Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Research Article
  • Published:

Development of safe and efficient novel nonviral gene transfer using ultrasound: enhancement of transfection efficiency of naked plasmid DNA in skeletal muscle

Gene Therapy volume 9, pages 372–380 (2002)Cite this article

Abstract

Although clinical trials of stimulation of angiogenesis by transfection of angiogenic growth factors using naked plasmid DNA or adenoviral vector have been successful, there are still unresolved problems for human gene therapy such as low transfection efficiency and safety. From this viewpoint, it is necessary to develop safe and efficient novel nonviral gene transfer methods. As therapeutic ultrasound induces cell membrane permeabilization, ultrasound irradiation might increase the transfection efficiency of naked plasmid DNA into skeletal muscle. Thus, we examined the transfection efficiency of naked plasmid DNA using ultrasound irradiation with echo contrast microbubble (Optison) in vitro and in vivo experiments. First, we examined the feasibility of ultrasound-mediated transfection of naked plasmid DNA into skeletal muscle cells. Luciferase plasmid mixed with or without Optison was transfected into cultured human skeletal muscle cells using ultrasound (1 MHz; 0.4 W2) for 30 s. Interestingly, luciferase activity was markedly increased in cells treated with Optison, while little luciferase activity could be detected without Optison (P < 0.01). Electron microscopy demonstrated the transient formation of holes (less than 5 μM) in the cell surface, which could possibly explain the rapid migration of the transgene into the cells. Next, we studied the in vivo transfection efficiency of naked plasmid DNA using ultrasound with Optison into skeletal muscle. Two days after transfection, luciferase activity in skeletal muscle transfected with Optison using ultrasound was significantly increased about 10-fold as compared with plasmid alone. Successful transfection was also confirmed by β-galactosidase staining. Finally, we examined the feasibility of therapeutic angiogenesis using naked hepatocyte growth factor (HGF) plasmid in a rabbit ischemia model using the ultrasound–Optison method. Five weeks after transfection, the angiographic score and the number of capillary density in rabbits transfected with Optison using ultrasound was significantly increased as compared with HGF plasmid alone (P < 0.01), accompanied by a significant increase in blood flow and blood pressure ratio (P < 0.01). Overall, the ultrasound transfection method with Optison enhanced the transfection efficiency of naked plasmid DNA in vivo as well as in vitro. Transfection of HGF plasmid by the ultrasound-Optison method could be useful for safe clinical gene therapy to treat peripheral arterial disease without a viral vector system.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

Similar content being viewed by others

References

  1. Second European Consensus Document on Chronic Critical Leg Ischemia. Circulation 1991; 84 (4 Suppl.): 1–26

  2. Dormandy J. et al. Fate of the patient with chronic leg ischaemia. A review article J Cardiovasc Surg Torino 1989 30: 50 50

    CAS  Google Scholar 

  3. Rutherford R.B. et al. Suggested standards for reports dealing with lower extremity ischemia. Ad Hoc Committee on Reporting Standards. Society for Vascular Surgery/North American Chapter, International Society for Cardiovascular Surgery J Vasc Surg 1986 4: 80 80

    Article  Google Scholar 

  4. Isner J.M. et al. Clinical evidence of angiogenesis after arterial gene transfer of ph VEGF165 in patient with ischaemic limb Lancet 1996 348: 370 370

    Article  CAS  Google Scholar 

  5. Baumgartner I. et al. Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia Circulation 1998 97: 1114 1114

    Article  CAS  Google Scholar 

  6. Isner J.M. et al. Treatment of thromboangiitis obliterans (Buerger's disease) by intramuscular gene transfer of vascular endothelial growth factor: preliminary clinical results J Vasc Surg 1998 28: 964 964

    Article  CAS  Google Scholar 

  7. Baumgartner I. et al. Lower-extremity edema associated with gene transfer of naked DNA encoding vascular endothelial growth factor Ann Intern Med 2000 132: 880 880

    Article  CAS  Google Scholar 

  8. Losordo D.W. et al. Gene therapy for myocardial angiogenesis: initial clinical results with direct myocardial injection of phVEGF165 as sole therapy for myocardial ischemia Circulation 1998 98: 2800 2800

    Article  CAS  Google Scholar 

  9. Vale P.R. et al. Left ventricular electromechanical mapping to assess efficacy of phVEGF165 gene transfer for therapeutic angiogenesis in chronic myocardial ischemia Circulation 2000 102: 965 965

    Article  CAS  Google Scholar 

  10. Rosengart T.K. et al. Angiogenesis gene therapy: phase 1 assessment of direct intramyocardial administration of an adenovirus vector expressing VEGF121 cDNA to individuals with clinically significant severe coronary artery disease Circulation 1999 100: 468 468

