A clinical trial carried out in several countries has just ended. This test, called ’open phase 2-3’, confirms the results obtained in 2009 in France by the teams of Patrick Aubourg and Nathalie Cartier. Florian Eichler, principal investigator of the study, presents the results:

“Hematopoietic stem cell gene therapy for cerebral adrenoleukodystrophy”

“Cerebral adrenoleukodystrophy (ALD) affects 30 to 40% of boys aged 4 to 8 born with a mutation in the ABCD1 gene. These boys with ALD quickly begin to lose their walk and speech. In the New England Journal of Medicine, researchers just described a clinical trial using a lentivirus to infuse a normal copy of the ABCD1 gene into the bone marrow of boys with ALD. The corrected protein stopped the progression of the disease. It is the first effective gene therapy treatment to stop fatal brain disease.

Hematopoietic stem cell gene therapy for cerebral adrenoleukodystrophy

Florian Eichler, MD, Christine Duncan, MD, Patricia L. Musolino, MD, Ph.D., Paul J. Orchard, MD, Satiro De Oliveira, MD, Adrian J. Thrasher, MD, Myriam Armant, Ph.D., Colleen Dansereau, MSN, RN, Troy C. Lund, MD, Weston P. Miller, MD, Gerald V. Raymond, MD, Raman Sankar, MD, Ami J. Shah, MD, Caroline Sevin, MD, Ph.D., H. Bobby Gaspar, MD, Paul Gissen, MD, Hernan Amartino, MD, Drago Bratkovic, MD, Nicholas JC Smith, MD, Asif M. Paker, MD, Esther Shamir, MPH, Tara O'Meara, BS, David Davidson, MD, Patrick Aubourg, MD, and David A. Williams, MD


In X-linked adrenoleukodystrophy, mutations in the ABCD1 gene lead to loss of function of the ALD protein. Cerebral adrenoleukodystrophy is characterised by demyelination and neurodegeneration. The progression of the disease, which leads to loss of neurological function and death, can only be stopped with an allogeneic hematopoietic stem cell transplant.


We recruited boys with cerebral adrenoleukodystrophy in a phase 2-3, open-label, single-arm safety and efficacy study. When screened, patients had to be at an early stage of the disease and show a signal on magnetic resonance imaging (MRI) with the contrast agent gadolinium. The experimental therapy involved the infusion of autologous CD34 + cells transduced with the lentiviral vector elivaldogene tavalentivec (Lenti-D). In this interim analysis, patients were assessed for graft-versus-host disease onset, death, and major functional disabilities, as well as for changes in neurological function and the extent of visible lesions by MRI. The main end goal  was to be alive and have no major functional disability 24 months after the infusion.


A total of 17 boys received Lenti-D gene therapy. At the time of the interim analysis, the median follow-up was 29.4 months (range: 21.6 to 42.0 months). Following the transplant, all patients had cells marked with the gene, with no evidence of preferential integration near known oncogenes or clonal outgrowth. A measurable ALD protein was observed in all patients. No treatment-related death or graft-versus-host disease has been reported; 15 of 17 patients (88%) were alive and without major functional disability, with minimal clinical symptoms. One patient, who had had rapid neurological deterioration, died from the progression of the disease. Another patient, who had signs of disease progression on MRI, withdrew from the study to undergo an allogeneic stem cell transplant and later died of transplant-related complications.


Initial results from this study suggest that Lenti-D gene therapy may be a safe and effective alternative to allogeneic stem cell transplantation in boys with early-stage cerebral adrenoleukodystrophy. Further follow-up is necessary to fully assess the duration of response and long-term safety. (Funded by Bluebird Bio et al., STARBEAM ClinicalTrials.gov, NCT01896102; ClinicalTrialsRegister.eu number, 2011-001953-10.)

Reference: Eichler, F., Duncan, C., Musolino, PL, Orchard, PJ, De Oliveira, S., Thrasher, AJ, Armant, M., Dansereau, C., Lund, TC, Miller, WP, et al . (2017). Hematopoietic Stem-Cell Gene Therapy for Cerebral Adrenoleukodystrophy. N. Engl. J. Med. Oct 26; 377 (17): 1630-1638

Aicardi-Goutières syndrome is a genetic disorder associated with inappropriate activation of the immune system.

