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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., Competing Interests: Declaration of Competing Interest No honorarium, grant, or other form of payment was received to produce the manuscript. LAA is a consultant to Biogen, Takeda Pharmaceuticals, Orchard Therapeutics, is a site subinvestigator for the Takeda trial, and serves on the scientific advisory board of Cure MLD and MLD Foundation; JLB is a site principal investigator for the Takeda SHP611 trial; JJB has received consulting fees from Sobi, Omeros, Bluebird Bio, Sanofi, SmartImmune, Merck and Bluerock; EM has no conflicts of interest to disclose; RAN has no conflicts of interest to disclose; JAB is a site principal investigator for the Takeda SHP611 trial; AB is site subinvestigator for the Takeda SHP611 trial and received travelling support by Orchard-Tx; BB is a site principal investigator for the Takeda SHP611 trial and is a consultant to Aro, AlltRNA, Orchard Therapeutics, Astella, Passage Bio, Biomarin, PTC Therapeutics, JCR Pharma, Takeda; received honoraria from Astra Zeneca, Biomarin, Chiesi, Horizon, JCR Pharma; grant funding from Biomarin Pharmaceutical, Takeda, Homology Medicines, Denali Therapeutics, Sangamo, JCR Pharma, and Ultragenyx; AD has participated in an advisory board organized by Orchard Therapeutics; FE has <1% equity in Swan Bio, and royalties from AAV9 license for AMN; receives consulting fees from Leal Therapeutics, Swan Bio, Ionis, Minoryx, UptoDate, Origen, Takeda Therapeutics and Third Rock Ventures; founder and consultant of Swan Bio and serves on the chair on the advisory board of European Leukodystrophy Association, and as a board member at United Leukodystrophy Foundation; EE has participated in several advisory boards arranged by Orchard Therapeutics; LE serves on the advisory board for Ionis pharmaceuticals; ME is Chief Medical Officer at Forge Biologics; AF receives research support from SwanBio, Autobahn Therapeutics, Poxel Therapeutics, Vertex Pharmaceuticals, and Maryland Stem Cell Research Fund, was a Data and Safety Monitoring Board (DSMB) member for BlueBird Bio, and coinvented a patent currently licensed to Ashvattha; JF is a consultant for GeneDx, Educational Consultant on the Impact of Exome and Genome Sequencing in Well-Phenoptyped Populations and is Chair of the Maryland Secretary's Advisory Council on Hereditary and Congenital Disorders; AG has received payment or honoraria from Spark Therapeutics and Orchard Therapeutics, consulting fees from Takeda; chair of the MN Rare Disease Advisory Council and the Clinical and Laboratory Standards Institute; SK is a clinical trial site principal investigator for Ionis and was a consultant for Veristat; MCP is the site principal investigator for clinical trials funded by Azafaros, Glycomine, Idorsia, Maggie's Pearl, Takeda, and Zevra, and has consulted for Azafaros, Takeda, and Zevra; serves as editor in chief of the Journal of Child Neurology and an editor for JIMD, and royalties as section editor for up to date; PO is a consultant to Orchard Therapeutics, serves on a DSMB for Ionis, and has clinical trial support from Immusoft and Allovir; JOM is a consultant to Novoglia and site principal investigator for Vigil Neuroscience; JDS is a consultant to Biogen and Cycle Pharma; LS is a consultant to Vico Therapeutics and a site principal investigator for trials of Vigil Neuroscience, Stealth Biotherapeutics and PTC Therapeutics; CS is PI of the Takeda clinical trial and consultant for Orchard Therapeutics; INS has no conflicts of interest to disclose; DR is site PI for the Takeda trial; JPR has no conflicts of interest to disclose; KVH is a consultant for Bluebird bio and Poxel, a site PI for trials funded by Minoryx, Bluebird bio, IONIS and a trial advisor for Calico; MW has received research support from Abeona Therapeutics, Alexion Pharmaceuticals, the Ara Parseghian Medical Research Foundation, BioMarin Pharmaceutical, Cure Sanfilippo Foundation, Dana's Angels Research Trust, Firefly Fund, Mirum Pharma, Noah's Hope, Orchard Therapeutic, Passage Bio, Sanofi Genzyme, Sio Gene Therapies, Takeda Pharmaceutical, Travere Therapeutics, and Ultragenyx Pharmaceutical; has received consulting fees from Sanofi Genzyme; AZ is a site subinvestigator for the Takeda trial; FF is License holder of OTL-200, I Orchard Therapeutics trial, advisor Orchard, Takeda, funding Telethon Foundation, GSK, Orchard; LL has no conflicts of interest to disclose; AV is an advisor to Takeda, Passagebio, Orchard; and is site PI Takeda trial., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Metachromatic leukodystrophy (MLD) is a neuro-metabolic disorder due to arylsulfatase A deficiency, causing demyelination of the central and peripheral nervous system. Hematopoietic cell transplantation (HCT) can provide a symptomatic and survival benefit for pre-symptomatic and early symptomatic patients by stabilizing CNS disease. This case series, however, illustrates the occurrence of severely progressive polyneuropathy shortly after HCT in two patients with late-infantile, one with late-juvenile, and one with adult MLD, leading to the inability to walk or sit without support. The patients had demyelinating polyneuropathy before HCT, performed at the ages of 2 years in the first two patients and at 14 and 23 years in the other two patients. The myeloablative conditioning regimen consisted of busulfan, fludarabine and, in one case, rituximab, with anti-thymocyte globulin, cyclosporine, steroids, and/or mycophenolate mofetil for GvHD prophylaxis. Polyneuropathy after HCT progressed parallel with tapering immunosuppression and paralleled bouts of infection and graft-versus-host disease (GvHD). Differential diagnoses included MLD progression, neurological GvHD or another (auto)inflammatory cause. Laboratory, electroneurography and pathology investigations were inconclusive. In two patients, treatment with immunomodulatory drugs led to temporary improvement, but not sustained stabilization of polyneuropathy. One patient showed recovery to pre-HCT functioning, except for a Holmes-like tremor, for which a peripheral origin cannot be excluded. One patient showed marginal response to immunosuppressive treatment and died ten months after HCT due to respiratory failure. The extensive diagnostic and therapeutic attempts highlight the challenge of characterizing and treating progressive polyneuropathy in patients with MLD shortly after HCT. We advise to consider repeat electro-neurography and possibly peripheral nerve biopsy in such patients. Nerve conduction blocks, evidence of the presence of T lymphocytes and macrophages in the neuronal and surrounding nerve tissue, and beneficial effects of immunomodulatory drugs may indicate a partially (auto)immune-mediated pathology. Polyneuropathy may cause major residual disease burden after HCT. MLD patients with progressive polyneuropathy could potentially benefit from a more intensified immunomodulatory drug regime following HCT, especially at times of immune activation., (© 2024. The Author(s).)

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 ]., Competing Interests: YK has received research grants and honoraria from Takeda Pharmaceutical Company Limited. NS has received research grants and honoraria for lectures from Takeda Pharmaceutical Company Limited and JCR Pharmaceuticals. The other authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Wolters Kluwer Health, Inc. on Behalf of JBI.)

