|
|
|
|
|
|
|
|
|||||
|
Programmes de recherche financés par la Fondation ELA en 2005
Basic Research Study of the role of cdk2 in oligodendroglial development For 30% of patients suffering from leukodystrophies of genetic origin, the etiology of their disease remains unknown despite all of the research currently possible. The present project’s goal is to improve our knowledge of the role played by genes that are essential to the development of the oligodendroglial line, up until the stage of the myelinating oligodendrocyte. The identification of new genes fundamental to the normal development of white substance is a means of identifying new molecular targets whose potential dysfunction might be implicated in the physiopathology of leukodystrophies of undetermined etiology. The hypomyelination or dysmyelination that can be observed in leukodystrophies implies that errors occur at specific stages of the complex sequence of events that transforms an oligodendrocyte precursor cell (OPC) into a mature oligodendrocyte capable of producing myelin. We are concerned here with the earliest events of this sequence: the proliferation of OPCs, their exit from the cell cycle, and the initiation of the differentiation process leading to mature oligodendrocytes. With this in mind, we attempt to achieve two main goals through our investigations: Diseases linked to EIF2B: Physiopathology and treatment approaches, conservation between yeasts and humans Leukodystrophies, hereditary diseases causing degeneration of the brain’s white substance, represent a very heterogeneous group of disorders. Their detection has been transformed by the introduction of nuclear magnetic resonance imaging techniques (MRI), allowing the emergence of novel forms, characterized based on the comparison of clinical and MRI data. Amongst these novel disorders, CACH syndrome (childhood ataxia with central hypomyelination) or VWM (vanishing white matter) is characterized by an onset in early childhood (2-5 years old) and a particular pattern of white substance deterioration in the brain, resulting in a signal that over time becomes identical to that of cerebrospinal fluid. This is an autosomal recessive hereditary disorder, causing rapid decline and resulting in death 2 to 5 years from the onset of clinical signs. Brain MRIs allow detection at a presymptomatic stage. No treatment exists. Five genes (EIF2B1 to 5) code for the five protein subunits of a factor (eIF2B) present in all cells of the organism and implicated in the regulation of protein synthesis, particularly in response to cellular stress. The clinical spectrum of leukodystrophies linked to mutations in these genes is quite broad, ranging from forms with prenatal onset and rapid decline followed by death within a few days to forms with adult onset and a slower decline or even a lack of symptoms. There is a certain correlation between the type of mutation found and the age of onset for a disorder. In mutated lymphocytes of patients with the disorder, we have demonstrated a decrease in the principal action of the eIF2B protein complex (gives GTP to the factor eIF2). This decrease is greatest for early ages of onset. As factor eIF2B is conserved in all animal species, we have concurrently developed a yeast model mimicking the functional abnormalities found in mutated human cells and tested it with a group of 2000 molecules that may have therapeutic applications. Role of Slit proteins in the recruitment and remyelinization potential The existence of precursor cells within the central nervous system offers the possibility of new therapeutic strategies for the treatment of demyelinating pathologies. The subventricular zone (SVZ) of the lateral ventricles is a germinative zone in which these cells are found. One of the properties of the SVZ neural precursors is that they migrate in chains along the rostral migration stream (RMS) over long distances to become part of the olfactory bulb. In the context of demyelination, these cells take a detour from their normal migration stream and become part of areas in need of myelination, where they differentiate into oligodendrocytes. Identification of transcriptional factors controlling the specification of oligodendrocyte progenitors The study of embryonic development has demonstrated the importance of certain molecules in either the production, proliferation, or migration of oligodendrocytes, all of which are disrupted in diseases involving de- and dysmyelination, including the various forms of leukodystrophy. Our recent research involving embryonic mouse brain has allowed the identification of new molecules which regulate oligodendrocyte development . Notably, we created transgenic mouse strains in which the oligodendroglial cells are fluorescent. They can thus be seen and isolated by fluorescence-activated cell sorting for the purpose of making cell cultures or performing molecular genetic analysis. Our project is to use these living tools in order to identify the key genes controlling the production of oligodendrocytes. We will thus select oligodendroglial cells in the earliest stages of their development and analyze their transcriptome using the DNA microchip technique. This research will contribute to a better understanding of the transcriptional control of oligodendrocyte specification. It will provide fundamental data that can be used to promote myelin repair and recuperation of the oligodendrocyte compartiment. It will also offer new tools for diagnosing abnormalities in oligodendrogenesis Myelin Repair
