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
Sebahattin Cirak, Virginia Arechavala-Gomeza, Michela Guglieri, Lucy Feng, Silvia Torelli, Karen Anthony, Stephen Abbs, Maria Elena Garralda, John Bourke, Dominic J Wells, George Dickson, Matthew J A Wood, Steve D Wilton, Volker Straub, Ryszard Kole, Stephen B Shrewsbury, Caroline Sewry, Jennifer E Morgan, Kate Bushby, Francesco Muntoni, Sebahattin Cirak, Virginia Arechavala-Gomeza, Michela Guglieri, Lucy Feng, Silvia Torelli, Karen Anthony, Stephen Abbs, Maria Elena Garralda, John Bourke, Dominic J Wells, George Dickson, Matthew J A Wood, Steve D Wilton, Volker Straub, Ryszard Kole, Stephen B Shrewsbury, Caroline Sewry, Jennifer E Morgan, Kate Bushby, Francesco Muntoni
Abstract
Background: We report clinical safety and biochemical efficacy from a dose-ranging study of intravenously administered AVI-4658 phosphorodiamidate morpholino oligomer (PMO) in patients with Duchenne muscular dystrophy.
Method: We undertook an open-label, phase 2, dose-escalation study (0·5, 1·0, 2·0, 4·0, 10·0, and 20·0 mg/kg bodyweight) in ambulant patients with Duchenne muscular dystrophy aged 5-15 years with amenable deletions in DMD. Participants had a muscle biopsy before starting treatment and after 12 weekly intravenous infusions of AVI-4658. The primary study objective was to assess safety and tolerability of AVI-4658. The secondary objectives were pharmacokinetic properties and the ability of AVI-4658 to induce exon 51 skipping and dystrophin restoration by RT-PCR, immunohistochemistry, and immunoblotting. The study is registered, number NCT00844597.
Findings: 19 patients took part in the study. AVI-4658 was well tolerated with no drug-related serious adverse events. AVI-4658 induced exon 51 skipping in all cohorts and new dystrophin protein expression in a significant dose-dependent (p=0·0203), but variable, manner in boys from cohort 3 (dose 2 mg/kg) onwards. Seven patients responded to treatment, in whom mean dystrophin fluorescence intensity increased from 8·9% (95% CI 7·1-10·6) to 16·4% (10·8-22·0) of normal control after treatment (p=0·0287). The three patients with the greatest responses to treatment had 21%, 15%, and 55% dystrophin-positive fibres after treatment and these findings were confirmed with western blot, which showed an increase after treatment of protein levels from 2% to 18%, from 0·9% to 17%, and from 0% to 7·7% of normal muscle, respectively. The dystrophin-associated proteins α-sarcoglycan and neuronal nitric oxide synthase were also restored at the sarcolemma. Analysis of the inflammatory infiltrate indicated a reduction of cytotoxic T cells in the post-treatment muscle biopsies in the two high-dose cohorts.
Interpretation: The safety and biochemical efficacy that we present show the potential of AVI-4658 to become a disease-modifying drug for Duchenne muscular dystrophy.
Funding: UK Medical Research Council; AVI BioPharma.
Copyright © 2011 Elsevier Ltd. All rights reserved.
Figures
References
- Bushby K, Finkel R, Birnkrant DJ, for the DMD Care Considerations Working Group Diagnosis and management of Duchenne muscular dystrophy, part 1: diagnosis, and pharmacological and psychosocial management. Lancet Neurol. 2010;9:77–93.
- Hoffman EP, Brown RH, Jr, Kunkel LM. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell. 1987;51:919–928.
- Bushby KM, Gardner-Medwin D, Nicholson LV. The clinical, genetic and dystrophin characteristics of Becker muscular dystrophy. II. Correlation of phenotype with genetic and protein abnormalities. J Neurol. 1993;240:105–112.
- Sazani P, Graziewicz MA, Kole R. Splice switching oligonucleotides as potential therapeutics. In: Crooke ST, editor. Antisense drug technology, principles, strategies and applications. CBC Press; Boca Raton, FL, USA: 2008. pp. 89–114.
- Lu QL, Rabinowitz A, Chen YC. Systemic delivery of antisense oligoribonucleotide restores dystrophin expression in body-wide skeletal muscles. Proc Natl Acad Sci USA. 2005;102:198–203.
- Yokota T, Lu QL, Partridge T. Efficacy of systemic morpholino exon-skipping in Duchenne dystrophy dogs. Ann Neurol. 2009;65:667–676.
- Fletcher S, Honeyman K, Fall AM, Harding PL, Johnsen RD, Wilton SD. Dystrophin expression in the mdx mouse after localised and systemic administration of a morpholino antisense oligonucleotide. J Gene Med. 2006;8:207–216.
- van Deutekom JC, Janson AA, Ginjaar IB. Local dystrophin restoration with antisense oligonucleotide PRO051. N Engl J Med. 2007;357:2677–2686.
- Kinali M, Arechavala-Gomeza V, Feng L. 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;8:918–928.
- Goemans NM, Tulinius M, van den Akker JT. Systemic administration of PRO051 in Duchenne's muscular dystrophy. N Engl J Med. 2011;364:1513–1522.
