Clinical outcome assessments of disease burden and progression in late-onset GM2 gangliosidoses.

The late-onset GM2 gangliosidoses, comprising late-onset Tay-Sachs and Sandhoff diseases, are rare, slowly progressive, neurogenetic disorders primarily characterized by neurogenic weakness, ataxia, and dysarthria. The aim of this longitudinal study was to characterize the natural history of late-onset GM2 gangliosidoses using a number of clinical outcome assessments to measure different aspects of disease burden and progression over time, including neurological, functional, and quality of life, to inform the design of future clinical interventional trials. Patients attending the United States National Tay-Sachs & Allied Diseases Family Conference between 2015 and 2019 underwent annual clinical outcome assessments. Currently, there are no clinical outcome assessments validated to assess late-onset GM2 gangliosidoses; therefore, instruments used or designed for diseases with similar features, or to address various aspects of the clinical presentations, were used. Clinical outcome assessments included the Friedreich's Ataxia Rating Scale, the 9-Hole Peg Test, and the Assessment of Intelligibility of Dysarthric Speech. Twenty-three patients participated in at least one meeting visit (late-onset Tay-Sachs, n = 19; late-onset Sandhoff, n = 4). Patients had high disease burden at baseline, and scores for the different clinical outcome assessments were generally lower than would be expected for the general population. Longitudinal analyses showed slow, but statistically significant, neurological progression as evidenced by worsening scores on the 9-Hole Peg Test (2.68%/year, 95% CI: 0.13-5.29; p = 0.04) and the Friedreich's Ataxia Rating Scale neurological examination (1.31 points/year, 95% CI: 0.26-2.35; p = 0.02). Time since diagnosis to study entry correlated with worsening scores on the 9-Hole Peg Test (r = 0.728; p < 0.001), Friedreich's Ataxia Rating Scale neurological examination (r = 0.727; p < 0.001), and Assessment of Intelligibility of Dysarthric Speech intelligibility (r = -0.654; p = 0.001). In summary, patients with late-onset GM2 gangliosidoses had high disease burden and slow disease progression. Several clinical outcome assessments suitable for clinical trials showed only small changes and standardized effect sizes (change/standard deviation of change) over 4 years. These longitudinal natural history study results illustrate the challenge of identifying responsive endpoints for clinical trials in rare, slowly progressive, neurogenerative disorders where arguably the treatment goal is to halt or decrease the rate of decline rather than improve clinical status. Furthermore, powering such a study would require a large sample size and/or a long study duration, neither of which is an attractive option for an ultra-rare disease with no available treatment. These findings support the development of potentially more sensitive late-onset GM2 gangliosidoses-specific rating instruments and/or surrogate endpoints for use in future clinical trials.

Efficacy of Adeno-Associated Virus Serotype 9-Mediated Gene Therapy for AB-Variant GM2 Gangliosidosis.

GM2 gangliosidoses are a group of neurodegenerative lysosomal storage disorders that are characterized by the accumulation of GM2 gangliosides (GM2), leading to rapid neurological decline and death. The hydrolysis of GM2 requires the specific synthesis, processing, and combination of products of three genes-HEXA, HEXB, and GM2A-within the cell's lysosomes. Mutations in these genes result in Tay-Sachs disease, Sandhoff disease, or AB-variant GM2 gangliosidosis (ABGM2), respectively. ABGM2, the rarest of the three types, is characterized by a mutation in the GM2A gene, which encodes the GM2 activator (GM2A) protein. Being a monogenic disease, gene therapy is a plausible and likely effective method of treatment for ABGM2. This study aimed at assessing the effects of administering a one-time intravenous treatment of single-stranded Adeno-associated virus serotype 9 (ssAAV9)-GM2A viral vector at a dose of 1 × 1014 vector genomes (vg) per kilogram per mouse in an ABGM2 mouse model (Gm2a-/-). ssAAV9-GM2A was administered at 1-day (neonatal) or 6-weeks of age (adult-stage). The results demonstrated that, in comparison to Gm2a-/- mice that received a vehicle injection, the treated mice had reduced GM2 accumulation within the central nervous system and had long-term persistence of vector genomes in the brain and liver. This proof-of-concept study is a step forward towards the development of a clinically therapeutic approach for the treatment of patients with ABGM2.

Quantitative longitudinal natural history of 8 gangliosidoses-conceptual framework and baseline data of the German 8-in-1 disease registry. A cross-sectional analysis.

PURPOSE: Gangliosidoses are a group of inherited neurogenetic autosomal recessive lysosomal storage disorders usually presenting with progressive macrocephaly, developmental delay, and regression, leading to significant morbidity and premature death. A quantitative definition of the natural history would support and enable clinical development of specific therapies. METHODS: Single disease registry of 8 gangliosidoses (NCT04624789). Cross-sectional analysis of baseline data in N = 26 patients. Primary end point: disease severity assessed by the 8-in-1 score. Secondary end points: first neurologic sign or symptom observed (1) by parents and (2) by physicians, diagnostic delay, as well as phenotypical characterization. Tertiary end points: neurologic outcomes (development, ataxia, dexterity) and disability. RESULTS: The 8-in-1 score quantitatively captured severity of disease. Parents recognized initial manifestations (startle reactions) earlier than physicians (motor developmental delay and hypotonia). Median diagnostic delay was 3.16 (interquartile range 0.69-6.25) years. In total, 8 patients presented with late-infantile phenotypes. CONCLUSION: Data in this registry raise awareness of these rare and fatal conditions to accelerate diagnosis, inform counseling of afflicted families, define quantitative end points for clinical trials, and can serve as historical controls for future therapeutic studies. We provide further insight into the rare late-infantile phenotype for GM2-gangliosidosis. Longitudinal follow up is planned.

CRISPR/nCas9-Based Genome Editing on GM2 Gangliosidoses Fibroblasts via Non-Viral Vectors.

The gangliosidoses GM2 are a group of pathologies mainly affecting the central nervous system due to the impaired GM2 ganglioside degradation inside the lysosome. Under physiological conditions, GM2 ganglioside is catabolized by the ß-hexosaminidase A in a GM2 activator protein-dependent mechanism. In contrast, uncharged substrates such as globosides and some glycosaminoglycans can be hydrolyzed by the ß-hexosaminidase B. Monogenic mutations on HEXA, HEXB, or GM2A genes arise in the Tay-Sachs (TSD), Sandhoff (SD), and AB variant diseases, respectively. In this work, we validated a CRISPR/Cas9-based gene editing strategy that relies on a Cas9 nickase (nCas9) as a potential approach for treating GM2 gangliosidoses using in vitro models for TSD and SD. The nCas9 contains a mutation in the catalytic RuvC domain but maintains the active HNH domain, which reduces potential off-target effects. Liposomes (LPs)- and novel magnetoliposomes (MLPs)-based vectors were used to deliver the CRISPR/nCas9 system. When LPs were used as a vector, positive outcomes were observed for the ß-hexosaminidase activity, glycosaminoglycans levels, lysosome mass, and oxidative stress. In the case of MLPs, a high cytocompatibility and transfection ratio was observed, with a slight increase in the ß-hexosaminidase activity and significant oxidative stress recovery in both TSD and SD cells. These results show the remarkable potential of CRISPR/nCas9 as a new alternative for treating GM2 gangliosidoses, as well as the superior performance of non-viral vectors in enhancing the potency of this therapeutic approach.

GM2 Gangliosidoses: Clinical Features, Pathophysiological Aspects, and Current Therapies.

GM2 gangliosidoses are a group of pathologies characterized by GM2 ganglioside accumulation into the lysosome due to mutations on the genes encoding for the ß-hexosaminidases subunits or the GM2 activator protein. Three GM2 gangliosidoses have been described: Tay-Sachs disease, Sandhoff disease, and the AB variant. Central nervous system dysfunction is the main characteristic of GM2 gangliosidoses patients that include neurodevelopment alterations, neuroinflammation, and neuronal apoptosis. Currently, there is not approved therapy for GM2 gangliosidoses, but different therapeutic strategies have been studied including hematopoietic stem cell transplantation, enzyme replacement therapy, substrate reduction therapy, pharmacological chaperones, and gene therapy. The blood-brain barrier represents a challenge for the development of therapeutic agents for these disorders. In this sense, alternative routes of administration (e.g., intrathecal or intracerebroventricular) have been evaluated, as well as the design of fusion peptides that allow the protein transport from the brain capillaries to the central nervous system. In this review, we outline the current knowledge about clinical and physiopathological findings of GM2 gangliosidoses, as well as the ongoing proposals to overcome some limitations of the traditional alternatives by using novel strategies such as molecular Trojan horses or advanced tools of genome editing.

