Exploring the effect of disease causing mutations in metal binding sites of human ARSA in metachromatic leukodystrophy.

The arylsulfatase A (ARSA) gene is observed to be deficient in patients with metachromatic leukodystrophy (MLD), a type of lysosomal storage disease. MLD is a severe neurodegenerative disorder characterized by an autosomal recessive inheritance pattern. This study aimed to map the most deleterious mutations at the metal binding sites of ARSA and the amino acids in proximity to the mutated positions. We utilized an array of computational tools, including PredictSNP, MAPP, PhD-SNP, PolyPhen-1, PolyPhen-2, SIFT, SNAP, and ConSurf, to identify the most detrimental mutations potentially implicated in MLD collected from UniProt, ClinVar, and HGMD. Two mutations, D29N and D30H, as being extremely deleterious based on assessments of pathogenicity, conservation, biophysical characteristics, and stability analysis. The D29 and D30 are located at the metal-interacting regions of ARSA and found to undergo post-translational modification, specifically phosphorylation. Henceforth, the in-depth effect of metal binding upon mutation was examined using molecular dynamics simulations (MDS) before and after phosphorylation. The MDS results exhibited high deviation for the D29N and D30H mutations in comparison to the native, and the same was confirmed by significant residue fluctuation and reduced compactness. These structural alterations suggest that such mutations may influence protein functionality, offering potential avenues for personalized therapeutic and providing a basis for potential mutation-specific treatments for severe MLD patients.

Biochemical and molecular analysis of pediatric patients with metachromatic leukodystrophy in South China: functional characterization of five novel ARSA variants.

Metachromatic leukodystrophy (MLD) is a rare hereditary neurodegenerative disease caused by deficiency of the lysosomal enzyme arylsulfatase A (ARSA). This study described the clinical and molecular characteristics of 24 Chinese children with MLD and investigated functional characterization of five novel ARSA variants. A retrospective analysis was performed in 24 patients diagnosed with MLD at Guangzhou Women and Children's Medical Center in South China. Five novel mutations were further characterized by transient expression studies. We recruited 17 late-infantile, 3 early-juvenile, 4 late-juvenile MLD patients. In late-infantile patients, motor developmental delay and gait disturbance were the most frequent symptoms at onset. In juvenile patients, cognitive regression and gait disturbance were the most frequent chief complaints. Overall, 25 different ARSA mutations were identified with 5 novel mutations.The most frequent alleles were p.W320* and p.G449Rfs. The mutation p.W320*, p.Q155=, p.P91L, p.G156D, p.H208Mfs*46 and p.G449Rfs may link to late-infantile type. The novel missense mutations were predicted damaging in silico. The bioinformatic structural analysis of the novel missense mutations showed that these amino acid replacements would cause severe impairment of protein structure and function. In vitro functional analysis of the six mutants, showing a low ARSA enzyme activity, clearly demonstrated their pathogenic nature. The mutation p.D413N linked to R alleles. In western blotting analysis of the ARSA protein, the examined mutations retained reduced amounts of ARSA protein compared to the wild type. This study expands the spectrum of genotype of MLD. It helps to the future studies of genotype-phenotype correlations to estimate prognosis and develop new therapeutic approach.

Consensus guidelines for the monitoring and management of metachromatic leukodystrophy in the United States.

Metachromatic leukodystrophy (MLD) is a fatal, progressive neurodegenerative disorder caused by biallelic pathogenic mutations in the ARSA (Arylsulfatase A) gene. With the advent of presymptomatic diagnosis and the availability of therapies with a narrow window for intervention, it is critical to define a standardized approach to diagnosis, presymptomatic monitoring, and clinical care. To meet the needs of the MLD community, a panel of MLD experts was established to develop disease-specific guidelines based on healthcare resources in the United States. This group developed a consensus opinion for best-practice recommendations, as follows: (i) Diagnosis should include both genetic and biochemical testing; (ii) Early diagnosis and treatment for MLD is associated with improved clinical outcomes; (iii) The panel supported the development of newborn screening to accelerate the time to diagnosis and treatment; (iv) Clinical management of MLD should include specialists familiar with the disease who are able to follow patients longitudinally; (v) In early onset MLD, including late infantile and early juvenile subtypes, ex vivo gene therapy should be considered for presymptomatic patients where available; (vi) In late-onset MLD, including late juvenile and adult subtypes, hematopoietic cell transplant (HCT) should be considered for patients with no or minimal disease involvement. This document summarizes current guidance on the presymptomatic monitoring of children affected by MLD as well as the clinical management of symptomatic patients. Future data-driven evidence and evolution of these recommendations will be important to stratify clinical treatment options and improve clinical care.