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 non‑neonatal 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., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Abstract: Metachromatic leukodystrophy (MLD) is a rare genetic disorder caused by pathogenic variants of the ARSA gene, leading to a deficiency of the arylsulfatase A enzyme (ARSA) and consecutive accumulation of galactosylceramide-3-0-sulfate in the nervous system. The condition leads to severe neurological deficits and subsequently results in profound intellectual and motoric disability. Especially, the adult form of MLD, which occurs in individuals aged >16 years, poses significant challenges for treating physicians because of the rarity of cases, limited therapeutic options, and different allogeneic hematopoietic cell transplantation (allo-HCT) protocols worldwide. Here, we report the results of allo-HCT treatment in 4 patients with a confirmed adult MLD diagnosis. Bone marrow or mobilized peripheral progenitor cells were infused after a reduced intensity conditioning regime consisting of fludarabine and treosulfan. In 3 patients, allo-HCT was followed by an infusion of mesenchymal cells to further consolidate ARSA production. We observed a good tolerability and an increase in ARSA levels up to normal range values in all patients. A full donor chimerism was detected in 3 patients within the first 12 months. In a 1-year follow-up, patients with complete donor chimerism showed a neurological stable condition. Only 1 patient with an increasing autologous chimerism showed neurological deterioration and a decline in ARSA levels in the first year. In summary, allo-HCT offers a therapeutic option for reconstituting ARSA enzyme levels in adult patients with MLD, with tolerable side effects., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Introduction: Metachromatic leukodystrophy (MLD) is a rare autosomal recessive lysosomal storage disorder resulting from arylsulfatase A enzyme deficiency, leading to toxic sulfatide accumulation. As a result affected individuals exhibit progressive neurodegeneration. Treatments such as hematopoietic stem cell transplantation (HSCT) and gene therapy are effective when administered pre-symptomatically. Newborn screening (NBS) for MLD has recently been shown to be technically feasible and is indicated because of available treatment options. However, there is a lack of guidance on how to monitor and manage identified cases. This study aims to establish consensus among international experts in MLD and patient advocates on clinical management for NBS-identified MLD cases., Methods: A real-time Delphi procedure using eDELPHI software with 22 experts in MLD was performed. Questions, based on a literature review and workshops, were answered during a seven-week period. Three levels of consensus were defined: A) 100%, B) 75-99%, and C) 50-74% or >75% but >25% neutral votes. Recommendations were categorized by agreement level, from strongly recommended to suggested. Patient advocates participated in discussions and were involved in the final consensus., Results: The study presents 57 statements guiding clinical management of NBS-identified MLD patients. Key recommendations include timely communication by MLD experts with identified families, treating early-onset MLD with gene therapy and late-onset MLD with HSCT, as well as pre-treatment monitoring schemes. Specific knowledge gaps were identified, urging prioritized research for future evidence-based guidelines., Discussion: Consensus-based recommendations for NBS in MLD will enhance harmonized management and facilitate integration in national screening programs. Structured data collection and monitoring of screening programs are crucial for evidence generation and future guideline development. Involving patient representatives in the development of recommendations seems essential for NBS programs., Competing Interests: Declaration of competing interest L. A. Adang is a consultant to Takeda Pharmaceuticals, Orchard Therapeutics, and is a site sub-investigator for the Takeda trial. G. Bernard is/was a consultant for Calico (2023-present), Orchard Therapeutics (2023-present), Passage Bio Inc (2020–2022) and Ionis (2019). She is/was a site investigator for the Alexander's disease trial of Ionis (2021-present), Metachromatic leukodystrophy of Shire/Takeda (2020–2021), Krabbe (2021–2023) and GM1 gene therapy trials of Passage Bio (2021-present), GM1 natural history study from the University of Pennsylvania sponsored by Passage Bio (2021-present) and Adrenoleukodystrophy/Hematopoietic stem cell transplantation natural history study of Bluebird Bio (2019), a site sub-investigator for the MPS II gene therapy trial of Regenxbio (2021-present) and the MPS II clinical trial of Denali (2022-present). She has received an unrestricted educational grant from Takeda (2021–2022). Orchard Therapeutics: V. Calbi works as consultant for Orchard Therapeutics. F. Fumagalli is investigator of gene therapy clinical trials for MLD sponsored by Orchard Therapeutics, the licence holder of investigational medicinal product arsa-cel. Dr. Fumagalli has acted as ad-hoc consultants for Orchard Therapeutics advisory boards. The MLD gene therapy was licensed to GlaxoSmithKline in 2014, and then to Orchard Therapeutics in 2018. Telethon and Ospedale San Raffaele are entitled to receive milestone payments and royalties for such a therapy. M. Langeveld is involved in a premarketing studies with Sanofi-Genzyme, Protalix/Chiesi and Idorsia. Financial arrangements were made through AMC Research BV. No fees, travel support or grants were obtained from Pharmaceutical Industry. F. Mochel has participated in advisory boards arranged by Minoryx Therapeutics and Vigil Neuroscience. Her research work is funded by the French Ministry of Health (PHRC), the French Ministry of Research (ANR), the Paris Brain Institute and Minoryx Therapeutics. L. Schöls is a consultant to Vico Therapeutics and a site principal investigator for trials of Vigil Neuroscience, Stealth Biotherapeutics and PTC Therapeutics. C. Sevin is PI of the Takeda clinical trial and consultant for Orchard Therapeutics. A. Zerem is a site sub-investigator for the Takeda clinical trial and local PI for the Ionis clinical trial. Institutional research support from Shire plc and Orchard. Advisor and coinvestigator for trials in MLD (Shire/Takeda, Orchard) without personal payments. N. I. Wolf is researcher at the MLD initiative, which is a publicly funded academic registry and collaborative platform for metachromatic leukodystrophy and local PI for several multicenter trials for leukodystrophies (Takeda, Ionis, VigilNeuro). She does consultancies for Takeda, Ionis, VigilNeuro, Eli Lilly, PassageBio, Sana Biotech, Sanofi, without personal payments., (© 2024 Published by Elsevier Ltd on behalf of European Paediatric Neurology Society.)

Background: For decades, early allogeneic stem cell transplantation (HSCT) has been used to slow neurological decline in metachromatic leukodystrophy (MLD). There is lack of consensus regarding who may benefit, and guidelines are lacking. Clinical practice relies on limited literature and expert opinions. The European Reference Network for Rare Neurological Diseases (ERN-RND) and the MLD initiative facilitate expert panels for treatment advice, but some countries are underrepresented. This study explores organizational and clinical HSCT practices for MLD in Europe and neighboring countries to enhance optimization and harmonization of cross-border MLD care., Methods: A web-based EUSurvey was distributed through the ERN-RND and the European Society for Blood and Marrow Transplantation Inborn Errors Working Party. Personal invitations were sent to 89 physicians (43 countries) with neurological/metabolic/hematological expertise. The results were analyzed and visualized using Microsoft Excel and IBM SPSS statistics., Results: Of the 30 countries represented by 42 respondents, 23 countries offer HSCT for MLD. The treatment is usually available in 1-3 centers per country (18/23, 78%). Most countries have no or very few MLD patients transplanted during the past 1-5 years. The eligibility criteria regarding MLD subtype, motor function, IQ, and MRI largely differ across countries., Conclusion: HSCT for MLD is available in most European countries, but uncertainties exist in Eastern and South-Eastern Europe. Applied eligibility criteria and management vary and may not align with the latest scientific insights, indicating physicians' struggle in providing evidence-based care. Interaction between local physicians and international experts is crucial for adequate treatment decision-making and cross-border care in the rapidly changing MLD field., (© 2024. The Author(s).)

More than 50 leukodystrophies have been described. This group of inherited disorders affects myelin development and/or maintenance and can manifest from birth to adulthood. Neuroinflammation is a hallmark of some leukodystrophies, explaining in part the therapeutic benefit of hematopoietic stem cell transplantation (HSCT). Indeed, in addition to supplying the CNS with myelomonocyte donor cells expressing the deficient protein or enzyme, HSCT allows the restoration of normal microglia function, which may act on neuroinflammation. In this chapter, we explore the rationale, indication, and outcome of HSCT in Cerebral Adrenoleukodystrophy (CALD), Metachromatic Leukodystrophy (MLD), Krabbe Disease (KD), and Adult-onset Leukoencephalopathy with Axonal Spheroids and Pigmented Glia (ALSP), which are among the most frequent leukodystrophies. For these leukodystrophies, HSCT may modify notably the natural history and improve CNS-related deficits, provided that the procedure is performed early into the disease course. In addition, we discuss the recent development of ex vivo gene therapy for CALD and MLD as a promising alternative to allograft., (Copyright © 2024 Elsevier B.V. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

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., (© 2024 Japanese Society of Developmental Biologists.)

