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Which US Microbubble Contrast Agent Is Best for Gene Therapy?

Imaging Sciences Department, Imperial College, Hammersmith Hospital Campus, London W12 0HS, England m.blomley@imperial.ac.uk

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The use of microbubbles and ultrasound shows promise for gene therapy. Early data suggest skeletal muscle as a promising target for microbubble ultrasound, but work needs to be done to develop the technique and clarify the underlying mechanisms. In this issue of Radiology, Li and colleagues provide intriguing data that show that the type of microbubbles used may be of crucial importance (1).

The Science

Despite an immense research investment (over 600 registered clinical trials: wiley.co.uk/genetherapy/clinical/), the development of gene therapy as a clinical tool has been frustratingly slow. The biggest single barrier is safe and efficient delivery of DNA to the target cells. Both viral and nonviral methods have been developed. Viruses can be efficient, but there are numerous problems associated with them, including immunogenicity and potential mutagenicity (2). Physical methods such as electroporation offer an alternative by creating transient increases in membrane permeability. This allows the use of safer nonviral plasmid or naked DNA, but unfortunately the efficiency of such methods is generally rather low, and it is unlikely that current methods could safely be applied to clinical practice (2).

Microbubble ultrasound offers a promising alternative. It has been known since the 1980s that ultrasound can enhance gene transfer by increasing cell permeability (3). It is noteworthy that the second and corresponding author, Katsuro Tachibana, is a pioneer of this approach (4,5). This sonoporation is potentiated by gas-filled microbubbles, which were originally developed as ultrasound contrast agents. It is believed that the main mechanism is that microbubbles act to focus ultrasound energy by lowering the threshold for ultrasound bioeffects.

Li and colleagues studied three microbubbles, all of which have been developed for patient use, with different shell and gas contents. They used a physiotherapy ultrasound system and worked with plasmid DNA encoding a nontherapeutic reporter gene (a gene that encodes an easily detectable protein marker). They first studied the effects on cells in suspension. They found that Optison (albumin shell with a perfluorocarbon gas; Nycomed-Amersham, Oslo, Norway) behaved differently than the air-based agents Albunex (Molecular Biosystems, San Diego, Calif, distributed by Mallinckrodt, St Louis, Mo) and Levovist (Schering, Berlin, Germany). While Optison was much more effective at killing cells than were the other agents, when the concentration was reduced to correct for this, it was eight times better at transfection (ie, uptake of DNA into muscle fibers with consequent expression of the reporter gene). They then injected microbubbles with DNA into the quadriceps muscle of mice and applied ultrasound. With Optison, a marked increase was seen in the efficiency of transfection, while no difference was seen by using the other microbubbles or ultrasound alone.

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The Practice

Clinical use.—Microbubble ultrasound strategies for gene therapy are in an early stage, and more preclinical development must precede any ethical patient study. The work described in this study needs to be extended to therapeutic gene transfer, and for clinical use an intravenous injection strategy would be greatly preferable. Although transfer efficiency was increased by an order of magnitude, this is still substantially below a level that would be useful to treat diseases such as Duchenne muscular dystrophy. However, the potential of microbubble ultrasound has only just started to be realized, and it is certainly plausible that these technologies could be applied to this and other organ targets in humans within the next few years. From a clinician’s perspective, the fact that commercially available microbubbles were used may be helpful.

Future opportunities and challenges.—As the authors point out, there are ways in which the effectiveness of these approaches could be improved. Microbubbles can be engineered to carry antibodies or other ligands or DNA to target tissues (8). This creates the exciting possibility of using them as drug delivery vehicles, as well as focusers of acoustic cavitation, and in principle allow for a point and shoot approach as they accumulate in the target tissue and ultrasound is then applied.

Summary

Using a model of interstitial injection of plasmid DNA into muscle, the authors have shown a marked difference between perfluorocarbon and air-containing microbubbles. Further work to explain these differences would be of great value in developing microbubble ultrasound for gene therapy.

FOOTNOTES

See also the article by Li et al in this issue.

REFERENCES

1. Li T, Tachibana K, Kuroki M, Kuroki M. Gene transfer with echo-enhanced contrast agents: comparison between Albunex, Optison, and Levovist in mice—initial results. Radiology 2003; 229:423-428.[Abstract/Free Full Text]
2. Thomas CE, Ehrhardt A, Kay MA. Progress and problems with the use of viral vectors for gene therapy. Nat Rev Genet 2003; 4:346-358.[CrossRef][Medline]
3. Fechheimer M, Boylan JF, Parker S, Sisken JE, Patel GL, Zimmer SG. Transfection of mammalian cells with plasmid DNA by scrape loading and sonication loading. Proc Natl Acad Sci USA 1987; 84:8463-8467.[Abstract/Free Full Text]
4. Tachibana K, Uchida T, Ogawa K, Yamashita N, Tamura K. Induction of cell-membrane porosity by ultrasound. Lancet 1999; 353:1409.
5. 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 2002; 9:372-380.[CrossRef][Medline]
6. Lu QL, Liang HD, Partridge T, Blomley MJ. Microbubble ultrasound improves the efficiency of gene transduction in skeletal muscle in vivo with reduced tissue damage. Gene Ther 2003; 10:396-405.[CrossRef][Medline]
7. Danialou G, Comtois AS, Dudley RW, et al. Ultrasound increases plasmid-mediated gene transfer to dystrophic muscles without collateral damage. Mol Ther 2002; 6:687-693.[CrossRef][Medline]
8. Leong-Poi H, Christiansen J, Klibanov AL, Kaul S, Lindner JR. Noninvasive assessment of angiogenesis by ultrasound and microbubbles targeted to alpha(v)-integrins. Circulation 2003; 107:455-460.[Abstract/Free Full Text]

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				V R Stewart and P S Sidhu New directions in ultrasound: microbubble contrast. Br. J. Radiol., March 1, 2006; 79(939): 188 - 194. [Full Text] [PDF]

				S. Sonoda, K. Tachibana, E. Uchino, A. Okubo, M. Yamamoto, K. Sakoda, T. Hisatomi, K.-H. Sonoda, Y. Negishi, Y. Izumi, et al. Gene Transfer to Corneal Epithelium and Keratocytes Mediated by Ultrasound with Microbubbles Invest. Ophthalmol. Vis. Sci., February 1, 2006; 47(2): 558 - 564. [Abstract] [Full Text] [PDF]

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