Current Research Update

HIGHLIGHTS OF THE PAST YEAR AND PLANS FOR THE FUTURE

The Nancy Davis Center Without Walls (NDCWW) is made of seven groups with complementary expertise in multiple sclerosis (MS) research. The NDCWW exchanges scientific information and collaborates at multiple levels. Several new and exciting scientific achievements in the past year have continued to fuel the NDCWW’s commitment to find a cure for MS. Scientific meetings provide an open forum for discussion and presentation of novel ideas and findings. Centers with specific expertise provide valuable support to others, with each having a unique background. This constant exchange process is nurturing an outstandingly rich research activity. During these meetings, 30 key investigators of the 7 institutions shared information prior to publication. The specific scientific accomplishments of individual centers are contained in the individual reports. The highlights are presented below.

UNIVERSITY OF CALIFORNIA, SAN FRANCISCO (UCSF)

Understanding the genetic events leading to MS is key to defining the basic underlying etiology of this disease. However, despite intensive long-standing efforts by many research groups across the globe, the knowledge of MS genetics remains incomplete. We believe that the availability of more efficient tools in molecular genetics together with a growing understanding of the landscape structure of the human genome, provide us with a new experimental paradigm for the deconstruction of this complex problem. In light of clinical and locus heterogeneity as well as modest individual gene effects, achieving adequate statistical power constitutes an important requisite to generate unequivocal results. Therefore, a large and well-characterized dataset is fundamental for the success of genetic studies in MS. Our recent progress includes:

1. Collecting large numbers of samples
To achieve our research objectives, we collect blood samples to extract genetic material from a large number of families with one or more affected individuals. Since the initiation of this effort, we have completed the ascertainment and collection of biological specimens from over 11,700 individuals, including 4,500 affected with MS. By analyzing their genetic makeup, we will be able to understand the rules of MS inheritance, and consequently define the basic etiology of MS, improve risk assessment, and influence therapeutics. During the past year, we extended significantly our library of biological materials.

2. Completing the MS genetics map
The UCSF MS Genetics Group, a founding member of the International MS Genetics Consortium (IMSGC), has brought together leading scientific groups worldwide to advance MS genetics research and to share valuable resources. A genome-wide association study was carried out and published in late 2007 by the UCSF team in conjunction with the IMSGC. We identified the Interleukin-7 Receptor (IL-7R), the Interelukin-2 Receptor (IL-2R) and the Lymphocyte Function-Associated Antigen 3 (LFA3 – also known as CD58) as susceptibility genes for MS.
Following this success of identifying the first non-HLA associated MS susceptibility genes, the UCSF group led a new scientific analysis of an US MS cohort. One of the most significant findings in this study was an association for a gene called GPC5. This gene may play a role in the control of cell division and/or growth regulation. We also identified significant associations between several neural genes and different MS traits, including the amount of brain tissue involved (e.g. lesion load), brain atrophy and age of onset. Over the course of the last 2 years, together with colleagues from around the world, we identified 10 novel genes influencing disease risk. This, however, represents just a fraction of the entire MS genetic architecture. Our current goal is to understand how these genes influence MS susceptibility and/or disease progression, and translate this information to the clinic to improve the therapeutic management of MS.

3. Relationships between genetics and MRI measures
Using state of the art MRI imaging, we characterize the clinical expression of MS in a way that has never been possible before. As a first step, we focused on the relationship between the strongest susceptibility gene known in MS (HLA-DRB1*1501) and disease severity. We found that patients carrying this gene had increased white matter lesions visible on brain MRI scans, increased axonal injury, increased brain atrophy and increased cognitive impairment compared to patients who did not carry that gene.

4. Testing promising therapies for MS
The group at UCSF is conducting the first trial of neuroprotection in MS in collaboration with OHSU. Patients with early MS who receive a drug that protects nerve cells or placebo are monitored for two years with state of the art MRI to determine drug efficacy.

5. Understanding pediatric MS
The group at UCSF has identified difference in the spinal fluid and brain MRI scans obtained at the time the disease starts in pediatric patients that will result in earlier diagnosis and care. Also these differences inform us on underling disease mechanisms at play in younger patients.

YALE MULTIPLE SCLEROSIS PROGRAM

The goal of MS research at Yale is to advance neurorehabilitation in MS through novel molecular and cellular approaches that will provide neuroprotection and neural repair, to preserve and restore function in people with nervous system disorders. Our recent progress includes:

1. Showing that bone marrow-derived stem cells support remyelination
Our group is attempting to demonstrate that bone marrow-derived stem cells, delivered intravenously, support remyelination within injured spinal cord in rodent animal models and non-human primates. This study brings us closer to novel stem cell-based therapies for MS.

