Animal models of Duchenne muscular dystrophy: from basic mechanisms to gene therapy

Abstract

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disorder. It is caused by loss-of-function mutations in the dystrophin gene. Currently, there is no cure. A highly promising therapeutic strategy is to replace or repair the defective dystrophin gene by gene therapy. Numerous animal models of DMD have been developed over the last 30 years, ranging from invertebrate to large mammalian models. mdx mice are the most commonly employed models in DMD research and have been used to lay the groundwork for DMD gene therapy. After ~30 years of development, the field has reached the stage at which the results in mdx mice can be validated and scaled-up in symptomatic large animals. The canine DMD (cDMD) model will be excellent for these studies. In this article, we review the animal models for DMD, the pros and cons of each model system, and the history and progress of preclinical DMD gene therapy research in the animal models. We also discuss the current and emerging challenges in this field and ways to address these challenges using animal models, in particular cDMD dogs.

Keywords: Animal model; Canine DMD; Duchenne muscular dystrophy; Dystrophin; Gene therapy.

Fig. 1.

Schematic outline of dystrophin and the dystrophin-associated glycoprotein complex (DAGC). Dystrophin contains N-terminal (NT), middle rod, cysteine-rich (CR) and C-terminal (CT) domains. The middle rod domain is composed of 24 spectrin-like repeats (numerical numbers in the cartoon, positively charged repeats are marked in white color) and four hinges (H1, H2, H3 and H4). Dystrophin has two actin-binding domains located at NT and repeats 11–15, respectively. Repeats 1–3 interact with the negatively charged lipid bilayer. Repeats 16 and 17 form the neuronal nitric oxide synthase (nNOS)-binding domain. Dystrophin interacts with microtubule through repeats 20–23. Part of H4 and the CR domain bind to the β-subunit of dystroglycan (βDG). The CT domain of dystrophin interacts with syntrophin (Syn) and dystrobrevin (Dbr). Dystrophin links components of the cytoskeleton (actin and microtubule) to laminin in the extracellular matrix. Sarcoglycans and sarcospan do not interact with dystrophin directly but they strengthen the entire DAGC, which consists of dystrophin, DG, sarcoglycans, sarcospan, Syn, Dbr and nNOS.

Fig. 2.

DMD gene therapy and dystrophin mutations in animal models. (A) The 14-kb dystrophin cDNA and the principle of DMD gene therapy. The numbers in the cDNA refers to exon number. The DNA sequence position of the main dystrophin domains and of the dystrophin-associated protein-binding sites (see Fig. 1) is also shown. Frame-interrupting (out-of-frame) mutation leads to severe DMD. In-frame mutation results in mild Becker muscular dystrophy (BMD). The primary goal of DMD gene therapy is to ameliorate muscle pathology and to improve muscle function. Gene therapy can convert the DMD phenotype to the benign BMD phenotype. Gene therapy might also prevent or slow down the development of muscle disease if affected individuals are treated early enough. (B) Domain structure of dystrophin and location of the mutations in representative mouse and dog models. ABD, actin-binding domain; CKCS, Cavalier King Charles spaniel; CR, cysteine-rich domain; CT, C-terminal domain; Dbr, dystrobrevin; DG, dystroglycan; GRMD, golden retriever muscular dystrophy; GSHP, German shorthaired pointer; nNOS, neuronal nitric oxide synthase; NT, N-terminal domain; Syn, syntrophin; UTR, untranslated region. See supplementary material Table S1 for a description on each model.

Fig. 3.

Representative animal models for DMD. (A) Representative pictures of selected DMD mouse and dog models. mdx mice do not show symptoms (see 6-month-old photo) until very old (see 23-month-old photo). Aged mdx mice are also prone to rhabdomyosarcoma (a tumor of muscle origin; red arrow). Utrophin/dystrophin and integrin/dystrophin double-knockout (dko) mice are much smaller than the age-matched wild-type (BL10 and BL6) mice. A 5-month-old affected dog shows limb muscle atrophy and is reluctant to exercise. At the age of 2 years old, the affected dog displays severe clinical disease, whereas its normal sibling remains healthy. (B) Lifespan comparison among affected humans, affected dogs and various mouse models.

Fig. 4.

Structure of abbreviated dystrophins. (A) Naturally occurring dystrophin isoforms. In the topmost schematic, blue boxes denote exons. The full-length dystrophin (Dp427) transcripts have three isoforms, including brain Dp427 (B), muscle Dp427 (M) and Purkinje cell Dp427 (P). Smaller dystrophin isoforms are produced from promoters located in different introns (intron positions are marked for each isoform). Dp260 is expressed in the retina, Dp140 in the brain and kidney, Dp116 in Schwann cells, and Dp71 and Dp40 are expressed from the same promoter except Dp71 is ubiquitously expressed whereas Dp40 only exists in the brain. Except for Dp140, all other dystrophin isoforms have unique N-terminal sequences not present in the full-length protein. (B) Structure of representative mini- and micro-dystrophins. The full-length dystrophin protein is shown uppermost, and features the same terminology as that used in Fig. 1.

Fig. 5.

Multiple-exon skipping. The uppermost diagram is the intron/exon structure of the dystrophin gene. Blue boxes denote exons. The top box shows the golden retriever muscular dystrophy dog (GRMD) mutation and exon skipping for GRMD. A point mutation in intron 6 alters normal splicing, and the resulting transcript (gray) is out-of-frame. Skipping exons 6, 7 and 8 yields an in-frame transcript. The bottom box shows the mdx52 mutation and exon skipping in mdx52. Deletion of exon 52 disrupts the reading frame and results in a premature stop. Removing exons 45 to 55 from the mutated transcript generates an in-frame transcript.