Infantile gangliosidoses: Mapping a timeline of clinical changes.

BACKGROUND: Infantile gangliosidoses include GM1 gangliosidosis and GM2 gangliosidosis (Tay-Sachs disease, Sandhoff disease). To date, natural history studies in infantile GM2 (iGM2) have been retrospective and conducted through surveys. Compared to iGM2, there is even less natural history information available on infantile GM1 disease (iGM1). There are no approved treatments for infantile gangliosidoses. Substrate reduction therapy using miglustat has been tried, but is limited by gastrointestinal side effects. Development of effective treatments will require identification of meaningful outcomes in the setting of rapidly progressive and fatal diseases. OBJECTIVES: This study aimed to establish a timeline of clinical changes occurring in infantile gangliosidoses, prospectively, to: 1) characterize the natural history of these diseases; 2) improve planning of clinical care; and 3) identify meaningful future treatment outcome measures. METHODS: Patients were evaluated prospectively through ongoing clinical care. RESULTS: Twenty-three patients were evaluated: 8 infantile GM1, 9 infantile Tay-Sachs disease, 6 infantile Sandhoff disease. Common patterns of clinical change included: hypotonia before 6months of age; severe motor skill impairment within first year of life; seizures; dysphagia and feeding-tube placement before 18months of age. Neurodevelopmental testing scores reached the floor of the testing scale by 20 to 28months of age. Vertebral beaking, kyphosis, and scoliosis were unique to patients with infantile GM1. Chest physiotherapy was associated with increased survival in iGM1 (p=0.0056). Miglustat combined with a low-carbohydrate ketogenic diet (the Syner-G regimen) in patients who received a feeding-tube was associated with increased survival in infantile GM1 (p=0.025). CONCLUSIONS: This is the first prospective study of the natural history of infantile gangliosidoses and the very first natural history of infantile GM1. The homogeneity of the infantile gangliosidoses phenotype as demonstrated by the clinical events timeline in this study provides promising secondary outcome measure candidates. This study indicates that overall survival is a meaningful primary outcome measure for future clinical trials due to reliable timing and early occurrence of this event. Combination therapy approaches, instead of monotherapy approaches, will likely be the best way to optimize clinical outcomes. Combination therapy approaches include palliative therapies (e.g., chest physiotherapy) along with treatments that address the underlying disease pathology (e.g. miglustat or future gene therapies).

Direct Intracranial Injection of AAVrh8 Encoding Monkey ß-N-Acetylhexosaminidase Causes Neurotoxicity in the Primate Brain.

GM2 gangliosidoses, including Tay-Sachs disease and Sandhoff disease, are lysosomal storage disorders caused by deficiencies in ß-N-acetylhexosaminidase (Hex). Patients are afflicted primarily with progressive central nervous system (CNS) dysfunction. Studies in mice, cats, and sheep have indicated safety and widespread distribution of Hex in the CNS after intracranial vector infusion of AAVrh8 vectors encoding species-specific Hex α- or ß-subunits at a 1:1 ratio. Here, a safety study was conducted in cynomolgus macaques (cm), modeling previous animal studies, with bilateral infusion in the thalamus as well as in left lateral ventricle of AAVrh8 vectors encoding cm Hex α- and ß-subunits. Three doses (3.2 × 1012 vg [n = 3]; 3.2 × 1011 vg [n = 2]; or 1.1 × 1011 vg [n = 2]) were tested, with controls infused with vehicle (n = 1) or transgene empty AAVrh8 vector at the highest dose (n = 2). Most monkeys receiving AAVrh8-cmHexα/ß developed dyskinesias, ataxia, and loss of dexterity, with higher dose animals eventually becoming apathetic. Time to onset of symptoms was dose dependent, with the highest-dose cohort producing symptoms within a month of infusion. One monkey in the lowest-dose cohort was behaviorally asymptomatic but had magnetic resonance imaging abnormalities in the thalami. Histopathology was similar in all monkeys injected with AAVrh8-cmHexα/ß, showing severe white and gray matter necrosis along the injection track, reactive vasculature, and the presence of neurons with granular eosinophilic material. Lesions were minimal to absent in both control cohorts. Despite cellular loss, a dramatic increase in Hex activity was measured in the thalamus, and none of the animals presented with antibody titers against Hex. The high overexpression of Hex protein is likely to blame for this negative outcome, and this study demonstrates the variations in safety profiles of AAVrh8-Hexα/ß intracranial injection among different species, despite encoding for self-proteins.

