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.

[1]  H. Kazazian,et al.  Homozygous beta-thalassemia without anemia. , 1989, Blood.

[2]  S. Rivella,et al.  Therapeutic haemoglobin synthesis in beta-thalassaemic mice expressing lentivirus-encoded human beta-globin. , 2000, Nature.

[3]  Michel Sadelain,et al.  Therapeutic haemoglobin synthesis in β-thalassaemic mice expressing lentivirus-encoded human β-globin , 2000, Nature.

[4]  Paul Shinn,et al.  HIV-1 Integration in the Human Genome Favors Active Genes and Local Hotspots , 2002, Cell.

[5]  Tom H. Pringle,et al.  The human genome browser at UCSC. , 2002, Genome research.

[6]  A. Poustka,et al.  Parameter estimation for the calibration and variance stabilization of microarray data , 2003, Statistical applications in genetics and molecular biology.

[7]  Cameron S. Osborne,et al.  LMO2-Associated Clonal T Cell Proliferation in Two Patients after Gene Therapy for SCID-X1 , 2003, Science.

[8]  D. Haussler,et al.  Ultraconserved Elements in the Human Genome , 2004, Science.

[9]  J. Holmes,et al.  Unintegrated Lentivirus DNA Persistence and Accessibility to Expression in Nondividing Cells: Analysis with Class I Integrase Mutants , 2004, Journal of Virology.

[10]  Gordon K Smyth,et al.  Statistical Applications in Genetics and Molecular Biology Linear Models and Empirical Bayes Methods for Assessing Differential Expression in Microarray Experiments , 2011 .

[11]  B. Fehse,et al.  Clonal Dominance of Hematopoietic Stem Cells Triggered by Retroviral Gene Marking , 2005, Science.

[12]  Angelique M. Nelson,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. , 2006, Blood.

[13]  Yang Du,et al.  Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1 , 2006, Nature Medicine.

[14]  E. Papapetrou,et al.  Gene transfer into human hematopoietic progenitor cells with an episomal vector carrying an S/MAR element , 2006, Gene Therapy.

[15]  Marius Wernig,et al.  Treatment of Sickle Cell Anemia Mouse Model with iPS Cells Generated from Autologous Skin , 2007, Science.

[16]  Christine Kinnon,et al.  Gammaretrovirus-mediated correction of SCID-X1 is associated with skewed vector integration site distribution in vivo. , 2007, The Journal of clinical investigation.

[17]  Shulan Tian,et al.  Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells , 2007, Science.

[18]  T. Ichisaka,et al.  Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors , 2007, Cell.

[19]  Hanno Glimm,et al.  High-resolution insertion-site analysis by linear amplification–mediated PCR (LAM-PCR) , 2007, Nature Methods.

[20]  S. Nishikawa,et al.  A ROCK inhibitor permits survival of dissociated human embryonic stem cells , 2007, Nature Biotechnology.

[21]  Bruce Aronow,et al.  Vector integration is nonrandom and clustered and influences the fate of lymphopoiesis in SCID-X1 gene therapy. , 2007, The Journal of clinical investigation.

[22]  F. Bushman,et al.  HIV integration site selection: analysis by massively parallel pyrosequencing reveals association with epigenetic modifications. , 2007, Genome research.

[23]  R. Stewart,et al.  Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells , 2007, Science.

[24]  G. Keller,et al.  Identification and targeting of the ROSA26 locus in human embryonic stem cells , 2007, Nature Biotechnology.

[25]  Luca Biasco,et al.  Multilineage hematopoietic reconstitution without clonal selection in ADA-SCID patients treated with stem cell gene therapy. , 2007, The Journal of clinical investigation.

[26]  E. S. Venkatraman,et al.  A faster circular binary segmentation algorithm for the analysis of array CGH data , 2007, Bioinform..

[27]  E. Bouhassira,et al.  Globin switches in yolk sac-like primitive and fetal-like definitive red blood cells produced from human embryonic stem cells. , 2008, Blood.

[28]  George Q. Daley,et al.  Reprogramming of human somatic cells to pluripotency with defined factors , 2008, Nature.

[29]  A. Trounson,et al.  Genetic modification of human embryonic stem cells for derivation of target cells. , 2008, Cell stem cell.

[30]  T. Ichisaka,et al.  Induction of Pluripotent Stem Cells From Adult Human Fibroblasts by Defined Factors , 2008 .

[31]  R. Pedersen,et al.  Robust, Persistent Transgene Expression in Human Embryonic Stem Cells Is Achieved with AAVS1‐Targeted Integration , 2008, Stem cells.

[32]  Christine Kinnon,et al.  Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients. , 2008, The Journal of clinical investigation.

[33]  M. Sadelain,et al.  Stem cell engineering for the treatment of severe hemoglobinopathies. , 2008, Current molecular medicine.

[34]  M. Bossé,et al.  OP9 Stroma Augments Survival of Hematopoietic Precursors and Progenitors During Hematopoietic Differentiation from Human Embryonic Stem Cells , 2008, Stem cells.

[35]  R. Jaenisch,et al.  Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases , 2009, Nature Biotechnology.

[36]  Michel Sadelain,et al.  Stoichiometric and temporal requirements of Oct4, Sox2, Klf4, and c-Myc expression for efficient human iPSC induction and differentiation , 2009, Proceedings of the National Academy of Sciences.

[37]  A. Consiglio,et al.  Disease-corrected haematopoietic progenitors from Fanconi anaemia induced pluripotent stem cells , 2009, Nature.

[38]  A. Schnerch,et al.  Characterization of human embryonic stem cells with features of neoplastic progression , 2009, Nature Biotechnology.

[39]  A. Viale,et al.  Modeling Pathogenesis and Treatment of Familial Dysautonomia using Patient Specific iPSCs , 2009, Nature.

[40]  M. Sadelain,et al.  Harnessing endogenous miR-181a to segregate transgenic antigen receptor expression in developing versus post-thymic T cells in murine hematopoietic chimeras. , 2008, The Journal of clinical investigation.

[41]  Prashant Mali,et al.  Gene targeting of a disease-related gene in human induced pluripotent stem and embryonic stem cells. , 2009, Cell stem cell.

[42]  Pei-Rong Wang,et al.  Globin phenotype of erythroid cells derived from human induced pluripotent stem cells. , 2010, Blood.

[43]  Michel Sadelain,et al.  A Genetic Strategy for Single and Combinatorial Analysis of miRNA Function in Mammalian Hematopoietic Stem Cells , 2009, Stem cells.

[44]  Jérôme Larghero,et al.  Transfusion independence and HMGA2 activation after gene therapy of human β-thalassaemia , 2010, Nature.

[45]  篠原 隆司,et al.  Induction of pluripotent stem cell cells from germ cells , 2012 .