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

Abstract

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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References

  1. 1

    Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2

    Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007).

    CAS  PubMed  Google Scholar 

  3. 3

    Park, I.H. et al. Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451, 141–146 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Hanna, J. et al. Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science 318, 1920–1923 (2007).

    CAS  PubMed  Google Scholar 

  5. 5

    Raya, A. et al. Disease-corrected haematopoietic progenitors from Fanconi anaemia induced pluripotent stem cells. Nature 460, 53–59 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Schroder, A.R. et al. HIV-1 integration in the human genome favors active genes and local hotspots. Cell 110, 521–529 (2002).

    CAS  PubMed  Google Scholar 

  7. 7

    Hacein-Bey-Abina, S. et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 302, 415–419 (2003).

    CAS  PubMed  Google Scholar 

  8. 8

    Ott, M.G. et al. Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1–EVI1, PRDM16 or SETBP1. Nat. Med. 12, 401–409 (2006).

    CAS  PubMed  Google Scholar 

  9. 9

    Howe, S.J. et al. Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients. J. Clin. Invest. 118, 3143–3150 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Cavazzana-Calvo, M. et al. Transfusion independence and HMGA2 activation after gene therapy of human beta-thalassaemia. Nature 467, 318–322 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11

    Bejerano, G. et al. Ultraconserved elements in the human genome. Science 304, 1321–1325 (2004).

    CAS  PubMed  Google Scholar 

  12. 12

    Kustikova, O. et al. Clonal dominance of hematopoietic stem cells triggered by retroviral gene marking. Science 308, 1171–1174 (2005).

    CAS  PubMed  Google Scholar 

  13. 13

    May, C. et al. Therapeutic haemoglobin synthesis in beta-thalassaemic mice expressing lentivirus-encoded human beta-globin. Nature 406, 82–86 (2000).

    CAS  PubMed  Google Scholar 

  14. 14

    Sadelain, M., Boulad, F., Lisowki, L., Moi, P. & Riviere, I. Stem cell engineering for the treatment of severe hemoglobinopathies. Curr. Mol. Med. 8, 690–697 (2008).

    CAS  PubMed  Google Scholar 

  15. 15

    Papapetrou, E.P. et al. Stoichiometric and temporal requirements of Oct4, Sox2, Klf4, and c-Myc expression for efficient human iPSC induction and differentiation. Proc. Natl. Acad. Sci. USA 106, 12759–12764 (2009).

    CAS  PubMed  Google Scholar 

  16. 16

    Chang, K.H. et al. Definitive-like erythroid cells derived from human embryonic stem cells coexpress high levels of embryonic and fetal globins with little or no adult globin. Blood 108, 1515–1523 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17

    Qiu, C., Olivier, E.N., Velho, M. & Bouhassira, E.E. Globin switches in yolk sac-like primitive and fetal-like definitive red blood cells produced from human embryonic stem cells. Blood 111, 2400–2408 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    Chang, K.H. et al. Globin phenotype of erythroid cells derived from human induced pluripotent stem cells. Blood 115, 2553–2554 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    Giudice, A. & Trounson, A. Genetic modification of human embryonic stem cells for derivation of target cells. Cell Stem Cell 2, 422–433 (2008).

    CAS  PubMed  Google Scholar 

  20. 20

    Hockemeyer, D. et al. Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat. Biotechnol. 27, 851–857 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Zou, J. et al. Gene targeting of a disease-related gene in human induced pluripotent stem and embryonic stem cells. Cell Stem Cell 5, 97–110 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

    Smith, J.R. et al. Robust, persistent transgene expression in human embryonic stem cells is achieved with AAVS1-targeted integration. Stem Cells 26, 496–504 (2008).

    CAS  PubMed  Google Scholar 

  23. 23

    Irion, S. et al. Identification and targeting of the ROSA26 locus in human embryonic stem cells. Nat. Biotechnol. 25, 1477–1482 (2007).

