Using Viral Vectors to Deliver Drugs, Discover Therapeutic Targets
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Autosomal-dominant mutations in the LRRK2 gene are the most common known genetic cause of Parkinson disease. The most common LRRK2 mutation that causes Parkinson disease is the G2019S mutation in the activation loop of the kinase. The G2019S mutation causes hyperactivity.
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UAB researchers are again pushing the frontier of Parkinson disease (PD) treatment. UAB is one of nine nationally recognized American Parkinson Disease Association (APDA) Centers for Advanced Research. The neurodegenerative disorder strikes 1% to 2% of the population aged ≥60 years, disabling more than 1 million people in the United States with a total cost to society that exceeds $6 billion annually. Prevalence will increase with the aging of the Baby Boomer generation.
Current medical therapies for PD provide effective symptom control in the early stages of illness, but inevitable disease progression results in significant disability and diminished quality of life for patients and their families. Mortality among PD patients is two to five times greater than in unaffected age-matched controls. A recent study found mortality among PD patients increased regardless of disease duration, age at onset, and smoking status (Neurology. 2008;70:1423-1430).
UAB Research Directions
UAB researchers are investigating two new cutting-edge approaches to PD treatment. Both center on the process of neurodegeneration in the brain region linked to the characteristic bradykinesia, rigidity, resting tremor, and other motor deficits in PD. A defining characteristic of PD is selective loss of midbrain dopaminergic neurons in the substantia nigra. Dopamine relays the neuronal signals that produce smooth, purposeful movement. By the time symptoms manifest, 60% to 80% of these dopaminergic neurons are damaged or destroyed.
CERE-120
A new treatment for PD that appears to protect and rejuvenate dopaminergic neurons may be only 5 years out if the drug CERE-120 works as effectively and safely as early studies indicate. UAB neurologist, eminent PD researcher, and Department of Neurology Chair Ray L. Watts, MD, is principal investigator at UAB for a double-blind randomized controlled phase 2 trial underway at UAB and eight other PD research centers. Ceregene Inc, a San Diego biotechnology company, makes the experimental drug.
CERE-120 is a gene transfer agent comprised of an adeno-associated virus (AAV) that carries genetic instructions for making neurturin (NTN), a neurotrophic growth-factor protein that restores function and protects neurons. In patients with PD, NTN is depleted. The protein has demonstrated neuroprotective and neuroregenerative properties for dopaminergic neurons in the substantia nigra of rodent and nonhuman primate models of nigrostriatal degeneration (Mol Ther 2007;15:62-68) and (Brain Res. 2002;947:271-283). In a phase 1 open-label trial, CERE-120 was well tolerated and achieved a persistent mean improvement in symptoms of 36% (Lancet Neurol. 2008;7[5]:400-408).
The ongoing challenge in harnessing the potential of growth factors such as NTN for treating neurodegenerative diseases lies in achieving persistent localized delivery to targeted neurons. Attaining adequate protein levels in a specific brain region has been difficult because proteins do not cross the blood-brain barrier. Previous attempts to deliver growth factors directly into the brain do not appear to reach therapeutic levels and often result in undesirable side effects.
Viral vectors may be the key to targeted delivery. "Incorporating the gene for NTN into a vector facilitates controlled sustained delivery while minimizing NTN in regions outside the nigrostriatal system," Watts says. "We avoid the side effects endemic to high doses of nontargeted therapy. We have a clear opportunity to improve parkinsonism by enhancing the function of the remaining dopaminergic neurons, potentially protecting them from further neurodegeneration and slowing the rate of disease progression."
Primary funding for the trial is through Ceregene, but the Michael J. Fox Foundation (MJFF) for Parkinson's Research, which helped fund the phase 1 study of CERE-120, has provided $1.9 million to assist in funding the study's second phase. The MJFF is dedicated to developing a cure for Parkinson disease within this decade through an aggressively funded research agenda.
In the phase 2 trial so far, investigators at UAB and other sites have enrolled 58 PD patients: two-thirds in an active treatment group and the remaining in a control group. Participants in the active group receive CERE-120 via highly targeted bilateral stereotactic injections into the putamen. The control group will undergo imitation surgery, and investigators will follow participants for 12 months. A report on the results is expected before the end of 2008 or early 2009.