A growing body of evidence suggests that an accumulation of nucleic acid (RNA and DNA), possibly derived from ancient viruses included in our own cells, would elicit an immune response orchestrated by type I interferon. Aicardi-Goutières syndrome is a serious disease that requires the development of treatments. The development of effective therapeutic approaches will be promoted by a better knowledge of its mechanisms. Following proof of principle studies carried out in diseased Trex1-null mice and thanks to new knowledge concerning the proteins associated with Aicardi-Goutières  syndrome, strategies of immediate interest include blocking type I interferon and other components of the inflammatory pathways, disrupting the production of ’reverse transcription’ products and decreasing white blood cells.

There are already treatments related to each of these possibilities. In particular, we will talk about a clinical trial of reverse transcriptase inhibitors, which is starting in France. Aicardi-Goutières syndrome is no exception to the difficulties of recruiting for the performance of controlled studies of rare diseases affecting small populations. It might be useful to consider using a historical cohort as a control population in a therapeutic trial. This is why it is currently crucial to pay special attention to the natural course of this pathology.

In addition, criteria for evaluating the effectiveness of treatments must be established and consideration should be given to their best possible use. Manifestations of this syndrome - eg. imaging observations and clinical outcomes - are often difficult to measure objectively. Therefore, it is necessary to establish the relevance and specificity of the biomarkers for these future clinical trials. From this point of view, we are particularly interested in the detection of an “interferon signature” in almost all the cases of Aicardi-Goutières syndrome analyzed to date.

Treatment will most likely be of benefit in the early stages of the disease. Early diagnosis will therefore be of crucial importance. However, the characteristics of the later-onset disease (eg, frostbite in some patients) mean that treatment is also likely to have a role in some older patients. Uncertainties regarding the suitability of treatment for Aicardi-Goutières syndrome based on genetic type will begin to dissipate as our understanding of the protein function associated with this disease progresses.

The development of high throughput sequencing (Next Generation Sequencing, NGS) in the 2000s marked a major turning point in genome studies. Exome technology makes it possible to sequence all exons in the genome. The exon is the part of the gene that is translated into proteins. Leukodystrophies are a group of genetic diseases that affect white matter and primarily its major component, myelin. Despite advances in gene identification, 60% of patients remain without any genetic abnormalities found. We performed exome sequencing in a cohort of 80 patients with leukodystrophies of undetermined cause.

We have identified the causal mutation in 56% of cases, the remaining 44% are being analyzed with several candidate variants. This analysis allowed us to divide our cohort into three subgroups. A group with mutations in known genes for leukodystrophies (15%), a 2nd group with mutations in genes already involved in another genetic disease and with a very atypical clinical picture (9%), and a 3rd group with mutations in potentially candidate genes (32%).

The identification of mutations in known leukodystrophy genes by exome sequencing has demonstrated the major interest in developing a chip that brings together all known leukodystrophy genes for diagnostic application.

Leukodystrophies can be defined as genetic abnormalities primarily affecting the white matter of the central nervous system. It is typically  myelin and glial cells (oligodendrocytes and astrocytes) that are affected.  In most cases, there is a gradual deterioration.

Leukodystrophies can be classified into 2 main groups: hypomyelination pathologies and other pathologies. Research into leukodystrophies began in Amsterdam in 1987, shortly after the introduction of Magnetic Resonance Imaging (MRI) in medicine. MRI has been found to be a very useful tool in distinguishing different forms of leukodystrophies with consistent and distinct types of abnormalities detectable on MRI. It soon became evident that in more than half of the patients ( ~60%) with leukodystrophy, as revealed on MRI, a specific diagnosis could not be made.

Since then, many  newer leukodystrophies have been defined and the responsible mutated genes have been identified, first by genetic linkage analysis and, more recently, by full exome sequencing. The following list of “new” leukodystrophies, which are other conditions associated with an identified genetic abnormality, is incomplete:

Alexander disease (GFAP mutations)

Vanishing white matter / CACH (EIF2B1-5)

Megalencephalic leukoencephalopathy with subcortical cysts / MLC (MLC1 and GLIALCAM) Acardi-Goutières syndrome (TREX1, RNASEH2B, RNASEH2C and RNASEH2A, RNASET2, SAMHD1, ADAR1 and IFIH1)

Mitochondrial tRNA synthetase defects: LBSL (DARS2), LTBL (EARS2), AARS2 related leukoencephalopathy

Cytoplasmic tRNA synthetase defects: HBSL (DARS), RARS-related leukoencephalopathy ClC-2 chloride channel defect (CLCN2)

Mitochondrial leukoencephalopathies (complex I: NDUFV1, NDUFS1, NDUFS4, NDUFS7, NDUFS8, NUPBL; complex II: SDHAF1, SDHA, SDHB; complex III: LYRM7; complex IV: SURF1, APOPT1; other: NFU1, BOLRX3, LIAS, LIAS)

Peroxisomal leukoencephalopathies (PEX1, PEX2, PEX3, PEX5, PEX6, PEX10, PEX12, PEX13, PEX14, PEX16, PEX19, PEX26, PHYH, PAHX, PEX7, DHAPAT, AGPS, ABCD1)

Diffuse neuroaxonal spheroidal hereditary leukoencephalopathy (CSF1R) Autosomal dominant leukoencephalopathy in adults (LMNB1).

