Cancer cells are just one target of ultrashort pulsed electric fields. By lowering the power and altering their target, for example, we can also use the pulses in gene therapy. For instance, in proof-of-principle experiments, we used the pulses to insert new genes into chromosomes in the nuclei of cells—one of the key challenges of gene therapy.
For various reasons, the enormous potential of gene therapy has largely eluded medical researchers. Basically, the techniques have proven difficult for physicians to execute and dangerous for patients subjected to them. A prominent example of gene therapy in humans was a trial in Europe in the 1990s to treat severe combined immune deficiency syndrome. Commonly called ”bubble boy” syndrome, the disease is caused by an inherited defect in a single gene, which cripples the body’s defense against infection.
To combat the disease, doctors introduced a corrected copy of the gene into the nuclei of the children’s immune-cell-generating tissue. Encouragingly, the therapy defeated the disease, but unfortunately, three of the first 11 patients developed leukemia, caused by the way the new gene inserted itself into their existing DNA. Despite the setbacks, medical scientists have not given up on gene therapy for bubble-boy syndrome and are also trying it out for nerve damage from diabetes, heart failure, hemophilia, and a host of other diseases.
Another reason to insert new genes into people is to immunize them against a particular disease. Ordinary vaccines provide immunity because they are made up of crippled or dead versions of a disease-causing microbe. Exposure to a neutered version of the microbe enables our immune systems to recognize the chemical characteristics of the weakened microbe and to mount a fast, effective defense against the real version. However, the vaccine must be refrigerated, and if the microbe is not weakened enough—and this only very rarely occurs—it can cause rather than protect against the disease.
Partly because of these drawbacks, researchers have become intrigued with the idea of injecting a person with the DNA that codes for one of the infectious bug’s proteins. Some of the person’s own cells take up the DNA, produce the protein, and trigger the immune system to learn to recognize and defend against any microbe carrying that protein.
Among the chief technical difficulties with these DNA vaccines, as well as with gene therapies, is getting the DNA into cells. Simply injecting a dollop of DNA into someone is not good enough, because the cell membrane is such a strong barrier against DNA. One popular solution is to actually genetically engineer the DNA into a virus. Viruses infect us by ”sneaking” their genetic material through the cell membrane and tricking the cell into copying it. So scientists have sought to include the DNA they want into harmless viruses, with which they then infect the patient in the hopes that the virus will deliver the new gene to the place it needs to go.
The problem is that the virus can stitch the new gene into a bad spot in the cell’s own DNA, disrupting an important chemical program and causing disease, as happened when the immune-deficient children developed leukemia. Or the virus itself can cause a runaway immune system reaction that can kill the patient, as seems to have happened in a gene therapy trial several years ago at the University of Pennsylvania, in Philadelphia.
Pulsed electricity may offer a safer solution. First we can use strong, but rather long-lasting, electric fields to induce electroporation, the state we mentioned earlier in which the cell’s outer membrane temporarily becomes porous. This works, to a point, because although the new DNA can now enter the cell, it must still get past the nucleus’s membrane for the cell to decode it.
Because the ultrashort pulses we’ve worked with appear to affect subcellular membranes, such as the double membrane that bounds the nucleus, we figured they might help genes make it through that last step of their journey by opening pores in the nuclear membrane. As a test, we tried to insert a certain gene from a jellyfish into bone marrow cells in a test tube. If this gene makes it into the nucleus and is decoded, it produces a protein that glows green.
By itself, electroporation improved the amount of the gene that was taken into the cells’ nuclei by 260 percent, as measured by the number of cells glowing and the strength of the green glow. But following electroporation with a nanoseconds-long pulse aimed at opening the cells’ nuclei increased gene uptake by a whopping 900 percent—potentially enough to improve the efficiency and safety of gene therapies or DNA vaccinations.
