Journal: Molecular Genetics and Metabolism Reports

Article Title: Intravenous delivery of a chemically modified sulfamidase efficiently reduces heparan sulfate storage and brain pathology in mucopolysaccharidosis IIIA mice

doi: 10.1016/j.ymgmr.2019.100510

Figure Lengend Snippet: Structural characteristics of chemically modified glycans of CM-rhSulfamidase (A) Example of predicted bond breaks on mannose after periodate oxidation and borohydride reduction. (B) Mass spectra prior and after chemical modification. Mass spectra of doubly charged ion(s) corresponding to tryptic peptide T13, NITR, with Man-6 glycan attached to N131. S=Single bond break; D = double (two) bond breaks, e.g. S. × 3 = 3 single bond breaks. A: Prior to chemical modification. B, C, D: After chemical modification of three batches of CM-rhSulfamidase.

Article Snippet: Concentrations of CM-rhSulfamidase and rhSulfamidase and presence of antibodies against CM-rhSulfamidase, i.e. anti-drug antibodies (ADA), in serum, were determined by Meso Scale Discovery electrochemiluminescence (MSD-ECL) based bridging format immunoassays on MSD Multi-array streptavidin coated plates.

Techniques: Modification, Glycoproteomics

Journal: Molecular Genetics and Metabolism Reports

Article Title: Intravenous delivery of a chemically modified sulfamidase efficiently reduces heparan sulfate storage and brain pathology in mucopolysaccharidosis IIIA mice

doi: 10.1016/j.ymgmr.2019.100510

Figure Lengend Snippet: In vitro characterization of CM-rhSulfamifdase and rhSulfamidase in primary MPS IIIA patient fibroblasts. (A) Endocytosis rate as a function of concentration. (B) M6P inhibition of endocytosis. (C) Concentration-dependent reduction of HS storage. (D) Intracellular retention and stability.

Article Snippet: Concentrations of CM-rhSulfamidase and rhSulfamidase and presence of antibodies against CM-rhSulfamidase, i.e. anti-drug antibodies (ADA), in serum, were determined by Meso Scale Discovery electrochemiluminescence (MSD-ECL) based bridging format immunoassays on MSD Multi-array streptavidin coated plates.

Techniques: In Vitro, Concentration Assay, Inhibition

Journal: Molecular Genetics and Metabolism Reports

Article Title: Intravenous delivery of a chemically modified sulfamidase efficiently reduces heparan sulfate storage and brain pathology in mucopolysaccharidosis IIIA mice

doi: 10.1016/j.ymgmr.2019.100510

Figure Lengend Snippet: Plasma concentration versus time following i.v. administration of 12 mg/kg rhSulfamidase or CM-rhSulfamidase in WT mice. Log-linear mean plasma concentration versus time profiles of rhSulfamidase and CM-rhSulfamidase in male C57BL/6 mice following a single 12 mg/kg i.v. dose ( n = 3 samples per time point; Standard deviation error bars are included but are smaller than the symbol size).

Article Snippet: Concentrations of CM-rhSulfamidase and rhSulfamidase and presence of antibodies against CM-rhSulfamidase, i.e. anti-drug antibodies (ADA), in serum, were determined by Meso Scale Discovery electrochemiluminescence (MSD-ECL) based bridging format immunoassays on MSD Multi-array streptavidin coated plates.

Techniques: Clinical Proteomics, Concentration Assay, Standard Deviation

Journal: Molecular Genetics and Metabolism Reports

Article Title: Intravenous delivery of a chemically modified sulfamidase efficiently reduces heparan sulfate storage and brain pathology in mucopolysaccharidosis IIIA mice

doi: 10.1016/j.ymgmr.2019.100510

Figure Lengend Snippet: PK parameters of rhSulfamidase and CM-rhSulfamidase dosed 12 mg/kg i.v. in C57BL/6 mice.

Article Snippet: Concentrations of CM-rhSulfamidase and rhSulfamidase and presence of antibodies against CM-rhSulfamidase, i.e. anti-drug antibodies (ADA), in serum, were determined by Meso Scale Discovery electrochemiluminescence (MSD-ECL) based bridging format immunoassays on MSD Multi-array streptavidin coated plates.

