Lysosomal Acid Β-glucosidase Research Articles

Gaucher disease prevalence in 600 patients affected by monoclonal gammopathy of undetermined significance.

Gaucher disease (GD) is a rare autosomal recessive inherited disorder caused by the lysosomal enzyme acid β-glucosidase deficiency. Many patients experience a critical delay in the diagnosis of up to 8-10 years due to its rarity and variability in signs and symptoms, with the consultation of several specialists. This prospective observational study analyzed the prevalence of GD in 600 patients with monoclonal gammopathy of uncertain significance (MGUS) from January 2018 until February 2022. The mean age of participants was 66 years, with a mean monoclonal component of 0.58 g/dL. In 433 MGUS patients with available data, anemia (hemoglobin level < 10 g/dL) was present in 31 patients (7%), and thrombocytopenia (platelet count <100.000/mm3 ) in 24 (5.5%). Of 600 MGUS patients tested for acid β-glucosidase enzyme activity, 7 patients (1.2%) had activity below 2.5 nmol/h/mL. In comparison, GBA gene analysis was executed in 110 patients. It revealed 4 patients (0.7%) affected by GD (3 patients with compound heterozygous mutation and 1 with homozygous mutation), with a prevalence of 1 every 150 MGUS patients. Furthermore, 12 out of the remaining 106 evaluated patients (11%) were carriers of a single heterozygous mutation while having regular enzyme activity. The clinical heterogeneity of GD and frequent lack of awareness among physicians often lead to diagnostic delays and severe clinical manifestations. The role of MGUS in the presence of at least one clinical sign, such as low platelet count, organomegaly, bone pain, or bleeding tendency, could aid in initiating GD screening with DBS, thus reducing the period between symptom onset and the diagnosis of this rare disease.

IPSC-derived neural precursor cells engineering GBA1 recovers acid β-glucosidase deficiency and diminishes α-synuclein and neuropathology

Mutations in GBA1, encoding the lysosomal acid β-glucosidase (GCase), cause neuronopathic Gaucher disease (nGD) and promote Parkinson disease (PD). The mutations on GBA1 include deletion and missense mutations that are pathological and lead to GCase deficiency in Gaucher disease. Both nGD and PD lack disease-modifying treatments and are critical unmet medical needs. In this study, we evaluated a cell therapy treatment using mouse iPSC-derived neural precursor cells (NPCs) engineered to overexpress GCase (termed hGBA1-NPCs). The hGBA1-NPCs secreted GCase that was taken up by adjacent mouse Gba -/- neurons and improved GCase activity, reduced GCase substrate accumulation, and improved mitochondrial function. Short-term invivo effects were evaluated in 9H/PS-NA mice, an nGD mouse model exhibiting neuropathology and α-synuclein aggregation, the typical PD phenotypes. Intravenously administrated hGBA1-NPCs were engrafted throughout the brain and differentiated into neural lineages. GCase activity was increased in various brain regions of treated 9H/PS-NA mice. Compared with vehicle, hGBA1-NPC-transplanted mice showed ∼50% reduction of α-synuclein aggregates in the substantia nigra, significant reduction of neuroinflammation and neurodegeneration in the regions of NPC migration, and increased expression of neurotrophic factors that support neural cell function. Together, these results support the therapeutic benefit of intravenous delivery of iPSC-derived NPCs overexpressing GCase in mitigating nGD and PD phenotypes and establish the feasibility of combined cell and gene therapy for GBA1-associated PD.

Venglustat combined with imiglucerase for neurological disease in adults with Gaucher disease type 3: the LEAP trial.

Gaucher disease type 3 is a chronic neuronopathic disorder with wide-ranging effects, including hepatosplenomegaly, anaemia, thrombocytopenia, skeletal disease and diverse neurological manifestations. Biallelic mutations in GBA1 reduce lysosomal acid β-glucosidase activity, and its substrates, glucosylceramide and glucosylsphingosine, accumulate. Enzyme replacement therapy and substrate reduction therapy ameliorate systemic features of Gaucher disease, but no therapies are approved for neurological manifestations. Venglustat is an investigational, brain-penetrant, glucosylceramide synthase inhibitor with potential to improve the disease by rebalancing influx of glucosylceramide with impaired lysosomal recycling. The Phase 2, open-label LEAP trial (NCT02843035) evaluated orally administered venglustat 15 mg once-daily in combination with maintenance dose of imiglucerase enzyme replacement therapy during 1 year of treatment in 11 adults with Gaucher disease type 3. Primary endpoints were venglustat safety and tolerability and change in concentration of glucosylceramide and glucosylsphingosine in CSF from baseline to Weeks 26 and 52. Secondary endpoints included change in plasma concentrations of glucosylceramide and glucosylsphingosine, venglustat pharmacokinetics in plasma and CSF, neurologic function, infiltrative lung disease and systemic disease parameters. Exploratory endpoints included changes in brain volume assessed with volumetric MRI using tensor-based morphometry, and resting functional MRI analysis of regional brain activity and connectivity between resting state networks. Mean (SD) plasma venglustat AUC0-24 on Day 1 was 851 (282) ng•h/ml; Cmax of 58.1 (26.4) ng/ml was achieved at a median tmax 2.00 h. After once-daily venglustat, plasma concentrations (4 h post-dose) were higher compared with Day 1, indicating ∼2-fold accumulation. One participant (Patient 9) had low-to-undetectable venglustat exposure at Weeks 26 and 52. Based on mean plasma and CSF venglustat concentrations (excluding Patient 9), steady state appeared to be reached on or before Week 4. Mean (SD) venglustat concentration at Week 52 was 114 (65.8) ng/ml in plasma and 6.14 (3.44) ng/ml in CSF. After 1 year of treatment, median (inter-quartile range) glucosylceramide decreased 78% (72, 84) in plasma and 81% (77, 83) in CSF; median (inter-quartile range) glucosylsphingosine decreased 56% (41, 60) in plasma and 70% (46, 76) in CSF. Ataxia improved slightly in nine patients: mean (SD, range) total modified Scale for Assessment and Rating of Ataxia score decreased from 2.68 [1.54 (0.0 to 5.5)] at baseline to 1.55 [1.88 (0.0 to 5.0)] at Week 52 [mean change: -1.14 (95% CI: -2.06 to -0.21)]. Whole brain volume increased slightly in patients with venglustat exposure and biomarker reduction in CSF (306.7 ± 4253.3 mm3) and declined markedly in Patient 9 (-13894.8 mm3). Functional MRI indicated stronger connectivity at Weeks 26 and 52 relative to baseline between a broadly distributed set of brain regions in patients with venglustat exposure and biomarker reduction but not Patient 9, although neurocognition, assessed by Vineland II, deteriorated in all domains over time, which illustrates disease progression despite the intervention. There were no deaths, serious adverse events or discontinuations. In adults with Gaucher disease type 3 receiving imiglucerase, addition of once-daily venglustat showed acceptable safety and tolerability and preliminary evidence of clinical stability with intriguing but intrinsically inconsistent signals in selected biomarkers, which need to be validated and confirmed in future research.

