Combination HSCT and intravenous AAV-mediated gene therapy in a canine model proves pivotal for translation of Krabbe disease therapy.

Hematopoietic stem cell transplantation (HSCT) is the only approved treatment for presymptomatic infantile globoid cell leukodystrophy (GLD [Krabbe disease]). However, correction of disease is not complete, and outcomes remain poor. Herein we evaluated HSCT, intravenous (IV) adeno-associated virus rh10 vector (AAVrh10) gene therapy, and combination HSCT + IV AAVrh10 in the canine model of GLD. While HSCT alone resulted in no increase in survival as compared with untreated GLD dogs (∼16 weeks of age), combination HSCT + IV AAVrh10 at a dose of 4E13 genome copies (gc)/kg resulted in delayed disease progression and increased survival beyond 1 year of age. A 5-fold increase in AAVrh10 dose to 2E14 gc/kg, in combination with HSCT, normalized neurological dysfunction up to 2 years of age. IV AAVrh10 alone resulted in an average survival to 41.2 weeks of age. In the peripheral nervous system, IV AAVrh10 alone or in addition to HSCT normalized nerve conduction velocity, improved ultrastructure, and normalized GALC enzyme activity and psychosine concentration. In the central nervous system, only combination therapy at the highest dose was able to restore galactosylceramidase activity and psychosine concentrations to within the normal range. These data have now guided clinical translation of systemic AAV gene therapy as an addition to HSCT (NCT04693598, NCT05739643).

Evaluation of safety and early efficacy of AAV gene therapy in mouse models of vanishing white matter disease.

Leukoencephalopathy with vanishing white matter (VWM) is a progressive incurable white matter disease that most commonly occurs in childhood and presents with ataxia, spasticity, neurological degeneration, seizures, and premature death. A distinctive feature is episodes of rapid neurological deterioration provoked by stressors such as infection, seizures, or trauma. VWM is caused by autosomal recessive mutations in one of five genes that encode the eukaryotic initiation factor 2B complex, which is necessary for protein translation and regulation of the integrated stress response. The majority of mutations are in EIF2B5. Astrocytic dysfunction is central to pathophysiology, thereby constituting a potential therapeutic target. Herein we characterize two VWM murine models and investigate astrocyte-targeted adeno-associated virus serotype 9 (AAV9)-mediated EIF2B5 gene supplementation therapy as a therapeutic option for VWM. Our results demonstrate significant rescue in body weight, motor function, gait normalization, life extension, and finally, evidence that gene supplementation attenuates demyelination. Last, the greatest rescue results from a vector using a modified glial fibrillary acidic protein (GFAP) promoter-AAV9-gfaABC(1)D-EIF2B5-thereby supporting that astrocytic targeting is critical for disease correction. In conclusion, we demonstrate safety and early efficacy through treatment with a translatable astrocyte-targeted gene supplementation therapy for a disease that has no cure.

Life-Limiting Peripheral Organ Dysfunction in Feline Sandhoff Disease Emerges after Effective CNS Gene Therapy.

OBJECTIVE: GM2 gangliosidosis is usually fatal by 5 years of age in its 2 major subtypes, Tay-Sachs and Sandhoff disease. First reported in 1881, GM2 gangliosidosis has no effective treatment today, and children succumb to the disease after a protracted neurodegenerative course and semi-vegetative state. This study seeks to further develop adeno-associated virus (AAV) gene therapy for human translation. METHODS: Cats with Sandhoff disease were treated by intracranial injection of vectors expressing feline ß-N-acetylhexosaminidase, the enzyme deficient in GM2 gangliosidosis. RESULTS: Hexosaminidase activity throughout the brain and spinal cord was above normal after treatment, with highest activities at the injection sites (thalamus and deep cerebellar nuclei). Ganglioside storage was reduced throughout the brain and spinal cord, with near complete clearance in many regions. While untreated cats with Sandhoff disease lived for 4.4 ± 0.6 months, AAV-treated cats lived to 19.1 ± 8.6 months, and 3 of 9 cats lived >21 months. Correction of the central nervous system was so effective that significant increases in lifespan led to the emergence of otherwise subclinical peripheral disease, including megacolon, enlarged stomach and urinary bladder, soft tissue spinal cord compression, and patellar luxation. Throughout the gastrointestinal tract, neurons of the myenteric and submucosal plexuses developed profound pathology, demonstrating that the enteric nervous system was inadequately treated. INTERPRETATION: The vector formulation in the current study effectively treats neuropathology in feline Sandhoff disease, but whole-body targeting will be an important consideration in next-generation approaches. ANN NEUROL 2023;94:969-986.

