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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 β-hexosaminidase A in a GM2 activator protein-dependent mechanism. In contrast, uncharged substrates such as globosides and some glycosaminoglycans can be hydrolyzed by the β-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 β-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 β-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.

Sandhoff disease is one of the GM2‐gangliosidoses which is caused by a mutation in the HEXB preventing the breakdown of GM2‐ganglioside. We report a novel HEXB variant in a family with a history of a dead girl with Sandhoff disease which was not found in controls.

Thalamic infusion of adeno-associated viral (AAV) vectors has been shown to have therapeutic effects in neuronopathic lysosomal storage diseases. Preclinical studies in sheep model of Tay-Sachs disease demonstrated that bilateral thalamic injections of AAV gene therapy are required for maximal benefit. Translation of thalamic injection to patients carries risks in that (1) it has never been done in humans, and (2) dosing scale-up based on brain weight from animals to humans requires injection of larger volumes. To increase the safety margin of this infusion, a flexible cannula was selected to enable simultaneous bilateral thalamic infusion in infants while monitoring by imaging and/or to enable awake infusions for injection of large volumes at low infusion rates. In this study, we tested various infusion volumes (200-800 μL) and rates (0.5-5 μL/min) to determine the maximum tolerated combination of injection parameters. Animals were followed for ∼1 month postinjection with magnetic resonance imaging (MRI) performed at 14 and 28 days. T1-weighted MRI was used to quantify thalamic damage followed by histopathological assessment of the brain. Trends in data show that infusion volumes of 800 μL (2 × the volume required in sheep based on thalamic size) resulted in larger lesions than lower volumes, where the long infusion times (between 13 and 26 h) could have contributed to the generation of larger lesions. The target volume (400 μL, projected to be sufficient to cover most of the sheep thalamus) created the smallest lesion size. Cannula placement alone did result in damage, but this is likely associated with an inherent limitation of its use in a small brain due to the length of the distal rigid portion and lack of stable fixation. An injection rate of 5 μL/min at a volume ∼1/3 of the thalamus (400-600 μL) appears to be well tolerated in sheep both clinically and histopathologically.

Lysosphingolipids are an N-deacetylated form of sphingolipids. Lysosphingolipids are considered as potential biomarkers of lysosomal storage disorders (LSD) such as Gaucher disease (GD), Fabry disease (FD), Krabbe disease (KD), and Nimann-Peak disease (NPD) as well as extremely rare LSD as GM1 and GM2 gangliosidoses. To date, there are various methods for assessing lysosphingolipid levels in blood plasma. However, one of the most promising is liquid chromatography tandem mass spectrometry (LC-MS/MS) that allows simultaneous assessment of the concentration of several lysosphingolipids (HexSph, LysoGb3, LysoGM1, LysoGM2, LysoSM and LysoSM509) in plasma, urine, dried blood spots. Despite the fact that the assessment of lysosphingolipids in blood plasma is the gold standard, the LC/MS/MS method in dried blood spots is a fast and reliable method for evaluation of lysosphingolipid profile in patients as a consequence identifying LSDs such as GD (HexSph), FD (LysoGb3), prosaposin deficiency (HexSph, LysoGb3), NPD type A / B and C (LysoSM and LysoSM509). Diagnostics of a number of LSDs by measuring of the lysosphingolipid concentrations in blood by LC/MS/MS is currently being carried out at the Research Center for Medical Genetic in Moscow. We used this method to assess the lysosphingolipid levels in Parkinson's disease associated with a mutations in the gene encoding lysosomal enzyme called glucocerebrosidase (GBA-PD). For the first time, increased level of HexSph (galactosylsphingosine (GalSph) and glucosylsphingosine (GlcSph) in blood of GBA-PD patients was shown that proposes us to make an assumption about the mechanism of GBA-PD pathogenesis. The possible involvement of these metabolites in the stabilization of neurotoxic forms of alpha synuclein in PD is discussed.

GM2 gangliosidoses are a family of severe neurodegenerative disorders resulting from a deficiency in the β-hexosaminidase A enzyme. These disorders include Tay-Sachs disease and Sandhoff disease, caused by mutations in the HEXA gene and HEXB gene, respectively. The HEXA and HEXB genes are required to produce the α and β subunits of the β-hexosaminidase A enzyme, respectively. Using a Sandhoff disease mouse model, we tested for the first time the potential of a comparatively lower dose (2.04 × 1013 vg/kg) of systemically delivered single-stranded adeno-associated virus 9 expressing both human HEXB and human HEXA cDNA under the control of a single promoter with a P2A-linked bicistronic vector design to correct the neurological phenotype. A bicistronic design allows maximal overexpression and secretion of the Hex A enzyme. Neonatal mice were injected with either this ssAAV9-HexB-P2A-HexA vector or a vehicle solution via the superficial temporal vein. An increase in survival of 56% compared with vehicle-injected controls and biochemical analysis of the brain tissue and serum revealed an increase in enzyme activity and a decrease in brain GM2 ganglioside buildup. This is a proof-of-concept study showing the “correction efficacy” of a bicistronic AAV9 vector delivered intravenously for GM2 gangliosidoses. Further studies with higher doses are warranted. Keywords: Sandhoff disease, Tay-Sachs disease, AAV9, gene therapy, hexosaminidase

