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Uptake and lysosomal delivery of recombinant human alpha-n-acetylglucosamine-6-sulfatase to mucopolysaccharidosis IIID fibroblasts
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Abstracts / Molecular Genetics and Metabolism 117 (2016) S14–S124
progressive symmetric arthritis, presence of subcutaneous nodules at the joints, scalp and/or spine, and an unusual, hoarse cry or voice (due to nodule formation on the larynx) in Farber patients. Currently, Farber disease can be treated by hematopoietic stem cell transplantation, which has shown variable results and carries a severe burden for the patients. The fact that ceramide induces macrophage-driven inflammation leading to local and systemic symptoms, inspired the use of antiinflammatory agents as symptomatic treatments for Farber. Here we present 5 patients with Farber disease, who demonstrated a significant clinical response to tocilizumab (an interleukin-6 receptor inhibitor) measureable by changes in standard inflammatory markers (ex. erythrocyte sedimentation rate), and improvement of symptoms such as pain and physical impairment. The degree to which each patient responded was variable, and in no case has the disease ceased to progress. The implications of these findings are threefold: antiinflammatory therapy is indicated in Farber disease since macrophage-driven inflammation causes symptoms and tissue damage, antiinflammatory therapies should be considered to prevent tissue damage and relieve symptoms in diseases where inflammation plays a role, and that the use of such therapies in pediatric rheumatology may lead to a delayed or missed diagnosis of Farber disease. doi:10.1016/j.ymgme.2015.12.364
207 Uptake and lysosomal delivery of recombinant human alpha-n-acetylglucosamine-6-sulfatase to mucopolysaccharidosis IIID fibroblasts Derek R. Moena, Xiaoyi Zhangb, Kan Shin-hsinb, Jill Wooda, Sean Ekinsa, Tsui-Fen Choub, Patricia Dicksonb, aPhoenix Nest, Inc., Brooklyn, NY, United States, bLos Angeles Biomedical Research Institute at HarborUCLA, Torrance, CA, United States Sanfilippo syndrome (mucopolysaccharidosis type III; MPS III) is a devastating neurodegenerative lysosomal disorder of childhood. There is no cure or effective treatment available. The fundamental cause of MPS III is an inherited mutation in one of the 4 enzymes required to catabolize heparan sulfate (HS), a glycosaminoglycan which plays important structural and functional roles in the brain and elsewhere. We now propose to develop an enzyme replacement treatment (ERT) for MPS IIID that will ameliorate or reverse the catastrophic and fatal neurologic decline caused by this disease. The symptoms of MPS IIID are largely localized to the brain, therefore, our strategy proposes to deliver recombinant human alpha-Nacetylglucosamine-6-sulfatase (rhGNS) intrathecally to effectively treat the underlying causes of the neurologic symptoms that dominate MPS III pathology. We have purified ~100 ug rhGNS from 1500 mL media of CHO cells (specific activity N200,000 nmol/24 h/mg), demonstrated maximal enzymatic activity at pH 5.6 (low activity at neutral pH), which is closer to lysosomal pH, demonstrated good enzymatic activity at 37 °C, and showed it was stable for over one month at 4 °C in artificial cerebrospinal fluid storage buffer. Additionally, we have demonstrated intracellular enzymatic activity of rhGNS in MPS IIID fibroblasts when rhGNS is added to the media, shown it colocalizes with lysosomal markers using confocal microscopy, and confirmed radiolabelled HS is diminished (33-65%) to WT levels in MPS IIID fibroblasts treated with rhGNS. These results demonstrate scale-up is feasible in preparation for proof of concept studies in the MPS IIID knock-out mouse. Our ultimate goal is bring this ERT to the clinic which will likely have a transformational effect on patients and families. doi:10.1016/j.ymgme.2015.12.365
208 2-year experience of newborn screening of Pompe disease in a Japanese region Ken Momosaki, Kimitoshi Nakamura, Shinichiro Yoshida, Fumio Endo, Kumamoto University, Kumamoto, Japan Background: The high incidence (3.3-3.9%) of acid α-glucosidase pseudodeficiency, c.