Techniques:

Journal: Molecular Genetics and Metabolism Reports

Article Title: Intravenous delivery of a chemically modified sulfamidase efficiently reduces heparan sulfate storage and brain pathology in mucopolysaccharidosis IIIA mice

doi: 10.1016/j.ymgmr.2019.100510

Figure Lengend Snippet: Effect of CM-rhSulfamidase on HS levels in CNS of MPS IIIA mice. Relative levels of HS and a HS-derived tetrasaccharide, GlcNS-UA-GlcNAc-UA (+1S), in CSF and brain homogenate samples from MPS IIIA mice at 29 weeks of age after i.v. administration of vehicle or CM-rhSulfamidase at 3, 6, 12 or 17 mg/kg once weekly for 20 weeks ( n = 2–8). Naïve MPS IIIA and WT mice were included as controls. (A) Results are shown as group mean (± SEM). One-way ANOVA and post hoc Dunnett's test versus vehicle; * p < .05, ** p < .01, *** p < .001, and **** p < .0001. (B) Individual data are presented. Pearson's correlation is illustrated with r 2 and p values in the figure.

Article Snippet: Concentrations of CM-rhSulfamidase and rhSulfamidase and presence of antibodies against CM-rhSulfamidase, i.e. anti-drug antibodies (ADA), in serum, were determined by Meso Scale Discovery electrochemiluminescence (MSD-ECL) based bridging format immunoassays on MSD Multi-array streptavidin coated plates.

Techniques: Derivative Assay

Journal: Molecular Genetics and Metabolism Reports

Article Title: Intravenous delivery of a chemically modified sulfamidase efficiently reduces heparan sulfate storage and brain pathology in mucopolysaccharidosis IIIA mice

doi: 10.1016/j.ymgmr.2019.100510

Figure Lengend Snippet: HS levels in brain of MPS IIIA mice 24 h, 8 and 16 weeks after discontinuation of treatment with CM-rhSulfamidase. Relative levels of HS in CSF and brain homogenate samples from male MPS IIIA mice at 30 to 46 weeks of age after i.v. administration of vehicle or CM-rhSulfamidase at 22 mg/kg once weekly for 20 weeks ( n = 3–8). Results are shown as group mean (± SEM).

Article Snippet: Concentrations of CM-rhSulfamidase and rhSulfamidase and presence of antibodies against CM-rhSulfamidase, i.e. anti-drug antibodies (ADA), in serum, were determined by Meso Scale Discovery electrochemiluminescence (MSD-ECL) based bridging format immunoassays on MSD Multi-array streptavidin coated plates.

Techniques:

Journal: Molecular Genetics and Metabolism Reports

Article Title: Intravenous delivery of a chemically modified sulfamidase efficiently reduces heparan sulfate storage and brain pathology in mucopolysaccharidosis IIIA mice

doi: 10.1016/j.ymgmr.2019.100510

Figure Lengend Snippet: Autofluorescing intracellular inclusions in brain of male MPS IIIA mice at 19 weeks of age after i.v. administration of vehicle or CM-rhSulfamidase at 6 or 18 mg/kg once weekly for 10 weeks ( n = 5–6). Naïve MPS IIIA and WT mice were included as controls. (A) Representative photomicrographs from the cerebral cortex layer IV/V acquired at 10× (numerical aperture N.A. 0.45) using long exposure (600 ms) epifluorescence microscopy. Scale bar 100 μm. Signal intensities shown in a pseudocolor scale (0–255). (B) AF inclusions quantified as sum of signal intensity arbitrary unit (a.u.) per area unit in cortex, hippocampus and cerebellum. Results are shown as group mean (± SEM) sum of signal per mm 2 . One-way ANOVA and post hoc Dunnett's test versus vehicle; * p < .05, ** p < .01, *** p < .001, and **** p < .0001. .

Article Snippet: Concentrations of CM-rhSulfamidase and rhSulfamidase and presence of antibodies against CM-rhSulfamidase, i.e. anti-drug antibodies (ADA), in serum, were determined by Meso Scale Discovery electrochemiluminescence (MSD-ECL) based bridging format immunoassays on MSD Multi-array streptavidin coated plates.

