Could Exon Skipping Help Dystrophic Boys to Run, Hop, and Jump?
Authors: Terence Partridge
Journal: Molecular Therapy (2014)

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.1Le Guiner C Montus M Servais L Cherel Y Francois V Thibaud J-L et al.Forelimb treatment in a large cohort of dystrophic dogs supports delivery of a recombinant AAV for exon skipping in Duchenne patients.Mol Ther. 2014; 22: 1923-1935Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar 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 murine2Alter J Lou F Rabinowitz A Yin H Rosenfeld J Wilton SD et al.Systemic delivery of morpholino oligonucleotide restores dystrophin expression bodywide and improves dystrophic pathology.Nat Med. 2006; 12: 175-177Crossref PubMed Scopus (432) Google Scholar and canine models of DMD.3Yokota T Lu QL Partridge T Kobayashi M Nakamura A Takeda S et al.Efficacy of systemic morpholino exon-skipping in Duchenne dystrophy dogs.Ann Neurol. 2009; 65: 667-676Crossref PubMed Scopus (324) Google Scholar In humans, too, the principle has been demonstrated by direct intramuscular injection of constructs targeting human exon 51 based on the 2′-O-methyl4van Deutekom JC Janson AA Ginjaar IB Frankhuizen WS Aartsma-Rus A Bremmer-Bout M et al.Local dystrophin restoration with antisense oligonucleotide PRO051.N Engl J Med. 2007; 357: 2677-2686Crossref PubMed Scopus (699) Google Scholar or morpholino backbones.5Kinali M Arechavala-Gomeza V Feng L Cirak S Hunt D Adkin C et al.Local restoration of dystrophin expression with the morpholino oligomer AVI-4658 in Duchenne muscular dystrophy: a single-blind, placebo-controlled, dose-escalation, proof-of-concept study.Lancet Neurol. 2009; 8: 918-928Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar However, systemic delivery of these same agents has achieved only marginal elevation of dystrophin in humans,6Goemans NM Tulinius M van den Akker JT Burm BE Ekhart PF Heuvelmans N et al.Systemic administration of PRO051 in Duchenne's muscular dystrophy.N Engl J Med. 2011; 364: 1513-1522Crossref PubMed Scopus (595) Google Scholar,7Cirak S Arechavala-Gomeza V Guglieri M Feng L Torelli S Anthony K et al.Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study.Lancet. 2011; 378: 595-605Abstract Full Text Full Text PDF PubMed Scopus (699) Google Scholar,8Mendell JR Rodino-Klapac LR Sahenk Z Roush K Bird L Lowes LP et al.Eteplirsen for the treatment of Duchenne muscular dystrophy.Ann Neurol. 2013; 74: 637-647Crossref PubMed Scopus (538) Google Scholar,9Hoffman EP Connor EM Orphan drug development in muscular dystrophy: update on two large clinical trials of dystrophin rescue therapies.Discov Med. 2013; 16: 233-239PubMed Google Scholar 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.10Lu QL Cirak S Partridge T What can we learn from clinical trials of exon skipping for DMD?.Mol Ther Nucleic Acids. 2014; 3: e152Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar 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)3Yokota T Lu QL Partridge T Kobayashi M Nakamura A Takeda S et al.Efficacy of systemic morpholino exon-skipping in Duchenne dystrophy dogs.Ann Neurol. 2009; 65: 667-676Crossref PubMed Scopus (324) Google Scholar,11Vulin A Barthelemy I Goyenvalle A Thibaud JL Beley C Griffith G et al.Muscle function recovery in golden retriever muscular dystrophy after AAV1-U7 exon skipping.Mol Ther. 2012; 20: 2120-2133Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar 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 demonstrations11Vulin A Barthelemy I Goyenvalle A Thibaud JL Beley C Griffith G et al.Muscle function recovery in golden retriever muscular dystrophy after AAV1-U7 exon skipping.Mol Ther. 2012; 20: 2120-2133Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar,12Goyenvalle A Vulin A Fougerousse F Leturcq F Kaplan JC Garcia L et al.Rescue of dystrophic muscle through U7 snRNA-mediated exon skipping.Science. 2004; 306: 1796-1799Crossref PubMed Scopus (410) Google Scholar 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 animal11Vulin A Barthelemy I Goyenvalle A Thibaud JL Beley C Griffith G et al.Muscle function recovery in golden retriever muscular dystrophy after AAV1-U7 exon skipping.Mol Ther. 2012; 20: 2120-2133Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar 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. 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.11Vulin A Barthelemy I Goyenvalle A Thibaud JL Beley C Griffith G et al.Muscle function recovery in golden retriever muscular dystrophy after AAV1-U7 exon skipping.Mol Ther. 2012; 20: 2120-2133Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar,13Le Hir M Goyenvalle A Peccate C Precigout G Davies KE Voit T et al.AAV genome loss from dystrophic mouse muscles during AAV-U7 snRNA-mediated exon-skipping therapy.Mol Ther. 2013; 21: 1551-1558Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar 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.14Wu B Xiao B Cloer C Shaban M Sali A Lu P et al.One-year treatment of morpholino antisense oligomer improves skeletal and cardiac muscle functions in dystrophic mdx mice.Mol Ther. 2011; 19: 576-583Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar,15Malerba A Sharp PS Graham IR Arechavala-Gomeza V Foster K Muntoni F et al.Chronic systemic therapy with low-dose morpholino oligomers ameliorates the pathology and normalizes locomotor behavior in mdx mice.Mol Ther. 2011; 19: 345-354Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar 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,11Vulin A Barthelemy I Goyenvalle A Thibaud JL Beley C Griffith G et al.Muscle function recovery in golden retriever muscular dystrophy after AAV1-U7 exon skipping.Mol Ther. 2012; 20: 2120-2133Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar 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.16Flanigan KM Campbell K Viollet L Wang W Gomez AM Walker CM et al.Anti-dystrophin T cell responses in Duchenne muscular dystrophy: prevalence and a glucocorticoid treatment effect.Hum Gene Ther. 2013; 24: 797-806Crossref PubMed Scopus (82) Google Scholar 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.9Hoffman EP Connor EM Orphan drug development in muscular dystrophy: update on two large clinical trials of dystrophin rescue therapies.Discov Med. 2013; 16: 233-239PubMed Google Scholar,10Lu QL Cirak S Partridge T What can we learn from clinical trials of exon skipping for DMD?.Mol Ther Nucleic Acids. 2014; 3: e152Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar,17Hoffman EP McNally EM Exon-skipping therapy: a roadblock, detour, or bump in the road?.Sci Transl Med. 2014; 6: 230fs14Crossref PubMed Scopus (28) Google Scholar 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.18Partridge TA The mdx mouse model as a surrogate for Duchenne muscular dystrophy.FEBS J. 2013; 280: 4177-4186Crossref PubMed Scopus (129) Google Scholar 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.19Anthony K Arechavala-Gomeza V Ricotti V Torelli S Feng L Janghra N et al.Biochemical characterization of patients with in-frame or out-of-frame DMD deletions pertinent to exon 44 or 45 skipping.JAMA Neurol. 2014; 71: 32-40Crossref PubMed Scopus (52) Google Scholar 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. The author would like to thank Luis Garcia for valuable discussion.