Sulfatase Modifying Factor Research Articles

Structure and expression of sulfatase and sulfatase modifying factor genes in the diamondback moth, Plutella xylostella.

The diamondback moth, Plutella xylostella (L.), uses sulfatases (SULF) to counteract the glucosinolate-myrosinase defensive system that cruciferous plants have evolved to deter insect feeding. Sulfatase activity is regulated by post-translational modification of a cysteine residue by sulfatase modifying factor 1 (SUMF1). We identified 12 SULF genes (PxylSulfs) and two SUMF1 genes (PxylSumf1s) in the P. xylostella genome. Phylogenetic analysis of SULFs and SUMFs from P. xylostella, Bombyx mori, Manduca sexta, Heliconius melpomene, Danaus plexippus, Drosophila melanogaster, Tetranychus urticae and Homo sapiens showed that the SULFs were clustered into five groups, and the SUMFs could be divided into two groups. Profiling of the expression of PxylSulfs and PxylSumfs by RNA-seq and by quantitative real-time polymerase chain reaction showed that two glucosinolate sulfatase genes (GSS), PxylSulf2 and PxylSulf3, were primarily expressed in the midgut of 3rd- and 4th-instar larvae. Moreover, expression of sulfatases PxylSulf2, PxylSulf3 and PxylSulf4 were correlated with expression of the sulfatases modifying factor PxylSumf1a. The findings from this study provide new insights into the structure and expression of SUMF1 and PxylSulf genes that are considered to be key factors for the evolutionary success of P. xylostella as a specialist herbivore of cruciferous plants.

Intra cerebral administration of AAVrh-10 carrying human SGSH and SUMF1 cDNAs in children with MPSIIIA disease: Long term follow-up of a phase I–II trial

Objective: Mucopolysaccharidosis type IIIA is a severe degenerative disease due to an autosomal recessive genetic defect in the gene coding for the lysosomal N- sulfoglycosamine sulfohydrolase (SGSH) whose catalytic site is activated by a sulfatase modifying factor (SUMF1). No effective treatment exists and an intra-cerebral gene therapy was developed using an AAV-2/rh.10-SGSH-IRES-SUMF1 vector that was administered in four children in a phase I/II clinical trial. The first 3 patients were close to 6 years and the last was 2.7 years old (median age=6,1 y) at treatment. All children were ambulatory but had already decreased cognitive abilities with brain atrophy detectable on MRIs at the time of treatment. In order to prevent elimination of transduced cells, an immunosuppressive treatment (Mycophenolate mofetil, Tacrolimus) was initiated before surgery and maintained either 8 weeks (Mycophenolate-mofetil) or throughout the follow-up (Tacrolimus, with progressive dose reduction). Methods: Yearly, neurological and cognitive performance (PEP, Vineland, TBAQ tests) as well as brain imaging were performed. Biological follow-up was done every 4 months and adverse events (AE) and severe adverse events (SAE) were recorded regularly. Results: Overall safety evaluation showed a good tolerance of the treatment with a total number of 6 SAE and 209 AE not related to the treatment nor to the procedure. Cognitive evolution, assessed by serial motor and cognitive assessment showed a persistent decline in abilities in the four included patients during follow-up, even if a transitory improvement of mood and sleep disorders was observed in all. Follow-up of brain MRI showed an increase of brain atrophy in all children. Conclusion: This first gene therapy trial using AAVrh-10 carrying human SGSH and SUMF1 cDNAs in MPSIIIA proved to be safe and well tolerated, but clinical and cognitive outcome of the four included children followed natural history of MPSIIIA.

Hydrocephalus as a rare clinical symptom in a child with multiple sulfatase deficiency

Objective: Multiple Sulfatase Deficiency (MSD) is a rare autosomal recessive inborn error of metabolism caused by mutations in the sulfatase modifying factor 1 gene. Retarded psychomotor development, macrocephaly, coarse face, hepatosplenomegaly, ichtyosis and skeletal findings like scoliosis and dysostosis multiplex are the most common findings of the disease. Methods: A 4-year-old girl who was admitted to our hospital for vomiting and feding difficulty since 10 days. On examination, she had coarse face, ichtyosis, hypertrichosis, and hepatosplenomegaly. Also, she had generalized axial hypotonia with decreased tone in the four extremities, and deep tendon reflexes were absent. Results: X-rays of the elbow and vertebral column were compatible with dysostosis multiplex. Electromyography, and echocardiogram were normal. Cerebral magnetic resonance imaging (MRI) revealed large ventricles suggested hydrocephalus, and ventriculoperitoneal shunt performed. MRI did not demonstrate any white matter changes in the brain. We analyzed a novel homozygous p.S155P (c.463T>C) in the SUMF1 gene.Conclusion: Hydrocephalus is rare symptom that may occur in MSD as in other MPS. It's important to consider the possibility of hydrocephalus when additional acute neurological findings such as increased intracranial pressure were developed besides chronic neurological findings.

