Iduronate-2-sulfatase Enzyme Activity Research Articles

Enhanced osteoblastic differentiation of parietal bone in a novel murine model of mucopolysaccharidosis type II

Mucopolysaccharidosis type II (MPS II, OMIM 309900) is an X-linked disorder caused by a deficiency of lysosomal enzyme iduronate-2-sulfatase (IDS). The clinical manifestations of MPS II involve cognitive decline, bone deformity, and visceral disorders. These manifestations are closely associated with IDS enzyme activity, which catalyzes the stepwise degradation of heparan sulfate and dermatan sulfate. In this study, we established a novel Ids-deficient mice and further assessed the enzyme's physiological role. Using DNA sequencing, we found a genomic modification of the Ids genome, which involved the deletion of a 138-bp fragment spanning from intron 2 to exon 3, along with the insertion of an adenine at the 5′ end of exon 3 in the mutated allele. Consistent with previous data, our Ids-deficient mice showed an attenuated enzyme activity and an enhanced accumulation of glycosaminoglycans. Interestingly, we noticed a distinct enlargement of the calvarial bone in both neonatal and young adult mice. Our examination revealed that Ids deficiency led to an enhanced osteoblastogenesis in the parietal bone, a posterior part of the calvarial bone originating from the paraxial mesoderm and associated with an enhanced expression of osteoblastic makers, such as Col1a and Runx2. In sharp contrast, cell proliferation of the parietal bone in these mice appeared similar to that of wild-type controls. These results suggest that the deficiency of Ids could be involved in an augmented differentiation of calvarial bone, which is often noticed as an enlarged head circumference in MPS II-affected individuals.

Sustained long-term disease correction in a murine model of MPSII following stem cell gene therapy

Mucopolysaccharidosis type II (MPSII) is a pediatric lysosomal storage disease caused by deficiencies in the IDS (iduronate-2-sulfatase) gene resulting in accumulation of glycosaminoglycans, multisystem disease, and profound neurodegeneration in severe forms. Although enzyme replacement therapy is available for somatic forms of disease, the inability of native IDS to pass the blood-brain barrier renders it ineffective for the brain. We previously demonstrated the short-term efficacy of a brain-targeted hematopoietic stem cell gene therapy approach to treat MPSII mice using lentiviral IDS fused to the blood-brain-barrier-crossing peptide ApoEII (IDS.ApoEII) in comparison with a lentivirus expressing native IDS and an unmanipulated bone marrow transplant. Here we evaluated the longevity of disease correction for 12-16months following treatment. We observed sustained IDS enzyme activity in organs of long-term IDS.ApoEII-treated MPSII mice, similar to those analyzed 6months post-treatment, with continued clearance of storage material in the brain and peripheral organs, maintained correction of astrogliosis, microgliosis, and correction of altered cytokines and chemokines. IDS.ApoEII also significantly reduced retinal atrophy, characteristic of MPSII. Overall, IDS.ApoEII resulted in systemic prevention of the MPSII phenotype, with no observed toxicity following treatment. This provides evidence of the sustained efficacy and safety of this treatment ahead of a recently opened clinical trial.

New Genetic Analysis in Cases with Hunter Syndrome in Whom IDS Gene Mutations Could Not Be Detected: RNA Sequencing

Introduction: Mucopolysaccharidosis-II (MPSII) is diagnosed based on a deficiency in iduronate 2-sulfatase enzyme activity. Detection of a hemizygous pathogenic variant in the iduronate 2-sulfatase (IDS) gene confirms the diagnosis in a male proband. Case Presentation: We report a five-year-old boy with MPSII in whom no mutation was detected in the IDS gene by next-generation sequencing (Miseq-Illumina) covering the coding regions of the gene. Therefore, we tried to detect the mutation in the IDS gene using RNA sequencing that has recently been used. Conclusions: In some diseases diagnosed by clinical and biochemical methods, mutations cannot be detected even with advanced genetic methods, such as next-generation sequencing. In these cases, we emphasize that mutations should be investigated using other methods, including RNA sequencing.

Fusion of Rabies Virus Glycoprotein or gh625 to Iduronate-2-Sulfatase for the Treatment of Mucopolysaccharidosis Type II.