Experiences of patients with metachromatic leukodystrophy, adrenoleukodystrophy, or Krabbe disease and the experiences of their family members: a qualitative systematic review.

OBJECTIVE: This review aimed to synthesize the experiences of patients with metachromatic leukodystrophy, adrenoleukodystrophy, or Krabbe disease and the experiences of their families. INTRODUCTION: Leukodystrophies are metabolic diseases caused by genetic mutations. There are multiple forms of the disease, varying in age of onset and symptoms. The progression of leukodystrophies worsens central nervous system symptoms and significantly affects the lives of patients and their families. INCLUSION CRITERIA: Qualitative studies on the experiences of patients with leukodystrophies and their family members were included. These experiences included treatments such as enzyme replacement therapy and hematopoietic stem cell transplantation; effects of tracheostomy and gastrostomy; burdens on the family, coordinating care within the health care system, and family planning due to genetic disorders. This review considered studies in any setting. METHODS: MEDLINE (Ovid), CINAHL Plus (EBSCOhost), APA PsycINFO (EBSCOhost), Scopus, and MedNar databases were searched on November 18, 2022. Study selection, critical appraisal, data extraction, and data synthesis were conducted in accordance with the JBI methodology for systematic reviews of qualitative evidence, and synthesized findings were evaluated according to the ConQual approach. RESULTS: Eleven studies were eligible for synthesis, and 45 findings were extracted corresponding with participants' voices. Of these findings, 40 were unequivocal and 5 were credible. The diseases in the included studies were metachromatic leukodystrophy and adrenoleukodystrophy; no studies were identified for patients with Krabbe disease and their families. These findings were grouped into 11 categories and integrated into 3 synthesized findings, including i) providing care by family members and health care providers as physical symptoms progress, which relates to the effects of the characteristics of progressive leukodystrophies; ii) building medical teamwork to provide appropriate support services, comprising categories related to the challenges experienced with the health care system for patients with leukodystrophy and their families; and iii) coordinating family functions to accept and cope with the disease, which included categories related to family psychological difficulties and role divisions within the family. According to the ConQual criteria, the second synthesized finding had a low confidence level, and the first and third synthesized findings had a very low confidence level. CONCLUSIONS: The synthesized findings of this review provide evidence on the experiences of patients with metachromatic leukodystrophy or adrenoleukodystrophy and their families. These findings indicate that there are challenges in managing a patient's physical condition and coordinating the health care system and family functions. REVIEW REGISTRATION: PROSPERO CRD42022318805. SUPPLEMENTAL DIGITAL CONTENT: A Japanese-language version of the abstract of this review is available [ http://links.lww.com/SRX/A49 ].

Improving newborn screening test performance for metachromatic leukodystrophy: Recommendation from a pre-pilot study that identified a late-infantile case for treatment.