Preclinical studies in Krabbe disease: A model for the investigation of novel combination therapies for lysosomal storage diseases.

Krabbe disease (KD) is a lysosomal storage disease (LSD) caused by mutations in the galc gene. There are over 50 monogenetic LSDs, which largely impede the normal development of children and often lead to premature death. At present, there are no cures for LSDs and the available treatments are generally insufficient, short acting, and not without co-morbidities or long-term side effects. The last 30 years have seen significant advances in our understanding of LSD pathology as well as treatment options. Two gene therapy-based clinical trials, NCT04693598 and NCT04771416, for KD were recently started based on those advances. This review will discuss how our knowledge of KD got to where it is today, focusing on preclinical investigations, and how what was discovered may prove beneficial for the treatment of other LSDs.

Gene-based therapeutics for rare genetic neurodevelopmental psychiatric disorders.

We are in an emerging era of gene-based therapeutics with significant promise for rare genetic disorders. The potential is particularly significant for genetic central nervous system disorders that have begun to achieve Food and Drug Administration approval for select patient populations. This review summarizes the discussions and presentations of the National Institute of Mental Health-sponsored workshop "Gene-Based Therapeutics for Rare Genetic Neurodevelopmental Psychiatric Disorders," which was held in January 2021. Here, we distill the points raised regarding various precision medicine approaches related to neurodevelopmental and psychiatric disorders that may be amenable to gene-based therapies.

Therapeutic benefit after intracranial gene therapy delivered during the symptomatic stage in a feline model of Sandhoff disease.

Sandhoff disease (SD) is an autosomal recessive lysosomal storage disease caused by defects in the ß-subunit of ß-N-acetylhexosaminidase (Hex), the enzyme that catabolizes GM2 ganglioside. Hex deficiency causes neuronal storage of GM2 and related glycoconjugates, resulting in progressive neurodegeneration and death, typically in infancy. No effective treatment exists for human patients. Adeno-associated virus (AAV) gene therapy led to improved clinical outcome and survival of SD cats treated before the onset of disease symptoms. Most human patients are diagnosed after clinical disease onset, so it is imperative to test AAV-gene therapy in symptomatic SD cats to provide a realistic indication of therapeutic benefits that can be expected in humans. In this study, AAVrh8 vectors injected into the thalamus and deep cerebellar nuclei of symptomatic SD cats resulted in widespread central nervous system enzyme distribution, although a substantial burden of storage material remained. Cats treated in the early symptomatic phase showed delayed disease progression and a significant survival increase versus untreated cats. Treatment was less effective when administered later in the disease course, although therapeutic benefit was still possible. Results are encouraging for the treatment of human patients and provide support for the development AAV-gene therapy for human SD.

Cerebrospinal fluid and serum glycosphingolipid biomarkers in canine globoid cell leukodystrophy (Krabbe Disease).

Globoid cell leukodystrophy (GLD, Krabbe disease, Krabbe's disease) is caused by genetic mutations in the gene encoding, galactosylceramidase (GALC). Deficiency of this enzyme results in central and peripheral nervous system pathology, and is characterized by loss of myelin and an infiltration of globoid cells. The canine model of GLD provides a translational model which faithfully recapitulates much of the human disease pathology. Targeted lipidomic analysis was conducted in serum and cerebrospinal fluid (CSF) over the lifetime of GLD affected and normal canines, and in brain tissue at humane endpoint to better understand disease progression and identify potential biomarkers of disease. Psychosine, a substrate of GALC and primary contributor to the pathology in GLD, was observed to be significantly elevated in the serum and CSF by 2 or 4 weeks of age, respectively, and steadily increased over the lifetime of affected animals. Importantly, psychosine concentration strongly correlated with disease severity. Galactosylceramide, glucosylceramide, and lactosylceramide were also found to be elevated in the CSF of affected animals and increased with age. Psychosine and galactosylceramide were found to be significantly increased in brain tissue at humane endpoint. This study identified several biomarkers which may be useful in the development of therapeutics for GLD.

