A TALEN-based approach to developing safer, more effective treatments for people with Epidermolysis Bullosa
The goal of the project was to develop a treatment based on a patient’s own cells, corrected in the laboratory for the gene defect, and returned to the EB individual with the hope that this would have the advantage of the absence of rejection of cells from unrelated donors.
The project combined two technologies – induced pluripotent stem (iPS) cells and transcription activator-like effector nuclease (TALEN). iPS cells are a type of stem cell generated from other adult cells – they have the ability to become another type of cell in the body. TALEN act as ‘molecular scissors’ to target and remove sections of the faulty gene for Collagen VII, which causes the skin fragility in Recessive Dystrophic Epidermolysis Bullosa (RDEB).
Recessive Dystrophic Epidermolysis Bullosa (RDEB) is characterized by a functional deficit of Collagen VII protein due to gene defects in the Collagen VII 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.
The project reported 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.
Osborn, M.J., et al. TALEN-based Gene Correction for Epidermolysis Bullosa. Molecular Therapy 2013;21(6):1151-1159