A pentasaccharide for monitoring pharmacodynamic response to gene therapy in GM1 gangliosidosis.

BACKGROUND: GM1 gangliosidosis is a rare, fatal, neurodegenerative disease caused by mutations in the GLB1 gene and deficiency in ß-galactosidase. Delay of symptom onset and increase in lifespan in a GM1 gangliosidosis cat model after adeno-associated viral (AAV) gene therapy treatment provide the basis for AAV gene therapy trials. The availability of validated biomarkers would greatly improve assessment of therapeutic efficacy. METHODS: The liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to screen oligosaccharides as potential biomarkers for GM1 gangliosidosis. The structures of pentasaccharide biomarkers were determined with mass spectrometry, as well as chemical and enzymatic degradations. Comparison of LC-MS/MS data of endogenous and synthetic compounds confirmed the identification. The study samples were analyzed with fully validated LC-MS/MS methods. FINDINGS: We identified two pentasaccharide biomarkers, H3N2a and H3N2b, that were elevated more than 18-fold in patient plasma, cerebrospinal fluid (CSF), and urine. Only H3N2b was detectable in the cat model, and it was negatively correlated with ß-galactosidase activity. Following intravenous (IV) AAV9 gene therapy treatment, reduction of H3N2b was observed in central nervous system, urine, plasma, and CSF samples from the cat model and in urine, plasma, and CSF samples from a patient. Reduction of H3N2b accurately reflected normalization of neuropathology in the cat model and improvement of clinical outcomes in the patient. INTERPRETATIONS: These results demonstrate that H3N2b is a useful pharmacodynamic biomarker to evaluate the efficacy of gene therapy for GM1 gangliosidosis. H3N2b will facilitate the translation of gene therapy from animal models to patients. FUNDING: This work was supported by grants U01NS114156, R01HD060576, ZIAHG200409, and P30 DK020579 from the National Institutes of Health (NIH) and a grant from National Tay-Sachs and Allied Diseases Association Inc.

GM1 gangliosidosis: patients with different phenotypic features and novel mutations.

OBJECTIVES: GM1-gangliosidosis is an autosomal recessive lysosomal storage disorder caused by beta-galactosidase deficiency encoded by GLB1. It is mainly characterized by progressive neurodegeneration due to accumulation of glycosphingolipids in central nervous system and classified into 3 forms according to the age of onset and severity of symptoms. CASE PRESENTATION: In this study, we described the demographic, clinical, molecular, biochemical characteristics of 4 patients from 3 unrelated families diagnosed with GM1-gangliosidosis. The ages of the patients included in the study were between 5 months and 10 years old and all were male. All families had third degree consanguinity. Two of the patients were diagnosed as infantile type and the other two siblings were diagnosed as juvenile type. Infantile type patients had coarse facial appearance, developmental delay and early neurodegeneration. Juvenile type patients had mild motor and cognitive developmental delays at the beginning, but they did not have coarse facial features. Cherry-red macula and cardiac involvement were detected in only one infantile patient, while hepatomegaly was present in both infantile type patients. Beta galactosidase enzyme levels were extremely low in all patients and two novel variants were identified in GLB1. CONCLUSIONS: In this study, we identified four patients with different phenotypic features and two new mutations. GM1 gangliosidosis shows clinical heterogeneity according to age of onset. In some patients, developmental delay can be seen before the loss of gained functions. Therefore, this disorder should be kept in mind in patients with developmental delay who have not yet started neurodegeneration. There is no curative treatment for the disease yet, but ongoing gene therapy studies are promising for curing the disease in the future.

