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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Available from: Chan-Jung Chang, Sep 29, 2015
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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.
    EMBO Molecular Medicine 05/2014; 6(6). DOI:10.15252/emmm.201303374 · 8.67 Impact Factor
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    • "In most diseases, there is no formal proof of a causal relationship between the genetic mutation and the disease phenotype because experimental genetic studies are not possible in humans, as opposed to animal models, such as Drosophila and mouse. However, demonstration of a causal relationship between a genetic mutation and a disease phenotype can be verified using genome editing technologies such as the helper-dependent adenoviral vector [90], zinc-finger nucleases [91], transcription activator-like effector nucleases [92,93], or the CRISPR-Cas9 system [94,95] or its improved method [96]. These technologies can be used to perform rescue experiments with gene corrections, as well as recapitulation of the disease phenotype by introducing disease-related mutations into control iPS cells [82]. "
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    ABSTRACT: In 2006, we demonstrated that mature somatic cells can be reprogrammed to a pluripotent state by gene transfer, generating induced pluripotent stem (iPS) cells. Since that time, there has been an enormous increase in interest regarding the application of iPS cell technologies to medical science, in particular for regenerative medicine and human disease modeling. In this review article, we outline the current status of applications of iPS technology to cell therapies (particularly for spinal cord injury), as well as neurological disease-specific iPS cell research (particularly for Parkinson's disease and Alzheimer's disease). Finally, future directions of iPS cell research are discussed including a) development of an accurate assay system for disease-associated phenotypes, b) demonstration of causative relationships between genotypes and phenotypes by genome editing, c) application to sporadic and common diseases, and d) application to preemptive medicine.
    Molecular Brain 03/2014; 7(1):22. DOI:10.1186/1756-6606-7-22 · 4.90 Impact Factor
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    • "One such site in the human genome is the AAVS1 " safe harbor " locus which encodes the PPP1R12C gene and when targeted with transgenes results in stable gene expression (Dekelver et al., 2010; Hockemeyer et al., 2009). It has been shown that transgenic constructs targeted to the AAVS1 locus maintain expression in hematopoietic cells differentiated from human induced pluripotent stem (iPS) cells when using constitutive promoters (Garçon et al., 2013; Zou et al. 2011) or when using erythroid specific promoters (Chang & Bouhassira, 2012). Therefore, the AAVS1 locus offers a well-characterized site for transgene expression for use in human pluripotent stem cells. "
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    ABSTRACT: Human pluripotent stem cells offer a powerful system to study human biology and disease. Here, we report a system to both express transgenes specifically in ES cell derived hematopoietic cells and knockdown gene expression stably throughout the differentiation of ES cells. We characterize a CD43 promoter construct that when inserted into the AAVS1 "safe harbor" locus utilizing a zinc finger nuclease specifically drives GFP expression in hematopoietic cells derived from a transgenic ES cell line and faithfully recapitulates endogenous CD43 expression. In addition, using the same gene targeting strategy we demonstrate that constitutive expression of short hairpin RNAs within a microRNA backbone can suppress expression of PU.1, an important regulator of myeloid cell development. We show that PU.1 knockdown cell lines display an inhibition in myeloid cell formation and skewing towards erythroid development. Overall, we have generated a powerful system to track hematopoietic development from pluripotent stem cells and study gene function through hematopoietic specific gene expression and constitutive gene knockdown.
    Stem Cell Research 02/2014; 12(3):630-637. DOI:10.1016/j.scr.2014.02.004 · 3.69 Impact Factor
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