Inhibition of lysosomal acid β-glucosidase induces cell apoptosis via impairing mitochondrial clearance in pancreatic cancer

Sphingolipid metabolism plays an important role in the formation of cellular membranes and is associated with malignant potential and chemosensitivity of cancer cells. Sphingolipid degradation depends on multiple lysosomal glucosidases. We focused on acid β-glucosidase (GBA), a lysosomal enzyme the deficiency of which is related to mitochondrial dysfunction. We analyzed the function of GBA in pancreatic ductal adenocarcinoma (PDAC). Human PDAC cell lines (PANC-1, BxPC-3 and AsPC-1) were examined under conditions of GBA knockdown via the short interfering RNA (siRNA) method. We assessed the morphological changes, GBA enzyme activity, GBA protein expression, cell viability, reactive oxygen species (ROS) generation, mitochondrial membrane potential (MMP) and mitophagy flux of PDAC cells. The GBA protein level and enzyme activity differed among cell lines. GBA knockdown suppressed cell proliferation and induced apoptosis, especially in PANC-1 and BxPC-3 cells, with low GBA enzyme activity. GBA knockdown also decreased the MMP and impaired mitochondrial clearance. This impaired mitochondrial clearance further induced dysfunctional mitochondria accumulation and ROS generation in PDAC cells, inducing apoptosis. The antiproliferative effects of the combination of GBA suppression and gemcitabine were higher than those of gemcitabine alone. These results showed that GBA suppression exerts a significant antitumor effect and may have therapeutic potential in the clinical treatment of PDAC.

Acid β-glucosidase insufficiency activates the C-X-C Motif Chemokine Ligand 9/CXCR3 axis leading to T cell mediated inflammation in Gaucher disease

Abstract Gaucher disease (GD) is caused by GBA1 mutations that lead to decreased activity of lysosomal acid β-glucosidase and abnormal tissue accumulation of its substrate, glucosylceramide (GC). Monocyte lineage cells, e.g., macrophages (Mφs) and dendritic cells (DCs) are prominent disease effectors due to their massive accumulation of GC resulting in “Gaucher cells.” Interaction of Gaucher cells and T lymphocytes trigger massive secretion of IFNγ, TNFα, IL6, IL12p40, IL12p70, IL23, and IL17A/F, that leads to the tissue destruction in GD. The exact mechanisms that trigger excess tissue T cells recruitment in GD are still unclear. CXCR3+ T cells are enriched at inflammatory sites in patients with rheumatoid arthritis and multiple sclerosis. Using D409V/null (Gba19V/−) GD mouse model, this study uncovered the role of CXCR3 receptor and its C-X-C Motif Chemokine Ligand 9 (CXCL9) in induction of excess tissue recruitment of T cells in GD. Intracellular FACS staining of Mφs and DCs from Gba19V/− mice showed elevated amounts of CXCL9. CD4+ T and the CD8+ T cells purified from Gba19V/− mice showed increased amounts of CXCR3. Ex vivo and in vivo chemotaxis experiments, evaluated CXCL9’s recruitment of Gba19V/− T cells. Blockade of T-cell CXCL9 receptor using antibodies to CXCR3 caused marked reduction in their CXCL9-mediated chemotaxis in Gba19V/− mice. These data suggest that Gba1 defects and the resultant deficiency of acid β-glucosidase activate CXCL9-CXCR3 axis for enhanced tissue Tells recruitment of T cells in GD. Collectively, our results indicate a critical role for CXCR3 mediated T cell transmigration to sites of inflammation. Molecular targeting of CXCL9/CXCR3 axis may provide needed anti-inflammatory therapies in human patients with GD. Supported by Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229, USA

Mechanistic Insight into the Mode of Action of Acid β-Glucosidase Enhancer Ambroxol.