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disease caused by a mutation in the IDS gene, resulting in deficiency of the enzyme iduronate-2-sulfatase (IDS) causing heparan sulfate (HS) and dermatan sulfate (DS) accumulation in all cells. This leads to skeletal and cardiorespiratory disease with severe neurodegeneration in two thirds of sufferers. Enzyme replacement therapy is ineffective at treating neurological disease, as intravenously delivered IDS is unable to cross the blood-brain barrier (BBB). Hematopoietic stem cell transplant is also unsuccessful, presumably due to insufficient IDS enzyme production from transplanted cells engrafting in the brain. We used two different peptide sequences (rabies virus glycoprotein [RVG] and gh625), both previously published as BBB-crossing peptides, fused to IDS and delivered via hematopoietic stem cell gene therapy (HSCGT). HSCGT with LV.IDS.RVG and LV.IDS.gh625 was compared with LV.IDS.ApoEII and LV.IDS in MPS II mice at 6 months post-transplant. Levels of IDS enzyme activity in the brain and peripheral tissues were lower in LV.IDS.RVG- and LV.IDS.gh625-treated mice than in LV.IDS.ApoEII- and LV.IDS-treated mice, despite comparable vector copy numbers. Microgliosis, astrocytosis, and lysosomal swelling were partially normalized in MPS II mice treated with LV.IDS.RVG and LV.IDS.gh625. Skeletal thickening was normalized by both treatments to wild-type levels. Although reductions in skeletal abnormalities and neuropathology are encouraging, given the low levels of enzyme activity compared with control tissue from LV.IDS- and LV.IDS.ApoEII-transplanted mice, the RVG and gh625 peptides are unlikely to be ideal candidates for HSCGT in MPS II and are inferior to the ApoEII peptide that we have previously demonstrated to be more effective at correcting MPS II disease than IDS alone.

Non-invasive intravenous administration of AAV9 transducing iduronate sulfatase leads to global metabolic correction and prevention of neurologic deficits in a mouse model of Hunter syndrome

Hunter syndrome is a rare x-linked recessive genetic disorder that affects lysosomal metabolism due to deficiency of iduronate-2-sulfatase (IDS), with subsequent accumulation of glycosaminoglycans heparan and dermatan sulfates (GAG). Enzyme replacement therapy is the only FDA-approved remedy and is an expensive life-time treatment that alleviates some symptoms of the disease without neurocognitive benefit. We previously reported successful treatment in a mouse model of mucopolysaccharidosis type II (MPS II) using adeno-associated viral vector serotype 9 encoding human IDS (AAV9.hIDS) via intracerebroventricular injection. As a less invasive and more straightforward procedure, here we report intravenously administered AAV9.hIDS in a mouse model of MPS II. In animals administered 1.5 × 1012 vg of AAV9.hIDS at 2 months of age, we observed supraphysiological levels of IDS enzyme activity in the circulation (up to 9100-fold higher than wild-type), in the tested peripheral organs (up to 560-fold higher than wild-type), but only 4% to 50% of wild type levels in the CNS. GAG levels were normalized on both sides of the blood-brain-barrier (BBB) in most of tissues tested. Despite low levels of the IDS observed in the CNS, this treatment prevented neurocognitive decline as shown by testing in the Barnes maze and by fear conditioning. This study demonstrates that a single dose of IV-administered AAV9.hIDS may be an effective and non-invasive procedure to treat MPS II that benefits both sides of the BBB, with implications for potential use of IV-administered AAV9 for other neuronopathic lysosomal diseases.

Phenotypic and genetic characteristics of 130 patients with mucopolysaccharidosis type II: A single-center retrospective study in China.