- Arechavala-Gomeza V, Graham IR, Popplewell LJ. Comparative analysis of antisense oligonucleotide sequences for targeted skipping of exon 51 during dystrophin pre-mRNA splicing in human muscle. Hum Gene Ther. 2007;18:798–810.
- Nguyen TM, Morris GE. Use of epitope libraries to identify exon-specific monoclonal antibodies for characterization of altered dystrophins in muscular dystrophy. Am J Hum Genet. 1993;52:1057–1066.
- Arechavala-Gomeza V, Kinali M, Feng L. Immunohistological intensity measurements as a tool to assess sarcolemma-associated protein expression. Neuropathol Appl Neurobiol. 2010;36:265–274.
- Arechavala-Gomeza V, Kinali M, Feng L. Revertant fibres and dystrophin traces in Duchenne muscular dystrophy: implication for clinical trials. Neuromuscul Disord. 2010;20:295–301.
- Neri M, Torelli S, Brown S. Dystrophin levels as low as 30% are sufficient to avoid muscular dystrophy in the human. Neuromuscul Disord. 2007;17:913–918.
- Mazzone ES, Messina S, Vasco G. Reliability of the North Star Ambulatory Assessment in a multicentric setting. Neuromuscul Disord. 2009;19:458–461.
- Mayhew JE, Florence JM, Mayhew TP. Reliable surrogate outcome measures in multicenter clinical trials of Duchenne muscular dystrophy. Muscle Nerve. 2007;35:36–42.
- McDonald CM, Widman LM, Walsh DD, Walsh SA, Abresch RT. Use of step activity monitoring for continuous physical activity assessment in boys with Duchenne muscular dystrophy. Arch Phys Med Rehabil. 2005;86:802–808.
- McDonald CM, Henricson EK, Han JJ. The 6-minute walk test in Duchenne/Becker muscular dystrophy: longitudinal observations. Muscle Nerve. 2010;42:966–974.
- Lai Y, Thomas GD, Yue Y. Dystrophins carrying spectrin-like repeats 16 and 17 anchor nNOS to the sarcolemma and enhance exercise performance in a mouse model of muscular dystrophy. J Clin Invest. 2009;119:624–635.
- Krieger CC, Bhasin N, Tewari M. Exon-skipped dystrophins for treatment of Duchenne muscular dystrophy: mass spectrometry mapping of most exons and cooperative domain designs based on single molecule mechanics. Cytoskeleton (Hoboken) 2010;67:796–807.
- Pescatori M, Broccolini A, Minetti C. Gene expression profiling in the early phases of DMD: a constant molecular signature characterizes DMD muscle from early postnatal life throughout disease progression. FASEB J. 2007;21:1210–1226.
- Kobayashi YM, Rader EP, Crawford RW. Sarcolemma-localized nNOS is required to maintain activity after mild exercise. Nature. 2008;456:511–515.
- Arahata K, Engel AG. Monoclonal antibody analysis of mononuclear cells in myopathies. I: quantitation of subsets according to diagnosis and sites of accumulation and demonstration and counts of muscle fibers invaded by T cells. Ann Neurol. 1984;16:193–208.
- Muntoni F, Di Lenarda A, Porcu M. Dystrophin gene abnormalities in two patients with idiopathic dilated cardiomyopathy. Heart. 1997;78:608–612.
- Helderman-van den Enden AT, Straathof CS, Aartsma-Rus A. Becker muscular dystrophy patients with deletions around exon 51; a promising outlook for exon skipping therapy in Duchenne patients. Neuromuscul Disord. 2010;20:251–254.
- Morandi L, Mora M, Confalonieri V. Dystrophin characterization in BMD patients: correlation of abnormal protein with clinical phenotype. J Neurol Sci. 1995;132:146–155.
- Saengpattrachai M, Ray PN, Hawkins CE, Berzen A, Banwell BL. Grandpa and I have dystrophinopathy?: approach to asymptomatic hyperCKemia. Pediatr Neurol. 2006;35:145–149.
- Aoki Y, Nakamura A, Yokota T. In-frame dystrophin following exon 51-skipping improves muscle pathology and function in the exon 52-deficient mdx mouse. Mol Ther. 2011;18:1995–2005.
- Sazani P, Weller DL, Shrewsbury SB. Safety pharmacology and genotoxicity evaluation of AVI-4658. Int J Toxicol. 2010;29:143–156.
- Malerba A, Sharp PS, Graham IR. Chronic systemic therapy with low-dose morpholino oligomers ameliorates the pathology and normalizes locomotor behavior in mdx mice. Mol Ther. 2011;19:345–354.
- Kinali M, Arechavala-Gomeza V, Cirak S. Muscle histology vs MRI in Duchenne muscular dystrophy. Neurology. 2010;76:346–353.
- Garrood P, Hollingsworth KG, Eagle M. MR imaging in Duchenne muscular dystrophy: quantification of T1-weighted signal, contrast uptake, and the effects of exercise. J Magn Reson Imaging. 2009;30:1130–1138.
- Wu B, Xiao B, Cloer C. One-year treatment of morpholino antisense oligomer improves skeletal and cardiac muscle functions in dystrophic mdx mice. Mol Ther. 2010;19:576–583.
Source: PubMed