Novel Biomarkers of Human GM1 Gangliosidosis Reflect the Clinical Efficacy of Gene Therapy in a Feline Model.

GM1 gangliosidosis is a fatal neurodegenerative disease that affects individuals of all ages. Favorable outcomes using adeno-associated viral (AAV) gene therapy in GM1 mice and cats have prompted consideration of human clinical trials, yet there remains a paucity of objective biomarkers to track disease status. We developed a panel of biomarkers using blood, urine, cerebrospinal fluid (CSF), electrodiagnostics, 7 T MRI, and magnetic resonance spectroscopy in GM1 cats-either untreated or AAV treated for more than 5 years-and compared them to markers in human GM1 patients where possible. Significant alterations were noted in CSF and blood of GM1 humans and cats, with partial or full normalization after gene therapy in cats. Gene therapy improved the rhythmic slowing of electroencephalograms (EEGs) in GM1 cats, a phenomenon present also in GM1 patients, but nonetheless the epileptiform activity persisted. After gene therapy, MR-based analyses revealed remarkable preservation of brain architecture and correction of brain metabolites associated with microgliosis, neuroaxonal loss, and demyelination. Therapeutic benefit of AAV gene therapy in GM1 cats, many of which maintain near-normal function >5 years post-treatment, supports the strong consideration of human clinical trials, for which the biomarkers described herein will be essential for outcome assessment.

Canine and Feline Models of Human Genetic Diseases and Their Contributions to Advancing Clinical Therapies
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For many lethal or debilitating genetic disorders in patients there are no satisfactory therapies. Several barriers exist that hinder the developments of effective therapies including the limited availability of clinically relevant animal models that faithfully recapitulate human genetic disease. In 1974, the Referral Center for Animal Models of Human Genetic Disease (RCAM) was established by Dr. Donald F. Patterson and continued by Dr. Mark E. Haskins at the University of Pennsylvania with the mission to discover, understand, treat, and maintain breeding colonies of naturally occurring hereditary disorders in dogs and cats that are orthologous to those found in human patients. Although non-human primates, sheep, and pig models are also available within the medical community, naturally occurring diseases are rarely identified in non-human primates, and the vast behavioral, clinicopathological, physiological, and anatomical knowledge available regarding dogs and cats far surpasses what is available in ovine and porcine species. The canine and feline models that are maintained at RCAM are presented here with a focus on preclinical therapy data. Clinical studies that have been generated from preclinical work in these models are also presented.

Clinical, electrophysiological, and biochemical markers of peripheral and central nervous system disease in canine globoid cell leukodystrophy (Krabbe's disease).

Globoid cell leukodystrophy (GLD), or Krabbe's disease, is a debilitating and always fatal pediatric neurodegenerative disease caused by a mutation in the gene encoding the hydrolytic enzyme galactosylceramidase (GALC). In the absence of GALC, progressive loss of myelin and accumulation of a neurotoxic substrate lead to incapacitating loss of motor and cognitive function and death, typically by 2 years of age. Currently, there is no cure. Recent convincing evidence of the therapeutic potential of combining gene and cell therapies in the murine model of GLD has accelerated the requirement for validated markers of disease to evaluate therapeutic efficacy. Here we demonstrate clinically relevant and quantifiable measures of central (CNS) and peripheral (PNS) nervous system disease progression in the naturally occurring canine model of GLD. As measured by brainstem auditory-evoked response testing, GLD dogs demonstrated a significant increase in I-V interpeak latency and hearing threshold at all time points. Motor nerve conduction velocities (NCVs) in GLD dogs were significantly lower than normal by 12-16 weeks of age, and sensory NCV was significantly lower than normal by 8-12 weeks of age, serving as a sensitive indicator of peripheral nerve dysfunction. Post-mortem histological evaluations confirmed neuroimaging and electrodiagnostic assessments and detailed loss of myelin and accumulation of storage product in the CNS and the PNS. Additionally, cerebrospinal fluid psychosine concentrations were significantly elevated in GLD dogs, demonstrating potential as a biochemical marker of disease. These data demonstrate that CNS and PNS disease progression can be quantified over time in the canine model of GLD with tools identical to those used to assess human patients. © 2016 Wiley Periodicals, Inc.

