TALEN-based Gene Correction for Epidermolysis Bullosa

1] Division of Blood and Marrow Transplantation, Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota, USA [2] Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota, USA.
Molecular Therapy (Impact Factor: 6.23). 04/2013; 21(6). DOI: 10.1038/mt.2013.56
Source: PubMed


Recessive dystrophic epidermolysis bullosa (RDEB) is characterized by a functional deficit of type VII collagen protein due to gene defects in the type VII collagen gene (COL7A1). Gene augmentation therapies are promising, but run the risk of insertional mutagenesis. To abrogate this risk, we explored the possibility of using engineered transcription activator-like effector nucleases (TALEN) for precise genome editing. We report the ability of TALEN to induce site-specific double-stranded DNA breaks (DSBs) leading to homology-directed repair (HDR) from an exogenous donor template. This process resulted in COL7A1 gene mutation correction in primary fibroblasts that were subsequently reprogrammed into inducible pluripotent stem cells and showed normal protein expression and deposition in a teratoma-based skin model in vivo. Deep sequencing-based genome-wide screening established a safety profile showing on-target activity and three off-target (OT) loci that, importantly, were at least 10 kb from a coding sequence. This study provides proof-of-concept for TALEN-mediated in situ correction of an endogenous patient-specific gene mutation and used an unbiased screen for comprehensive TALEN target mapping that will cooperatively facilitate translational application.Molecular Therapy (2013); doi:10.1038/mt.2013.56.

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    • "First-generation TALEN scaffolds have been described with different N-terminal segments (NTSs) and C-terminal segments (CTSs) flanking the TALE central repeat domains (Kim et al., 2013a; Miller et al., 2011; Mussolino et al., 2011; Sun et al., 2012b). Some of them have been applied for generating human stem cell-based disease models (Ding et al., 2013) and treating human diseases (Choi et al., 2013; Osborn et al., 2013; Sun and Zhao, 2013a), but their efficacy of modifying human genomes is limited in targeting certain loci (Kim et al., 2013a; Reyon et al., 2012). Although a second-generation TALEN platform called GoldyTALEN has been reported with improved genome editing efficacy in zebrafish (Bedell et al., 2012) and livestock (Carlson et al., 2012), its efficacy in modifying human genomes has not been demonstrated. "
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    ABSTRACT: Transcription activator-like effector nucleases (TALENs) have rapidly emerged as a powerful genome editing tool. The site-specific DNA double-strand breaks generated by TALENs in the human chromosome can induce homologous recombination or non-homologous end joining, resulting in desired genetic modifications. In this study, we report the development of a TALEN variant, SunnyTALEN, with >2.5-fold improved genome editing efficacy in human cells. The corresponding scaffold increases the rate of genetic modification at all the 13 tested loci of human genome and is compatible with heterodimer TALEN architectures. This enhanced and high-efficiency TALEN variant represents a novel second-generation TALEN system and has great potential for biological and therapeutic applications. Biotechnol. Bioeng. © 2013 Wiley Periodicals, Inc.
    Full-text · Article · Apr 2014 · Biotechnology and Bioengineering
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    • "However, recent findings about the off-target effects of CRISPRs confound their wide-spread application [9]-[11]. In contrast, TALENs exhibit high targeting specificity with little off-target effect except in one study [8], [12], [13]. Transcription activator-like effectors (TALEs) are important virulence factors first identified in plant pathogenic bacteria Xanthomonas spp.[5], [14], [15]. "
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    ABSTRACT: The development of human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) facilitates in vitro studies of human disease mechanisms, speeds up the process of drug screening, and raises the feasibility of using cell replacement therapy in clinics. However, the study of genotype-phenotype relationships in ESCs or iPSCs is hampered by the low efficiency of site-specific gene editing. Transcription activator-like effector nucleases (TALENs) spurred interest due to the ease of assembly, high efficiency and faithful gene targeting. In this study, we optimized the TALEN design to maximize its genomic cutting efficiency. We showed that using optimized TALENs in conjunction with single-strand oligodeoxynucleotide (ssODN) allowed efficient gene editing in human cells. Gene mutations and gene deletions for up to 7.8 kb can be accomplished at high efficiencies. We established human tumor cell lines and H9 ESC lines with homozygous deletion of the microRNA-21 (miR-21) gene and miR-9-2 gene. These cell lines provide a robust platform to dissect the roles these genes play during cell differentiation and tumorigenesis. We also observed that the endogenous homologous chromosome can serve as a donor template for gene editing. Overall, our studies demonstrate the versatility of using ssODN and TALEN to establish genetically modified cells for research and therapeutic application.
    Full-text · Article · Apr 2014 · PLoS ONE
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    • "Taking this a step farther, others have combined NILV with a rare cutting nuclease for targeted recombination at specific sites by HR[84]. Still others have used NILV as templates for HR along with engineered zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs)129130131132. Increasing clinical utility is expected as these systems are optimized to reduce off-target integrations and increase the efficiency of delivery. "

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