[1726G N A; 2065G N A] homozygote (AA homozygote), in Asian populations complicates newborn mass screening for Pompe disease. Combination mutation (one pseudodeficiency mutation and one pathogenic mutation) also could remarkably reduce GAA enzyme activities. Purpose: In this study, we examined GAA acitivities with DBS among newborns in a Japanese area and also count the incidence of GAA mutation among these newborns. Method: We screened 44,621 DBSs from the NBS program in Kumamoto, Japan from April 2013 to August 2015. We assayed A α Glu activity in DBS with two different methods; 4-methylumbelliferone (4-MU) method and Ba/Zn method. Definitive diagnosis was done by the measurement of AαGlu activity in fibroblasts using resolving power of glycogen AND GAA mutation analysis using next-generation sequencing technology. Genetic analysis was also performed by Sanger methods. Results: A total of 112 (0.25%) newborns were recalled for second DBSs with low enzyme activity. A α Glu activity in fibroblasts was carried out for 28 samples with abnormal low enzyme activity (GAA b 4.0), revealed 10 subjects with remarkable low GAA activity equally to patients. 4 of 10 subjects has pathogenic heterozygous mutations previously reported. And 2 has new mutation also in one allele. There were no subjects with 2 independent pathogenic mutations. These subjects also has pseudodeficiency allele. Conclusions: The incidence of heterozygotes of GAA mutation was revealed and we continue to investigate of influence about combination mutations. doi:10.1016/j.ymgme.2015.12.366
209 Fabry disease and membranous nephropathy: Case report Jose T. Monte Netoa, Daniel Santos Rocha Sobral Filhoa, Roosevelt Valente Chavesa, Nayze L.S. Aldemana, Marlene Antonia Reisb, Semiramis J. Hadad Montea, aUniversidade Federal do Piaui, Teresina, Brazil, bUniversidade Federal do Triangulo Mineiro, Uberaba, Brazil Fabry disease (FD) is a multisystem lysosomal disorder, X-linked, caused by a mutation in the GLA gene, resulting in deficient activity of alpha-galactosidase A enzyme (α-GAL). It causes lysosomal accumulation of glycosphingolipids, especially the GB-3 in various cells, leading to progressive multiple organ dysfunction. Renal involvement, common in FD, produces significant morbidity and adverse prognosis. Membranous glomerulonephritis (MN) is an important cause of nephrotic syndrome in adults, characterized by the presence of spikes, thickening of the glomerular basement membrane (GBM) and the subepithelial IgG and C3 deposition. The association between these glomerulopathies has not been previously described. A 61 years old male was referred to nephrologist to investigate generalized edema and massive proteinuria. He referred a previous diagnosis of cardiomyopathy and heart failure treatment. Physical examination was marked by widespread edema. Complementary tests showed nephrotic proteinuria (7.0 g/24 hr), hypoalbuminemia (2.2 g/dL) and dyslipidemia featuring the Nephrotic Syndrome. The findings of renal biopsy were consistent with the
Abstracts / Molecular Genetics and Metabolism 117 (2016) S14–S124
progressive symmetric arthritis, presence of subcutaneous nodules at the joints, scalp and/or spine, and an unusual, hoarse cry or voice (due to nodule formation on the larynx) in Farber patients. Currently, Farber disease can be treated by hematopoietic stem cell transplantation, which has shown variable results and carries a severe burden for the patients. The fact that ceramide induces macrophage-driven inflammation leading to local and systemic symptoms, inspired the use of antiinflammatory agents as symptomatic treatments for Farber. Here we present 5 patients with Farber disease, who demonstrated a significant clinical response to tocilizumab (an interleukin-6 receptor inhibitor) measureable by changes in standard inflammatory markers (ex. erythrocyte sedimentation rate), and improvement of symptoms such as pain and physical impairment. The degree to which each patient responded was variable, and in no case has the disease ceased to progress. The implications of these findings are threefold: antiinflammatory therapy is indicated in Farber disease since macrophage-driven inflammation causes symptoms and tissue damage, antiinflammatory therapies should be considered to prevent tissue damage and relieve symptoms in diseases where inflammation plays a role, and that the use of such therapies in pediatric rheumatology may lead to a delayed or missed diagnosis of Farber disease. doi:10.1016/j.ymgme.2015.12.364
207 Uptake and lysosomal delivery of recombinant human alpha-n-acetylglucosamine-6-sulfatase to mucopolysaccharidosis IIID fibroblasts Derek R. Moena, Xiaoyi Zhangb, Kan Shin-hsinb, Jill Wooda, Sean Ekinsa, Tsui-Fen Choub, Patricia Dicksonb, aPhoenix Nest, Inc., Brooklyn, NY, United States, bLos Angeles Biomedical Research Institute at HarborUCLA, Torrance, CA, United States Sanfilippo syndrome (mucopolysaccharidosis type III; MPS III) is a devastating neurodegenerative lysosomal disorder of childhood. There is no cure or effective treatment available. The fundamental cause of MPS III is an inherited mutation in one of the 4 enzymes required to catabolize heparan sulfate (HS), a glycosaminoglycan which plays important structural and functional roles in the