Reading Frame Correction by Targeted Genome Editing Restores Dystrophin Expression in Cells From Duchenne Muscular Dystrophy Patients

A recent paper from Gersbach et al. (Molecular Therapy, 4^th June, 2013) entitled “Reading Frame Correction by Targeted Genome Editing Restores Dystrophin Expression in Cells From Duchenne Muscular Dystrophy Patients” highlights the recent advances in approaches to correct genetic mutations in patient derived cells using an engineered nuclease.

The study exploits the effect of two separate TALEN’s, (an engineered fusion protein known as a transcription activator-like effector nucleases) that work together to selectively identify and bind specific sequences of DNA, ultimately leading to the creation of a double strand break in the region that the TALEN’s bind. This targeted break in the DNA stimulates the cells own repair mechanisms and leads to the formation of multiple new forms of the original DNA that contains microinsertions and microdeletions at the previous DNA breakpoint.

In Duchenne muscular dystrophy, the deletion of certain “in-frame” exons (particularly in the long central rod domain) has been associated with the much milder form of the disease known as Becker muscular dystrophy. This flexibility of function of the dystrophin protein is important, because it is critical to the approach used by Gersbach et al. The micro changes in the DNA (insertions and deletions) imparted by the TALEN approach need to produce a functional form of novel dystrophin.

To demonstrate its utility, the team designed TALEN’s that targeted exon 51 of the dystrophin gene and transfected cells with the TALEN encoded plasmids. They identified a specific TALEN pair, designated TN3/8, which were active and well tolerated in the cellular assay.

TN3/8 was used to correct the reading frame and restore protein expression in immortalized human myoblast cells derived from a Duchenne patient with a reading frame-disrupting deletion of exon 48 to exon 50. Similar dose-dependent results were obtained in primary DMD dermal fibroblasts carrying a reading frame-disrupting deletion of exon 46 to exon 50.

This is an exciting and powerful example of gene editing technology that could provide a permanent gene correction approach and offer an alternative strategy to antisense based exon skipping drugs.

Reading Frame Correction by Targeted Genome Editing Restores Dystrophin Expression in Cells From Duchenne Muscular Dystrophy Patients

David G Ousterout¹, Pablo Perez-Pinera¹, Pratiksha I Thakore¹, Ami M Kabadi¹, Matthew T Brown¹, Xiaoxia Qin², Olivier Fedrigo², Vincent Mouly³, Jacques P Tremblay⁴ and Charles A Gersbach^1,2,5

1.     ¹Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA

2.     ²Institute for Genome Sciences and Policy, Duke University, Durham, North Carolina, USA

3.     ³Thérapie des maladies du muscle strié/Institut de Myologie UM76, Université Pierre et Marie Curie, INSERM U974; CNRS UMR 7215, Paris, France

4.     ⁴Unité de Recherche en Génétique Humaine, Centre de Recherche de CHUL, CHUQ, Faculté de Médecine, Université Laval, Québec, Québec, Canada

5.     ⁵Department of Orthopaedic Surgery, Duke University Medical Center, Durham, North Carolina, USA

Correspondence: Charles A Gersbach, Department of Biomedical Engineering, Room 136 Hudson Hall, Box 90281, Duke University, Durham, North Carolina 27708-0281, USA. E-mail:charles.gersbach@duke.edu

Received 18 February 2013; Accepted 7 April 2013
Advance online publication 4 June 2013

Abstract:

Genome editing with engineered nucleases has recently emerged as an approach to correct genetic mutations by enhancing homologous recombination with a DNA repair template. However, many genetic diseases, such as Duchenne muscular dystrophy (DMD), can be treated simply by correcting a disrupted reading frame. We show that genome editing with transcription activator-like effector nucleases (TALENs), without a repair template, can efficiently correct the reading frame and restore the expression of a functional dystrophin protein that is mutated in DMD. TALENs were engineered to mediate highly efficient gene editing at exon 51 of the dystrophin gene. This led to restoration of dystrophin protein expression in cells from Duchenne patients, including skeletal myoblasts and dermal fibroblasts that were reprogrammed to the myogenic lineage by MyoD. Finally, exome sequencing of cells with targeted modifications of the dystrophin locus showed no TALEN-mediated off-target changes to the protein-coding regions of the genome, as predicted by in silico target site analysis. This strategy integrates the rapid and robust assembly of active TALENs with an efficient gene-editing method for the correction of genetic diseases caused by mutations in non-essential coding regions that cause frameshifts or premature stop codons.