2. Demonstrating that human bone marrow-derived stem cells improve functional outcome in neurologic diseases
We have demonstrated that human bone marrow-derived stem cells (hMSCs) improve functional outcome in stroke models. We now plan studies on neuroprotection and repair by hMSCs and olfactory ensheathing cells in models of MS. These studies will provide basis for human clinical studies.

3. Confirming that sodium channels play an important role in immune cells in MS
We are involved in delineating the role of sodium channels in immune cells in MS. MS arises, in part, from immune attack on myelin and neurons. Macrophages and microglia are two cell types that participate in this attack. We have made novel finding that sodium channels are present in and regulate the function of macrophages and microglia. This opens up the possibility of finding new therapies that will halt the inflammatory assault on the nervous system in MS.

4. Testing promising therapies in clinical trials
Our clinical program provides care to a large number of patients with MS and addresses complex medical problems, irrespective of their economic background. Our clinical activities are aimed at contributing toward the development of novel treatment approaches for MS. Ongoing clinical trials, many of which have been initiated through the networking of investigators at the various Nancy Davis Centers, include Phase I through Phase III clinical trials, selected for their scientific merit in developing new therapies for MS.

CLEVELAND CLINIC FOUNDATION

Key issues in MS treatment include the underlying mechanisms causing disease, and the related issue of how to repair damaged brain tissue in MS patients. To better pursue these two issues, we recruited Drs. Richard Ransohoff and Bruce Trapp for expanded roles in the NDCWW. Dr. Ransohoff is an expert in the role of a group of immune communicating molecules – chemokines – in biology and disease, and Dr. Trapp is an expert in myelin biology with a strong commitment to understanding the pathogenesis of neurodegenerative diseases.

1. Chemokines are important in the MS disease process

We believe that chemokines may be important in the MS disease process – both inflammation mediated tissue damage, and the repair process. Dr. Ransohoff and colleagues are using sophisticated gene-targeted mice to determine which model system will permit the most powerful insights into the function of individual chemokines and receptors. It has become clear that certain chemokine receptors are essential to development of brain inflammation in the animal model of MS. This information will identify which of the chemokine receptors should be targeted for novel treatments. The information from these studies will also provide valuable information needed to track inflammation in MS patients. The studies supported by the NDCWW address the role of CXCR2 in the disease. These studies are directed at testing the hypothesis that the chemokine receptor CXCR2, which is expressed both by infiltrating white blood cells, and by myelinating cells in EAE lesions, carries out two deleterious functions during demyelination. First, CXCR2 helps white blood cells enter lesions. Second, CXCR2 stops remyelinating cells from entering lesions to carry out repair. Dr. Ransohoff is studying both effects, which can be separated by sophisticated gene knock out mice and radiation chimeras.

2. Immune cells residing in the brain protect nerve cells

Pioneering work in Dr. Bruce Trapp’s laboratory has identified transected axons in demyelinated white matter lesions and transected neurites in cortical lesions in MS brains. Dr. Trapp’s work has helped us to characterize cortical lesions in multiple sclerosis patients. Dr. Trapp has developed the hypothesis that activated microglia, the resident immune cells of the central nervous system, play an important neuroprotective role in the CNS. Using animal models, Dr. Trapp and colleagues have successfully shown that activated microglia induces synaptic stripping and may upregulate a neuroprotective response in the cerebral cortex. Using a combination of gene expression profiling and proteomic approaches, Dr. Trapp’s laboratory is identifying candidate molecules and pathways that play roles in this microglial activation and associated neuroprotective response in the CNS. Identification of these molecules will ultimately result in developing therapeutic targets to promote neuroprotection and halt neurological disability in MS patients.

HARVARD MEDICAL SCHOOL

In the past year and throughout the next, we are:
1. Continuing to develop new blood tests for multiple sclerosis
It is generally believed that MS is a disease in which white blood cells go from the bloodstream into the brain and cause damage. A major goal of MS research is a blood test that could be used to measure the abnormal white blood cells and use this blood test as a basis for therapy and monitoring. At the Harvard Center, we continue to work on the development of new blood tests. In the past year, we have identified a serum marker for MS that can be carried out on a few drops of frozen blood and a marker that may be able to distinguish relapsing-remitting from progressive phases of the disease.

2. Continuing to advance MRI research
MRI has been a major advance in MS because it allows us to see the disease process in patients and to follow the disease progression and response to therapy. In the past year, we continued working to develop new and sophisticated ways to measure the MS process by MRI and are now studying what MS looks like on 3 Tesla imaging which is more powerful than 1.5 Tesla imaging. We continue to study changes in the gray matter of the brain, which may be better linked to disability.