Promoting the formation of primate oligodendrocyte progenitors LThe discovery approximately ten years ago of the existence of neural stem cells able to proliferate and generate all of the cell types in the central nervous system, including oligodendrocytes, offers cellular therapy treatment possibilities for diseases affecting myelin. In rodents, this strategy has allowed myelin repair in various animal models for dysmyelinating diseases. However, in primates, the difficulty of obtaining cells of oligodendrocyte lineage in vitro has long been an obstacle to this type of approach. The aim of our project is thus to optimize the process of obtaining oligodendroglial cells from stem cells or CNS precursors in primates. Firstly, we attempted to determine if there was an area in the embryonic brain more likely than others to produce oligodendrocyte precursors. To do so, we conducted in vitro investigations, finely dissecting the various areas of the anterior brain of 6- to 12-week-old human embryos and culturing them in order to determine their differentiation potential. We have shown that the telencephalon (cortex, lateral ganglionic eminence, and medial ganglionic eminence) produce primarily neuronal cells, whereas the diencephalon (thalamus) produces a large number of cells engaged in the differentiation pathway leading to oligodendrocytes. The latter area of the brain is thus of particular interest for performing transplantations in dysmyelinating disease models, because it allows us to increase the number of target cells. These results will be verified in vivo by transplanting cells from various brain areas studied in the shiverer mouse, an animal model of dysmyelinating diseases. At the same time, we have conducted research aimed at increasing the proportion of target cells obtained in vitro. Cells from various areas of the brain in 6- to 12-week-old human embryos were subjected to factors known for their involvement in oligodendrocyte differentiation during development. Unfortunately, the results obtained up to this point indicate that molecules known for their positive effects on oligodendrogenesis in rodents, such as Sonic Hedgehog (Shh), Noggin, and Cystatine C inhibitors, have been unable to induce the differentiation of immature cells along the differentiation pathway that interests us. Other molecules such as IGF-1 are currently being tested. We have also analyzed the effect of overexpression of the transcription factors Olig 1 and 2 on the biogenesis of oligodendrocytes. Our results indicate that the overexpression of Olig 2 doubles the number of primate oligodendrocyte precursors, which indicates that this strategy, based on controlling certain genes, is more effective than the one based on Sonic Hedgehog and other molecules and used in rodents. Study of the myelinizing potential of cells from different telencephalic regions in human and non-human primates: The discovery approximately ten years ago of the existence of neural stem cells able to proliferate and generate all of the cell types in the central nervous system, including oligodendrocytes, offers cellular therapy treatment possibilities for diseases affecting myelin . In rodents, this strategy has allowed myelin repair in various animal models for dysmyelinating diseases. However, in primates, the difficulty of obtaining cells of oligodendrocyte lineage in vitro has long been an obstacle to this type of approach. The aim of this project is thus to optimize the process of obtaining oligodendrocyte precursors from neural stem cells in primates. To do so, we plan on a three-step approach: firstly, a histological study using human embryos from abortions and macaque embryos will allow us to describe the emergence of oligodendrocyte precursors during the anterior brain’s early development. Secondly, the three principal telencephalic areas (cortex, lateral ganglionic eminence, and medial ganglionic eminence) will be finely dissected and cultured in order to evaluate their respective potential for generating cells of oligodendrocyte lineage in vitro. Finally, cells from these three areas will be transplanted in animal models for dysmyelinating diseases in order to evaluate their respective potential for repairing lesions and producing myelin in vivo. Concurrent to these three steps, we will evaluate the effect of various factors on differentiation in vitro of stem cells and precursor cells from human and non-human embryos in order to optimize the process of obtaining oligodendrocyte precursors. The differentiated cells will then be screened and transplanted