- Email: [email protected]
Distribution of chemically modified rhSulfamidase to CNS monitored by brain microdialysis and repeated CSF sampling after intravenous administration in rat
Abstracts / Molecular Genetics and Metabolism 126 (2019) S17–S156
effects of EGT and ABX on autophagy-lysosomal pathways were examined in various primary cells derived from patients with GD. Overall, both compounds increased GCase activity, but this effect was only observed in some patients. GCase expression analysis demonstrated elevated protein but not mRNA levels after ABX and EGT treatments. There was also an increase in autophagic vesicle formation and autophagosome accumulation as evidenced by changing levels of LC3A/BII. There was an increase in LysoTracker signal and mitochondria levels, but RT-PCR demonstrated no change in LC3 and LAMP1 in primary fibroblasts, PBMC and macrophages. Decreasing RNA levels of pro-inflammatory cytokines CCL17, IL10, IFNγ were observed in macrophages following both treatments, while ABX particularly inhibited CCL2 levels. While the small molecules, EGT and ABX have completely different mechanisms of action, they both lead to ALP activation and decreased proinflammatory cytokine levels that could be among the reasons for improved GCase activity in select cell lines.
doi:10.1016/j.ymgme.2018.12.191
176 Effects of genetic background on disease phenotypes in a mouse model of Fabry disease Siamak Jabbarzadeh-Tabrizia, Taniqua Daya, Mouna Tarouaa, Michel Boutinb, Christiane Auray-Blaisb, Raphael Schiffmanna, Jin-Song Shena, aBaylor Research Institute, Dallas, TX, United States, bFaculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada Knockout mouse model of Fabry disease (Ohshima, 1997) is widely used, and has been proven an invaluable tool to investigate disease pathogenesis and therapies. However, the importance of genetic background of this mouse line has been underestimated, and in many publications WT control mice were chosen incorrectly. Our aim was to examine the effects of genetic background on biochemical and functional abnormalities of Fabry mice. We characterized Fabry mice in B6/129 mixed background (~75% B6) and Fabry mice on a purely C57BL/6 strain (10 generations backcrossing), compared with WT mice on an identical genetic background. Both Fabry-B6/129 and Fabry-B6 developed progressive cardiac and renal hypertrophy and impaired thermosensation. However, Fabry-B6/129 exhibited earlier onset and more severe phenotypes than Fabry-B6. Cardiac and/or renal hypertrophy appeared at age of 6 months in Fabry-B6/129, but at 12 months in Fabry-B6. At 12 months, kidney-to-body weight ratio increased by 50% in Fabry-B6/129 (vs. WT), but only 15% in Fabry-B6. The onset of the thermosensation abnormality was at age 2 months in Fabry-B6/129 and 7 months in Fabry-B6. Cardiac expression of hypertrophy marker, atrial natriuretic peptide (ANP), was upregulated in Fabry-B6/129, but not in Fabry-B6. Despite these differences, globotriaosylceramide (Gb3) and lyso-Gb3 levels in the heart, kidney and dorsal root ganglia were similar between Fabry-B6/129 and Fabry-B6. However, Gb3 level in liver (organ unaffected in Fabry disease) was twofold higher in Fabry-B6/129 compared to Fabry-B6. Additionally, several parameters including body weight, organ weight, kidney Gb3 and cardiac ANP level were different between WT-B6/129 and WT-B6. In conclusion: 1) genetic background has a significant impact on the severity and onset of phenotypes in Fabry mice 2) the lack of correlation between phenotype and Gb3/lyso-Gb3 levels in disease-relevant organs
S79
suggests that the effect of genetic background on the phenotype is determined by other genetic and epigenetic factors.
