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Mosaicism of podocyte involvement is related to podocyte injury in females with Fabry disease
Abstracts / Molecular Genetics and Metabolism 114 (2015) S11–S130
RR
doi:10.1016/j.ymgme.2014.12.182
EC
TE
DP
A phase I–II trial (NCT00920647) evaluating the effects of investigational recombinant iduronate-2-sulfatase formulated for intrathecal administration (idursulfase-IT) in cognitively impaired children with Hunter syndrome (MPS II), and its extension (NCT01506141), has been ongoing since 2010. In addition to safety assessments, changes in glycosaminoglycan (GAG) concentrations in cerebrospinal fluid (CSF) and change from baseline in standardized neurocognitive assessments are being assessed. Sixteen cognitively impaired children with MPS II were originally enrolled. Four patients per dose group received either no treatment, 10 mg, 30 mg, or 1 mg idursulfase-IT monthly for 6 months while continuing intravenous idursulfase 0.5 mg/kg weekly. Patients (currently n = 14) continued into an ongoing extension study, receiving either 10 or 30 mg monthly idursulfase-IT doses. To date, the range of patient exposure time to idursulfase-IT is approximately 2 to 4.5 years. Mean CSF GAG concentration was reduced by about 90% in the 10- and 30-mg groups and by about 80% in the 1-mg group at month 6. Long-term follow-up indicates that these low CSF GAG values are maintained, despite occasional skipped doses or, in 1 patient, the development of neutralizing CSF antibodies. Only 5/16 patients were originally cognitively testable over time using the Differential Abilities Scale (2nd edition). Currently, there are 4 patients with cognitive data obtained by this assessment. Although the cognitive data shows large variability, all 4 patients, including 1 patient who was switched from 1 to 10 mg, appear to have experienced stabilization or decreased rate of decline. These data support further development of idursulfase-IT for the stabilization or decreased decline of cognitive function in moderately affected MPS II patients with the severe phenotype. These studies are funded by Shire.
OF
Joseph Muenzera, Christian J. Hendrikszb, Margot B. Steina, Zheng Fana, Shauna Kearneyb, Johan Hortonb, Suresh Vijayaraghavanb, Saikat Santrab, Guirish A. Solankib, Luying Panc, Nan Wangc, Mary Ann Mascellic, Kenneth Sciarappac, Ann J. Barbierc, aUniversity of North Carolina Chapel Hill, Chapel Hill, NC, USA, bBirmingham Children's Hospital NHS Foundation Trust, Birmingham, UK, cShire, Lexington, MA, USA
Results: Absolute GL3 volume per podocyte decreased in all cases from baseline (3126 ± 2091 μm3) to 1 year (797 ± 291 μm3), (paired t-test p = 0.03). This reduction paralleled reduction in podocyte size from baseline to 1 year (p = 0.03). In contrast, volume fraction of GL3 per podocyte was not statistically different between baseline and 1 year biopsies. There was a strong relationship between the absolute GL3 volume per podocyte or podocyte volume at baseline and % reduction in the absolute GL3 volume in podocytes at 1 year (r = 0.90, p = 0.01), indicating that biopsies with larger podocytes and with more abundant GL3 in podocytes at baseline showed the most prominent GL3 loss at 1 year. While 0 [0–10]% of podocytes at baseline contained no GL3 inclusions, this increased to 15 [4–25]% at 1 year. This increase did not correlate with change in volume fraction of GL3 in podocytes, arguing that this phenomenon was likely not due to random sectioning. Volume fraction of GL3 approached to 0 in endothelial and mesangial cells at 1 year post-ERT. Conclusion: These data, for the first time, document reduced GL3 in podocytes after 1 year of ERT in male patients with Fabry disease. The unchanged volume fraction of GL3 in podocytes explains why scoring methods failed to detect this in previous studies as our finding depended on reductions in podocyte size, a parameter very difficult to detect by subjective methods. Although further studies are needed to determine if this response is maintained with longer ERT duration, these results suggest that shorter studies may be adequate to detect important early treatment effects in Fabry disease. Increased number of podocytes with no GL3 following ERT is also novel and may suggest podocyte regeneration.
