1] Division of Blood and Marrow Transplantation, Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota, USA  Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota, USA.
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.
"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. "
"However, recent findings about the off-target effects of CRISPRs confound their wide-spread application -. In contrast, TALENs exhibit high targeting specificity with little off-target effect except in one study , , . Transcription activator-like effectors (TALEs) are important virulence factors first identified in plant pathogenic bacteria Xanthomonas spp., , . "
[Show abstract][Hide abstract] 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.
PLoS ONE 04/2014; 9(4):e93575. DOI:10.1371/journal.pone.0093575 · 3.23 Impact Factor
"Epidermolysis bullosa is a family of monogenic disorders characterized by severe blistering of the skin.1,2,3 The need for effective therapy is currently unmet and, despite attempts at protein,1,2,3,4 bone marrow transplant,5 cell6,7 and induced pluripotent stem cells–based treatments,8,9 palliative care remains the only option available. The most severe form of EB, often lethal by early childhood, is Herlitz-junctional EB (H-JEB), caused by mutations in the heterotrimer laminin-332, which plays a nonredundant role in epidermal–dermal adhesion by serving as a stable anchoring contact between the epidermal integrin receptors α3β1 and α6β6, and collagen VII, the main component of the dermal anchoring fibrils.2 "
[Show abstract][Hide abstract] ABSTRACT: Definitive correction of disease causing mutations in somatic cells by homologous recombination (HR) is an attractive therapeutic approach for the treatment of genetic diseases. However, HR-based somatic gene therapy is limited by the low efficiency of gene targeting in mammalian cells and replicative senescence of primary cells ex-vivo, forcing investigators to explore alternative strategies such as retro- and lentiviral gene transfer, or genome editing in induced pluripotent stem cells (iPSC). Here, we report correction of mutations at the LAMA3 locus in primary keratinocytes derived from a patient affected by recessive inherited Herlitz junctional epidermolysis bullosa (H-JEB) disorder using recombinant adeno-associated virus (rAAV)-mediated HR. We identified a highly recombinogenic AAV serotype, AAV-DJ, that mediates efficient gene targeting in keratinocytes at clinically relevant frequencies with a low rate of random integration. Targeted H-JEB patient cells were selected based on restoration of adhesion phenotype, which eliminated the need for foreign sequences in repaired cells, enhancing the clinical use and safety profile of our approach. Corrected pools of primary cells assembled functional laminin-332 heterotrimer and fully reversed the blistering phenotype both in vitro and in skin grafts. The efficient targeting of the LAMA3 locus by AAV-DJ using phenotypic selection, together with the observed low frequency of off-target events, makes AAV-DJ based somatic cell targeting a promising strategy for ex-vivo therapy for this severe and often lethal epithelial disorder.Molecular Therapy (2014); doi:10.1038/mt.2013.290.
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