Category: Metachromatic leukodystrophy (MLD)

Metachromatic Leukodystrophy (MLD) is a rare demyelinating disease, due to arylsulfatase A (ARSA) deficiency, an enzyme involved in the catabolism of sulfatides, the main component of the myelin sheath. This deficiency leads to progressive demyelination of central nervous system and peripheral neuropathy. The most frequent form of the disease is the late infantile form of the disease which is characterized by a rapid progression of the disease, especially after the first onset of symptom. Ex vivo gene therapy develop by the group of Alessandra Biffi, and based on engraftment of lentiviral transduced hematopoietic stem cells has been shown to be effective for presymptomatic forms of MLD but not effective in early symptomatic patients, probably due to the rapid evolution and the time needed for the engrafment. On the other hand, enzyme replacement was shown as potentially effective but needed a chronic delivery. In the group, we previously proposed a gene therapy approach for MLD based on intracerebral delivery of an AAVrh.10 encoding ARSA. We had established proof of concept in mouse model of the disease and scale up study in non-human primates (NHP) leading us to propose a clinical trial which included 4 patients from 2013 to 2016. Despite ARSA expression in the brain and detection in cerebrospinal fluid (CSF) we failed to obtained any therapeutical benefits in these patients. Our point is that expressing ARSA in the CNS is fundamental to rapidly stop the disease progression, however, it could be essential also to bring back in patient healthy microglial cells. This project aim at establishing the proof of concept of rapid, sustained and important ARSA expression in the whole CNS (brain, spinal cord) and potentially also in the peripheral nerve. To achieve this aim, we propose to use a novel serotype of AAV: AAVPHP.eB that has been engineered to efficiently cross the blood brain barrier (BBB) after IV delivery. The project will be divided in two main parts, first a rapid study in the MLD mouse model both for pre and post symptomatic treatment, in order to evaluate efficiency of AAVPHP.eB-ARSA vector for biodistribution, ARSA expression, sulfatides correction, Purkinje cell prevention and behavioral testing. In a second phase, we want to test this vector in NHP thought several routes of delivery: intravenous (IV), intrathecal (IT) and intracerebroventricular (ICV), especially by potential coupling of these routes of administration to optimize CNS targeting. This with the final aim to propose a clinical trial for symptomatic MLD patients.

Project financed by ELA up to: 100 000 €

Hematopoietic stem cell gene therapy (HSC GT) is an experimental treatment based on the transplantation of autologous hematopoietic precursor cells genetically modified to express high levels of ARSA enzyme. This treatment benefits late infantile and early juvenile MLD children if treated in the pre-symptomatic/early symptomatic stage of the disease but is less effective in MLD children treated in the progressive phase of disease, who experience severe neurological deterioration. In order to recognize the reasons behind this different clinical outcome we need a better understanding of the therapeutic mechanisms of correction of MLD neural cells by the progeny of transplanted hematopoietic cells that engraft in the brain.

In this project the investigators aim to fill this gap of knowledge taking advantage of unique and clinically relevant in vitro human disease models, i.e. neural and blood cells derived from healthy donors, untreated and gene therapy treated MLD patients. We expect to clarify how and to which extent the ARSA enzyme is transported from metabolically competent donor blood cells to MLD neurons and glia. Also, they will provide hints on the contribution of complementary mechanisms of correction that could be exploited to enhance the benefit of HSC GT and broaden its accessibility to larger cohorts of MLD patients who presently not meet the inclusion criteria of these experimental treatments.

Project financed by ELA up to: 60 000 €

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 €