Metachromatic leukodystrophy (MLD) is a devastating rare neurodegenerative disease. Typically, loss of motor and cognitive skills precedes early death. The disease is characterised by deficient lysosomal arylsulphatase A (ARSA) activity and an accumulation of undegraded sulphatide due to pathogenic variants in the ARSA gene. Atidarsagene autotemcel (arsa-cel), an ex vivo haematopoietic stem cell gene therapy was approved for use in the UK in 2021 to treat early-onset forms of pre- or early-symptomatic MLD. Optimal outcomes require early diagnosis, but in the absence of family history this is difficult to achieve without newborn screening (NBS). A pre-pilot MLD NBS study was conducted as a feasibility study in Manchester UK using a two-tiered screening test algorithm. Pre-established cutoff values (COV) for the first-tier C16:0 sulphatide (C16:0-S) and the second-tier ARSA tests were evaluated. Before the pre-pilot study, initial test validation using non­neonatal diagnostic bloodspots demonstrated ARSA pseudodeficiency status was associated with normal C16:0-S results for age (n = 43) and hence not expected to cause false positive results in this first-tier test. Instability of ARSA in bloodspot required transfer of NBS bloodspots from ambient temperature to -20°C storage within 7-8 days after heel prick, the earliest possible in this UK pre-pilot study. Eleven of 3687 de-identified NBS samples in the pre-pilot were positive for C16:0-S based on the pre-established COV of ≥170 nmol/l or ≥ 1.8 multiples of median (MoM). All 11 samples were subsequently tested negative determined by the ARSA COV of <20% mean of negative controls. However, two of 20 NBS samples from MLD patients would be missed by this C16:0-S COV. A further suspected false negative case that displayed 4% mean ARSA activity by single ARSA analysis for the initial test validation was confirmed by genotyping of this NBS bloodspot, a severe late infantile MLD phenotype was predicted. This led to urgent assessment of this child by authority approval and timely commencement of arsa-cel gene therapy at 11 months old. Secondary C16:0-S analysis of this NBS bloodspot was 150 nmol/l or 1.67 MoM. This was the lowest result reported thus far, a new COV of 1.65 MoM is recommended for future pilot studies. Furthermore, preliminary data of this study showed C16:1-OH sulphatide is more specific for MLD than C16:0-S. In conclusion, this pre-pilot study adds to the international evidence that recommends newborn screening for MLD, making it possible for patients to benefit fully from treatment through early diagnosis.

A systematic review on the birth prevalence of metachromatic leukodystrophy.

BACKGROUND: Metachromatic leukodystrophy (MLD) is an autosomal recessive lysosomal storage disease caused by deficiency in arylsulfatase A (ASA) activity arising primarily from ASA gene (ARSA) variants. Late-infantile, juvenile and adult clinical subtypes are defined by symptom onset at ≤ 2.5, > 2.5 to < 16 and ≥ 16 years, respectively. Epidemiological data were sought to address knowledge gaps and to inform decisions regarding the clinical development of an investigational drug. METHODS: To synthesize all available estimates of MLD incidence and birth prevalence worldwide and in selected countries, Ovid MEDLINE and Embase were searched systematically (March 11, 2022) using a population, intervention, comparator, outcome, time and setting framework, complemented by pragmatic searching to reduce publication bias. Where possible, results were stratified by clinical subtype. Data were extracted from non-interventional studies (clinical trials, non-clinical studies and case reports were excluded; reviews were used for snowballing only). RESULTS: Of the 31 studies included, 14 reported birth prevalence (13 countries in Asia-Pacific, Europe, the Middle East, North America and South America), one reported prevalence and none reported incidence. Birth prevalence per 100,000 live births ranged from 0.16 (Japan) to 1.85 (Portugal). In the three European studies with estimates stratified by clinical subtypes, birth prevalence was highest for late-infantile cases (0.31-1.12 per 100,000 live births). The distribution of clinical subtypes reported in cases diagnosed over various time periods in 17 studies varied substantially, but late-infantile and juvenile MLD accounted for at least two-thirds of cases in most studies. CONCLUSIONS: This review provides a foundation for further analysis of the regional epidemiology of MLD. Data gaps indicate the need for better global coverage, increased use of epidemiological measures (e.g. prevalence estimates) and more stratification of outcomes by clinical and genetic disease subtype.

The burden of disease in metachromatic leukodystrophy: results of a caregiver survey in the UK and Republic of Ireland.