Tay-Sachs and Sandhoff diseases (GM2 gangliosidosis) are autosomal recessive disorders of lysosomal function that cause fatal and progressive neurodegeneration in infants and young children. Impaired hydrolysis catalysed by β-hexosaminidase A (HexA) leads to the accumulation of its specific substrate, GM2 ganglioside, in neuronal lysosomes. Despite the development of a florid storage phenotype, the role of autophagy and its regulation by the mammalian target of rapamycin (mTOR) has yet to be explored in the neuropathogenesis. Accordingly, we investigated the effects on autophagy and lysosomal integrity using skin fibroblasts obtained from patients with Tay-Sachs and Sandhoff diseases. Pathological autophagosomes, with enhanced expression of the p62/SQSTM1 protein, suggested impaired autophagic flux, an abnormality confirmed by electron microscopy and biochemical studies revealing the accelerated release of mature cathepsins and HexA into the cytosol, indicating increased lysosomal permeability. GM2 fibroblasts showed inappropriately diminished mTOR signalling with reduced basal mTOR activity. Accordingly, provision of a positive nutrient signal by L-arginine supplementation partially restored mTOR activity and ameliorated the cytopathological abnormalities - and immediately suggests an avenue for therapeutic exploration in this cruel disease. We also contend that the expression of autophagy/lysosome/mTOR-associated molecules may prove useful peripheral biomarkers for facile monitoring of systemic treatment of GM2 gangliosidosis and neurodegenerative disorders that affect the lysosomal function and disrupt autophagy

Sphingolipids are lipids derived from an 18-carbons unsaturated amino alcohol, the sphingosine. Ceramide, sphingomyelins, sphingosine-1-phosphates, gangliosides and globosides, are part of this group of lipids that participate in important cellular roles such as structural part of plasmatic and organelle membranes maintaining their function and integrity, cell signaling response, cell growth, cell cycle, cell death, inflammation, cell migration and differentiation, autophagy, angiogenesis, immune system. The metabolism of these lipids involves a broad and complex network of reactions that convert one lipid into others through different specialized enzymes. Impairment of sphingolipids metabolism has been associated with several disorders, from several lysosomal storage diseases, known as sphingolipidoses, to polygenic diseases such as diabetes and Parkinson and Alzheimer diseases. Sphingolipids are mainly located in central nervous system, and their abundance and distribution depend on brain development state and cell type. Although several studies have expanded the knowledge about sphingolipids function both in health and disease, it is still necessary to continue their study to understand the cellular implications of novel sphingolipids. These studies will also contribute to the diagnosis and treatment of diseases in which these molecules are part of their pathophysiology. In this review, we summarize the main sphingolipid characteristics and current knowledge about the synthesis, catabolism, regulatory pathways, participation in action potential and ionic channels, among other relevant functions.

