December 2004 Progress Report 
To: All Supporters of The Myelin Project (TMP) From: Augusto Odone As 2004 draws to a close, we would like to report on both The Myelin Project's most recent activities and the advances of the research we are sponsoring. The 15th Annual Meeting of The Myelin Project Work Group was held in Nice (France) on September 10-12, 2004. Impeccably organized by the European Leukodystrophy Association (ELA), and masterly chaired by Dr. Sam Ludwin, the meeting provided the 33 attending researchers a forum for comparing notes on the progress of their research and for forging new collaborations. Please find below an account of research progress based in part on our notes of the meeting in Nice (a summary of the notes is presented in the Appendix) and in part on developments before and after the meeting. Research Research sponsored or co-sponsored by The Myelin Project (TMP) advanced at a brisk pace in 2004. In addition, 2004 has witnessed several new initiatives directed at myelin repair and at combating individual myelin diseases such as multiple sclerosis (MS) and the leukodystrophies. Multiple Sclerosis In Nice, Dr. Gianvito Martino presented the findings of his experiment designed to transplant human fetal neural stem cells (hfNSCs) in 15 marmosets, with some of the animals receiving the cells intravenously, and other intrathecally. Prior to the transplantation, the animals were immunized using recombinant human myelin oligodendrocyte glycoprotein (MOG) to induce experimental allergig encephalopathy (EAE) and then underwent MRI to assess the extent of their white matter abnormalities. During and after transplantation the marmosets received cyclosporine, an immunosuppressive drug, to prevent rejection of the hfNSCs. Preliminary results have been encouraging. None of the marmosets died due to the procedure. Furthermore, MRI and post-mortem neuropathological analyses showed amelioration of the experimental disease. Further analyses are necessary to assess the feasibility of translating such procedure to humans affected by multiple sclerosis. In coordination with Dr. Martino, Dr. Annik Baron is moving forward with her experiment, "Autologous Schwann Cell Transplantation in the Macaque." Schwann Cells (SCs) derived from peripheral nerve biopsies of adult macaques were genetically modified to express the tracer gene in order to facilitate identification and morphological evaluation at the autopsy stage. After being expanded, they were transplanted into rodent models with MOG-induced focal lesions in the spinal cord, to assess the efficacy of the mode of administration, tracing, and functionality. If this part of the experiment is successful, the next step will be to transplant the cells into the monkeys. Dr. Christian Confavreux is completing the preparation of the progesterone clinical trial in postpartum women. The trial is scheduled to start at the beginning of next year. Please see our June 2004 progress report for background information on the trial at http://myelin.org/progressreports.htm. In consultation with The Myelin Project, ELA has awarded Dr. Confavreux a US$ 300,000 grant to cover the expenses required for the commencement of the study scheduled in January 2005. Dr. Su-Chun Zhang reported on the progress of his study, aimed at transplanting oligodendrocyte precursors (OPs) produced from human stem cells into the brain of neonatal shiverer mice. Serial histological analyses showed that the OPs survived and differentiated into oligodendrocytes, the cells responsible for myelin formation in the central nervous system (CNS). However, myelin basic protein-positive profiles were observed only three months following transplantation, which suggests a slow maturation process of human OPs. Dr. Jeffery Kocsis transplanted highly purified olfactory ensheathing cells (OECs) from transgenic pigs into a demyelinated lesion of the African green monkey spinal cord. Robust remyelination was found four weeks post-transplantation in 62.5% of the lesions. This is the first demonstration of xenotransplantation of OECs into the primate spinal cord resulting in remyelination. We have received proposals from Dr. Bert 't Hart of the Biochemical Primate Research Center in the Netherlands and Dr. Yakov Ron of Robert Wood Johnson Medical School, New Jersey. The former proposal aims at inducing specific immune tolerance of the CNS myelin in marmosets as a therapy for MOG-induced EAE. The latter intends to achieve the same result by testing an immunotherapy protocol in which genetically modified autologous B-cells would be transplanted in mice to achieve tolerance against the major self-antigens in MS. We are currently reviewing these proposals. Leukodystrophies Dr. Johannes Berger of the Brain Research Institute at the University of Vienna has asked The Myelin Project to co-finance an experiment based on a pharmacological gene therapy approach to X-ALD. In this disorder, a gene called ABCD1 is impaired and is unable to code for the protein responsible for transporting the very long chain fatty acids (VLCFAs) into the peroxisomes where these acids are metabolized. ABCD2 is another gene that produces a transport protein but in cells not involved in the ALD pathology. In his experiment, Dr. Berger plans to