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 (Ca 2+ ) 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 (EC 50 ∼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 (IC 50 ∼100 nM). In addition, sulfatide caused impairments of ROS production, mitochondrial stress, and Ca 2+ 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 Ca 2+ release from the endoplasmic reticulum, thus enhancing intracellular Ca 2+ concentration. PARP-1 inhibition with Olaparib reduces Ca 2+ 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., (© 2023. The Author(s).)

A deficiency of human arylsulfatase A (hASA) causes metachromatic leukodystrophy (MLD), a lysosomal storage disease characterized by sulfatide accumulation and central nervous system (CNS) demyelination. Efficacy of enzyme replacement therapy (ERT) is increased by genetic engineering of hASA to elevate its activity and transfer across the blood-brain barrier (BBB), respectively. To further improve the enzyme's bioavailability in the CNS, we mutated a cathepsin cleavage hot spot and obtained hASAs with substantially increased half-lives. We then combined the superstabilizing exchange E424A with the activity-promoting triple substitution M202V/T286L/R291N and the ApoEII-tag for BBB transfer in a trimodal modified neoenzyme called SuPerTurbo-ASA. Compared with wild-type hASA, half-life, activity, and M6P-independent uptake were increased more than 7-fold, about 3-fold, and more than 100-fold, respectively. ERT of an MLD-mouse model with immune tolerance to wild-type hASA did not induce antibody formation, indicating absence of novel epitopes. Compared with wild-type hASA, SuPerTurbo-ASA was 8- and 12-fold more efficient in diminishing sulfatide storage of brain and spinal cord. In both tissues, storage was reduced by ∼60%, roughly doubling clearance achieved with a 65-fold higher cumulative dose of wild-type hASA previously. Due to its enhanced therapeutic potential, SuPerTurbo-ASA might be a decisive advancement for ERT and gene therapy of MLD., Competing Interests: Declaration of interests An international patent application PCT/EP2022/053785 is pending for SuPerTurbo-ASA, claiming the priority of the meanwhile withdrawn EP patent application EP 21157364.4 filed on 16/02/2021. Applicant is the Rheinische Friedrich-Wilhelms-Universität, Bonn, Regina-Pacis-Weg 3, 53113 Bonn, Germany. V.G. and U.M. are inventors., (Copyright © 2023 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Objective: To understand the benefit-risk profile for historical and current treatments for MLD., Methods: A systematic review was conducted on the effectiveness, safety, and costs of MLD treatments: allogeneic haematopoietic stem cell transplantation (HSCT) and atidarsagene autotemcel (arsa-cel) according to best practice., Results: A total of 6940 titles and abstracts were retrieved from the literature searches and 26 from other sources. From these, 35 manuscripts reporting on a total of 12 studies were selected for inclusion in the review. There were no controlled multi-armed trials. However, we provide observations comparing two interventional therapies (alloHSCT and arsa-cel) and each of these to standard/supportive care (natural history). There were no benefits for survival, gross motor function and cognitive function for LI patients receiving alloHSCT, as patients experienced disease progression similar to LI natural history. For juvenile patients receiving alloHSCT, no differences in survival were observed versus natural history, however stabilisation of cognitive and motor function were reported for some patients (particularly for pre- or minimally-symptomatic LJ patients), while others experienced disease progression. Furthermore, alloHSCT was associated with severe complications such as treatment-related mortality, graft versus host disease, and re-transplantation in both LI and EJ treated patients. Most LI and EJ patients treated with arsa-cel appeared to have normal development, preservation, or slower progression of gross motor function and cognitive function, in contrast to the rapid decline observed in natural history patients. A survival benefit for arsa-cel versus natural history and versus alloHSCT was observed in LI patients.LI and EJ patients treated with arsa-cel had better gross motor function and cognitive function compared to alloHSCT, which had limited effect on motor and cognitive decline. No data has been reported for arsa-cel treatment of LJ patients., Conclusions: Overall, this systematic review indicates that compared to NHx and HSCT, treatment with arsa-cel results in clinically relevant benefits in LI and EJ MLD patients by preserving cognitive function and motor development in most patients, and increased survival for LI patients. Nevertheless, further research is required to confirm these findings, given they are based on results from non-RCT studies., (© 2023. Institut National de la Santé et de la Recherche Médicale (INSERM).)

Background: Metachromatic leukodystrophy (MLD) is a rare lysosomal storage disease caused by deficient activity of arylsulfatase A (ASA). Treatment options for patients are limited; gene therapy based on haematopoietic stem cell transplantation is the only approved treatment for some subtypes of MLD. Any therapeutic benefit of treatments must be meaningful for patients and their families. We evaluated the clinical meaningfulness of slowing the decline in gross motor function as measured by the Gross Motor Function Classification in MLD (GMFC-MLD) from the caregiver perspective via semi-structured telephone interviews with caregivers of children with late-infantile MLD. We also evaluated the perceived significance of declines in communication abilities measured by the Expressive Language Function Classification in MLD (ELFC-MLD). This work could help to inform the endpoints of a phase 2 clinical trial (NCT03771898) assessing the efficacy of intrathecal recombinant human ASA in MLD., Results: Twelve caregivers were recruited, reporting on 12 children with MLD. Children had a mean age of 6.1 years; mean age at symptom onset was 17.6 months. Most children (10/12) progressed from walking without support (categories 0-1) to a loss of locomotion (categories 5-6) in ≤ 2 years. Caregivers felt that GMFC-MLD and ELFC-MLD accurately described motor and language declines in their children, respectively. Most caregivers (10/12) reported that the idea of delaying disease progression would be meaningful. Further, a slowing of motor function decline in GMFC-MLD, from category 1 to category 3 or from category 2 to category 4 over 2 years, was seen as meaningful by all caregivers asked; however, only 3/12 caregivers reported that delayed decline would be meaningful if baseline category was ≥ 3. Caregivers also reported that delaying expressive language decline at any level that did not indicate a complete loss of expressive language (indicated by categories 1-3) would be meaningful., Conclusions: Caregivers of children with MLD felt that a delayed decline in gross motor function, as assessed by the GMFC-MLD, would be meaningful, supporting the selection of primary and secondary endpoints for the phase 2 clinical trial. Communication abilities were another area of significance for consideration in future clinical trial design., (© 2023. The Author(s).)

Objective: Metachromatic leukodystrophy is a lysosomal storage disease caused by deficient arylsulfatase A. It is characterized by progressive demyelination and thus mainly affects the white matter. Hematopoietic stem cell transplantation may stabilize and improve white matter damage, yet some patients deteriorate despite successfully treated leukodystrophy. We hypothesized that post-treatment decline in metachromatic leukodystrophy might be caused by gray matter pathology., Methods: Three metachromatic leukodystrophy patients treated with hematopoietic stem cell transplantation with a progressive clinical course despite stable white matter pathology were clinically and radiologically analyzed. Longitudinal volumetric MRI was used to quantify atrophy. We also examined histopathology in three other patients deceased after treatment and compared them with six untreated patients., Results: The three clinically progressive patients developed cognitive and motor deterioration after transplantation, despite stable mild white matter abnormalities on MRI. Volumetric MRI identified cerebral and thalamus atrophy in these patients, and cerebellar atrophy in two. Histopathology showed that in brain tissue of transplanted patients, arylsulfatase A expressing macrophages were clearly present in the white matter, but absent in the cortex. Arylsulfatase A expression within patient thalamic neurons was lower than in controls, the same was found in transplanted patients., Interpretation: Neurological deterioration may occur after hematopoietic stem cell transplantation in metachromatic leukodystrophy despite successfully treated leukodystrophy. MRI shows gray matter atrophy, and histological data demonstrate absence of donor cells in gray matter structures. These findings point to a clinically relevant gray matter component of metachromatic leukodystrophy, which does not seem sufficiently affected by transplantation., (© 2023 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

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., (Copyright © 2023 the authors.)