Background: Mucopolysaccharidosis Type II (MPS II) is a rare, progressive and ultimately fatal X-linked lysosomal storage disorder caused by mutations in the iduronate-2-sulfatase (IDS) gene. This report conducted a retrospective analysis to investigate the clinical characteristics, genotypes and management strategies in a large cohort of Chinese patients with MPS II. Methods: In this study, we explored 130 Chinese patients with MPS II between September 2008 and April 2022. Clinical manifestations, auxiliary examination, IDS pathogenic gene variants and IDS enzyme activity, surgical history were analysed in the study. Results: A total of 130 patients were enrolled and the mean age at diagnosis was 5years old. This study found the most common symptoms in our patients were claw-like hands, followed by coarse facial features, birthmarks (Mongolian spot), delayed development, inguinal or umbilical hernia. The most commonly cardiac manifestations were valve abnormalities, which were mitral/tricuspid valve regurgitation (71.9%) and aortic/pulmonary valve regurgitation (36.8%). We had found 43 different IDS pathogenic gene variants in 55 patients, included 16 novel variants. The variants were concentrated in exon 9 (20% = 11/55), exon 3 (20% = 11/55) and exon 8 (15% = 8/55). A total of 50 patients (38.5%) underwent surgical treatment, receiving a total of 63 surgeries. The average age of first surgery was 2.6years, and the majority of surgery (85.7%, 54/63) was operated before 4years old. The most common and earliest surgery was hernia repair. Three patients were died of respiratory failure. Conclusion: This study provided additional information on the clinical, cardiac ultrasound and surgical procedure in MPS II patients. Our study expanded the genotype spectrum of MPS II. Based on these data, characterization of MPS II patients group could be used to early diagnosis and treatment of the disease.

Phenotypic Correction of Murine Mucopolysaccharidosis Type II by Engraftment of Ex Vivo Lentiviral Vector-Transduced Hematopoietic Stem and Progenitor Cells.

Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is an X-linked recessive lysosomal disease caused by deficiency of iduronate-2-sulfatase (IDS). The absence of IDS results in the accumulation of the glycosaminoglycans (GAGs) heparan sulfate and dermatan sulfate. Currently, the only approved treatment option for MPS II is enzyme replacement therapy (ERT), Elaprase. However, ERT is demanding for the patient and does not ameliorate neurological manifestations of the disease. Using an IDS-deficient mouse model that phenocopies the human disease, we evaluated hematopoietic stem and progenitor cells (HSPCs) transduced with a lentiviral vector (LVV) carrying a codon-optimized human IDS coding sequence regulated by a ubiquitous MNDU3 promoter (MNDU3-IDS). Mice treated with MNDU3-IDS LVV-transduced cells showed supraphysiological levels of IDS enzyme activity in plasma, peripheral blood mononuclear cells, and in most analyzed tissues. These enzyme levels were sufficient to normalize GAG storage in analyzed tissues. Importantly, IDS levels in the brains of MNDU3-IDS-engrafted animals were restored to 10-20% than that of wild-type mice, sufficient to normalize GAG content and prevent emergence of cognitive deficit as evaluated by neurobehavioral testing. These results demonstrate the potential effectiveness of ex vivo MNDU3-IDS LVV-transduced HSPCs for treatment of MPS II.

Mutational spectrum of the iduronate-2-sulfatase gene in Mexican patients with Hunter syndrome.

Hunter syndrome, or mucopolysaccharidosis type II (MPS II), is caused by deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS), which is responsible for degrading heparan and dermatan sulfate. The IDS gene is located on chromosome Xq28; pathological variants in this gene mostly consist of missense mutations and small and larger deletions, which produce different phenotypes. However, there is only one record in our population concerning the molecular mechanism of this disease; a genotype-phenotype description is not available. There were included 24 unrelated male patients; clinical features were recorded at a database, fluorometric IDS enzyme activity testing was done for each individual, followed by Sanger sequencing to identify mutations. The mutational spectrum was found in 16 out of 24 Mexican patients with MPS II, and its range of phenotypes was described. The most frequent variants were of the missense type. The most affected exons were exon 3 (c.275T>G, c.284_287del, c.325T>C), exon 8 (c.1035G>C, c.550G>A), exon 9 (c.1403G>C, c.1229_1229del), and exon 7 (c.979A>C; this variant has not been previously reported). Exon 5 (c.438C>T, a non-pathogenic variant) was the least frequent. It was also found that the most severely affected patients were those with large deletions (2 out of 24) [rsaIDS: IDSP1 (P164)x0, FMR1, AFF2 (P164)x2] involving genes and pseudogenes. We found 2 patients with a synonymous mutation in exon 4. Our results confirmed reports in the literature, since the most frequent variants were reported in exons 3 and 8. However, this result varies from one previous report in our population, which mentions large deletions and rearrangements as the most frequent alterations, since complex rearrangements were not found. According to what has been previously found, the most severely affected patients are those in which a whole gene has been deleted.

Newborn Screening Program for Mucopolysaccharidosis Type II and Long-Term Follow-Up of the Screen-Positive Subjects in Taiwan.

Background: Mucopolysaccharidosis II (MPS II) is an X-linked disorder resulting from a deficiency in lysosomal enzyme iduronate-2-sulfatase (IDS), which causes the accumulation of glycosaminoglycans (GAGs) in the lysosomes of many tissues and organs, leading to progressive cellular dysfunction. An MPS II newborn screening program has been available in Taiwan since 2015. The aim of the current study was to collect and analyze the long-term follow-up data of the screen-positive subjects in this program. Methods: From August 2015 to April 2022, 548,624 newborns were screened for MPS II by dried blood spots using tandem mass spectrometry, of which 202 suspected infants were referred to our hospital for confirmation. The diagnosis of MPS II was confirmed by IDS enzyme activity assay in leukocytes, quantitative determination of urinary GAGs by mass spectrometry, and identification of the IDS gene variant. Results: Among the 202 referred infants, 10 (5%) with seven IDS gene variants were diagnosed with confirmed MPS II (Group 1), 151 (75%) with nine IDS gene variants were classified as having suspected MPS II or pseudodeficiency (Group 2), and 41 (20%) with five IDS gene variants were classified as not having MPS II (Group 3). Long-term follow-up every 6 months was arranged for the infants in Group 1 and Group 2. Intravenous enzyme replacement therapy (ERT) was started in four patients at 1, 0.5, 0.4, and 0.5 years of age, respectively. Three patients also received hematopoietic stem cell transplantation (HSCT) at 1.5, 0.9, and 0.6 years of age, respectively. After ERT and/or HSCT, IDS enzyme activity and the quantity of urinary GAGs significantly improved in all of these patients compared with the baseline data. Conclusions: Because of the progressive nature of MPS II, early diagnosis via a newborn screening program and timely initiation of ERT and/or HSCT before the occurrence of irreversible organ damage may lead to better clinical outcomes. The findings of the current study could serve as baseline data for the analysis of the long-term effects of ERT and HSCT in these patients.

Production of therapeutic iduronate-2-sulfatase enzyme with a novel single-stranded RNA virus vector.

The Sendai virus vector has received a lot of attention due to its broad tropism for mammalian cells. As a result of efforts for genetic studies based on a mutant virus, we can now express more than 10 genes of up to 13.5 kilo nucleotides in a single vector with high protein expression efficiency. To prove this benefit, we examined the efficacy of the novel ribonucleic acid (RNA) virus vector harboring the human iduronate-2-sulfatase (IDS) gene with 1,653 base pairs, a causative gene for mucopolysaccharidosis type II, also known as a disorder of lysosomal storage disorders. As expected, this novel RNA vector with the human IDS gene exhibited its marked expression as determined by the expression of enhanced green fluorescent protein and IDS enzyme activity. While these cells exhibited a normal growth rate, the BHK-21 transformant cells stably expressing the human IDS gene persistently generated an active human IDS enzyme extracellularly. The human IDS protein produced failed to be incorporated into the lysosome when cells were pretreated with mannose-6-phosphate, demonstrating that this human IDS enzyme has potential for therapeutic use by cross-correction. These results suggest that our novel RNA vector may be applicable for further clinical settings.

Effect of Mutated ids Overexpression on IDS Enzyme Activity and Developmental Phenotypes in Zebrafish Embryos: A Valuable Index for Assessing Critical Point-Mutations Associated with Mucopolysaccharidosis Type II Occurrence in Humans.

Mucopolysaccharidosis type II (MPS II) is an X-linked disorder resulting from a deficiency in iduronate 2-sulfatase (IDS), which is reported to be caused by gene mutations in the iduronate 2-sulfatase (IDS) gene. Many IDS mutation sites have not yet had their causal relationship with MPS II characterized. We employed a gain-of-function strategy whereby we microinjected different mutated zebrafish ids (z-ids) mRNAs corresponded to human IDS gene into zebrafish embryos, and then measured their total IDS enzymatic activity and observed the occurrence of defective phenotypes during embryonic development. We examined three known mutation sites for human IDS genes (h-IDS) associated with MPS II symptoms, including h-IDS-P86L, -S333L and -R468W, which corresponded to z-ids-P80L, -S327L and -R454W. When these three mutated z-ids mRNAs were overexpressed in zebrafish embryos, the IDS enzymatic activity of the total proteins extracted from the injected embryos was not increased compared with the endogenous IDS of the untreated embryos, which suggests that the IDS enzymatic activity of these three mutated z-ids was totally lost, as expected. Additionally, we observed defective phenotypes in these injected embryos, resulting from the failed IDS enzyme breakdown, which, in turn, has a dominant negative effect on the endogenous wild-type IDS function. These phenotypes were similar to the clinical symptoms observed in MPS II pathogenesis. We further studied six uncharacterized IDS mutation sites as identified by the Taiwanese MPS newborn screening programs. We propose a novel IDS enzyme activity assay combined with phenotypic observation in zebrafish embryos, as an alternative platform for quickly providing a valuable index for preliminarily assessment of any identified IDS point mutation gene that has not yet been characterized, in the context of its role in MPS II development.

