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 €