Objective: The objective of this review is to synthesize the experiences of patients with metachromatic leukodystrophy (MLD), adrenoleukodystrophy (ALD), and Krabbe disease and the experiences of their family members., Introduction: MLD, ALD, and Krabbe disease are rare disorders that are classified as lysosomal storage or peroxisomal disorders, with similar presentations as leukodystrophy. As these diseases cause cognitive and neurological decline due to the progression of leukodystrophy associated with demyelination, they have significant impact on the lives of patients and their families. It is important to identify the impact and challenges of these diseases on patients' lives and on their families, as well as to synthesize qualitative studies regarding their experiences., Inclusion Criteria: We will consider studies including patients with MLD, ALD, or Krabbe disease and their family members. These experiences will include the challenges, dissatisfactions, and frustrations with symptoms and treatments; complications of hematopoietic stem cell transplantation; and the increased caregiver burden with disease progression. This is important since the impacts of disease progression are experienced in a variety of settings beyond the hospital, such as in the community and at home., Methods: The search strategy will follow JBI methodology and be conducted in 3 steps: an initial limited search, a comprehensive database search, and a reference search of the included articles. MEDLINE, CINAHL Plus, PsycINFO, and Scopus will be searched with no restriction on language or publication dates. The study selection, critical appraisal, data extraction, and data synthesis will be performed according to JBI guidelines for systematic reviews of qualitative research. Final syntheses will be assessed using the ConQual approach., Systematic Review Registration Number: PROSPERO CRD42022318805., Competing Interests: The other authors declare no conflict of interest., (Copyright © 2022 JBI.)

Metachromatic leukodystrophy (MLD) is a rare autosomal recessive disorder of sphingolipid metabolism, due to a deficiency of the enzyme arylsulfatase A (ARSA). The main clinical signs of the disease are secondary to central and peripheral nervous system demyelination. MLD is subdivided into early- and late-onset subtypes based upon the onset of neurological disease. The early-onset subtype is associated with a more rapid progression of the disease that leads to death within the first decade of life. Until recently, no effective treatment was available for MLD. The blood-brain barrier (BBB) prevents systemically administered enzyme replacement therapy from reaching target cells in MLD. The evidence for the efficacy of hematopoietic stem cell transplantation is limited to the late-onset MLD subtype. Here, we review the preclinical and clinical studies that facilitated the approval of the ex vivo gene therapy atidarsagene autotemcel for early-onset MLD by the European Medicines Agency (EMA) in December 2020. This approach was studied in an animal model first and then in a clinical trial, eventually proving its efficacy in preventing disease manifestations in presymptomatic patients and stabilizing its progression in paucisymptomatic subjects. This new therapeutic consists of patients' CD34+ hematopoietic stem/progenitor cells (HSPCs) transduced with a lentiviral vector encoding functional ARSA cDNA. The gene-corrected cells get reinfused into the patients after a cycle of chemotherapy conditioning., (Copyright 2023 Clarivate.)

Metachromatic leukodystrophy (MLD) is a rare inherited lysosomal disorder. The condition progresses relentlessly, with severe disability typically established within 6-14 years of symptom onset. There is no cure, and limited treatment options are available to slow disease progression. We describe the case of a 23-year-old woman with forgetfulness, unsteady gait, and falls. Neurologic examination revealed intermittent dystonic posturing of the right upper and lower limb when walking. The Addenbrooke's Cognitive Examination (ACE) score was 70/100. MRI sequences demonstrated frontal-predominant atrophy and extensive white matter hyperintensity. Differential diagnoses such as autoimmune, inflammatory, and neoplastic diseases were excluded, and a genetic diagnosis was considered. Lysosomal enzyme testing showed low arylsulfatase with elevated urinary sulfatides, and genetic testing revealed a homozygous pathogenic mutation in the ARSA gene securing a diagnosis of adult-onset MLD. A male sibling also had early cognitive impairment and was found to have the same mutation. Hematopoietic stem cell transplantation (HSCT) was offered after discussion with experts. The male sibling died of multiple complications after HSCT. The index patient is now 24 months after HSCT, and disease progression has halted. This case highlights the challenges in the accurate diagnosis of adult-onset leukoencephalopathies and explores potential treatment strategies. A stepwise approach to the differential diagnosis of white matter diseases is demonstrated. HSCT may be an effective treatment, but the significant complication rate needs to be carefully considered., (© 2022 American Academy of Neurology.)

Background: Metachromatic leukodystrophy (MLD), a relentlessly progressive and ultimately fatal condition, is a rare autosomal recessive lysosomal storage disorder caused by a deficiency of the enzyme arylsulfatase A (ARSA). Historically management has been palliative or supportive care. Hematopoietic stem cell transplantation is poorly effective in early-onset MLD and benefit in late-onset MLD remains controversial. Hematopoietic stem cell gene therapy, Libmeldy (atidarsagene autotemcel), was recently approved by the European Medicines Agency for early-onset MLD. Treatment benefit is mainly observed at an early disease stage, indicating the need for early diagnosis and intervention. This study contributes insights into the caregiver language used to describe initial MLD symptomatology, and thereby aims to improve communication between clinicians and families impacted by this condition and promote a faster path to diagnosis., Results: Data was collected through a moderator-assisted online 60-min survey and 30-min semi-structured follow-up telephone interview with 31 MLD caregivers in the United States (n = 10), France (n = 10), the United Kingdom (n = 5), and Germany (n = 6). All respondents were primary caregivers of a person with late infantile (n = 20), juvenile (n = 11) or borderline late infantile/juvenile (n = 1) MLD (one caregiver reported for 2 children leading to a sample of 32 individuals with MLD). Caregivers were asked questions related to their child's initial signs and symptoms, time to diagnosis and interactions with healthcare providers. These results highlight the caregiver language used to describe the most common initial symptoms of MLD and provide added context to help elevate the index of suspicion of disease. Distinctions between caregiver descriptions of late infantile and juvenile MLD in symptom onset and disease course were also identified., Conclusions: This study captures the caregiver description of the physical, behavioral, and cognitive signs of MLD prior to diagnosis. The understanding of the caregiver language at symptom onset sheds light on a critical window of often missed opportunity for earlier diagnosis and therapeutic intervention in MLD., (© 2022. The Author(s).)

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder primarily affecting the white matter of the nervous system that results from a deficiency of the arylsulfatase A (ARSA). Mesenchymal stem cells (MSCs) are able to secrete ARSA and have shown beneficial effects in MLD patients. In this retrospective analysis, 10 pediatric MLD patients [mesenchymal stem cell group (MSCG)] underwent allogeneic hematopoietic stem cell transplantation (HSCT) and received two applications of 2 × 10 6 MSCs/kg bodyweight at day +30 and +60 after HSCT between 2007 and 2018. MSC safety, occurrence of graft-versus-host disease (GvHD), blood ARSA levels, chimerism, cell regeneration and engraftment, magnetic resonance imaging (MRI) changes, and the gross motor function were assessed within the first year of HSCT. The long-term data included clinical outcomes and safety aspects of MSCs. Data were compared to a control cohort of seven pediatric MLD patients [control group (CG)] who underwent HSCT only. The application of MSC in pediatric MLD patients after allogeneic HSCT was safe and well tolerated, and long-term potentially MSC-related adverse effects up to 13.5 years after HSCT were not observed. Patients achieved significantly higher ARSA levels (CG: median 1.03 nmol·10 -6 and range 0.41-1.73 | MSCG: median 1.58 nmol·10 -6 and range 0.44-2.6; P  < 0.05), as well as significantly higher leukocyte ( P  < 0.05) and thrombocyte ( P  < 0.001) levels within 365 days of MSC application compared to CG patients. Statistically significant effects on acute GvHD, regeneration of immune cells, MRI changes, gross motor function, and clinical outcomes were not detected. In conclusion, the application of MSCs in pediatric MLD patients after allogeneic HSCT was safe and well tolerated. The two applications of 2 × 10 6 /kg allogeneic MSCs were followed by improved engraftment and hematopoiesis within the first year after HSCT. Larger, prospective trials are necessary to evaluate the impact of MSC application on engraftment and hematopoietic recovery.

