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.
Crossing the Divide
The different stages of cell division - a process so orderly as to appear almost choreographed - have long been known. While tremendous advances in cell biology and genetics over the past few years have shed light on this process, its underlying molecular machinery is still not fully understood. The final step of this elegant performance, the separation into two cells, is called cytokinesis. Recently, Dr. Sima Lev of the Weizmann Institute of Science's Neurobiology Department discovered that a protein called Nir2 is essential for normal cytokinesis in human cells.
As a postdoctoral researcher at New York University, Lev discovered a protein called Pyk2, which plays an important role in cell signaling. Looking for proteins that interact with Pyk2, she discovered a protein family consisting of three members, which she called the "Nir"family. She then isolated the genes responsible for producing the proteins.
Highly conserved throughout evolution, the Nirs are found in fish, worms, flies, and mammals. Lev decided to dedicate her work todetermining the Nir proteins'function in the body. "No one in the world was working on the Nirs,"she says, "and I strongly believed that they had an important cellular function."
Upon her return to the Weizmann Institute, Lev spent nearly three years evaluating many possible roles for the protein, with team members Vladimir Litvak, Donghua Tian, and Shari Carmon. The breakthrough came with their identification of a particular fragment of Nir2, consisting of 219 amino acids out of the protein's full 1,244. When they expressed this fragment in human cells, it had adramatic effect on their shape and caused severe defects in cytokinesis. The cells failed to separate, forming long bridges between asymmetrical daughter cells. The fragment, Lev concluded, was inhibiting cytokinesis in some way, but its precise role remained obscure.
In a pinch
The scientists looked closely at dividing cells to determine exactly where the Nir2 protein was located during the process. They found that during normal cytokinesis Nir2 is present at the "cleavage furrow,"the pinched area of the cell at which the break into two daughter cells will eventually take place. But it's not alone. Beside it is an enzyme called Rho-GTPase, which plays a long-established role in cytokinesis. What, Lev wondered, was Nir2 doing there?
She found that Nir2's protein fragment is able to inhibit the activity of the Rho enzyme. She therefore designated it "Rid"(Rho inhibitory domain). It was already known that inactivation of the Rho enzyme is necessary for the final separation into two daughter cells, but it was not known what triggered Rho to move into an inactive state. Lev contends that Nir2 essentially subcontracts Rid to inhibit the activity of Rho when appropriate. If she is right, Nir2 - by hosting Rid - is vital for breaking the contractile ring between two daughter cells and thus is essential for successful cytokinesis.
The physical evidence supports Lev's assertion. When she cut off the end of Nir2 containing Rid, she saw that cytokinesis was severely impaired. The cells struggled to divide and eventually gave up, resulting in unseparated cells with multiple nuclei. The absence of Rid apparently short-circuited the cells'ability to separate.
Lev's findings shed new light on the Nir proteins as well as on the process of cytokinesis. But many open questions remain regarding the clinical implications of her results. It is well known that cytokinesis plays a critical role in animal development, and that defects in this stage of cell division can lead to instability of the genome, a phenomenon associated with cancer. In addition, recent experiments have shown that mouse embryos which lack the Nir2 protein do not survive. Thus her findings may provide insights into the necessity of this protein for normal embryonic development. "The challenge now,"says Lev, "is to translate these results into practical medicine."
Dr. Lev's research is supported by Mr. and Mrs. Nathan Baltor, Bensalem, PA; Minna James Heineman Stiftung, Germany; the Carl and Micaela Einhorn-Dominic Institute for Brain Research; and the Nella and Leon Benoziyo Center for Neurosciences. She holds the Helena Rubinstein Career Development Chair.