RO
179 Long-term biomarker and cognitive follow-up of children with Hunter syndrome receiving intrathecal enzyme replacement therapy
S83
CO
180 Enzyme replacement therapy in Fabry disease reduces podocyte globotriaosylceramide (GL3) content within a year Behzad Najafiana, Michael Mauerb, aUniversity of Washington, Seattle, WA, USA, bUniversity of Minnesota, Minneapolis, MN, USA
UN
Background: Chronic kidney disease is a common complication of Fabry disease. Recent studies support a role for podocyte injury in Fabry nephropathy. Previous studies using flawed methodologies failed to show GL3 clearance after 1 year of ERT. We hypothesized that this failure could be at least partially due to concomitant shrinkage of podocyte cytoplasm proportional to GL3 loss. We aimed to re-evaluate podocyte response to ERT using unbiased electron microscopic morphometry. Methods: Paired biopsies at baseline (ERT naïve) and after ~1 year of ERT (1 mg/kg Fabrazyme every 2 weeks) from 6 males with Fabry disease (age 31 [18–46], median [range]) were studied. Fractional volumes of GL3 inclusions per podocytes, mesangial and endothelial cells were estimated using point counting. Average podocyte volume and absolute volume of GL3 inclusions per podocytes were estimated using the point sampled intercept method.
doi:10.1016/j.ymgme.2014.12.183
181 Mosaicism of podocyte involvement is related to podocyte injury in females with Fabry disease Behzad Najafian, University of Washington, Seattle, WA, USA Background: Fabry disease. An X-linked deficiency of α-galactosidase A coded by the GLA gene, leads to intracellular globotriaosylceramide
Abstracts / Molecular Genetics and Metabolism 114 (2015) S11–S130
antibodies (NAb), twenty-nine had the same NAb status at both labs. The NAb titers for positive samples from both labs were very positively correlated with a Spearman correlation coefficient of 0.88. The two samples having conflicting status in the two laboratories both had very low titers, attributable to normal inter-assay variability. In summary, the anti-velaglucerase alfa antibody assays at both the Shire and LabCorp were highly comparable to each other and can be considered as interchangeable methods for the measurement of total and neutralizing anti-velaglucerase alfa antibodies. doi:10.1016/j.ymgme.2014.12.185
OF
183 Novel live cell screening platform for small molecules to enhance enzyme activity in Gaucher's disease John J. Naleway, Fiona K. Harlan, Cinthia Costa Jones, Jason S. Lusk, Marker Gene Technologies, Inc., Eugene, OR, USA Defects in lysosomal enzymes are associated with a number of genetic diseases. These include mutations within the GBA1 gene encoding acid beta-glucosidase involved in Gaucher's disease as well as Parkinson's disease. A new screening platform utilizing novel fluorogenic probes for monitoring lysosomal enzyme activity using a live cell format has been developed. This new platform utilizes three different assay formats: a 384-well fluorescence microplate based assay for high throughput screening, a flow cytometry based analysis for secondary screening of identified hits and a fluorescence microscopic analysis method for verification of preliminary hits. These assays are based upon fluorogenic beta-glucosidase substrates that (1) have a low pKa value for optimum fluorescence at the lower physiological pH within the lysosome; (2) contain a cleavable substrate group to detect specific enzyme activity; and (3) contain a targeting group for specific analysis in the lysosome. The application of this novel platform has been demonstrated by screening of the Library of Pharmacologically Active Compounds (LOPAC) against immortalized leukocyte and fibroblast patient cell lines. Primary screening identified 9 possible hits that considerably increased enzyme activity above that of no drug controls. Secondary screening in flow cytometry and by microscopy analysis both verified the activity of these potential hits without compromising the viability of the cells. Confirmed hits were then counter screened using a Thermal Shift Assay to confirm specific activity. The results of this screening have implications for identification of potential new therapeutics for Gaucher's disease and allied syndromes. This work was supported in part by NIH Grant 5R44NS073225-04.
DP
(GL-3) accumulation. Although less common than in males, chronic kidney disease occurs in ~15% of females. Recent studies highlight the importance of podocyte injury in Fabry nephropathy development and progression. We hypothesized that the greater the % of podocytes with active wild-type GLA gene (due to X-inactivation of the mutant copy) the less is the overall podocyte injury. Methods: Kidney biopsies from 11 treatment-naive females with Fabry disease, ages 14 [8–39] (median [range]) years were studied by electron microscopy and compared with 4 treatment-naive male patients. Results: In females, 50 [13–100]% of podocytes (PC) per glomerulus had no GL-3 inclusions, this consistent with a non-Fabry podocyte phenotype (NFPC). In PC with GL-3 inclusions [Fabry podocyte phenotype (FPC)], GL-3 volume density per podocyte was not different between females and males, consistent with little or no cross-correction between FPC and NFPC. However, Vv(Inc/Endo) and Vv(Inc/Mes) were remarkably (~ 20 times) less in females compared to males. %NFPC per glomerulus (%NFPC/glom) correlated with age in females (r = 0.69, p = 0.04), suggesting a survival disadvantage for FPC over time. Age-adjusted %NFPC/glom was inversely related to foot process width (FPW) (r = −0.80, p = 0.02), an indicator of PC injury. GL-3 volume density in FPC in females correlated directly with FPW. Conclusions: These findings support important relationships between podocyte mosaicism and podocyte injury in female Fabry patients. Kidney biopsy, by providing information about podocyte mosaicism, may help to stratify females with Fabry disease for kidney disease risk and to guide treatment decisions.