Metachromatic leukodystrophy is a lethal metabolic leukodystrophy, with emerging treatments for early disease stages. Biomarkers to measure disease activity are required for clinical assessment and treatment follow-up. This retrospective study compared neurofilament light chain and glial fibrillary acidic protein (GFAP) levels in CSF (n = 11) and blood (n = 92) samples of 40 patients with metachromatic leukodystrophy (aged 0-42 years) with 38 neurologically healthy children (aged 0-17 years) and 38 healthy adults (aged 18-45 years), and analysed the associations between these levels with clinical phenotype and disease evolution in untreated and transplanted patients. Metachromatic leukodystrophy subtype was determined based on the (expected) age of symptom onset. Disease activity was assessed by measuring gross motor function deterioration and brain MRI. Longitudinal analyses with measurements up to 23 years after diagnosis were performed using linear mixed models. CSF and blood neurofilament light chain and GFAP levels in paediatric controls were negatively associated with age (all P < 0.001). Blood neurofilament light chain level at diagnosis (median, interquartile range; picograms per millilitre) was significantly increased in both presymptomatic (14.7, 10.6-56.7) and symptomatic patients (136, 40.8-445) compared to controls (5.6, 4.5-7.1), and highest among patients with late-infantile (456, 201-854) or early-juvenile metachromatic leukodystrophy (291.0, 104-445) and those ineligible for treatment based on best practice (291, 57.4-472). GFAP level (median, interquartile range; picogram per millilitre) was only increased in symptomatic patients (591, 224-1150) compared to controls (119, 78.2-338) and not significantly associated with treatment eligibility (P = 0.093). Higher blood neurofilament light chain and GFAP levels at diagnosis were associated with rapid disease progression in late-infantile (P = 0.006 and P = 0.051, respectively) and early-juvenile patients (P = 0.048 and P = 0.039, respectively). Finally, blood neurofilament light chain and GFAP levels decreased during follow-up in untreated and transplanted patients but remained elevated compared with controls. Only neurofilament light chain levels were associated with MRI deterioration (P < 0.001). This study indicates that both proteins may be considered as non-invasive biomarkers for clinical phenotype and disease stage at clinical assessment, and that neurofilament light chain might enable neurologists to make better informed treatment decisions. In addition, neurofilament light chain holds promise assessing treatment response. Importantly, both biomarkers require paediatric reference values, given that their levels first decrease before increasing with advancing age., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain.)

Metachromatic leukodystrophy (MLD) is a rare genetic disorder caused by mutations in the Arylsulfatase-A ( ARSA ) gene. The enzyme plays a key role in sulfatide metabolism in brain cells, and its deficiency leads to neurodegeneration. The clinical manifestations of MLD include stagnation and decline of motor and cognitive function, leading to premature death with limited standard treatment options. Here, we describe a mutation-agnostic hematopoietic stem and progenitor cell (HSPC) gene therapy using CRISPR-Cas9 and AAV6 repair template as a prospective treatment option for MLD. Our strategy achieved efficient insertions and deletions (>87%) and a high level of gene integration (>47%) at the ARSA locus in human bone marrow-derived HSPCs, with no detectable off-target editing. As a proof of concept, we tested our mutation-agnostic therapy in HSPCs derived from two MLD patients with distinct mutations and demonstrated restoration of ARSA enzyme activity (>30-fold improvement) equivalent to healthy adults. In summary, our investigation enabled a mutation-agnostic therapy for MLD patients with proven efficacy and strong potential for clinical translation.

Competing Interests: JK is a consultant for Neurogene, a company developing gene therapy for Krabbe disease. She is also developing a cell therapy using a microglial/macrophage cell manufactured from cord blood and under study funded by the Marcus Foundation. This technology was licensed by her institution to CryoCell International.

Background: Effective treatment for metachromatic leukodystrophy (MLD) remains a substantial unmet medical need. In this study we investigated the safety and efficacy of atidarsagene autotemcel (arsa-cel) in patients with MLD., Methods: This study is an integrated analysis of results from a prospective, non-randomised, phase 1/2 clinical study and expanded-access frameworks. 29 paediatric patients with pre-symptomatic or early-symptomatic early-onset MLD with biochemical and molecular confirmation of diagnosis were treated with arsa-cel, a gene therapy containing an autologous haematopoietic stem and progenitor cell (HSPC) population transduced ex vivo with a lentiviral vector encoding human arylsulfatase A (ARSA) cDNA, and compared with an untreated natural history (NHx) cohort of 31 patients with early-onset MLD, matched by age and disease subtype. Patients were treated and followed up at Ospedale San Raffaele, Milan, Italy. The coprimary efficacy endpoints were an improvement of more than 10% in total gross motor function measure score at 2 years after treatment in treated patients compared with controls, and change from baseline of total peripheral blood mononuclear cell (PBMC) ARSA activity at 2 years after treatment compared with values before treatment. This phase 1/2 study is registered with ClinicalTrials.gov, NCT01560182., Findings: At the time of analyses, 26 patients treated with arsa-cel were alive with median follow-up of 3·16 years (range 0·64-7·51). Two patients died due to disease progression and one due to a sudden event deemed unlikely to be related to treatment. After busulfan conditioning, all arsa-cel treated patients showed sustained multilineage engraftment of genetically modified HSPCs. ARSA activity in PBMCs was significantly increased above baseline 2 years after treatment by a mean 18·7-fold (95% CI 8·3-42·2; p<0·0001) in patients with the late-infantile variant and 5·7-fold (2·6-12·4; p<0·0001) in patients with the early-juvenile variant. Mean differences in total scores for gross motor function measure between treated patients and age-matched and disease subtype-matched NHx patients 2 years after treatment were significant for both patients with late-infantile MLD (66% [95% CI 48·9-82·3]) and early-juvenile MLD (42% [12·3-71·8]). Most treated patients progressively acquired motor skills within the predicted range of healthy children or had stabilised motor performance (maintaining the ability to walk). Further, most displayed normal cognitive development and prevention or delay of central and peripheral demyelination and brain atrophy throughout follow-up; treatment benefits were particularly apparent in patients treated before symptom onset. The infusion was well tolerated and there was no evidence of abnormal clonal proliferation or replication-competent lentivirus. All patients had at least one grade 3 or higher adverse event; most were related to conditioning or to background disease. The only adverse event related to arsa-cel was the transient development of anti-ARSA antibodies in four patients, which did not affect clinical outcomes., Interpretation: Treatment with arsa-cel resulted in sustained, clinically relevant benefits in children with early-onset MLD by preserving cognitive function and motor development in most patients, and slowing demyelination and brain atrophy., Funding: Orchard Therapeutics, Fondazione Telethon, and GlaxoSmithKline., Competing Interests: Declaration of interests FFu, VC, MGNS, AA, CB, FB, FFe, MM, FT, VG, SR, ESF, MPC, and MEB are investigators of gene therapy clinical trials for MLD sponsored by Orchard Therapeutics, the licence holder of investigational medicinal product arsa-cel. FFu and VC have acted as ad-hoc consultants for an Orchard Therapeutics advisory board. The MLD gene therapy was licensed to GlaxoSmithKline in 2014, and then to Orchard Therapeutics in 2018. Telethon and Ospedale San Raffaele are entitled to receive milestone payments and royalties for such a therapy. MSe and AB left San Raffaele Hospital and their role as principal investigators of the pivotal study on November, 2014, and September, 2015, respectively. AA subsequently became study principal investigator and responsible physician for treatment under expanded access frameworks. AB is currently a member of the scientific advisory board of Orchard Therapeutics and holds stock in Orchard Therapeutics. PMVR and CDS have a contract with Orchard Therapeutics to perform statistical analyses of gene therapy clinical trials for MLD. SMa and FM have a service contract with Ospedale San Raffaele. SMa has a contract with Orchard Therapeutics to perform ARSA activity on CSF in the clinical trial NCT03392987. JG-S, LCS, and GFD are former employees and hold stock in Orchard Therapeutics, which sponsored the clinical trial. All other authors declare that they have no financial interest related to the work described in the manuscript., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by an arylsulfatase A (ARSA) deficiency and characterized by severe neurological symptoms resulting from demyelination within the central and peripheral nervous systems. We investigated the feasibility and efficacy of intrathecal administration of a type 9 adeno-associated viral vector encoding ARSA (AAV9/ARSA) for the treatment of 6-week-old MLD model mice, which are presymptomatic, and 1-year-old mice, which exhibit neurological abnormalities. Immunohistochemical analysis following AAV9/ARSA administration showed ARSA expression within the brain, with highest activities in the cerebellum and olfactory bulbs. In mice treated at 1 year, alcian blue staining and quantitative analysis revealed significant decreases in stored sulfatide. Behaviorally, mice treated at 1 year showed no improvement in their ability to traverse narrow balance beams as compared to untreated mice. By contrast, MLD mice treated at 6 weeks showed significant decreases in stored sulfatide throughout the entire brain and improved ability to traverse narrow balance beams. These findings suggest intrathecal administration of an AAV9/ARSA vector is a promising approach to treating genetic diseases of the central nervous system, including MLD, though it may be essential to begin therapy before the onset of neurological symptoms., (© 2021. The Author(s).)