Ambroxol (ABX) is a mucolytic agent used for the treatment of respiratory diseases. Bioactivity has been demonstrated as an enhancement effect on lysosomal acid β-glucosidase (β-Glu) activity in Gaucher disease (GD). The positive effects observed have been attributed to a mechanism of action similar to pharmacological chaperones (PCs), but an exact mechanistic description is still pending. The current study uses cell culture and in vitro assays to study the effects of ABX on β-Glu activity, processing, and stability upon ligand binding. Structural analogues bromohexine, 4-hydroxybromohexine, and norbromohexine were screened for chaperone efficacy, and in silico docking was performed. The sugar mimetic isofagomine (IFG) strongly inhibits β-Glu, while ABX exerts its inhibitory effect in the micromolar range. In GD patient fibroblasts, IFG and ABX increase mutant β-Glu activity to identical levels. However, the characteristics of the banding patterns of Endoglycosidase-H (Endo-H)-digested enzyme and a substantially lower half-life of ABX-treated β-Glu suggest different intracellular processing. In line with this observation, IFG efficiently stabilizes recombinant β-Glu against thermal denaturation in vitro, whereas ABX exerts no significant effect. Additional β-Glu enzyme activity testing using Bromohexine (BHX) and two related structures unexpectedly revealed that ABX alone can refunctionalize β-Glu in cellula. Taken together, our data indicate that ABX has little in vitro ability to act as PC, so the mode of action requires further clarification.

C-X-C Motif Chemokine Ligand 9 and Its CXCR3 Receptor Are the Salt and Pepper for T Cells Trafficking in a Mouse Model of Gaucher Disease.

Gaucher disease is a lysosomal storage disease, which happens due to mutations in GBA1/Gba1 that encodes the enzyme termed as lysosomal acid β-glucosidase. The major function of this enzyme is to catalyze glucosylceramide (GC) into glucose and ceramide. The deficiency of this enzyme and resultant abnormal accumulation of GC cause altered function of several of the innate and adaptive immune cells. For example, augmented infiltration of T cells contributes to the increased production of pro-inflammatory cytokines, (e.g., IFNγ, TNFα, IL6, IL12p40, IL12p70, IL23, and IL17A/F). This leads to tissue damage in a genetic mouse model (Gba19V/−) of Gaucher disease. The cellular mechanism(s) by which increased tissue infiltration of T cells occurs in this disease is not fully understood. Here, we delineate role of the CXCR3 receptor and its exogenous C-X-C motif chemokine ligand 9 (CXCL9) in induction of increased tissue recruitment of CD4+ T and CD8+ T cells in Gaucher disease. Intracellular FACS staining of macrophages (Mϕs) and dendritic cells (DCs) from Gba19V/− mice showed elevated production of CXCL9. Purified CD4+ T cells and the CD8+ T cells from Gba19V/− mice showed increased expression of CXCR3. Ex vivo and in vivo chemotaxis experiments showed CXCL9 involvement in the recruitment of Gba19V/− T cells. Furthermore, antibody blockade of the CXCL9 receptor (CXCR3) on T cells caused marked reduction in CXCL9- mediated chemotaxis of T cells in Gba19V/− mice. These data implicate abnormalities of the CXCL9-CXCR3 axis leading to enhanced tissue recruitment of T cells in Gaucher disease. Such results provide a rationale for blockade of the CXCL9/CXCR3 axis as potential new therapeutic targets for the treatment of inflammation in Gaucher disease.

Long-Term Hematologic Response to Oral Eliglustat in Treatment-Naive Patients with Gaucher Disease Type 1: Final Results from a Phase 2 and the Phase 3 Engage Trials

Introduction:Gaucher disease is caused by deficient activity of the lysosomal enzyme acid β-glucosidase, leading to accumulation of glucosylceramide primarily in macrophages (Gaucher cells). Thrombocytopenia, anemia, hepatosplenomegaly, and skeletal disease are common presenting symptoms and hematologists often identify and manage the disease. Eliglustat is an oral substrate reduction therapy approved as first-line treatment for adults with Gaucher disease type 1 (GD1) who have poor, intermediate, or extensive CYP2D6-metabolizer phenotypes (>90% of patients). Unlike enzyme replacement therapy (the historic standard of care), which supplements lysosomal acid β-glucosidase in mononuclear phagocytes and is administered in biweekly infusions, oral eliglustat reduces glucosylceramide accumulation by partially inhibiting glucosylceramide synthase.Methods:We report on improvements in hemoglobin concentration and platelet count in two completed clinical trials of eliglustat in treatment-naïve adults with GD1: an open-label Phase 2 trial (N=26, NCT00358150), for which 8-year data are available, and a randomized, placebo-controlled Phase 3 trial (ENGAGE, N=40, NCT00891202) for which 4.5-year data are available. Both trials were sponsored by Sanofi Genzyme.Results:In the Phase 2 trial, the primary endpoint was met after 1 year, with 77% of patients improving in at least 2 of 3 main efficacy parameters (spleen volume, hemoglobin level, and platelet count) (Lukina et al. Blood 2010;116:816). Improvements continued or were maintained throughout the trial. Among patients with 8 years of data (n=19), mean (±SD) hemoglobin level and mean platelet count increased by 2.1±1.7 g/dL (from 11.3±1.6 to 13.4±1.3 g/dL) and 110% (from 67.5±21.1 to 130.7±59.8 x109/L), respectively, after 8 years of eliglustat. In the Phase 3 ENGAGE trial, all primary and secondary endpoints were met, with clinically meaningful improvements from baseline in platelet and hemoglobin levels and spleen and liver volumes at 9 months in eliglustat- but not placebo-treated patients (Mistry et al. JAMA 2015;313;695). During the trial extension, in which all patients received eliglustat, patients switching from placebo had the same magnitude and time course of improvement during the first 9 months as was seen with eliglustat patients in the primary analysis and all patients continued to improve over time with continued eliglustat treatment. Among patients with 4.5 years of eliglustat treatment (n=14), mean (±SD) hemoglobin level increased by 1.4±1.31 g/dL (from 12.0±2.0 g/dL to 13.4±1.9 g/dL) and mean platelet count increased by 87% (from 69.6±18.5 to 122.6±34.1 x109/L). In both trials, hematologic improvements were accompanied by clinically significant decreases in spleen and liver volumes, improvements in bone parameters, and substantive reductions in Gaucher disease biomarkers. In both trials, eliglustat was generally well-tolerated, with three withdrawals due to adverse events in the Phase 2 trial (one of which was considered related to eliglustat) and no withdrawals due to adverse events during the ENGAGE trial. Most adverse events in both trials were non-serious, considered unrelated to eliglustat, and either mild or moderate in severity.Conclusions:In previously untreated patients with GD1, clinically meaningful improvements in hematologic parameters continued or were maintained over time after 8 years of treatment with eliglustat in the Phase 2 trial and after 4.5 years of eliglustat in the Phase 3 ENGAGE trial. DisclosuresLau:Sanofi Genzyme: Other: Principal Investigator in the eliglustat Phase 2 and ENGAGE clinical trials . Lukina:Sanofi Genzyme: Honoraria, Other: Principal investigator in the eliglustat Phase 2, ENGAGE, ENCORE, and EDGE clinical trials; receives travel reimbursement; Shire: Honoraria, Other: travel reimbursement. Mistry:Sanofi Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Principal investigator in the eliglustat ENGAGE and ENCORE clinical trials; received travel reimbursement, Research Funding. Wu:Sanofi Genzyme: Employment. Gaemers:Sanofi Genzyme: Employment. Peterschmitt:Sanofi Genzyme: Employment.