The GM1 and GM2 Gangliosidoses: Natural History and Progress toward Therapy.

The gangliosidoses are lysosomal storage disorders caused by accumulation of GM1 or GM2 gangliosides. GM1 gangliosidosis has both central nervous system and systemic findings; while, GM2 gangliosidosis is restricted primarily to the central nervous system. Both disorders have autosomal recessive modes of inheritance and a continuum of clinical presentations from a severe infantile form to a milder, chronic adult form. Both are devastating diseases without cure or specific treatment however, with the use of supportive aggressive medical management, the lifespan and quality of life has been extended for both diseases. Naturally occurring and engineered animal models that mimic the human diseases have enhanced our understanding of the pathogenesis of disease progression. Some models have shown significant improvement in symptoms and lifespan with enzyme replacement, substrate reduction, and anti-inflammatory treatments alone or in combination. More recently gene therapy has shown impressive results in large and small animal models. Treatment with FDA-approved glucose analogs to reduce the amount of ganglioside substrate is used as off-label treatments for some patients. Therapies also under clinical development include small molecule chaperones and gene therapy.

[Molecular pathogenesis and therapeutic approach of GM2 gangliosidosis].

Tay-Sachs and Sandhoff diseases (GM2 gangliosidoses) are autosomal recessive lysosomal storage diseases caused by gene mutations in HEXA and HEXB, each encoding human lysosomal ß-hexosaminidase α-subunits and ß-subunits, respectively. In Tay-Sachs disease, excessive accumulation of GM2 ganglioside (GM2), mainly in the central nervous system, is caused by a deficiency of the HexA isozyme (αß heterodimer), resulting in progressive neurologic disorders. In Sandhoff disease, combined deficiencies of HexA and HexB (ßß homodimer) cause not only the accumulation of GM2 but also of oligosaccharides carrying terminal N-acetylhexosamine residues (GlcNAc-oligosaccharides), resulting in systemic manifestations including hepatosplenomegaly as well as neurologic symptoms. Hence there is little clinically effective treatment for these GM2 gangliosidoses. Recent studies on the molecular pathogenesis in Sandhoff disease patients and disease model mice have shown the involvement of microglial activation and chemokine induction in neuroinflammation and neurodegeneration in this disease. Experimental and therapeutic approaches, including recombinant enzyme replacement, have been performed using Sandhoff disease model mice, suggesting the future application of novel techniques to treat GM2 gangliosidoses (Hex deficiencies), including Sandhoff disease as well as Tay-Sachs disease. In this study, we isolated astrocytes and microglia from the neonatal brain of Sandhoff disease model mice and demonstrated abnormalities of glial cells. Moreover, we demonstrated the therapeutic effect of an intracerebroventricular administration of novel recombinant human HexA carrying a high content of M6P residue in Sandhoff disease model mice.

Gene transfer corrects acute GM2 gangliosidosis--potential therapeutic contribution of perivascular enzyme flow.

The GM2 gangliosidoses are fatal lysosomal storage diseases principally affecting the brain. Absence of ß-hexosaminidase A and B activities in the Sandhoff mouse causes neurological dysfunction and recapitulates the acute Tay-Sachs (TSD) and Sandhoff diseases (SD) in infants. Intracranial coinjection of recombinant adeno-associated viral vectors (rAAV), serotype 2/1, expressing human ß-hexosaminidase α (HEXA) and ß (HEXB) subunits into 1-month-old Sandhoff mice gave unprecedented survival to 2 years and prevented disease throughout the brain and spinal cord. Classical manifestations of disease, including spasticity-as opposed to tremor-ataxia-were resolved by localized gene transfer to the striatum or cerebellum, respectively. Abundant biosynthesis of ß-hexosaminidase isozymes and their global distribution via axonal, perivascular, and cerebrospinal fluid (CSF) spaces, as well as diffusion, account for the sustained phenotypic rescue-long-term protein expression by transduced brain parenchyma, choroid plexus epithelium, and dorsal root ganglia neurons supplies the corrective enzyme. Prolonged survival permitted expression of cryptic disease in organs not accessed by intracranial vector delivery. We contend that infusion of rAAV into CSF space and intraparenchymal administration by convection-enhanced delivery at a few strategic sites will optimally treat neurodegeneration in many diseases affecting the nervous system.

