Though one thousand warrior-filled ships were sent to the ancient city of Troy, the city's layout and impregnable walls saved it from conquest. The turning point, as related in Homer's Iliad, was the famous scheme devised by a shrewd Greek strategist.
The task of gene therapists is no less challenging. Armed with beneficial genes, the tools for the potential recovery of ailing patients, gene therapists must navigate them to the appropriate cells within a ten-trillion-cell body. The scientists then have to pass two barriers: They must smuggle the genes first into these cells, and then into the cell's inner sanctum, the nucleus, which houses the genetic material. Having tackled the first barrier, scientists are now pondering how to overcome the second. The nucleus's "walls"are lined with "sentries"that keep a watchful eye out for anything peculiar.
At first, viruses seemed well suited to the task because that is exactly what they have been doing for billions of years - penetrating the walls of the cell nuclei to maintain and propagate their genetic material. By replacing the harmful parts of their genetic material with genes intended to help treat a disease, scientists had hoped to use engineered viruses as delivery vehicles.
However, this method proved problematic: The viruses were sometimes toxic, induced immune and inflammatory responses, and even turned off healthy genes, including genes that suppress tumor growth. And as if that weren't enough, these engineered viruses sometimes underwent mutations that equipped them with additional damaging properties.
As a result, scientists began to experiment with synthetic systems, which are safer than viruses and provide better control. But while scientists could sneak DNA into the cell using these methods, they were usually unable to effectively transport DNA into the nucleus. Past attempts succeeded only in slipping an "unreadable,"and therefore ineffective, form of DNA into the nucleus. (DNA contains genes, which must be "read"to produce the proteins they encode.) And even those attempts were too costly to render gene therapy practicable.
This is where Dr. Ziv Reich of the Weizmann Institute's Biological Chemistry Department and his team came in. They crafted a method for smuggling DNA through the walls of the nucleus under the very noses of the cell's guardians. The method bears an uncanny resemblance to the Trojan horse.
Finding a camouflage
There are two main routes into the nucleus. Molecules less than 8-9 nanometers (one nanometer is a billionth of a meter) in diameter can pass freely through its large pores. Larger molecules, however, must possess special "papers"- called nuclear localization signals (NLSs) - which DNA molecules, around 10 nanometers in diameter, don't carry.
To overcome this problem, Reich's team devised an approach in which DNA hitchhikes on a protein that does possess NLS. Reich modifies DNA to include specific binding sites for these proteins, which then transport the DNA into the nucleus, where it is "read."The bound proteins pose no obstacle to the reading process since their natural function is to bind to DNA inside the nucleus. Such modifications can be made readily, accurately, and inexpensively.
In addition, this study contains an unexpected twist. Since a variety of proteins in the cell possess NLS, Reich's group had to decide which family of proteins would play host to the hitchhiking DNA. "We chose the NFkB family,"says Reich, "because they bind strongly to DNA and are inducible, meaning that we can control their entry into the nucleus by sending special signals to the cell."
The NFkB family of proteins proved even more promising than anticipated. While Reich was studying their potential as DNA transportation vehicles, these proteins were discovered to play a central role in several autoimmune diseases, such as asthma and atherosclerosis, as well as in certain types of blood cancers - especially Hodgkin's disease, a common lymphoma. Whereas NFkB proteins normally enter the nucleus only when an appropriate signal is received, in Hodgkin's cells, they continually shuttle back and forth between the diseased cell's nucleus and its cytoplasm.
This distinction, believes Adi Mesika, one of Reich's doctoral students, could be used to induce Hodgkin's cells to destroythemselves. DNA that contains instructions for self-destruction could infiltrate the nucleus, piggybacking on aberrant NFkB proteins. Since NFkB proteins would not enter the nucleus of healthy cells, onlydiseased cells would self-destruct. This ability to preferentially destroy Hodgkin's cells will soon be investigated in collaboration with physicians on cells obtained from patients diagnosed with this disease.
In parallel to the NFkB approach, another of Reich's students, Saroj Shekhawat, is looking for other proteins that can do the job. The aim, explains Shekhawat, is to find proteins that are specific to certain tissues or organs, or proteins that are active in disease, allowing the targeting of DNA to ailing cells. "We are also looking for proteins that will condense the DNA, making it smaller and therefore easier to import through the nucleus's channels,"he says.
If successful, Reich's special delivery service might be an important factor in developing future gene therapies.