Sulfatase modifying factor 1 (SUMF1) is associated with Chronic Obstructive Pulmonary Disease

BackgroundIt has been observed that mice lacking the sulfatase modifying factor (Sumf1) developed an emphysema-like phenotype. However, it is unknown if SUMF1 may play a role in Chronic Obstructive Pulmonary Disease (COPD) in humans. The aim was to investigate if the expression and genetic regulation of SUMF1 differs between smokers with and without COPD.MethodsSUMF1 mRNA was investigated in sputum cells and whole blood from controls and COPD patients (all current or former smokers). Expression quantitative trait loci (eQTL) analysis was used to investigate if single nucleotide polymorphisms (SNPs) in SUMF1 were significantly associated with SUMF1 expression. The association of SUMF1 SNPs with COPD was examined in a population based cohort, Lifelines. SUMF1 mRNA from sputum cells, lung tissue, and lung fibroblasts, as well as lung function parameters, were investigated in relation to genotype.ResultsCertain splice variants of SUMF1 showed a relatively high expression in lung tissue compared to many other tissues. SUMF1 Splice variant 2 and 3 showed lower levels in sputum cells from COPD patients as compared to controls. Twelve SNPs were found significant by eQTL analysis and overlapped with the array used for genotyping of Lifelines. We found alterations in mRNA expression in sputum cells and lung fibroblasts associated with SNP rs11915920 (top hit in eQTL), which validated the results of the lung tissue eQTL analysis. Of the twelve SNPs, two SNPs, rs793391 and rs308739, were found to be associated with COPD in Lifelines. The SNP rs793391 was also confirmed to be associated with lung function changes.ConclusionsWe show that SUMF1 expression is affected in COPD patients compared to controls, and that SNPs in SUMF1 are associated with an increased risk of COPD. Certain COPD-associated SNPs have effects on either SUMF1 gene expression or on lung function. Collectively, this study shows that SUMF1 is associated with an increased risk of developing COPD.

Sulfatase modifying factor 1 trafficking through the cells: from endoplasmic reticulum to the endoplasmic reticulum.

Sulfatase modifying factor 1 (SUMF1) is the gene mutated in multiple sulfatase deficiency (MSD) that encodes the formylglycine-generating enzyme, an essential activator of all the sulfatases. SUMF1 is a glycosylated enzyme that is resident in the endoplasmic reticulum (ER), although it is also secreted. Here, we demonstrate that upon secretion, SUMF1 can be taken up from the medium by several cell lines. Furthermore, the in vivo engineering of mice liver to produce SUMF1 shows its secretion into the blood serum and its uptake into different tissues. Additionally, we show that non-glycosylated forms of SUMF1 can still be secreted, while only the glycosylated SUMF1 enters cells, via a receptor-mediated mechanism. Surprisingly, following its uptake, SUMF1 shuttles from the plasma membrane to the ER, a route that has to date only been well characterized for some of the toxins. Remarkably, once taken up and relocalized into the ER, SUMF1 is still active, enhancing the sulfatase activities in both cultured cells and mice tissues.

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.

A non-conserved miRNA regulates lysosomal function and impacts on a human lysosomal storage disorder.

Sulfatases are key enzymatic regulators of sulfate homeostasis with several biological functions including degradation of glycosaminoglycans (GAGs) and other macromolecules in lysosomes. In a severe lysosomal storage disorder, multiple sulfatase deficiency (MSD), global sulfatase activity is deficient due to mutations in the sulfatase-modifying factor 1 (SUMF1) gene, encoding the essential activator of all sulfatases. We identify a novel regulatory layer of sulfate metabolism mediated by a microRNA. miR-95 depletes SUMF1 protein levels and suppresses sulfatase activity, causing the disruption of proteoglycan catabolism and lysosomal function. This blocks autophagy-mediated degradation, causing cytoplasmic accumulation of autophagosomes and autophagic substrates. By targeting miR-95 in cells from MSD patients, we can effectively increase residual SUMF1 expression, allowing for reactivation of sulfatase activity and increased clearance of sulfated GAGs. The identification of this regulatory mechanism opens the opportunity for a unique therapeutic approach in MSD patients where the need for exogenous enzyme replacement is circumvented.