doi:10.1016/j.ymgme.2018.12.192
177 Distribution of chemically modified rhSulfamidase to CNS monitored by brain microdialysis and repeated CSF sampling after intravenous administration in rat Juliette Jansona, Gudrun Anderssona, Lars Bergquista, Maria Erikssona, Joost H.A. Folgeringb, aSwedish Orphan Biovitrum, Stockholm, Sweden, b Charles River Laboratories, Groningen, Netherlands Mucopolysaccharidosis type IIIA (MPS IIIA) is an autosomal recessive disorder caused by absence or deficiency of the lysosomal enzyme sulfamidase. Glycans of recombinant human sulfamidase were chemically modified to generate CM-rhSulfamidase with retained stability and catalytic activity, but with significantly reduced uptake in peripheral tissues. For the enzyme to modify the neuropathology of MPS IIIA disease, it has to reach the lysosomes of cells in the brain, hence pass the blood brain barrier (BBB). To monitor BBB passage of CM-rhSulfamidase in a time dependent manner, concentration in brain interstitial fluid was explored using push-pull microdialysis combined with CSF and serum collection. Push-pull microdialysis is a sampling technique facilitating continuous measurement of larger molecules (e.g. proteins), here applied in awake, freely-moving Sprague Dawley rats. Having established membrane recovery, surgery was performed on rats to implant guide cannulas for the microdialysis probes using a stereotaxic frame to pin-point exact location in the prefrontal cortex, and cannulas for repeated CSF and serum sampling in the cisterna magna and jugular vein, respectively. After the animals fully recovered, CM-rhSulfamidase was dosed intravenously and brain interstitial fluid was sampled by microdialysis, CSF and serum via their respective cannulas. Concentrations of CMrhSulfamidase were determined by an immunoassay and data were subjected to quantitative pharmacokinetic analysis. While maximum serum concentration was observed shortly after administration, a delay was observed of CM-rhSulfamidase entering CSF and brain interstitial fluid. Maximum CSF and brain interstitial fluid concentrations differed somewhat between rats, but after reaching the maximum level reduction in concentration occurred at a similar rate between individual rats. Mean CM-rhSulfamidase concentrations versus time relationships were similar in brain interstitial fluid and CSF. Brain exposure was confirmed by determination of brain homogenate concentrations. In conclusion, CM-rhSulfamidase biodistribution to CNS was quantified using a combined push-pull brain microdialysis, serum and CSF approach.
doi:10.1016/j.ymgme.2018.12.193
178 Adding enzyme replacement therapy after hematopoietic stem cell transplantation results in increased metabolic correction in MPS VI Jeanine R. Jarnes-Utz, Elizabeth Braunlin, Paul Orchard, Chester B. Whitley, University of Minnesota, Minneapolis, MN, United States
effects of EGT and ABX on autophagy-lysosomal pathways were examined in various primary cells derived from patients with GD. Overall, both compounds increased GCase activity, but this effect was only observed in some patients. GCase expression analysis demonstrated elevated protein but not mRNA levels after ABX and EGT treatments. There was also an increase in autophagic vesicle formation and autophagosome accumulation as evidenced by changing levels of LC3A/BII. There was an increase in LysoTracker signal and mitochondria levels, but RT-PCR demonstrated no change in LC3 and LAMP1 in primary fibroblasts, PBMC and macrophages. Decreasing RNA levels of pro-inflammatory cytokines CCL17, IL10, IFNγ were observed in macrophages following both treatments, while ABX particularly inhibited CCL2 levels. While the small molecules, EGT and ABX have completely different mechanisms of action, they both lead to ALP activation and decreased proinflammatory cytokine levels that could be among the reasons for improved GCase activity in select cell lines.