Similar developments have been observed in the field of hypomyelination-related diseases, in which many new pathologies have been defined and the responsible genetic abnormalities have been identified. It is striking that, despite these incredible advances and the accumulation of knowledge over the past 25 years, physicians and families continue to experience  that no diagnosis is made in about 50% of patients with a leukodystrophy. It is therefore important to question what is real. When looking at older data sets for unclassified leukodystrophies (eg, van der Knaap et al., Radiology 1999), the majority of reported cases are now diagnosed. The “new” unclassified cases are different and, based on the MRI results, should not be considered to be leukodystrophy. Indeed, many of them are primary neuronal disorders and some correspond to acquired, non-genetic lesions. Incorrect classification for leukodystrophy confuses patients and their families as well as physicians and can result in unnecessary efforts to apply inappropriate tests.

RNA polymerase III leukodystrophy (POLR3-HLD), also known as 4H leukodystrophy, is a hypomyelinating leukodystrophy resulting in a spectrum of neurological and extra-neurological manifestations with an age of onset typically in early childhood. Several leukodystrophies described in the 2000s are now grouped under this leukodystrophy since they have similar clinical characteristics and are caused by mutations in the same genes: the 4H syndrome (Hypomyelination, Hypodontia and Hypogonadotropic Hypogonadism), ADDH (Ataxia, Delayed Dentition and Hypomyelination ), TACH leukodystrophy (Tremor-Ataxia with Central Hypomyelination ), leukodystrophy with oligodontia and HCAHC syndrome ( Hypomyelination with Cerebellar Atrophy and Hypoplasia of the Corpus Callosum ).

A large study on more than 100 patients has allowed us to better understand the spectrum of this disease.

The neurological clinical features of POLR3-HLD include: significant cerebellar manifestations (ataxia or problem with balance, dysarthria or difficulty pronouncing words correctly, dysmetria  or imprecision of movements), with or without tremor, pyramidal manifestations such as spasticity ( stiffness) and brisk reflexes, as well as so-called extra-pyramidal dystonia-like manifestations (stiffness in the arms and legs, which fluctuates with associated abnormal emotions and postures). Non-neurological features of the disease include:

dental abnormalities (examples: small teeth, missing teeth, delayed eruption of teeth, abnormalities in the order of tooth eruption, etc.), eye abnormalities (myopia), endocrine abnormalities (example: small size) and pubertal abnormalities (puberty arrest or absence of puberty). POLR3-HLD is caused by recessive mutations in the POLR3A and POLR3B genes. To date, more than 100 patients with this disease have mutations in one or the other of these genes. The POLR3A and POLR3B genes encode the two largest subunits of an enzyme called RNA polymerase III, and together form the active center of the 17-subunit complex. No patient has two null mutations, that is, two mutations that would result in the complete absence of the protein for which the gene codes. This is not surprising given the essential role of RNA polymerase III: the transcription of DNA encoding small RNAs such as transfer RNAs, 5S, U6 and 7SK. These small RNAs are important for the survival of the cell.


Work has begun on the link between mutations in the POLR3A or POLR3B / hypomyelinating leukodystrophy gene. The representation of the mutations found on three-dimensional modeling of polymerase III suggests that the mutations may have an effect on the assembly of the enzyme by altering the interactions between the subunits, or even have an effect on the binding of DNA with the complex, thereby resulting in abnormal transcription of DNA into RNA.

Preliminary results suggest that, at least for one mutation, the first hypothesis seems correct, that a mutation in the POLR3A gene leads to deficient assembly of the complex and, in this case, to migration failure of the complex in the nucleus of the cell, where the polymerase does its job.

Our hypothesis is that mutations in POLR3A or POLR3B lead to an abnormal transcription of certain RNAs, such as transfer RNAs, important for the development of myelin (myelination). We have set up several types of experiments to study these small RNAs from fibroblasts (skin cells from patients and healthy controls) and it seems that there are small variations in the transcription of some RNAs. However, as the skin was unaffected in our patients, and now having mice with mutations in POLR3A, we will now repeat these experiments with myelin and gray matter from diseased mouse brains.

