Category: Leukodystrophy

In Hypomyelination with Brain stem and Spinal cord involvement and Leg spasticity (HBSL), the rate and degree of spread of symptoms throughout the body are linked to survival outcome. However, the cause for the pathology in HBSL is unknown. Using mini 3D brain oligodendrocytes generated from HBSL patient skin cells, we will study how interactions between oligodendrocytes and their support cells (neurons, astrocytes, and microglia) might lead to the development of disease over time. We will apply the cutting-edge technology of spatial transcriptomics to generate “cell-to-cell network maps” to inform whether we can manipulate cell networks to prevent demyelination. Finally, this research project aims to test nutraceutical supplementation as a therapeutic potential to ameliorate and or reverse established HBSL pathology.

Project financed by ELA up to: 65 846 €

Metachromatic Leukodystrophy (MLD) is a genetic disorder caused by mutations in the ARSA gene. The absence of this enzyme leads to accumulation of sulfatides in neural and glial cells leading to progressive neurodegeneration and eventually death. Our firstin-human clinical trial for the treatment of MLD based on hematopoietic stem cell (HSC) gene therapy (GT) provided evidences on the feasibility, safety and efficacy of the procedure prompting its authorization with the name of Libmeldy. With this therapy, patients’ HSCs are genetically corrected and reinfused to reconstitute the entire hematopoietic system acting as “Trojan horses” for the delivery of the ARSA enzyme to the affected tissues. This treatment provide benefit when provided to pre- or early symptomatic MLD patients. Unfortunately, it is difficult to predict if a candidate MLD patient with genetic mutations of the ARSA gene will ever develop symptoms of the disease. Therefore, the clinical decision making for treatment is based on the case-by-case evaluation of patient’s clinical history and several clinical biochemical and instrumental assays. Moreover, treated patients must be monitored for the safety and efficacy of the therapy which include additional molecular analyses addressing the clonal composition and behavior of genetically modified blood cells after transplantation. Although these analyses are extremely useful to define the efficacy and safety of GT, the identification of additional biomarkers more predictive of disease burden and useful as treatment response indicators is strongly needed.

We recently developed LiBIS-seq (liquid-integration-site-sequencing), a PCR technique optimized to exploit blood plasma cell free DNA (cfDNA) for the study of the clonal composition of genetically modified cells in circulation and in solid tissues. cfDNA is constituted by short DNA fragments released in body fluids by dying cells residing in different tissues of the body and for this reason its analysis provided more reliable safety predictions of the GT approaches than the methods based only on the analysis of circulating cells. Moreover, cfDNA concentrations in plasma of MLD GT patients were above pathological levels before the treatment and decrease progressively after transplantation in patients with a positive therapeutic outcome. Hence, the level of cfDNA in MLD patients may reflect the number of dying cells because of the accumulation of toxic lysosomal compounds.

For these reasons, we aim to validate blood plasma cfDNA as new and non-invasive outcome measure of disease severity and as surrogate biomarker of the efficacy of GT treatment for MLD. Specifically, we will use state of the art technologies to investigate the epigenome an integrome on cfDNA and cells collected overtime from early juvenile and late infantile MLD patients treated by GT in our institution. These analyses will define the efficacy and safety of the treatment at the clonal level, determine which cell types are contributing to cfDNA production in MLD GT patients and in natural history patients unravelling tissue-specific toxicities over the course of the disease. If successful, the identification of a reliable disease biomarker will allow for a more accurate diagnosis evaluation and a more personalized therapeutic intervention for MLD.

