Mannose 6-phosphate receptor-mediated transport of sulfamidase across the blood-brain barrier in the newborn mouse.
ABSTRACT: Mucopolysaccharidosis type IIIA (MPS IIIA), which is a lysosomal storage disorder (LSD) caused by inherited deficiency of sulfamidase, is characterized by severe, progressive central nervous system (CNS) dysfunction. Enzyme replacement therapy (ERT) to treat CNS storage is challenging, because the access of enzymes to the brain is restricted by the blood-brain barrier (BBB). In a prior study, we found that phosphorylated beta-glucuronidase (P-GUS) could be transcytosed across the BBB in newborn mice by the mannose 6-phosphate (M6P) receptor. In order to determine whether sulfamidase can utilize this pathway, we examined brain influx and the specificity of uptake of sulfamidase after intravenous (i.v.) injection in 2-day-old and 8-week-old mice. [(131)I]Sulfamidase was transported across the BBB in neonates at rates higher than that of simultaneously injected [(125)I]albumin. In contrast, the transport of [(131)I]sulfamidase was negligible in 8-week-old mice, thereby showing that the BBB transport mechanism is developmentally downregulated. Capillary depletion revealed that 83.7% of the [(131)I]sulfamidase taken up by the brain was in the parenchyma, demonstrating transfer across the capillary wall. The uptake of [(131)I]sulfamidase into the brain was significantly reduced by co-injections of M6P and P-GUS. That is, the transport of sulfamidase into the brain parenchyma in early postnatal life is mediated by the M6P receptor, which is shared with P-GUS and is likely accessible to other M6P-containing lysosomal enzymes.
Project description:Mucopolysaccharidosis type VII is a lysosomal storage disorder resulting from inherited deficiency of beta-glucuronidase (GUS). Mucopolysaccharidosis type VII is characterized by glycosaminoglycan storage in most tissues, including brain. In these disorders, enzyme delivery across the blood-brain barrier (BBB) is the main obstacle to correction of lysosomal storage in the CNS. Prior studies suggested mouse brain is accessible to GUS in the first 2 weeks of life but not later. To explore a possible role for the mannose 6-phosphate/insulin-like growth factor II receptor in GUS transport across the BBB in neonatal mice, we compared brain uptake of phosphorylated GUS (P-GUS) and nonphosphorylated GUS (NP-GUS) in newborn and adult mice. (131)I-P-GUS was transported across the BBB after i.v. injection in 2-day-old mice. The brain influx rate (K(in)) of (131)I-P-GUS in 2-day-old mice was 0.21 microl/g.min and decreased with age. By 7 weeks of age, transport of (131)I-P-GUS was not significant. Capillary depletion revealed that 62% of the (131)I-P-GUS in brain was in brain parenchyma in 2-day-old mice. In addition, uptake of (131)I-P-GUS into brain was significantly reduced by coinjection of unlabeled P-GUS or M6P in a dose-dependent manner. In contrast, the K(in) of (131)I-NP-GUS (0.04 microl/g.min) was significantly lower than (131)I-P-GUS in 2-day-old mice. Transcardiac brain perfusion confirmed that neither (131)I-P-GUS nor (131)I-NP-GUS crossed the BBB in adult mice. These results indicate that (131)I-P-GUS transport into brain parenchyma in early postnatal life is mediated by the mannose 6-phosphate/insulin-like growth factor II receptor. This receptor-mediated transport is not observed in adult mice.
Project description:For most lysosomal storage diseases (LSDs) affecting the CNS, there is currently no cure. The BBB, which limits the bioavailability of drugs administered systemically, and the short half-life of lysosomal enzymes, hamper the development of effective therapies. Mucopolysaccharidosis type IIIA (MPS IIIA) is an autosomic recessive LSD caused by a deficiency in sulfamidase, a sulfatase involved in the stepwise degradation of glycosaminoglycan (GAG) heparan sulfate. Here, we demonstrate that intracerebrospinal fluid (intra-CSF) administration of serotype 9 adenoassociated viral vectors (AAV9s) encoding sulfamidase corrects both CNS and somatic pathology in MPS IIIA mice. Following vector administration, enzymatic activity increased throughout the brain and in serum, leading to whole body correction of GAG accumulation and lysosomal pathology, normalization of behavioral deficits, and prolonged survival. To test this strategy in a larger animal, we treated beagle dogs using intracisternal or intracerebroventricular delivery. Administration of sulfamidase-encoding AAV9 resulted in transgenic expression throughout the CNS and liver and increased sulfamidase activity in CSF. High-titer serum antibodies against AAV9 only partially blocked CSF-mediated gene transfer to the brains of dogs. Consistently, anti-AAV antibody titers were lower in CSF than in serum collected from healthy and MPS IIIA-affected children. These results support the clinical translation of this approach for the treatment of MPS IIIA and other LSDs with CNS involvement.