Gene therapy (GT) has rapidly attracted renewed interest as a treatment for otherwise incurable diseases, with several GT products already on the market and many more entering clinical testing for selected indications. Clonal tracking techniques based on vector integration enable monitoring of the fate of engineered cells in the blood of patients receiving GT and allow assessment of the safety and efficacy of these procedures. However, owing to the limited number of cells that can be tested and the impracticality of studying cells residing in peripheral organs without performing invasive biopsies, this approach provides only a partial snapshot of the clonal repertoire and dynamics of genetically modified cells and reduces the predictive power as a safety readout. In this study, we developed liquid biopsy integration site sequencing, or LiBIS-seq, a polymerase chain reaction technique optimized to quantitatively retrieve vector integration sites from cell-free DNA released into the bloodstream by dying cells residing in several tissues. This approach enabled longitudinal monitoring of in vivo liver-directed GT and clonal tracking in patients receiving hematopoietic stem cell GT, improving our understanding of the clonal composition and turnover of genetically modified cells in solid tissues and, in contrast to conventional analyses based only on circulating blood cells, enabling earlier detection of vector-marked clones that are aberrantly expanding in peripheral tissues., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Metachromatic leukodystrophy, a fatal pediatric neurodegenerative lysosomal storage disease caused by mutations in the arylsulfatase A ( ARSA ) gene, is characterized by intracellular accumulation of sulfatides in the lysosomes of cells of the central nervous system (CNS). In previous studies, we have demonstrated efficacy of AAVrh.10hARSA, an adeno-associated virus (AAV) serotype rh.10 vector coding for the human ARSA gene to the CNS of a mouse model of the disease, and that catheter-based intraparenchymal administration of AAVrh.10hARSA to the CNS of nonhuman primates (NHPs) white matter results in widespread expression of ARSA. As a formal dose-escalating safety/toxicology study, we assessed the safety of intraparenchymal delivery of AAVrh.10hARSA vector to 12 sites in the white matter of the CNS of NHPs at 2.85 × 10 10 (total low dose, 2.4 × 10 9 genome copies [gc]/site) and 1.5 × 10 12 (total high dose, 1.3 × 10 11 gc/site) gc, compared to AAVrh.10Null (1.5 × 10 12 gc total, 1.3 × 10 11 gc/site) as a vector control, and phosphate buffered saline for a sham surgical control. No significant adverse effects were observed in animals treated with low dose AAVrh.10hARSA. However, animals treated with the high dose AAVrh.10ARSA and the high dose Null vector had highly localized CNS abnormalities on magnetic resonance imaging scans at the sites of catheter infusions, and histopathology demonstrated that these sites were associated with infiltrates of T cells, B cells, microglial cells, and/or macrophages. Although these findings had no clinical consequences, these safety data contribute to understanding the dose limits for CNS white matter direct intraparenchymal administration of AAVrh.10 vectors for treatment of CNS disorders.

Leukodystrophies and genetic leukoencephalopathies comprise a growing group of inherited white matter disorders. Diagnostic rates have improved with increased utilization of next generation sequencing. As treatment options continue to advance for leukodystrophies, so will candidacy for inclusion in the United States' newborn Recommended Universal Screening Panel as was achieved for X-linked adrenoleukodystrophy. Stem cell therapies have become standard of care for selected leukodystrophies. However, transplantation-related risks remain high and outcomes are not fully satisfactory. Transduction of autologous hematopoietic stem cells with lentiviral vectors, referred to as ex vivo gene therapy, circumvents some, but not all, of the risks of traditional transplantation and has recently been demonstrated to be safe and efficective in clinical studies of X-linked adrenoleukodystrophy and metachromatic leukodystrophy. Gene therapy, through direct infusion of adeno-associated virus vectors, has emerged as a safer alternative for many monogenetic pediatric neurological disorders. Numerous preclinical studies have shown safety and efficacy of adeno-associated virus gene therapy in leukodystrophies allowing expanded access treatment for Canavan disease prior to initiation of a clinical trial. For inherited white matter disorders resulting from overexpression of a protein, such as Pelizaeus-Merzbacher disease, emerging RNA therapies have shown success in preclinical studies and promise for rapid translation to the clinic. Lastly, small molecule and protein therapies remain a long-term treatment option for a number of leukodystrophies, including intrathecal enzyme replacement therapy for metachromatic leukodystrophy. Herein we review recent advances in diagnosis and treatment of inherited white matter disorders., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Cerebrospinal fluid (CSF) has emerged as a sensitive matrix for the screening of biomarkers for diagnosis and clinical follow-up of diseases with neurological manifestations, including some lysosomal storage disorders. In this study, we assessed the range of values of arylsulfatase A (ARSA) activity in the CSF of pediatric and adult donors, and in pediatric patients who underwent gene therapy for metachromatic leukodystrophy (MLD)., Methods: A cohort of 56 CSF samples was included in the study: pediatric donors (n = 36), adult donors (n = 9), and MLD patients (n = 11) at different timepoints [pre-gene therapy (GT), post-GT + 1 Year, post-GT + 2 Years, post-GT + 3 Years]. We have used our fluorometric assay for the determination of ARSA activity. The total protein content in the samples was also evaluated., Results: We discovered that ARSA activity was higher in pediatric donors (geometric mean: 1.039 nmol/mg/h; 95% range: 0.859-1.258 nmol/mg/h) compared to adults (geometric mean: 0.305 nmol/mg/h; 95% range: 0.214-0.435 nmol/mg/h). No ARSA activity was detected in the CSF of MLD patients pre-GT, whereas ARSA activity was stably expressed and almost restored to range of values of pediatric donors in MLD patients post-GT + 3 Years with a geometric mean of 0.822 nmol/mg/h (95% range: 0.580-1.165 nmol/mg/h)., Conclusions: This study establishes range of values of ARSA activity in the CSF for MLD clinical practice. The observed ranges of ARSA activities in CSF exhibited an unpredicted age dependence and, in turn, revealed the need of using pediatric ARSA activity for evaluating the restoration of the enzyme activity during the therapy of MLD., (© American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Enzyme replacement therapies, allogeneic bone marrow transplantation and gene therapies are treatment options for lysosomal storage diseases caused by inherited deficiencies of soluble lysosomal enzymes. Independent from the approach, the enzyme must be delivered to lysosomes of deficient patient cells. Little is known about the dissemination of enzyme within a tissue where cells compete for uptake via different receptor systems, binding affinities and endocytic rates. To evaluate dissemination and lysosomal targeting of a lysosomal enzyme in the CNS, we analysed receptor-mediated endocytosis of arylsulfatase A (ASA) by different types of brain-derived cell lines and primary murine brain cells. For ASA expressed by chinese hamster ovary cells for enzyme replacement therapy of metachromatic leukodystrophy, endocytic rates decline from microglia to neurons and astrocytes and to oligodendrocytes. Only immature oligodendrocytes endocytose significant amounts of enzyme. Uptake by non-microglial cells is due to mannose 6-phosphate receptors, whereas several receptor systems participate in endocytosis by microglial cells. Interestingly, ASA expressed by microglial cells cannot be taken up in a mannose 6-phosphate dependent manner. The resulting failure to correct non-microglial cells corroborates in vivo data and indicates that therapeutic effects of allogeneic bone marrow transplantation and hematopoietic stem cell gene therapy on metachromatic leukodystrophy are independent of metabolic cross-correction of neurons, astrocytes and oligodendrocytes by receptor-mediated endocytosis., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Metachromatic leukodystrophy(MLD) is an autosomal recessive hereditary neurodegenerative lysosomal storage disorder caused by the mutations in arylsulfatase A gene (ARSA), which results in the deficiency of ARSA enzyme. The common clinical characteristics of MLD are abnormal gait, and then gradually appears ataxia, spastic quadriplegia, optic atrophy, cortical blindness, and dementia. We describe two patients in China who were diagnosed with MLD and find that the four ARSA gene mutations (c.1115G>A, c.302G>T, c.893 G> T, and c.302G>T) are associated with MLD, in which c.893 G>T and c.302G>T are novel mutations by gene sequence and clinical manifestations, to further understand the relationship between MLD and ARSA gene.

