Abstract
Duchenne muscular dystrophy (DMD) is caused by mutations that disrupt the reading frame of the human DMD gene. Selective removal of exons flanking an out-of-frame DMD mutation can result in an in-frame mRNA transcript that may be translated into an internally deleted, Becker muscular dystrophy (BMD)-like, but functionally active dystrophin protein with therapeutic activity. Antisense oligonucleotides (AOs) can be designed to bind to complementary sequences in the targeted mRNA and modify pre-mRNA splicing to correct the reading frame of a mutated transcript so that gene expression is restored. AO-induced exon skipping producing functional truncated dystrophin exon has been demonstrated in animal models of DMD both in vitro and in vivo, and in DMD patient cells in vitro in culture, and in DMD muscle explants. More recently, AO-mediated exon skipping has been confirmed in DMD patients in Phase I clinical trials. However, it should be noted that personalized molecular medicine may be necessary, since the various reading frame-disrupting mutations are spread across the DMD gene. The different deletions that cause DMD would require skipping of different exons, which would require the optimization and clinical trial workup of many specific AOs. This chapter describes the methodologies available for the optimization of AOs, and in particular phosphorodiamidate morpholino oligomers (PMOs), for the targeted skipping of specific exons on the DMD gene.
Key words
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Monaco AP, Bertelson CJ, Liechti-Gallati S, Moser H, Kunkel LM. (1988) An explanation for the phenotypic differences between patients bearing partial deletions of the DMD locus. Genomics 2, 90–95.
Bertoni C. (2008) Clinical approaches in the treatment of Duchenne muscular dystrophy (DMD) using oligonucleotides. Front Biosci 13, 517–527.
Trollet C, Athanasopoulos T, Popplewell L, Malerba A, Dickson G. (2009) Gene therapy for muscular dystrophy: current progress and future prospects. Expert Opin Biol Ther 9, 849–866.
Aartsma-Rus A, Janson AA, Kaman WE, Bremmer-Bout M, Den Dennen JT, Baas F, et al. (2003) Therapeutic antisense-induced exon skipping in cultured muscle cells from six different DMD patients. Hum Mol Genet 12, 907–914.
van Deutekom JC, Bremmer-Bout M, Janson AA, Ginjaar IB, Baas F, den Dunnen JT, et al. (2001) Antisense-induced exon skipping restores dystrophin expression in DMD patient derived muscle cells. Hum Mol Genet 10, 1547–1554.
Arechavala-Gomeza V, Graham IR, Popplewell LJ, Adams AM, Aartsma-Rus A, Kinali M, et al. (2007) Comparative analysis of antisense oligonucleotide sequences for targeted skipping of exon 51 during dystrophin pre-mRNA splicing in human muscle. Hum Gene Ther 18, 798–810.
Aartsma-Rus A, Janson AA, Kaman WE, Bremmer-Bout M, den Dunnen JT, Baas F, et al. (2003) Therapeutic antisense-induced exon skipping in cultured muscle cells from six different DMD patients. Hum Mol Genet 12, 907–914.
Aartsma-Rus A, Janson AA, Kaman WE, Bremmer-Bout M, van Ommen GJ, den Dunnen JT et al. (2004) Antisense-induced multiexon skipping for Duchenne muscular dystrophy makes more sense. Am J Hum Genet 74, 83–92.
Aartsma-Rus A, Janson AA, van Ommen GJ, van Deutekom JCT. (2007) Antisense-induced exon skipping for duplications in Duchenne muscular dystrophy. BMC Med Genet 8, 43–51.
Bremmer-Bout M, Aartsma-Rus A, de Meijer EJ, Kaman WE, Janson AA, Vossen RH, et al. (2004) Targeted exon skipping in transgenic hDMD mice: a model for direct preclinical screening of human-specific antisense oligonucleotides. Mol Ther 10, 232–240.
Graham IR, Hill VJ, Manoharan M, Inamati GB, Dickson G. (2004) Towards a therapeutic inhibition of dystrophin exon 23 splicing in mdx mouse muscle induced by antisense oligoribonucleotides (splicomers): target sequence optimisation using oligonucleotide arrays. J Gene Med 6, 1149–1158.
Lu QL, Rabinowitz A, Chen YC, Yokota T, Yin H, Alter J, et al. (2005) Systemic delivery of antisense oligoribonucleotide restores dystrophin expression in body-wide skeletal muscles. Proc Natl Acad Sci U S A 102, 198–203.