Project description:Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder (LSD) characterized by severe central nervous system (CNS) degeneration. The disease is caused by mutations in the SGSH gene coding for the lysosomal enzyme sulfamidase. Sulfamidase deficiency leads to accumulation of heparan sulfate (HS), which triggers aberrant cellular function, inflammation and eventually cell death. There is currently no available treatment against MPS IIIA. In the present study, a chemically modified recombinant human sulfamidase (CM-rhSulfamidase) with disrupted glycans showed reduced glycan receptor mediated endocytosis, indicating a non-receptor mediated uptake in MPS IIIA patient fibroblasts. Intracellular enzymatic activity and stability was not affected by chemical modification. After intravenous (i.v.) administration in mice, CM-rhSulfamidase showed a prolonged exposure in plasma and distributed to the brain, present both in vascular profiles and in brain parenchyma. Repeated weekly i.v. administration resulted in a dose- and time-dependent reduction of HS in CNS compartments in a mouse model of MPS IIIA. The reduction in HS was paralleled by improvements in lysosomal pathology and neuroinflammation. Behavioral deficits in the MPS IIIA mouse model were apparent in the domains of exploratory behavior, neuromuscular function, social- and learning abilities. CM-rhSulfamidase treatment improved activity in the open field test, endurance in the wire hanging test, sociability in the three-chamber test, whereas other test parameters trended towards improvements. The unique properties of CM-rhSulfamidase described here strongly support the normalization of clinical symptoms, and this candidate drug is therefore currently undergoing clinical studies evaluating safety and efficacy in patients with MPS IIIA.
Project description:Mucopolysaccharidosis IIIA (MPS IIIA) is a neurodegenerative lysosomal storage disorder characterised by progressive loss of learned skills, sleep disturbance and behavioural problems. Reduced activity of lysosomal sulfamidase results in accumulation of heparan sulfate and secondary storage of glycolipids in the brain. Intra-cisternal sulfamidase infusions reduce disease-related neuropathology; however, repeated injections may subject patients to the risk of infection and tissue damage so alternative approaches are required. We undertook a proof-of-principle study comparing the ability of slow/continual or repeat/bolus infusion to ameliorate neuropathology in MPS IIIA mouse brain. Six-week-old MPS IIIA mice were implanted with subcutaneously located mini-osmotic pumps filled with recombinant human sulfamidase (rhSGSH) or vehicle, connected to lateral ventricle-directed cannulae. Pumps were replaced at 8 weeks of age. Additional MPS IIIA mice received intra-cisternal bolus infusions of the same amount of rhSGSH (or vehicle), at 6 and 8 weeks of age. Unaffected mice received vehicle via each strategy. All mice were euthanised at 10 weeks of age and the brain was harvested to assess the effect of treatment on neuropathology. Mice receiving pump-delivered rhSGSH exhibited highly significant reductions in lysosomal storage markers (lysosomal integral membrane protein-2, GM3 ganglioside and filipin-positive lipids) and neuroinflammation (isolectin B4-positive microglia, glial fibrillary acidic protein-positive astroglia). MPS IIIA mice receiving rhSGSH via bolus infusion displayed reductions in these markers, but the effectiveness of the strategy was inferior to that seen with slow/pump-based delivery. Continual low-dose infusion may therefore be a more effective strategy for enzyme delivery in MPS IIIA.