Gaucher’s Disease with Rare Genotype- A Case Report

Gaucher’s Disease (GD) is a rare inherited Lysosomal Storage Disorder (LSD) caused by autosomal recessive inheritance of homozygous mutations in the Glucocerebrosidase (GBA) gene encoding the lysosomal enzyme acid β-glucosidase. GD is usually under-diagnosed in developing countries due to limitations in diagnostic resources for genetic evaluation. However, under such circumstances a meticulous Bone Marrow Examination (BME) and clinical correlation gives clue to the diagnosis of GD. Hence, the authors here report a case of 2-years-old male child who presented with abdominal distension and fullness with refractory anaemia for which he was transfused blood in the past. The ultrasound sonography revealed disproportionate hepatosplenomegaly. The clinical differential diagnoses were haematolymphoid malignancy and haemolytic anaemia. Haemoglobin (Hb) electrophoresis revealed no structurally abnormal Hb while the BME revealed hallmark Gaucher’s cells. To confirm the diagnosis of GD, enzymatic assay was done which revealed reduced β (beta) glucosidase and elevated plasma chitotriosidase levels followed by genetic work up which revealed a rare genotype with compound heterozygous mutation in GBA gene having uncommon variants.

Effect of Expression of Human Glucosylceramidase 2 Isoforms on Lipid Profiles in COS-7 Cells.

Glucosylceramide (GlcCer) is a major membrane lipid and the precursor of gangliosides. GlcCer is mainly degraded by two enzymes, lysosomal acid β-glucosidase (GBA) and nonlysosomal β-glucosidase (GBA2), which may have different isoforms because of alternative splicing. To understand which GBA2 isoforms are active and how they affect glycosphingolipid levels in cells, we expressed nine human GBA2 isoforms in COS-7 cells, confirmed their expression by qRT-PCR and Western blotting, and assayed their activity to hydrolyze 4-methylumbelliferyl-β-D-glucopyranoside (4MUG) in cell extracts. Human GBA2 isoform 1 showed high activity, while the other isoforms had activity similar to the background. Comparison of sphingolipid levels by ultra-high resolution/accurate mass spectrometry (UHRAMS) analysis showed that isoform 1 overexpression increased ceramide and decreased hexosylceramide levels. Comparison of ratios of glucosylceramides to the corresponding ceramides in the extracts indicated that GBA2 isoform 1 has broad specificity for the lipid component of glucosylceramide, suggesting that only one GBA2 isoform 1 is active and affects sphingolipid levels in the cell. Our study provides new insights into how increased breakdown of GlcCer affects cellular lipid metabolic networks.

Biomarker Response to Oral Eliglustat in Adults with Gaucher Disease Type 1: Results from 4 Completed Clinical Trials

