The Why Files The Why Files --

Gene Therapy: The eyes have it!


Some successes in animals
For humans, gene therapy remains mainly a hope, but the animal research is moving ahead on treatments for problems like pain, muscular dystrophy and cancer.

Large tumor mass on control animal compared to much smaller one on the gene-therapy animals.

Control brains compared to brains treated with gene therapy, stained to show the tumor (the white area marked "T"). MBP = myelin basic protein, the stain used to make the tumor show up. Courtesy Pedro Lowenstein.

Curing cancer?
Cancer has long been a key target for gene-therapy experiments. Since the immune system normally controls many tiny tumors, why not "teach" it to attack bigger ones? One promising target is glioblastoma, a brain cancer that is almost always fatal. According to Pedro Lowenstein, director of the Board of Governors' Gene Therapeutics Research Institute at Cedars-Sinai Medical Center in Los Angeles, "Although the surgeon can usually cut glioblastoma out, they can never cut out everything, and the tumor always recurs," because it spreads easily to adjacent regions of the brain, he says.

But using gene therapy to trigger an immune response in the brain must overcome major limits on the brain's immune system. In February, Lowenstein, his wife, Maria Castro, also of Cedars-Sinai, and other colleagues announced further progress on a dual-pronged gene therapy that brings new immune cells into the brain, and then instructs them to target tumor cells (see #6 and #7 in the bibliography).

"We want to use the immune system to kill the tumors," says Lowenstein, who is also professor of medicine and pharmacology at the University of California at Los Angeles, "but the problem with doing that in the brain is that it normally lacks a specific type of immune cell called the dendritic cell or antigen-presenting cell."

Antigen-presenting cells start adaptive immunity, which is the branch of the immune system that can "learn" to identify and kill invading cells. Lowenstein's group has found that it's possible to bring antigen-presenting cells through the blood-brain barrier into the brain, by using a powerful natural chemical with the mind-numbing moniker "Fms-like tyrosine kinase 3 ligand."

The second prong of the treatment uses a second virus to ferry in a second gene that activates a drug that kills fast-dividing brain tumor cells. As these cells die, they release fragments called antigens that the dendritic cells pick up and then "present" as targets to other immune-system cells. "Our theory is that by setting up an immune response directly within the brain, we are teaching the immune system to attack these tumors," Lowenstein says.

The evidence so far is promising, Lowenstein says. The cancer they are testing in rats is so lethal that control animals die within about 15 days of getting the tumor, and yet 70 percent of the animals that get the combination immuno-therapy survive indefinitely.

Graph shows sudden deaths at 15 days for the control animals, stable survival for experimental animals.

All the rats that receive a tumor transplant died if they received only the control injection, but about 70 percent of rats survived after the combined gene therapy. Courtesy Pedro Lowenstein.

"We have looked at the animals that survive long term, and the tumors are gone," says Lowenstein. "It's not that this is slowing the growth -- the tumor is really gone." To prove that the immune system protected the animals, the researchers placed a new tumor on the other side of brain several months after the gene treatment. The new tumor never got established, Lowenstein says.

Finally, the researchers have found that the animal's behavior returns to normal after the treatment -- apparently because the tumors ceased compressing the brain. This type of collateral damage is a major problem in human brain tumors.

These results are promising, but, as Lowenstein notes, "Mice are not people. Until we actually inject any of these new treatments in people, we have absolutely no idea what will happen. We talk about animal models, but the only model is a human patient with a brain tumor. Glioblastoma and pancreatic cancer are the two worst cancers; they kill patients faster than anything else. I have done as much as I can to convince myself it will work, and now I need to test it in humans. I desperately want to know, because if it will not work, we can move on to something else."

A gain with pain?
Could gene therapy treat the constant pain of cancer? Perhaps, according to research with endorphins, natural molecules related to opium, the king of the pain relievers that are produced in the body. Gene therapists have caused endorphins to briefly appear in lab animals, but only until the immune system wrecks the virus-carrying gene.

Picture is heavily speckled with green, showing widespread uptake of the new gene. Injecting a gene-transfer virus near the primary sensory nerve established long-lasting pain control in a rat. The green protein marks cells that took up the new gene, which makes endorphin, a relative of opium. Courtesy Andreas Beutler.

Andreas Beutler, an assistant professor of medicine, hematology and medical oncology at Mt. Sinai School of Medicine in New York City, has adapted both the vector and the injection procedure to test pain control in rats. The researchers injected a virus carrying a gene for one endorphin inside the spinal column, near the primary sensory nerve. These nerves are logical targets for damping pain, says Beutler, because all sensory information passes through them before reaching the brain.