upregulate the expression of the ABCD2 gene in ALD-specific cells to assess whether this would compensate for the impaired ABCD1. After Dr. Berger's proposal had been reviewed by several ALD specialists, The Myelin Project-ELA have agreed to grant the proponent Ä 281,000 (appr. US$ 366,000) out of an estimated Ä 386,000 (appr. US $503,000) total cost; the remaining funds would be provided by the EU. Dr. Patrick Aubourg of HÙpital Saint-Vincent-de-Paul in Paris plans to conduct a clinical trial in 2005 based on the transplantation of autologous hematopoietic stem cells that are transduced with the ALD gene. He intends to use a unique HIV-based vector that would allow the transplanted cells to live longer and express the transduced normal gene efficiently. Patients included in this trial will be children who are candidates for allogeneic bone marrow transplantation but without an HLA-matched donor. If successful, the trial would represent a major advance in the treatment of ALD. The experiment is being financed by organizations other than TMP. Dr. Hugo Moser is refining his study on the use of Lorenzo's Oil as a preventive therapy in childhood ALD; he plans to submit soon a manuscript to a major neurological journal. At the same time, he is setting up a controlled clinical trial with the Oil with the participation of 200 AMN patients, 100 of whom will receive the Oil and 100 a placebo. The Myelin Project has helped Dr. Moser put together the funding for the initial phase of the trial, which is scheduled to commence December 2004. So far we have attracted the interest of ELA and CRODA Universal to the study; they have transferred $75,000 and $ 50,000, respectively, to a special AMN account we set up at the Kennedy Krieger Institute. In addition, Scientific Hospital Supplies will donate the Oil required for the trial. For our part, we will contribute $25,000 toward this endeavor. Aside from these corporate contributions, the AMN initiative has been supported by several individuals, most notably through the services of Mr. Chris Kaag, whose mini-triathlon raised over $12,000. In a Canavan disease trial, Dr. Paola Leone of the Robert Woods Johnson Medical School, New Jersey has successfully transferred the aspartoacylase gene (ASPA) directly into the white matter lesions of ten affected children. Improvement in cognitive and motor function was observed in varying degrees. She hopes for even better results in the Phase II trial, which will test the efficacy of this treatment in a larger group of children. For all this progress, an overview of the advances in MS and the leukodystrophies reveal some constraints. One of them is that because of the differences among species, findings in animal models often do not apply to human conditions. Second, the difficulties in producing large quantities of stem cells hamper progress in transplantation research. Last, current therapeutic approaches to the leukodystrophies seem to work only for children who are asymptomatic at the time of therapy. Notwithstanding these constraints, we are encouraged by recent advances, which mark a transition from in vitro and animal research toward the clinic. The advances, together with the problems encountered, will provide a guide from this point on. Policies On the policy front, TMP will continue to direct its resources toward translational (as opposed to basic) research. We are pleased to learn that other organizations such as NIH and the MS Society are moving in the same direction. We believe we helped bring about these policy changes to some extent by financing innovative experiments (such as the Yale Schwann cell trial in MS) considered high-risk by others. We will continue to provide seed or trigger funds to finance pilot studies and jump-start experiments or trials whose major costs would then be borne by larger organizations. We have already moved in that direction in the past. The funds we have conveyed to the Kennedy Krieger Institute in Baltimore will allow Dr. Moser to start the AMN trial on schedule; the bulk of the funds would then be financed by the NIH and the FDA. Similarly, the funds we have given to Dr. Confavreux will allow him to start the progesterone trial next December. The balance of the funds required by this trial would come from French MS associations and private donors. TMP intends to augment the leverage of its own funds for new research endeavors. This means continuing to join forces with other organizations interested in co-financing experiments directed at finding therapies for myelin diseases-organizations such as the Hovde Foundation, Myelin Repair Foundation, PMD Foundation, Boston Cure Project, Stop-ALD Foundation, Hunter's Hope Foundation, and, of course, ELA. In between our annual meetings, The Myelin Project plans to hold a series of international meetings, each focused on an individual myelin disease, similar to the one we organized earlier this year on PMD. We propose metachromatic leukodystrophy (MLD) as the target disease for one such meeting in 2005. Finances and Fundraising As our next mid-year report will indicated in more details, TMP reserves are dwindling under the impact of increased experiment/trial funding on the one hand, and of declining revenues on the other. To continue its activities, TMP needs additional revenues to replenish, and possibly expand its finances. Since we do not receive public money, we rely solely on the generosity or fund-raising activities of our supporters. With regard to the latter, we would like to encourage our followers to organize fundraising events (golf, soccer and tennis tournaments, marathons and the like) in their respective communities. Please be generous with your donations and/or your time. We would like to extend to all our supporters our best wishes for the holidays and for 2005, which we hope will witness important breakthroughs in our research for the benefit of MS patients and leukodystrophy children. APPENDIX Summary Notes of the Meeting in Nice 