RO
S84
TE
doi:10.1016/j.ymgme.2014.12.184
182 An inter-laboratory comparison study of detection and characterization of anti-velaglucerase Alfa antibodies
EC
Diana Najarian, Kelly Hilton, Thomas McCauley, Yongchang Qiu, Shire, Lexington, MA, USA
UN
CO
RR
Analysis of serum samples for anti-drug antibody (ADA) status, titer and neutralizing capacity in Gaucher patients receiving velaglucerase alfa has been performed at the Shire laboratory using a complex tiered testing scheme involving the use of multiple technology platforms. In order to further facilitate the monitoring of anti-drug antibodies in Gaucher patients, a similar method with a much simpler testing scheme was developed at Shire and validated at a different testing facility (LabCorp). Here we report the evaluation of the correlation and degree of agreement between the two methods run at two different facilities. Patient samples were initially screened using an electrochemiluminescent (ECL) bridging assay. In the Shire laboratory, putative positive status was confirmed in a radio-immunoprecipitation (RIP) assay (IgG), and RIP negative samples were further tested by an ECL bridging assay (IgM and IgA). At LabCorp, samples identified as putative positive in the screening assay were further tested in a competitive ligand binding confirmatory assay. At both sites, confirmed positive samples were diluted for titer determination, and were also characterized using an enzyme activity-based neutralization assay. Among the fifty samples tested for anti-drug antibodies (ADA) at both facilities, forty-eight (96%) had the same antibody status. The titers for twenty-two antibody positive samples from the two titer datasets were highly correlated with a Spearman correlation coefficient of 0.95. Two samples were identified as positive at Shire with very low titers, and were identified as negative at LabCorp. Of the thirty-one patient samples tested for the presence of neutralizing anti-velaglucerase alfa
doi:10.1016/j.ymgme.2014.12.186
RR
doi:10.1016/j.ymgme.2014.12.182
EC
TE
DP
A phase I–II trial (NCT00920647) evaluating the effects of investigational recombinant iduronate-2-sulfatase formulated for intrathecal administration (idursulfase-IT) in cognitively impaired children with Hunter syndrome (MPS II), and its extension (NCT01506141), has been ongoing since 2010. In addition to safety assessments, changes in glycosaminoglycan (GAG) concentrations in cerebrospinal fluid (CSF) and change from baseline in standardized neurocognitive assessments are being assessed. Sixteen cognitively impaired children with MPS II were originally enrolled. Four patients per dose group received either no treatment, 10 mg, 30 mg, or 1 mg idursulfase-IT monthly for 6 months while continuing intravenous idursulfase 0.5 mg/kg weekly. Patients (currently n = 14) continued into an ongoing extension study, receiving either 10 or 30 mg monthly idursulfase-IT doses. To date, the range of patient exposure time to idursulfase-IT is approximately 2 to 4.5 years. Mean CSF GAG concentration was reduced by about 90% in the 10- and 30-mg groups and by about 80% in the 1-mg group at month 6. Long-term follow-up indicates that these low CSF GAG values are maintained, despite occasional skipped doses or, in 1 patient, the development of neutralizing CSF antibodies. Only 5/16 patients were originally cognitively testable over time using the Differential Abilities Scale (2nd edition). Currently, there are 4 patients with cognitive data obtained by this assessment. Although the cognitive data shows large variability, all 4 patients, including 1 patient who was switched from 1 to 10 mg, appear to have experienced stabilization or decreased rate of decline. These data support further development of idursulfase-IT for the stabilization or decreased decline of cognitive function in moderately affected MPS II patients with the severe phenotype. These studies are funded by Shire.
OF
Joseph Muenzera, Christian J. Hendrikszb, Margot B. Steina, Zheng Fana, Shauna Kearneyb, Johan Hortonb, Suresh Vijayaraghavanb, Saikat Santrab, Guirish A. Solankib, Luying Panc, Nan Wangc, Mary Ann Mascellic, Kenneth Sciarappac, Ann J. Barbierc, aUniversity of North Carolina Chapel Hill, Chapel Hill, NC, USA, bBirmingham Children's Hospital NHS Foundation Trust, Birmingham, UK, cShire, Lexington, MA, USA
Results: Absolute GL3 volume per podocyte decreased in all cases from baseline (3126 ± 2091 μm3) to 1 year (797 ± 291 μm3), (paired t-test p = 0.03). This reduction paralleled reduction in podocyte size from baseline to 1 year (p = 0.03). In contrast, volume fraction of GL3 per podocyte was not statistically different between baseline and 1 year biopsies. There was a strong relationship between the absolute GL3 volume per podocyte or podocyte volume at baseline and % reduction in the absolute GL3 volume in podocytes at 1 year (r = 0.90, p = 0.01), indicating that biopsies with larger podocytes and with more abundant GL3 in podocytes at baseline showed the most prominent GL3 loss at 1 year. While 0 [0–10]% of podocytes at baseline contained no GL3 inclusions, this increased to 15 [4–25]% at 1 year. This increase did not correlate with change in volume fraction of GL3 in podocytes, arguing that this phenomenon was likely not due to random sectioning. Volume fraction of GL3 approached to 0 in endothelial and mesangial cells at 1 year post-ERT. Conclusion: These data, for the first time, document reduced GL3 in podocytes after 1 year of ERT in male patients with Fabry disease. The unchanged volume fraction of GL3 in podocytes explains why scoring methods failed to detect this in previous studies as our finding depended on reductions in podocyte size, a parameter very difficult to detect by subjective methods. Although further studies are needed to determine if this response is maintained with longer ERT duration, these results suggest that shorter studies may be adequate to detect important early treatment effects in Fabry disease. Increased number of podocytes with no GL3 following ERT is also novel and may suggest podocyte regeneration.
RO
179 Long-term biomarker and cognitive follow-up of children with Hunter syndrome receiving intrathecal enzyme replacement therapy
S83
CO
180 Enzyme replacement therapy in Fabry disease reduces podocyte globotriaosylceramide (GL3) content within a year Behzad Najafiana, Michael Mauerb, aUniversity of Washington, Seattle, WA, USA, bUniversity of Minnesota, Minneapolis, MN, USA
UN
Background: Chronic kidney disease is a common complication of Fabry disease. Recent studies support a role for podocyte injury in Fabry nephropathy. Previous studies using flawed methodologies failed to show GL3 clearance after 1 year of ERT. We hypothesized that this failure could be at least partially due to concomitant shrinkage of podocyte cytoplasm proportional to GL3 loss. We aimed to re-evaluate podocyte response to ERT using unbiased electron microscopic morphometry. Methods: Paired biopsies at baseline (ERT naïve) and after ~1 year of ERT (1 mg/kg Fabrazyme every 2 weeks) from 6 males with Fabry disease (age 31 [18–46], median [range]) were studied. Fractional volumes of GL3 inclusions per podocytes, mesangial and endothelial cells were estimated using point counting. Average podocyte volume and absolute volume of GL3 inclusions per podocytes were estimated using the point sampled intercept method.
doi:10.1016/j.ymgme.2014.12.183
181 Mosaicism of podocyte involvement is related to podocyte injury in females with Fabry disease Behzad Najafian, University of Washington, Seattle, WA, USA Background: Fabry disease. An X-linked deficiency of α-galactosidase A coded by the GLA gene, leads to intracellular globotriaosylceramide
Abstracts / Molecular Genetics and Metabolism 114 (2015) S11–S130
antibodies (NAb), twenty-nine had the same NAb status at both labs. The NAb titers for positive samples from both labs were very positively correlated with a Spearman correlation coefficient of 0.88. The two samples having conflicting status in the two laboratories both had very low titers, attributable to normal inter-assay variability. In summary, the anti-velaglucerase alfa antibody assays at both the Shire and LabCorp were highly comparable to each other and can be considered as interchangeable methods for the measurement of total and neutralizing anti-velaglucerase alfa antibodies. doi:10.1016/j.ymgme.2014.12.185
OF
183 Novel live cell screening platform for small molecules to enhance enzyme activity in Gaucher's disease John J. Naleway, Fiona K. Harlan, Cinthia Costa Jones, Jason S. Lusk, Marker Gene Technologies, Inc., Eugene, OR, USA Defects in lysosomal enzymes are associated with a number of genetic diseases. These include mutations within the GBA1 gene encoding acid beta-glucosidase involved in Gaucher's disease as well as Parkinson's disease. A new screening platform utilizing novel fluorogenic probes for monitoring lysosomal enzyme activity using a live cell format has been developed. This new platform utilizes three different assay formats: a 384-well fluorescence microplate based assay for high throughput screening, a flow cytometry based analysis for secondary screening of identified hits and a fluorescence microscopic analysis method for verification of preliminary hits. These assays are based upon fluorogenic beta-glucosidase substrates that (1) have a low pKa value for optimum fluorescence at the lower physiological pH within the lysosome; (2) contain a cleavable substrate group to detect specific enzyme activity; and (3) contain a targeting group for specific analysis in the lysosome. The application of this novel platform has been demonstrated by screening of the Library of Pharmacologically Active Compounds (LOPAC) against immortalized leukocyte and fibroblast patient cell lines. Primary screening identified 9 possible hits that considerably increased enzyme activity above that of no drug controls. Secondary screening in flow cytometry and by microscopy analysis both verified the activity of these potential hits without compromising the viability of the cells. Confirmed hits were then counter screened using a Thermal Shift Assay to confirm specific activity. The results of this screening have implications for identification of potential new therapeutics for Gaucher's disease and allied syndromes. This work was supported in part by NIH Grant 5R44NS073225-04.
