Genome editing for Duchenne muscular dystrophy: a glimpse of the future?

Abstract

Mutations in Dystrophin, one of the largest proteins in the mammalian body, are causative for a severe form of muscle disease, Duchenne Muscular Dystrophy (DMD), affecting not only skeletal muscle, but also the heart. In particular, exons 45-52 constitute a hotspot for DMD mutations. A variety of molecular therapies have been developed, comprising vectors encoding micro- and minidystrophins as well as utrophin, a protein with partially overlapping functions. With the advent of the CRISPR-Cas9-nuclease, genome editing offers a novel option of correction of the disease-cuasing mutations. Full restoration of the healthy gene by homology directed repair is a rare event. However, non-homologous end-joining (NHEJ) may restore the reading frame by causing exon excision. This approach has first been demonstrated in mice and then translated to large animals (dogs, pigs). This review discusses the potential opportunities and limitations of genome editing in DMD, including the generation of appropriate animal models as well as new developments in genome editing tools.

Fig. 1. Sketch of the experimental workflow: Patient cells lacking DMD exon 52 were converted to iPS cells and subsequently to muscle cells.

An intein-split version of Cas9 was encoded into two AAV vectors, together with two gRNAs excising exon 52. Demonstrating that the AAV-Cas9-gRNAs excising exon 51 (AAV-Cas9-gE51) enabled expression of a shortened but stable dystrophin (51-52, the same approach was successfully used in the porcine model of DMD (lacking exon 52), serving as basis for further development of a therapeutic agent.

Fig. 2. Effects of DMD treatment: DMD muscle fibres without treatment display hallmarks of muscle decay, such as centralized nuclei, fibrosis, capillary rarefaction and continuous inflammation (no treatment).

Upon current and novel pharmacologic treatment, inflammation and muscle fibre decay may be decelerated, without alteration of the underlying mechanical strain leading to cell death, e.g., by necroptosis. In contrast, therapeutic strategies aiming at dystrophin re-expression (AONs = antisense olignucleotides targeting exon skipping) correct the disease-causing deficit of dystrophin, either temporally (AONs), or for prolonged intervals (AAV-microdystrophin) or permanently (AAV-Cas9).