Advances in genetic therapeutic strategies for Duchenne muscular dystrophy

Abstract

What is the topic of this review? This review highlights recent progress in genetically based therapies targeting the primary defect of Duchenne muscular dystrophy. What advances does it highlight? Over the last two decades, considerable progress has been made in understanding the mechanisms underlying Duchenne muscular dystrophy, leading to the development of genetic therapies. These include manipulation of the expression of the gene or related genes, the splicing of the gene and its translation, and replacement of the gene using viral approaches. Duchenne muscular dystrophy is a lethal X-linked disorder caused by mutations in the dystrophin gene. In the absence of the dystrophin protein, the link between the cytoskeleton and extracellular matrix is destroyed, and this severely compromises the strength, flexibility and stability of muscle fibres. The devastating consequence is progressive muscle wasting and premature death in Duchenne muscular dystrophy patients. There is currently no cure, and despite exhaustive palliative care, patients are restricted to a wheelchair by the age of 12 years and usually succumb to cardiac or respiratory complications in their late 20s. This review provides an update on the current genetically based therapies and clinical trials that target or compensate for the primary defect of this disease. These include dystrophin gene-replacement strategies, genetic modification techniques to restore dystrophin expression, and modulation of the dystrophin homologue, utrophin, as a surrogate to re-establish muscle function.

Figure 1. The dystrophin and utrophin‐associated protein complexes

A, structure of the dystrophin‐associated protein complex (DAPC) at the muscle membrane. The DAPC acts as a link between myofibres and the extracellular matrix to provide stability at the sarcolemma. The central rod domain of dystrophin contains 24 spectrin repeats and four hinges. The N‐terminal domain (NTD) and specific spectrin repeats bind to cytosolic F‐actin to aid in shock absorbance that results from elastic recoil during muscle contraction or stretch. The cysteine‐rich domain (CRD) links dystrophin to the sarcolemmal‐bound β‐dystroglycan, which in turn binds to α‐dystroglycan to form the dystroglycan complex. This complex is further strengthened by binding to the sarcoglycans (α, β, δ and γ) and sarcospan at the sarcolemma as well as laminin α2 at the extracellular matrix. The C‐terminal domain (CTD) of dystrophin binds several cytosolic proteins, such as α‐dystrobrevin and syntrophins (α and β). These syntrophins can recruit neuronal nitric oxide synthase (nNOS) to the sarcolemma via their PDZ domains to regulate blood flow to the muscle. In addition, spectrin repeats 16/17 in dystrophin are also able to recruit nNOS. Dystrophin interacts indirectly with microtubules through ankyrin‐B and directly via spectrin repeats 20–23. Together, dystrophin and its associated proteins protect the sarcolemma from contraction‐induced injury. B, structure of the utrophin‐associated protein complex (UAPC) at the neuromuscular junction. The UAPCs have similar protective functions compared with the DAPCs, because utrophin shows 80% sequence homology to dystrophin. However, utrophin lacks the sequence corresponding to spectrin‐like repeats 15 and 19 of dystrophin and binds actin only through the NTD. Utrophin is unable to recruit nNOS directly via its spectrin repeats, although nNOS can still be recruited indirectly through the syntrophins. At the neuromuscular junction, UAPC also binds to Raspyn and is involved in the clustering of acetylcholine receptors (AChRs) to the membrane. In addition, the CTD of utrophin binds to Multiple asters (MAST), which associates with microtubules. The UAPC is linked to the extracellular matrix of the neuromuscular junction via laminins α4, α5 and β2.

Figure 2. Dystrophin and approaches to therapy

A, full‐length wild‐type dystrophin consists of an actin‐binding N‐terminal domain (NTD), hinge domains (H1–H4) and a cysteine‐rich domain (CRD) next to a carboxy‐terminal domain (CTD). Spectrin repeats (R1–R24) make up the rod domain. B and C, a mildly affected Becker muscular dystrophy (BMD) patient with exons 17–48 deleted, resulting in 46% of dystrophin deleted, has been reported (B) and forms the basis of mini‐dystrophin (C). D, in dystrophin containing a nonsense mutation causing a premature stop codon, Translarna allows read through or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) can correct the mutation, restoring functional dystrophin. E, utrophin does not contain the same number of spectrin‐like repeats and can bind actin only through the NTD. Localization of nNOS to the sarcolemma is not possible with utrophin and some of the dystrophin mini‐genes, as observed for some mildly affected BMD patients. F, in DMD patients with a deletion of exon 50, exons 49 and 51 are out of frame. This leads to unstable pre‐mRNA, which is degraded without the protein being produced. G and F, using antisense oligonucleotides (such as Drisapersen or eteplirsen), skipping of exon 51 is promoted (G), resulting in restoration of the open reading frame (H).