3. Confirming the CLIMB natural history study
One of our major goals is to find a cure for MS. In order to do this, we must learn how people with MS are doing in the new era of treatment and MRI imaging. Towards this end we have established a natural history study called CLIMB (Comprehensive Longitudinal Investigation of Multiple Sclerosis at Brigham and Women’s Hospital) in which we obtain yearly neurologic exams, MRI scans, blood tests, cognitive testing and quality of life assessments. We have now entered over 830 patients in the CLIMB study, some of whom have been followed for 5 years. We are investigating the percentage of patients who are controlled or not controlled on current medications and are developing probability curves of how a patient is projected to do with their MS based on clinical exam, MRI imaging and blood tests.

4. Investigating the role of neural stem cells and neuroprotection in MS

A population of cells exists in the central nervous system of adults capable of self renewal and repair. They are neural stem cells and have the capability of becoming astrocytes, neurons, and myelin forming cells (oligodendrocytes). We are studying how these cells regenerate in the animal model of MS, called experiment allergic encephalomyelitis and studying compounds to induce them. We are also studying new compounds that can protect the nerve cells from being damaged in this animal model and may be applicable to the progressive forms of MS. One such drug is the antibiotic minocycline. We have also found new chemical compounds that slow progression in the animal model of progressive MS.

5. Investigating new MS drugs
• Oral Anti-CD3: One of our major goals is to find oral therapies for MS. Our lead drug in this area is using a monoclonal antibody that is usually given intravenously that we found acts orally. We have now treated normal subjects and get strong immune effects. This is an exciting result and we plan to dose MS patients in the coming year.
• CTLA4-Ig: This is a monoclonal antibody that has shown promise in initial trials of relapsing-remitting MS and we are planning to begin a phase II double-blind trial of this antibody.

JOHNS HOPKINS MULTIPLE SCLEROSIS PROGRAM

During the 2008–2009 year for the Johns Hopkins MS Center in the Nancy Davis Center Without Walls we have continued to enhance our understanding of the mechanisms underlying damage to the nerve fibers themselves (called axons) and are developing strategies to measure axon damage using non-invasive imaging methods. These projects will lead to therapeutic interventions aimed at arresting disease progression in MS.

1. Understanding how nerves degenerate
It is now well recognized that there is both demyelination and damage to nerve fibers (axons) in MS. Axon damage appears to correlate better with permanent disability in MS since loss of myelin only slows nerve conduction and it potentially repairable, but axon loss (akin to cutting a wire) disrupts the signal permanently. Since axons in the brain and spinal cord nerves do not regenerate it is critical to stop this process early. There are likely two mechanisms by which axons are damaged in MS, 1) as a result of inflammatory cells that migrate to the nerve tissue and release toxins, and 2) as a result of chronic loss of myelin, which not only provides a protective coating around nerves, but also is a source of active growth support to the axon. We have established models of both the inflammatory and absent myelin processed in order to understand the underlying cellular and molecular mechanisms by which axon damage and loss occur. We have mice with altered genes such that they cannot produce a myelin protein called MAG (myelin associated glycoprotein). MAG is the innermost part of the myelin sheath around axons and is the part that directly contacts the surface of axons. We discovered that the loss of MAG in these mice results in a very slow but progressive loss of axons in the spinal cords of these mice. This process appears very similar to what we think happens in the progressive stages of MS. We showed that by adding a soluble form of MAG (MAG-Fc) that we can protect axons from toxin induced damage. We also discovered that this occurs through the β-1 integrin signaling pathway. By understanding how myelin proteins normally protect axons, we may be able to design new neuroprotective therapies. We also found that mice lacking the MAG protein have much more severe axonal loss when they are induced to have EAE than do the EAE wildtype (genetically normal) mice. We are exploring whether this is related to enhance activation and invasion of immune cells or lack or protection of the nerve axons.

2. Visualizing axons and myelin with magnetic resonance imaging
A second major focus in our group has been to develop non-invasive methods of imaging axons and myelin in order to better quantify these processes in MS patients and to have an objective outcome measure in future clinical trial of drugs aimed at preventing axon damage (neuroprotection) or for remyelination (neurorepair). A method called diffusion tensor imaging (DTI) has shown promise in allowing us to visualize the integrity of nerve fiber pathways and we have found that this information better predicts damage than conventional MRIs, which mostly measure inflammation. Using the fiber tracking software that we have developed, we can simultaneously obtain information about myelin integrity using a second technique called magnetization transfer imaging (MTI). We are running parallel studies in animals and humans. In the animal studies we can take out the brains and spinal cords and determine under the microscope exactly what the DTI/MT measures tell us about axons and myelin stability so that in the human studies we can better interpret the data. We showed that these new MRI techniques can reliably detect damage to nerves in the spinal cord. We also found that spinal cord MRI abnormalities can be localized to specific functional pathways in that carry information related to sensation and power and that our new MRI methods predict these functional changes much more reliably than older methods.