Cerebrospinal Fluid Calbindin D Concentration as a Biomarker of Cerebellar Disease Progression in Niemann-Pick Type C1 Disease.

Niemann-Pick type C (NPC) 1 disease is a rare, inherited, neurodegenerative disease. Clear evidence of the therapeutic efficacy of 2-hydroxypropyl-ß-cyclodextrin (HPßCD) in animal models resulted in the initiation of a phase I/IIa clinical trial in 2013 and a phase IIb/III trial in 2015. With clinical trials ongoing, validation of a biomarker to track disease progression and serve as a supporting outcome measure of therapeutic efficacy has become compulsory. In this study, we evaluated calcium-binding protein calbindin D-28K (calbindin) concentrations in the cerebrospinal fluid (CSF) as a biomarker of NPC1 disease. In the naturally occurring feline model, CSF calbindin was significantly elevated at 3 weeks of age, prior to the onset of cerebellar dysfunction, and steadily increased to >10-fold over normal at end-stage disease. Biweekly intrathecal administration of HPßCD initiated prior to the onset of neurologic dysfunction completely normalized CSF calbindin in NPC1 cats at all time points analyzed when followed up to 78 weeks of age. Initiation of HPßCD after the onset of clinical signs (16 weeks of age) resulted in a delayed reduction of calbindin levels in the CSF. Evaluation of CSF from patients with NPC1 revealed that calbindin concentrations were significantly elevated compared with CSF samples collected from unaffected patients. Off-label treatment of patients with NPC1 with miglustat, an inhibitor of glycosphingolipid biosynthesis, significantly decreased CSF calbindin compared with pretreatment concentrations. These data suggest that the CSF calbindin concentration is a sensitive biomarker of NPC1 disease that could be instrumental as an outcome measure of therapeutic efficacy in ongoing clinical trials.

Mucopolysaccharidosis-like phenotype in feline Sandhoff disease and partial correction after AAV gene therapy.

Sandhoff disease (SD) is a fatal neurodegenerative disease caused by a mutation in the enzyme ß-N-acetylhexosaminidase. Children with infantile onset SD develop seizures, loss of motor tone and swallowing problems, eventually reaching a vegetative state with death typically by 4years of age. Other symptoms include vertebral gibbus and cardiac abnormalities strikingly similar to those of the mucopolysaccharidoses. Isolated fibroblasts from SD patients have impaired catabolism of glycosaminoglycans (GAGs). To evaluate mucopolysaccharidosis-like features of the feline SD model, we utilized radiography, MRI, echocardiography, histopathology and GAG quantification of both central nervous system and peripheral tissues/fluids. The feline SD model exhibits cardiac valvular and structural abnormalities, skeletal changes and spinal cord compression that are consistent with accumulation of GAGs, but are much less prominent than the severe neurologic disease that defines the humane endpoint (4.5±0.5months). Sixteen weeks after intracranial AAV gene therapy, GAG storage was cleared in the SD cat cerebral cortex and liver, but not in the heart, lung, skeletal muscle, kidney, spleen, pancreas, small intestine, skin, or urine. GAG storage worsens with time and therefore may become a significant source of pathology in humans whose lives are substantially lengthened by gene therapy or other novel treatments for the primary, neurologic disease.

Therapeutic response in feline sandhoff disease despite immunity to intracranial gene therapy.

Salutary responses to adeno-associated viral (AAV) gene therapy have been reported in the mouse model of Sandhoff disease (SD), a neurodegenerative lysosomal storage disease caused by deficiency of ß-N-acetylhexosaminidase (Hex). While untreated mice reach the humane endpoint by 4.1 months of age, mice treated by a single intracranial injection of vectors expressing human hexosaminidase may live a normal life span of 2 years. When treated with the same therapeutic vectors used in mice, two cats with SD lived to 7.0 and 8.2 months of age, compared with an untreated life span of 4.5 ± 0.5 months (n = 11). Because a pronounced humoral immune response to both the AAV1 vectors and human hexosaminidase was documented, feline cDNAs for the hexosaminidase α- and ß-subunits were cloned into AAVrh8 vectors. Cats treated with vectors expressing feline hexosaminidase produced enzymatic activity >75-fold normal at the brain injection site with little evidence of an immune infiltrate. Affected cats treated with feline-specific vectors by bilateral injection of the thalamus lived to 10.4 ± 3.7 months of age (n = 3), or 2.3 times as long as untreated cats. These studies support the therapeutic potential of AAV vectors for SD and underscore the importance of species-specific cDNAs for translational research.