Techniques: Epifluorescence Microscopy

Journal: Molecular Genetics and Metabolism Reports

Article Title: Intravenous delivery of a chemically modified sulfamidase efficiently reduces heparan sulfate storage and brain pathology in mucopolysaccharidosis IIIA mice

doi: 10.1016/j.ymgmr.2019.100510

Figure Lengend Snippet: Immunohistochemical detection of the lysosomal integral membrane protein (LIMPII) in MPS IIIA mouse brain after repeated i.v. administration of vehicle or CM-rhSulfamidase. Naïve MPS IIIA and WT mice were included as controls. (A) Representative photomicrographs from the hippocampal cell layer in male mice treated with CM-rhSulfamidase at 22 mg/kg for 20 weeks ( n = 8). Paraffin sections counterstained with hematoxylin acquired at 20× (numerical aperture N.A. 0.40). Scale bar 40 μm. (B) LIMPII-positivity quantifications from cortex, hippocampus and cerebellum in male mice treated with CM-rhSulfamidase at 6 or 18 mg/kg for 10 weeks ( n = 5–6), using image segmentation analysis. Results are shown as group mean (± SEM) of object density per mm 2 . One-way ANOVA and post hoc Dunnett's test versus vehicle; * p < .05, ** p < .01, *** p < .001, and **** p < .0001.

Article Snippet: Concentrations of CM-rhSulfamidase and rhSulfamidase and presence of antibodies against CM-rhSulfamidase, i.e. anti-drug antibodies (ADA), in serum, were determined by Meso Scale Discovery electrochemiluminescence (MSD-ECL) based bridging format immunoassays on MSD Multi-array streptavidin coated plates.

Techniques: Immunohistochemical staining, Membrane

Journal: Molecular Genetics and Metabolism Reports

Article Title: Intravenous delivery of a chemically modified sulfamidase efficiently reduces heparan sulfate storage and brain pathology in mucopolysaccharidosis IIIA mice

doi: 10.1016/j.ymgmr.2019.100510

Figure Lengend Snippet: Neuroinflammation in the MPS IIIA mouse brain after repeated i.v. administration of vehicle or CM-rhSulfamidase. Naïve MPS IIIA and WT mice were included as controls. (A) Representative bright field photomicrographs from the cortex in male mice treated with CM-rhSulfamidase at 22 mg/kg for 20 weeks ( n = 8). Paraffin sections counterstained with hematoxylin acquired at 20× (numerical aperture N.A. 0.40). Scale bar 50 μm. (B—C) Quantification of GFAP positive astrocytes and CD11b positive microglia in cortex and hippocampus in male mice treated with CM-rhSulfamidase at 7 mg/kg for 25 weeks ( n = 8). Results are shown as group mean values (± SEM) of object density per mm 2 (GFAP) or mean object size of each positive object (CD11b). One-way ANOVA and post hoc Dunnett's test versus vehicle; * p < .05, ** p < .01, *** p < .001, and **** p < .0001.

Article Snippet: Concentrations of CM-rhSulfamidase and rhSulfamidase and presence of antibodies against CM-rhSulfamidase, i.e. anti-drug antibodies (ADA), in serum, were determined by Meso Scale Discovery electrochemiluminescence (MSD-ECL) based bridging format immunoassays on MSD Multi-array streptavidin coated plates.

Techniques:

Journal: Molecular Genetics and Metabolism Reports

Article Title: Intravenous delivery of a chemically modified sulfamidase efficiently reduces heparan sulfate storage and brain pathology in mucopolysaccharidosis IIIA mice