Project financed by ELA up to: 100 000 €

Deficiency in the enzyme aspartoacylase leads to a severe leukodystrophy, called Canavan disease, which is characterized by the accumulation of the brain metabolite N-acetylaspartate (NAA). NAA is synthesized and released mainly by neurons. Many neurons use NAA also to synthesize the small neuropeptide N-acetylaspartylglutamate (NAAG). Extracellular NAAG secreted by neurons can be degraded to NAA and glutamate by an enzyme expressed by astrocytes. Thus, degradation of NAAG is another source of NAA. The current project will further investigate the role of NAAG in the NAA accumulation observed in Canavan disease. This will be done using different transgenic mouse models, including a mouse model of Canavan disease, that differ in their capability to synthesize NAAG. In the project, histological and biochemical methods will be used to evaluate the influence of the genetic changes in these mouse models on the accumulation of NAA and the development of brain spongiform degeneration and myelin loss. The aim of this project is to examine whether reduced NAAG synthesis leads to reduced NAA accumulation and reduced pathological changes in the Canavan disease mouse model. If this would be the case, inhibition of NAAG synthesizing enzymes would potentially be an alternative or additional option for a substrate reduction therapy of Canavan disease.

Project financed by ELA up to: 9 700 €

For its correct development and function, nervous tissue synthesizes various key components but is also reliant on essential nutrients. Choline is one of such nutrients that is necessary for the synthesis of phospholipids and the generation of the neurotransmitter acetylcholine. A novel disorder was recently identified and shown to be caused by mutations on SLC44A1, the gene that encodes the choline transporter-like protein 1. With an early childhood-onset presentation, this novel leukoencephalopathy is characterized by severe white matter involvement, optic nerve atrophy, ataxia, dysarthria, tremors, and patients have delayed motor and speech development. In order to have a valid animal model for the disorder, we generated the first Slc44a1 mutant mice to characterize the underlying pathology and disease mechanisms caused by a defect in choline transport. Using the Slc44a1 mutants, we will determine how choline dysregulation impairs oligodendrocyte differentiation, myelination, and neuron function. Our aims are to:

• Determine the neuropathology caused by impaired choline transport
• Establish the proteomic and metabolic changes caused by choline deficiency
• Evaluate the therapeutic potential of choline supplementation

Combined, this project addresses several unmet scientific and medical needs that are set to have a significant beneficial impact on scientific and societal communities.

Project financed by ELA up to: 81 000 €

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare genetic disorder characterized by an abnormal head, loss of motor functions, epilepsy and mild mental decline. The disease is caused by mutations in two genes named MLC1 and GLIALCAM. There are no therapies for MLC patients, only palliative treatment. We recently demonstrated the efficiency of gene therapy in correcting MLC-typical myelin vacuolation. Now we aim to demonstrate that gene therapy is also able to correct the motor impairment present in the MLC knock-out mouse models. Thus, we propose to administer the therapeutic AAV vectors to the affected cells in the brain of these mice, and to look for longitudinal, non-invasive tests to assess for in vivo indicators of disease correction in the animal models that could finally be translated to clinical trials for MLC patients.

Project financed by ELA up to: 44 414 €

Leukodystrophies are a major source of handicap at all ages, but children are affected most. We have studied leukodystrophies since 1987. Initially, our research focused on describing new leukodystrophies and finding the underlying gene defects. Much of our subsequent research efforts concerned the new leukodystrophy vanishing white matter (VWM), also called childhood ataxia with CNS hypomyelination (CACH). The disease occurs at all ages, but mainly starts in young children (2-6 years). Children with VWM experience progressive neurological handicap that prevent fever and head trauma, as these events trigger a fast worsening of the disease.

Several years ago we found that the gene defect for VWM lies in an enzyme complex that is crucial for protein synthesis. Since then we have studied how the disease works (disease mechanisms), most of all to find openings for potential treatment. We found that cells in the white matter of the brain do not develop into mature cells that can execute their normal function of myelination and white matter repair properly. The problem with maturing into functional cells can well explain the severe white matter damage that we observe in patients. Our recent studies have demonstrated that a basic stress pathway is abnormally activated in VWM white matter cells. We have evidence that abnormal activation of this stress pathway causes the disease. In the proposed study we will target and inhibit an important and toxic component of this stress pathway with antisense oligonucleotides and test the effects in a representative disease model. The proposed work has the potential to open up new treatment options relatively fast.

Our vision on treatment of VWM is that effective treatment of this complex disease is most likely not achieved with any single therapeutic modality. We think that the treatment should target the disease at multiple levels, including reduction of stress pathways, reduction of the toxicity of the diseased white matter, provision of healthy white matter cells and possibly gene therapy.

Project financed by ELA up to: 50 000 €

X-linked adrenoleukodystrophy (ALD) is the most common leukodystrophy. All ALD patients have a mutation in the ABCD1 gene that leads to a build-up of saturated very long-chain fatty acid (VLCFA) in tissues, including adrenal glands, spinal cord and brain. ALD is characterized by a striking and unpredictable clinical spectrum, even within families. In childhood, around 50% of affected boys develop adrenal disease before the age of 10 and 30-35% of affected boys develop a fatal inflammatory brain disease (cerebral ALD). If ALD is diagnosed in an early stage, cerebral ALD can be halted or reversed by a bone-marrow transplant. In adulthood, virtually all males and >80% of women develop a chronically slow progressive spinal cord disease (myeloneuropathy) for which no disease modifying therapy is available. Unfortunately, transplanted boys can still develop spinal cord disease in adulthood, because the transplant is only effective at halting the inflammatory component of the disease without addressing the underlying biochemical defect. This therapeutic gap highlights the need to develop effective treatments aimed at the normalization of VLCFA levels in the brain and spinal cord.

Using skin cells from ALD patients we have demonstrated that saturated VLCFA induce cellular stress, with prolonged exposure resulting in cell death. Remarkably, this is not observed with mono-unsaturated VLCFA (fatty acids with a double bond in the fatty acid chain). The enzyme stearoyl-CoA desaturase-1 (SCD1) coverts saturated fatty acids into mono-unsaturated fatty acids. In a previous ELA supported project we identified and characterized the drug TO901317 (an LXR agonist) as a small-molecule that activates SCD1 activity. Treatment of ALD cells with TO901317 completely corrects VLCFA levels and treatment of the ALD mouse with TO901317 added to the food resulted in a reduction in VLCFA levels in adrenals, spinal cord and brain.

Unfortunately, TO901317 (and other LXR agonists) as potential treatment for ALD have notable limitations due to off-target effects that result in serious side effects. This is largely due to LXR agonists not being specific for SCD1. We therefore hypothesize that specifically increasing SCD1 activity will circumvent the detrimental effect of LXR activation and offer a therapeutic strategy to counteract VLCFA-induced lipotoxicity in ALD.

Strategies to specifically increase SCD1, either pharmacologically or genetically have not been reported to date. Therefore, the overall goal of the proposal is to identify genetic and pharmacological regulators of SCD1 abundance and/or activity. Specifically, in this project we aim to:

• Generate cell lines in which the SCD1 enzyme is tagged with a fluorescent protein to allow monitoring of SCD1 in

1. live cells at a single cell resolution.

• Map enzymes that control SCD1 abundance using a genome-wide CRISPR/Cas9 genetic screen.
• Identify small molecules that increase SCD1 abundance and activity. Addressing these aims will increase our basic understanding of VLCFA metabolism in ALD.

Moreover, results from these experiments have the potential to inform on novel therapeutic strategies to treat ALD patients.

Project financed by ELA up to: 97 045 €

Alexander disease (AxD) is a rare autosomal dominant disorder caused by point mutations in the gene encoding for the glial fibrillary acidic protein (GFAP), the major intermediate filament protein in astrocytes. Accumulation of GFAP protein in Rosenthal fibers leads to astrocytic dysfunctions and altered development and homeostasis of affected brain tissues. No cures are currently available for AxD patients.

