Which US Microbubble Contrast Agent Is Best for Gene Therapy?

The Setting

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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While fascinating, this work raises several questions. Although the authors’ explanation that perfluorocarbon microbubbles like Optison last longer in an ultrasound field may be important, this is unlikely to be the only explanation. Interestingly, authors of two recent studies in which a similar skeletal muscle model was used have suggested that Optison and Definity (Bristol-Myers Squibb, New York, NY) (both microbubbles containing perfluorocarbons) may potentiate gene therapy even without ultrasound (6,7).

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.