UNIVERSITY OF SOUTHERN CALIFORNIA (USC)

The team at the USC Center remains engaged in the study of stem cells, protective effect of pregnancy, vaccine treatment for MS, role of viruses in MS, and their ability to participate in brain repair and regeneration. In the past year and throughout the next, we are:

1. Studying the ability of stem cells to repair and regenerate
Our efforts in this project involve new strategies to control the development of stem cells into the specialized brain cells needed to repair damage to myelin and promote healthy brain function. We have successfully transplanted stem cells into the brains of mice with MS-like disease and shown that these cells remain alive for over two months. Our current strategies are to enhance the function of these transplanted cells so that they can facilitate repair and recovery.

2. Evaluating protective effect of pregnancy in MS
The study of pregnancy in MS also remains a primary focus of USC investigators, with a goal of understanding the protective effect of pregnancy on MS, and what might be responsible for the increased risk for relapse that occurs after delivery. Recent data suggest an indirect neuroprotective function in immune cells isolated from pregnancy, and that the balance in the immune response is dramatically changed during and after pregnancy.

3. Developing a vaccine for the treatment of MS
We continue our efforts to develop a successful vaccine for the treatment of MS using heat shock protein, myelin complexes. We will test these complexes to see if they can treat animal models of MS.

4. Determining the role of viruses in MS
The USC team is continuing to study both endogenous viruses (HERV) and viral infections such as Epstein-Barr Virus.

5. Clinical trials for promising agents for MS
As with our partners in the Nancy Davis Centers Without Walls centers, we participate in programs aimed at a search for the cure of MS. This involves active study of disease mechanisms and the conduct of clinical trials. Our own trial with cell based gene therapy is in collaboration with UCSF. Several studies, especially those involving immune modulation, involve our colleagues at Oregon, Harvard, Cleveland Clinics, UCSF, and Hopkins. All the centers are also involved in trying to identify the risk factors that would indicate who may develop serious virus infection as a complication of Tysabri.

OREGON HEALTH SCIENCE UNIVERSITY (OHSU)

The highlights of 2008/09 are as follows:

1. Completing the first clinical trial of a genetically engineered protein to treat MS
This year we completed the first ever clinical trial of a new treatment for MS invented at OHSU. This treatment involves administering a bioengineered protein, called recombinant T cell ligand (RTL) 1000, by intravenous injection. RTC 1000 shifts the immune system from a “pro-inflammatory” to a regulatory state and thereby may control MS. This initial study involved OHSU, Yale and three other sites. Thirty people participated in this initial study and we determined that a dose of 60 mg could be given intravenously safely. This initial study sets the stage for doing a larger trial to determine whether giving 60 mg of RTL1000 once a month can be done safely and whether it prevents new MS lesions from developing in the brain.

2. Researching how blocking a protein in mitochondria protects nerve fibers
Mitochondria are the energy “factories” in cells. We believe that mitochondria in the nerve fibers or axons in MS become injured and that this leads to the loss of axons and permanent disability in MS. We discovered that inactivating a specific mitochondrial protein, called cyclophilin D, led to a dramatic protection of axons in a mouse model of MS. This novel finding points the way to a new approach to treating MS by blocking this protein with a drug. This year we showed that neurons in which cyclophilin D is inactivated are protected from injury caused by damaging increases in calcium. This supports our idea that blocking cyclophilin D with a drug would be protective in MS.

3. Continuing to develop lipoic acid as a treatment for MS
We were the first to demonstrate that the natural anti-oxidant, lipoic acid, was highly effective at treating the mouse model of MS and the first to begin testing lipoic acid in MS subjects. This year we showed that giving 1200 mg of lipoic acid orally to people with MS gave blood levels similar to that of mice given a therapeutic dose of lipoic acid. We are now planning to test lipoic acid as a treatment for optic neuritis; if successful, this trial would suggest that lipoic acid would be a good oral treatment of relapses of MS and therefore an alternative to steroids.

4. Studying whether energy production in the brains of people with MS is impaired
We believe that the mitochondria in the brain in MS do not produce normal amounts of ATP, the energy “packets” of all cells. If true, this may explain why nerve fibers die in MS and may also explain fatigue in MS. More importantly, it would indicate that treating people with MS with drugs and natural products that enhance mitochondrial function would be beneficial. Using a high field (7T) MRI scanner, we have established how we can measure ATP in the brain. We are now starting a study comparing ATP levels in the brain in people with MS and will compare these results with healthy controls.