Natural history of infantile G(M2) gangliosidosis.

OBJECTIVE: G(M2) gangliosidoses are caused by an inherited deficiency of lysosomal ß-hexosaminidase and result in ganglioside accumulation in the brain. Onset during infancy leads to rapid neurodegeneration and death before 4 years of age. We set out to quantify the rate of functional decline in infantile G(M2) gangliosidosis on the basis of patient surveys and a comprehensive review of existing literature. METHODS: Patients with infantile G(M2) gangliosidosis (N = 237) were surveyed via questionnaire by the National Tay Sachs & Allied Diseases Association (NTSAD). These data were supplemented by survival data from the NTSAD database and a literature survey. Detailed retrospective surveys from 97 patients were available. Five patients who had received hematopoietic stem cell transplantation were evaluated separately. The mortality rate of the remaining 92 patients was comparable to that of the 103 patients from the NTSAD database and 121 patients reported in the literature. RESULTS: Common symptoms at onset were developmental arrest (83%), startling (65%), and hypotonia (60%). All 55 patients who had learned to sit without support lost that ability within 1 year. Individual functional measures correlated with each other but not with survival. Gastric tube placement was associated with prolonged survival. Tay Sachs and Sandhoff variants did not differ. Hematopoietic stem cell transplantation was not associated with prolonged survival. CONCLUSIONS: We studied the timing of regression in 97 cases of infantile G(M2) gangliosidosis and conclude that clinical disease progression does not correlate with survival, likely because of the impact of improved supportive care over time. However, functional measures are quantifiable and can inform power calculations and study design of future interventions.

Juvenile-onset G(M2)-gangliosidosis in an African-American child with nystagmus.

G(M2)-gangliosidosis is a neurodegenerative lysosomal disease with several clinical variants. We describe a 2-year-old black child with juvenile-onset disease, who presented with abnormal eye movements and cherry-red spots of the maculae. Mutation analysis of the HEXA gene revealed the patient to be a compound heterozygote (M1V/Y37N). The M1V mutation was previously described in an African-American child with acute infantile G(M2)-gangliosidosis. The Y37N mutation is novel. This combination of mutations is consistent with juvenile-onset disease, and provides further evidence for the association of the M1V mutation with individuals of black ancestry. The presence of oculomotor abnormalities is an unusual finding in this form of G(M2)-gangliosidosis, and adds to the phenotypic spectrum.

Therapeutic evaluation of GM2 gangliosidoses by ELISA using anti-GM2 ganglioside antibodies.

BACKGROUND: GM2 gangliosidoses, including Tay-Sachs disease, Sandhoff disease and the AB variant, comprise deficiencies of beta-hexosaminidase isozymes and GM2 ganglioside activator protein associated with accumulation of GM2 ganglioside (GM2) in lysosomes and neurosomatic clinical manifestations. A simple assay system for intracellular quantification of GM2 is required to evaluate the therapeutic effects on GM2-gangliosidoses. METHODS: We newly established a cell-ELISA system involving anti-GM2 monoclonal antibodies for measuring GM2 storage in fibroblasts from Tay-Sachs and Sandhoff disease patients. RESULTS: We succeeded in detecting the corrective effect of enzyme replacement on elimination of GM2 in the cells with this ELISA system. CONCLUSIONS: This simple and sensitive system should be useful as additional diagnosis tool as well as therapeutic evaluation of GM2 gangliosidoses.

[Recent advances in the diagnosis and treatment of lysosomal storage diseases].

Lysosomal storage diseases are a group of genetic disorders that result from the defect in lysosomal function. Signs and symptoms are variable, it is difficult to diagnose this group of disease merely by the clinical manifestation. The diagnosis usually is made by measuring the activity of the corresponding enzyme. Gene mutational analysis is useful for the diagnosis of some of the lysosome storage diseases. The treatment has focused on the replacement of the defective enzyme responsible for the disease and the hematopoietic stem cell transplantation. Both of them have achieved exciting outcomes in some of the diseases.