Intrathecal recombinant human 4-sulfatase reduces accumulation of glycosaminoglycans in dura of mucopolysaccharidosis VI cats.

INTRODUCTION: Mucopolysaccharidosis VI (MPS-VI) is caused by a deficiency in N-acetylgalactosamine-4-sulfatase activity, resulting in lysosomal accumulation of partially degraded glycosaminoglycans (GAGs). Compressive myelopathy in early-onset MPS-VI patients has been partly attributed to thickening of the dura mater following engorgement with GAG. In this study, we therefore tested whether the dural abnormalities could be prevented in a feline model of the disorder. RESULTS: All intrathecal injections (IT-INJs) were well tolerated. MPS-VI cats treated with IT-INJ of recombinant human N-acetylgalactosamine-4-sulfatase (rhASB) exhibited reduced vacuolation in the dural fibroblasts, diminished levels of sulfated-N-acetylhexosamine (HNAc(+S)) in the cerebrospinal fluid (CSF) and no hind-limb paresis. Serum anti-rhASB antibodies remained low in MPS-VI cats treated with intravenous enzyme replacement therapy (IV-ERT) and increased slightly in normal cats treated with IT-INJ of rhASB alone. Anti-rhASB antibodies in CSF remained undetectable. DISCUSSION: These data indicate that repeated IT-INJ of rhASB can safely prevent GAG storage in MPS-VI dura. METHODS: Cats were assigned to three groups: (i) receiving weekly IV-ERT of rhASB from birth plus six monthly IT-INJs of rhASB from age 2 months; (ii) receiving six monthly IT-INJs of vehicle; or (iii) untreated. Additional normal cats received five fortnightly IT-INJs of rhASB or vehicle alone.

Repeated intrathecal injections of recombinant human 4-sulphatase remove dural storage in mature mucopolysaccharidosis VI cats primed with a short-course tolerisation regimen.

All MPS-VI cats treated thus far with weekly intravenous enzyme replacement therapy (IV ERT) with recombinant human N-acetylgalactosamine-4-sulphatase (rhASB) from 3 months of age onwards developed circulating anti-rhASB antibodies. In view of this, the possibility of inducing immune tolerance by using a short-course tolerisation regimen was tested. Starting at 4 months of age, MPS-VI (n=5) and unaffected cats (n=2) received cyclosporine and azathioprine over a 22-day period plus weekly IV ERT with 0.1mg/kg rhASB. After a 4-week resting period, these cats were administered weekly IV ERT with 1mg/kg rhASB until 11 or 17 months of age. Four unaffected cats (n=4) received weekly IV ERT only. Health, growth and seroconversion were regularly monitored. Four out of five MPS-VI cats tolerated rhASB well, as indicated by negligible or low antibody titres and absence of hypersensitivity reactions. One MPS-VI cat exhibited elevated antibody titres and hypersensitivity reactions during some IV treatments. The two unaffected cats that received the tolerisation regimen remained seronegative, however, only half of the unaffected cats not submitted to this regimen seroconverted. Only minor side-effects were attributed to the short-course of cyclosporine and azathioprine. Two MPS-VI cats also well-tolerated four weekly intrathecal injections of rhASB and consequently exhibited less oligosaccharide fragments in cerebrospinal fluid and less vacuolation within their dura mater. These data indicate that a relatively high rate of immunotolerance towards rhASB can be achieved in MPS-VI cats with a short-course tolerisation regimen ultimately permitting removal of lysosomal storage within the dura mater with the use of intrathecal therapy.

Effect of cisternal sulfamidase delivery in MPS IIIA Huntaway dogs--a proof of principle study.