Researchers will evaluate efficacy by the change from baseline in the Unified Parkinson's Disease Rating Scale motor score among the active and control groups. "Based on what we have seen thus far, we are cautiously optimistic about this new treatment approach," Watts says. "CERE-120's potential to improve symptoms in PD as well as slow disease progression is very significant. If all goes well, we expect the phase 3 trial to commence in 2009."
LRRK2: A Therapeutic Target
Recent research has confirmed that genetic factors play a role in the etiology of PD. Specifically, investigators working in laboratories around the world have found mutations in the leucine-rich repeat kinase 2 (LRRK2) gene in sporadic PD patients, although the mutations are most prevalent among patients with a family history of PD (Neurology. 2005;65:664-665).
LRRK2 is expressed in the brain (in the basal ganglia and hippocampus) and in the kidneys and leukocytes, where it potentially could be used as a biomarker. Clarification of the role of the LRRK2 gene in PD will aid in understanding the mechanisms of disease development.
"Mutations in the LRRK2 gene appear to increase the enzymatic activity of the encoded LRRK2 protein—in this case an enzyme activity associated with the phosphorylation of one or more unknown protein targets. The mutations that increase LRRK2 kinase activity are now recognized as the most common known cause of PD," says UAB neurology researcher Andrew B. West, PhD. West and colleagues demonstrated that PD-associated mutations overactivate the enzymatic activity of the LRRK2 protein in test tubes. The corresponding activity in neurons may be the cause of neurodegeneration.
"These are significant revelations because protein kinases, like LRRK2, are excellent targets for small-molecule drugs, and LRRK2 is the first clear target in neurodegeneration that is potentially modifiable," says neurologist David G. Standaert, MD, PhD, director of UAB's Center for Neurodegeneration and Experimental Therapeutics.
If LRRK2 activity is required for neurodegeneration, efforts can focus on developing drugs that inactivate its associated activity to halt or slow PD. The MJFF awarded UAB investigators a Critical Challenges grant to determine if the kinase activity associated with LRRK2 is responsible for neurodegeneration in PD.
West's and Standaert's laboratories are working to move in vitro research into animal models to solidify LRRK2 as an appropriate drug target, but that endeavor requires the development of customized tools. First, researchers needed a way to express the LRRK2 protein that harbors PD-linked mutations. During the course of several years, and after thousands of sequence reads and dozens of functional assays, West and colleagues succeeded. "We now possess the LRRK2 protein-encoded constructs capable of definitively assessing the impact of LRRK2 kinase activity toward neurotoxicity," he says. West and colleagues built these constructs with two mutations, one that down-regulates kinase activity (D1994A) and one that strongly activates kinase activity (G2019S). These are the molecular tools required to determine whether altered kinase activity of LRRK2 is associated with PD-relevant neurodegeneration.
The second challenge was to create viral vector systems capable of delivering the LRRK2 gene. It encodes a protein of exceptional size that heretofore has hindered its use in many research applications. West and collaborators William J. Bowers, PhD, of the University of Rochester, and Joel N. Glasgow, PhD, of the UAB Gene Therapy Center, are developing specialized virus-based gene transfer technology using modified herpes simplex virus and adenovirus-based vectors. The project will capitalize on Standaert's long-term efforts to identify vectors with an optimized selection for dopaminergic neurons important in PD. Adenovirus development has identified a vector with enhanced neuronal gene transfer due to a structural substitution of the vector's native cell-attachment protein for one derived from canine adenovirus type 2. This customized adenovirus vector potentially can deliver the genes encoding modified and wild-type LRRK2 proteins to the entire substantia nigra to assess definitively whether the kinase activity associated with LRRK2 is responsible for neurodegeneration in PD.
West and Standaert hypothesize that the kinase-enhanced mutation might induce neurodegeneration, indicating LRRK2 protein kinase inhibitors as a viable therapeutic approach. They also hope to define endpoints beyond cell death to begin to understand the molecular mechanisms underlying PD and other potential avenues of therapeutic intervention.
"This is an exciting time in Parkinson research, and at UAB we are on the leading edge of discovery and clinical trials testing new treatments," Watts says.
For more information contact Dr. Ray Watts, Dr. David Standaert, Dr. Joel Glasgow, or Dr. Andrew West at 1-800-UAB-MIST or email mist@uabmc.edu