INTRODUCTION: Gaucher disease type 1 (GD1) is caused by hereditary deficiency in activity of lysosomal acid β-glucosidase and lysosomal dysfunction due to abnormal recycling and storage of glycosphingolipids especially in tissue macrophages. Clinical manifestations can include anemia, thrombocytopenia, hepatosplenomegaly, and debilitating bone disease. Hematologists care for most patients with GD1 but may be unaware of the importance of plasma biomarkers not only as tissue response surrogates that are useful for monitoring treatment efficacy but also as pathogenic molecules that can directly contribute to disease manifestations. These biomarkers include chitotriosidase, macrophage inflammatory protein 1β (MIP-1β), glucosylceramide, glucosylsphingosine, and monosialodihexosyl ganglioside (GM3). Here we show differing changes in biomarker levels after inception of eliglustat therapy in treatment-naïve GD1 patients and in patients previously treated with enzyme replacement therapy (ERT). Eliglustat is an oral inhibitor of glucosylceramide biosynthesis approved by FDA as a first-line treatment of adults with GD1 who have extensive, intermediate, or poor CYP2D6 metabolizer phenotypes (&gt;90% of patients). METHODS: We evaluated percent change from baseline in biomarker levels during time on eliglustat in the 4 completed clinical trials of oral eliglustat for GD1 adults (Sanofi Genzyme): Phase 2/NCT00358150 (treatment-naïve patients with moderate to severe baseline disease, N=26); Phase 3 ENGAGE/NCT00891202 (treatment-naïve patients with mild to moderate baseline disease, N=40); Phase 3 ENCORE/NCT00943111 (switch patients at prespecified therapeutic goals stabilized after a mean of 10 years of ERT], N=159); Phase 3 EDGE/NCT01074944 (mixture of treatment-naïve [N=22] and ERT-switch patients [N=148] with mild to moderate baseline disease and variable prior time on ERT). All percentages were calculated in the subset of patients with biomarkers measured at baseline and the reported timepoint. The attrition of patients with data at the longer-term time points is attributable primarily to a combination of 2-year enrollment periods, trial design, and protocol-mandated switches to commercial therapy for US patients. Most patients (81%) continued until eliglustat became commercially available or completion of their trial; 9 patients (2.3% overall) withdrew due to adverse events reported as drug-related. For Phase 2, ENGAGE, and ENCORE, chitotriosidase genotypes were obtained and values were normalized; patients homozygous for the CHIT null mutation were excluded and values for heterozygous patients were doubled. For EDGE patients, chitotriosidase genotype data were not obtained but patients with ≥4 zero values (n=12) were excluded. RESULTS: In treatment-naïve patients, all biomarkers were elevated at baseline. Median levels of glucosylceramide, GM3, and MIP-1β normalized within 1 year of starting eliglustat. Chitotriosidase and glucosylsphingosine levels markedly decreased but did not normalize. After 3-8 years on treatment, median percent reduction in chitotriosidase and glucosylsphingosine (Phase 2 and ENGAGE) was 85-92% (Table) but values did not normalize. In patients switching from ERT, median chitotriosidase was still modestly elevated at baseline. Median values for other biomarkers were normal or near normal. After 1 year of treatment, chitotriosidase decreased (Table). Median glucosylceramide, GM3, and MIP-1β remained normal or normalized. After 3-4 years, GM3 and MIP-1β remained normal. Median percent reduction in chitotriosidase from baseline was 60-63% but values did not normalize. Eliglustat was generally well-tolerated in all 4 trials; 97% of adverse events were mild/moderate and 86% considered unrelated to eliglustat by the investigator. CONCLUSIONS: Gaucher disease biomarkers decreased substantially in treatment-naïve patients after eliglustat treatment. In switch patients, including very stable patients after a mean of 10 years on ERT, chitotriosidase and MIP-1β also decreased substantively after eliglustat treatment. Future longitudinal follow-up of eliglustat-treated patients should indicate whether the biomarker and plasma glycosphingolipid responses reported here in stable patients at long-term therapeutic goals are associated with immunological and clinical improvements. Disclosures Weinreb: Shire HGT: Consultancy, Honoraria, Speakers Bureau; Sanofi Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer Corporation: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Cox:Sanofi Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Reimbursement, Research Funding. Mistry:Sanofi Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Reimbursement, Research Funding; NIH NIMS: Research Funding; Shire: Honoraria, Other: Travel Reimbursement, Research Funding; Pfizer: Honoraria, Other: Travel Reimbursement; Synageva: Honoraria, Other: Travel Reimbursement. Charrow:BioMarin: Research Funding; Shire: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amicus: Research Funding. Lukina:JSC GENERIUM: Research Funding; Sanofi Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Reimbursement, Research Funding; Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Reimbursement. Foster:Sanofi Genzyme: Employment. Peterschmitt:Sanofi Genzyme: Employment.

The Long-Term Adverse Event Profile of Oral Eliglustat for the Treatment of Gaucher Disease Type 1: Pooled Analysis of Data from 393 Patients in 4 Completed Trials

Background:Gaucher disease type 1 (GD1) is a multi-systemic disorder resulting from deficient activity of the lysosomal enzyme acid β-glucosidase leading to lysosomal accumulation of glucosylceramide (GL-1), primarily in macrophages (Gaucher cells). Thrombocytopenia, anemia, hepatosplenomegaly, and skeletal disease are common presenting symptoms, and hematologists often identify and manage the disease. Eliglustat, an oral substrate reduction therapy, is a first-line treatment for adults with GD1 who have extensive, intermediate, or poor CYP2D6 metabolizer phenotypes (>90% of patients). The clinical trial program for eliglustat is the largest to date for Gaucher disease, and encompasses 2 trials in treatment-naïve patients, 1 trial in stable patients switching to eliglustat after long-term enzyme replacement therapy (the historic standard of care), and a dose regimen study in mostly switch patients.Methods:We analyzed pooled treatment-emergent adverse event (AE) data from all 393 patients with GD1 from 29 countries who received at least 1 eliglustat dose in 4 completed clinical trials sponsored by Sanofi Genzyme: Phase 2 (NCT00358150, N=26), Phase 3 ENGAGE (NCT00891202, N=40), Phase 3 ENCORE (NCT00943111, N=157), and Phase 3 EDGE (NCT01074944, N=170).Results:Mean overall treatment duration in all 393 patients from the 4 completed trials was 3.6 years (maximum: 9.3 years), representing 1400 patient-years of eliglustat exposure. Overall, 81% (n=319) of patients remained in their trial until the availability of commercial eliglustat or study completion. Twenty-five (6%) patients withdrew due to AEs, including 9 patients (2%) with AEs considered related to eliglustat: mild thrombocytopenia (1 patient); mild ventricular tachycardia (1 patient); mild vertigo (1 patient); mild lethargy and mild exfoliative rash (1 patient); mild nausea, mild headache, and moderate anemia (1 patient); moderate arrhythmia (1 patient); moderate palpitations (1 patient); moderate gastroesophageal reflux disease and moderate dyspepsia (1 patient); severe upper abdominal pain (1 patient). Other reasons for study withdrawals were: patient wished to withdraw (n=25, 6%), pregnancy (n=15, 4%), noncompliance (n=3, 1%), and “other” (n=6, 2%). There were 2 on-treatment deaths, neither of which were considered related to eliglustat (downhill skiing accident and cardiac arrest due to hemorrhage after blunt abdominal trauma). Most AEs were mild or moderate (97%) in severity and considered unrelated to eliglustat (86%). Four AEs considered related to eliglustat were reported in ≥5% of patients: dyspepsia (6%), headache (5%), abdominal pain upper (5%), and dizziness (5%). Most of these common related AEs were mild or moderate, occurred only once per patient, and lasted less than 2 weeks. A total of 77 (20%) patients reported at least 1 serious AE, the majority of which were due to hospitalizations for intercurrent illnesses (e.g., appendicitis) and underlying diseases for which Gaucher patients are at increased risk (e.g., femur fracture, joint dislocation, hepatocellular carcinoma, and cholecystitis). Eight patients (2%) had at least 1 serious AE considered related by the investigator: ventricular tachycardia; atrioventricular block and atrioventricular block second degree; peripheral neuropathy; intestinal obstruction; syncope (2 patients); syncope and muscular weakness; arrhythmia. Most were mild or moderate, recovered/resolved, and did not lead to study withdrawal. Most reported cardiac AEs were findings detected in asymptomatic patients during protocol-defined periodic 24‐hour Holter monitoring, were classified as mild, and did not lead to study withdrawal. No clinically significant prolongations of the QTcF interval were observed during extensive electrocardiographic and Holter monitoring in the clinical studies. No differences were observed for the overall frequency of AEs by age group, CYP2D6 metabolizer phenotype, or upper 10th percentile for plasma eliglustat exposure. The proportion of patients reporting AEs decreased over time on eliglustat.Conclusions:This pooled analysis of AE data from one Phase 2 and three Phase 3 completed eliglustat trials demonstrates long-term safety and tolerability of eliglustat in the treatment of adults with GD1. DisclosuresPeterschmitt:Sanofi Genzyme: Employment. Freisens:Sanofi Genzyme: Employment. Hou:Sanofi Genzyme: Employment. Underhill:Sanofi Genzyme: Employment. Foster:Sanofi Genzyme: Employment. Gaemers:Sanofi Genzyme: Employment.