Efficient engraftment of genetically modified cells is necessary to ameliorate central nervous system involvement of murine model of mucopolysaccharidosis type II by hematopoietic stem cell targeted gene therapy

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disease (LSD) caused by a deficiency of the iduronate-2-sulfatase (IDS) that catabolizes glycosaminoglycans (GAGs). Abnormal accumulations of GAGs in somatic cells lead to various manifestations including central nervous system (CNS) disease. Enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation (HSCT) are the currently available therapy for MPS II, but both therapies fail to improve CNS manifestations. We previously showed that hematopoietic stem cell targeted gene therapy (HSC-GT) with lethal irradiation improved CNS involvement in a murine model of MPS II which lacks the gene coding for IDS. However, the strong preconditioning, with lethal irradiation, would cause a high rate of morbidity and mortality. Therefore, we tested milder preconditioning procedures with either low dose irradiation or low dose irradiation plus an anti c-kit monoclonal antibody (ACK2) to assess CNS effects in mice with MPS II after HSC-GT. Mice from all the HSC-GT groups displayed super-physiological levels of IDS enzyme activity and robust reduction of abnormally accumulated GAGs to the wild type mice levels in peripheral organs. However, only the mice treated with lethal irradiation showed significant cognitive function improvement as well as IDS elevation and GAG reduction in the brain.These results suggest that an efficient engraftment of genetically modified cells for HSC-GT requires strong preconditioning to ameliorate CNS involvement in cases with MPS II.

Mucopolysaccharidosis type II (Hunter syndrome): Clinical and biochemical aspects of the disease and approaches to its diagnosis and treatment.

Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is a rare X-linked lysosomal storage disease caused by mutations of the gene encoding the lysosomal enzyme iduronate-2-sulfatase (IDS), the role of which is to hydrolytically remove O-linked sulfates from the two glycosaminoglycans (GAGs) heparan sulfate (HS) and dermatan sulfate (DS). HS and DS are linear, heterogeneous polysaccharides composed of repeating disaccharide subunits of l-iduronic acid (IdoA) or d-glucuronic acid, (1→4)-linked to d-glucosamine (for HS), or (1→3)-linked to 2-acetamido-2-deoxy-d-galactose (N-acetyl-d-galactosamine) (for DS). In healthy cells, IDS cleaves the sulfo group found at the C-2 position of terminal non-reducing end IdoA residues in HS and DS. The loss of IDS enzyme activity leads to progressive lysosomal storage of HS and DS in tissues and organs such as the brain, liver, spleen, heart, bone, joints and airways. Consequently, this leads to the phenotypic features characteristic of the disease. This review provides an overview of the disease profile and clinical manifestation, with a particular focus on the biochemical basis of the disease and chemical approaches to the development of new diagnostics, as well as discussing current treatment options and emerging new therapies.

Molecular diagnosis of 65 families with mucopolysaccharidosis type II (Hunter syndrome) characterized by 16 novel mutations in the IDS gene: Genetic, pathological, and structural studies on iduronate-2-sulfatase