BACKGROUND: Metachromatic Leukodystrophy (MLD) is a rare, autosomal recessive lysosomal storage disease characterised by the progressive loss of motor function and severe decline in cognitive function. Limited information is available on the burden MLD places on patients and their families and the medical and social support these patients need. Three UK-based MLD patient organisations commissioned an online survey, and follow-up semi-structured interviews to describe and quantify these burdens across MLD subtypes, stage of disease (including end of life) and treatment status (untreated, gene therapy or hematopoietic stem cell transplant [HSCT]). RESULTS: A total of 24 patients were included in the study: thirteen late infantile (LI), six early juvenile (EJ), two late juvenile (LJ) and three adult onset (AO). Six patients had received gene therapy and one had received an HSCT. MLD patients receiving no disease modifying treatment bore a high symptom burden: 94% were wheelchair dependent, 88% required tube feeding, 88% were incontinent, 82% had lost their speech and all the children were either unable to attend education or needed specialist provision. Patients were reliant on numerous medical interventions and assistive equipment. All early-onset patients (LI and EJ) were wheelchair dependent, and tube fed, with all EJ patients having lost all speech. The caregiving responsibilities of parents impacted their employment, finances, relationships and health. Patients treated with gene therapy or HSCT were more mobile and were able to eat normally and two thirds of the children were able to attend mainstream school. CONCLUSIONS: The impact of illness that patients and their caregivers faced was extensive, and the level of care, amount of medication, number of hospital visits and educational support required were substantial. Financial constraints often brought about by inability to work also placed considerable strain on families. The study increases understanding of the burden of MLD on patients and their families, and the level of unmet need in the treatment of the disease.

Progress in leukodystrophies with zebrafish.

Inherited leukodystrophies are genetic disorders characterized by abnormal white matter in the central nervous system. Although individually rare, there are more than 400 distinct types of leukodystrophies with a cumulative incidence of 1 in 4500 live births. The pathophysiology of most leukodystrophies is poorly understood, there are treatments for only a few, and there is significant morbidity and mortality, suggesting a critical need for improvements in this field. A variety of animal, cell, and induced pluripotent stem cell-derived models have been developed for leukodystrophies, but with significant limitations in all models. Many leukodystrophies lack animal models, and extant models often show no or mixed recapitulation of key phenotypes. Zebrafish (Danio rerio) have become increasingly used as disease models for studying leukodystrophies due to their early onset of disease phenotypes and conservation of molecular and neurobiological mechanisms. Here, we focus on reviewing new zebrafish disease models for leukodystrophy or models with recent progress. This includes discussion of leukodystrophy with vanishing white matter disease, X-linked adrenoleukodystrophy, Zellweger spectrum disorders and peroxisomal disorders, PSAP deficiency, metachromatic leukodystrophy, Krabbe disease, hypomyelinating leukodystrophy-8/4H leukodystrophy, Aicardi-Goutières syndrome, RNASET2-deficient cystic leukoencephalopathy, hereditary diffuse leukoencephalopathy with spheroids-1 (CSF1R-related leukoencephalopathy), and ultra-rare leukodystrophies. Zebrafish models offer important potentials for the leukodystrophy field, including testing of new variants in known genes; establishing causation of newly discovered genes; and early lead compound identification for therapies. There are also unrealized opportunities to use humanized zebrafish models which have been sparsely explored.

Complex genotypes in family with metachromatic leukodystrophy: Effect of trans and cis mutations distribution on the phenotype variability.

Metachromatic leukodystrophy (MLD) is a severe metabolic disorder caused by the deficient activity of arylsulfatase A due to ARSA gene mutations. According to the age of onset, MLD is classified into three forms: infantile, juvenile, and adult. In our study, we aimed to perform a genetic analysis for two siblings with juvenile MLD for a better characterization of the molecular mechanisms behind the disease. A consanguineous family including two MLD patients (PII.1 and PII.2) was enrolled in our study. The diagnosis was made based on the clinical and neuroimaging investigations. The sequencing of ARSA gene was performed followed by in silico analysis. Besides, the cis/trans distribution of the variants was verified through a PCR-RFLP. The ARSA gene sequencing revealed three known variants, two exonic c.1055A > G and c.1178C > G and an intronic one (c.1524 + 95A > G) in the 3'UTR region. All variants were present at heterozygous state in the two siblings and their mother. The assessment of the cis/trans distribution showed the presence of these variants in cis within the mother, while PII.2 and PII.2 present the c.1055A > G/c.1524 + 95A > G and the c.1178C > G in trans. Additionally, PII.1 harbored a de novo novel missense variant c.1119G > T, whose pathogenicity was supported by our predictive results. Our genetic findings, supported by a clinical examination, confirmed the affection of the mother by the adult MLD. Our results proved the implication of the variable distribution of the found variants in the age of MLD onset. Besides, we described a variable severity between the two siblings due to the de novo pathogenic variant. In conclusion, we identified a complex genotype of ARSA variants within two MLD siblings with a variable severity due to a de novo variant present in one of them. Our results allowed the establishment of an adult MLD diagnosis and highlighted the importance of an assessment of the trans/cis distribution in the cases of complex genotypes.