Single Institutional Experience with GM1 Gangliosidosis: Clinical and Laboratory Results of 14 Patients

Background: GM1 gangliosidosis is an autosomal recessive lysosomal storage disease caused by biallelic mutations in the GLB1 gene. Neurodegeneration, hypotonia, visceromegaly, macular cherry-red spots, skeletal dysplasia, and coarse and dysmorphic face are the major clinical features. Aims: To evaluate the demographic and clinical data of patients with GM1 gangliosidosis in a single center. Study Design: A retrospective clinical study. Methods: This study included patients followed at Hacettepe University Ihsan Dogramaci Children's Hospital Pediatric Metabolism Unit with the diagnosis of GM1 gangliosidosis between 1988 and 2021. Hospital records of the patients were reviewed for demographic, clinical, and laboratory findings. Results: Fourteen patients were included in the study and 10 (71.4%) were male. The age at onset of clinical symptoms was between 0 and 5 months, and the median time to diagnosis after the first symptom was 4.3 (0-13) months. Motor delay (54%) was the most common initial symptom. The median follow-up period was 14.8 (0.4-92.2) months. Twelve patients (85.7%) died, and all deaths occurred before the age of 24 months. The median survival was 21.3 (95% confidence interval, 15.5-24.9) months. Higher leukocyte beta-galactosidase activity correlated with later age at onset (ρ = 0.575), later age at diagnosis (ρ = 0.618), and longer diagnostic delay (ρ = 0.702) (ρ < 0.05). Conclusion: Median survival in patients with GM1 gangliosidosis is less than 24 months. Beta-galactosidase enzyme activity may be associated with clinical onset and time of diagnosis in these patients.

AAV9-coGLB1 Improves Lysosomal Storage and Rescues Central Nervous System Inflammation in a Mutant Mouse Model of GM1 Gangliosidosis.

BACKGROUND: GM1 gangliosidosis (GM1) is an autosomal recessive disorder characterized by the deficiency of beta-galactosidase (ß-gal), a ubiquitous lysosomal enzyme that catalyzes the hydrolysis of GM1 ganglioside. OBJECTIVE: The study aims to explore the application of the AAV9-coGLB1 for effective treatment in a GM1 gangliosidosis mutant mouse model. METHODS: We designed a novel adeno-associated virus 9 (AAV9) vector expressing ß-gal (AAV9- coGLB1) to treat GM1 gangliosidosis. The vector, injected via the caudal vein at 4 weeks of age, drove the widespread and sustained expression of ß-gal for up to 32 weeks in the Glb1G455R/G455R mutant mice (GM1 mice). RESULTS: The increased levels of ß-gal reduced the pathological damage occurring in GM1 mice. Histological analyses showed that myelin deficits and neuron-specific pathology were reduced in the cerebral cortex region of AAV9-coGLB1-treated mice. Immunohistochemical staining showed that the accumulation of GM1 ganglioside was also reduced after gene therapy. The reduction of the storage in these regions was accompanied by a decrease in activated microglia. In addition, AAV9 treatment reversed the blockade of autophagic flux in GM1 mice. CONCLUSION: These results show that AAV9-coGLB1 reduces the pathological signs of GM1 gangliosidosis in a mouse model.

Intravenous delivery of adeno-associated viral gene therapy in feline GM1 gangliosidosis.

GM1 gangliosidosis is a fatal neurodegenerative disease caused by a deficiency of lysosomal ß-galactosidase. In its most severe form, GM1 gangliosidosis causes death by 4 years of age, and no effective treatments exist. Previous work has shown that injection of the brain parenchyma with an adeno-associated viral (AAV) vector provides pronounced therapeutic benefit in a feline GM1 model. To develop a less invasive treatment for the brain and increase systemic biodistribution, intravenous injection of AAV9 was evaluated. AAV9 expressing feline ß-galactosidase was intravenously administered at 1.5×1013 vector genomes/kg body weight to six GM1 cats at ∼1 month of age. The animals were divided into two cohorts: (i) a long-term group, which was followed to humane end point; and (ii) a short-term group, which was analysed 16 weeks post-treatment. Clinical assessments included neurological exams, CSF and urine biomarkers, and 7 T MRI and magentic resonance spectroscopy (MRS). Post-mortem analysis included ß-galactosidase and virus distribution, histological analysis and ganglioside content. Untreated GM1 animals survived 8.0 ± 0.6 months while intravenous treatment increased survival to an average of 3.5 years (n = 2) with substantial improvements in quality of life and neurological function. Neurological abnormalities, which in untreated animals progress to the inability to stand and debilitating neurological disease by 8 months of age, were mild in all treated animals. CSF biomarkers were normalized, indicating decreased CNS cell damage in the treated animals. Urinary glycosaminoglycans decreased to normal levels in the long-term cohort. MRI and MRS showed partial preservation of the brain in treated animals, which was supported by post-mortem histological evaluation. ß-Galactosidase activity was increased throughout the CNS, reaching carrier levels in much of the cerebrum and normal levels in the cerebellum, spinal cord and CSF. Ganglioside accumulation was significantly reduced by treatment. Peripheral tissues such as heart, skeletal muscle, and sciatic nerve also had normal ß-galactosidase activity in treated GM1 cats. GM1 histopathology was largely corrected with treatment. There was no evidence of tumorigenesis or toxicity. Restoration of ß-galactosidase activity in the CNS and peripheral organs by intravenous gene therapy led to profound increases in lifespan and quality of life in GM1 cats. These data support the promise of intravenous gene therapy as a safe, effective treatment for GM1 gangliosidosis.