The list of effects that scientists have achieved using nanoseconds-long pulses is growing rapidly, though their actual use as a medical treatment is still years away. For example, brief pulses cause platelets, cellular fragments in the blood, to begin the complicated cascade of steps needed to form clots. Though the experiments were performed in a test tube rather than on a human being, we hope the effect might one day be used in healing wounds.
In other research, E. Stephen Buescher, a professor of pediatrics at Eastern Virginia Medical School, did a fascinating set of experiments with white blood cells that also might ultimately help heal wounds. In it, he observed the effect of ultrashort pulses on the release of calcium inside cells from internal stores. Calcium acts as a kind of signal transducer in many cells, translating an external signal such as a hormone into some cellular action, such as manufacturing a protein.
In a type of white blood cell whose purpose is to seek out foreign material and digest it, for example, the release of calcium allows the cell to follow an invader’s chemical trail. When Buescher subjected these cells, called leukocytes, to nanoseconds-long, 12-kV/cm electric fields, the cells immediately, but briefly, spilled calcium from their internal stores into their own cytosol. In experiments where the cells were actively crawling over a microscope slide, hot on the simulated trail of an invader, pulsing stopped them in their tracks and then sent them marching off in the direction of the electric field. One day doctors might use such an effect to recruit immune cells to the site of an infection.
The list of cells and the effects of pulsed power on them goes on and will only get longer as more laboratories begin work in bioelectrics. Scientists at Kumamoto University, in Japan, for example, are studying the subcellular effects of high-power RF pulses. Those at Karlsruhe University, in Germany, are testing nanopulses for killing bacteria. And researchers at the University of Southern California are studying how the pulses cause dying cells to signal other cells to consume them. Whether or not pulsed power becomes a cancer treatment, a gene therapy technology, or an infection fighter, ultrashort electric fields have already proved a powerful research tool. And the mark they ultimately make on medicine may be in allowing scientists unprecedented access to the internal workings of cells.
Still, we hope for more practical—and potentially lucrative—possibilities. While treatments for cancer and genetic diseases would be revolutionary, somewhat more prosaic applications are in the offing. We at Old Dominion University have recently used nanosecond pulsed electric fields to destroy fat cells. Think of it as electric liposuction. Hey, if it helps pay for the research needed to fight dread diseases, we’re all for it.
About the Authors
Karl H. Schoenbach, an IEEE Fellow, holds the Frank Batten Endowed Chair in Bioelectric Engineering at Old Dominion University, in Norfolk, Va. There he directs the Frank Reidy Research Center for Bioelectrics.
Richard Nuccitelli is a biophysicist at Frank Reidy who has studied the role of ion currents and ion concentration changes in the regulation of cell physiology for 30 years. He was the lead investigator on the melanoma project.
Steven J. Beebe is a faculty member in the department of physiological sciences and pediatrics at Eastern Virginia Medical School, in Norfolk, and is on the staff at Frank Reidy. He has studied mechanisms for signal transduction and apoptosis regulation for decades.
The authors would like to thank Peter F. Blackmore, E. Stephen Buescher, Ravindra P. Joshi, Juergen F. Kolb, and R. James Swanson.
To Probe Further
Proceedings of the IEEE devoted its July 2004 issue to pulsed power technology and its applications. The issue includes a more detailed look at bioelectrics: ”Ultrashort Electrical Pulses Open a New Gateway Into Biological Cells,” by Karl H. Schoenbach et al., pp. 112237.
For more on the effects of nanosecond pulses on cell biology, see ”Nanosecond Pulsed Electric Fields Modulate Cell Function Through Intracellular Signal Transduction Mechanisms,” by Stephen J. Beebe et al., Physiological Measurements , Vol. 25, 2004, pp. 107793.
For the latest on the authors’ experiments on melanoma, see ”Nanosecond Pulsed Electric Fields Cause Melanomas to Self-destruct,” by Richard Nuccitelli et al., Biochemical and Biophysical Research Communications , 5 May 2006, pp. 35160.