Skip to main content
SpringerLink
Log in
Book cover

Duchenne Muscular Dystrophy pp 185–192Cite as

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

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1687))

Abstract

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.

This is a preview of subscription content, log in via an institution.

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Hoffman EP, Brown RH Jr, Kunkel LM (1987) Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 51:919–928

    Article  CAS  PubMed  Google Scholar 

  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 45:498–506

    CAS  PubMed  PubMed Central  Google Scholar 

  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 226:445–449

    Article  CAS  PubMed  Google Scholar 

  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 98:42–47

    Article  CAS  PubMed  Google Scholar 

  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 9:1009–1014

    Article  CAS  PubMed  Google Scholar 

  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 12:175–177

    Article  CAS  PubMed  Google Scholar 

  7. Dowling JJ (2016) Eteplirsen therapy for Duchenne muscular dystrophy: skipping to the front of the line. Nat Rev Neurol 12:675–676

    Article  CAS  PubMed  Google Scholar 

  8. Young CS, Pyle AD (2016) Exon skipping therapy. Cell 167:1144

    Article  CAS  PubMed  Google Scholar 

  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 84:8463–8467

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Greenleaf WJ, Bolander ME, Sarkar G, Goldring MB, Greenleaf JF (1998) Artificial cavitation nuclei significantly enhance acoustically induced cell transfection. Ultrasound Med Biol 24:587–595

    Article  CAS  PubMed  Google Scholar 

  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 6:576–583

    Article  CAS  PubMed  Google Scholar 

  12. Duvshani-Eshet M, Machluf M (2005) Therapeutic ultrasound optimization for gene delivery: a key factor achieving nuclear DNA localization. J Control Release 108:513–528

    Article  CAS  PubMed  Google Scholar 

  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 9:372–380

    Article  CAS  PubMed  Google Scholar 

  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 105:1233–1239

    Article  CAS  PubMed  Google Scholar 

  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 229:423–428

    Article  PubMed  Google Scholar 

  16. Unger EC, Porter T, Culp W, Labell R, Matsunaga T, Zutshi R (2004) Therapeutic applications of lipid-coated microbubbles. Adv Drug Deliv Rev 56:1291–1314

    Article  CAS  PubMed  Google Scholar 

  17. Klibanov AL, Maruyama K, Torchilin VP, Huang L (1990) Amphipathic polyethyleneglycols effectively prolong the circulation time of liposomes. FEBS Lett 268:235–237

    Article  CAS  PubMed  Google Scholar 

  18. Blume G, Cevc G (1990) Liposomes for the sustained drug release in vivo. Biochim Biophys Acta 1029:91–97

    Article  CAS  PubMed  Google Scholar 

  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 1066:29–36

    Article  CAS  PubMed  Google Scholar 

  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 1128:44–49

    Article  CAS  PubMed  Google Scholar 

  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 117:130–136

    Article  CAS  PubMed  Google Scholar 

  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 132:124–130

    Article  CAS  PubMed  Google Scholar 

  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 11:1053–1061

    Article  CAS  PubMed  Google Scholar 

  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 12:1801–1811

    Article  Google Scholar 

  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 81:1189–1192

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgment

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.

Author information

Authors and Affiliations

  1. Department of Drug Delivery and Molecular Biopharmaceutics, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo, 192-0392, Japan

    Yoichi Negishi, Yuko Ishii, Kei Nirasawa, Eri Sasaki & Yoko Endo-Takahashi

  2. Laboratory of Drug and Gene Delivery Research, Faculty of Pharma-Sciences, Teikyo University, 2-11-1 Kaga, Itabashi-ku, Tokyo, 173-8605, Japan

    Ryo Suzuki & Kazuo Maruyama

Authors
  1. Yoichi Negishi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yuko Ishii
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kei Nirasawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Eri Sasaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yoko Endo-Takahashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ryo Suzuki
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kazuo Maruyama
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yoichi Negishi .

Editor information

Editors and Affiliations

  1. Istituto di Anatomia Umana e Biologia Cellulare, Università Cattolica del Sacro Cuore, Roma, Italy

    Camilla Bernardini

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Negishi, Y. et al. (2018). PMO Delivery System Using Bubble Liposomes and Ultrasound Exposure for Duchenne Muscular Dystrophy Treatment. In: Bernardini, C. (eds) Duchenne Muscular Dystrophy. Methods in Molecular Biology, vol 1687. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7374-3_13

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7374-3_13

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7373-6

  • Online ISBN: 978-1-4939-7374-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation