Category: Clinical trial

X-linked adrenoleukodystrophy (ALD) is the most common peroxisomal disease. The product of the affected gene is a peroxisomal membrane protein (ADLP), a member of the large family of ABC transporters (ATP-binding cassette, ATP binding cassette): ABCD1. The ABCD1 protein-deficient mouse model of X-ALD exhibits lateonset, progressive axonal degeneration resembling AMN in humans. We observe an early onset of oxidative stress accompanied by lesions, one year before the onset of the disease.

In these mice, treatment with a combination of antioxidants including α-tocopherol, N-acetyl-cysteine ​​and α-lipoic acid reverses oxidative damage, improves energy deficit as well as axonal degeneration and damage. Oxidative stress therefore appears to be underlying axonal degeneration in adrenoleukodystrophy; it is one of the hallmarks of the pathogenesis of X-ALD.

Our group has helped to demonstrate the glutathione imbalance present in the blood of patients with X-ALD, thus highlighting the importance of imbalance of forms of glutathione (its oxidised form versus its reduced form) as a characteristic and potential biomarker of this disease.

More recently, a new drug, called EPI-743 (alphatocotrienol quinone), has been designed to reconstitute glutathione in reduced form (anti-oxidant form). The exact mechanism of action of EPI-743 has not yet been determined, but EPI-743 has been shown to be  potent in vitro protector of cells taken from patients with diseases of the mitochondrial respiratory chain, such as Friedreich’s ataxia, Leigh’s syndrome (SURF1) and Leber’s inherited optic neuropathy.

Six patients with Leber’s hereditary optic neuropathy are currently being treated with EPI-743 in open-label emergency treatment studies. Improvements in visual acuity, visual field and color vision have been observed.

Several pediatric patients with Leigh syndrome, Kearns-Sayre syndrome, POLG1 deficiency, MELAS syndrome and Friedreich’s ataxia are currently being treated with EPI-743 in open-label clinical studies. Clinical improvement associated with biomarkers was observed.

In a recently completed Phase 2A trial, EPI-743 was associated with signs of neurological improvement in 10 included children with Leigh syndrome.

Our goal is to determine the efficacy and safety of EPI-743 in an open study in 10 patients with X-linked adrenomyeloneuropathy (AMN).

The main objective of this study is to evaluate the effect of EPI-743 administered orally to subjects with AMN on the change in the association between clinical ALD score in adults ( AACS, Adult ALD Clinical Score) and the dependency rating scale (mRS-9Q) between baseline and the end of 48 weeks of treatment.

The secondary objectives of this study are evaluated from baseline to up to 48 weeks of treatment. They  are:

• to assess the safety of EPI-743 for 48 consecutive weeks;
• to assess the effect of EPI-743 on motor functions (6 minute walk test distance (6MWT), time required to travel 25 feet (T25FW);
• to assess the effect of EPI-743 on disease progression as assessed by the Clinical Overall Impression Improvement Scale (CGIIMP);
• to assess the effect of EPI-743 on the change in quality of life (SF-36 questionnaire);
• to assess the effect of EPI-743 on the evolution of the auditory evoked potential of the brainstem;
• to assess the effect of EPI-743 on changes in hormone levels;
• to explore the effect of EPI-743 on altering components of the glutathione cycle.

Frédéric Sedel – MedDay – 2015

Preliminary data has shown that the drug MD1003 can stop the progression of the disease in patients with primary or secondary progressive multiple sclerosis (MS) and improve their symptoms. Among the 23 patients with progressive MS treated for an average of 9.2 months with the drug MD1003, 21 patients (91.3% of them) improved. The positive effects of the drug MD1003 may be linked to an increase in energy production in demyelinated neurons and stimulation of myelin repair. Analysis of two clinical trials involving 250 patients with progressive MS is currently underway and should confirm the previous results.

Adrenomyeloneuropathy (AMN) and progressive MS have similarities including a secondary energy problem leading to progressive axonal degeneration. One patient with AMN was treated with the drug MD1003 for 5 months and showed clinical improvement comparable to the effects seen in progressive MS. The objectives of the trial are to assess the efficacy and safety of MD1003 in patients with AMN. Sixty patients from 4 different centers (two in France, one in Spain, and one in Germany) will initially be divided into 2 groups: a group of 20 patients will receive a placebo while the second group of 40 patients will receive the drug MD1003. The placebo study will last 12 months and will be followed by a 12-month extension phase where all patients will receive the drug. The primary end point will measure, before and after 12 months of treatment, the average change observed during the 2MWT walking test measuring the distance covered in 2 minutes. Secondary end points will include:

• the TW25FW walk test measuring walking time over a distance of approximately 8 meters,
• time to get up and walk (or timed chair test)
• the Euroqol ED-5D and MOS SF-36 quality of life questionnaires
• a Qualiveen urinary function questionnaire.

Exploratory analyzes will also be carried out in certain centers, such as MRIs, measurement of nerve conduction speed and assessment of muscle strength. The recruitment of all patients will be completed in mid-June 2015.

In March 2010, a clinical trial based on the transplantation of autologous hematopoietic stem cells modified with a drug vector encoding normal human arylsulfatase A (ARSA) was authorised by the Italian authorities. The clinical protocol includes patients suffering from the form late infantile (TI) and early juvenile (JP), in the pre-symptomatic stage and, for patients with the JP form, in the pre-symptomatic and early symptomatic stage, as this patient profile should provide a reasonable perspective in terms of clinical benefit.

The objectives of the study are to assess the safety and efficacy of the treatment by monitoring any side effects and by measuring the scientific workshops of the patients’ motor and cognitive abilities as well as the demyelination of the nervous system.

Recruitment for the study is now complete, with twenty patients included (of which nineteen were treated). Ten of these patients had a biochemical, molecular and family history consistent with a diagnosis of MLD IT and ten others had a clinical and / or family history consistent with the JP form of the disease.

Preliminary data indicates that, overall, the transplant procedure was followed by good bone marrow recovery with no unexpected short to medium term side effects. In addition, reconstitution of ARSA activity is observed in the reconstituted blood cells and in the cerebrospinal fluid of patients.

For patients with the IT form for whom we have an informative follow-up (observation of at least 1.5 years after gene therapy [TG]), we report preliminary evidence of a therapeutic benefit associated with the procedure.

Indeed, the clinical state, the motor and cognitive capacities and the myelination of the central nervous system of these children are very clearly superior to the expected damage given the known evolution of the disease from which they suffer, their age, and the course of the illness of their affected older brother or sister.

The magnitude of the observed benefits may be influenced by the time interval between TG and expected onset of illness.

Two patients with the IT form treated shortly before the onset of the illness or at the time of onset showed some first signs of illness within 6-12 months of TG and were then stabilised.

Patients treated long enough before symptoms appear show virtually no clinical signs of the disease, have habitual motor and cognitive development, and enjoy a normal quality of life for their age. We are not yet sure whether TG can protect children with the IT form of the disease from peripheral demyelination.

Observation of patients with the JP form treated to date does not yet allow conclusions to be drawn in terms of therapeutic benefit since their disease was at a different stage at the time of treatment and due to short follow-up.

These data is  generally very encouraging, but will need to be confirmed by continued long-term monitoring.

We are currently studying with the pharmaceutical company GSK, one of our partners in this research, the Italian Telethon Foundation and the San Raffaele Hospital, the possibility of carrying out an appropriate program, in accordance with the Italian legislation, which would allow other children  suffering from MLD to receive this treatment, although it has not yet received a marketing authorisation. Various points will have to be discussed before being able to propose such a programme. In particular, it will be necessary to determine whether the preliminary data from this study allows us to have a favourable benefit / risk balance in order to be able to offer this treatment to children outside the framework of a clinical trial.

If authorised, this experimental treatment will not be generalised, and this therapeutic approach will have to be evaluated for each patient.

Metachromatic leukodystrophy or MLD is a lysosome overload disease primarily affecting the central nervous system. These diseases constitute a family of genetic diseases caused by missing or defective enzymes. Many of the symptoms of some of these conditions can be effectively treated by intravenous administration of synthetic enzymes. These enzymes are relatively large in size to be able to pass from the bloodstream to the central nervous system when given intravenously. Therefore, it is unlikely that they can act on central nervous system disorders associated with some forms of lysosome overload disease including MLD. The Shire Laboratory has designed a clinical research program to study the direct delivery of synthetic human arylsulfatase, or rhASA, into the central nervous system to overcome its deficiency. This clinical trial for MLD is in a early phase of development.

In animal models, the enzyme rhASA has been detected in all areas of the brain. The administered enzyme is deposited in the lysosomes of oligodendrocytes, the myelin cells of the brain where sulfatides accumulate abnormally during the disease.

Weekly administration of rhASA intrathecally (i.e. by direct injection into the area surrounding the spinal cord) in another animal model of MLD has been shown to decrease sulfatides in the central nervous system, as well as in the spinal cord (near the injection site) and in the deep tissues of the brain. These series of experiments demonstrate that the administration of rhASA by the intrathecal route can reach the target tissues of the central nervous system.

The data from these studies is useful for further research in humans. However, results observed in animals do not automatically correlate with what is observed in humans.

The Shire Laboratory is currently conducting a clinical trial to assess the safety of a synthetic form of ASA in MLD patients. This study, in addition to safety, will assess possible changes in coarse and fine motor skills, swallowing, cognition, adaptive behavior and the peripheral nervous system. This study is planning an extension phase that will assess longer term safety and clinical effect. The Shire laboratory is also studying the natural history of the disease in order to better understand its course in sick children and adults.

Metachromatic leukodystrophy (MLD) is a neurodegenerative disease caused by a defect in the activity of the enzyme Arylsulfatase A (ARSA), leading to the accumulation of sulfatides in cells of the central and peripheral nervous system, especially cells that make the myelin, but also neurons. The late infantile form, the most severe and most frequent form, begins around 1-2 years of age. It is characterised by very rapid motor and cognitive degradation leading to bedridden condition and early death. Today there is no treatment for this form of the disease when children are symptomatic.

Intracerebral gene therapy may allow rapid and sustained expression of the ARSA enzyme in the brain, a necessary condition to stop the neurodegenerative process in a timely manner. We have developed a clinical protocol for the intracerebral administration of a drug vector containing the therapeutic gene ARSA (AAVrh.10 / ARSA). After validating the effectiveness of this protocol in disease model mice, we optimised and validated the neurosurgical procedure in primates to allow simultaneous delivery of the drug vector to 12 brain regions. We have shown that injection of the AAVrh.10 / ARSA vector results in significant overexpression of ARSA throughout the primate brain without any deleterious effects.

We have obtained from the authorisations necessary to start a phase I-II clinical trial (tolerance and efficacy) in affected children from the ANSM (National Agency for the Safety of Medicines and Health Products) and the committee for the protection of persons, MLD.  This trial, currently open for recruitment, will include five children (aged 6 months to 5 years inclusive) with early forms of MLD (late infantile, precocious juvenile), at the very beginning of their disease. Safety and efficacy parameters will be evaluated for 2 years, a period which should be sufficient to assess the safety and therapeutic efficacy of this treatment. The first two patients were included in this clinical protocol and received the drug vector intracerebrally.

Krabbe disease or globoid cell leukodystrophy (LDG) is a genetic leukodystrophy caused by mutations in the gene encoding galactocerebrosidase (GALC). Both healthy parents of an affected person carry one copy of a pathological mutation in the GALC gene.

When a person inherits two copies of a mutated gene, they exhibit very low GALC activity, which results in a significant build-up of galactose-containing lipids, which are mainly found in the white matter or myelin of the central and peripheral nervous system. (SNC and SNP). This pathology, in addition to the abnormality in the lipid composition of myelin, also has an inflammatory component.

Although Krabbe disease primarily affects infants, the diagnosis is also made in older patients. In some patients, the diagnosis may not be made until late, when certain symptoms lead to genetic testing. However, this late diagnosis may limit the success of any treatment aimed at preventing or repairing damage to the nervous system.

In order to allow faster diagnosis, some states in the United States have introduced newborn screening for Krabbe disease. Some subjects identified in this screening because of reduced GALC activity have disease-causing mutations and certain changes called polymorphisms (normal changes in the gene that can lower the level of activity measured without causing disease).

Being able to determine when and if the subject will have Krabbe disease is critical to the success of the program. Clinical evaluation and neurodiagnostic studies are essential to determine when to initiate treatment. Treatment options are currently limited. Hematopoietic stem cell transplantation (HSCT), when performed in infants who are very mildly affected or before symptoms appear, can prolong life. However, these patients experience significant expressive language problems and increasing problems with walking. Although this treatment is now considered the norm, it is evident that more effective treatments are needed.

Several animal models also show low activity of GALC. They are used to test different treatments, to make sure they are both safe and effective, before they are evaluated in clinical trials in patients.

Numerous studies have been carried out on the mouse model called twitcher (twi), since 1984. These studies have focused on:

• bone marrow transplant (GMO),
• gene therapy using different viral vectors to provide a correct copy of the defective GALC gene,
• enzyme replacement therapy aimed at compensating for the deficiency in GALC activity,
• medicines aimed at slowing the synthesis of galactolipids, reducing the immune response or correcting the synthesis of the mutant GALC protein,
• neuronal stem cell therapy to stimulate remyelination
• and combinations of these treatments. Some treatments have resulted in only a small lifespan, others have not been deemed safe for use in patients Some studies have involved injecting different drug carriers directly into the brains of twi mice, sometimes in combination with other treatments. With some drug carriers, the gains have been modest.We tested an AAVrh10-type drug vector containing the GALC gene, called AAVrh10-GALC. The injection of this vector into the brain and into a blood vessel (intravenous, IV) in 2-day-old mice (PND2) has been shown to be promising: it has significantly extended the lifespan of the treated mice (approximately 40 days to 150 days and more). These mice are fertile and show few signs of the disease until very old age.

In order to streamline the treatment procedures, we decided to inject the vector AAVrh10-GALC IV into 10-day old mice. We chose this age because at this point in the development of the mice, myelination of the nerves begins and it is possible to inject a larger volume of the vector compared to younger mice. The single injection prolonged the life of the mice by an average of 25 to 35 days compared to untreated mice, although some lived longer than 150 days.

In addition, this single injection ensured GALC activity in the brain and medulla, as well as very strong activity in the sciatic nerve, a critical tissue not treated by other procedures. These mice remained fertile, exhibited normal mobility, did not suffer from tremors, and their weight gain was normal until a few weeks before they weakened and died. Myelination of the brain, spinal cord and sciatic nerve has been shown to be normal.

Since HSCT is the standard treatment in patients with Krabbe’s disease, we decided to combine GMO with a single injection of the AAVrh10-GALC vector given the next day in mice aged 9-10 days. These mice have normal weight, normal movements, and some currently live more than 200 days.

This approach should be the subject of further studies in order to determine the optimal time between GMO and the injection of the AAV vector and in order to determine the minimum dose of the drug vector necessary to deliver the gene into the target tissues. All of this is being evaluated in the mouse model.

Studies are also underway in the dog model, before being able to validate these procedures to be able to offer a clinical trial to patients with Krabbe disease.

The elF2B protein plays an important role in the normal cellular process of protein synthesis and in its regulation. Protein synthesis is the process by which cells make all the components they need, following the instructions provided by the genome (DNA), relayed by a messenger RNA molecule, and using the building blocks called amino acids, that come from our food. The elF2B protein is therefore needed everywhere in the body. Since CACH / VWM only affects certain tissues, it can be concluded that, in general, cells have sufficient amounts of the elF2B protein to function. The brain and myelin are an obvious exception to this rule; these tissues are therefore particularly sensitive to impairment of the function of elF2B. We do not yet know the reasons for this.

Currently, the most widely recognised hypothesis stems from the known role of elF2B in the cellular stress response. Various studies have shown that the regulation of the elF2B protein is essential for cells in response to several stressors.

We know that when stress is prolonged or too intense, cells that cannot resolve stress switch to a process called cell death or apoptosis. In experimental models, this process contributes to causing pathology similar to that associated with elF2B mutations in CACH / VWM.

In our lab, we are studying the function of the elF2B protein using a simple cell model system. We have experimentally demonstrated how elF2 acts as a major activator of protein synthesis. The elF2B protein is a class of proteins called ’guanine nucleotide exchange factor (or GEF)’, which acts as a molecular switch to switch to its partner, another protein called elf2. The elF2 protein binds to GTP (on state) and GDP (off state) and must be activated at the start of each cycle of protein synthesis. GTP is a co-factor that provides energy. When the elF2b protein is active, cells can make proteins, and when it is not, the level of protein synthesis drops. We have shown that part of the elF2B protein (called the epsilon subunit) has GEF function, while other parts of this protein are important in regulating this activity in response to stress.

In recent work, we have discovered that elF2B performs a second related function and that it is needed to move a third translation factor (named elF5) from elF2 before elF2B can perform its GEF function. We have found that some elF2b mutations affect GEF function while others affect elF5 displacement function.

Another unexpected finding is the fact that the elF2B protein is larger than we previously thought. It exists in cells as a complex made up of two subunits. We are currently working to elucidate the structure of elF2b using a technique called electron cryomicroscopy. We hope that this will provide us with important elements for understanding how it works and the effect of mutations on its functioning.

In our studies, we purify elF2 and elF2B protein complexes from cells. We have modified our methods so that we can produce human elF2 in a yeast cell-based expression system. We have demonstrated that the elF2 protein we produce works as well as a protein produced by other more laborious means and that it can be used in a diagnostic test as part of the procedures used to confirm that a patient has CACH / VWM. During studies carried out in collaboration with Pr Odile Boespflug-Tanguy’s team, we used blood cells isolated from patients as a source of elF2B and our human elF2 to show that patients’ cells have elF2b GEF activity lower than that of healthy controls.

We are currently discovering new and interesting insights into the role and biology of elF2B. We have developed a strategy to purify elF2. After some modifications, our biochemical assay can be used with other diagnostic tests used in the hospital to confirm the diagnosis of CACH / VWM.

Alexander’s disease is a disease linked to mutations in the GFAP gene. The mechanisms that cause these mutations to cause disease are not yet understood. In addition, the age of onset, the severity of symptoms and the rate of progression vary widely between patients, and the data available to us are too limited to explain this heterogeneity. In recent years, we have published the results of two studies that attempt to answer these questions using mouse models of the disease and brain samples taken during autopsies from patients with Alexander disease.

In the first study, published last year in the Journal of Neuroscience, we identified a potential link between the mechanisms responsible for neurodegeneration in Alexander disease and other more common neurodegenerative diseases. These results relate to a specific protein called TDP-43, which regulates the expression of other genes. This protein was already known in amyotrophic lateral sclerosis (ALS) and about 50% of cases of frontotemporal lobar degeneration (DLFT), a type of dementia in these cases referred to as DLFT-TDP. In ALS and DLFT-TDP, the TDP-43 protein becomes insoluble, is biochemically altered, and improperly localised in neurons and glia of the brain and spinal cord.

The TDP-43 protein had previously been detected in the Rosenthal fibers of certain brain tumors. We therefore tested for its presence in Rosenthal fibers characteristic of Alexander disease. We have found that the TDP-43 protein is indeed altered and incorrectly localised in the brains of patients with Alexander disease as well as in mouse models of the disease, which also contain Rosenthal fibers. TDP-43 is now the focus of attention of researchers working with Alexander disease.

In the second study, published late last year in Brain Research, we examined the possible role of environmental factors, such as head trauma, in modifying the severity of symptoms in patients with Alexander disease. Often, families and patients report rapid onset of new symptoms or rapid progression of pre-existing symptoms after seemingly minor head injuries. Head trauma is a known risk factor for epilepsy. Using our mouse models, we first performed electroencephalograms and found that even without injury, mice show signs of subconvulsive epilepsy.

We then subjected these mice to slightly traumatic head injuries, but enough to increase the risk of epilepsy. However, we were unable to show that GFAP mutant mice differed from control mice in terms of risk of epilepsy following trauma.

Upcoming research topics include the development of new animal models and the development or identification of drugs that could alter the course of the disease.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare and still incurable genetic leukodystrophy characterised by a slowly progressive course leading to motor and cognitive deficits as well as epilepsy. The clinical condition of patients is often worsened after trauma or certain infections. Patient care requires intensive parental, educational and social support.

About 80% of people with MLC carry mutations in the MLC1 gene encoding a protein whose function is not yet fully understood; a minority of patients (around 15%) have mutations in the Hepacam / Glialcam gene which encodes a cell adhesion molecule.

These two proteins are very strongly expressed in a population of brain cells called astrocytes. Astrocytes are essential for homeostasis and brain function, including maintaining water and ion balance. Studies by our research group and others suggest that MLC1 may regulate the exchange of ions and water. We recently began to study the role of MLC1 in the intracellular processes controlling the response of astrocytes to stress conditions (osmotic, inflammatory and oxidative stress). The results obtained will provide us with fundamental knowledge to study how mutations in MLC1 alter the functionality of astrocytes and lead to brain damage.

For this, we obtained inducible pluripotent stem cells from skin fibroblasts of people suffering from MLC and we are currently in the process of differentiating them into astrocytes carrying pathological mutations of MLC. This model should allow us to gain new insights into molecules and pathways that may become pharmacological targets to restore astrocyte function(s) and ultimately correct neurological deficits, which would pave the way for the development of treatments that can cure MLC or improve the quality of life of people with it.

Leukodystrophy called Canavan disease (CD, Canavan Disease) is a neurodegenerative disease. Patients with Canavan disease develop mental retardation, suffer from epilepsy, and die prematurely. Brain damage in patients with CD is characterised by progressive central nervous system (CNS) vacuolation, edema and loss of oligodendrocytes, cells responsible for myelin formation in the CNS.

In 1996, CD was the first neurogenic brain disease to be treated with gene therapy. CD is caused by a loss-of-function mutation in the gene encoding aspartoacylase (ASPA), an enzyme in oligodendrocytes.

Under normal conditions, ASPA breaks down N-acetyl-Laspartate (NAA) into aspartate and acetate.

The biochemical consequence of ASPA deficiency is an accumulation of NAA and its derivative NAAG in the brain, blood and urine. NAA is the most abundant free amino acid in the CNS and its role in the brain is not fully understood. The metabolism of NAA is extremely segmented: production of NAA, catalysed by the 8-like N acetyltransferase (NAT8l) enzyme, takes place in neurons, while its degradation is restricted to oligodendrocytes.

The etiology of CD is believed to be related to the cytotoxic and osmotic effects of excess NAA, but also to potential hypomyelination due to lack of NAA-derived acetate. However, no experimental data has come to support these hypotheses.

To assess the contribution of NAA to the complex pathology of CD, we created and characterised mouse models with varying levels of NAA. Here we show that, overall, loss of function of ASA or increase of NAA, by themselves, is well tolerated. However, oligodendrocytes suffering from ASPA deficiency are extremely vulnerable to NAA toxicity in vivo.

Our data as well as several publications concerning the correction of ASPA gene expression in neurons show the need to improve the means of gene expression for targeted reintroduction of ASPA into oligodendrocytes.

We therefore modified drug vectors to direct ASPA gene therapy specifically to oligodendrocytes from symptomatic ASPA-deficient mice.

We observe that this treatment stops and reverses the progression of the disease, but in an incomplete way. Our hypothesis is that future approaches to gene therapy for CD should include strategies aimed primarily at restoring the expression of ASPA by oligodendrocytes associated with further decrease in NAA.