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Gene therapy decreases damage to rats' brains after seizure
STANFORD -- Stanford scientists have shown for the first time that gene therapy can protect some brain cells from severe damage after strokes, seizures or other sudden brain injuries.
By using an altered form of herpes simplex virus to deliver a key gene to the brain cells of rats, the researchers demonstrated that they can reduce significantly the number of neurons that are killed, even when the treatment is administered after the brain trauma has begun.
The study, led by Stanford biological sciences Professor Robert Sapolsky, was published in the Aug. 1 issue of the Proceedings of the National Academy of Sciences. It shows that gene therapy can change the physiology of neurons quickly enough to reduce the number of neurons catastrophically damaged by a brain seizure: Under the introduced gene's direction, the neurons produced extra supplies of a needed protein in time to protect some of the cells.
In a second article, now in press, the researchers describe a similar treatment for experimentally induced strokes in rats. (A seizure is a surge of electrical impulses, analogous to a lightning storm in the brain. A stroke is caused when the brain's blood supply is blocked, cutting off delivery of oxygen and nutrients.)
This is the second published research to show a therapeutic effect using herpes simplex virus to deliver a gene to the brain. Last November, another research group published a study showing effects on a slow, degenerative nervous system disorder. That work, by Matthew J. During at Yale and colleagues at Harvard, Wesleyan and Washington University, showed that a different gene delivered by a herpes simplex virus vector could prevent behavioral changes in rats with a version of Parkinson's disease.
"These are baby steps," Sapolsky emphasized, referring to both studies. "There are a tremendous number of hurdles to be overcome before gene therapies with viral vectors could be used in the human brain."
The Stanford researchers used herpes simplex virus because it is one of the few vectors that can carry therapeutic genes into neurons. Virologist Dora Ho, a senior research associate in Sapolsky's laboratory, altered mutated forms of the virus so that instead of viral DNA, they contained packets of glucose transporter gene. Once inside the neuron, the virus used the cell's machinery to carry out that gene's coded instructions and produce many copies of glucose transporter protein. The protein helps the cell to import glucose from the bloodstream, supplying the energy for all cell functions.
Working first in cell culture, Ho showed that when the neurons are induced to overexpress the glucose transporter gene, they are protected from certain kinds of injury. Previous work in Sapolsky's laboratory had shown the reason for that protection: When neurons are buffered with extra glucose, they draw on that energy to remove excess excitatory amino acids and calcium, substances that flood the cells during a seizure or stroke and can damage them beyond repair.
In the study reported in PNAS, graduate student Matthew Lawrence showed that therapy with the glucose transporter gene could prevent brain damage in living rats. Working with biology undergraduate Rajesh Dash, Lawrence injected both sides of the rats' hippocampus with kainic acid, a chemical that causes brain seizures. Next, they injected one side of the hippocampus with Ho's viral vector, and the other side with a control vector, which did not express the glucose transporter gene and did not protect against seizure damage. Upon autopsy, they found that significantly fewer neurons were destroyed by the seizure on the side that was protected with glucose transporter gene.
Targeting the brain
Gene therapy for the brain poses special challenges. In other parts of the body, most conventional approaches to gene therapy take advantage of cell division to insert new genes into the cell as its nucleus is dividing. But the neurons in adult brains do not divide to form new cells. The Stanford and Yale research groups both used herpes simplex virus as a vector, because it is one of a small class of "neurotrophic" viruses that are able to deliver DNA to cells that are no longer dividing. Herpes simplex even has a preference for neurons.
However, researchers have not yet found a way to deliver enough gene-altering vectors to the proper place in the brain so that a sufficient number of neurons would be protected to save a human from stroke or seizure damage. There is also a potential that some of the viruses used in the gene-delivery system could themselves damage the cells. To prevent this, the Stanford team used a mutated form of the virus that they had previously tested to show that it would have no toxic effects of its own in the rats' brains.
Scientists in many labs are exploring ways to stop the swift succession of events that leads to neuron death after a seizure, stroke or brain injury. The emphasis is on stopping damage: The response to these sudden "necrotic" events must be rapid, and it must protect as many cells as possible from dying. A neuron, once lost, is irreplaceable -- if the cells cannot be saved in the first hours or days after the injury, they never will re-grow.
Many of the experimental approaches to necrotic brain damage use conventional drugs. But gene therapy has potential advantages. It may have fewer side effects than drugs because it harnesses the neuron's normal cell machinery to do its work. And at least in theory, a viral vector could be designed to hide in the nucleus of cells. Once triggered by a stroke or trauma, the therapeutic genes would go to work producing neuroprotective substances.
A collaborative effort
Sapolsky said his research team deserves the lion's share of the credit for the work in his laboratory, which represents the culmination of a line of studies about the energetics of neuron death. He said that Ho, who also completed her graduate studies at Stanford, has been in the forefront of work using viruses to deliver therapeutic genes to various types of cells. Her research was among the first to show that herpes simplex virus could deliver genes to the brain and change the physiology of neurons.
Lawrence, the lead author in the PNAS study, took on the challenge of testing whether the viral gene therapy could protect rats' brains against seizure and stroke. Lawrence also trained the fourth member of the team, undergraduate biology major Dash, who graduated from Stanford in June with a Firestone Medal for excellence in biological research. Sapolsky said Lawrence and Dash were "uniquely skilled at taking what had been learned at the molecular level and showing what it meant inside the living brain."
Members of Sapolsky's lab also are researching gene therapies to control other steps in the cascade of crises that leads to the death of a neuron. One virus-delivered gene controls the cells' production of toxic oxygen radicals. Another, dubbed BCL-2, appears to turn off the mechanism for apoptosis, or programmed cell death. Another study will examine whether protecting neurons has any practical effect. The research team will look at whether rats that receive gene therapy after a seizure retain more memory than unprotected rats.
Even though it is too soon to tell if they will lead to a practical therapy for human brain injury, gene therapy studies are helping to open new lines of inquiry into how brain cells die after a sudden injury, what cell functions might prevent neuron death, and what genes control those functions. And Sapolsky said the fact that they are saving neurons gives the work a special urgency.
"Nothing is as irreparable as brain injury," Sapolsky said, "and nothing hits more at the center of who we are. If any part of the body is damaged, it can be painful and disabling. But if the brain is damaged, you are compromised as to your existence -- your sense of self."
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