Mongolian spots in GM1 gangliosidosis: a pictorial report.

GM1 gangliosidosis is a lysosomal storage disorder, characterized by psychomotor deterioration, visceromegaly, facial coarseness, retinal cherry-red spots, and skeletal abnormalities. We report six unrelated patients with GM1 gangliosidosis with extensive Mongolian spots on the trunk and extremities that provided clue to clinical diagnosis. All patients exhibited psychomotor delay, coarse facies, hepatosplenomegaly, hypotonia, and dysostosis multiplex. Four patients had retinal cherry-red spots. The condition was confirmed by identification of very low activities of beta-galactosidase enzyme in peripheral leukocytes and biallelic pathogenic variants in the GLB1 gene. We identified one novel (c.1479G>T) and two known (c.75 + 2dup and c.1369C>T) pathogenic variants in homozygous state in them. Our work ascertains extensive Mongolian spots as a diagnostic handle for early recognition of GM1 gangliosidosis. Though a known feature of GM1 gangliosidosis, considerable variation in the prevalence and ethnic differences are observed. This report illustrates the Mongolian spots pictorially in Indian patients.

Morquio B Disease. Disease Characteristics and Treatment Options of a Distinct GLB1-Related Dysostosis Multiplex.

Morquio B disease (MBD) is an autosomal recessive GLB1-gene-related lysosomal storage disease, presenting with a peculiar type of dysostosis multiplex which is also observed in GALNS-related Morquio A disease. MBD may present as pure skeletal phenotype (pure MBD) or in combination with the neuronopathic manifestations seen in type 2 (juvenile) or type 3 (late onset) GM1 gangliosidosis (MBD plus). The main skeletal features are progressive growth impairment, kyphoscoliosis, coxa/genua valga, joint laxity, platyspondyly and odontoid hypoplasia. The main neuronopathic features are dystonia, ataxia, and intellectual/developmental/speech delay. Spinal cord compression occurs as a complication of spinal dysostosis. Chronic pain is reported, along with mobility issues and challenges with daily living and self-care activities, as the most common health concern. The most commonly reported orthopedic surgeries are hip and knee replacements. Keratan sulphate-derived oligosaccharides are characteristic biomarkers. Residual ß-galactosidase activities measured against synthetic substrates do not correlate with the phenotype. W273 L and T500A are the most frequently observed GLB1 variants in MBD, W273L being invariably associated with pure MBD. Cytokines play a role in joint destruction and pain, providing a promising treatment target. In the future, patients may benefit from small molecule therapies, and gene and enzyme replacement therapies, which are currently being developed for GM1 gangliosidosis.

A Single Injection of an Optimized Adeno-Associated Viral Vector into Cerebrospinal Fluid Corrects Neurological Disease in a Murine Model of GM1 Gangliosidosis.