in dysmyelinating models. These investigations conducted in primates should ultimately allow transplantations to be made in animal models pathologically similar to humans. They represent a step forward towards cellular therapy for leukodystrophies affecting myelin in humans. Gene Identification Identification of a gene for leucodystrophy of unknown etiology In approximately 30% of leukodystrophies, the etiology remains unknown. In these cases, none of the supplementary tests allow the identification of an abnormality specific to a particular form of the disease. The likelihood of multiple causes in these leukodystrophies of undetermined etiology makes it particularly complicated to study them using genetic techniques usually employed for localizing the gene(s) involved. We have been able to localize one of these genes on chromosome 11, having discovered a small chromosomal rearrangement (microdeletion) in a young man (index case) suffering from both a leukodystrophy of unknown etiology and oculo-cutaneous albinism. This deletion covers a chromosomic region measuring 1.7 Mb and contains a certain number of candidate genes which might explain the occurrence of this leukodystrophy. Several very interesting genes – GRM5, CTSC, and LOC143680 – were thus localized within the deleted region. A negative search for a mutation conducted simultaneously in the index case and in a population of 48 patients suffering from leukodystrophies of undetermined etiology allowed us to eliminate the possibility that these three genes might play a role. This work was part of a collaborative effort involving the INSERM U384 team (Prof Boespflüg-Tanguy). Next, we began the analysis of candidate genes situated outside of the deletion but whose function might be impaired due to a position effect. There are two candidate genes whose function makes them very interesting. While involvement seems unlikely for one of them, additional analysis on the other is necessary before definitive conclusions can be drawn. Elucidation of the role of the MLC1 protein in brain physiology and its implication in MLC pathogenesis; looking for a second gene linked to MLC1 Megalencephalic leukodystrophy with subcortical cysts (MLC) is a rare form of leukodystrophy. The
clinical features include early-onset gait difficulties followed by neurological signs and progressive mental deterioration. This syndrome is characterized by an increase in brain size (megalencephaly), reflected by an increase in cranial circumference during the first year of life. Magnetic resonance imaging (MRI) shows early and severe white substance involvement, despite relatively mild neurological findings during the early stages of the disease. In the later stages of MLC, there is a slow cognitive deterioration, aggravating the disability caused by the disease. Looking for a gene implicated in a new form of leucodystrophy causing liver damage, deafness, and cardiomyopathy Leukodystrophies represent a very heterogeneous group of hereditary diseases which affect white substance, composed primarily of myelin. Molecular flaws implicated in Pelizaeus-Merzbacher-like disease LPelizaeus-Merzbacher disease (PMD) is a leukodystrophy of genetic origin affecting the central nervous system (CNS) and myelin, its primary structural element. This disease is characterized by a defect in the formation of the myelin sheath by oligodendrocytes, the myelinating cells of the CNS. The disease causes a very early-onset disturbance in motor development of variable severity. The PLP1 gene, which codes for the main structural proteins of SNC myelin, is implicated in PMD. The most frequently encountered abnormality is a duplication of the entire PLP1 gene, which causes an abnormal increase in the quantity of PLP protein. This is detrimental to the process of myelination carried out by oligodendrocytes.
Physiopathology Analysis of myelin membrane transmission pathways in diseases linked to PLP A subclass of inherited leukodystrophies, such as Pelizaeus-Merzbacher Disease (PMD) and Spastic Paraplegia Type-2 (SPG2), is caused by mutation of the gene coding for the major myelin protein PLP. Molecular pathology of PLP-missense mutations has largely been studied in heterologous cells. We studied trafficking and turnover of wild-type and mutant PLP protein in oligodendroglial cells. Since myelin membrane transport in general is not well understood, one part of our project is focusing on the characterisation of myelin trafficking pathways. In the second part, we investigate mistrafficking of the PLP missense mutations msd and rsh, which model severe PMD and mild SPG2, respectively. We found that both mutations alter oligodendroglial cell morphology in correlation with the severity of the mediated disease and largely lead to absence of PLP/DM20 from the cell surface. While msd PLP/DM20 was retained in the ER, rsh PLP/DM20 was able to overcome ER retention and resided in late endosomes/lysosomes. Interestingly, cholesterol binding and lipid raft association of the mutant PLP/DM20 was impaired. Furthermore, turnover of mutant PLP/DM20 was increased compared to wild-type protein, demonstrating that mutant protein degrades rapidly. Thus, it’s not the accumulation of mutant PLP/DM20 protein, which harms oligodendroglial cells. Degradation of misfolded PLP/DM20 was preferentially performed by the proteasome, but lysosomal degradation of rsh PLP/DM20 may occur in addition. We conclude, that distinct trafficking defects are associated with PLP-missense mutations. More importantly, accumulation and deposition of misfolded protein is not a pathological feature of the leukodystrophies PMD and SPG2. We suggest that impaired cholesterol and lipid raft interaction is responsible for missorting of rsh PLP/DM20 to the lysosomes and thus may account for dysmyelination in SPG2. Molecular characterization of the genetic relationship observed between pABC1 (an ortholog of ALDP) and PEX2 in a model system Several aspects of peroxisomal diseases, most notably the relationship between the affected gene and multiple disease characteristics, have yet to be elucidated. In order to better understand these complex phenotypes, it is essential to work on “simple” model systems that can be easily manipulated in the laboratory. Filamentous fungi are an interesting model because they lie somewhere between uni- and multicellular eukaryotes and allow the study of developmental problems that cannot be modeled by unicellular yeasts. In Podospora anserina (whose genome has been sequenced), we have demonstrated that a pex2/peroxisome biogenesis mutant (Zellweger syndrome in humans) causes a differentiation defect. This defect can be “short-circuited” by expression of either the human peroxisomal ABC transporter PMP70 or the P. anserina peroxisomal ABC transporter pABC1; part of ABC transporter function is thus conserved from humans to P. anserina. Such a relationship between ABC transporters (ALDP and PMP70) and PEX2 is also observed in humans and remains totally unexplained for the moment. Our recent results suggest that this relationship between ABC transporters and a peroxisomal membrane protein is specific to PEX2. Role of phytanic acid in nervous system pathogenesis Refsum disease is caused by the accumulation of a branched-chain fatty acid, named phytanic acid. Patients with Refsum disease cannot break-down this fatty acid, which is derived from dietary sources, and therefore start accumulating phytanic acid in their tissues including the nervous system. At a certain point (mostly after the early childhood years) the high levels of phytanic acid start causing problems in the patients. The main clinical symptoms are retinitis pigmentosa, peripheral polyneuropathy and cerebellar ataxia. It is, however, not known yet how the accumulation of phytanic acid causes these problems. We aim to investigate this mechanism by using a mouse model for Refsum disease. Mice were created with the same defect as patients with Refsum disease. Rhizomelic chondrodysplasia punctata and the role of plasmalogens in its pathogenesis, including the abnormal formation of myelin Rhizomelic chondrodysplasia punctata is a peroxisomal disorder in which the main biochemical defect is the impairment in plasmalogen biosynthesis. Plasmalogens are of great importance in all cell membranes especially in the myelin sheets of both the peripheral and the central nervous system. The aim of this project is to find answers on the following questions: Physiopathogenesis of X-linked ALD in murine models, strategies for treatment Adrenoleukodystrophy (ALD) is a genetic X chromosome-linked disease that begins either in childhood (CCALD), adolescence, or adulthood (AMN). It is characterized by demyelination of the central nervous system and adrenal failure. In its cerebral forms (the most severe), the onset of the disease occurs between the ages of 5 and 12. Decline is rapid, followed by a bed-ridden state, then death. The biochemical deficiency occurs in the peroxisomal oxidation of very long chain fatty acids (VLCFA) which accumulate in the white substance of the brain and other tissues. This accumulation most likely has a destabilizing effect on membranes and thus on myelin, and a toxic effect on the adrenal glands. The gene mutated by the disease was cloned in our laboratory (Mandel/Aubourg). It codes a transport protein (ALDP) found in the membrane of peroxisomes. We show that knocking this gene out in mice produces symptoms resembling those of the adult form of the human disease (AMN) rather than those of the early-onset form (CCALD). This mouse knocked out for the ALDP gene is a good animal model for studying the causes behind this disease and testing therapeutic strategies. Analysis of degenerative mechanisms induced by VLCFAs X-ALD is a severe hereditary disease which mainly affects boys. Accumulation of fatty acids with 22 or more carbon atoms is characteristic for this disease. A failure in the enzyme cascade responsible for the degradation of these fatty acids is found to be responsible for this accumulation in the body of patients. In the current