Note that the involvement of transfer RNAs is also suspected in other hereditary diseases involving the cerebral white matter such as LBSL (Leukoencephalopathy with Brainstem and Spinal cord involvement and Lactate elevation) and in two other hypomyelinating leukodystrophies called HBSL ( Hypomyelination with brainstem and spinal cord abnormalities and leg spasticity) and RARS-associated hypomyelination, caused by mutations in the DARS and RARS genes, respectively.

Finally, we have now started the experiments in POLR3A mice, exhibiting motor difficulties. We are going to study its characteristics, its brain. This will allow us to advance our understanding of the pathophysiology of the disease.

The discovery of the associated POLR3-HLD genes has enabled many patients and their families to obtain molecular diagnosis and appropriate genetic counseling. Clinical, radiological, and pathophysiological studies are still ongoing in order to better understand the extent of clinical and radiological manifestations, genetic abnormalities and of course, the pathophysiology of this group of diseases in order to be able to develop therapeutic strategies.

Pelizeus-Merzbacher's disease (PMD) is an inherited disease affecting the central nervous system characterized by physical and mental impairment. In the majority of cases, PMD is due to an increase in the expression of the PLP1 gene (gene encoding Protein Proteolipid 1 or lipophilin, the primary constituent of myelin). Currently, there is no cure for patients with PMD.

As part of an experimental therapeutic approach, we were able to reduce the gene expression of PLP1. We have been able to improve the course of the disease by administering an experimental progesterone receptor antagonist in mice mimicking the most common form of PMD.

In order to facilitate the application of our results to patients with PMD, we will attempt to answer the following questions in PMD mice:

Do progesterone antagonists currently authorised in other indications reduce PLP1 gene expression?

How can we optimise the treatment to obtain the best possible therapeutic effects?

What is the long-term impact of treatment acting on the progesterone receptor?

Can we identify other potential classes of drugs that reduce the expression ofPLP1?

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.

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.

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.

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.

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.

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.

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.

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.

Zellweger spectrum diseases (MSZ) result from abnormalities in the functions of cellular structures called peroxisomes.

They are also called peroxisome biogenesis disorders or generalised peroxisomal disorders.

Within the cell, the peroxisome performs several important functions necessary for the proper functioning of various organs, such as the nervous system, liver, and adrenal glands.

The severity of diseases on the Zellweger spectrum can vary from relatively moderate to severe, following a continuum of at least three pathologies: Zellweger syndrome, the most severe form, neonatal adrenoleukodystrophy, and infantile Refsum disease, the lesser form. These different pathologies were originally named before their biochemical and molecular basis had been determined.

The diagnosis of MSZ can be made by biochemical blood and / or urine examinations and confirmed by culture of skin fibroblasts. Specific biochemical tests are as follows: in blood: very long chain fatty acids, phytanic acid and pristanic acid, bile acids, plasma pipecolic acid, and red blood cell plasma genes; in urine: pipecolic acid, bile acids, oxalate.

Mutations in twelve different PEX genes, encoding peroxins, proteins involved in the transport of peroxisome enzymes, have been identified in diseases of the Zellweger spectrum. PEX1 is the most common cause of MSZ, present in approximately 70% of people.

The clinical course of infantile Refsum's disease is variable.

It can include delayed intellectual and motor development, hearing loss, visual impairment, liver dysfunction, and moderate craniofacial abnormalities. The disease may get attention initially because the child fails a hearing test and / or has vision problems. Liver dysfunction may be seen first in a child with bleeding episodes caused by a coagulation abnormality in relation to vitamin K. These children may also have adrenal insufficiency. The overall clinical course may be stable, but the disease is often slowly progressive, with deterioration of hearing, sight and ability to walk. Some subjects can develop leukodystrophy with the consequence of the loss of acquired skills. Other people may have mainly sensory deficits in adulthood or only ataxia (abnormal movements).

Since people with Zellweger spectrum diseases can reach adulthood, clinical manifestations of these conditions should be monitored and treated such as: diet and nutrition;

hearing aids;

vision correction;

for the liver, supplementation with fat-soluble vitamins;

for adrenal insufficiency, supplementation with cortisol. Experimental treatments are being studied, such as the administration of bile acids (cholic acid), docosahexaenoic acid and a diet low in phytanic acid. So far, treatment for Zellweger's spectrum diseases remains primarily symptomatic and supportive.

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.

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.