GM1 gangliosidosis is a rare neurodegenerative lysosomal storage disease caused by loss-of-function mutations in the gene encoding beta-galactosidase (ß-gal). There are no approved treatments for GM1 gangliosidosis. Previous studies in animal models have demonstrated that adeno-associated viral (AAV) vector-mediated gene transfer to the brain can restore ß-gal expression and prevent the onset of neurological signs. We developed an optimized AAV vector expressing human ß-gal and evaluated the efficacy of a single intracerebroventricular injection of this vector into the cerebrospinal fluid (CSF) of a murine disease model. The AAV vector administration into the CSF increased ß-gal activity in the brain, reduced neuronal lysosomal storage lesions, prevented the onset of neurological signs and gait abnormalities, and increased survival. These findings demonstrate the potential therapeutic activity of this vector and support its subsequent development for the treatment of GM1 gangliosidosis.

The skeletal phenotype of intermediate GM1 gangliosidosis: Clinical, radiographic and densitometric features, and implications for clinical monitoring and intervention.

GM1 gangliosidosis is a lysosomal storage disorder caused by mutations in GLB1 encoding a lysosomal ß-galactosidase. This disease is a continuum from the severe infantile form with rapid neurological decline to the chronic adult form, which is not life-limiting. The intermediate or type 2 form can be further classified into late infantile and juvenile forms. The frequency and severity of skeletal outcomes in late infantile and juvenile patients have not been characterized. Our goals are to describe the radiological skeletal abnormalities, bone mineral density (BMD), and frequency of fractures in patients with intermediate GM1 gangliosidosis. We evaluated 13 late infantile and 21 juvenile patients as part of an ongoing natural history study. Average time from onset of symptoms to diagnosis was 1.9 and 6.3 years for late infantile and juvenile patients, respectively. All late infantile patients had odontoid hypoplasia and pear-shaped vertebral bodies, the frequency of which was significantly different than in patients with juvenile disease (none and 14%, respectively). Juvenile patients had irregular endplates of the vertebral bodies (15/21), central indentation of endplates (10/21), and squared and flat vertebral bodies (10/21); all allowed radiographic differentiation from late infantile patients. Lumbar spine, femoral neck, and total hip BMD were significantly decreased (-2.1, -2.2, and -1.8 Z-scores respectively). Lumbar spine BMD peaked at 19 years, while distal forearm BMD peaked at 30 years. Despite low BMD, no patients exhibited fractures. We have demonstrated that all late infantile patients have some degree of odontoid hypoplasia suggesting the need for cervical spine evaluation particularly prior to anesthesia, whereas juvenile patients had variable skeletal involvement often affecting activities of daily living. Type 2 GM1 gangliosidosis patients have skeletal abnormalities that are both an early indication of their diagnosis, and require monitoring and management to ensure the highest possible quality of life.

Identification of a novel GLB1 mutation in a consanguineous Pakistani family affected by rare infantile GM1 gangliosidosis.

Monosialotetrahexosylganglioside (GM1) is a rare lysosomal storage disorder caused by the deficiency of beta-galactosidase (ß-Gal) encoded by galactose beta 1 (GLB1). It is clinically characterized by developmental delay attributed to multifold accumulation of GM1 gangliosides in nerve cells. In this study, we present a case of infantile GM1 gangliosidosis in a consanguineous Pakistani family. The child was presented with developmental delay, hepatosplenomegaly and recurrent chest infections at 7.5 months of age. Radiological and biochemical investigations including magnetic resonance imaging (MRI), bonemarrow biopsy and urine oligosaccharide analyses suggested lysosomal storage disorder. Significantly low levels of ß-Gal enzyme confirmed the diagnosis of GM1 gangliosidosis. DNA sequencing of GLB1 identified a homozygous 2-bp deletion c.881-882delAT (p.Tyr294Terfs) in exon 8. In silico analysis supported the deleterious effect of the variant. This study extends GLB1 mutation spectrum and should benefit genetic counselling and prenatal diagnosis of the affected family.

SAAMP 2.0: An algorithm to predict genotype-phenotype correlation of lysosomal storage diseases.