Mucopolysaccharidosis type IIIA (MPS IIIA) results from lack of functional sulfamidase (SGSH), a lysosomal enzyme. Its substrate, heparan sulfate, and other secondarily-stored compounds subsequently accumulate primarily within the central nervous system (CNS), resulting in progressive mental deterioration and early death. Presently there is no treatment. As a potential therapeutic strategy, recombinant human sulfamidase (rhSGSH) was administered into the CSF (via the cerebellomedullary cistern) of three adult MPS IIIA dogs either twice with a 4 day interval, or weekly for up to 4 weeks. The dogs were euthanased 24 h post-injection along with one untreated unaffected and two MPS IIIA controls. We have examined the three dimensional pattern of distribution of enzyme in the CNS and its ability to reduce primary substrate storage. High concentrations of rhSGSH protein, with up to 39-fold normal enzyme activity levels were detected within widespread areas of the CNS. RhSGSH protein was also detectable by immunohistochemistry in neurons and glia in all three enzyme-treated dogs. In both weekly-treated dogs, relative levels of a heparan sulfate-derived disaccharide, measured using tandem mass spectrometry, were lower in many brain regions when compared to untreated MPS IIIA controls. A moderately severe meningitis was also present as well as antibodies to rhSGSH in CSF/plasma. These findings demonstrate proof of principle that MPS IIIA can be treated by intracisternal enzyme replacement warranting further experiments in animals tolerant to rhSGSH. This enzyme delivery method may represent a means of treating neuropathology in MPS IIIA and other lysosomal storage disorders affecting the CNS.

Long-term intra-articular administration of recombinant human N-acetylgalactosamine-4-sulfatase in feline mucopolysaccharidosis VI.

Degenerative joint disease (DJD) is one aspect of mucopolysaccharidosis VI (MPS VI) pathology that has proven resistant to systemic enzyme replacement therapy (ERT). In this study the effect of repeated intra-articular injections (IA INJ) of recombinant human acetylgalactosamine-4-sulfatase (rh4S) on DJD was examined. Four MPS VI cats received i.v. ERT weekly from birth plus IA INJ (0 or 500 microg of rh4S per joint; monthly or every three months) while three MPS VI cats received IA INJ only. After 10 months, shoulders, elbows and knees were compared. Taken individually, an improvement in joint appearance was observed between the joints that received rh4S monthly or every three months compared with the contralateral joints treated with buffer or at lower frequency. Within articular cartilage of joints treated more frequently, the depth of clearance of lysosomal storage tended to be greater and uronic acid content was reduced reflecting the removal of glycosaminoglycans. Synovium in treated joints also showed less storage. No abnormal clinical signs were observed after the IA INJ and negligible antibody titres were measured throughout the study. No clear benefit was observed by combining IA INJ with weekly ERT and the most significant improvement in joint appearance resulted from increased IA INJ frequency. These data support the view that intra-articular therapy may be a good option for preventing the development of the severe articular pathology in MPS VI.

Characterization of a C57BL/6 congenic mouse strain of mucopolysaccharidosis type IIIA.

The original mucopolysaccharidosis type IIIA (MPS IIIA) mice were identified in a mixed background with contributions from four different strains. To ensure long-term stability and genetic homogeneity of this lysosomal storage disease (LSD) model, the aim of this study was to develop and characterize a C57BL/6 congenic strain. The B6.Cg-Sgsh(mps3a) strain compares favorably with the original mixed donor strain, exhibiting low liver sulfamidase activity and significant brain heparan sulfate-derived disaccharide elevation from birth. A rapid increase in brain disaccharide levels occurred after birth, with a plateau reached by 13 weeks of age at 110x the levels observed in brains of age-matched unaffected mice. Typical lysosomal inclusions were observed in cerebral cortical and cerebellar neurons and in liver hepatocytes and Kupffer cells. Ubiquitin-positive spheroids and GM(2)-ganglioside were also detected in brain. Using the Morris water maze in male mice, impaired memory and spatial learning was evident at 20 weeks of age in B6.Cg-Sgsh(mps3a) MPS IIIA mice. Other behavioral changes include motor, cognitive and sensory deficits, and aggression. Male B6.Cg-Sgsh(mps3a) MPS IIIA mice exhibited more behavioral abnormalities than B6.Cg-Sgsh(mps3a) MPS IIIA females, as observed previously in the original mixed background strain. Affected mice generally survive to 9 to 12 months of age, before death or euthanasia for humane reasons. Overall, minor differences were apparent between the new congenic and previously described mixed MPS IIIA strains. Availability of an in-bred strain will ensure more reproducible experimental outcomes thereby assisting in our goal of developing effective therapies for LSD with central nervous system disease.

