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.
Bibliographical noteFunding Information:
This study was supported in part by grants from Epidermolysis Bullosa Research Fund, Jackson Gabriel Silver Foundation, DebRA International, University of Minnesota Academic Health Center, Pioneering Unique Cures for Kids foundation, Children’s Cancer Research Fund, Minneapolis, Minnesota, and the United States of America Department of Defense. D.F.V. was supported by National Institutes of Health (R01 GM098861). J.K.J. was supported by a National Institutes of Health Director’s Pioneer Award DP1 OD006862, and the Jim and Ann Orr MGH Research Scholar Award. M.L.M was supported by a National Science Foundation Graduate Research Fellowship. D.F.V. is a listed inventor on a patent application titled “TAL effector–mediated DNA modification,” which is co-owned by Iowa State Univ. and the Univ. of Minnesota, and has been licensed to Cellectis, a European biotechnology company. J.K.J. has a financial interest in Transposagen Biopharmaceuticals. J.K.J.’s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. The other authors declare no conflict of interest.