In new research that builds upon previous work with atrophied brain cells, UCSD School of Medicine researchers have found that essential brain fibers that shrivel up and disappear in aged monkeys can be restored to normal levels with infusion of tissue that has been genetically altered to produce nerve growth factor (NGF), a naturally occurring substance found in all vertebrate animals. Like telephone wires, these fibers, called axons, are vital for transmitting messages to and from neurons within the brain.

According to the UCSD researchers, the new findings provide additional support for the potential use of gene therapy to treat loss of memory and cognitive function in Alzheimer's patients. Two patients have been enrolled in a clinical trial now underway, and another six are being recruited to evaluate this procedure in humans.

The latest research findings are published in the February 13, 2001 issue of the Proceedings of the National Academy of Science (PNAS) by investigators in the lab of Mark Tuszynski, M.D., Ph.D., UCSD associate professor of neurosciences and a neurologist with the VA Medical Center, and by the California Regional Primate Research Center at University of California Davis.

The study's first author, UCSD assistant professor James Conner, Ph.D., notes that "our research shows that as a result of normal aging, monkeys lose 25% of their axons, the threadlike projections that neurons send out into the cortex, the outer layer of the brain where short term memory is retrieved and intellectual processing occurs. Following gene therapy, the axons were restored to levels seen in young monkeys, and sometimes exceeded those levels."

Previous research by the Tuszynski lab focused on the neuron cell bodies located in the cholinergic system, which originates deep within the brain. The cholinergic system has been identified as important for supporting memory and cognitive function. This system profoundly degenerates in the course of Alzheimer's disease.

In work published in PNAS in September 1999, the UCSD researchers found that 40% of cholinergic neuron cell bodies had atrophied in normal monkey aging, but were returned to nearly normal size and quantity following the surgical transplant of cells genetically altered to produce NGF. After genetically modifying primate skin cells to produce and deliver NGF, the investigators used a long needle to inject the modified cells deep into the brain, adjacent to the atrophied neurons. Three months later, the researchers found that the neurons had been restored to normal levels.

"Although the neuron cells in the cholinergic system sit in the base of the brain, their ability to communicate with other cells and sustain normal function depends on the axons that link them to other parts of the brain," Conner says. "In our current study, we utilized the same gene therapy procedure, implanting the NGF next to the atrophied neurons. Just as the NGF has pumped up the neuron cells, it also stimulates growth of the critical axon connections."

The researchers worked with 18 rhesus monkeys, including young adults approximately 9.5 years old (comparable to 20-year-old humans), and older monkeys ranging in age from 22 to 27.5 years (comparable to 60- to 70-year old humans). Some of the older monkeys were "control" animals and did not receive the NGF.

The Tuszynski team reports that another significant and somewhat surprising research finding was that the direction of axon growth was normal. Because NGF has properties that attract nerve fibers, the researchers worried that axons might grow toward the NGF implant, rather than out into the cortex. However, NGF was found to stimulate neuron cell bodies to produce the proteins and other substances needed to function normally, with appropriate outward extension of axons.
"These findings provide further support for our human clinical trials," Tuszynski says. "Now we know that delivering nerve growth factor deep within the brain can benefit the connections out in the cortex and not worsen them by causing a retraction of axons toward the implant."

Since receiving approval for human clinical trials from the U.S. Food and Drug Administration and review by the National Institutes of Health's Recombinant DNA Advisory Committee in late 1999, the UCSD team has received more than 500 inquiries from individuals interested in the therapy. Of these, two individuals have been selected after rigorous screening to receive the NGF gene transplant within the next few months. An additional six volunteers are still sought for the clinical trial, which is the first time that gene therapy will be used in the brain for a condition other than cancer.

To be eligible, a participant must meet the following criteria:
¥ A neurologist-certified diagnosis of "probable Alzheimer's disease";
¥ Early stage of the disease (generally within two years of diagnosis);
¥ Completely normal speaking ability and completely normal ability to understand what others are saying;
¥ An ability to understand the potential risks of participation in the study;
¥ The ability to travel to and from San Diego, California up to nine times in the first year of study; and
¥ The willingness to discontinue use of the drugs cognex or aricept for the first 18 months of the trial.

Skin cells taken from each participant will be genetically modified in test tubes to produce NGF, then surgically implanted three months later in the brain. Each participant will be monitored for 18 months following surgery.

Tuszynski notes that this is a Phase 1 clinical trial. Called a "Safety/Toxicity" study by the FDA, the Phase 1 trial will determine whether the experimental procedure is safe for humans. At the same time, it should give researchers a preliminary sense of whether this therapy will be effective in combating the loss of memory and cognitive function with Alzheimer's disease in humans.

"If we see a fraction of the effects in humans that we see in primates, we may have something here," Tuszynski says.

For more information about the clinical trial, call the UCSD Alzheimer's Disease Research Center at 858-622-5800. Additional information is also available on the web site http://obsidian.ucsd.edu/~tuszynski/clinical_study.htm.

In addition to Tuszynski and Conner, authors of the paper in PNAS are M.A.Darracq, UCSD Department of Neurosciences, and Jeff Roberts, California Regional Primate Research Center, University of California Davis.

---University of Califormia, San Diego

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