Families of children with special health care needs may travel substantial distances to access specialized health care. However, it is not known how race/ethnicity, insurance status, and access to disease-specific specialty care affect travel distances. This analysis examines patients aged 18 years or younger who were discharged from a Pediatric Health Information System (PHIS) children's hospital (n = 52) with a diagnosis of an inherited leukodystrophy between October 1, 2015, and September 30, 2018 (n = 950 patients). Leukodystrophies are rare but very serious neurological illnesses, with elevated mortality and morbidity rates. Bivariate and hierarchical generalized linear models reveal that white children, privately insured children, and children visiting leukodystrophy specialist centers travel farther for children's hospital care. These findings indicate that socially privileged families travel greater distances to obtain specialized health care, which could affect clinical outcomes.

Competing Interests: POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose.

Adeno-associated virus (AAV)-based vectors have transformed into powerful elements of genetic medicine with proven therapeutic efficacy and a good safety profile. Over the years, efforts to transduce hematopoietic stem cells (HSCs) with AAV2 vectors have, however, been challenging. While there was evidence that AAV2 delivered vector genomes to primitive, quiescent, multipotential, self-renewing, in vivo engrafting HSCs, transgene expression was elusive. In this study, we review the evolution of AAV transduction of HSC, starting with AAV2 vectors leading to the isolation of a family of naturally occurring AAVs from human CD34 + HSC, the AAVHSC. The stem cell-derived AAVHSCs have turned out to have remarkable potentials for genetic therapies well beyond the hematopoietic system. AAVHSCs have tropism for a wide variety of peripheral tissues, including the liver, muscle, and the retina. They cross the blood-brain barrier and transduce cells of the central nervous system. Preclinical gene therapy studies underway using AAVHSC vectors are discussed. We review the notable ability of AAVHSCs to mediate efficient, seamless homologous recombination in the absence of exogenous nuclease activity and discuss the therapeutic implications. We also discuss early results from an AAVHSC-based clinical gene therapy trial that is underway for the treatment of phenylketonuria. Thus, the stem cell-derived AAVHSC, offer a multifaceted platform for in vivo gene therapy and genome editing for the treatment of inherited diseases.

Objective: In metachromatic leukodystrophy, a lysosomal storage disorder due to decreased arylsulfatase A activity, hematopoietic stem cell transplantation may stop brain demyelination and allow remyelination, thereby halting white matter degeneration. This is the first study to define the effects and therapeutic mechanisms of hematopoietic stem cell transplantation on brain tissue of transplanted metachromatic leukodystrophy patients., Methods: Autopsy brain tissue was obtained from eight (two transplanted and six nontransplanted) metachromatic leukodystrophy patients, and two age-matched controls. We examined the presence of donor cells by immunohistochemistry and microscopy. In addition, we assessed myelin content, oligodendrocyte numbers, and macrophage phenotypes. An unpaired t-test, linear regression or the nonparametric Mann-Whitney U-test was performed to evaluate differences between the transplanted, nontransplanted, and control group., Results: In brain tissue of transplanted patients, we found metabolically competent donor macrophages expressing arylsulfatase A distributed throughout the entire white matter. Compared to nontransplanted patients, these macrophages preferentially expressed markers of alternatively activated, anti-inflammatory cells that may support oligodendrocyte survival and differentiation. Additionally, transplanted patients showed higher numbers of oligodendrocytes and evidence for remyelination. Contrary to the current hypothesis on therapeutic mechanism of hematopoietic cell transplantation in metachromatic leukodystrophy, we detected no enzymatic cross-correction to resident astrocytes and oligodendrocytes., Interpretation: In conclusion, donor macrophages are able to digest accumulated sulfatides and may play a neuroprotective role for resident oligodendrocytes, thereby enabling remyelination, albeit without evidence of cross-correction of oligo- and astroglia. These results emphasize the importance of immunomodulation in addition to the metabolic correction, which might be exploited for improved outcomes., (© 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals, Inc on behalf of American Neurological Association.)

The leukodystrophies are a heterogeneous group of inherited diseases characterized by progressive demyelination of the central nervous system leading to devastating neurologic symptoms and premature death. Hematopoietic stem cell transplantation (HSCT) has been successfully used to treat certain leukodystrophies, including adrenoleukodystrophy, globoid leukodystrophy (Krabbe disease), and metachromatic leukodystrophy, over the past 30 years. To date, these complex patients have primarily been transplanted at a limited number of pediatric centers. As the number of cases identified through pregnancy and newborn screening is increasing, additional centers will be required to treat these children. Hunter's Hope created the Leukodystrophy Care Network in part to create and standardize high-quality clinical practice guidelines to guide the care of affected patients. In this report the clinical guidelines for the care of pediatric patients with leukodystrophies undergoing treatment with HSCT are presented. The initial transplant evaluation, determination of patient eligibility, donor selection, conditioning, supportive care, and post-transplant follow-up are discussed. Throughout these guidelines the need for early detection and treatment and the role of the partnership between families and multidisciplinary providers are emphasized., (Copyright © 2019 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

The leukodystrophies are a family of heritable disorders characterised by white matter degeneration, accompanied by variable clinical symptoms including loss of motor function and cognitive decline. Now thought to include over 50 distinct disorders, there are a vast array of mechanisms underlying the pathology of these monogenic conditions and, accordingly, a range of animal models relating to each disorder. While both murine and zebrafish models continue to aid in the development of potential therapies, many of these models fail to truly recapitulate the human condition - thus leaving substantial weaknesses in our understanding of leukodystrophy pathogenesis. Additionally, the heterogeneity in leukodystrophy presentation - both in patients and in vivo models - often results in a narrow focus on single disorders in isolation across much of the literature. Thus, this review aims to synthesise prominent research regarding the most common leukodystrophies in order to provide an overview of key animal models and their utility in developing novel treatments. We begin by discussing the ongoing revolution across the leukodystrophy field following the rise of next generation sequencing, before focusing more extensively on existing animal models from the mouse and zebrafish fields. Finally, we explore how these preclinical models have shaped the development of therapeutic strategies currently in development. We propose future directions for the field and suggest a more critical view of the dogma which has underpinned leukodystrophy research for decades., (© 2019 Federation of European Biochemical Societies.)

