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Dose-Dependent Restoration of Dystrophin Expression in Cardiac Muscle of Dystrophic Mice by Systemically Delivered Morpholino

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Dose-Dependent Restoration of Dystrophin Expression in Cardiac Muscle of Dystrophic Mice by Systemically Delivered Morpholino Gene Therapy (2010) 17, 132–140 & 2010 Macmillan Publishers Limited All rights reserved 0969-7128/10 $32.00 www.nature.com/gt ORIGINAL ARTICLE Dose-dependent restoration of dystrophin expression in cardiac muscle of dystrophic mice by systemically delivered morpholino B Wu, P Lu, E Benrashid, S Malik, J Ashar, TJ Doran and QL Lu McColl-Lockwood Laboratory for Muscular Dystrophy Laboratory, Neuromuscular/ALS Center, Department of Neurology, Carolinas Medical Center, Charlotte, NC, USA We have earlier shown that antisense morpholino oligomers rescues dystrophin expression dose dependently in both are able to restore dystrophin expression by systemic skeletal and cardiac muscles. Therapeutic levels of delivery in body-wide skeletal muscles of dystrophic mdx dystrophin were achieved in cardiac muscle albeit at higher mice. However, the levels of dystrophin expression vary doses than in skeletal muscles. Up to 50 and 30% normal considerably and, more importantly, no dystrophin levels of dystrophin were induced by single systemic delivery expression has been achieved in cardiac muscle. In this of3gkg–1 of morpholino in skeletal and cardiac muscles, study, we investigate the efficiency of morpholino-induced respectively. High doses of morpholino treatment reduced exon skipping in cardiomyoblasts and myocytes in vitro, and the serum levels of creatine kinase without clear toxicity. in cardiac muscle in vivo by dose escalation. We showed These findings suggest that effective rescue of dystrophin in that morpholino induces targeted exon skipping equally cardiac muscles can be achieved by morpholino for the effectively in both skeletal muscle myoblasts and treatment of Duchenne muscular dystrophy. cardiomyoblasts. Effective exon skipping was achieved in Gene Therapy (2010) 17, 132–140; doi:10.1038/gt.2009.120; cardiomyocytes in culture. In the mdx mice, morpholino published online 17 September 2009 Keywords: morpholino; exon skipping; DMD; heart Introduction appears not to be critical for its functions. For instance, deletion of exons from 17 to 49 was associated with only Duchenne muscular dystrophy (DMD) affects one in a mild clinical phenotype.6 Similarly, an artificially approximately 3500 male births and is characterized by constructed microdystrophin with deletion from exon rapid progression of muscle degeneration. The disease is 18 to 58 remains largely functional.7 caused by nonsense and frame-shift mutations in the Antisense oligonucleotide (AON)-mediated exon skip- DMD gene at Xp21.1, resulting in lack of dystrophin ping is able to restore the reading frame of dystrophin production.1 In-frame mutations in the dystrophin gene gene disrupted by DMD mutations. This can lead to the also cause a milder form of Becker muscular dystrophy production of truncated but functional dystrophin, and with expression of truncated but partially functional can reverse a DMD to a milder Becker muscular dystrophin proteins.2–4 The amount of dystrophin in dystrophy or near normal phenotypes.8–11 The thera- Becker muscular dystrophy varies, but can reach 30% or peutic potential of antisense therapy for DMD was higher of normal levels, in contrast to only a few initially showed in the dystrophic mdx mouse that revertant fibers in the majority of DMD.5 The DMD gene harbors a nonsense point mutation in the exon 23, consists of 79 exons spanning 42.3 million base pairs. leading to general absence of dystrophin in the muscles. The muscle form of dystrophin protein has 3685 amino Specifically designed 20O methyl phosphorothioate acids (427 kDa) that can be divided into four structural AONs delivered by intramuscular injections were able domains: amino terminal, rod, cysteine-rich and carboxy to skip the exon 23 effectively in tibialis anterior (TA) terminal domains. The majority (approximately 60%) of muscles and produce functional amount of proteins.8 the DMD mutations occur within the rod domain of The same 20O methyl phosphorothioate AON was shown dystrophin, which spans about half the length of the to induce dystrophin expression in body-wide muscles protein. However, the rod domain of the dystrophin gene when delivered systemically.9 More recently, regular systemic administration of the phosphorodiamidate Correspondence: Dr QL Lu, McColl-Lockwood Laboratory for morpholino oligomers targeting the same mouse dystro- Muscular Dystrophy Laboratory, Neuromuscular/ALS Center, phin exon 23 induced effective exon skipping and Department of Neurology, Carolinas Medical Center, 1000 Blythe produced functional levels of dystrophin in skeletal Boulevard, Charlotte, NC 28231, USA. 10 E-mail: [email