Article

Zinc-finger nuclease-mediated correction of -thalassemia in iPS cells

Department of Medicine, Hematology, Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY, United States.
Blood (Impact Factor: 10.45). 09/2012; 120(19). DOI: 10.1182/blood-2012-03-420703

ABSTRACT

Induced pluripotent stem cell technology holds vast promises for a cure to the hemoglobinopathies. Constructs and methods to safely insert therapeutic genes to correct the genetic defect need to be developed. Site-specific insertion is a very attractive method for gene therapy because the risks of insertional mutagenesis are eliminated provided that a "safe harbor" is identified, and because a single set of validated constructs can be used to correct a large variety of mutations simplifying eventual clinical use. We report here the correction of α-thalassemia major hydrops fetalis in transgene-free iPS cells using zinc finger-mediated insertion of a globin transgene in the AAVS1 site on human chromosome 19. Homozygous insertion of the best of the four constructs tested led to complete correction of globin chain imbalance in erythroid cells differentiated from the corrected iPS cells.

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    • "Interestingly, in contrast with hepatocyte differentiation , the uninsulated inducible tdT cassette could be efficiently activated in hESC-derived motor neurons and mesodermal cells, and ubiquitous reporter expression was seen in cells of the three germ layers in teratomas in vivo, although we cannot definitely state that inhibition does not occur in hepatocytes in vivo. Even if our studies did not show faithful activity of the HB9p, the studies using the inducible system are consistent with other reports demonstrating successful neural and hematopoietic lineage identification by promoter constructs in the AAVS1 locus without the need of insulators (Chang and Bouhassira, 2012; Sullivan et al., 2014; Tiyaboonchai et al., 2014; Wainger et al., 2014) and with previous studies in vivo reporting stable transgene expression (Hockemeyer et al., 2011; Qian et al., 2014). Why cHS4 insulators were not able to block the AAVS1-mediated inhibition of the TRE during hepatocyte differentiation, and potentially the hepatoblast/hepatocyte lineage-specific promoters, but were functional when used with the OCT4p and NF-kB sensor remains unknown. "
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    ABSTRACT: Tools for rapid and efficient transgenesis in "safe harbor" loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs). We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE) in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage. However, we observed unexpected and variable transgene expression inhibition in vitro, due to DNA methylation and other unknown mechanisms, both in undifferentiated hESC and differentiating hepatocytes. Therefore, the AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro, and using it for transgenesis in hPSC will require careful assessment of the function of individual transgenes.
    Full-text · Article · Oct 2015 · Stem Cell Reports
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    • "Interestingly, in contrast with hepatocyte differentiation , the uninsulated inducible tdT cassette could be efficiently activated in hESC-derived motor neurons and mesodermal cells, and ubiquitous reporter expression was seen in cells of the three germ layers in teratomas in vivo, although we cannot definitely state that inhibition does not occur in hepatocytes in vivo. Even if our studies did not show faithful activity of the HB9p, the studies using the inducible system are consistent with other reports demonstrating successful neural and hematopoietic lineage identification by promoter constructs in the AAVS1 locus without the need of insulators (Chang and Bouhassira, 2012; Sullivan et al., 2014; Tiyaboonchai et al., 2014; Wainger et al., 2014) and with previous studies in vivo reporting stable transgene expression (Hockemeyer et al., 2011; Qian et al., 2014). Why cHS4 insulators were not able to block the AAVS1-mediated inhibition of the TRE during hepatocyte differentiation, and potentially the hepatoblast/hepatocyte lineage-specific promoters, but were functional when used with the OCT4p and NF-kB sensor remains unknown. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Tools for rapid and efficient transgenesis in "safe harbor" loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs). We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE) in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage. However, we observed unexpected and variable transgene expression inhibition in vitro, due to DNA methylation and other unknown mechanisms, both in undifferentiated hESC and differentiating hepatocytes. Therefore, the AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro, and using it for transgenesis in hPSC will require careful assessment of the function of individual transgenes.
    Full-text · Article · Oct 2015 · Stem Cell Reports
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    • "Additionally, no detectable transcriptional perturbations of the PPP1R12C and its flanking genes were observed after integration of transgenes in this locus, indicating that AAVS1 may represent a safe landing path for therapeutic transgene insertion in the human genome (Lombardo et al, 2011). These observations, together with the development of artificial zinc finger nucleases (ZFNs) that efficiently and selectively target the AAVS1 locus, have facilitated gene editing strategies aiming at inserting therapeutic transgenes in this locus, not only in immortalized cell lines but also in several primary human cell types, including induced pluripotent stem cells (hiPSCs; Hockemeyer et al, 2009; DeKelver et al, 2010; Lombardo et al, 2011; Zou et al, 2011b; Chang & Bouhassira, 2012). Because a defective FA pathway not only predisposes FA patients to cancer (Rosenberg et al, 2008) but also to the early development of bone marrow failure due to the progressive extinction of the HSCs (Larghero et al, 2002; Jacome et al, 2006), our final aim in these studies was the generation of gene-edited, disease-free FA-HSCs, obtained from non-hematopoietic tissues of the patient. "
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    ABSTRACT: Gene targeting is progressively becoming a realistic therapeutic alternative in clinics. It is unknown, however, whether this technology will be suitable for the treatment of DNA repair deficiency syndromes such as Fanconi anemia (FA), with defects in homology-directed DNA repair. In this study, we used zinc finger nucleases and integrase-defective lentiviral vectors to demonstrate for the first time that FANCA can be efficiently and specifically targeted into the AAVS1 safe harbor locus in fibroblasts from FA-A patients. Strikingly, up to 40% of FA fibroblasts showed gene targeting 42 days after gene editing. Given the low number of hematopoietic precursors in the bone marrow of FA patients, gene-edited FA fibroblasts were then reprogrammed and re-differentiated toward the hematopoietic lineage. Analyses of gene-edited FA-iPSCs confirmed the specific integration of FANCA in the AAVS1 locus in all tested clones. Moreover, the hematopoietic differentiation of these iPSCs efficiently generated disease-free hematopoietic progenitors. Taken together, our results demonstrate for the first time the feasibility of correcting the phenotype of a DNA repair deficiency syndrome using gene-targeting and cell reprogramming strategies.
    Full-text · Article · May 2014 · EMBO Molecular Medicine
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