doi:10.1016/j.ymgme.2018.12.191
176 Effects of genetic background on disease phenotypes in a mouse model of Fabry disease Siamak Jabbarzadeh-Tabrizia, Taniqua Daya, Mouna Tarouaa, Michel Boutinb, Christiane Auray-Blaisb, Raphael Schiffmanna, Jin-Song Shena, aBaylor Research Institute, Dallas, TX, United States, bFaculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada Knockout mouse model of Fabry disease (Ohshima, 1997) is widely used, and has been proven an invaluable tool to investigate disease pathogenesis and therapies. However, the importance of genetic background of this mouse line has been underestimated, and in many publications WT control mice were chosen incorrectly. Our aim was to examine the effects of genetic background on biochemical and functional abnormalities of Fabry mice. We characterized Fabry mice in B6/129 mixed background (~75% B6) and Fabry mice on a purely C57BL/6 strain (10 generations backcrossing), compared with WT mice on an identical genetic background. Both Fabry-B6/129 and Fabry-B6 developed progressive cardiac and renal hypertrophy and impaired thermosensation. However, Fabry-B6/129 exhibited earlier onset and more severe phenotypes than Fabry-B6. Cardiac and/or renal hypertrophy appeared at age of 6 months in Fabry-B6/129, but at 12 months in Fabry-B6. At 12 months, kidney-to-body weight ratio increased by 50% in Fabry-B6/129 (vs. WT), but only 15% in Fabry-B6. The onset of the thermosensation abnormality was at age 2 months in Fabry-B6/129 and 7 months in Fabry-B6. Cardiac expression of hypertrophy marker, atrial natriuretic peptide (ANP), was upregulated in Fabry-B6/129, but not in Fabry-B6. Despite these differences, globotriaosylceramide (Gb3) and lyso-Gb3 levels in the heart, kidney and dorsal root ganglia were similar between Fabry-B6/129 and Fabry-B6. However, Gb3 level in liver (organ unaffected in Fabry disease) was twofold higher in Fabry-B6/129 compared to Fabry-B6. Additionally, several parameters including body weight, organ weight, kidney Gb3 and cardiac ANP level were different between WT-B6/129 and WT-B6. In conclusion: 1) genetic background has a significant impact on the severity and onset of phenotypes in Fabry mice 2) the lack of correlation between phenotype and Gb3/lyso-Gb3 levels in disease-relevant organs
S79
suggests that the effect of genetic background on the phenotype is determined by other genetic and epigenetic factors.
doi:10.1016/j.ymgme.2018.12.192
177 Distribution of chemically modified rhSulfamidase to CNS monitored by brain microdialysis and repeated CSF sampling after intravenous administration in rat Juliette Jansona, Gudrun Anderssona, Lars Bergquista, Maria Erikssona, Joost H.A. Folgeringb, aSwedish Orphan Biovitrum, Stockholm, Sweden, b Charles River Laboratories, Groningen, Netherlands Mucopolysaccharidosis type IIIA (MPS IIIA) is an autosomal recessive disorder caused by absence or deficiency of the lysosomal enzyme sulfamidase. Glycans of recombinant human sulfamidase were chemically modified to generate CM-rhSulfamidase with retained stability and catalytic activity, but with significantly reduced uptake in peripheral tissues. For the enzyme to modify the neuropathology of MPS IIIA disease, it has to reach the lysosomes of cells in the brain, hence pass the blood brain barrier (BBB). To monitor BBB passage of CM-rhSulfamidase in a time dependent manner, concentration in brain interstitial fluid was explored using push-pull microdialysis combined with CSF and serum collection. Push-pull microdialysis is a sampling technique facilitating continuous measurement of larger molecules (e.g. proteins), here applied in awake, freely-moving Sprague Dawley rats. Having established membrane recovery, surgery was performed on rats to implant guide cannulas for the microdialysis probes using a stereotaxic frame to pin-point exact location in the prefrontal cortex, and cannulas for repeated CSF and serum sampling in the cisterna magna and jugular vein, respectively. After the animals fully recovered, CM-rhSulfamidase was dosed intravenously and brain interstitial fluid was sampled by microdialysis, CSF and serum via their respective cannulas. Concentrations of CMrhSulfamidase were determined by an immunoassay and data were subjected to quantitative pharmacokinetic analysis. While maximum serum concentration was observed shortly after administration, a delay was observed of CM-rhSulfamidase entering CSF and brain interstitial fluid. Maximum CSF and brain interstitial fluid concentrations differed somewhat between rats, but after reaching the maximum level reduction in concentration occurred at a similar rate between individual rats. Mean CM-rhSulfamidase concentrations versus time relationships were similar in brain interstitial fluid and CSF. Brain exposure was confirmed by determination of brain homogenate concentrations. In conclusion, CM-rhSulfamidase biodistribution to CNS was quantified using a combined push-pull brain microdialysis, serum and CSF approach.
doi:10.1016/j.ymgme.2018.12.193
178 Adding enzyme replacement therapy after hematopoietic stem cell transplantation results in increased metabolic correction in MPS VI Jeanine R. Jarnes-Utz, Elizabeth Braunlin, Paul Orchard, Chester B. Whitley, University of Minnesota, Minneapolis, MN, United States