    CAS  PubMed  Google Scholar 

  24. 24

    Safaya, S., Rieder, R.F., Dowling, C.E., Kazazian, H.H. Jr. & Adams, J.G. 3rd Homozygous beta-thalassemia without anemia. Blood 73, 324–328 (1989).

    CAS  PubMed  Google Scholar 

  25. 25

    Werbowetski-Ogilvie, T.E. et al. Characterization of human embryonic stem cells with features of neoplastic progression. Nat. Biotechnol. 27, 91–97 (2009).

    CAS  PubMed  Google Scholar 

  26. 26

    Ji, J. et al. OP9 stroma augments survival of hematopoietic precursors and progenitors during hematopoietic differentiation from human embryonic stem cells. Stem Cells 26, 2485–2495 (2008).

    CAS  PubMed  Google Scholar 

  27. 27

    Deichmann, A. et al. Vector integration is nonrandom and clustered and influences the fate of lymphopoiesis in SCID-X1 gene therapy. J. Clin. Invest. 117, 2225–2232 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28

    Aiuti, A. et al. Multilineage hematopoietic reconstitution without clonal selection in ADA-SCID patients treated with stem cell gene therapy. J. Clin. Invest. 117, 2233–2240 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Schwarzwaelder, K. et al. Gammaretrovirus-mediated correction of SCID-X1 is associated with skewed vector integration site distribution in vivo. J. Clin. Invest. 117, 2241–2249 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

    Lee, G. et al. Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs. Nature 461, 402–406 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    Papapetrou, E.P., Kovalovsky, D., Beloeil, L., Sant'angelo, D. & Sadelain, M. Harnessing endogenous miR-181a to segregate transgenic antigen receptor expression in developing versus post-thymic T cells in murine hematopoietic chimeras. J. Clin. Invest. 119, 157–168 (2009).

    CAS  PubMed  Google Scholar 

  32. 32

    Saenz, D.T. et al. Unintegrated lentivirus DNA persistence and accessibility to expression in nondividing cells: analysis with class I integrase mutants. J. Virol. 78, 2906–2920 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33

    Watanabe, K. et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681–686 (2007).

    CAS  PubMed  Google Scholar 

  34. 34

    Papapetrou, E.P., Ziros, P.G., Micheva, I.D., Zoumbos, N.C. & Athanassiadou, A. Gene transfer into human hematopoietic progenitor cells with an episomal vector carrying an S/MAR element. Gene Ther. 13, 40–51 (2006).

    CAS  PubMed  Google Scholar 

  35. 35

    Schmidt, M. et al. High-resolution insertion-site analysis by linear amplification-mediated PCR (LAM-PCR). Nat. Methods 4, 1051–1057 (2007).

    CAS  PubMed  Google Scholar 

  36. 36

    Wang, G.P., Ciuffi, A., Leipzig, J., Berry, C.C. & Bushman, F.D. HIV integration site selection: analysis by massively parallel pyrosequencing reveals association with epigenetic modifications. Genome Res. 17, 1186–1194 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37

    Kent, W.J. et al. The human genome browser at UCSC. Genome Res. 12, 996–1006 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Papapetrou, E.P., Korkola, J.E. & Sadelain, M. A genetic strategy for single and combinatorial analysis of miRNA function in mammalian hematopoietic stem cells. Stem Cells 28, 287–296 (2009).

    Google Scholar 

  39. 39

    Huber, W., von Heydebreck, A., Sueltmann, H., Poustka, A. & Vingron, M. Parameter estimation for the calibration and variance stabilization of microarray data. Stat. Appl. Genet. Mol. Biol. 2, Article 3 (2003).

    Google Scholar 

  40. 40

    Smyth, G.K. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3, Article 3 (2004).

    Google Scholar 

  41. 41

    Venkatraman, E.S. & Olshen, A.B. A faster circular binary segmentation algorithm for the analysis of array CGH data. Bioinformatics 23, 657–663 (2007).

    CAS  PubMed  Google Scholar 

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Acknowledgements

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.

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Contributions

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). https://doi.org/10.1038/nbt.1717

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