brain and elsewhere. We now propose to develop an enzyme replacement treatment (ERT) for MPS IIID that will ameliorate or reverse the catastrophic and fatal neurologic decline caused by this disease. The symptoms of MPS IIID are largely localized to the brain, therefore, our strategy proposes to deliver recombinant human alpha-Nacetylglucosamine-6-sulfatase (rhGNS) intrathecally to effectively treat the underlying causes of the neurologic symptoms that dominate MPS III pathology. We have purified ~100 ug rhGNS from 1500 mL media of CHO cells (specific activity N200,000 nmol/24 h/mg), demonstrated maximal enzymatic activity at pH 5.6 (low activity at neutral pH), which is closer to lysosomal pH, demonstrated good enzymatic activity at 37 °C, and showed it was stable for over one month at 4 °C in artificial cerebrospinal fluid storage buffer. Additionally, we have demonstrated intracellular enzymatic activity of rhGNS in MPS IIID fibroblasts when rhGNS is added to the media, shown it colocalizes with lysosomal markers using confocal microscopy, and confirmed radiolabelled HS is diminished (33-65%) to WT levels in MPS IIID fibroblasts treated with rhGNS. These results demonstrate scale-up is feasible in preparation for proof of concept studies in the MPS IIID knock-out mouse. Our ultimate goal is bring this ERT to the clinic which will likely have a transformational effect on patients and families. doi:10.1016/j.ymgme.2015.12.365
208 2-year experience of newborn screening of Pompe disease in a Japanese region Ken Momosaki, Kimitoshi Nakamura, Shinichiro Yoshida, Fumio Endo, Kumamoto University, Kumamoto, Japan Background: The high incidence (3.3-3.9%) of acid α-glucosidase pseudodeficiency, c.[1726G N A; 2065G N A] homozygote (AA homozygote), in Asian populations complicates newborn mass screening for Pompe disease. Combination mutation (one pseudodeficiency mutation and one pathogenic mutation) also could remarkably reduce GAA enzyme activities. Purpose: In this study, we examined GAA acitivities with DBS among newborns in a Japanese area and also count the incidence of GAA mutation among these newborns. Method: We screened 44,621 DBSs from the NBS program in Kumamoto, Japan from April 2013 to August 2015. We assayed A α Glu activity in DBS with two different methods; 4-methylumbelliferone (4-MU) method and Ba/Zn method. Definitive diagnosis was done by the measurement of AαGlu activity in fibroblasts using resolving power of glycogen AND GAA mutation analysis using next-generation sequencing technology. Genetic analysis was also performed by Sanger methods. Results: A total of 112 (0.25%) newborns were recalled for second DBSs with low enzyme activity. A α Glu activity in fibroblasts was carried out for 28 samples with abnormal low enzyme activity (GAA b 4.0), revealed 10 subjects with remarkable low GAA activity equally to patients. 4 of 10 subjects has pathogenic heterozygous mutations previously reported. And 2 has new mutation also in one allele. There were no subjects with 2 independent pathogenic mutations. These subjects also has pseudodeficiency allele. Conclusions: The incidence of heterozygotes of GAA mutation was revealed and we continue to investigate of influence about combination mutations. doi:10.1016/j.ymgme.2015.12.366
209 Fabry disease and membranous nephropathy: Case report Jose T. Monte Netoa, Daniel Santos Rocha Sobral Filhoa, Roosevelt Valente Chavesa, Nayze L.S. Aldemana, Marlene Antonia Reisb, Semiramis J. Hadad Montea, aUniversidade Federal do Piaui, Teresina, Brazil, bUniversidade Federal do Triangulo Mineiro, Uberaba, Brazil Fabry disease (FD) is a multisystem lysosomal disorder, X-linked, caused by a mutation in the GLA gene, resulting in deficient activity of alpha-galactosidase A enzyme (α-GAL). It causes lysosomal accumulation of glycosphingolipids, especially the GB-3 in various cells, leading to progressive multiple organ dysfunction. Renal involvement, common in FD, produces significant morbidity and adverse prognosis. Membranous glomerulonephritis (MN) is an important cause of nephrotic syndrome in adults, characterized by the presence of spikes, thickening of the glomerular basement membrane (GBM) and the subepithelial IgG and C3 deposition. The association between these glomerulopathies has not been previously described. A 61 years old male was referred to nephrologist to investigate generalized edema and massive proteinuria. He referred a previous diagnosis of cardiomyopathy and heart failure treatment. Physical examination was marked by widespread edema. Complementary tests showed nephrotic proteinuria (7.0 g/24 hr), hypoalbuminemia (2.2 g/dL) and dyslipidemia featuring the Nephrotic Syndrome. The findings of renal biopsy were consistent with the