Long-Term Effects of Oral Eliglustat on Skeletal Manifestations of Gaucher Disease Type 1: Results from Four Completed Clinical Trials

Background:In Gaucher disease type 1 (GD1), deficient lysosomal acid β-glucosidase activity leads to accumulation of glucosylceramide and formation of lipid-laden macrophages known as Gaucher cells. Patients experience a range of disease manifestations including anemia, thrombocytopenia, hepatosplenomegaly, and debilitating bone complications that impair quality of life. Hematologists are central to disease recognition and management. Most patients, even those without overt symptoms of bone involvement, have radiologic evidence of bone disease. Eliglustat, an oral substrate-reduction therapy, is a first-line treatment for adults with GD1 who have extensive, intermediate or poor CYP2D6 metabolizer phenotypes (>90% of patients). Oral eliglustat partially inhibits glucosylceramide synthase, thereby reducing glucosylceramide accumulation, unlike enzyme replacement therapy (ERT), the historic standard of care for GD1, which augments acid β-glucosidase activity in mononuclear phagocytes and is administered in biweekly infusions.Methods:We analyzed bone mineral density T- and Z-scores and occurrence of bone pain and bone crises in 4 completed clinical trials sponsored by Sanofi Genzyme that involved 393 GD1 patients treated with eliglustat for 4-8 years: Phase 2 (NCT00358150), Phase 3 ENGAGE (NCT00891202), Phase 3 ENCORE (NCT00943111), and Phase 3 EDGE (NCT01074944). Healthy reference scores are >-1 for T-scores and >-2 for Z-scores. Bone marrow burden (BMB) scores and changes in macrophage inflammatory protein 1β (MIP-1β) were also analyzed in the 3 Phase 3 trials. BMB, an MRI-based scoring system to evaluate marrow infiltration of both the lumbar spine and femur, and MIP-1β, a biomarker produced by inflammatory phagocytes surrounding Gaucher cells, are associated with active and evolving bone disease in GD1.Results:In treatment-naïve patients (Phase 2, N=26; ENGAGE, N=40), mean spine T-scores moved from the osteopenic range (>-2.5 to ≤ -1) at baseline to the healthy reference range after 2-3 years of eliglustat, with continued improvement thereafter in both trials. Mean±SEM spine T-score increased from -1.55±0.28 to -0.59±0.34 (n=14; mean±SEM increase from baseline: 0.96±0.32), after 8 years in Phase 2, and increased to -0.53±0.27 in ENGAGE patients with bone data at 4.5 years (n=9; mean±SEM increase from baseline: 0.53±0.16). Spine Z-scores for both trials showed similar improvements; femur T- and Z-scores in both trails remained in the normal range. In the ENCORE trial of patients stabilized after a mean of 10 years of ERT (N=157), T- and Z-scores (spine and femur) remained in the reference ranges for up to 4 years; least square mean spine Z-scores improved by 0.29 (P<0.0001). In EDGE (N=170, mostly ERT switch), randomized patients stabilized on eliglustat during a 6- to 18-month Lead-In Period maintained normal T- and Z-scores (spine and femur) through the dose regimen arm of the study and the succeeding extension period. Mean total BMB scores improved from 10 (in the marked to severe range) to 8 (moderate range) in ENGAGE (treatment-naive patients), and remained stable at 8 throughout ENCORE and EDGE (switch/mostly switch patients). The proportion of patients experiencing recent (≤4 weeks) bone pain decreased after treatment in all 4 trials, and reported pain became less severe. No patient in Phase 2 or ENGAGE had a bone crisis while on eliglustat. Bone crises were reported in 3/157 (1.9%) ENCORE patients over the trial duration. In EDGE, the only trial to enroll patients with active bone disease, 6/170 (3.5%) patients had bone crises during the trial, 3 of whom had bone crises during the 6 months prior to trial entry. Bone crises decreased with increasing time on eliglustat, with only 1 bone crisis reported during the 2- to 3-year trial extension. In ENGAGE (treatment-naïve), median MIP-1β levels elevated 3-fold at baseline normalized. In ENCORE and EDGE (switch/mostly switch), MIP-1β levels remained in the normal range. Across the 4 trials, most adverse events were mild/moderate (97%) and considered unrelated to eliglustat (86%); 9 (2.3% overall) patients withdrew due to adverse events considered drug-related.Conclusions:Long-term eliglustat treatment induced continued improvement in bone parameters in treatment-naïve patients. These parameters remained stable in trials involving patients who had been previously treated. Eliglustat was generally well-tolerated. DisclosuresMistry:Sanofi Genzyme: Honoraria, Other: Principal Investigator for the eliglustat ENGAGE and ENCORE trials; ReceivedTravel Reimbursement; Member of the ICCG North American Advisory Board; , Research Funding. Charrow:Sanofi Genzyme: Honoraria, Other: Principal investigator, eliglustat EDGE trial; Member of advisory boards, Research Funding. Cox:Sanofi Genzyme: Honoraria, Other: Principal investigator for the eliglustat ENCORE trial; received travel reimbursement, Research Funding. Lukina:Sanofi Genzyme: Honoraria, Other: Principal investigator in the eliglustat Phase 2, ENGAGE, ENCORE, and EDGE trials; receives travel reimbursement; member of advisory boards, Research Funding. Marinakis:Sanofi Genzyme: Other: Principal investigator for the eliglustat EDGE trial, Research Funding. Foster:Sanofi Genzyme: Employment. Gaemers:Sanofi Genzyme: Employment. Peterschmitt:Sanofi Genzyme: Employment.