Project description:Mucopolysaccharidosis III A (MPS IIIA) is an autosomal recessive lysosomal storage disorder caused by deficiency of the enzyme sulfamidase. The disorder results in accumulation of heparan sulfate, lysosomal enlargement and cellular and organ dysfunction. Patients exhibit progressive neurodegeneration and behavioral problems and no treatment is currently available. Enzyme replacement therapy is explored as potential treatment strategy for MPS IIIA patients and to modify the disease, sulfamidase must reach the brain. The glycans of recombinant human sulfamidase (rhSulfamidase) can be chemically modified to generate CM-rhSulfamidase. The chemical modification reduced the affinity to the cation-independent mannose-6-phosphate receptor with the aim a prolonged higher concentration in circulation and thus at the blood brain barrier. The pharmacokinetic properties in serum and the distribution to brain and to cerebrospinal fluid (CSF) of chemically modified recombinant human sulfamidase (CM-rhSulfamidase) were studied and compared to those of rhSulfamidase, after a single intravenous (i.v.) 30 mg/kg dose in awake, freely-moving male Sprague Dawley rats. Distribution to brain was studied by microdialysis of the interstitial fluid in prefrontal cortex and by repeated intra-individual CSF sampling from the cisterna magna. Push-pull microdialysis facilitated sampling of brain interstitial fluid to determine large molecule concentrations in awake, freely-moving male Sprague Dawley rats. Together with repeated serum and CSF sampling, push-pull microdialysis facilitated determination of CM-rhSulfamidase and rhSulfamidase kinetics after i.v. administration by non-compartments analysis and by a population modelling approach. Chemical modification increased the area under the concentration versus time in serum, CSF and brain interstitial fluid at least 7-fold. The results and the outcome of a population modelling approach of the concentration versus time data indicated that both compounds pass the BBB with an equilibrium established fairly rapid after administration. We suggest that prolonged high serum concentrations facilitated high brain interstitial fluid concentrations, which could be favorable to reach various target cells in the brain.
Project description:Mucopolysaccharidosis type IIIA (MPS-IIIA) is a lysosomal storage disorder (LSD) caused by inherited defect of sulfamidase, a lysosomal sulfatase. MPS-IIIA is one of the most common and severe forms of LSDs with CNS involvement. Presently there is no cure. Here we have developed a new gene delivery approach for the treatment of MPS-IIIA based on the use of a modified version of sulfamidase expression cassette. This cassette encodes both a chimeric sulfamidase containing an alternative signal peptide (sp) to improve enzyme secretion and sulfatase-modifying factor 1 (SUMF1) to increase sulfamidase post-translational activation rate. We demonstrate that improved secretion and increased activation of sulfamidase act synergistically to enhance enzyme biodistribution in wild-type (WT) pigs upon intrathecal adeno-associated virus serotype 9 (AAV9)-mediated gene delivery. Translating such gene delivery strategy to a mouse model of MPS-IIIA results in a rescue of brain pathology, including memory deficit, as well as improvement in somatic tissues. These data may pave the way for developing effective gene delivery replacement protocols for the treatment of MPS-IIIA patients.
Project description:Sulfamidase (SGSH) deficiency causes mucopolysaccharidosis type IIIA (MPS IIIA), a lysosomal storage disease (LSD) that affects the CNS. In earlier work in LSD mice and dog models, we exploited the utility of adeno-associated viruses (AAVs) to transduce brain ventricular lining cells (ependyma) for secretion of lysosomal hydrolases into the cerebrospinal fluid (CSF), with subsequent distribution of enzyme throughout the brain resulting in improved cognition and extending lifespan. A critical feature of this approach is efficient secretion of the expressed enzyme from transduced cells, for delivery by CSF to nontransduced cells. Surprisingly, we found that SGSH was poorly secreted from cells, resulting in retention of the expressed product. Using site-directed mutagenesis of native SGSH, we identified an improved secretion variant that also displayed enhanced uptake properties that were mannose-6-phosphate receptor independent. In studies in MPS IIIA-deficient mice, ependymal transduction with AAVs expressing variant SGSH improved spatial learning and reduced memory deficits, substrate accumulation, and astrogliosis. Secondary lysosomal enzyme elevations in the CSF and brain parenchyma were also resolved. In contrast, ependymal transduction with AAVs expressing wild-type SGSH had significantly lower CSF SGSH levels and limited impacts on behavior. These results demonstrate the utility of a previously undescribed SGSH variant for improved MPS IIIA brain gene therapy.