Identification and structural characterization of a pathogenic ARSA missense variant in two consanguineous families from Jammu and Kashmir (India) with late infantile metachromatic leukodystrophy.

BACKGROUND: Metachromatic leukodystrophy (MLD) is a rare lysosomal storage disorder caused by a deficiency of Arylsulfatase A (ARSA) enzyme activity. Its clinical manifestations include progressive motor and cognitive decline. ARSA gene mutations are frequent in MLD. METHODS AND RESULTS: In the present study, whole exome sequencing (WES) was employed to decipher the genetic cause of motor and cognitive decline in proband's of two consanguineous families from J&K (India). Clinical investigations using radiological and biochemical analysis revealed MLD-like features. WES confirmed a pathogenic variant in the ARSA gene. Molecular simulation dynamics was applied for structural characterization of the variant. CONCLUSION: We report the identification of a pathogenic missense variant (c.1174 C > T; p.Arg390Trp) in the ARSA gene in two cases of late infantile MLD from consanguineous families in Jammu and Kashmir, India. Our study utilized genetic analysis and molecular dynamics simulations to identify and investigate the structural consequences of this mutation. The molecular dynamics simulations revealed significant alterations in the structural dynamics, residue interactions, and stability of the ARSA protein harbouring the p.Arg390Trp mutation. These findings provide valuable insights into the molecular mechanisms underlying the pathogenicity of this variant in MLD.

Elucidating the Therapeutic Utility of Olaparib in Sulfatide-Induced Human Astrocyte Toxicity and Neuroinflammation.

Metachromatic leukodystrophy (MLD) is a severe demyelinating, autosomal recessive genetic leukodystrophy, with no curative treatment. The disease is underpinned by mutations in the arylsulfatase A gene (ARSA), resulting in deficient activity of this lysosomal enzyme, and consequential accumulation of galactosylceramide-3-O-sulfate (sulfatide) in the brain. Most of the effects in the brain have been attributed to the accumulation of sulfatides in oligodendrocytes and their cell damage. In contrast, less is known regarding sulfatide toxicity in astrocytes. Poly (ADP-ribose) polymerase (PARP) inhibitors are anti-cancer therapeutics that have proven efficacy in preclinical models of many neurodegenerative and inflammatory diseases, but have never been tested for MLD. Here, we examined the toxic effect of sulfatides on human astrocytes and restoration of this cell damage by the marketed PARP-1 inhibitor, Olaparib. Cultured human astrocytes were treated with increasing concentrations of sulfatides (5-100 µM) with or without Olaparib (100 nM). Cell viability assays were used to ascertain whether sulfatide-induced toxicity was rescued by Olaparib. Immunofluorescence, calcium (Ca2+) imaging, ROS, and mitochondrial damage assays were also used to explore the effects of sulfatides and Olaparib. ELISAs were performed and chemotaxis of peripheral blood immune cells was measured to examine the effects of Olaparib on sulfatide-induced inflammation in human astrocytes. Here, we established a concentration-dependent (EC50∼20 µM at 24 h) model of sulfatide-induced astrocyte toxicity. Our data demonstrate that sulfatide-induced astrocyte toxicity involves (i) PARP-1 activation, (ii) pro-inflammatory cytokine release, and (iii) enhanced chemoattraction of peripheral blood immune cells. Moreover, these sulfatide-induced effects were attenuated by Olaparib (IC50∼100 nM). In addition, sulfatide caused impairments of ROS production, mitochondrial stress, and Ca2+ signaling in human astrocytes, that were indicative of metabolic alterations and that were also alleviated by Olaparib (100 nM) treatment. Our data support the hypothesis that sulfatides can drive astrocyte cell death and demonstrate that Olaparib can dampen many facets of sulfatide-induced toxicity, including, mitochondrial stress, inflammatory responses, and communication between human astrocytes and peripheral blood immune cells. These data are suggestive of potential therapeutic utility of PARP inhibitors in the sphere of rare demyelinating diseases, and in particular MLD. Graphical abstract. Proposed mechanism of action of Olaparib in sulfatide-treated astrocytes. Human astrocytes treated for 24 h with sulfatides increase PARP-1 expression and die. PARP-1 overexpression is modulated by Ca2+ release from the endoplasmic reticulum, thus enhancing intracellular Ca2+ concentration. PARP-1 inhibition with Olaparib reduces Ca2+ influx and cell death. Olaparib also decreases IL-6, IL-8, IL-17, and CX3CL1 release from sulfatide-stimulated astrocytes, suggesting that PARP-1 plays a role in dampening neuroinflammation in MLD. This is confirmed by the reduction of immune cell migration such as lymphocytes, NK cells, and T cells towards sulfatide-treated astrocytes. Moreover, mitochondrial stress and ROS production induced by sulfatides are rescued by PARP-1 inhibition. Future studies will focus on the signaling cascades triggered by PARP-1-mediated currents in reactive astrocytes and Olaparib as a potential therapeutic target for MLD.