BackgroundThe lack of approved treatments for the majority of rare diseases is reflective of the unique challenges of orphan drug development. Novel methodologies, including new functionally relevant endpoints, are needed to render the development process more feasible and appropriate for these rare populations, and thereby expedite the approval of promising treatments to address patients’ high unmet medical need. Here, we describe the development of an innovative master protocol and primary outcome assessment to investigate the modified amino acid N-acetyl-L-leucine (Sponsor Code: IB1001) in three seperate, multinational, phase II trials for three ultra-rare, autosomal-recessive, neurodegenerative disorders: Niemann-Pick disease type C (NPC), GM2 Gangliosidoses (Tay-Sachs and Sandhoff disease; “GM2”), and Ataxia Telangiectasia (A-T).Methods/designThe innovative IB1001 master protocol and novel CI-CS primary endpoints were developed through a close collaboration between the Industry Sponsor, Key Opinion Leaders, representatives of the Patient Communities, and National Regulatory Authorities. As a result, the open-label, rater-blinded study design is considerate of the practical limitations of recruitment and retention of subjects in these ultra-orphan populations. The novel primary endpoint, the Clinical Impression of Change in Severity© (CI-CS), accommodates the heterogenous clinical presentation of NPC, GM2, and A-T: at screening, the Principal Investigator appoints for each patient a primary anchor test (either the 8 Meter Walk Test (8MWT) or 9 Hole Peg Test of the Dominant Hand (9HPT-D)) based on his/her unique clinical symptoms. The anchor tests are videoed in a standardized manner at each visit to capture all aspects related to the patient’s functional performance. The CI-CS assessment is ultimately performed by independent, blinded raters who compare videos of the primary anchor test from three periods: baseline, the end of treatment, and the end of a post-treatment washout. Blinded to the timepoint of each video, the raters make an objective comparison scored on a 7-point Likert scale of the change in the severity of the patient’s neurological signs and symptoms from Video A to Video B. To investigate both the symptomatic and disease-modifying effects of treatment, N-acetyl-L-leucine is assessed during two treatment sequences: a 6-week parent study, and one-year extension phase.DiscussionThe novel CI-CS assessment, developed through a collaboration of all stakeholders, is advantageous in that it better ensures the primary endpoint is functionally relevant for each patient, is able to capture small but meaningful clinical changes critical to the patients’ quality of life (fine-motor skills; gait), and blinds the primary outcome assessment. The results of these three trials will inform whether N-acetyl-L-leucine is an effective treatment for NPC, GM2, and A-T, and can also serve as a new therapeutic paradigm for the development of future treatments for other orphan diseases.Trial registrationsThe three trials (IB1001-201 for Niemann-Pick disease type C (NPC); IB1001-202 for GM2 Gangliosidoses (Tay-Sachs and Sandhoff); IB1001-203 for Ataxia-Telangiectasia (A-T)) have been registered at www.clinicaltrials.gov (NCT03759639; NCT03759665; NCT03759678), www.clinicaltrialsregister.eu (EudraCT: 2018-004331-71; 2018-004406-25; 2018-004407-39) and https://www.germanctr.de (DR KS-ID: DRKS00016567; DRKS00017539; DRKS00020511).

Aims: Tay–Sachs and Sandhoff diseases (GM2 gangliosidosis) are autosomal recessive disorders of lysosomal function that cause progressive neurodegeneration in infants and young children. Impaired hydrolysis catalysed by β-hexosaminidase A (HexA) leads to the accumulation of GM2 ganglioside in neuronal lysosomes. Despite the storage phenotype, the role of autophagy and its regulation by mTOR has yet to be explored in the neuropathogenesis. Accordingly, we investigated the effects on autophagy and lysosomal integrity using skin fibroblasts obtained from patients with Tay–Sachs and Sandhoff diseases. Results: Pathological autophagosomes with impaired autophagic flux, an abnormality confirmed by electron microscopy and biochemical studies revealing the accelerated release of mature cathepsins and HexA into the cytosol, indicating increased lysosomal permeability. GM2 fibroblasts showed diminished mTOR signalling with reduced basal mTOR activity. Accordingly, provision of a positive nutrient signal by L-arginine supplementation partially restored mTOR activity and ameliorated the cytopathological abnormalities. Innovation: Our data provide a novel molecular mechanism underlying GM2 gangliosidosis. Impaired autophagy caused by insufficient lysosomal function might represent a new therapeutic target for these diseases. Conclusion: We contend that the expression of autophagy/lysosome/mTOR-associated molecules may prove useful peripheral biomarkers for facile monitoring of treatment of GM2 gangliosidosis and neurodegenerative disorders that affect the lysosomal function and disrupt autophagy., Spanish Association of families affected by Tay–Sachs and Sandhoff disease (ACTAYS), BBSRC project grant (BB/T016183/1), Cure and Action for Tay-Sachs (CATS) Foundation: (UK charity 1144543), National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre grant number IS-BRC-1215-20014.

GM2 gangliosidoses are a group of pathologies characterized by GM2 ganglioside accumulation into the lysosome due to mutations on the genes encoding for the β-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.

