Gene Therapists Aim for Parkinson's Disease

At first blush, gene therapy seems ill-suited to treating Parkinson's disease (PD). Scientists have linked few cases to missing or mutated genes and are generally clueless about the disease's cause. But the need for some relief from its debilitating symptoms is so great that gene therapy researchers have labored over the past decade to develop counter-strategies. These studies have had promising results. When the brains of rat and monkey PD models express certain transgenes, the animals show less of the disease's hallmark pathology, a deterioration of nerve fibers that secrete the neurotransmitter dopamine. The animals also exhibit fewer behavioral abnormalities than do controls. Armed with these findings, laboratory scientists and clinicians are now preparing to test gene therapy on humans with PD. Last spring, the National Institutes of Health Recombinant DNA Advisory Committee (RAC) reviewed a PD gene therapy protocol for the first time, endorsing various recommendations by a 12-0 vote. The protocol's investigators, at Jefferson Medical College in Philadelphia and Cornell University's Weill Medical College in New York, recently submitted it to the Food and Drug Administration. Other groups also intend to treat PD patients with gene therapy, but they say they won't seek official approval for months, if not years. Courtesy of Michael G. Kaplitt Michael G. Kaplitt Collectively, these groups have injected at least six candidate transgenes into three brain structures using two types of viruses. But until various clinical trials reveal which gene, if any, works and under what conditions, the researchers' buzzwords are safety and caution. Michael G. Kaplitt, who directs stereotactic and functional neurosurgery at New York Presbyterian Hospital and is co-principal investigator of the Jefferson/Weill proposal, admits that gene therapists might feel tempted to manipulate the Parkinsonian brain in a biologically revealing but possibly harmful way. They might, for example, want to introduce a gene into one side of the brain while leaving the other side, which is often also diseased, as an untreated control. Kaplitt recalls that, in proposing the PD protocol, "We really tried hard to start from the point of asking, 'What is the safest thing we could do for a patient first and still have some reasonable expectation of efficacy?'" Complex Brain Circuitry PD afflicts roughly a million Americans, with 65,000 new cases arising yearly, according to the Alexandria, Va.-based Parkinson's Action Network. Well known to cause hand tremors, the disease can also lead to severe disability and death. PD affects the brain's basal ganglia, which consist of five interconnected neuron clusters, or nuclei, on each side of the brain. The disease decimates dopamine-secreting neurons extending from one nucleus, the substantia nigra, to another, the striatum. The subthalamic nucleus (STN) is also affected. STN is normally inhibited by the neurotransmitter *-aminobutyric acid (GABA), released by a nucleus known as the globus pallidus. In PD, STN becomes hyperactive, overstimulating other basal ganglia nuclei. Surgeons treat PD by lesioning hyperactive brain areas or inserting electrical stimulators that block hyperactivity. The most prominent drug against the disease is L-dopa, which crosses the blood-brain barrier and is converted into dopamine by an intracellular enzyme. Over time, however, L-dopa often loses its efficacy, so that ever-increasing doses are necessary. Many patients then develop involuntary movement disorders known as dyskinesias, which presumably stem from excess dopamine. Courtesy of Thomas Jefferson Matthew J. During The original approach of Kaplitt and his co-principal investigator on the PD protocol, Matthew J. During, was to increase dopamine levels in the striatum. In 1994, they were part of a group that chemically destroyed rats' substantia nigra and then injected the striatum with a virus that expressed tyrosine hydroxylase (TH), an enzyme crucial to dopamine synthesis. Treated rats showed significant behavioral recovery.1 Nevertheless, During, director of Jefferson's CNS Gene Therapy Center and a gene therapy pioneer,2 says that his team was never committed to clinical use of a dopamine strategy. "The idea that unregulated dopamine could potentially cause runaway dyskinesias was a concern to us," he recalls. Another concern was that the striatum is not a routine surgical site. Kaplitt, however, remembers changing the team's strategy "on a dime" when he first heard two years ago about a now-notorious study in which dopaminergic neurons from human embryos were transplanted into PD patients. The transplants caused dyskinesias that persisted unexpectedly--and tragically--even after doctors halted L-dopa administration.3 Dampening Hyperactivity In their new approach, Kaplitt and During loaded nontoxic, nonpathogenic adeno-associated viruses with genes encoding glutamic acid decarboxylase (GAD), an enzyme that catalyzes GABA synthesis. By injecting recombinant viruses into the tiny STN--in humans, the nucleus contains only 300,000 neurons--the investigators hoped that virally infected neurons would make and secrete GABA. (Non-transduced cells produce the neurotransmitter glutamate.) The theory was that these neurons would inhibit both themselves and their targets and dampen PD symptoms. Rat and monkey experiments support this theory, according to documents submitted to RAC in April. RAC's consideration of the protocol was swift but probing. Committee members wondered, for example, whether an immune response to GAD could lead to diabetes. Some biologists have hypothesized that GAD stimulates the production of antibodies that attack insulin-generating cells in the pancreas. The investigators responded to RAC that their preclinical studies had been "spectacularly unsuccessful in effecting a diabetic ... phenotype with GAD immunization." Three weeks after a June 14 public hearing, RAC made recommendations, which Kaplitt says were all incorporated into the FDA application. RAC cautiously advised cutting the number of subjects from 