gene disruption in zebrafish using designed zinc‐finger nucleases
    Doyon, Doyon; McCammon, McCammon; Miller, Miller
  • High‐frequency modification of plant genes using engineered zinc‐finger nucleases
    Townsend, Townsend; Wright, Wright; Winfrey, Winfrey
  • Generation of transgene‐free lung disease‐specific human induced pluripotent stem cells using a single excisable lentiviral stem cell cassette
    Somers, Somers; Jean, Jean; Sommer, Sommer
  • Copy number variation and selection during reprogramming to pluripotency
    Hussein, Hussein; Batada, Batada; Vuoristo, Vuoristo
  • Somatic coding mutations in human induced pluripotent stem cells
    Gore, Gore; Li, Li; Fung, Fung
  • Gene targeting in human pluripotent stem cells with adeno‐associated virus vectors
    Mitsui, Mitsui; Suzuki, Suzuki; Aizawa, Aizawa

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