Gangliosidoses are progressive neurodegenerative disorders caused by the deficiency of enzymes involved in the breakdown of glycosphingolipids. There are not much data about gangliosidosis in India; hence, this study was planned. The aim is to study the clinical, biochemical, and molecular profile of gangliosidosis. A retrospective chart review, in the pediatric neurology department from January 2015 to March 2020, was performed. Children diagnosed with Gangliosidosis were included. The disorder was confirmed by reduced activity of enzymes and/or pathogenic or likely pathogenic variants in associated genes. We assessed age at presentation, gender, parental consanguinity, clinical manifestations, neuroimaging findings, enzyme level, and pathogenic or likely pathogenic variants. Clinical data for 32 children with gangliosidosis were analyzed, which included 12 (37.5%) with GM1 gangliosidosis, 8 (25%) with Tay-Sachs disease (TSD), 11 (34.37%) with Sandhoff disease (SD), and 1 AB variant of GM2 gangliosidosis that occurs due to GM2 ganglioside activator protein deficiency. Twenty-four (75%) children were the offspring of consanguineous parents. Thirty-one (97%) had developmental delay. The median age at presentation was 15.5 months. Nine (28.12%) had seizures. Five children (41.6%) with GM1 gangliosidosis and two with SD had extensive Mongolian spots. Ten children with GM1 gangliosidosis (83.3%) had coarse facial features. Cherry red spot was found in 24 out of 32 children (75%). All children with GM1 gangliosidosis and none with TSD had hepato-splenomegaly. Two children (2/8; 25%) with TSD and seven (7/11; 63%) with SD had microcephaly. One child with SD had coarse facies and three did not have hepato-splenomegaly. Neuroimaging findings revealed bilateral thalamic involvement in 20 (62.5%) patients and periventricular hypomyelination in all cases. One child had a rare AB variant of GM2 gangliosidosis. GM2 Gangliosidoses are more common compared with GM1 variety. All of them had infantile onset except one child with TSD. Microcephaly can be present while usually megalencephaly is reported in the literature. The absence of hepato-splenomegaly does not rule out SD. Extensive Mongolian spots can be seen in GM2 gangliosidosis. AB variant of GM2 gangliosidosis should be considered when the enzyme is normal in the presence of strong clinical suspicion.

The GM2 gangliosidoses, Tay-Sachs disease (TSD) and Sandhoff disease (SD), are fatal lysosomal storage disorders caused by mutations in the HEXA and HEXB genes, respectively. These mutations cause dysfunction of the lysosomal enzyme β-N-acetylhexosaminidase A (HexA) and accumulation of GM2 ganglioside (GM2) with ensuing neurodegeneration, and death by 5 years of age. Until recently, the most successful therapy was achieved by intracranial co-delivery of monocistronic adeno-associated viral (AAV) vectors encoding Hex alpha and beta-subunits in animal models of SD. The blood-brain barrier crossing properties of AAV9 enables systemic gene therapy; however, the requirement of co-delivery of two monocistronic AAV vectors to overexpress the heterodimeric HexA protein has prevented the use of this approach. To address this need, we developed multiple AAV constructs encoding simultaneously HEXA and HEXB using AAV9 and AAV-PHP.B and tested their therapeutic efficacy in 4- to 6-week-old SD mice after systemic administration. Survival and biochemical outcomes revealed superiority of the AAV vector design using a bidirectional CBA promoter with equivalent dose-dependent outcomes for both capsids. AAV-treated mice performed normally in tests of motor function, CNS GM2 ganglioside levels were significantly reduced, and survival increased by >4-fold with some animals surviving past 2 years of age.

Tay-Sachs disease and Sandhoff disease are severe hereditary neurodegenerative disorders caused by a deficiency of β-hexosaminidase A (HexA) enzyme, which results in the accumulation of GM2 gangliosides in the nervous system cells. In this work, we analyzed the efficacy and safety of cell-mediated gene therapy for Sandhoff disease and Sandhoff disease using a bicistronic lentiviral vector encoding cDNA of HexA α- and β-subunit genes separated by the nucleotide sequence of a P2A peptide (HEXA-HEXB). The functionality of the bicistronic construct containing the HEXA-HEXB genetic cassette was analyzed in a culture of HEK293T cells and human umbilical cord blood mononuclear cells (hUCBMCs). Our results showed that the enzymatic activity of HexA in the conditioned medium harvested from genetically modified HEK293T-HEXA-HEXB and hUCBMCs-HEXA-HEXB was increased by 23 and 8 times, respectively, compared with the conditioned medium of native cells. Western blot analysis showed that hUCBMCs-HEXA-HEXB secreted both completely separated HEXA and HEXB proteins, and an uncleaved protein containing HEXA + HEXB linked by the P2A peptide. Intravenous injection of genetically modified hUCBMCs-HEXA-HEXB to laboratory Wistar rats was carried out, and the HexA enzymatic activity in the blood plasma of experimental animals, as well as the number of live cells of immune system organs (spleen, thymus, bone marrow, lymph nodes) were determined. A significant increase in the enzymatic activity of HexA in the blood plasma of laboratory rats on days 6 and 9 (by 2.5 and 3 times, respectively) after the administration of hUCBMCs-HEXA-HEXB was shown. At the same time, the number of live cells in the studied organs remained unchanged. Thus, the functionality of the bicistronic genetic construct encoding cDNA of the HEXA and HEXB genes separated by the nucleotide sequence of the P2A peptide was shown in vitro and in vivo. We hypothesize that due to the natural ability of hUCBMCs to overcome biological barriers, such a strategy can restore the activity of the missing enzyme in the central nervous system of patients with GM2 gangliosidoses. Based on the obtained data, it can be concluded that intravenous administration of hUCBMCs with HexA overexpression is a promising method of the therapy for GM2 gangliosidoses. The animal protocol was approved by the Animal Ethics Committee of the Kazan Federal University (No. 23) on June 30, 2020.