In the proposed pilot project, we aim to develop gene-editing approaches to downregulate the expression of the mutated GFAP protein or correct mutations in the Gfap gene in an animal AxD model. This project will collect solid proof-of-concept data for the future progress of a novel and definitive gene therapy strategy for the treatment of AxD patients. In addition, we expect to outline novel editing platforms that could be applied prospectively for disease modeling studies and treatments of other leukodystrophies characterized by astrocyte degeneration or dysfunctional/maladaptive astrogliosis

Project financed by ELA up to: 100 000 €

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare disease mainly related to mutations in the MLC1 gene. Patients suffer from macrocephaly and motor and cognitive defects associated with progressive myelin degeneration. We have recently shown that MLC originates from early defects in the gliovascular unit, a specialized interface in the brain between blood vessels and astrocytic glial cells, including a defect in vessel contractility that impairs blood flow in the brain. We believe that these alterations are at the origin of MLC. Our project is now to develop an approach to restore the functions of the altered gliovascular unit in a mouse model of MLC and to test whether this strategy can reverse leukodystrophy, which would pave the way for therapeutic intervention in patients.

Project financed by ELA up to: 94 000 €

Globoid cell leukodystrophy (GLD) is a neurodegenerative inherited genetic disease, due to defects in an enzyme that normally degrades sphingolipids, a class of biological molecules present in the myelin sheath, necessary for nervous tissue function. The name of this enzyme is beta-galactosylceramidase (GALC). Impairment of GALC activity in GLD results in progressive demyelination and nerve tissue degeneration. Most individuals with infantile forms die before the age of two. Currently, there is no cure for GLD.

Several gene replacement therapy strategies have been attempted in pre-clinical models with varying degree of success, however, still facing the challenge of providing widespread enzymatic reconstitution and full correction of pathology in affected tissues, particularly the central and peripheral nervous systems (CNS and PNS). Previous evidence suggests effective transport across the blood brain barrier of several therapeutic proteins when fused with parts of other proteins, known to be able to reach CNS and PNS. Thus, GALC will be endowed with these additional portions, which may be sufficient to ensure its transport across the blood brain barrier.

In addition, we will equip GALC with small portions of highly secreted proteins to improve GALC secretion by cells. The long-term objective of the proposed research is thus to develop a safe and effective liver gene therapy for GLD. In vivo gene therapy to the liver indeed offers the attractive prospects of a minimally invasive, one-time potentially curative treatment for GLD, by providing sustained pervasive high amounts of a functional GALC enzyme to the CNS and PNS through the bloodstream.

Here we propose a project to obtain an early proof-of-principle in a mouse model of the disease. The CNS and PNS are naturally protected by a blood brain barrier that ensures selective passage of necessary substances, while blocking potentially dangerous molecules. Lentiviral vectors (LV) are attractive gene delivery vehicles for liver gene therapy thanks to their ability to insert their DNA into the host cell DNA. For this reason, LV are maintained following liver cell proliferation in liver growth and turnover, including potentially in newborns, that would be the target population in GLD. We have developed LV that allow stable expression of therapeutic proteins in the liver of mice and dogs, following systemic administration. More recently, we have generated engineered LV with increased resistance to capture by cells of the immune system, a process called phagocytosis. Thanks to this feature, these LV are more efficient at reaching the target cells in the liver.

Here we propose to generate LV expressing the above-described optimized GALC from the liver, administer them intravenously to newborn mice affected by GLD and assess survival and correction of the hallmarks of the GLD disease. We also propose to engineer LV to further escape phagocytosis by exposing on their surface additional inhibitors of phagocytosis. If successful, the work proposed here will allow the generation of novel LV with increased protection from phagocytosis and efficiency of gene transfer into liver cells that will facilitate development and manufacturing to quality and scale required for use in humans and alleviate concerns of possible LV particle dose dependent acute toxicity. These LV, transporting the engineered GALC enzymes, may become a feasible, sustainable, safe and effective liver gene therapy for GLD and potentially for other leukodystrophies.

Project financed by ELA up to: 96 000 €