Correction of murine mucopolysaccharidosis type IIIA central nervous system pathology by intracerebroventricular lentiviral-mediated gene delivery.

Mucopolysaccharidoses (MPS) are inborn metabolic disorders caused by a deficiency of glycosaminoglycan degrading enzymes. Although intravenous enzyme replacement therapy is a viable approach for the treatment of non-neuronopathic forms of MPS, its effectiveness in the central nervous system (CNS) is limited by the blood-brain barrier. Alternatively, enzyme replacement therapies and other therapies that directly target the brain represent approaches that circumvent the blood-brain barrier and, in the case of gene therapies, are intended to negate the need for repetitive dosing. In the present study, gene therapy was targeted to the brains of young adult mice affected by mucopolysaccharidosis type IIIA (MPS IIIA) by bilateral delivery of two different therapeutic lentivirus vectors to the cerebral lateral ventricles. One vector expressed codon optimised murine sulphamidase, whereas the other co-expressed sulphamidase and sulfatase modifying factor-1. Six months after gene delivery, bladder distension was prevented in all treated animals, and behavioural deficits were improved. Therapeutic enzyme activity from the most efficacious vector, which was also the simpler vector, ranged from 0.5- to four-fold normal within the brains of treated animals, and the average amount of integrated vector ranged from 0.1-1 gene copies per cell. Consequently, levels of ganglioside and lysosomal β-hexosaminidase, both of which are characteristically elevated in MPS IIIA, were significantly reduced, or were normalised. The present study demonstrates the efficacy of the intraventricular injection as a tool to target the brain with therapeutic genes in adult MPS IIIA mice, and provides evidence supporting this approach as a potentially effective means of treating CNS pathology in MPS IIIA patients.

Intracerebral Administration of Adeno-Associated Viral Vector Serotype rh.10 Carrying Human SGSH and SUMF1 cDNAs in Children with Mucopolysaccharidosis Type IIIA Disease: Results of a Phase I/II Trial

Mucopolysaccharidosis type IIIA is a severe degenerative disease caused by an autosomal recessive defect of a gene encoding a lysosomal heparan-N-sulfamidase, the N-sulfoglycosamine sulfohydrolase (SGSH), the catalytic site of which is activated by a sulfatase-modifying factor (SUMF1). Four children (Patients 1-3, aged between 5.5 and 6 years; Patient 4 aged 2 years 8 months) received intracerebral injections of an adeno-associated viral vector serotype rh.10-SGSH-IRES-SUMF1 vector in a phase I/II clinical trial. All children were able to walk, but their cognitive abilities were abnormal and had declined (Patients 1-3). Patients 1-3 presented with brain atrophy. The therapeutic vector was delivered in a frameless stereotaxic device, at a dose of 7.2×10(11) viral genomes/patient simultaneously via 12 needles as deposits of 60 μl over a period of 2 hr. The vector was delivered bilaterally to the white matter anterior, medial, and posterior to the basal ganglia. Immunosuppressive treatment (mycophenolate mofetil and tacrolimus) was initiated 15 days before surgery and maintained for 8 weeks (mycophenolate mofetil) or throughout follow-up (tacrolimus, with progressive dose reduction) to prevent elimination of transduced cells. Safety data collected from inclusion, during the neurosurgery period and over the year of follow-up, showed good tolerance, absence of adverse events related to the injected product, no increase in the number of infectious events, and no biological sign of toxicity related to immunosuppressive drugs. Efficacy analysis was necessarily preliminary in this phase I/II trial on four children, in the absence of validated surrogate markers. Brain atrophy evaluated by magnetic resonance imaging seemed to be stable in Patients 1 and 3 but tended to increase in Patients 2 and 4. Neuropsychological evaluations suggested a possible although moderate improvement in behavior, attention, and sleep in Patients 1-3. The youngest patient was the most likely to display neurocognitive benefit.

Elosulfase alfa.