GM2 Gangliosidoses are a group of autosomal recessive genetic disorders caused by intra-lysosomal deposition of ganglioside GM2 mainly in the neuronal cells. GM2-Activator protein deficiency is an extremely rare type of GM2 gangliosidosis (AB variant) caused by the mutation of GM2A.We report a case of a female child who presented with clinical features similar to classical Tay-Sachs disease, but with normal beta hexosaminidase enzyme levels. Molecular study revealed a novel homozygous intronic mutation which confirmed the diagnosis of GM2 Activator protein deficiency. GM2 Activator protein deficiency is a mimic of Classical Tay-Sachs disease and should be a differential diagnosis in children who present with neuroregression, cherry red spots without hepatosplenomegaly and with normal beta hexosaminidase enzyme levels.

Consistent with a tentative diagnosis of neuronal ceroid lipofuscinosis (NCL), autofluorescent cytoplasmic storage bodies were found in neurons from the brains of 2 related Shiba Inu dogs with a young‐adult onset, progressive neurodegenerative disease. Unexpectedly, no potentially causal NCL‐related variants were identified in a whole‐genome sequence generated with DNA from 1 of the affected dogs. Instead, the whole‐genome sequence contained a homozygous 3 base pair (bp) deletion in a coding region of HEXB. The other affected dog also was homozygous for this 3‐bp deletion. Mutations in the human HEXB ortholog cause Sandhoff disease, a type of GM2 gangliosidosis. Thin‐layer chromatography confirmed that GM2 ganglioside had accumulated in an affected Shiba Inu brain. Enzymatic analysis confirmed that the GM2 gangliosidosis resulted from a deficiency in the HEXB encoded protein and not from a deficiency in products from HEXA or GM2A, which are known alternative causes of GM2 gangliosidosis. We conclude that the homozygous 3‐bp deletion in HEXB is the likely cause of the Shiba Inu neurodegenerative disease and that whole‐genome sequencing can lead to the early identification of potentially disease‐causing DNA variants thereby refocusing subsequent diagnostic analyses toward confirming or refuting candidate variant causality.

β-hexosaminidase is an enzyme responsible for the degradation of gangliosides, glycans, and other glycoconjugates containing β-linked hexosamines that enter the lysosome. GM2 gangliosidoses, such as Tay-Sachs and Sandhoff, are lysosomal storage disorders characterized by β-hexosaminidase deficiency and subsequent lysosomal accumulation of its substrate metabolites. These two diseases result in neurodegeneration and early mortality in children. A significant difference between these two disorders is the accumulation in Sandhoff disease of soluble oligosaccharide metabolites that derive from N- and O-linked glycans. In this paper we describe our results from a longitudinal biochemical study of a feline model of Sandhoff disease and an ovine model of Tay-Sachs disease to investigate the accumulation of GM2/GA2 gangliosides, a secondary biomarker for phospholipidosis, bis-(monoacylglycero)-phosphate, and soluble glycan metabolites in both tissue and fluid samples from both animal models. While both Sandhoff cats and Tay-Sachs sheep accumulated significant amounts of GM2 and GA2 gangliosides compared to age-matched unaffected controls, the Sandhoff cats having the more severe disease, accumulated larger amounts of gangliosides compared to Tay-Sachs sheep in their occipital lobes. For monitoring glycan metabolites, we developed a quantitative LC/MS assay for one of these free glycans in order to perform longitudinal analysis. The Sandhoff cats showed significant disease-related increases in this glycan in brain and in other matrices including urine which may provide a useful clinical tool for measuring disease severity and therapeutic efficacy. Finally, we observed age-dependent increasing accumulation for a number of analytes, especially in Sandhoff cats where glycosphingolipid, phospholipid, and glycan levels showed incremental increases at later time points without signs of peaking. This large animal natural history study for Sandhoff and Tay-Sachs is the first of its kind, providing insight into disease progression at the biochemical level. This report may help in the development and testing of new therapies to treat these disorders.