20 to six and eliminating saline-injected controls. The original protocol called for treating the STNs on both sides of the brain; RAC recommended treating patients with unilateral PD who would receive recombinant viruses only on the diseased side of their brains. What if gene therapy fails to relieve PD symptoms? Kaplitt, a neurosurgeon, notes that he could then easily insert an electrical stimulator into STN because its location would already have been mapped for the viral injection. Conversely, if gene therapy works too well, yielding excessive GABA, STN could be lesioned, an operation that has long been performed. Boosting Dopamine Unlike Kaplitt and During, other gene therapy researchers remain intent on replacing the dopamine lost during PD. Krys S. Bankiewicz, an associate neurosurgery professor at the University of California, San Francisco, studies L-amino-acid decarboxylase (AADC), the enzyme that converts L-dopa into dopamine. His team injected viruses containing the AADC gene into the striatum of a PD rat model. When the rats received L-dopa, their behavior was more normal after gene therapy than before.4 This strategy offers two potential clinical advantages. First, by prescribing the appropriate doses of L-dopa, physicians could fine-tune the dopamine amount made by transduced neurons. Second, because striatal neurons would be unusually responsive to L-dopa, doctors could prescribe far lower doses, hopefully preventing dyskinesias from arising. Other investigators are examining additional enzymes involved in dopamine synthesis and release. Ronald J. Mandel, an associate neuroscience professor at the University of Florida in Gainesville, has created recombinant viruses that encode TH and GTP cyclohydrolase 1 (GCH-1). Together, these enzymes catalyze L-dopa synthesis. By injecting these viruses into the striatum, surgeons could restrict the area of L-dopa exposure. In contrast, patients now take the drug orally, and it circulates throughout their bodies. Besides TH, AADC, and GCH-1, Un Jung Kang, an associate neurology professor at the University of Chicago's Pritzker School of Medicine, also works on the vesicular monoamine transporter (VMAT), a protein that helps store dopamine in intracellular vesicles. By adding genes that encode VMAT and the dopamine-generating enzymes to neuron-targeting viruses, Kang hopes to achieve a more sustained release of the neurotransmitter. Another benefit: While free dopamine inhibits its further synthesis by a feedback mechanism, dopamine sequestered in vesicles can't have this inhibitory effect. Whatever genes they study, researchers agree that dopamine overproduction by transduced neurons must be avoided at all costs. The prevalent strategy is to control transgene transcription by using promoters regulated by tetracycline or its analog, doxycycline. Adding these drugs to an animal's or patient's diet turns the gene on or off. The problem with this system, notes Mandel, is its "leakiness." Transduced cells will produce a small amount of a transgene's protein product, even when the presence or lack of tetracycline in the diet should ideally keep the protein from being made. Keeping Neurons Alive Another way of boosting dopamine levels in the striatum is by preventing the further deterioration of the substantia nigra (SN) neurons that make this neurotransmitter. This is the strategy adopted by Jeffrey H. Kordower, director of the Research Center for Brain Repair at Chicago's Rush Presbyterian-St. Luke's Medical Center. He and his colleagues added the gene for glial-derived neurotrophic factor (GDNF) to lentiviruses, which are gutted--and presumably safe--HIV-1 viruses. (Mandel has added the GDNF gene to adeno-associated viruses.) Previous studies had shown that GDNF nourishes and protects dopaminergic neurons. After injecting the recombinant lentiviruses into the nigrostriatal region of primate PD models, Kordower's team noted several felicitous results: less neurodegeneration; the sprouting of new nerve fibers; a reversal of behavioral deficits; and more dopamine synthesized per neuron.5 Kordower foresees GDNF gene therapy being applied to PD patients who haven't yet lost too many SN neurons. But the Jefferson/Weill team's RAC submission questions this scenario. Such patients, the document states, "have not yet exhausted benefits from medical therapy." Consequently, regulators might be loath to subject these patients to risky brain surgery, and the patients might balk at the operations. GDNF gene therapy raises another issue: Which brain region should be treated with recombinant viruses--the dopamine-requiring striatum or the dopamine-producing SN, which is a more dangerous site for surgery? Martha C. Bohn, a pediatrics professor at Northwestern University Medical School in Chicago, has investigated this question. When she injected recombinant viruses into rat striatum, dopamine-dependent behaviors were protected; injections into SN didn't have this effect. But because PD destroys many of the nerve fibers that terminate in the striatum, Bohn concludes, "It's possible we're going to have to put [GDNF] in both sites." Douglas Steinberg (dougste@attglobal.net) is a freelance writer in New York. References 1. M.G. Kaplitt et al., "Long-term gene expression and phenotypic correction using adeno- associated virus vectors in the mammalian brain," Nature Genetics, 8:148-54, 1994. 2. D. Steinberg, "Gene therapy targets Canavan disease," The Scientist, 15[18]: 20, Sep. 17, 2001. 3. C.R. Freed et al., "Transplantation of embryonic dopamine neurons for severe Parkinson's disease," New England Journal of Medicine, 344:710-19, March 8, 2001. 4. R. Sánchez-Pernaute et al., "Functional effect of adeno-associated virus mediated gene transfer of aromatic L-amino acid decarboxylase into the striatum of 6-OHDA-lesioned rats," Journal of Molecular Therapy, 4:324-30, October 2001. 5. J.H. Kordower et al., "Neurodegeneration prevented by lentiviral vector delivery of GDNF in primate models of Parkinson's disease," Science, 290:5492, 2000.

Gene Therapists Aim for Parkinson's Disease

Interested in reading more?

Become a Member of