Mucopolysaccharidosis type II (MPS II: also called as Hunter syndrome) is an X-linked recessive lysosomal storage disorder characterized by the accumulation of extracellular glycosaminoglycans due to the deficiency of the enzyme iduronate-2-sulfatase (IDS). Previous observations suggested that MPS II can be classified into two distinct disease subtypes: (1) severe type of MPS II involves a decline in the cognitive ability of a patient and (2) attenuated type of MPS II exhibits no such intellectual phenotype. To determine whether such disease subtypes of MPS II could be explained by genetic diagnosis, we analyzed mutations in the IDS gene of 65 patients suffering from MPS II among the Japanese population who were diagnosed with both the accumulation of urinary glycosaminoglycans and a decrease in their IDS enzyme activity between 2004 and 2014. Among the specimens examined, we identified the following mutations: 33 missense, 8 nonsense, 7 frameshift, 4 intronic changes affecting splicing, 8 recombinations involving IDS-IDS2, and 7 other mutations including 4 large deletions. Consistent with the previous data, the results of our study showed that most of the attenuated phenotype was derived from the missense mutations of the IDS gene, whereas mutations associated with a large structural alteration including recombination, splicing, frameshift, and nonsense mutations were linked to the severe phenotype of MPS II. Furthermore, we conducted a homology modeling study of IDS P120R and N534I mutant as representatives of the causative mutation of the severe and attenuated type of MPS II, respectively. We found that the substitution of P120R of the IDS enzyme was predicted to deform the α-helix generated by I119-F123, leading to the major structural alteration of the wild-type IDS enzyme. In sharp contrast, the effect of the structural alteration of N534I was marginal; thus, this mutation was pathogenically predicted to be associated with the attenuated type of MPS II. These results suggest that a combination of the genomic diagnosis of the IDS gene and the structural prediction of the IDS enzyme could enable the prediction of a phenotype more effectively.

86. AAV9 Mediated Correction of Iduronate-2-Sulfatase Deficiency in the Central Nervous System to Prevent the Onset of Neurologic Deficits in a Murine Model of Mucopolysaccharidosis Type II

Mucopolysaccharidosis type II (MPS II; Hunter Syndrome) is an X-linked recessive inherited lysosomal storage disease caused by deficiency of iduronate-2-sulfatase (IDS) and subsequent accumulation of glycosaminoglycans (GAGs) dermatan and heparan sulphate. Affected individuals exhibit a range in severity of manifestations such as organomegaly, skeletal dysplasias, cardiopulmonary obstruction, neurocognitive deficit, and shortened life expectancy. There is no cure for MPS II at the moment. Current standard of care is enzyme replacement therapy (ELAPSRASE; idursulfase), which is used to manage disease progression. However, enzyme replacement therapy (ERT) does not show neurologic improvement. As hematopoetic stem cell transplantation (HSCT) has not shown neurologic benefit for MPS II, there is currently no clinical recourse for patients exhibiting neurologic manifestations of this disease, and new therapies are desperately needed. We have been developing the use of AAV9 vectors for delivering the human IDS coding sequence (AAV9.hIDS) into the central nervous system of MPS II mice to restore IDS levels in the brain and prevent the emergence of neurocognitive deficits in the treated animals. A series of CMV-enhancer, beta actin-regulated vectors were generated that encode human IDS with or without the human sulfatase modifying factor-1 (SUMF-1), required for activation of the sulfatase active site. Three routes of administration; Intrathecal (IT), Intracerebroventricular (ICV) and Intravenous (IV) were used in these experiments. We found no significant difference in the enzyme level between mice that were treated with AAV9 vector transducing hIDS alone and mice that were treated with AAV9 vector encoding human IDS and SUMF-1, regardless of the route of administration. IT-administrated NOD. SCID (IDS Y+) and C57BL/6 (IDS Y+) did not show elevated IDS activity in the brain and spinal cord when compared to untreated animals, while plasma showed ten-fold higher (NOD. SCID) and 150-fold higher (C57BL/6) levels than untreated animals. IDS-deficient mice intravenously administered AAV9-hIDS exhibited IDS activities in all organs that were comparable to wild type. Moreover, the plasma of IV injected animals showed enzyme activity that was 100-fold higher than wild type. IDS-deficient mice administered AAV9-hIDUA intracerebroventricularly showed IDS activities comparable to wild type in most areas of the brain and peripheral tissues, while some portions of the brain showed two- to four-fold higher activity than wild type. Furthermore, IDS activity in plasma was 200-fold higher than wild type. Surprisingly, IDS enzyme activity in the plasma of all administrated animals showed persistence for at least 12 weeks post injection; therefore, IDS enzyme was not immunogenic at least on the C57BL/6 murine background. We also conducted additional neurobehavioral testing using the Barnes maze to differentiate neurocognitive deficits of untreated MPS II animals from that of wild type littermates. We found that the learning capability of affected animals is distinctively slower than that observed in littermates. Thus, Barnes maze will be used to address the benefit of these therapies in the MPS II murine model in future experiments. These results indicate potential of therapeutic benefit of AAV9 mediated human IDS gene transfer to the CNS to prevent neurologic deficiency in the MPS II.