AAV-based in vivo gene therapy for neurological disorders.

Recent advancements in gene supplementation therapy are expanding the options for the treatment of neurological disorders. Among the available delivery vehicles, adeno-associated virus (AAV) is often the favoured vector. However, the results have been variable, with some trials dramatically altering the course of disease whereas others have shown negligible efficacy or even unforeseen toxicity. Unlike traditional drug development with small molecules, therapeutic profiles of AAV gene therapies are dependent on both the AAV capsid and the therapeutic transgene. In this rapidly evolving field, numerous clinical trials of gene supplementation for neurological disorders are ongoing. Knowledge is growing about factors that impact the translation of preclinical studies to humans, including the administration route, timing of treatment, immune responses and limitations of available model systems. The field is also developing potential solutions to mitigate adverse effects, including AAV capsid engineering and designs to regulate transgene expression. At the same time, preclinical research is addressing new frontiers of gene supplementation for neurological disorders, with a focus on mitochondrial and neurodevelopmental disorders. In this Review, we describe the current state of AAV-mediated neurological gene supplementation therapy, including critical factors for optimizing the safety and efficacy of treatments, as well as unmet needs in this field.

CD8+ T cell depletion prevents neuropathology in a mouse model of globoid cell leukodystrophy.

Globoid cell leukodystrophy (GLD) or Krabbe's disease is a fatal genetic demyelinating disease of the central nervous system caused by loss-of-function mutations in the galactosylceramidase (galc) gene. While the metabolic basis for disease is known, the understanding of how this results in neuropathology is not well understood. Herein, we report that the rapid and protracted elevation of CD8+ cytotoxic T lymphocytes occurs coincident with clinical disease in a mouse model of GLD. Administration of a function-blocking antibody against CD8α effectively prevented disease onset, reduced morbidity and mortality, and prevented CNS demyelination in mice. These data indicate that subsequent to the genetic cause of disease, neuropathology is driven by pathogenic CD8+ T cells, thus offering novel therapeutic potential for treatment of GLD.

Efficacy and Safety of a Krabbe Disease Gene Therapy.

Krabbe disease is a lysosomal storage disease caused by mutations in the gene that encodes galactosylceramidase, in which galactosylsphingosine (psychosine) accumulation drives demyelination in the central and peripheral nervous systems, ultimately progressing to death in early childhood. Gene therapy, alone or in combination with transplant, has been developed for almost two decades in mouse models, with increasing therapeutic benefit paralleling the improvement of next-generation adeno-associated virus (AAV) vectors. This effort has recently shown remarkable efficacy in the canine model of the disease by two different groups that used either systemic or cerebrospinal fluid (CSF) administration of AAVrh10 or AAV9. Building on our experience developing CSF-delivered, AAV-based drug products for a variety of neurodegenerative disorders, we conducted efficacy, pharmacology, and safety studies of AAVhu68 delivered to the CSF in two relevant natural Krabbe animal models, and in nonhuman primates. In newborn Twitcher mice, the highest dose (1 × 1011 genome copies [GC]) of AAVhu68.hGALC injected into the lateral ventricle led to a median survival of 130 days compared to 40.5 days in vehicle-treated mice. When this dose was administered intravenously, the median survival was 49 days. A single intracisterna magna injection of AAVhu68.cGALC at 3 × 1013 GC into presymptomatic Krabbe dogs increased survival for up to 85 weeks compared to 12 weeks in controls. It prevented psychosine accumulation in the CSF, preserved peripheral nerve myelination, ambulation, and decreased brain neuroinflammation and demyelination, although some regions remained abnormal. In a Good Laboratory Practice-compliant toxicology study, we administered the clinical candidate into the cisterna magna of 18 juvenile rhesus macaques at 3 doses that displayed efficacy in mice. We observed no dose-limiting toxicity and sporadic minimal degeneration of dorsal root ganglia (DRG) neurons. Our studies demonstrate the efficacy, scalability, and safety of a single cisterna magna AAVhu68 administration to treat Krabbe disease. ClinicalTrials.Gov ID: NCT04771416.