    Article  CAS  Google Scholar 

  11. Rosengart T.K. et al. Six-month assessment of a phase 1 trial of angiogenic gene therapy for the treatment of coronary artery disease using direct intramyocardial administration of an adenovirus vector expressing the VEGF121 cDNA Ann Surg 1999 230: 466 466

    Article  CAS  Google Scholar 

  12. Taniyama Y. et al. Therapeutic angiogenesis induced by human hepatocyte growth factor gene in rat and rabbit hind limb ischemia models: preclinical study for treatment of peripheral arterial disease Gene Therapy 2000 8: 181 181

    Article  Google Scholar 

  13. Tabata H., Silver M., Isner J.M. . Arterial gene transfer of acidic fibroblast growth factor for therapeutic angiogenesis in vivo: critical role of secretion signal in use of naked DNA Cardiovasc Res 1997 35: 470 470

    Article  CAS  Google Scholar 

  14. Ueno H. et al. Adenovirus-mediated expression of the secreted form of basic fibroblast growth factor (FGF-2) induces cellular proliferation and angiogenesis in vivo Arterioscler Thromb Vasc Biol 1997 17: 2453 2453

    Article  CAS  Google Scholar 

  15. Takahashi J.C. et al. Adenovirus-mediated gene transfer of basic fibroblast growth factor induces in vitro angiogenesis Atherosclerosis 1997 132: 199 199

    Article  CAS  Google Scholar 

  16. Vincent K.A. et al. Angiogenesis is induced in a rabbit model of hindlimb ischemia by naked DNA encoding an HIF-1alpha/VP16 hybrid transcription factor Circulation 2000 102: 2255 2255

    Article  CAS  Google Scholar 

  17. Giordano F.J. et al. Intracoronary gene transfer of fibroblast growth factor-5 increases blood flow and contractile function in an ischemic region of the heart Nat Med 1996 2: 534 534

    Article  CAS  Google Scholar 

  18. Aoki M. et al. Angiogenesis induced by hepatocyte growth factor in non-infarcted myocardium and infarcted myocardium: up-regulation of essential transcription factor for angiogenesis, ets Gene Therapy 2000 7: 417 417

    Article  CAS  Google Scholar 

  19. Ueda H. et al. Gene transfection of hepatocyte growth factor attenuates reperfusion injury in the heart Ann Thorac Surg 1999 67: 1726 1726

    Article  CAS  Google Scholar 

  20. Safi J. Jr. et al. Adenovirus-mediated acidic fibroblast growth factor gene transfer induces angiogenesis in the nonischemic rabbit heart Microvasc Res 1999 58: 238 238

    Article  CAS  Google Scholar 

  21. Schwarz E.R. et al. Evaluation of the effects of intramyocardial injection of DNA expressing vascular endothelial growth factor (VEGF) in a myocardial infarction model in the rat – angiogenesis and angioma formation J Am Coll Cardiol 2000 35: 1323 1323

    Article  CAS  Google Scholar 

  22. Lazarous D.F. et al. Adenoviral-mediated gene transfer induces sustained pericardial VEGF expression in dogs: effect on myocardial angiogenesis Cardiovasc Res 1999 44: 294 294

    Article  CAS  Google Scholar 

  23. Lee L.Y. et al. Focal angiogen therapy using intramyocardial delivery of an adenovirus vector coding for vascular endothelial growth factor 121 Ann Thorac Surg 2000 69: 14 14

    Article  CAS  Google Scholar 

  24. Gowdak L.H. et al. Adenovirus-mediated VEGF(121) gene transfer stimulates angiogenesis in normoperfused skeletal muscle and preserves tissue perfusion after induction of ischemia Circulation 2000 102: 565 565

    Article  CAS  Google Scholar 

  25. Varenne O. et al. Percutaneous adenoviral gene transfer into porcine coronary arteries: is catheter-based gene delivery adapted to coronary circulation? Hum Gene Ther 1999 10: 1105 1105

    Article  CAS  Google Scholar 

  26. Barr E. et al. Efficient catheter-mediated gene transfer into the heart using replication-defective adenovirus Gene Therapy 1994 1: 51 51

    CAS  Google Scholar 

  27. Dzau V.J., Mann M.J., Morishita R., Kaneda Y. . Fusigenic viral liposome for gene therapy in cardiovascular diseases Proc Natl Acad Sci USA 1996 93: 11421 11421

    Article  CAS  Google Scholar 

  28. Marshall E. . Gene therapy death prompts review of adenovirus vector Science 1999 286: 2244 2244

    Article  CAS  Google Scholar 

  29. Aoki M. et al. Survival of grafts of genetically modified cardiac myocytes transfected with FITC-labeled oligodeoxynucleotides and β-galactosidase gene in non-infarcted area, but not myocardial infarcted area Gene Therapy 1997 4: 120 120

    Article  CAS  Google Scholar 

  30. Tyrrell M.R., Wolfe J.H. . Critical leg ischaemia: an appraisal of clinical definitions. Joint Vascular Research Group Br J Surg 1993 80: 177 177