DP
(GL-3) accumulation. Although less common than in males, chronic kidney disease occurs in ~15% of females. Recent studies highlight the importance of podocyte injury in Fabry nephropathy development and progression. We hypothesized that the greater the % of podocytes with active wild-type GLA gene (due to X-inactivation of the mutant copy) the less is the overall podocyte injury. Methods: Kidney biopsies from 11 treatment-naive females with Fabry disease, ages 14 [8–39] (median [range]) years were studied by electron microscopy and compared with 4 treatment-naive male patients. Results: In females, 50 [13–100]% of podocytes (PC) per glomerulus had no GL-3 inclusions, this consistent with a non-Fabry podocyte phenotype (NFPC). In PC with GL-3 inclusions [Fabry podocyte phenotype (FPC)], GL-3 volume density per podocyte was not different between females and males, consistent with little or no cross-correction between FPC and NFPC. However, Vv(Inc/Endo) and Vv(Inc/Mes) were remarkably (~ 20 times) less in females compared to males. %NFPC per glomerulus (%NFPC/glom) correlated with age in females (r = 0.69, p = 0.04), suggesting a survival disadvantage for FPC over time. Age-adjusted %NFPC/glom was inversely related to foot process width (FPW) (r = −0.80, p = 0.02), an indicator of PC injury. GL-3 volume density in FPC in females correlated directly with FPW. Conclusions: These findings support important relationships between podocyte mosaicism and podocyte injury in female Fabry patients. Kidney biopsy, by providing information about podocyte mosaicism, may help to stratify females with Fabry disease for kidney disease risk and to guide treatment decisions.
RO
S84
TE
doi:10.1016/j.ymgme.2014.12.184
182 An inter-laboratory comparison study of detection and characterization of anti-velaglucerase Alfa antibodies
EC
Diana Najarian, Kelly Hilton, Thomas McCauley, Yongchang Qiu, Shire, Lexington, MA, USA
UN
CO
RR
Analysis of serum samples for anti-drug antibody (ADA) status, titer and neutralizing capacity in Gaucher patients receiving velaglucerase alfa has been performed at the Shire laboratory using a complex tiered testing scheme involving the use of multiple technology platforms. In order to further facilitate the monitoring of anti-drug antibodies in Gaucher patients, a similar method with a much simpler testing scheme was developed at Shire and validated at a different testing facility (LabCorp). Here we report the evaluation of the correlation and degree of agreement between the two methods run at two different facilities. Patient samples were initially screened using an electrochemiluminescent (ECL) bridging assay. In the Shire laboratory, putative positive status was confirmed in a radio-immunoprecipitation (RIP) assay (IgG), and RIP negative samples were further tested by an ECL bridging assay (IgM and IgA). At LabCorp, samples identified as putative positive in the screening assay were further tested in a competitive ligand binding confirmatory assay. At both sites, confirmed positive samples were diluted for titer determination, and were also characterized using an enzyme activity-based neutralization assay. Among the fifty samples tested for anti-drug antibodies (ADA) at both facilities, forty-eight (96%) had the same antibody status. The titers for twenty-two antibody positive samples from the two titer datasets were highly correlated with a Spearman correlation coefficient of 0.95. Two samples were identified as positive at Shire with very low titers, and were identified as negative at LabCorp. Of the thirty-one patient samples tested for the presence of neutralizing anti-velaglucerase alfa
doi:10.1016/j.ymgme.2014.12.186