In this appendix, we wish to report on the Nice presentations, or certain aspects of them that are not covered in the main text of our letter. We apologize for some unavoidable overlaps between these two sections of our paper. Dr. Jack Antel of the Montreal Neurological Institute talked about Magnetization Transfer Ratio (MTR) as a tool to assess change in MS lesions. Abnormalities of MTR values in the normal-appearing white matter are better indicators of the development of patient disability than MRI findings in the lesions. In particular, lower MTR values predict a poorer long-term clinical outcome. Dr. Giancarlo Comi of San Raffaele Hospital, Milan discussed the physiology of recovery in MS. He noted that repeated episodes of demyelination lead to axonal degeneration and a failure of endogenous remyelination. Early axonal damage is followed by an initial period of compensation due to the redundancy of the CNS and brain plasticity. Neurophysiological and functional magnetic resonance studies demonstrate that these compensatory mechanisms fail with the increase of axonal degeneration. At this point, patients enter the progressive phase of the disease. Dr. Comi also mentioned that the degenerative phase is at least partially due to acute inflammation. He further described how several factors affect recovery after the occurrence of an acute lesion. These factors include lesion characteristics, pre-existing damage and efficiency of remyelination. He stressed the favorable role played by steroids in MS recovery. In this connection, he cited Dr. Richert of the Children's National Medical Center, Washington, DC and others who observed a reduction in tissue damage and accelerated lesion recovery in RRMS patients who were treated with intravenous methylprednisolone. Dr. Inderjit Singh of the Medical University of South Carolina presented his findings on the anti-inflammatory mechanism of Lovastatin. He reported that Lovastatin down-regulates 140 and up-regulates 18 immune-related genes. These altered genes encode for leukocyte-specific markers and receptors, histocompatibility complex and various other components of the immune system. He also indicated that certain transcription factors, which were upregulated, might be mediating Lovastatin's anti-inflammatory and immunomodulatory effects. These findings provide new insight into the molecular events associated with the protection afforded by statins in the treatment of demyelinating diseases such as MS. Dr. Singh believes that these anti-inflammatory effects also favorably influence the proliferation and development of transplanted cells. Dr. Robin Franklin of the University of Cambridge talked about a substance called superparamagnetic iron oxide (SPIO). This substance can be used to label glial cells, especially Schwann cells (SCs) and olfactory ensheathing cells (OECs), so that when transplanted, the cells can be detected by MRI. He also described a new MRI-detectable agent called Magnetic Microspheres (MM), which like SPIO does not affect the function of glial cells and allows them to retain their remyelinating capabilities. Dr. Antonio Uccelli of the University of Genoa, Italy discussed the transplantation of mesenchymal stem cells (MSCs) as a new potential approach to cell therapy in demyelinating diseases. He pointed out that MSCs, which represent a subset of stem cells obtained from human bone marrow have unique immunomodulatory properties. Indeed MSCs can inhibit proliferation of T cells both by means of cell-to-cell contact mechanisms and through the release of soluble factors. When transplanted into mice, MSCs were shown to ameliorate MOG-induced EAE. This amelioration was obtained not only when MSCs were transplanted before disease onset but also early after the appearance of first symptoms. This effect was accompanied by a striking reduction of T-cell and macrophage infiltrates as well as of demyelination. Dr. Bill Blakemore of the University of Cambridge reviewed experiments that evaluated the effectiveness of transplant-mediated remyelination in areas of chronic demyelination. Specifically, he reviewed various animal models in which successful results had been achieved by transplanting oligodendrocyte precursor cells (OPCs), SCs and olfactory ensheathing cells (OECs). He pointed out that most models represented situations in which myelination or remyelination was already occurring and therefore differed from chronic MS lesions where remyelination was absent. In his experiments in the Taiep rat, which suffers from chronic demyelination, he showed that