Lysosomal storage diseases (LSDs) are a group of genetic disorders, resulting from deficiencies of lysosomal enzyme. Genotype-phenotype correlation is essential for timely and proper treatment allocation. Recently, by integrating prediction outcomes of 7 bioinformatics tools, we developed a SAAMP algorithm to predict the impact of individual amino-acid substitution. To optimize this approach, we evaluated the performance of these bioinformatics tools in a broad array of genes. PolyPhen and PROVEAN had the best performances, while SNP&GOs, PANTHER and I-Mutant had the worst performances. Therefore, SAAMP 2.0 was developed by excluding 3 tools with worst performance, yielding a sensitivity of 94% and a specificity of 90%. To generalize the guideline to proteins without known structures, we built the three-dimensional model of iduronate-2-sulfatase by homology modeling. Further, we investigated the phenotype severity of known disease-causing mutations of the GLB1 gene, which lead to 2 LSDs (GM1 gangliosidosis and Morquio disease type B). Based on the previous literature and structural analysis, we associated these mutations with disease subtypes and proposed a theory to explain the complicated genotype-phenotype correlation. Collectively, an updated guideline for phenotype prediction with SAAMP 2.0 was proposed, which will provide essential information for early diagnosis and proper treatment allocation, and they may be generalized to many monogenic diseases.

Mosaic uniparental disomy results in GM1 gangliosidosis with normal enzyme assay.

Inherited metabolic disorders are traditionally diagnosed using broad and expensive panels of screening tests, often including invasive skin and muscle biopsy. Proponents of next-generation genetic sequencing have argued that replacing these screening panels with whole exome sequencing (WES) would save money. Here, we present a complex patient in whom WES allowed diagnosis of GM1 gangliosidosis, caused by homozygous GLB1 mutations, resulting in ß-galactosidase deficiency. A 10-year-old girl had progressive neurologic deterioration, macular cherry-red spot, and cornea verticillata. She had marked clinical improvement with initiation of the ketogenic diet. Comparative genomic hybridization microarray showed mosaic chromosome 3 paternal uniparental disomy (UPD). GM1 gangliosidosis was suspected, however ß-galactosidase assay was normal. Trio WES identified a paternally-inherited pathogenic splice-site GLB1 mutation (c.75+2dupT). The girl had GM1 gangliosidosis; however, enzymatic testing in blood was normal, presumably compensated for by non-UPD cells. Severe neurologic dysfunction occurred due to disruptive effects of UPD brain cells.

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.

Systemic AAV9 gene transfer in adult GM1 gangliosidosis mice reduces lysosomal storage in CNS and extends lifespan.

GM1 gangliosidosis (GM1) is an autosomal recessive lysosomal storage disease where GLB1 gene mutations result in a reduction or absence of lysosomal acid ß-galactosidase (ßgal) activity. ßgal deficiency leads to accumulation of GM1-ganglioside in the central nervous system (CNS). GM1 is characterized by progressive neurological decline resulting in generalized paralysis, extreme emaciation and death. In this study, we assessed the therapeutic efficacy of an adeno-associated virus (AAV) 9-mßgal vector infused systemically in adult GM1 mice (ßGal(-/-)) at 1 × 10(11) or 3 × 10(11) vector genomes (vg). Biochemical analysis of AAV9-treated GM1 mice showed high ßGal activity in liver and serum. Moderate ßGal levels throughout CNS resulted in a 36-76% reduction in GM1-ganglioside content in the brain and 75-86% in the spinal cord. Histological analyses of the CNS of animals treated with 3 × 10(11) vg dose revealed increased presence of ßgal and clearance of lysosomal storage throughout cortex, hippocampus, brainstem and spinal cord. Storage reduction in these regions was accompanied by a marked decrease in astrogliosis. AAV9 treatment resulted in improved performance in multiple tests of motor function and behavior. Also the majority of GM1 mice in the 3 × 10(11) vg cohort retained ambulation and rearing despite reaching the humane endpoint due to weight loss. Importantly, the median survival of AAV9 treatment groups (316-576 days) was significantly increased over controls (250-264 days). This study shows that moderate widespread expression of ßgal in the CNS of GM1 gangliosidosis mice is sufficient to achieve significant biochemical impact with phenotypic amelioration and extension in lifespan.

Chaperone therapy update: Fabry disease, GM1-gangliosidosis and Gaucher disease.