project we want to investigate if fatty acids with C ≥ 22 are toxic for brain cells. Because living human brain cells are not amenable for such experiments, we use brain cells in culture from rat, which can be isolated. Cells are kept alive on glass plates accessible for microscopy. By using fluorescent dyes sensitive to ions, we follow the reactions of rat brain cells to the application of fatty acids. Function and regulation of the ABCD2 (ALDR) gene X-linked adrenoleukodystrophy (X-ALD) is the most commonplace genetic peroxisomal disease. In biochemical terms, X-ALD is characterized by the accumulation of very long chain fatty acids, which may be the cause of the progressive cerebral degeneration and the adrenal damage often observed in patients. X-ALD is associated with mutations in the ABCD1 gene (ALD) which codes for a protein thought to transport VLCFA, allowing them to be broken down in the peroxisome. A very homologous gene (ABCD2, ALDR) is partially redundant; its overexpression in fibroblasts from X-ALD patients or in the disease’s animal model (XALD mice) can compensate for the ALDP protein deficiency. The ultimate goal of our research is to increase the cellular production of ALDRP protein in the brain or the adrenals by means of a pharmacological treatment. Our project involves characterizing the regulation patterns for the expression of the ALDR gene. Identification of new exons in the human PLP1 gene: Characterization and functional study of new transcripts, study of their implication in human pathology PLP and DM20, the primary proteins of myelin in the central nervous system (CNS), are coded by the same gene: the proteolipid protein gene or PLP1. These proteins play a major role in the stabilization and compaction of the myelin sheath around the axon, allowing rapid transmission of nerve impulses. In mice, a novel exon in the Plp1 gene has been shown to exist and results in the expression of two additional proteins: sr-PLP and sr-DM20. Their role has not yet been clearly established but it appears to be different than that of “traditional” proteins. Periventricular leucomalacy and cerebral abnormalities in premature babies: Using tractography to describe the mechanisms involved Mortality in premature infants has enormously decreased in recent years, resulting in increased survival rates for very low-weight newborns. These newborns are at a high risk for cerebral lesions resulting in serious motor and cognitive sequelae. The cerebral lesions, called periventricular leukomalacias, are found primarily in the periventricular white matter.A better understanding of the physiopathology of these lesions is crucial for prevention and for improving the early treatment of affected children. Very low-weight newborns frequently present more diffuse white substance lesions. The current hypothesis is that early on-set, diffuse lesions in white substance affect the development and growth of other cerebral regions (cerebellum, corticospinal tracts, optic radiations, etc.), because white substance serves to connect the various regions of the brain. Certain features presented by children born prematurely, such as fine motor and coordination problems, can be attributed to the cerebellum. My team has focused on cerebellar lesions in premature infants and has been able to demonstrate reduced growth in the cerebellar hemisphere opposite a unilateral lesion in periventricular white substance. To pursue my research on the repercussions of periventricular leukomalacies on cerebellar development, I will analyze quantitative MRI (magnetic resonance imaging) data from children born prematurely who present isolated lesions in periventricular white substance (without cerebellar lesions). New techniques for using MRI brain data will be employed. With these techniques, the volumes of various central nervous system compartments (white substance, grey substance, and cerebrospinal fluid) can be measured and the bundles of myelin fibers can be represented visually (3D MRI and tractography). The results of the morphological analyses will then be correlated with the neurological and intellectual development of these children. We hypothesize that the isolated white substance lesions will have an impact on cerebro-cerebellar connectivity and on the organization and microstructure of the cerebellum. We will also attempt to determine whether cerebro-cerebellar connectivity causes specific disturbances in neurological and intellectual development. By focusing on cerebro-cerebellar connections and the connections between periventricular white substance and the cerebellar hemispheres, this research will provide direct data on the establishment and organization of brain connections. Furthermore, it will provide data on myelin’s development, formation, structure, and topographical organization. This research will also be a means of further developing the data analysis techniques made possible by early MRI of the developing brain. These techniques can be used with other white substance pathologies, most notably in leukodystrophy research. Study of the physiopathological mechanisms involved in Alexander disease, Alexander disease (AXD) is a disorder of the myelin in the central nervous system. It is caused by mutations in the gene coding for GFAP (glial fibrillary acidic protein), a protein that, along with other proteins, formes the filaments that make up the framework of astrocytes. Astrocytes are non-myelinating glial cells and thus very different from oligodendrocytes, which produce myelin in the central nervous system. Their star shape and long processes allow them to interact with all the other cells of the central nervous system. Astrocytes play a very important role in both neuronal and oligodendroglial function; astrocyte damage due to a mutation in an important structural protein will thus have harmful functional consequences for all the cells of the central nervous system. Identification and characterization of specific biomarkers for MLD and ALD Two distinct structures have been described in the brain: grey substance which contains
neuronal somata, the generators of brain electricity, and white substance, which contains the complex wiring system that allows the propagation of nerve impulses. In order to accelerate the transmission of nerve impulses, certain “wires” are covered in myelin, a fatty, insulating sheath. Myelin acts as an insulating casing around nerve fibers and allows the conduction of messages. Leukodystrophies, the degenerative hereditary diseases affecting the brain’s white substance, represent a very heterogeneous group of disorders. In leukodystrophies, (1) the formation of myelin is deficient, (2) the formation is initially unaffected and later becomes defective, or (3) there is hypermyelination. These disorders are called “orphan diseases” because they are rare and poorly understood by the general public, and because there is not a great deal of medical research pertaining to them. There are numerous forms of leukodystrophy, the most frequent being adrenoleukodystropy (ALD) and metachromatic leukodystrophy (MLD). In both forms, the clinical symptoms are quite variable and no specific biological marker is currently available to facilitate diagnosis by laboratory analysis and to monitor the disease’s evolution during treatment. This is especially critical given the development of new therapeutic approaches such as gene therapy that require rigorous medical follow-up. Treatment Approaches NMR follow-up of PLP-mutated mice: Use in the therapeutic evaluation of neuroreparative substances Leukodystrophies represent a heterogeneous group of genetic disorders characterized by preferential involvement of central nervous system white substance, composed primarily of myelin. Amongst the diseases of myelin formation, the most well-studied forms are those linked to mutations in the gene for proteolipid protein (PLP). In humans and in several animal mutants, they can cause conditions of widely varying severity. The most severe forms involve an absence (or paucity) of myelin (Pelizaeus-Merzbacher disease, jimpy mice, and md rats). The mildest forms involve functional impairment of the myelin-axon interaction (X-linked spastic paraplegia and mice knocked out for the PLP gene). For these disorders, the therapeutic challenge is not only myelin repair; the fight against axonal degeneration (neuroprotection or neurorepair) is also of prime importance. In order to identify substances of therapeutic value in humans, it is imperative to test as many new molecules as possible on the central nervous system (CNS) of small animals, both healthy specimens and mutants. The mouse species offers the largest number of strains manifesting the same pathologies as those observed in humans. It is thus imperative to master tools which allow non-invasive and non-destructive follow-up of the level of neuronal impairment in the central nervous system (CNS) of these animals. The identification in vivo of quantitative and qualitative markers of axonal degeneration in mice is a major line of research and aims to evaluate the effectiveness of experimental therapeutic protocols. Correction of myelin deficiencies due to PLP: Pelizaeus-Merzbacher disease (PMD) is linked to mutations in the gene for myelin proteolipids (PLP). Proteolipids (PLP/DM20) make up ~50% of all myelin proteins. In the brain, myelin is produced by glial cells called oligodendrocytes and forms an insulating sheath around neuronal axons, allowing rapid conduction of nerve impulses. PMD is caused by acquiring supplementary copies of the PLP gene (duplication) in the majority of cases and by gene sequence abnormalities resulting in the production of defective protein (mutation) in a smaller number of cases. There is currently no suitable treatment for this disease. Gene therapy for AMN and cerebral ALD using hematopoietic stem cells corrected ex vivo with a lentiviral vector We have obtained sufficient pre-clinical results to request authorization from AFSSAPS [French agency ensuring the safety of health products] to conduct a clinical trial for gene therapy that aims to evaluate the tolerance and effectiveness of autotransplantation of CD34+ cells genetically corrected ex vivo with a lentiviral vector in 5 patients suffering from cerebral ALD who are candidates for allogenic bone marrow transplantation but lack donors. We are waiting for official authorization from AFSSAPS to start our trial. 