Genomic safe harbors permit high β-globin transgene expression in thalassemia induced pluripotent stem cells


Realizing the therapeutic potential of human induced pluripotent stem (iPS) cells will require robust, precise and safe strategies for genetic modification, as cell therapies that rely on randomly integrated transgenes pose oncogenic risks. Here we describe a strategy to genetically modify human iPS cells at 'safe harbor' sites in the genome, which fulfill five criteria based on their position relative to contiguous coding genes, microRNAs and ultraconserved regions. We demonstrate that 10% of integrations of a lentivirally encoded β-globin transgene in β-thalassemia-patient iPS cell clones meet our safe harbor criteria and permit high-level β-globin expression upon erythroid differentiation without perturbation of neighboring gene expression. This approach, combining bioinformatics and functional analyses, should be broadly applicable to introducing therapeutic or suicide genes into patient-specific iPS cells for use in cell therapy.

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Figure 1: Safe harbor selection strategy and characterization of thal-iPS cell lines.
Figure 2: Single-vector copy, clonality and mapping of the integration site.
Figure 3: β-globin expression in the erythroid progeny of single-vector-copy thal-iPS cell clones.

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We thank X. Wang and N. Wu for assistance with HPLC analysis; L. Ferro, E. Reed, J. Miller, M. Leversha and M. Tomishima for technical assistance; F. Boulad, Memorial Sloan-Kettering Cancer Center New York for bone marrow specimens; and A. Athanassiadou for advice on β-thalassemia genotyping. pCMVΔR8.91N/N was kindly provided by E. Poeschla, Mayo Clinic, Rochester, Minnesota. This work was supported by the Starr Foundation (Tri-Institutional Stem Cell Initiative, Tri-SCI-018), the New York State Stem Cell Science, NYSTEM (N08T-060) and National Heart, Blood, and Lung Institute grant HL053750 (M.S.). F.D.B., S.L.R. and N.M. were supported by National Institutes of Health grants AI052845 and AI082020 (F.D.B.). G.L. was supported by a New York Stem Cell Foundation Druckenmiller fellowship.

Author information




E.P.P. conceived and designed the study, designed and performed experiments, analyzed data and wrote the manuscript; G.L. performed iPS cell differentiation experiments; N.M. performed bioinformatics analyses; M.S. and C.L. analyzed microarray data; L.M.S.T. provided technical assistance; K.K. performed histological analyses of teratomas; S.L.R. generated and analyzed integration site data; P.G. provided skin biopsy samples from β-thalassemia patients; A.V. generated microarray data; I.R., F.D.B. and L.S. analyzed data; M.S. conceived and designed the study, analyzed data and wrote the manuscript.

Corresponding author

Correspondence to Michel Sadelain.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1–9 and Supplementary Figs. 1–20 (PDF 3117 kb)

Supplementary Movie 1

Beating putative cardiomyocytes derived from iPS cell line thal1.52. (MOV 20336 kb)

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Papapetrou, E., Lee, G., Malani, N. et al. Genomic safe harbors permit high β-globin transgene expression in thalassemia induced pluripotent stem cells. Nat Biotechnol 29, 73–78 (2011).

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