doi: 10.1016/j.ymgmr.2019.100510

Figure Lengend Snippet: Effect of CM-rhSulfamidase on behavioral impairments in MPS IIIA mice. (A) Exploratory activity assessed by the open field test using male mice from Study C at 27 to 28 weeks of age. Graphs illustrate active time (speed >5 m/s), hyperactive time (speed >15 m/s), total distance travelled, time spent in the periphery (thigmotaxis), rearing number, and time spent rearing. A total experiment duration of 5 min was used. (B) Neuromuscular function assessed by the wire hanging test using male mice from Study D at 34 weeks of age. Graph illustrates total time the animals were able to hold themselves on the wire grid. The result was the same if body weight was taken into account. (C) Social ability assessed by the three-chamber social interaction test using male mice from Study C at 29 to 30 weeks of age. Graphs illustrate the delta of the interaction duration during the social approach (stranger versus empty) and social novelty (stranger versus familiar) sessions of the test. An experiment duration of 10 min was used for the different sessions. (D) Learning ability assessed by the Barnes maze test using male mice from Study C at 28 to 29 weeks of age. Graphs illustrate the latency to find the target hole per day during the learning trials of the test and relative duration at the target hole during the probe trial (PT). Results are shown as group mean (± SEM) of n = 8–16. One- or two-way ANOVA and post hoc Dunnett's test versus vehicle; * p < .05, ** p < .01, and *** p < .001.

Article Snippet: Concentrations of CM-rhSulfamidase and rhSulfamidase and presence of antibodies against CM-rhSulfamidase, i.e. anti-drug antibodies (ADA), in serum, were determined by Meso Scale Discovery electrochemiluminescence (MSD-ECL) based bridging format immunoassays on MSD Multi-array streptavidin coated plates.

Techniques: Activity Assay

Journal: Molecular Genetics and Metabolism Reports

Article Title: Intravenous delivery of a chemically modified sulfamidase efficiently reduces heparan sulfate storage and brain pathology in mucopolysaccharidosis IIIA mice

doi: 10.1016/j.ymgmr.2019.100510

Figure Lengend Snippet: CM-rhSulfamidase concentration ratio in brain homogenate versus serum at 24 h after the last of 20 i.v. doses given once weekly to MPS IIIA mice (Study B). Individual data with mean are presented.

Article Snippet: Concentrations of CM-rhSulfamidase and rhSulfamidase and presence of antibodies against CM-rhSulfamidase, i.e. anti-drug antibodies (ADA), in serum, were determined by Meso Scale Discovery electrochemiluminescence (MSD-ECL) based bridging format immunoassays on MSD Multi-array streptavidin coated plates.

Techniques: Concentration Assay

Journal: Molecular Genetics and Metabolism Reports

Article Title: Intravenous delivery of a chemically modified sulfamidase efficiently reduces heparan sulfate storage and brain pathology in mucopolysaccharidosis IIIA mice

doi: 10.1016/j.ymgmr.2019.100510

Figure Lengend Snippet: Immunohistological localization of Sulfamidase in the cerebral cortex of male MPS IIIA mice treated with vehicle or CM-rhSulfamidase at 6 or 18 mg/kg once weekly for 10 weeks ( n = 4–6). Naïve MPS IIIA mice and WT mice were included as controls. (A) Representative inverted greyscale photomicrographs of CM-rhSulfamidase fluorescent (−647) signal from the superficial cortical layers of a vehicle- or CM-rhSulfamidase-treated mouse (18 mg/kg). The arrowhead points to the strong meningeal signal and the arrow to an immunopositive arteriole. Scale bar 100 μm. (B) Quantification of strong signals classified to a “vascular” mask (left graph), weaker signal intensities classified as “parenchymal” (mid graph) and sum of vascular and parenchymal signals (right graph). (C) Quantification of signal above threshold localized to AF inclusions (left) and to an expanded rim from the AF masked objects (3 pixels ≈ 1.94 μm) as depicted schematically to the bottom right. All values normalized to the total AF object area ( i.e. mean sum of signal per autofluorescing area unit). Results are shown as group mean (± SEM). One-way ANOVA and post hoc Dunnett's test versus vehicle; * p < .05, *** p < .001, and **** p < .0001. One outlier was excluded from the vehicle group from all datasets based on Grubb's outlier analysis.

Article Snippet: Concentrations of CM-rhSulfamidase and rhSulfamidase and presence of antibodies against CM-rhSulfamidase, i.e. anti-drug antibodies (ADA), in serum, were determined by Meso Scale Discovery electrochemiluminescence (MSD-ECL) based bridging format immunoassays on MSD Multi-array streptavidin coated plates.

Techniques: