Howden, SE, Gore, A, Li, Z, Fung, HL, Nisler, BS, Nie, J et al.. Genetic correction and analysis of induced pluripotent stem cells from a patient with gyrate atrophy. Proc Natl Acad Sci USA 108: 6537-6542

Morgridge Institute for Research, Madison, WI 53715, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 04/2011; 108(16):6537-42. DOI: 10.1073/pnas.1103388108
Source: PubMed


Gene-corrected patient-specific induced pluripotent stem (iPS) cells offer a unique approach to gene therapy. Here, we begin to assess whether the mutational load acquired during gene correction of iPS cells is compatible with use in the treatment of genetic causes of retinal degenerative disease. We isolated iPS cells free of transgene sequences from a patient with gyrate atrophy caused by a point mutation in the gene encoding ornithine-δ-aminotransferase (OAT) and used homologous recombination to correct the genetic defect. Cytogenetic analysis, array comparative genomic hybridization (aCGH), and exome sequencing were performed to assess the genomic integrity of an iPS cell line after three sequential clonal events: initial reprogramming, gene targeting, and subsequent removal of a selection cassette. No abnormalities were detected after standard G-band metaphase analysis. However, aCGH and exome sequencing identified two deletions, one amplification, and nine mutations in protein coding regions in the initial iPS cell clone. Except for the targeted correction of the single nucleotide in the OAT locus and a single synonymous base-pair change, no additional mutations or copy number variation were identified in iPS cells after the two subsequent clonal events. These findings confirm that iPS cells themselves may carry a significant mutational load at initial isolation, but that the clonal events and prolonged cultured required for correction of a genetic defect can be accomplished without a substantial increase in mutational burden.

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    • "Multiple studies have proved the utility of this approach (reviewed in Hanna et al., 2010; Han et al., 2011; Bellin et al., 2012; Imaizumi and Okano, 2014). Moreover, recent studies showed that mutations in patient-derived iPSCs can be corrected by genomic manipulations (Raya et al., 2009; Zou et al., 2009; Howden et al., 2011). However, many ASD-and schizophrenia-associated mutations exhibit incomplete penetrance, suggesting that genetic background effects significantly influence the clinical presentation and could contribute to the observed phenotypes. "
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    ABSTRACT: Heterozygous mutations of the NRXN1 gene, which encodes the presynaptic cell-adhesion molecule neurexin-1, were repeatedly associated with autism and schizophrenia. However, diverse clinical presentations of NRXN1 mutations in patients raise the question of whether heterozygous NRXN1 mutations alone directly impair synaptic function. To address this question under conditions that precisely control for genetic background, we generated human ESCs with different heterozygous conditional NRXN1 mutations and analyzed two different types of isogenic control and NRXN1 mutant neurons derived from these ESCs. Both heterozygous NRXN1 mutations selectively impaired neurotransmitter release in human neurons without changing neuronal differentiation or synapse formation. Moreover, both NRXN1 mutations increased the levels of CASK, a critical synaptic scaffolding protein that binds to neurexin-1. Our results show that, unexpectedly, heterozygous inactivation of NRXN1 directly impairs synaptic function in human neurons, and they illustrate the value of this conditional deletion approach for studying the functional effects of disease-associated mutations. Copyright © 2015 Elsevier Inc. All rights reserved.
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    • "RPE differentiated from the uncorrected GA hiPSCs showed very low levels of OAT enzymatic activity, whereas RPE from gene-corrected hiPSCs had OAT activity comparable to human prenatal RPE and RPE differentiated from hESC and control hiPSC lines (Meyer et al., 2011). In addition, it was shown that the process of OAT gene repair did not add to the mutational load, nor did it increase genetic instability (Howden et al., 2011). However, the cost and effort that would be required to produce and test clinical grade, gene-corrected cells makes this option unwieldy with currently available technology. "
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    ABSTRACT: Human pluripotent stem cells have made a remarkable impact on science, technology and medicine by providing a potentially unlimited source of human cells for basic research and clinical applications. In recent years, knowledge gained from the study of human embryonic stem cells and mammalian somatic cell reprogramming has led to the routine production of human induced pluripotent stem cells (hiPSCs) in laboratories worldwide. hiPSCs show promise for use in transplantation, high throughput drug screening, "disease-in-a-dish" modeling, disease gene discovery, and gene therapy testing. This review will focus on the first application, beginning with a discussion of methods for producing retinal lineage cells that are lost in inherited and acquired forms of retinal degenerative disease. The selection of appropriate hiPSC-derived donor cell type(s) for transplantation will be discussed, as will the caveats and prerequisite steps to formulating a clinical Good Manufacturing Practice (cGMP) product for clinical trials.
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    • "In the context of clinic development, the high targeting efficiency to generate CP hESC lines supports the feasibility to genetically modify any clinic-grade hESCs under the GMP conditions. While the genetic modification of hESCs is a known safety concern in human therapy associated with random integration of the exogenous DNA into the human genome, the knockin approach used here should minimize such risk because homologous recombination can be achieved without any apparent random integration of the exogenous DNA (Howden et al., 2011; Song et al., 2010). To further address this concern before clinic application of the genetically modified hESCs, the "
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    ABSTRACT: Human embryonic stem cells (hESCs) hold great promise for cell therapy as a source of diverse differentiated cell types. One key bottleneck to realizing such potential is allogenic immune rejection of hESC-derived cells by recipients. Here, we optimized humanized mice (Hu-mice) reconstituted with a functional human immune system that mounts a vigorous rejection of hESCs and their derivatives. We established knockin hESCs that constitutively express CTLA4-Ig and PD-L1 before and after differentiation, denoted CP hESCs. We then demonstrated that allogenic CP hESC-derived teratomas, fibroblasts, and cardiomyocytes are immune protected in Hu-mice, while cells derived from parental hESCs are effectively rejected. Expression of both CTLA4-Ig, which disrupts T cell costimulatory pathways, and PD-L1, which activates T cell inhibitory pathway, is required to confer immune protection, as neither was sufficient on their own. These findings are instrumental for developing a strategy to protect hESC-derived cells from allogenic immune responses without requiring systemic immune suppression.
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