PMO Delivery System Using Bubble Liposomes and Ultrasound Exposure for Duchenne Muscular Dystrophy Treatment

  • Yoichi Negishi
  • Yuko Ishii
  • Kei Nirasawa
  • Eri Sasaki
  • Yoko Endo-Takahashi
  • Ryo Suzuki
  • Kazuo Maruyama
Part of the Methods in Molecular Biology book series (MIMB, volume 1687)


Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by progressive muscle degeneration, caused by nonsense or frameshift mutations in the dystrophin (DMD) gene. Antisense oligonucleotides can be used to induce specific exon skipping; recently, a phosphorodiamidate morpholino oligomer (PMO) has been approved for clinical use in DMD. However, an efficient PMO delivery strategy is required to improve the therapeutic efficacy in DMD patients. We previously developed polyethylene glycol (PEG)-modified liposomes containing ultrasound contrast gas, “Bubble liposomes” (BLs), and found that the combination of BLs with ultrasound exposure is a useful gene delivery tool. Here, we describe an efficient PMO delivery strategy using the combination of BLs and ultrasound exposure to treat muscles in a DMD mouse model (mdx). This ultrasound-mediated BL technique can increase the PMO-mediated exon-skipping efficiency, leading to significantly increased dystrophin expression. Thus, the combination of BLs and ultrasound exposure may be a feasible PMO delivery method to improve therapeutic efficacy and reduce the PMO dosage for DMD treatment.

Key words

Antisense oligonucleotides Exon skipping PMO Duchenne muscular dystrophy (DMD) Bubble liposome Ultrasound Delivery 



This study was supported in part by Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science and Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture, Japan.