369. AAV9 Mediated Correction of Iduronate-2-Sulfatase Deficiency in the Central Nervous System of Mucopolysaccharidosis Type II Mice

Mucopolysaccharidosis type II (MPS II; Hunter Syndrome) is an X-linked inherited lysosomal storage disease caused by deficiency of iduronate-2-sulfatase (IDS) and subsequent accumulation of glycosaminoglycans (GAGs) dermatan and heparan sulphate. Affected individuals exhibit a range in severity of manifestations such as organomegaly, skeletal dysplasias, cardiopulmonary obstruction, neurocognitive deficit, and shortened life expectancy. There is no cure for MPS II at the moment. Current standard of care is enzyme therapy (ELAPSRASE; idursulfase), which is used to manage disease progression. As hematopoetic stem cell transplantation (HSCT) has not shown neurologic benefit for MPS II, there is currently no clinical recourse for patients exhibiting neurologic manifestations of this disease, and new therapies are desperately needed.We have been developing the use of AAV9 vectors for delivery of the human IDS gene into the central nervous system of MPS II mice to restore IDS levels in the brain and prevent the emergence of neurocognitive deficits in the treated animals. A series of CBA-regulated vectors were generated that encode human IDS with or without the human sulfatase modifying factor-1 (SUMF-1), required for activation of the sulfatase active site. Intrathecal (IT) administration of these vectors into IDS positive mice (NOD.SCID and C57BL/6) did not result in a significant increase in IDS levels in the brains or in the organs of treated mice when compared to untreated animals. However, plasma showed higher enzyme levels in the intrathecally treated C57BL/6 mice than in untreated animals that persisted for at least 6 weeks post injection. There was no evidence for diminished activity that would be associated with immune response against human IDS protein. In contrast to the IT administered mice, MPS II mice administered AAV9-IDS vector intracerebroventricularly exhibited two- to four-fold higher levels of IDS enzyme activity in all portions of the brain in comparison with untreated, unaffected littermates. The ICV treated animals also showed lower levels of tissue GAG in the brain than untreated MPS II animals and similar levels as their wild-type littermates. IDS activity in the plasma of ICV treated mice was also significantly higher than that of wild type mice with no sign of immune response at least 6 weeks post administration. These results show the potential therapeutic benefit of AAV9-mediated IDS gene delivery to the CNS through the cerebrospinal fluid to address neurological manifestations of MPS II, Hunter syndrome.

Insulin receptor antibody‐iduronate 2‐sulfatase fusion protein: Pharmacokinetics, anti‐drug antibody, and safety pharmacology in Rhesus monkeys

Mucopolysaccharidosis (MPS) Type II is caused by mutations in the gene encoding the lysosomal enzyme, iduronate 2-sulfatase (IDS). The majority of MPSII cases affect the brain. However, enzyme replacement therapy with recombinant IDS does not treat the brain, because IDS is a large molecule drug that does not cross the blood-brain barrier (BBB). To enable BBB penetration, IDS has been re-engineered as an IgG-IDS fusion protein, where the IgG domain is a monoclonal antibody (MAb) against the human insulin receptor (HIR). The HIRMAb crosses the BBB via receptor-mediated transport on the endogenous BBB insulin receptor, and the HIRMAb domain of the fusion protein acts as a molecular Trojan horse to ferry the fused IDS into brain from blood. The present study reports on the first safety pharmacology and pharmacokinetics study of the HIRMAb-IDS fusion protein. Juvenile male Rhesus monkeys were infused intravenously (IV) weekly for 26 weeks with 0, 3, 10, or 30 mg/kg of the HIRMAb-IDS fusion protein. The plasma clearance of the fusion protein followed a linear pharmacokinetics profile, which was equivalent either with measurements of the plasma concentration of immunoreactive HIRMAb-IDS fusion protein, or with assays of plasma IDS enzyme activity. Anti-drug antibody (ADA) titers were monitored monthly, and the ADA response was primarily directed against the variable region of the HIRMAb domain of the fusion protein. No infusion related reactions or clinical signs of immune response were observed during the course of the study. A battery of safety pharmacology, clinical chemistry, and tissue histopathology showed no signs of adverse events, and demonstrate the safety profile of chronic treatment of primates with 3-30 mg/kg weekly IV infusion doses of the HIRMAb-IDS fusion protein.