repopulation of chronically demyelinated tissue by transplanted OPCs was extremely limited unless the CNS tissue was depleted of endogenous OPCs. He also noted that repopulation was not followed by remyelination unless an acute inflammatory environment was created in the OPC-repopulated tissue. Transplant-mediated remyelination would therefore be unlikely to succeed in areas of chronic demyelination. However, induced acute inflammation may activate the OPCs present in some chronically demyelinated MS lesions and generate remyelinating oligodendrocytes. Dr. Evan Snyder of the Burnham Institute, California, reported on the fate and therapeutic action of human umbilical cord cells (UCCs) in the brain of children with Krabbe disease and noted that umbilical cord blood transplant works well if done in the first two weeks of life. The embryonic stem cells derived from the donor's umbilical cord blood stimulate the host cells to differentiate and form neurons. He added that UCCs were found to be admixed with host cells in both white and gray matter after transplantation. He also talked about neuroinflammation like ischemia, lysosomal storage disorders, and demyelination and noted that these conditions stimulate homeostasis-promoting cells to migrate towards the injury site. Dr. Patrick Aubourg of Hospital Saint Vincent de Paul, Paris is planning a clinical trial in the beginning of 2005 envisioning transplantation of autologous CD34-positive cells (these subpopulation of cells contains hematopoietic stem cells, HSCs) in children with the cerebral form of adrenoleukodystrophy (ALD). The CD34+ cells will be transduced with the normal ALD gene using an HIV-based vector. The trial would evaluate the safety of the corrected cells' infusion, the efficacy of gene transfer in peripheral blood and bone marrow, as well as the effect of the procedure on the course of ALD. Transplantation of CD34+ cells genetically corrected ex vivo with a murine retroviral vector results in too low a percentage of corrected cells (less than 1%) in the peripheral blood when the therapeutic gene has no selective advantage. This is attributed to the high cytokine concentrations required by murine retroviral vectors to transduce HSCs. Because the cytokines also trigger the differentiation of HSCs, this led in the long term to loss of transgene expression. Dr. Aubourg pointed out that by contrast HIV-based vectors have the ability to cross the nuclear pores of non-dividing cells, thus minimizing the requirement of cytokines for HSC prestimulation. This was confirmed by a preclinical experiment in which the researcher achieved high transduction efficiency (at least 50%) and transgene expression with the use of an HIV-based vector with minimal cytokine use. Mr. Augusto Odone used the slot originally allocated to Dr. Moser (who was unable to attend the meeting) to illustrate the mechanism by which Lorenzo's Oil normalizes very long chain fatty acids (VLCFA) in ALD. Using an animated flash presentation, he explained that monounsaturated fatty acids contained in the Oil, namely, oleic and erucic acids, compete with the elongation of their saturated homologues, thus curtailing the biosynthesis of C24:0 and C26:0, the main villains in ALD according to several researchers. He mentioned that researchers are still investigating the impact of the normalization of VLCFA on the clinical course of the disease. Dr. Paola Leone of the Robert Woods Johnson Medical School, New Jersey described her study in which a recombinant adeno-associated virus (AAV) was used as a vector to transfer the aspartoacylase gene (ASPA) into patients with Canavan disease. Nine hundred billion genomic particles were administered neurosurgically directly into the affected regions of the brain of patients. It was expected that the introduction of this gene, which is defective in patients with Canavan disease, would result in the production of the enzyme aspartoacylase and the subsequent break down of N-acetyl-aspartate (NAA), high levels of which impair normal myelination. To date, this procedure has been conducted in ten children some of whom under the age of 2 years. Pre-and post-delivery biochemical, radiological, and neurological tests have shown significant improvement in all the children, with the improvement more marked in cognitive functions than in motor abilities. Spasticity was greatly reduced and some children were able to coordinate with both hands, which is considered a significant success. As expected, the children who were younger had a comparatively better outcome. Dr. Leone is now planning a phase II clinical trial to test the efficacy of the treatment in a larger group of children. In his overview of Pelizaeus Merzbacher Disease (PMD), Dr. James Garbern of Wayne State University School of Medicine confirmed that the disease is triggered by a mutation in the Plp1 gene that codes for proteolipid protein (PLP). A total absence of PLP results in a milder form of PMD than that observed in children who overproduce it. Therefore, gene-silencing techniques aimed at stopping PLP production could be a potential treatment strategy for children who overproduce the protein. Dr. Garbern suggested that cell transplantation could be another therapy. However he mentioned that it was