In the last decade, the gene therapy (GT) field experienced a renaissance, thanks to crucial understandings and innovations in vector design, stem cell manipulation, conditioning protocols, and cell/vector delivery. These efforts were successfully coupled with unprecedented clinical results of the trials employing the newly developed technology and with the novel establishment of academic-industrial partnerships. A renewed and strengthened interest is rising in the development of gene-based approaches for inherited neurometabolic disorders with severe neurological involvement. Inherited metabolic disorders are monogenetic diseases caused by enzymatic or structural deficiencies affecting the lysosomal or peroxisomal metabolic activity. The metabolic defect can primarily affect the central nervous system, leading to neuronal death, microglial activation, inflammatory demyelination, and axonal degeneration. This review provides an overview of the GT strategies currently under clinical investigation for neurometabolic lysosomal and peroxisomal storage diseases, such as adrenoleukodystrophy and metachromatic leukodystrophy, as well as novel emerging indications such as mucopolysaccharidoses, gangliosidoses, and neuronal ceroid lipofuscinoses, with a comprehensive elucidation of the main features and mechanisms at the basis of a successful GT approach for these devastating diseases.

Protein engineering is a means to optimize protein therapeutics developed for the treatment of so far incurable diseases including cancers and genetic disorders. Here we report on an engineering approach in which we successfully increased the catalytic rate constant of an enzyme that is presently evaluated in enzyme replacement therapies (ERT) of a lysosomal storage disease (LSD). Although ERT is a treatment option for many LSDs, outcomes are lagging far behind expectations for most of them. This has been ascribed to insufficient enzyme activities accumulating in tissues difficult to target such as brain and peripheral nerves. We show for human arylsulfatase A (hARSA) that the activity of a therapeutic enzyme can be substantially increased by reversing activity-diminishing and by inserting activity-promoting amino acid substitutions that had occurred in the evolution of hominids and non-human mammals, respectively. The potential of this approach, here designated as evolutionary redesign, was highlighted by the observation that murinization of only 1 or 3 amino acid positions increased the hARSA activity 3- and 5-fold, with little impact on stability, respectively. The two kinetically optimized hARSA variants showed no immunogenic potential in ERT of a humanized ARSA knockout mouse model of metachromatic leukodystrophy (MLD) and reduced lysosomal storage of kidney, peripheral and central nervous system up to 3-fold more efficiently than wild-type hARSA. Due to their safety profile and higher therapeutic potential the engineered hARSA variants might represent major advances for future enzyme-based therapies of MLD and stimulate analogous approaches for other enzyme therapeutics., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Allogeneic blood or marrow transplantation (BMT) is currently considered the standard of care for patients with specific inborn errors of metabolism (IEM). However, there is a paucity of studies describing long-term survival and cause-specific late mortality after BMT in these patients with individual types of IEM. We studied 273 patients who had survived ≥2 years after allogeneic BMT for IEM performed between 1974 and 2014. The most prevalent IEM in our cohort were X-linked adrenoleukodystrophy (ALD; 37.3%), Hurler syndrome (35.1%), and metachromatic leukodystrophy (MLD; 10.2%). Conditional on surviving ≥2 years after BMT, the overall survival for the entire cohort was 85.5 ± 2.4% at 10 years and 73.5 ± 3.7% at 20 years. The cohort had a 29-fold increased risk of late death compared with an age- and sex-matched cohort from the general US population (95% CI, 22- to 38-fold). The increased relative mortality was highest in the 2- to 5-year period after BMT (standardized mortality ratio [SMR], 207; 95% confidence interval [CI], 130 to 308) and declined with increasing time from BMT, but remained elevated for ≥21 years after BMT (SMR, 9; 95% CI, 4 to 18). Sequelae from the progression of primary disease were the most common causes of late mortality in this cohort (76%). The use of T cell-depleted grafts in patients with ALD and Hurler syndrome was a risk factor for late mortality. Younger age at BMT and use of busulfan and cyclosporine were protective in patients with Hurler syndrome. Our findings demonstrate relatively favorable overall survival in ≥2-year survivors of allogeneic BMT for IEM, although primary disease progression continues to be responsible for the majority of late deaths., (Copyright © 2018. Published by Elsevier Inc.)

Leukodystrophies (LD) are devastating inherited disorders leading to rapid neurological deterioration and premature death. Hematopoietic stem cell transplantation (HSCT) can halt disease progression for selected LD. Cord blood is a common donor source for transplantation of these patients because it is rapidly available and can be used without full HLA matching. However, precise recommendations allowing care providers to identify patients who benefit from HSCT are lacking. In this study, we define risk factors and describe the early and late outcomes of 169 patients with globoid cell leukodystrophy, X-linked adrenoleukodystrophy, and metachromatic leukodystrophy undergoing cord blood transplantation (CBT) at an European Society for Blood and Marrow Transplantation center or at Duke University Medical Center from 1996 to 2013. Factors associated with higher overall survival (OS) included presymptomatic status (77% vs 49%; P = .006), well-matched (≤1 HLA mismatch) CB units (71% vs 54%; P = .009), and performance status (PS) of >80 vs <60 or 60 to 80 (69% vs 32% and 55%, respectively; P = .003). For patients with PS≤60 (n = 20) or 60 to 80 (n = 24) pre-CBT, only 4 (9%) showed improvement. Of the survivors with PS >80 pre-CBT, 50% remained stable, 20% declined to 60 to 80, and 30% to <60. Overall, an encouraging OS was found for LD patients after CBT, especially for those who are presymptomatic before CBT and received adequately dosed grafts. Early identification and fast referral to a specialized center may lead to earlier treatment and, subsequently, to improved outcomes., Competing Interests: Conflict-of-interest disclosure: J.K. is Director of the Carolinas Cord Blood Bank and Medical Director of the CORD:USE Cord Blood Bank (no personal compensation). The remaining authors declare no competing financial interests.

Objective: To determine whether proton magnetic resonance spectroscopic imaging is useful in predicting clinical course of patients with metachromatic leukodystrophy (MLD), an inherited white matter disorder treatable with haematopoietic cell transplantation (HCT)., Methods: 21 patients with juvenile or adult MLD (12 HCT-treated) were compared with 16 controls in the same age range. Clinical outcome was determined as good, moderate or poor. Metabolites were quantified in white matter, and significance of metabolite concentrations at baseline for outcome prediction was assessed using logistic regression analysis. Evolution of metabolic changes was assessed for patients with follow-up examinations., Results: In this retrospective study, 16 patients with baseline scans were included, 5 with good, 3 with moderate and 8 with poor outcome, and 16 controls. We observed significant group differences for all metabolite concentrations in white matter (p<0.001). Compared with controls, patients had decreased N-acetylaspartate and glutamate, and increased myo-inositol and lactate, most pronounced in patients with poor outcome (post hoc, all p<0.05). Logistic regression showed complete separation of data. Creatine could distinguish poor from moderate and good outcome, the sum of glutamate and glutamine could distinguish good from moderate and poor outcome, and N-acetylaspartate could distinguish all outcome groups. For 13 patients (8 with baseline scans), one or more follow-up examinations were evaluated, revealing stabilisation or even partial normalisation of metabolites in patients with moderate and good outcome, clearly visible in the ratio of choline/N-acetylaspartate., Conclusion: In MLD, quantitative spectroscopic imaging at baseline is predictive for outcome and aids in determining eligibility for HCT., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Recent evidence indicates that hematopoietic stem and progenitor cells (HSPCs) can serve as vehicles for therapeutic molecular delivery to the brain by contributing to the turnover of resident myeloid cell populations. However, such engraftment needs to be fast and efficient to exert its therapeutic potential for diseases affecting the central nervous system. Moreover, the nature of the cells reconstituted after transplantation and whether they could comprise bona fide microglia remain to be assessed. We demonstrate that transplantation of HSPCs in the cerebral lateral ventricles provides rapid engraftment of morphologically, antigenically, and transcriptionally dependable microglia-like cells. We show that the cells comprised within the hematopoietic stem cell compartment and enriched early progenitor fractions generate this microglia-like population when injected in the brain ventricles in the absence of engraftment in the bone marrow. This delivery route has therapeutic relevance because it increases the delivery of therapeutic molecules to the brain, as shown in a humanized animal model of a prototypical lysosomal storage disease affecting the central nervous system.