Mucopolysaccharidosis type IVA (MPS IVA), also known as Morquio A syndrome, is an inherited, lysosomal storage disorder caused by genetic mutations in N-acetylgalactosamine-6-sulfatase (GALNS) enzyme gene. GALNS is essential for breakdown of glycosaminoglycans. The disease is characterized by the early onset of severe skeletal dysplasia resulting in significant disability by the second decade of life. Until recently there have been no available treatments other than surgery and palliative care. BioMarin Pharmaceutical developed elosulfase alfa, a recombinant human GALNS coproduced with sulfatase-modifying factor 1, as an enzyme replacement therapy for patients with MPS IVA. In clinical studies, enzyme replacement therapy with elosulfase alfa significantly improved physical endurance, respiratory function, growth and quality of life in patients with MPS IVA. Treatment increased clearance of glycosaminoglycans and induced gene expression consistent with improved chondrocyte function. Elosulfase alfa is approved for the treatment of MPS IVA in the U.S. and Europe.

Sos recruitment system for the analysis of the interaction between sulfatase-modifying factor 2 subtypes and interleukin-13

Interleukin (IL)-13 is a central mediator in allergic asthma. Our previous results have indicated that sulfatase-modifying factor 2 (SUMF2) interacts with IL-13 and inhibits its secretion. In this study, we investigated the interactions between SUMF2 subtypes and 2 types of IL-13. Wild type IL-13 (wh-IL-13) and its mutated counterpart (mh-IL-13) were analyzed and cloned into pSos yeast expression vectors. Protein was expressed in host cdc25H yeast strains. A quartet of agar growth plates was prepared for the yeast two-hybrid system, which was used to detect IL-13 and SUMF2 subtype interactions. Both yeast expression vectors, pSos/whIL-13 and pSos/whIL-13, and recombinant expression vectors for the 5 subtypes of SUMF2 (pMyr/SUMF2-Vx) were constructed. Our data showed that all of the SUMF2 subtypes bound to whIL-13 and mhIL-13 in the CytoTrap system. Five SUMF2 subtypes - SUMF2-V2, SUMF2-V3, SUMF2-V4, SUMF2-V5, and SUMF2-V7--interacted with whIL-13 and mhIL-13. These subtypes may contribute to allergic asthma by mediating IL-13 release.

Multiple sulfatase deficiency: A case series of four children

Multiple sulfatase deficiency is biochemically characterized by the accumulation of sulfated lipids and acid mucopolysaccharides. The gene sulfatase-modifying factor 1 (SUMF1), recently identified, encodes the enzyme responsible for post-translational modification of a cysteine residue, which is essential for the activity of sulfatases. We describe clinical findings and mutation analysis of four patients. The patients presented with hypotonia, developmental delay, coarse face, ichthyosis, and hepatosplenomegaly. The diagnosis was made through clinical findings, enzymatic assays, and mutation analysis. We were detected to be homozygous for a novel missense mutation c. 739G > C causing a p.G247R amino acid substitution in the SUMF1 protein.

Neonatal Gene Therapy With a Gamma Retroviral Vector in Mucopolysaccharidosis VI Cats

Mucopolysaccharidosis (MPS) VI is due to a deficiency in the activity of N-acetylgalactosamine 4-sulfatase (4S), also known as arylsulfatase B. Previously, retroviral vector (RV)-mediated neonatal gene therapy reduced the clinical manifestations of MPS I and MPS VII in mice and dogs. However, sulfatases require post-translational modification by sulfatase-modifying factors. MPS VI cats were injected intravenously (i.v.) with a gamma RV-expressing feline 4S, resulting in 5 ± 3 copies of RV per 100 cells in liver. Liver and serum 4S activity were 1,450 ± 1,720 U/mg (26-fold normal) and 107 ± 60 U/ml (13-fold normal), respectively, and were directly proportional to the liver 4S protein levels for individual cats. This study suggests that sulfatase-modifying factor (SUMF) activity in liver was sufficient to result in active enzyme despite overexpression of 4S. RV-treated MPS VI cats achieved higher body weights and longer appendicular skeleton lengths, had reduced articular cartilage erosion, and reduced aortic valve thickening and aortic dilatation compared with untreated MPS VI cats, although cervical vertebral bone lengths were not improved. This demonstrates that therapeutic expression of a functional sulfatase protein can be achieved with neonatal gene therapy using a gamma RV, but some aspects of bone disease remain difficult to treat.

