June 06, 2004
Technique Delivers Switchable Gene Into Rat Brains

Researchers at Northwestern University have developed a technique that delivers into rat brains genes that can be switched off with the use of an antibiotic.

Northwestern University neuroscientists have overcome a major obstacle in gene therapy research. They've devised a method that will safely deliver and regulate expression of therapeutic genes introduced into the central nervous system to treat Parkinson's disease and other neurodegenerative diseases.

The method, developed by Martha C. Bohn and colleagues, is described in the June issue of the journal Gene Therapy. Bohn is Medical Research Institute Council Professor of Pediatrics at the Children's Memorial Institute for Education and Research and professor of pediatrics and of molecular pharmacology and biological chemistry at Northwestern University Feinberg School of Medicine.

Jiang Lixin, a post-doctoral fellow in Bohn's laboratory, created three different viral vectors -- carrier molecules -- that used human fluorescent green protein to track gene delivery and expression in cells. The vectors, made with the harmless adeno-associated virus (AAV), carried the "tet-off" system, in which the introduced gene is continually expressed or "on" but can be temporarily "turned off" when a small dose of the tetracycline antibiotic derivative doxycycline is administered.

One vector, known as rAAVS3, displayed particularly tighter regulation in neurons when gene expression was measured at the protein and molecular RNA levels.

To assess regulation in the brain, the researchers injected the vector into the striatum of rats, the area in the brain where the neurotransmitter dopamine activates the nerve cells that control motor coordination.

In their experiments, Bohn and co-researchers found that up to 99 percent of the vector-introduced gene was turned off when the rats were given small doses of doxycycline. In Parkinson's disease, dopamine-producing neurons degenerate, resulting in gait problems, muscle rigidity and tremors

Several years ago Bohn's laboratory group discovered that glial cells in the embryonic brain stem secrete factors, or proteins, that promote survival and differentiation of dopamine neurons.

One of these proteins, called glial cell line-derived neurotrophic factor (GDNF), is a potent factor that promotes growth of not only dopamine neurons, but also motor neurons and several other types of neurons. GDNF may have therapeutic potential for several neurodegenerative diseases, including Parkinson's disease and Lou Gehrig's disease.

Bohn's laboratory was the first to show that introduction of a GDNF gene in a rodent model of Parkinson's disease halts the disease process.

"GDNF gene therapy has exciting potential to 'cure' Parkinson's disease, but since putting a gene into the brain may lead to expression and increased levels of GDNF protein for years, it will be important to have some way to turn off gene expression to arrest unanticipated side effects," Bohn said.

Bohn and her colleagues have been developing viral vectors that offer a safe means to deliver GDNF, as well as other therapeutic genes. The AAV vector that the researchers used in these experiments is safe and approved for use in several clinical trials in the brain of humans; however, no vector in which the gene can be turned off is yet approved for use in clinical trials.

"A crucial piece of our research is related to safety," Bohn said. "We were excited to find the right mechanism to deliver the gene into the nervous system and tightly control its expression using doxycycline, a drug already approved by the Food and Drug Administration and found to have no side effects."

Bohn cautioned that thorough safety and toxicity studies of the new vector are needed and that her laboratory group is not ready to assess its use in humans.

The mechanism these researchers are using delivers a gene that expresses unless the doxycycline is delivered. But for some therapeutic applications what will be needed is the ability to deliver a gene that will by default not express. Then delivery of a drug would be used to turn it on for some finite period of time. In some ceases one can imagine why it would be helpful to turn on a gene periodically. We really need a large variety of types of gene switches where a gene delivered by gene therapy can be flipped on to stay on or flipped off to stay off or stay on temporarily to stay off temporarily. Plus, we need better ways to deliver genes only into specific desired target cell types.

One might think that the bigger challenge of gene therapy is developing the gene or genes to deliver. But so far the biggest challenge has been in various aspects of delivery. We need better ways to get genes into cells, in only into specific cell types, in amounts no higher or lower than desired, and in ways that do not cause damage to the normal DNA in each cell. It is hard to guess at the rate gene therapy will advance because really good delivery mechanisms have turned out to be very difficult to develop.

Once genes with control switches can be easily and reliably delivered into brain neurons consider the James Bond angle: brains could be surreptitiously programmed to alter their behavior in response to some environmental exposure. Imagine some scent that carries a chemical that has a switch in it that turns genes in the brain on or off. A guy goes into a room, smells the perfume that Sydney Bristow of Alias is innocently (or not so innocently) wearing and suddenly he goes berzerk and starts trying to kill someone that the Covenant knows he hates.

There is also the large group control aspect. A country that need killer soldiers may need the soldiers to be mild mannered civilians between wars but homicidal killers when sent on missions. Well, flip a few switches and suddenly the special forces are chafing at the bit to inflict suffering and death. Or an entire society could be rendered docile during a coup attempt by putting something into the water supply to flip genetic switches that were gradually installed via insect-born vectors in all the brains in a capital city without being noticed.

Share |      Randall Parker, 2004 June 06 08:45 PM  Biotech Therapies

toby said at June 7, 2004 5:21 PM:

For the record, the TET system can be used in both repression and activation.

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