protected] muscles. Systemic effect of morpholino oligomers for 11 Received 23 April 2009; revised 23 July 2009; accepted 5 August exon skipping has now been shown in dystrophic dog. 2009; published online 17 September 2009 The dog harbors a splice site mutation in intron 6, Dose-dependent restoration of dystrophin expression BWuet al 133 leading to exclusion of exon 7 from the mRNA transcript. toxicity. Considerable reduction in the serum levels of Treating the dogs weekly or biweekly with a cocktail of creatine kinase was detected in mice treated with three morpholino oligomers (120–200 mg kg–1) systemi- morpholinoE23 at 0.03 g kg–1 and above. Improved cally resulted in further removal of exon 6 and 8 with muscle pathology was observed in the diaphragm after extensive dystrophin expression and significant func- treatment with 1.5 g kg–1 or higher dose of morpholi- tional stabilization by several criteria.11 Furthermore, noE23. These findings suggest that effective rescue of effective restoration of reading frame and production of dystrophin in cardiac muscles can be achieved by dystrophin have now been achieved in Phase I clinical unmodified morpholino with a dose probably tolerable trials targeting human dystrophin exon 51 in muscles of for long-term treatment. DMD patients by local injections with both 20O methyl phosphorothioate AONs and morpholino oligomers (personal communication).12,13 Results DMD affects body-wide muscles including the cardiac muscle. As DMD patients live longer because of MorpholinoE23 induces skipping of exon 23 efficiently improved multidisciplinary patient care, rescuing dys- in cardiac myoblast and myocytes trophin expression in cardiac muscle becomes more To examine whether cardiac myoblasts and myocytes critical for their longevity and quality of life.14–20 More could be effectively induced to skip dystrophin exon 23, importantly, a recent study suggests that restoration of we established cardiac myoblast culture from young mdx dystrophin in skeletal muscles only may exacerbate the mice (2 weeks old) for morpholinoE23 transfection failure of heart function if dystrophin expression cannot (Figure 1a). The nature of cardiac myoblasts was be effectively restored in cardiac muscle.16 However, confirmed by immunocytochemistry for Troponin I both unmodified 20O methyl phosphorothioate AONs (Figure 1b). The cells and C2C12 myoblasts as a control and morpholino oligomers have been unable to induce were first incubated with 10 mgml–1 fluorescein isothio- effective exon skipping and dystrophin expression in cyanate-labeled morpholino and the delivery was mon- cardiac muscle. This could severely limit the therapeutic itored directly under fluorescence microscope. Efficient values of the treatment to DMD patients with the current delivery of the labeled morpholino was clearly demon- chemistries on clinic trials.12,13 The mechanism(s) for the strated, as almost all cells showed fluorescence with lower antisense effect in cardiac muscle is not under- stronger signal within the nuclear areas (Figure 2a). The stood, but one likely contributing factor is the lower cells were then incubated with morpholinoE23 specific delivery efficiency of antisense oligomers to cardio- for targeting exon 23 of the mouse dystrophin. Exon myocytes than to myofibers in skeletal muscles.10 skipping was examined 1, 2 and 4 days after treatment. In this study, we investigated the efficiency of RT-PCR showed targeted skipping of exon 23 in both cell morpholino-induced exon skipping in cardiomyoblasts types. Approximately 30 and 50% of exon 23 skipping and myocytes in vitro, and in cardiac muscles in vivo by were observed with 10 and 50 mgml–1 morpholinoE23 dose escalation. Morpholino oligomer (morpholinoE23) respectively, in both skeletal and cardiac myoblasts was able to induce targeted exon 23 skipping in both (Figure 2b). Maximum efficiency of exon skipping was skeletal and cardiac myoblasts with similar efficiency. observed 4 days after 50 mgml–1 morpholinoE23 treat- Effective exon skipping was also achieved in freshly ment in both types of myoblasts (Figure 2c). isolated cardiomyocytes. In the mdx mice, morpholi- We also examined exon 23 skipping in freshly noE23 rescued dystrophin expression dose dependently prepared cardiomyocytes. Exon 23 skipping was clearly in both skeletal and cardiac muscles. However, potential detected 2 days after treatment with 50 mgml–1 morpho- therapeutic levels of dystrophin were achieved in cardiac linoE23. As expected, the isolated cardiomyocytes only muscles at considerable higher dosage than in skeletal survived for a limited time in culture and no exon muscles. MorpholinoE23 treatment, up to 3 g kg–1 deliv- skipping was detected by day 5 after the isolation (Figure ered systemically, produced approximately 50 and 30% 2d). These results, therefore, suggest that specific exon normal levels of dystrophin in skeletal and cardiac skipping can be
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