The Scientist 15[19]:18, Oct. 1 2001

http://www.the-scientist.com/yr2001/oct/research1_011001.html


RESEARCH

Gene Therapists Aim for Parkinson's Disease

For the first time, an NIH views a clinical protocol


 By Douglas Steinberg

 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.

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, over
stimulating 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.

     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 andrelease. 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
catalyzeL-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 ([log in to unmask]) 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.

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