Chaperone therapy is a newly developed molecular therapeutic approach to lysosomal diseases, a group of human genetic diseases causing severe brain damage. Based on early molecular studies during the last decade of the 20th century and early years of the 21st century, mainly on Fabry disease and GM1-gangliosidosis, we found some mutant enzyme proteins were unstable in the cell, and unable to express catalytic activities. Subsequently galactose and other active-site binding substrate analogs were found stabilized and enhance the mutant enzyme activity in culture cells. We concluded that the mutant misfolding enzyme protein and substrate analog competitive inhibitor (chemical chaperone) form a stable complex to be transported to the lysosome, to restore the catalytic activity of mutant enzyme after spontaneous dissociation under the acidic condition. This gene mutation-specific molecular interaction is a paradoxical phenomenon that an enzyme inhibitor in vitro serves as an enzyme stabilizer in situ. First we developed a commercially available compound 1-deoxygalactonojirimycin (DGJ) for Fabry disease, and confirmed the above molecular phenomenon. Currently DGJ has become a new candidate of oral medicine for Fabry disease, generalized vasculopathy involving the kidneys, heart and central nervous system in the middle age. This drug development has reached the phase 3 of human clinical study. Then we found two valienamine derivatives, N-octyl-4-epi-ß-valienamine (NOEV) and N-octyl-ß-valienamine (NOV), as promising therapeutic agents for human ß-galactosidase deficiency disorders (GM1-gangliosidosis and Morquio B disease) and ß-glucosidase deficiency disorders (phenotypic variations of Gaucher disease), respectively. Originally NOEV and NOV had been discovered as competitive inhibitors, and then their paradoxical bioactivities as chaperones were confirmed in cultured fibroblasts from patients with these disorders. Subsequently GM1-gangliosidosis model mice have been used for confirmation of clinical effectiveness, adverse effects and pharmacokinetic studies. Orally administered NOEV entered the brain through the blood-brain barrier, enhanced ß-galactosidase activity, reduced substrate storage, and improved neurological deterioration clinically. Computational analysis revealed pH-dependent enzyme-chaperone interactions. Our recent study indicated chaperone activity of a new DGJ derivative, MTD118, for ß-galactosidase complementary to NOEV. NOV also showed the chaperone effect toward several ß-glucosidase gene mutants in Gaucher disease. Furthermore a commercial expectorant drug ambroxol was found to be a chaperone for ß-glucosidase. A few Gaucher patients responded to this drug with remarkable improvement of oculomotor dysfunction and myoclonus. We hope chaperone therapy will become available for some patients with Fabry disease, GM1-gangliosidosis, Gaucher disease, and other lysosomal storage diseases particularly with central nervous system involvement.

Beta-galactosidase deficiencies and novel GLB1 mutations in three Chinese patients with Morquio B disease or GM1 gangliosidosis.

BACKGROUND: This paper aims to report GLB1 activities and mutation analysis of three patients from the mainland of China, one with Morquio B disease and two with GM1 gangliosidosis. METHODS: GLB1 activity and GLB1 gene mutation were analyzed in the three patients who were clinically suspected of having Morquio B disease or GM1 gangliosidosis. Novel mutations were analyzed by aligning GLB1 homologs, 100 control chromosomes, and the PolyPhen-2 tool. RESULTS: The enzymatic activity of GLB1 was found to be 5.03, 4.20, and 4.50 nmol/h/mg in the three patients, respectively. Patient 1 was a compound heterozygote for p.[Arg148Cys] and p.[Tyr485Cys] mutations in the GLB1 gene. Patient 2 was a compound heterozygote for p.[Tyr270Phe] and p.[Leu337Pro] mutations. Patient 3 was a homozygote for p.[Asp448Val] mutation. Three mutations (p.[Tyr485Cys], p.[Tyr270Phe] and p.[Leu337Pro]) were novel variants and were predicted to damage GLB1 function. CONCLUSIONS: The enzymatic activity and related gene analysis of ß-galactosidase should be performed in clinically suspected individuals to confirm diagnosis. The three novel mutations, p.[Tyr485Cys], p.[Tyr270Phe], and p.[Leu337Pro], are thought to be disease-causing mutations.

