Could Exon Skipping Help Dystrophic Boys to Run, Hop, and Jump?

Rescue of information from mutated genes, as opposed to replacing them, is a particularly promising approach to therapy for Duchenne muscular dystrophy (DMD). Because this disease commonly arises from relatively small mutations in the very large dystrophin gene, most of the structural information is often left intact and available for use if the interfering effect of the mutated region could be removed from the gene transcript. Practical application of this idea in DMD patients has been moved an important step forward in the recent publication by Le Guiner et al.1 demonstrating that the principle can be applied to safely achieve therapeutically effective levels of dystrophin protein in the canine model of DMD. The majority of DMD mutations are deletions, duplications, or nonsense mutations that disrupt the open reading frame of the dystrophin gene, causing premature termination of translation. Because most occur in the region encoding the long rodlike part of the dystrophin protein that bridges the functionally essential N- and C-terminal regions, it is in many cases possible to remove exons from the messenger RNA transcript so as to restore open reading frame while still retaining much of the function of the full-length protein. Such selective elimination of exons, termed exon skipping, has been investigated mainly via injection of antisense constructs designed to bind to regions associated with splicing of the target exon. These agents were based on the 2′-O-methyl or morpholino backbones, which are less susceptible to degradation than are natural polynucleotides. The approach was demonstrated to restore functionally effective levels of dystrophin on systemic delivery into the murine2 and canine models of DMD.3 In humans, too, the principle has been demonstrated by direct intramuscular injection of constructs targeting human exon 51 based on the 2′-O-methyl4 or morpholino backbones.5 However, systemic delivery of these same agents has achieved only marginal elevation of dystrophin in humans,6,7,8,9 probably as a result of inadequate dosages—a limitation imposed by toxicity in the case of 2′-O-methyl chemistry and by cost in the case of morpholinos, and in both cases reflecting inadequate delivery of the antisense agent via the systemic route.10 The approach used by Le Guiner et al. aims to maximize the efficiency of splicing by use of expression plasmids based on the U7snRNA modified to contain the antisense targeting sequences for the two exons (Figure 1)3,11 that must be removed to restore an open reading frame in the GRMD (golden retriever muscular dystrophy) dog. At the same time, efficient delivery of these plasmids is achieved by use of a myotropic adeno-associated virus (AAV) vector. The authors' report enlarges on previous demonstrations11,12 that the modified U7snRNA is amplified within the transduced myofibers and remains stably expressed, thus maintaining a constant supply of antisense sequences while the fiber remains alive. Herein lie both the strength and the main limitation of this approach. One clear advantage is the amount of the quasi-dystrophin produced, far greater than has been achieved with 2′-O-methyl or morpholino antisense reagents. Moreover, two or more antisense sequences can be packaged within a single vector, greatly facilitating the skipping of more than one exon, as is necessary in the GRMD animal11 and as may be desirable in some other instances. The main problem is that the first delivery elicits a neutralizing antibody response to the AAV vector that blocks subsequent administration. Figure 1 Rationale of exon skipping in the GRMD dog. Codon triplet boundaries often do not coincide with exon boundaries as in exon 6, 7, and 8 of the dystrophin gene (a); in the normal gene, the functional triplet codon is maintained by splicing the two nucleotides ... Because the quasi-dystrophin corresponds to that of a patient with Becker muscular dystrophy, it will not completely protect the muscle fiber from necrosis, and, when muscle fibers are ultimately lost, so too are the antisense plasmids they contain, leading to a progressive diminution of exon skipping.11,13 Both the 2′-O-methyl and morpholino reagents are also subject to loss, both from necrotic muscle fibers and by diffusion from healthy fibers. But these agents can, in principle, be readministered to maintain functional levels.14,15 An area of further concern is the possibility of a cell-mediated immune response against antigens associated with the AAV vector, although no evidence of this has been found so far in the dog,11 or perhaps against peptides in the newly produced dystrophin, especially that arising from the new exon junctions that might not be expressed in the occasional dystrophin-positive revertant muscle fiber.16 There is also a practical limitation on this approach because of the lack of access of the vector to the musculature above the tourniquet; limb-girdle muscles especially are important for ambulation and other day-to-day functions for a boy with DMD and would be excluded from the high-pressure delivery. Although the principal objective of the work by Le Guiner et al. was to establish safety and efficacy of the AAV-U7snRNA approach, preliminary to a human trial on treatment of boys with DMD, it also produced some interesting incidental information. The high levels of dystrophin produced by this protocol give great hope of normalizing muscle structure and function. Indeed, the authors found evidence of near-rectification of muscle histopathology by dystrophin expression at ≥30% of normal levels and restoration of muscle strength at ≥40%. These data provide experimental support for arguments advanced in the debate over the results of current human trials of antisense reagents, namely, that the levels achieved are too low to be of significant functional value and that doses must be escalated considerably if we are to reach a point where functional improvement can be correlated with amounts of dystrophin.9,10,17 Among the less contentious views on the topic is the idea that broad distribution of dystrophin is a major contributor to its beneficial effect. This view springs largely from the conventional model of the function of dystrophin as reinforcing the muscle surface against local work-induced trauma, by organizing a structural link between the internal cytoskeleton of the fiber and the overlying basement membrane. This notion is brought into question by the revelation of Le Guiner and colleagues' investigation that near-normal structure and function are achieved in muscles where ~60% of fiber surface does not contain dystrophin; this fraction of the surface should remain susceptible to mechanical trauma and continue to initiate myonecrotic lesions with ensuing muscle fiber loss and inflammation. Because this does not seem to be the case in the treated dog limbs, this simple local mechanical role of dystrophin and its associated complex would appear not to be the limiting determinant of muscle fiber health, and we should perhaps look to more diffuse mechanisms, such as regulation of calcium homeostasis, as playing a critical role in the maintenance by dystrophin of muscle fiber integrity. For a mechanical tissue, such as skeletal muscle, the stress–strain relationships do not translate reliably across the >103-fold difference between humans and mice.18 For this reason, the report by Le Guiner et al. of performing preclinical investigations on an animal of a size similar to that of a human is reassuring. Currently, exon skipping appears to be the most promising of potential therapies for DMD, but there is some debate as to the best route to achieving it. The protocol used by Le Guiner and colleagues demonstrates the efficiency and safety in the medium term of the AAV-U7snRNA approach for rescue of function in muscles of a single limb and provides a good basis for moving into similar studies in humans. For mutations for which exon skipping could simulate the specific in-frame deletions seen in some almost-asymptomatic patients with the Becker form of muscular dystrophy, the muscle fibers may be sufficiently conserved to preserve their content of therapeutic plasmid and give prolonged benefit, but this would not apply equally to all mutations.19 It is important, therefore, pending well-authenticated protocols of effective readministration of AAV vectors in humans, to continue investigation of other exon-skipping agents that could be readministered chronically and search for methods of achieving their better delivery.