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.

Towards combinatorial transcriptional engineering

“…However, the specificity of TALE based sTFs has come under scrutiny as some computational and experimental studies have reported off-target activity for TALE based nucleases in more complex genomes including the human genome (Fine et al, 2014;Osborn et al, 2013). Although the data from these studies concerns TALE nucleases, the authors emphasized the need for a detailed analysis of the targeting specificity of TALE based sTFs.…”

“…Orthogonal and highly specific (Blount et al, 2012) Off-target activity of TALENs in complex genomes (Bultmann et al, 2012;Deng et al, 2012;Fine et al, 2014;Osborn et al, 2013;Valton et al, 2012) Can antagonize transcription via steric hindrance without trans-repressor fusions (Li et al, 2015) Challenging to transport protein across cell membrane (Gaj et al, 2012;Liu et al, 2014) TALE-like proteins allow enhanced tunability and control of stringency for recognition (de Lange et al, 2015(de Lange et al, , 2014Stella et al, 2014) High recombination propensity of the respective DNA sequence. (Holkers et al, 2013) …”

Epidermal Stem Cells

“…Tüm bu araştırmalar ileriki zamanlarda uygun olan insan çalışmalarında da kullanılabilecek ve uzun süreli takipler sonucu rejeneratif tıp alanında devrim anlamında yeniliklere yol açabilecektir. (27). Ancak, tedavilerdeki umut verici bu gelişmelere karşın, diferansiye olmayan UPKH'lerden teratom gelişme riski halen vardır.…”

Management of chronic wounds in patients with dystrophic epidermolysis bullosa: challenges and solutions

“…Preclinical studies have shown remarkable results in terms of gene correction/excision using both in vivo and ex vivo approaches. [64][65][66][67][68][69][70] In addition, a previous study of ex vivo gene therapy in an individual with junctional EB demonstrated restoration of laminin 332 expression following retroviralmediated transfection of epidermal stem cells with the LAMB3 gene, with long-lasting phenotypic correction in the grafted skin. 71 A US trial is currently evaluating the safety and efficacy of a retroviral vector and genetically engineered epithelial graft cultured from the skin of patients with RDEB (ClinicalTrials.gov identifier: NCT01263379).…”