When the viral vector attaches to nerve cells near the injection, it carries the new genes into the cell nuclei (but not into the chromosomes), where it starts to make endorphin molecules that then block pain in nearby neurons. Because drugs that block opiates negated the pain control, Beutler and colleagues deduced that endorphins made by the new genes were indeed attenuating the pain. Beutler says the vector was designed to evade immune attack, and "once you achieve expression [activity] of the opioid gene, it's a very long-term expression."

Graph shows tolerance drops to about 1 gram, then up to about 3 grams after gene starts cranking out endorphins.

In this test of pain tolerance, the rat withdraws its paw under a weight of 12 grams. After a nearby nerve was cut, the rat becomes much more sensitive to weight on the paw. But after one month, gene-therapized rats (blue marks) tolerate more weight than control rats (green). Courtesy Andreas Beutler.

When measured by a standard test for sensitivity to pain, the effects were obvious, Beutler says. "If we got something comparable in humans, it would be very solid." In the rats, the analgesic effect lasted for all three months of the experiment, although it did taper off in the last month.

The next step, Beutler says, is to repeat the procedure with larger animals, and if successful, to start a clinical trial, probably on intractable cancer pain. Gene therapy is supposed to be a 21st-century treatment, but this version is a step "back to the future," since the ancient drug opium and its many relatives remain the acme of pain control. "The strongest drugs we have are opioids; for 5,000 years, in principle, there has been no change in them," says Beutler. "The mechanism is the same, the side effects are the same, and the limits are the same" (see #8 in the bibliography).

But by stimulating endorphins, which are chemically similar to opium, exactly where they will be most effective, gene therapy could take the oldest painkiller to an entirely new level.

Reversing muscular dystrophy?
In Duchenne muscular dystrophy, a broken gene causes muscles to waste away; many patients die in their 20s when they can no longer breathe. The disease affects about one boy in 3,500 in the United States, but very few girls.

One safety trial for Duchenne, which transferred a miniature version of the giant dystrophin gene to patients, is due to be completed in December, 2008.

A second tactic may involve inserting a gene for follistatin, a protein associated with muscle growth. In a study reported in March 2008 (see #9 in the bibliography), Brian Kaspar, an assistant professor of pediatrics at The Ohio State University, reported on the effects of follistatin genes in mice carrying many of the symptoms of Duchenne muscular dystrophy.

The researchers grafted a follistatin gene to a virus and injected it into the hind legs, as "a one-time injection to determine whether we could boost muscle mass and strength," Kaspar says. The virus delivered the follistatin genes into muscle cells, where they used the normal protein factories to crank out follistatin.

About eight weeks after the injections, the muscles were indeed bigger, stronger and healthier, says Kaspar, who is also with The Research Institute at Nationwide Children's Hospital Center for Gene Therapy in Columbus. "We found follistatin increased muscle size and actually delayed the inflammation and scarring in the muscle, compared to control-treated animals."

Control animals weighed 28 grams; follistatin animals 41 grams; others were in between these figures.

Effects of transferring follistatin gene (FS) compared to control animals (GFP) and two other genes (GASP1 and FLRG). Follistatin caused the greatest increase in muscle mass, body mass and grip strength. Courtesy Brian Kasper, Ohio State University.

Surprisingly, the benefits, but not the virus, could be detected beyond the injected muscles, Kaspar adds. "The greatest effect was in the muscles that we injected ... but follistatin is secreted into circulation and it acted on other skeletal muscles in the mouse."

The follistatin gene also increased total body weight by about 25 percent. And since animals treated after their muscular dystrophy was already visible benefited as much as animals treated one month after birth, Kaspar suggests that gene therapy may help older patients. "We were surprised to find we could treat them young, and get a robust benefit in muscle size and strength, and treat others at 200 days, and see an equivalent response," he says.

Kaspar and his colleagues are discussing safety issues with the Food and Drug Administration, with the goal of starting gene-therapy experiments in people with muscular dystrophy or other muscle diseases. After all, the study did fulfill the early promise of gene therapy: After a one-time injection, the benefits persisted for two years, until the study ended.

Gene therapy: What's taken so long?

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Megan Anderson, project assistant; Terry Devitt, editor; S.V. Medaris, designer/illustrator; David Tenenbaum, feature writer; Amy Toburen, content development executive

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