title: TALEN-based gene correction for epidermolysis bullosa.
abstract: 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.
authors.affiliation: Division of Blood and Marrow Transplantation, Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Osborn
authors.forename: Colby G
authors.initials: CG
authors.lastname: Starker
authors.forename: Amber N
authors.initials: AN
authors.lastname: McElroy
authors.forename: Beau R
authors.initials: BR
authors.lastname: Webber
authors.forename: Megan J
authors.initials: MJ
authors.lastname: Riddle
authors.forename: Lily
authors.initials: L
authors.lastname: Xia
authors.forename: Anthony P
authors.initials: AP
authors.lastname: DeFeo
authors.forename: Richard
authors.initials: R
authors.lastname: Gabriel
authors.forename: Manfred
authors.initials: M
authors.lastname: Schmidt
authors.forename: Christof
authors.initials: C
authors.lastname: von Kalle
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Carlson
authors.forename: Morgan L
authors.initials: ML
authors.lastname: Maeder
authors.forename: J Keith
authors.initials: JK
authors.lastname: Joung
authors.forename: John E
authors.initials: JE
authors.lastname: Wagner
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Voytas
authors.forename: Bruce R
authors.initials: BR
authors.lastname: Blazar
authors.forename: Jakub
authors.initials: J
authors.lastname: Tolar
mesh_terms: D001482:Base Composition; D002874:Chromosome Mapping; D024143:Collagen Type VII / Q000235:genetics / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D003851:Deoxyribonucleases / Q000235:genetics* / Q000378:metabolism; D016108:Epidermolysis Bullosa Dystrophica / Q000235:genetics* / Q000628:therapy*; D005347:Fibroblasts / Q000378:metabolism; D017353:Gene Deletion; D018390:Gene Targeting; D018014:Gene Transfer Techniques; D005808:Genes, Recessive; D056426:Genetic Loci; D015316:Genetic Therapy / Q000379:methods*; D005838:Genotype; D057809:HEK293 Cells; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D010641:Phenotype; D059767:Recombinational DNA Repair; D015203:Reproducibility of Results; D012641:Selection, Genetic; D015533:Transcriptional Activation
chemical_list: C569053:COL7A1 protein, human; D024143:Collagen Type VII; D003851:Deoxyribonucleases
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