crucial that the transplanted oligodendrocytes be in direct contact with axons in order to keep them healthy since degeneration of axons is probably a major factor in progression of neurologic disability. Dr. Garbern also stated that glial derived growth factors and other neuroprotective agents have been shown to increase the life span in patients with amyotrophic lateral sclerosis and could have a similar effect in other myelin degenerative disease, such as MS. Lastly, he mentioned that researchers at Sanofi-Aventis had developed an experimental drug, HP184, which increases nerve conduction velocity in the Long Evans shaker rat, which has a mutation in the myelin basic protein gene. Because there are similarities between these and mouse leukodystrophy models (e.g, the shiverer mouse), there is reason to believe this drug could be effective also in PMD and other myelin diseases. Dr. Franca Cambi of the University of Kentucky talked about studies directed at elucidating PLP splicing, a process by which the RNA non-coding segments, i.e., the introns, are separated from exons that carry the genetic code. Mutations that interfere with the splicing process are believed to be responsible for the underproduction of PLP in some patients with PMD. She said that identifying the affected gene sequences and regulating their function in splicing can, in principle, result in the normal production of PLP and might hold the key to future therapies. She is in the process of identifying those sequences and suggested cell-specific regulation strategies. In collaboration with Dr. Odile Boespflug-Tanguy of INSERM, France, Dr. Cambi is also working with RNA interference (RNAi) as a technique that inhibits overproduction of PLP. RNAi is a double-stranded RNA that is a potent inhibitor of its corresponding DNA. Two types of synthetic RNAi have been developed and proven successful in cell studies, and the technique is expected to eventually be helpful in those PMD children who overproduce PLP. Both researchers are now working towards fine-tuning the procedure so that only the defective gene is targeted. An efficient system to deliver these "drugs" will then be developed and tested in animal studies. Dr. Luigi Naldini from San Raffaele Telethon Institute for Gene Therapy, Italy shared the findings from experiments performed by his team in which they successfully used lentiviral vectors to transduce the arylsulfatase-A gene into autologous HSCs ex vivo and transplanted them into metachromatic leukodystrophy (MLD) mice. They achieved extensive repopulation of the CNS microglia and peripheral nervous system endoneurial macrophages by transgene expressing cells. Arylsulfatase enzyme activity was reconstituted to significant levels in the tissues. This prevented deficits in the development of learning and coordination, motor conduction impairment and neuropathological abnormalities. Drs. Maria Escolar and Joanne Kurtzberg presented the clinical and neurocognitive results of HSC transplants in patients suffering from infantile Krabbe disease that were conducted by the Pediatric Blood and Marrow Transplant Program at Duke University Medical Center and the Center for Child Development and Learning at the University of North Carolina at Chapel Hill. HSCs are cells that are present in the umbilical cord blood and the bone marrow. Of the 29 patients who received HSC transplants, all those who were asymptomatic and 50% of those who were symptomatic at the time of transplant survived. Patients who were asymptomatic at the time of transplantation did better in both cognitive and motor development. In those who were already symptomatic, the transplant arrested the progression of the disease and there was modest cognitive improvement. In a subset of eight children who were transplanted in the 1st month of life, the results were more favorable. These children were learning new skills and their overall development was good. However, there were differences in the extent of improvement depending on how far the disease had progressed (Krabbe disease often starts during the gestation period). Follow-up evaluations showed that three out of the eight were able to walk, three walked with walking aids and two couldn't walk at all. Umbilical cord blood transplant has also been conducted in 15 ALD and 14 MLD patients. In both diseases, the children who were highly symptomatic did not benefit from the transplant, either stabilizing at a level of poor function or showing signs of progressive disease, whereas there was some improvement in those who were minimally symptomatic. Asymptomatic patients did not develop clinical symptoms of leukodystrophy. Dr. Klaus Nave of Max Planck Institute, Germany discussed the techniques used by researchers to develop various mouse models of human diseases. He also talked about the role of cholesterol in myelin formation. He described a mouse model he created that lacked the ability to produce myelin in glia cells to study the effects of the absence of cholesterol. These mice had severe delayed myelination and exhibited a PMD-like phenotype underscoring the importance of cholesterol in myelination in both CNS and PNS. He intends to send us a proposal to further this research aimed to evolve a therapy for PMD and possibly ALD. | 
