Researchers Externally Regulate Gene Implanted In Brain

CHAPEL HILL, N.C. -- University of North Carolina researchers have used an antibiotic like an on-off switch to externally regulate a gene carried by a defective virus and implanted in the brain.

The accomplishment, made with laboratory rats, suggests that gene-transfer technology using the recombinant defective virus known as AAV (adeno-associated virus) may eventually prove feasible for gene therapy in human brain disorders such as Parkinson's disease and epilepsy.

A report of the new study appears Dec. 2 in the journal Gene Therapy.

The successful use of the tetracycline derivative doxycycline as a way to regulate a gene delivered to the brain via AAV also offers researchers a new tool for unraveling the complex molecular biology of neurological disorders and other conditions, including obesity, according to Dr. Richard J. Samulski, associate professor of pharmacology at the UNC-CH School of Medicine and director of the university's Gene Therapy Center.

"We're providing an offshoot from our clinically oriented efforts so that basic science researchers can now deliver a gene to an adult animal, turn a gene on and off and study its biology," Samulski says. He points out that the AAV gene transfer system used in the study may offer an alternative to the genetically engineered mouse strains that are developed to mimic human disease and gene defects.

"With this approach, you can establish if molecular principles that work in rodents would work in a larger animal, like dogs or monkeys. And that allows you to move into really relevant models quickly without having to be dependent on the genetic manipulation of embryos," Samulski says.

Rebecca P. Haberman, a UNC-CH graduate student in neurobiology, is the study's lead author. She is training in the laboratories of Samulski and Dr. Thomas J. McCown, research associate professor of psychiatry.

For the study, the UNC-CH team altered the AAV virus to serve as a vector for delivering two genetically engineered genes. One gene, coded for a "fusion protein," was comprised of specific protein regions from a herpes virus and from E. coli, an intestinal bacteria. The second was a "reporter gene," a DNA molecule that serves no biological function within the cell and is not normally present. Research has shown that tetracycline or any of its analogs triggers the fusion protein to suppress expression of a gene delivered by the virus.

The viral vector was then injected directly into the brains of laboratory rats. The protein expressed by the reporter gene served as a biochemical marker that the gene had been transferred successfully. Subsequent tests on brain tissue during three weeks of doxycycline administration in the animals' drinking water revealed highly significant reductions in the reporter gene's protein production. Protein production resumed during the weeks without the drug, demonstrating that the gene could be regulated in the brain by administration or withdrawal of doxycycline.

"Control of gene expression, its protein production, is of particular importance in gene therapy. And one of the hallmarks of AAV is that it can last for a very long time at a sustained level," Haberman says. She and her colleagues point out that AAV gene transfer technology might eventually make it possible to block or reduce seizure activity in epilepsy or to raise brain dopamine levels in Parkinson's disease.

Obesity may be another targeted disorder for AAV gene transfer. The UNC-CH researchers have begun studying animals that do not produce endogenous leptin, a molecule that plays an important role in obesity. "We already demonstrated that this particular [AAV] virus cassette sustains a reporter gene for long periods of time in the brain area where leptin is produced," McCown says. "This means we should be able to produce leptin in that part of the brain that regulates body weight and observe changes in weight by regulating the gene."

"I can envision the advent of a whole new generation of drugs we can put in the brain, drugs that will be able to turn endogenous genes on and off," Samulski predicts.