Glucosylceramide and Glucosylsphingosine Quantitation by Liquid Chromatography-Tandem Mass Spectrometry to Enable In Vivo Preclinical Studies of Neuronopathic Gaucher Disease.

Gaucher disease (GD) is caused by mutations in the GBA1 gene that encodes the lysosomal enzyme acid β-glucosidase (GCase). Reduced GCase activity primarily leads to the accumulation of two substrates, glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph). Current treatment options have not been shown to ameliorate the neurological pathology observed in the most severe forms of GD, clearly representing an unmet medical need. To better understand the relationship between GlcCer and GlcSph accumulation and ultimately their connection with the progression of neurological pathology, we developed LC-MS/MS methods to quantify GlcCer and GlcSph in mouse brain tissue. A significant challenge in developing these methods was the chromatographic separation of GlcCer and GlcSph from the far more abundant isobaric galactosyl epimers naturally occurring in white matter. After validation of both methods, we evaluated the levels of both substrates in five different GD mouse models, and found significant elevation of brain GlcSph in all five, while GlcCer was elevated in only one of the five models. In addition, we measured GlcCer and GlcSph levels in the brains of wild-type mice after administration of the GCase inhibitor conduritol β-epoxide (CBE), as well as the nonlysosomal β-glucosidase (GBA2) inhibitor N-butyldeoxygalactonojirimycin (NB-DGJ). Inhibition of GCase by CBE resulted in elevation of both sphingolipids; however, inhibition of GBA2 by NB-DGJ resulted in elevation of GlcCer only. Taken together, these data support the idea that GlcSph is a more selective and sensitive biomarker than GlcCer for neuronopathic GD in preclinical models.

GBA Analysis in Next-Generation Era: Pitfalls, Challenges, and Possible Solutions

Mutations in the gene encoding the lysosomal enzyme acid β-glucosidase (GBA) are responsible for Gaucher disease and represent the main genetic risk factor for developing Parkinson disease. In past years, next-generation sequencing (NGS) technology has been applied for the molecular analysis of the GBA gene, both as a single gene or as part of gene panels. However, the presence of complex gene-pseudogene rearrangements, resulting from the presence of a highly homologous pseudogene (GBAP1) located downstream of the GBA gene, makes NGS analysis of GBA challenging. Therefore, adequate strategies should be adopted to avoid misdetection of GBA recombinant mutations. Here, we validated a strategy for the identification of GBA mutations using parallel massive sequencing and provide an overview of the major drawbacks encountered during GBA analysis by NGS. We implemented a NGS workflow, using a set of 38 patients with Gaucher disease carrying different GBA alleles identified previously by Sanger sequencing. As expected, the presence of the pseudogene significantly affected data output. However, the combination of specific procedures for the library preparation and data analysis resulted in maximal repeatability and reproducibility, and a robust performance with 97% sensitivity and 100% specificity. In conclusion, the pipeline described here represents a useful approach to deal with GBA sequencing using NGS technology.

Spotlight on taliglucerase alfa in the treatment of pediatric patients with type 1 Gaucher disease.

Gaucher disease (GD) is a heritable storage disorder caused by functional defects of the lysosomal acid β-glucosidase and the accumulation of glucosylceramide within macrophages, resulting in multiple organ dysfunction. There are three commercially available enzyme replacement therapy (ERT) products for the treatment of GD type 1 (GD1): imiglucerase, velaglucerase alfa, and taliglucerase alfa. Imiglucerase and velaglucerase alfa are produced in different mammalian cell systems; imiglucerase requires postproduction deglycosylation to expose terminal α-mannose residues, which are required for mannose receptor-mediated uptake by target macrophages. These steps are critical to the success of ERT for the treatment of visceral and hematologic manifestations of GD. Taliglucerase alfa is the first US Food and Drug Administration-approved plant-cell-expressed recombinant human protein, using carrot root cell cultures. Furthermore, it does not require postproduction glycosidic modifications. It is indicated for treatment of adults with GD1 in the US, Israel, Australia, Canada, Chile, Brazil, and other countries, and it is additionally approved for the treatment of pediatric patients in the US, Australia, and Canada and for the treatment of hematologic manifestations in pediatric patients with Type 3 GD in Canada and other countries. Our review focuses on the role of taliglucerase alfa in the pediatric population. A literature search through PubMed (from 1995 up till November 2016) of English language articles was performed with the following terms: Gaucher disease, lysosomal storage disease, taliglucerase. Secondary and tertiary references were obtained by reviewing related articles as well as the website www.Clinicaltrials.gov. It has been demonstrated that taliglucerase alfa is efficacious, with a well-established safety profile in pediatric, ERT-naïve patients with symptomatic GD1, as well as for those patients previously treated with imiglucerase.

A rare form of Gaucher disease resulting from saposin C deficiency

Gaucher disease is mainly caused by the deficiency of lysosomal acid β-glucosidase. Gaucher disease caused by the deficiency of saposin C is rare. Here we report a patient mainly presenting with hepatosplenomegaly, thrombocytopenia and anemia. EEG examination revealed increased theta waves. Gaucher cells identified in his bone marrow and the highly elevated plasma chitotriosidase activity and glucosylsphingosine supported a diagnosis of Gaucher disease. However, the leukocyte β-glucosidase activity was in a normal range. Sanger sequencing revealed a novel maternal exonic mutation c.1133C>G (p.Pro378Arg) in exon 10 of the PSAP gene, which codes the Sap C domain of PSAP protein. To search for other underlying mutations in this patient, whole genome sequencing was applied and revealed a deletion involving exon 2 to 7 of PSAP gene. The deletion appears as a de novo event on paternal chromosome. We concluded that biallelic mutations of PSAP gene were the cause of this patient's Gaucher disease. Our finding expands the mutation spectrum of Gaucher disease with saposin C deficiency.

Identification of a feedback loop involving β-glucosidase 2 and its product sphingosine sheds light on the molecular mechanisms in Gaucher disease

The lysosomal acid β-glucosidase GBA1 and the non-lysosomal β-glucosidase GBA2 degrade glucosylceramide (GlcCer) to glucose and ceramide in different cellular compartments. Loss of GBA2 activity and the resulting accumulation of GlcCer results in male infertility, whereas mutations in the GBA1 gene and loss of GBA1 activity cause the lipid-storage disorder Gaucher disease. However, the role of GBA2 in Gaucher disease pathology and its relationship to GBA1 is not well understood. Here, we report a GBA1-dependent down-regulation of GBA2 activity in patients with Gaucher disease. Using an experimental approach combining cell biology, biochemistry, and mass spectrometry, we show that sphingosine, the cytotoxic metabolite accumulating in Gaucher cells through the action of GBA2, directly binds to GBA2 and inhibits its activity. We propose a negative feedback loop, in which sphingosine inhibits GBA2 activity in Gaucher cells, preventing further sphingosine accumulation and, thereby, cytotoxicity. Our findings add a new chapter to the understanding of the complex molecular mechanism underlying Gaucher disease and the regulation of β-glucosidase activity in general.

Stability is maintained in adults with Gaucher disease type 1 switched from velaglucerase alfa to eliglustat or imiglucerase: A sub-analysis of the eliglustat ENCORE trial

Gaucher disease type 1 is an autosomal recessive disorder caused by deficient activity of the lysosomal enzyme acid β-glucosidase resulting in accumulation of glucosylceramide and clinical manifestations of anemia, thrombocytopenia, hepatosplenomegaly, and skeletal disease. The historic standard of care is intravenous recombinant enzyme therapy with imiglucerase. Eliglustat, an oral substrate reduction therapy, is a first-line treatment for adults with Gaucher disease type 1 who have a compatible CYP2D6-metabolizer phenotype (≈95% of patients). The 12-month ENCORE trial (NCT00943111) found eliglustat non-inferior to imiglucerase in maintaining stability in adult Gaucher patients previously stabilized after ≥3years of enzyme therapy (imiglucerase or velaglucerase alfa). This post-hoc analysis examined safety and efficacy in the 30 ENCORE patients who were receiving velaglucerase alfa at study entry and were randomized to eliglustat (n=22) or imiglucerase (n=8). Efficacy and safety in velaglucerase alfa-transitioned patients were consistent with the full ENCORE trial population; 90% of patients switched to eliglustat and 88% of patients switched to imiglucerase met the composite endpoint (stable hemoglobin concentration, platelet count, spleen volume, and liver volume). Clinical stability was maintained for 12months in Gaucher disease type 1 patients in the ENCORE trial who switched from velaglucerase alfa to either eliglustat or imiglucerase.

Risk factors for fracture in imiglucerase-treated Gaucher disease type 1 patients in the ICGG Gaucher Registry

heterogeneous, inborn error of metabolism caused by a deficiency of the lysosomal enzyme acid β-glucosidase, leading to accumulation of glucosylceramide within lysosomes of tissue macrophages in multiple organs. ► Disease manifestations are multisystemic and include splenomegaly, hepatomegaly, anemia, thrombocytopenia, diverse skeletal lesions, bone pain and bone crises. ► Enzyme replacement therapy (ERT) with exogenous acid βglucosidase is standard of care, and can improve symptoms.1,2 and radiographic abnormalities seen in GD patients.3 Established bone manifestations of GD, however, continue to be of concern.3-5 Risk Factors for Fracture in Imiglucerase-treated Gaucher Disease Type 1 Patients in the ICGG Gaucher Registry Patrick Deegan1, Julie L. Batista2 1Lysosomal Disorders Unit, Department of Medicine, Addenbrooke's Hospital, Cambridge, United Kingdom; 2Sanofi Genzyme, Cambridge, MA, USA