Project description:BACKGROUND:With ongoing efforts to develop improved treatments for Sanfilippo Syndrome Type A (MPS-IIIA), a disease caused by the inability to degrade heparan sulfate in lysosomes, we sought to develop an enzymatic activity assay for the relevant enzyme, sulfamidase, that uses dried blood spots (DBS). METHODS:We designed and synthesized a new sulfamidase substrate that can be used to measure sulfamidase activity in DBS using liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS:Sulfamidase activity was readily detected in DBS using the new substrate and LC-MS/MS. Sulfamidase activity showed acceptable linearity proportional to the amount of enzyme and reaction time. Sulfamidase activity in 238 random newborns was well elevated compared to the range of activities measured in DBS from 8 patients previously confirmed to have MPS-IIIA. CONCLUSIONS:This is the first report of an assay capable of detecting sulfamidase in DBS. The new assay could be useful in diagnosis and potentially for newborn screening of MPS-IIIA.
Project description:Mutations in the lysosomal enzyme, N-sulfoglucosamine sulfohydrolase (SGSH), also called sulfamidase, cause accumulation of lysosomal inclusion bodies in the brain of children born with mucopolysaccharidosis type IIIA, also called Sanfilippo type A syndrome. Enzyme replacement therapy with recombinant SGSH does not treat the brain because the enzyme is a large molecule drug that does not cross the blood-brain barrier (BBB). A BBB-penetrating form of SGSH was produced by re-engineering the enzyme as an IgG fusion protein, where the IgG domain is a monoclonal antibody (mAb) against the human insulin receptor (HIR). The HIRMAb domain of the HIRMAb-SGSH fusion protein acts as a molecular Trojan horse to ferry the fused enzyme across the BBB. The HIRMAb-SGSH was produced in stably transfected host cells and purified to homogeneity by protein A chromatography. The fusion protein reacted with antibodies against either human IgG or SGSH on Western blotting. High affinity binding to the HIR was retained following SGSH fusion to the HIRMAb, with an EC50 of 0.33 ± 0.05 nM in an HIR binding ELISA. The SGSH enzyme activity of the HIRMAb-SGSH fusion protein was 4712 ± 388 units/mg protein based on a two-step fluorometric enzyme assay. The HIRMAb-SGSH was taken up by lysosomes in MPSIIIA fibroblasts, and treatment of these cells with the fusion protein caused an 83% reduction in sulfate incorporation into lysosomal glycosoaminoglycans. The HIRMAb-SGSH fusion protein was radiolabeled with the [(125)I]-Bolton-Hunter reagent and injected intravenously in the Rhesus monkey. The brain uptake of the fusion protein was high, ?1% injected dose/brain. Calculations, based on this level of brain uptake, suggest normalization of brain SGSH enzyme activity is possible following administration of therapeutic doses of the fusion protein. These studies describe a novel IgG-SGSH fusion protein that is a new noninvasive treatment of the brain in MPS type IIIA.
Project description:A versatile experimental model for the investigation of the blood-brain barrier (BBB), including the neuro-vascular unit, is the isolated brain microvessel preparation. Brain microvessels are primarily comprised of endothelial cells, but also include pericytes, pre-capillary arteriolar smooth muscle cells, astrocyte foot processes, and occasional nerve endings. These microvessels can be isolated from brain with a 3 h procedure, and the microvessels are free of brain parenchyma. Brain microvessels have been isolated from fresh animal brain, fresh human brain obtained at neurosurgery, as well as fresh or frozen autopsy human brain. Brain microvessels are the starting point for isolation of brain microvessel RNA, which then enables the production of BBB cDNA libraries and a genomics analysis of the brain microvasculature. Brain microvessels, combined with quantitative targeted absolute proteomics, allow for the quantitation of specific transporters or receptors expressed at the brain microvasculature. Brain microvessels, combined with specific antibodies and immune labeling of isolated capillaries, allow for the cellular location of proteins expressed within the neuro-vascular unit. Isolated brain microvessels can be used as an "in vitro" preparation of the BBB for the study of the kinetic parameters of BBB carrier-mediated transport (CMT) systems, or for the determination of dissociation constants of peptide binding to BBB receptor-mediated transport (RMT) systems expressed at either the animal or the human BBB. This review will discuss how the isolated brain microvessel model system has advanced our understanding of the organization and functional properties of the BBB, and highlight recent renewed interest in this 50 year old model of the BBB.