    Article  CAS  Google Scholar 

  31. Eneroth M., Persson B.M. . Amputation for occlusive arterial disease. A prospective multicentre study of 177 amputees Int Orthop 1992 16: 383 383

    Article  CAS  Google Scholar 

  32. Dawson I. et al. Late outcomes of limb loss after failed infrainguinal bypass J Vasc Surg 1995 21: 613 613

    Article  CAS  Google Scholar 

  33. Losordo D.W. et al. Use of the rabbit ear artery to serially assess foreign protein secretion after site specific arterial gene transfer in vivo: evidence that anatomic identification of successful gene transfer may underestimate the potential magnitude of transgene expression Circulation 1994 89: 785 785

    Article  CAS  Google Scholar 

  34. Huber P.E., Pfisterer P. . In vitro and in vivo transfection of plasmid DNA in the Dunning prostate tumor R3327-AT1 is enhanced by focused ultrasound Gene Therapy 2000 7: 1516 1516

    Article  CAS  Google Scholar 

  35. Greenleaf W.J. et al. Artificial cavitation nuclei significantly enhance acoustically induced cell transfection Ultrasound Med Biol 1998 24: 587 587

    Article  CAS  Google Scholar 

  36. Manome Y., Nakamura M., Ohno T., Furuhata H. . Ultrasound facilitates transduction of naked plasmid DNA into colon carcinoma cells in vitro and in vivo Hum Gene Ther 2000 11: 1521 1521

    Article  CAS  Google Scholar 

  37. Tachibana K. et al. Induction of cell-membrane porosity by ultrasound Lancet 1999 353: 1409 1409

    Article  CAS  Google Scholar 

  38. Lawrie A. et al. Microbubble-enhanced ultrasound for vascular gene delivery Gene Therapy 2000 7: 2023 2023

    Article  CAS  Google Scholar 

  39. Tachibana K., Tachibana S. . Albumin microbubble echo-contrast material as an enhancer for ultrasound accelerated thrombolysis Circulation 1995 92: 1148 1148

    Article  CAS  Google Scholar 

  40. Podell S. et al. Physical and biochemical stability of Optison, an injectable ultrasound contrast agent Biotechnol Appl Biochem 1999 30: 213 213

    CAS  PubMed  Google Scholar 

  41. Aoki M. et al. Efficient in vivo gene transfer into heart in rat myocardial infarction model using HVJ (hemagglutinating virus of Japan)–liposome method J Mol Cell Cardiol 1997 29: 949 949

    Article  CAS  Google Scholar 

  42. Morishita R. et al. Therapeutic angiogenesis induced by human recombinant hepatocyte growth factor in rabbit hind limb ischemia model as ‘cytokine supplement therapy’ Hypertension 1999 33: 1379 1379

    Article  CAS  Google Scholar 

  43. Hayashi S. et al. Potential role of hepatocyte growth factor, a novel angiogenic growth factor, in peripheral arterial disease: down-regulation of HGF in response to hypoxia in vascular cells Circulation 1999 100: II301 II301

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was partially supported by grants from the Japan Health Sciences Foundation, a Grant-in-Aid from The Ministry of Public Health and Welfare, a Grant-in-Aid for the Development of Innovative Technology, a Grant-in-Aid from Japan Promotion of Science, and through Special Coordination Funds of the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government.

Author information

Authors and Affiliations

  1. Department of Geriatric Medicine, Graduate School of Medicine, Osaka University, Suita, Japan

    Y Taniyama, K Hiraoka, S Yamamoto, T Nakamura, T Ogihara & R Morishita

  2. Division of Gene Therapy Science, Graduate School of Medicine, Osaka University, Japan

    Y Taniyama, M Aoki, Y Kaneda & R Morishita

  3. First Department of Anatomy, Fukuoka University, School of Medicine, Hakata, Japan

    K Tachibana

  4. Division of Biochemistry, Department of Oncology, Biomedical Research Center, Graduate School of Medicine, Osaka University, Japan

    K Matsumoto

Authors
  1. Y Taniyama
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. K Tachibana
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. K Hiraoka
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. M Aoki
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. S Yamamoto
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. K Matsumoto
    View author publications

    You can also search for this author inPubMed Google Scholar

  7. T Nakamura
    View author publications

    You can also search for this author inPubMed Google Scholar

  8. T Ogihara
    View author publications

    You can also search for this author inPubMed Google Scholar

  9. Y Kaneda
    View author publications

    You can also search for this author inPubMed Google Scholar

  10. R Morishita
    View author publications

    You can also search for this author inPubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Taniyama, Y., Tachibana, K., Hiraoka, K. et al. 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 (2002). https://doi.org/10.1038/sj.gt.3301678

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.gt.3301678

Keywords

This article is cited by

Search

Advanced search

Quick links