  1. 1.
    Hoffman EP, Brown RH Jr, Kunkel LM (1987) Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 51:919–928CrossRefPubMedGoogle Scholar
  2. 2.
    Koenig M, Beggs AH, Moyer M et al (1989) The molecular basis for Duchenne versus Becker muscular dystrophy: correlation of severity with type of deletion. Am J Hum Genet 45:498–506PubMedPubMedCentralGoogle Scholar
  3. 3.
    Pramono ZA, Takeshima Y, Alimsardjono H, Ishii A, Takeda S, Matsuo M (1996) Induction of exon skipping of the dystrophin transcript in lymphoblastoid cells by transfecting an antisense oligodeoxynucleotide complementary to an exon recognition sequence. Biochem Biophys Res Commun 226:445–449CrossRefPubMedGoogle Scholar
  4. 4.
    Mann CJ, Honeyman K, Cheng AJ, Ly T, Lloyd F, Fletcher S, Morgan JE, Partridge TA, Wilton SD (2001) Antisense induced exon skipping and synthesis of dystrophin in the mdx mouse. Proc Natl Acad Sci U S A 98:42–47CrossRefPubMedGoogle Scholar
  5. 5.
    Lu QL, Mann CJ, Lou F, Bou-Gharios G, Morris GE, Xue SA, Fletcher S, Partridge TA, Wilton SD (2003) Functional amounts of dystrophin produced by skipping the mutated exon in the mdx dystrophic mouse. Nat Med 9:1009–1014CrossRefPubMedGoogle Scholar
  6. 6.
    Alter J, Lou F, Rabinowitz A, Yin H, Rosenfeld J, Wilton SD, Partridge TA, Lu QL (2006) Systemic delivery of morpholino oligonucleotide restores dystrophin expression body wide and improves dystrophic pathology. Nat Med 12:175–177CrossRefPubMedGoogle Scholar
  7. 7.
    Dowling JJ (2016) Eteplirsen therapy for Duchenne muscular dystrophy: skipping to the front of the line. Nat Rev Neurol 12:675–676CrossRefPubMedGoogle Scholar
  8. 8.
    Young CS, Pyle AD (2016) Exon skipping therapy. Cell 167:1144CrossRefPubMedGoogle Scholar
  9. 9.
    Fechheimer M, Boylan JF, Parker S, Sisken JE, Patel GL, Zimmer SG (1987) Transfection of mammalian cells with plasmid DNA by scrape loading and sonication loading. Proc Natl Acad Sci U S A 84:8463–8467CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Greenleaf WJ, Bolander ME, Sarkar G, Goldring MB, Greenleaf JF (1998) Artificial cavitation nuclei significantly enhance acoustically induced cell transfection. Ultrasound Med Biol 24:587–595CrossRefPubMedGoogle Scholar
  11. 11.
    Schratzberger P, Krainin JG, Schratzberger G, Silver M, Ma H, Kearney M, Zuk RF, Brisken AF, Losordo DW, Isner JM (2002) Transcutaneous ultrasound augments naked DNA transfection of skeletal muscle. Mol Ther 6:576–583CrossRefPubMedGoogle Scholar
  12. 12.
    Duvshani-Eshet M, Machluf M (2005) Therapeutic ultrasound optimization for gene delivery: a key factor achieving nuclear DNA localization. J Control Release 108:513–528CrossRefPubMedGoogle Scholar
  13. 13.
    Taniyama Y, Tachibana K, Hiraoka K, Aoki M, Yamamoto S, Matsumoto K, Nakamura T, Ogihara T, Kaneda Y, Morishita R (2002) Development of safe and efficient novel nonviral gene transfer using ultrasound: enhancement of transfection efficiency of naked plasmid DNA in skeletal muscle. Gene Ther 9:372–380CrossRefPubMedGoogle Scholar
  14. 14.
    Taniyama Y, Tachibana K, Hiraoka K, Namba T, Yamasaki K, Hashiya N, Aoki M, Ogihara T, Yasufumi K, Morishita R (2002) Local delivery of plasmid DNA into rat carotid artery using ultrasound. Circulation 105:1233–1239CrossRefPubMedGoogle Scholar
  15. 15.
    Li T, Tachibana K, Kuroki M, Kuroki M (2003) Gene transfer with echo-enhanced contrast agents: comparison between Albunex, Optison, and Levovist in mice–initial results. Radiology 229:423–428CrossRefPubMedGoogle Scholar
  16. 16.
    Unger EC, Porter T, Culp W, Labell R, Matsunaga T, Zutshi R (2004) Therapeutic applications of lipid-coated microbubbles. Adv Drug Deliv Rev 56:1291–1314CrossRefPubMedGoogle Scholar
  17. 17.
    Klibanov AL, Maruyama K, Torchilin VP, Huang L (1990) Amphipathic polyethyleneglycols effectively prolong the circulation time of liposomes. FEBS Lett 268:235–237CrossRefPubMedGoogle Scholar
  18. 18.
    Blume G, Cevc G (1990) Liposomes for the sustained drug release in vivo. Biochim Biophys Acta 1029:91–97CrossRefPubMedGoogle Scholar
  19. 19.
    Allen TM, Hansen C, Martin F, Redemann C, Yau-Young A (1991) Liposomes containing synthetic lipid derivatives of poly(ethylene glycol) show prolonged circulation half-lives in vivo. Biochim Biophys Acta 1066:29–36CrossRefPubMedGoogle Scholar
  20. 20.
    Maruyama K, Yuda T, Okamoto A, Kojima S, Suginaka A, Iwatsuru M (1992) Prolonged circulation time in vivo of large unilamellar liposomes composed of distearoyl phosphatidylcholine and cholesterol containing amphipathic poly(ethylene glycol). Biochim Biophys Acta 1128:44–49CrossRefPubMedGoogle Scholar
  21. 21.
    Suzuki R, Takizawa T, Negishi Y, Hagisawa K, Tanaka K, Sawamura K, Utoguchi N, Nishioka T, Maruyama K (2007) Gene delivery by combination of novel liposomal bubbles with perfluoropropane and ultrasound. J Control Release 117:130–136CrossRefPubMedGoogle Scholar
  22. 22.
    Negishi Y, Endo Y, Fukuyama T, Suzuki R, Takizawa T, Omata D, Maruyama K, Aramaki Y (2008) Delivery of siRNA into the cytoplasm by liposomal bubbles and ultrasound. J Control Release 132:124–130CrossRefPubMedGoogle Scholar
  23. 23.
    Negishi Y, Ishii Y, Shiono H, Akiyama S, Sekine S, Kojima T, Mayama S, Kikuchi T, Hamano N, Endo-Takahashi Y, Suzuki R, Maruyama K, Aramaki Y (2014) Bubble liposomes and ultrasound exposure improve localized morpholino oligomer delivery into the skeletal muscles of dystrophic mdx mice. Mol Pharm 11:1053–1061CrossRefPubMedGoogle Scholar
  24. 24.
    Gebski BL, Mann CJ, Fletcher S, Wilton SD (2006) Morpholino antisense oligonucleotide induced dystrophin exon 23 skipping in mdx mouse muscle. Hum Mol Genet 12:1801–1811CrossRefGoogle Scholar
  25. 25.
    Bulfield G, Siller WG, Wight PA, Moore KJ (1984) X chromosome-linked muscular dystrophy (mdx) in the mouse. Proc Natl Acad Sci U S A 81:1189–1192CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Yoichi Negishi
    • 1
  • Yuko Ishii
    • 1
  • Kei Nirasawa
    • 1
  • Eri Sasaki
    • 1
  • Yoko Endo-Takahashi
    • 1
  • Ryo Suzuki
    • 2
  • Kazuo Maruyama
    • 2
  1. 1.Department of Drug Delivery and Molecular Biopharmaceutics, School of PharmacyTokyo University of Pharmacy and Life SciencesHachiojiJapan
  2. 2.Laboratory of Drug and Gene Delivery Research, Faculty of Pharma-SciencesTeikyo UniversityItabashi-kuJapan

Personalised recommendations