2/ Obtaining the authorization to start our trial on ALD gene therapy is underscored by the need to conduct additional experiments in ALD mice in order to evaluate the risk of leukemia. We will use a lentiviral vector that can insert itself anywhere in chromosomal DNA and “activate” a gene that could lead to leukemia. As a rule, this risk is much lower than in gene therapy trials with “bubble children” but it cannot be altogether dismissed. We have already performed experiments in 2 mice and obtained reassuring results. These experiments, long and expensive, involve performing transplants in ALD mice using bone marrow cells isolated from ALD mice who had themselves received transplants of genetically corrected cells using the same viral vector to be used in the trial. This experimental paradigm allows us to maximize the chances of observing leukemia. These experiments are part of a series of toxicological experiments and studies that we had to carry out to obtain authorization for our gene therapy trial. Gene therapy using AAV for the treatment of MLD: We are developing a gene therapy strategy for metachromatic leukodystrophy (MLD) based on the intracerebral transfer of the normal gene carried out directly in the brain. This strategy complements the one developed by L. Naldini in Milan and aims to allow autotransplantation of bone marrow cells genetically corrected ex vivo with a lentiviral vector. An identical strategy has been proposed for adrenoleukodystrophy and will soon be put into action. In MLD, however, the intracerebral transfer of the normal gene should allow a more rapid therapeutic effect in patients than the autotransplantation of bone marrow cells genetically corrected ex vivo. We know that bone marrow transplantation requires at least 12-18 months before a therapeutic effect is observed, whether it be for MLD, ALD, or other brain diseases where bone marrow transplantation is effective. This factor has to be taken into consideration given the rapidity and severity of the disease’s progression in children suffering from infantile and juvenile forms of MLD (onset between the ages of 1 and 5). Detection of dysmyelinization and repair of myelin using diffusion tensor MRI The importance of myelin in normal nervous tissue function and development is highlighted by its involvement in a large array of neurological diseases such as multiple sclerosis (MS) and leukodystrophies. Early detection of subtle changes in brain structure and accurate assessment of dys/demyelination by noninvasive imaging technique, the diffusion tensor magnetic resonance imaging (DT-MRI), may have an important impact on the diagnosis of myelin diseases, as well as on basic and clinical investigations. Moreover, the implementation of therapeutic strategies requires an accurate evaluation of the disease effects and its evolution. Recent advance in DT-MRI provided a superior sensitivity of the method to discriminate between injury arising at myelin or axonal levels. This step is essential for diagnosis as well as for treatment trials. However, any therapy should be tested in suitable animal models before clinical application. For this purpose, a transgenic mouse, capable to mimic different pathological conditions associated with human leukodystrophy was generated and described in our laboratory (Jalabi et al., 2005). The mouse expresses a truncated form of herpes simplex virus 1 thymidine kinase (HSV1-TK) gene in oligodendrocytes, made under the control of myelin basic protein (MBP) promoter. Therefore, oligodendrocyte apoptosis and subsequent dysmyelination can be induced in a temporal manner by injections with Ganciclovir (GCV), a nucleoside analog. Enzyme replacement therapy in a mouse model for MLD: Evaluation Human arylsulfatase A (ASA) has been successfully used for enzyme replacement therapy of ASA knockout mice representing an animal model for metachromatic leukodystrophy (MLD) (Matzner et al., 2005). Treatment reduced sulfatide storage in the CNS and improved the function of the nervous system. Due to the unexpected high efficacy, the cause of which is not yet understood, enzyme replacement emerged as a promising therapeutic option for MLD. Evaluation of cellular therapy in leucodystrophies with demyelinization Although in leukodystrophies demyelination occurs primarily in the central nervous system, nerve demyelination is also responsible for part of the disabling clinical symptoms in many of these disorders. Nevertheless, the nerve has received limited attention with respect to therapeutic perspectives. In this project we propose to evaluate the therapeutic potential of stem cell delivery, namely neural stem cells and bone marrow-derived cells (MSC), for nerve remyelination in leukodystrophies. This study will be performed using leukodystrophy mouse models with nerve demyelination specifically, the mouse models for metachromatic leukodystrophy and for Krabbe’s disease. These mice will receive stem cells locally in the nerve or through the blood-stream both prior major disease symptoms and after disease onset. Nerve regeneration after stem cell delivery will be evaluated through functional assays and imaging techniques. These studies are necessary to prove the regenerative capacity of cell therapy in the demyelinated nerve as well as they will provide the initial safety and efficacy assessments for in the future, further translating such procedures to humans affected by leukodystrophies. Up-regulation of ABCD2/ALDR and ABCD3/PMP70 as a result of a low-fat diet, statins and fibrates as potential means of treating X-ALD X-linked adrenoleukodystrophy (X-ALD) is characterized by nervous system disease, adrenal insufficiency and accumulation of very long chain fatty acids (VLCFA) due to an impaired ß-oxidation in peroxisomes. The clinical variability is high with an absence of genetic constitution – clinical characteristics correlation, even within a single kindred, suggesting the influence of modifier genes and/or environmental precipitating factors. Based on the finding that the cholesterol lowering drug lovastatin was able to normalize VLCFA levels in X-ALD fibroblasts by inducing VLCFA ß-oxidation, a trial was started in X-ALD/AMN-patients, which showed lovastatin-induced normalisation of blood VLCFA levels in two studies, whereas in a third study, using simvastatin instead of lovastatin, no effect on blood VLCFA was found. Apart from the different statins used, another difference between the two successful and one unsuccessful study is that in the two successful studies statin therapy was combined with a low-fat diet. For this reason, we will first study the therapeutic potential of a low-fat diet only, and of lovastatin in AMN-patients under condition of a low-fat and normal diet. In parallel to this work we will study the mechanism behind the statin-induced normalisation of VLCFA levels in fibroblasts. Gene therapy for MLD: Study of MLD’s natural history and direction of preclinical studies in order to submit a protocol to AFSSAPS The ultimate goal of our project is to submit a clinical protocol for gene therapy for metachromatic leukodystrophy (MLD) to AFSSAPS in 2007. MLD is a severe demyelinating disease for which no treatment is currently available. 2) Establish MLD’s “natural history”. In other words, I will define all of the clinical (neuropsychological), radiological, and biochemical markers detectable at a pre-symptomatic stage of the disease in patients suffering from late-onset infantile and juvenile forms of MLD. The objective of this essential step is to determine precise criteria for selecting patients for our clinical trial. We have already established a collaboration based on this approach with the teams of Prof L Naldini and Dr A Biffi (Milan, TIGET), who are organizing their clinical trial for the autotransplantation of hematopoietic stem cells corrected ex vivo in MLD patients and share our objectives. I will be involved full-time in these additional projects, dividing my work hours evenly between 2 responsibilities: 2) Acting as co-investigator for the clinical research project aimed at establishing the natural history of MLD, to be carried out at Saint-Vincent de Paul Hospital (Paris) under the direction of Prof P Aubourg. Administration of ZK230211 (anti-progesterone) to reduce overexpression of PLP in a mouse model for Pelizaeus-Merzbacher disease Pharmacological gene therapy for X-ALD Elongation of very long-chain fatty acids in X-ALD: X-linked adrenoleukodystrophy (X-ALD) is a serious progressive, genetic disorder, which affects the adrenal glands and the white matter of the nervous system. It has an incidence of 1 in 17.000 life births. X-ALD is characterized by an accumulation of very long-chain fatty acids (VLCFA) in all tissues of the body, especially the brain and the adrenal glands. While some of the VLCFAs come from the diet, they are derived mainly from production within the body itself. Excess VLCFAs are metabolized in small compartments in the cell which are named peroxisomes. Normally, there is a balance between the synthesis and the degradation of VLCFAs. In X-ALD, however, there appears to be an imbalance in this process. This is due to a defect in the peroxisomal break-down of the VLCFAs. The VLCFA accumulate in the body of X-ALD patients and they have a negative effect on cell functions and membrane stability. Ultimately, in the brain this results in the destruction of the myelin sheath that surrounds the nerves which causes neurological problems, and in the adrenal gland specific cells are destroyed which causes primary adrenocortical insufficiency. Investment in Equipment Purchase of 2 mass spectrometers Purchase of a 128-cage ventilated mouse rack CellGro culture media |
back to the list