Brazilian reference values for MPS II screening in dried blood spots — A fluorimetric assay

ObjectivesMucopolysaccharidosis II (MPS II), or Hunter Syndrome, is a lysosomal storage disorder that is caused by the deficiency or absence of iduronate-2-sulfatase (IDS) enzyme; in this disease, early diagnosis is essential to provide higher life expectancy for patients. This study validates a fluorimetric assay that is used to assess IDS enzyme activity using dried blood spot (DBS) samples and presents the reference interval for the Brazilian population. Design and methodsVenous blood sample was collected in heparin tubes for leukocyte extraction and DBS preparation. IDS activity in the leukocytes was analyzed, and the results were considered the gold standard reference for the categorization of volunteers as positive or negative controls (PC and NC, respectively). IDS activity in the DBS was analyzed using an adapted version of the leukocyte assay. Statistical analyses were performed using a ROC curve to determine cutoff values and using a parametric Student's t test to compare values between genders. To verify that the assay yielded consistent results, a Bland–Altman plot was prepared. ResultsLeukocyte IDS activity values ranged between 2.71 and 17.36nmol/mg protein/h in the NC group and between 0 and 0.11nmol/mg protein/h in the PC group. Based on the DBS assay, activities ranged between 1.83 and 16.86μmol/L blood/h in the NC group and between 0.58 and 4.32μmol/L blood/h in the PC group. ConclusionsReference values of IDS activity were determined in DBS with acceptable sensitivity and specificity. Therefore, the DBS assay described in this work may be a useful tool to screen MPS II patients in the Brazilian population.

Fast and robust protocol for prenatal diagnosis of mucopolysaccharidosis Type II

Introduction: Mucopolysaccharidosis type II (MPSII) is an X-linked lysosomal disorder caused by deficiency of iduronate-2-sulfatase ( IDS ). In this study, we proposed a new protocol for prenatal diagnosis, using DNA obtained from amniotic fluid cells that did not attach to the bottom of the culture flask after the first medium change. Methods: Four pregnant MPS II carriers were referred to the Medical Genetics Service of Hospital de Clinicas dePorto Alegre for a prenatal diagnosis and identification of the disease, which were performed by polymerase chain reaction (PCR) amplification, restriction fragment length polymorphism, and sequencing according to the mutation previously found in the family. Results: The analysis indicated the absence of mutation in three fetal materials and the presence of mutation in one case. Concomitantly, cytogenetic and biochemical analyses were performed after 12 days of cell culture, and only one case showed absence of enzyme activity, confirming the molecular analysis. Conclusions: This diagnostic protocol designed to provide more robust results and safer genetic counseling suggests that DNA obtained from floating amniotic fluid cells can be used as a source of fetal material to allow a faster alternative for prenatal care through molecular analysis. Determination of IDS gene mutation in fetal amniotic fluid cells together with IDS enzyme activity testing is a rapid, sensitive and accurate method for prenatal diagnosis of MPS II for high-risk pregnant women.

A novel iduronate 2-sulfatase mutation in a Chinese family with mucopolysaccharidosis type II

Mucopolysaccharidosis type II (MPS II; also known as Hunter syndrome) is an X-linked multisystem disorder resulting from the defective activity of the enzyme iduronate-2-sulfatase (IDS). Genetic testing is crucial in clarifying and diagnosing different types of MPS diseases. In this paper we report a novel IDS nonsense mutation resulting in MPS II in several patients from a Chinese family. IDS enzyme activity, polymerase chain reaction, and DNA sequencing were performed to confirm the diagnosis of MPS II. Three patients had no detectable IDS activity. Two genetic tests revealed a novel IDS nonsense mutation (c.1030G>T, p.E344X) inherited from their mothers. The nonsense mutation shortened the peptide chain from 550 to 344 amino acids, which is believed to be a disease-causing mutation. MPS II is inherited in an X-linked manner. The risk to sibs depends on the carrier status of the mother. Genetic testing is necessary to identify disease-causing mutation. With this information, carrier testing for at-risk female relatives and prenatal testing for pregnancies at increased risk become possible.