Mann CJ, Honeyman K, Cheng AJ, Ly T, Lloyd F, Fletcher S, et al. (2001). Antisense-induced exon skipping and synthesis of dystrophin in the mdx mouse. Proc Natl Acad Sci U S A 98, 42–47.
Jearawiriyapaisarn N, Moulton HM, Buckley B, Roberts J, Sazani P, Fucharoen S, et al. (2008) Sustained dystrophin expression induced by peptide-conjugated morpholino oligomers in the muscles of mdx mice. Mol Ther 16, 1624–1629.
van Deutekom JC, Janson AA, Ginjaar IB, Franzhuzen WS, Aartsma-Rus A, Bremmer-Bout M, et al. (2007) Local antisense dystrophin restoration with antisense oligonucleotide PRO051. N Engl J Med 357, 2677–2687.
Kinali M, Arechavala-Gomeza V, Feng L, Cirak S, Hunt D, Adkin C, et al. (2009) 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 8, 918–928.
Arora V, Devi GR, Iversen PL. (2004) Neutrally charged phosphorodiamidate morpholino antisense oligomers: uptake, efficacy and pharmacokinetics. Curr Pharm Biotechnol 5, 431–439.
Heemskerk HA, de Winter CL, de Kimpe SJ, van Kuik-Romeijn P, Heuvelmans N, Platenburg GJ et al. (2009) In vivo comparison of 2′O-methyl-PS and morpholino antisense oligonucleotides for DMD exon skipping. J Gene Med 11, 257–266.
Gebski BL, Mann CJ, Fletcher S, Wilton SD. (2003) Morpholino antisense oligonucleotide induced dystrophin exon 23 skipping in mdx mouse muscle. Hum Mol Genet 12, 1801–1811.
Alter J, Lou F, Rabinowitz A, Yin H, Rosenfeld J, Wilton SD, et al. (2006) Systemic delivery of morpholino oligonucleotide restores dystrophin expression bodywide and improves dystrophic pathology. Nat Med 12, 175–177.
Malerba A, Thorogood FC, Dickson G, Graham IR. (2009) Dosing regimen has a significant impact on the efficiency of morpholino oligomer-induced exon skipping in mdx mice. Hum Gene Ther 20, 955–965.
McClorey G, Fall AM, Moulton HM, Iversen PL, Rasko JE, Ryan M, et al. (2006) Induced dystrophin exon skipping in human muscle explants. Neuromuscul Disord 16, 583–590.
McClorey G, Moulton HM, Iversen PL, Fletcher S, Wilton SD. (2006) Antisense oligonucleotide-induced exon skipping restores dystrophin expression in vitro in a canine model of DMD. Gene Ther 13, 1373–1381.
Aartsma-Rus A, Fokkema I, Verschuuren J, Ginjaar I, van Deutekom J, van Ommen GJ, et al. (2009) Theoretic applicability of antisense-mediated exon skipping for Duchenne muscular dystrophy mutations. Hum Mutat 30, 292–299.
Aartsma-Rus A, De Winter CL, Janson AAM, Kaman WE, van Ommen G-JB, Den Dunnen JT, et al. (2005) Functional analysis of 114 exon-internal AONs for targeted DMD exon skipping: indication for steric hindrance of SR protein binding sites. Oligonucleotides 15, 284–297.
Wilton SD, Fall AM, Harding PL, McClorey G, Coleman C, Fletcher S. (2007) Antisense oligonucleotide-induced exon skipping across the human dystrophin gene transcript. Mol Ther 15, 1288–1296.
Aartsma-Rus A, van Vliet L, Hirschi M, Janson AA, Heemskerk H, de Winter CL, et al. (2009) Guidelines for antisense oligonucleotide design and insight into splice-modulating mechanisms. Mol Ther 17, 548–553.
Popplewell LJ, Trollet C, Dickson G, Graham IR. (2009) Design of phosphorodiamidate morpholino oligomers (PMOs) for the induction of exon skipping of the human DMD gene. Mol Ther 17, 554–561.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Popplewell, L.J., Graham, I.R., Malerba, A., Dickson, G. (2011). Bioinformatic and Functional Optimization of Antisense Phosphorodiamidate Morpholino Oligomers (PMOs) for Therapeutic Modulation of RNA Splicing in Muscle. In: Duan, D. (eds) Muscle Gene Therapy. Methods in Molecular Biology, vol 709. Humana Press. https://doi.org/10.1007/978-1-61737-982-6_10
Download citation
DOI: https://doi.org/10.1007/978-1-61737-982-6_10
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61737-981-9
Online ISBN: 978-1-61737-982-6
eBook Packages: Springer Protocols