Sulfatases are determinants of alveolar formation

Alveolar formation or alveolarization is orchestrated by a finely regulated and complex interaction between growth factors and extracellular matrix proteins. The lung parenchyma contains various extracellular matrix proteins including proteoglycans, which are composed of glycosaminoglycans (GAGs) linked to a protein core. Although GAGs are known to regulate growth factor distribution and activity according to their degree of sulfation the role of sulfated GAG in the respiratory system is not well understood. The degree of sulfation of GAGs is regulated in part, by sulfatases that remove sulfate groups. In vertebrates, the enzyme Sulfatase-Modifying Factor 1 (Sumf1) activates all sulfatases. Here we utilized mice lacking Sumf1−/− to study the importance of proteoglycan desulfation in lung development. The Sumf1−/− mice have normal lungs up until the onset of alveolarization at post-natal day 5 (P5). We detected increased deposition of sulfated GAG throughout the lung parenchyma and a decrease in alveolar septa formation. Moreover, stereological analysis showed that the alveolar volume is 20% larger in Sumf1−/− as compared to wild type (WT) mice at P10 and P30. Additionally, pulmonary function test was consistent with increased alveolar volume. Genetic experiments demonstrate that in Sumf1−/− mice arrest of alveolarization is independent of fibroblast growth factor signaling. In turn, the Sumf1−/− mice have increased transforming growth factor β (TGFβ) signaling and in vivo injection of TGFβ neutralizing antibody leads to normalization of alveolarization. Thus, absence of sulfatase activity increases sulfated GAG deposition in the lungs causing deregulation of TGFβ signaling and arrest of alveolarization.

HpSumf1 is involved in the activation of sulfatases responsible for regulation of skeletogenesis during sea urchin development

Sulfatases such as arylsulfatase and heparan sulfate 6-O-endosulfatase play important roles in morphogenesis during sea urchin development. For the activation of these sulfatases, Cα-formylglycine formation by sulfatase modifying factor (Sumf) is required. In this study, to clarify the regulatory mechanisms for the activation of sulfatases during sea urchin development, we examined the expression and function of the Hemicentrotus pulcherrimus homologs of Sumf1 and Sumf2 (HpSumf1 and HpSumf2, respectively). Expression of HpSumf1 but not HpSumf2 mRNA was dynamically changed during early development. Functional analyses of recombinant HpSumf1 and HpSumf2 using HEK293T cells expressing mouse arylsulfatase A (ArsA) indicated that HpSumf1 and HpSumf2 were both able to activate mammalian ArsA. Knockdown of HpSumf1 using morpholino antisense oligonucleotides caused abnormal spicule formation in the sea urchin embryo. Injection of HpSumf2 mRNA had no effect on skeletogenesis, while injection of HpSumf1 mRNA induced severe supernumerary spicule formation. Taken together, these findings suggest that HpSumf1 is involved in the activation of sulfatases required for control of skeletogenesis.

Efficacy of a Combined Intracerebral and Systemic Gene Delivery Approach for the Treatment of a Severe Lysosomal Storage Disorder

Multiple sulfatase deficiency (MSD), a severe autosomal recessive disease is caused by mutations in the sulfatase modifying factor 1 gene (Sumf1). We have previously shown that in the Sumf1 knockout mouse model (Sumf1(-/-)) sulfatase activities are completely absent and, similarly to MSD patients, this mouse model displays growth retardation and early mortality. The severity of the phenotype makes MSD unsuitable to be treated by enzyme replacement or bone marrow transplantation, hence the importance of testing the efficacy of novel treatment strategies. Here we show that recombinant adeno-associated virus serotype 9 (rAAV9) vector injected into the cerebral ventricles of neonatal mice resulted in efficient and widespread transduction of the brain parenchyma. In addition, we compared a combined, intracerebral ventricles and systemic, administration of an rAAV9 vector encoding SUMF1 gene to the single administrations-either directly in brain, or systemic alone -in MSD mice. The combined treatment resulted in the global activation of sulfatases, near-complete clearance of glycosaminoglycans (GAGs) and decrease of inflammation in both the central nervous system (CNS) and visceral organs. Furthermore, behavioral abilities were improved by the combined treatment. These results underscore that the "combined" mode of rAAV9 vector administration is an efficient option for the treatment of severe whole-body disorders.