Identification of a Novel ARSA Gene Mutation Through High-Throughput Molecular Diagnosis Method in Two Girls with Late Infantile Metachromatic Leukodystrophy.

Metachromatic leukodystrophy (MLD) is a rare leukoencephalopathy caused by pathogenic mutations in the ARSA gene. It manifests as severe motor symptoms, mental problems, and sometimes, seizures. We aimed to investigate the phenotypic manifestations and genetic causes of MLD in an Iranian family. We present the case of a 3-year-old girl who presented with hypotonia, muscular atrophy, and seizures. Neurological and neuromuscular examinations were performed to evaluate clinical characteristics. Whole exome sequencing (WES) was used to detect disease-causing variants. In silico analysis was performed to predict the pathogenicity of this variant. GROMACS software was utilized for molecular dynamic simulation (MDS). Neurological studies revealed marked slowing of motor conduction velocities and an increased motor unit action potential duration. Brain MRI scan revealed white matter abnormalities. By applying WES, we identified a novel homozygous missense variant (NM_000487.6, c.938G > C, p.R313P) in ARSA. Direct sequencing identified this homozygous variant in her asymptomatic younger sister, whereas both parents carried a heterozygous variant. This mutation has not been reported in genetic databases or in literature. In silico analysis predicted that any variation in this DNA position would cause disease, as it is highly conserved. The c.938G > C variant was classified as a pathogenic variant according to ACMG/AMP guidelines. MDS analysis indicated that c.938G > C had a significant impact on both the structure and stabilization of ARSA, ultimately resulting in impaired protein function. The identification of this variant expands the spectrum of ARSA gene mutations associated with MLD and highlights the importance of genetic testing for the diagnosis of MLD.

Predicting disease severity in metachromatic leukodystrophy using protein activity and a patient phenotype matrix.

BACKGROUND: Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by mutations in the arylsulfatase A gene (ARSA) and categorized into three subtypes according to age of onset. The functional effect of most ARSA mutants remains unknown; better understanding of the genotype-phenotype relationship is required to support newborn screening (NBS) and guide treatment. RESULTS: We collected a patient data set from the literature that relates disease severity to ARSA genotype in 489 individuals with MLD. Patient-based data were used to develop a phenotype matrix that predicts MLD phenotype given ARSA alleles in a patient's genotype with 76% accuracy. We then employed a high-throughput enzyme activity assay using mass spectrometry to explore the function of ARSA variants from the curated patient data set and the Genome Aggregation Database (gnomAD). We observed evidence that 36% of variants of unknown significance (VUS) in ARSA may be pathogenic. By classifying functional effects for 251 VUS from gnomAD, we reduced the incidence of genotypes of unknown significance (GUS) by over 98.5% in the overall population. CONCLUSIONS: These results provide an additional tool for clinicians to anticipate the disease course in MLD patients, identifying individuals at high risk of severe disease to support treatment access. Our results suggest that more than 1 in 3 VUS in ARSA may be pathogenic. We show that combining genetic and biochemical information increases diagnostic yield. Our strategy may apply to other recessive diseases, providing a tool to address the challenge of interpreting VUS within genotype-phenotype relationships and NBS.