Evaluation of N-nonyl-deoxygalactonojirimycin as a pharmacological chaperone for human GM1 gangliosidosis leads to identification of a feline model suitable for testing enzyme enhancement therapy.

Deficiencies of lysosomal ß-D-galactosidase can result in GM1 gangliosidosis, a severe neurodegenerative disease characterized by massive neuronal storage of GM1 ganglioside in the brain. Currently there are no available therapies that can even slow the progression of this disease. Enzyme enhancement therapy utilizes small molecules that can often cross the blood brain barrier, but are also often competitive inhibitors of their target enzyme. It is a promising new approach for treating diseases, often caused by missense mutations, associated with dramatically reduced levels of functionally folded enzyme. Despite a number of positive reports based on assays performed with patient cells, skepticism persists that an inhibitor-based treatment can increase mutant enzyme activity in vivo. To date no appropriate animal model, i.e., one that recapitulates a responsive human genotype and clinical phenotype, has been reported that could be used to validate enzyme enhancement therapy. In this report, we identify a novel enzyme enhancement-agent, N-nonyl-deoxygalactonojirimycin, that enhances the mutant ß-galactosidase activity in the lysosomes of a number of patient cell lines containing a variety of missense mutations. We then demonstrate that treatment of cells from a previously described, naturally occurring feline model (that biochemically, clinically and molecularly closely mimics GM1 gangliosidosis in humans) with this molecule, results in a robust enhancement of their mutant lysosomal ß-galactosidase activity. These data indicate that the feline model could be used to validate this therapeutic approach and determine the relationship between the disease stage at which this therapy is initiated and the maximum clinical benefits obtainable.

Tuning glycosidase inhibition through aglycone interactions: pharmacological chaperones for Fabry disease and GM1 gangliosidosis.

Competitive inhibitors of either α-galactosidase (α-Gal) or ß-galactosidase (ß-Gal) with high affinity and selectivity have been accessed by exploiting aglycone interactions with conformationally locked sp(2)-iminosugars. Selected compounds were profiled as potent pharmacological chaperones for mutant lysosomal α- and ß-Gal associated with Fabry disease and GM(1) gangliosidosis.

AAV-mediated gene delivery in adult GM1-gangliosidosis mice corrects lysosomal storage in CNS and improves survival.

BACKGROUND: GM1-gangliosidosis is a glycosphingolipid (GSL) lysosomal storage disease caused by a genetic deficiency of acid ß-galactosidase (ßgal), which results in the accumulation of GM1-ganglioside and its asialo-form (GA1) primarily in the CNS. Age of onset ranges from infancy to adulthood, and excessive ganglioside accumulation produces progressive neurodegeneration and psychomotor retardation in humans. Currently, there are no effective therapies for the treatment of GM1-gangliosidosis. METHODOLOGY/PRINCIPAL FINDINGS: In this study we examined the effect of thalamic infusion of AAV2/1-ßgal vector in adult GM1 mice on enzyme distribution, activity, and GSL content in the CNS, motor behavior, and survival. Six to eight week-old GM1 mice received bilateral injections of AAV vector in the thalamus, or thalamus and deep cerebellar nuclei (DCN) with pre-determined endpoints at 1 and 4 months post-injection, and the humane endpoint, or 52 weeks of age. Enzyme activity was elevated throughout the CNS of AAV-treated GM1 mice and GSL storage nearly normalized in most structures analyzed, except in the spinal cord which showed ∼50% reduction compared to age-matched untreated GM1 mice spinal cord. Survival was significantly longer in AAV-treated GM1 mice (52 wks) than in untreated mice. However the motor performance of AAV-treated GM1 mice declined over time at a rate similar to that observed in untreated GM1 mice. CONCLUSIONS/SIGNIFICANCE: Our studies show that the AAV-modified thalamus can be used as a 'built-in' central node network for widespread distribution of lysosomal enzymes in the mouse cerebrum. In addition, this study indicates that thalamic delivery of AAV vectors should be combined with additional targets to supply the cerebellum and spinal cord with therapeutic levels of enzyme necessary to achieve complete correction of the neurological phenotype in GM1 mice.