SUMF1 mutations affecting stability and activity of formylglycine generating enzyme predict clinical outcome in multiple sulfatase deficiency

Multiple Sulfatase Deficiency (MSD) is caused by mutations in the sulfatase-modifying factor 1 gene encoding the formylglycine-generating enzyme (FGE). FGE post translationally activates all newly synthesized sulfatases by generating the catalytic residue formylglycine. Impaired FGE function leads to reduced sulfatase activities. Patients display combined clinical symptoms of single sulfatase deficiencies. For ten MSD patients, we determined the clinical phenotype, FGE expression, localization and stability, as well as residual FGE and sulfatase activities. A neonatal, very severe clinical phenotype resulted from a combination of two nonsense mutations leading to almost fully abrogated FGE activity, highly unstable FGE protein and nearly undetectable sulfatase activities. A late infantile mild phenotype resulted from FGE G263V leading to unstable protein but high residual FGE activity. Other missense mutations resulted in a late infantile severe phenotype because of unstable protein with low residual FGE activity. Patients with identical mutations displayed comparable clinical phenotypes. These data confirm the hypothesis that the phenotypic outcome in MSD depends on both residual FGE activity as well as protein stability. Predicting the clinical course in case of molecularly characterized mutations seems feasible, which will be helpful for genetic counseling and developing therapeutic strategies aiming at enhancement of FGE.

Enzyme Replacement in a Human Model of Mucopolysaccharidosis IVA In Vitro and Its Biodistribution in the Cartilage of Wild Type Mice

Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome) is a lysosomal storage disorder caused by deficiency of N-acetylgalactosamine-6-sulfatase (GALNS), an enzyme that degrades keratan sulfate (KS). Currently no therapy for MPS IVA is available. We produced recombinant human (rh)GALNS as a potential enzyme replacement therapy for MPS IVA. Chinese hamster ovary cells stably overexpressing GALNS and sulfatase modifying factor-1 were used to produce active (∼2 U/mg) and pure (≥97%) rhGALNS. The recombinant enzyme was phosphorylated and was dose-dependently taken up by mannose-6-phosphate receptor (Kuptake = 2.5 nM), thereby restoring enzyme activity in MPS IVA fibroblasts. In the absence of an animal model with a skeletal phenotype, we established chondrocytes isolated from two MPS IVA patients as a disease model in vitro. MPS IVA chondrocyte GALNS activity was not detectable and the cells exhibited KS storage up to 11-fold higher than unaffected chondrocytes. MPS IVA chondrocytes internalized rhGALNS into lysosomes, resulting in normalization of enzyme activity and decrease in KS storage. rhGALNS treatment also modulated gene expression, increasing expression of chondrogenic genes Collagen II, Collagen X, Aggrecan and Sox9 and decreasing abnormal expression of Collagen I. Intravenous administration of rhGALNS resulted in biodistribution throughout all layers of the heart valve and the entire thickness of the growth plate in wild-type mice. We show that enzyme replacement therapy with recombinant human GALNS results in clearance of keratan sulfate accumulation, and that such treatment ameliorates aberrant gene expression in human chondrocytes in vitro. Penetration of the therapeutic enzyme throughout poorly vascularized, but clinically relevant tissues, including growth plate cartilage and heart valve, as well as macrophages and hepatocytes in wild-type mouse, further supports development of rhGALNS as enzyme replacement therapy for MPS IVA.

Adeno‐associated virus gene transfer in Morquio A disease – effect of promoters and sulfatase‐modifying factor 1

Mucopolysaccharidosis (MPS) IVA is an autosomal recessive disorder caused by deficiency of the lysosomal enzyme N-acetylgalatosamine-6-sulfate sulfatase (GALNS), which leads to the accumulation of keratan sulfate and chondroitin 6-sulfate, mainly in bone. To explore the possibility of gene therapy for Morquio A disease, we transduced the GALNS gene into HEK293 cells, human MPS IVA fibroblasts and murine MPS IVA chondrocytes by using adeno-associated virus (AAV)-based vectors, which carry human GALNS cDNA. The effects of the promoter and the cotransduction with the sulfatase-modifying factor 1 gene (SUMF1) on GALNS activity levels was evaluated. Downregulation of the cytomegalovirus (CMV) immediate early enhancer/promoter was not observed for 10 days post-transduction. The eukaryotic promoters induced equal or higher levels of GALNS activity than those induced by the CMV promoter in HEK293 cells. Transduction of human MPS IVA fibroblasts induced GALNS activity levels that were 15-54% of those of normal human fibroblasts, whereas in transduced murine MPS IVA chondrocytes, the enzyme activities increased up to 70% of normal levels. Cotransduction with SUMF1 vector yielded an additional four-fold increase in enzyme activity, although the level of elevation depended on the transduced cell type. These findings suggest the potential application of AAV vectors for the treatment of Morquio A disease, depending on the combined choice of transduced cell type, selection of promoter, and cotransduction of SUMF1.