Olaparib Attenuates Demyelination and Neuroinflammation in an Organotypic Slice Culture Model of Metachromatic Leukodystrophy.

Metachromatic leukodystrophy (MLD) is a severe demyelinating, autosomal recessive genetic leukodystrophy. The disease is underpinned by mutations in the arylsulfatase A gene (ARSA), resulting in deficient activity of the arylsulfatase A lysosomal enzyme and consequential accumulation of galactosylceramide-3-O-sulfate (sulfatide) in the brain. Using an ex vivo murine-derived organotypic cerebellar slice culture model, we demonstrate that sulfatide induces demyelination in a concentration-dependent manner. Interestingly, our novel data demonstrate that sulfatide-induced demyelination is underpinned by PARP-1 activation, oligodendrocyte loss, pro-inflammatory cytokine expression, astrogliosis, and microgliosis. Moreover, such sulfatide-induced effects can be attenuated by the treatment with the poly (ADP-ribose) polymerase 1 (PARP-1) inhibitor Olaparib (IC50∼100 nM) suggesting that this small molecule may be neuroprotective and limit toxin-induced demyelination. Our data support the idea that sulfatide is a key driver of demyelination and neuroinflammation in MLD and suggest that PARP-1 inhibitors have therapeutic utility in the sphere of rare demyelinating disease.

Safety and Efficacy of Intravenous and Intrathecal Delivery of AAV9-Mediated ARSA in Minipigs.

Metachromatic leukodystrophy (MLD) is a hereditary neurodegenerative disease characterized by demyelination and motor and cognitive impairments due to deficiencies of the lysosomal enzyme arylsulfatase A (ARSA) or the saposin B activator protein (SapB). Current treatments are limited; however, gene therapy using adeno-associated virus (AAV) vectors for ARSA delivery has shown promising results. The main challenges for MLD gene therapy include optimizing the AAV dosage, selecting the most effective serotype, and determining the best route of administration for ARSA delivery into the central nervous system. This study aims to evaluate the safety and efficacy of AAV serotype 9 encoding ARSA (AAV9-ARSA) gene therapy when administered intravenously or intrathecally in minipigs, a large animal model with anatomical and physiological similarities to humans. By comparing these two administration methods, this study contributes to the understanding of how to improve the effectiveness of MLD gene therapy and offers valuable insights for future clinical applications.

Single Systemic Administration of a Gene Therapy Leading to Disease Treatment in Metachromatic Leukodystrophy Arsa Knock-Out Mice.

Metachromatic leukodystrophy (MLD) is a rare, inherited, demyelinating lysosomal storage disorder caused by mutations in the arylsulfatase-A gene (ARSA). In patients, levels of functional ARSA enzyme are diminished and lead to deleterious accumulation of sulfatides. Herein, we demonstrate that intravenous administration of HSC15/ARSA restored the endogenous murine biodistribution of the corresponding enzyme, and overexpression of ARSA corrected disease biomarkers and ameliorated motor deficits in Arsa KO mice of either sex. In treated Arsa KO mice, when compared with intravenously administered AAV9/ARSA, significant increases in brain ARSA activity, transcript levels, and vector genomes were observed with HSC15/ARSA Durability of transgene expression was established in neonate and adult mice out to 12 and 52 weeks, respectively. Levels and correlation between changes in biomarkers and ARSA activity required to achieve functional motor benefit was also defined. Finally, we demonstrated blood-nerve, blood-spinal and blood-brain barrier crossing as well as the presence of circulating ARSA enzyme activity in the serum of healthy nonhuman primates of either sex. Together, these findings support the use of intravenous delivery of HSC15/ARSA-mediated gene therapy for the treatment of MLD.SIGNIFICANCE STATEMENT Herein, we describe the method of gene therapy adeno-associated virus (AAV) capsid and route of administration selection leading to an efficacious gene therapy in a mouse model of metachromatic leukodystrophy. We demonstrate the therapeutic outcome of a new naturally derived clade F AAV capsid (AAVHSC15) in a disease model and the importance of triangulating multiple end points to increase the translation into higher species via ARSA enzyme activity and biodistribution profile (with a focus on the CNS) with that of a key clinically relevant biomarker.