Recent Advancements in the Diagnosis and Treatment of Leukodystrophies.

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.

Krabbe disease: New hope for an old disease.

Krabbe disease (globoid cell leukodystrophy) is a lysosomal storage disease (LSD) characterized by progressive and profound demyelination. Infantile, juvenile and adult-onset forms of Krabbe disease have been described, with infantile being the most common. Children with an infantile-onset generally appear normal at birth but begin to miss developmental milestones by six months of age and die by two to four years of age. Krabbe disease is caused by a deficiency of the acid hydrolase galactosylceramidase (GALC) which is responsible for the degradation of galactosylceramides and sphingolipids, which are abundant in myelin membranes. The absence of GALC leads to the toxic accumulation of galactosylsphingosine (psychosine), a lysoderivative of galactosylceramides, in oligodendrocytes and Schwann cells resulting in demyelination of the central and peripheral nervous systems, respectively. Treatment strategies such as enzyme replacement, substrate reduction, enzyme chaperones, and gene therapy have shown promise in LSDs. Unfortunately, Krabbe disease has been relatively refractory to most single-therapy interventions. Although hematopoietic stem cell transplantation can alter the course of Krabbe disease and is the current standard-of-care, it simply slows the progression, even when initiated in pre-symptomatic children. However, the recent success of combinatorial therapeutic approaches in small animal models of Krabbe disease and the identification of new pathogenic mechanisms provide hope for the development of effective treatments for this devastating disease. This review provides a brief history of Krabbe disease and the evolution of single and combination therapeutic approaches and discusses new pathogenic mechanisms and how they might impact the development of more effective treatment strategies.

Canine Models of Inherited Musculoskeletal and Neurodegenerative Diseases.

Mouse models of human disease remain the bread and butter of modern biology and therapeutic discovery. Nonetheless, more often than not mouse models do not reproduce the pathophysiology of the human conditions they are designed to mimic. Naturally occurring large animal models have predominantly been found in companion animals or livestock because of their emotional or economic value to modern society and, unlike mice, often recapitulate the human disease state. In particular, numerous models have been discovered in dogs and have a fundamental role in bridging proof of concept studies in mice to human clinical trials. The present article is a review that highlights current canine models of human diseases, including Alzheimer's disease, degenerative myelopathy, neuronal ceroid lipofuscinosis, globoid cell leukodystrophy, Duchenne muscular dystrophy, mucopolysaccharidosis, and fucosidosis. The goal of the review is to discuss canine and human neurodegenerative pathophysiologic similarities, introduce the animal models, and shed light on the ability of canine models to facilitate current and future treatment trials.

Krabbe disease successfully treated via monotherapy of intrathecal gene therapy.

Globoid cell leukodystrophy (GLD; Krabbe disease) is a progressive, incurable neurodegenerative disease caused by deficient activity of the hydrolytic enzyme galactosylceramidase (GALC). The ensuing cytotoxic accumulation of psychosine results in diffuse central and peripheral nervous system (CNS, PNS) demyelination. Presymptomatic hematopoietic stem cell transplantation (HSCT) is the only treatment for infantile-onset GLD; however, clinical outcomes of HSCT recipients often remain poor, and procedure-related morbidity is high. There are no effective therapies for symptomatic patients. Herein, we demonstrate in the naturally occurring canine model of GLD that presymptomatic monotherapy with intrathecal AAV9 encoding canine GALC administered into the cisterna magna increased GALC enzyme activity, normalized psychosine concentration, improved myelination, and attenuated inflammation in both the CNS and PNS. Moreover, AAV-mediated therapy successfully prevented clinical neurological dysfunction, allowing treated dogs to live beyond 2.5 years of age, more than 7 times longer than untreated dogs. Furthermore, we found that a 5-fold lower dose resulted in an attenuated form of disease, indicating that sufficient dosing is critical. Finally, postsymptomatic therapy with high-dose AAV9 also significantly extended lifespan, signifying a treatment option for patients for whom HSCT is not applicable. If translatable to patients, these findings would improve the outcomes of patients treated either pre- or postsymptomatically.