Dr. Reich's research is supported by the Levine Institute of Applied Science; the Clore Center for Biological Physics; the Kekst Family Center for Medical Genetics; the Avron-Wilstaetter Minerva Center for Research in Photosynthesis; the Molecular Imaging Corporation, Phoenix, AZ; Ms. Lois Rosen, Los Angeles, CA; and Teva Pharmaceuticals, Israel. He holds the Abraham and Jennie Fialkow Career Development Chair.
The Body Electric
A brilliant flash of lightning streaks across the evening sky followed by a sharp crack of thunder. As you rush upstairs to close the bedroom window, the millions of cells making up your heart muscle continue their endless task of contracting and relaxing in concert, guided by a small group of conductor cells. You then turn up the heater, relishing the wave of hot air as it meets your skin.
Electricity is everywhere: in nature, appliances and the human body. A steady heartbeat, mental processing, the perception of sounds, sights and temperatures - all depend on meticulously orchestrated cellular communication pathways linked up through electrochemical signaling.
One of the body’s primary communication pathways consists of a tiny pipe located in the cell membrane that opens to allow ions (electrically charged atoms) to flow into or out of the cells according to specific signals. These pipes, known as ion channels, are “motivated” to open by electrical differences existing between a cell’s internal and external environment.
Cells maintain an electric charge that is negative relative to their external environment. When these channels open, this difference in energy propels positively charged ions through the channel, triggering a variety of cellular processes.
Most channels allow the passage of only one type of ion. Yet how do different channels open and close? What enables them to act like bouncers at a club, selectively determining which ions (such as sodium, calcium or potassium) will enter or exit the cell?
Prof. Eitan Reuveny of the Institute’s Biological Chemistry Department is studying this cellular feat in potassium channels - key ion channels affecting the electrical state of the heart, nerves and muscles.
Previous research had revealed that the potassium channel contains a selectivity filter that identifies and binds to potassium ions, filtering these ions from the intracellular solution. It does so with remarkable speed - tens of millions of ions are identified and travel through a single channel every second. The channel opens when an intracellular molecule, called a G protein, is activated, causing four of its subunits to rearrange themselves, thus permitting ion flow. The mechanism is similar to that of a door latch. Following activation, the G protein subunits bind to the channel, changing its formation in a way that essentially presses down a handle, pulling a door tongue inside and thus opening the channel.
To probe this “door-latch” mechanism, Reuveny is combining exciting new technologies from the worlds of biophysics and molecular genetics to do what was unthinkable just a few years ago: identify the tiny fluctuations of electric potential that occur within an individual cell. One of the technologies applied (which earned its developers, German cell physiologists Erwin Neher and Bert Sakmann, the 1991 Nobel Prize in Physiology or Medicine) is based on using microscopic glass pipettes a thousandth of a millimeter in diameter. By sucking in a tiny part of the cell membrane containing only one ion channel, the pipettes make it possible to measure the incredibly tiny current created as ions pass through.
Working with graduate students Rona Sadja, Karin Smadja, Noga Alagem and Inbal Riven, Reuveny began by introducing genetic modifications that in effect shortened the latch tongue, causing the channel to open even without G protein activation. Next, they tagged the channel with “reporter” proteins that lit up under certain optical conditions, making it possible to trace the actual movements of the channel latch.
Using this double strategy, the team succeeded in identifying the key molecular elements that open the potassium channel. Later research showed that these same elements also fulfill an important role in stabilizing the channel once it opens. The team’s findings were published in Neuron.
A better understanding of the rules governing potassium and other ion channels will clarify some of the most basic life processes. Insights into what goes wrong when cellular communication pathways break down may also lead to new therapies, from those targeting heart arrhythmia to diabetes and a range of neuronal disorders.
Potassium channels and diabetes
In related research, Prof. Reuveny is studying how fluctuations in the electrical activity of potassium channels trigger a “shut-down” response that controls insulin release. Research in this field might lead to a new therapy for hypoglycemia - a complication of Type 1 diabetes occurring when elevated levels of insulin flow into the blood, causing glucose to drop to dangerously low levels.
Prof. Reuveny’s research is supported by the Y. Leon Benoziyo Institute for Molecular Medicine; the Clore Center for Biological Physics; the Dr. Josef Cohn Minerva Center for Biomembrane Research; and the Buddy Taub Foundation.