Intra-articular enzyme administration for joint disease in feline mucopolysaccharidosis VI: enzyme dose and interval.

Degenerative joint changes have been reported in human mucopolysaccharidosis VI (MPS VI) and are a prominent feature of feline MPS VI. Joint disease has proven refractory to intravenous enzyme replacement therapy (ERT) in the MPS VI cat because enzyme is unable to reach cells in cartilage. In this study, enzyme was infused directly into the intraarticular space to determine whether joint tissues are able to respond to replacement enzyme. Clearance of glycosaminoglycans from chondrocytes was observed at a dose of 10 microg recombinant human N-acetylgalactosamine-4-sulfatase (rh4S), but greater clearance was observed with higher doses. The chondrocytes at the articular surface were cleared preferentially. Lysosomal vacuolation in cruciate ligament and synovial cells also decreased upon addition of rh4S. One month after injection of rh4S, a slight reaccumulation of storage was observed at the surface of the joint, but extensive reaccumulation was observed 2 mo after injection. These results indicate that by bypassing the synovium using intraarticular ERT, significant reduction in storage material in joint tissues can be achieved. Localized ERT in the joint space provides a mechanism for delivering enzyme directly to the articular cartilage and a potential therapy for joint pathology in MPS VI.

Development of an assay for the detection of mucopolysaccharidosis type VI patients using dried blood-spots.

BACKGROUND: Mucopolysaccharidosis type VI (MPS VI) is a lysosomal storage disorder (LSD), which is caused by a deficiency in the enzyme N-acetylgalactosamine 4-sulfatase (4-sulfatase). MPS VI is characterized by severe skeletal abnormalities, somatic tissue pathology and early death. Treatment possibilities include bone marrow transplantation (BMT) and enzyme replacement therapy (ERT; currently in phase III clinical trial). Early diagnosis of MPS VI will allow treatment before the onset of irreversible pathology. METHODS: Sensitive immune assays have been developed to detect 4-sulfatase protein and activity in normal control and MPS VI blood-spots. RESULTS: Dried blood-spots from MPS VI patients contained no detectable 4-sulfatase protein and activity, compared to 3.5-21 microg/L of 4-sulfatase protein and 291-1298 nmol/min/L of activity for normal human controls. In this evaluation study, the assay was sensitive and 100% specific, allowing reliable detection of individuals with MPS VI. CONCLUSIONS: The assays reported here have the potential to detect MPS VI patients using dried blood-spots.

Replacement therapy in Mucopolysaccharidosis type VI: advantages of early onset of therapy.

This study evaluates the immunological response following weekly 2h infusions of recombinant human N-acetylgalactosamine 4-sulfatase (rh4S) in Mucopolysaccharidosis VI (MPS VI) cats. The results of three trials (Trial "A": 9 month duration with onset at 3-5 months of age, n = 5; and Trials "B" and "C": 6 month duration starting at birth, n = 9) were compared. No detrimental effects were noted throughout Trials B and C. Temporary hypersensitivity reactions (e.g., vomiting, diarrhoea) occurred in four cats in Trial A and were alleviated by increasing the dose of antihistamine premedication and the duration of infusion. All cats in Trial A developed antibodies to rh4S (range of final titres: 1041-134,931). All cats treated from birth showed negligible titres (range: < 50-598). In vitro inhibition of rh4S activity (up to 47%) was demonstrated with plasma from four cats with elevated titres. Significant reduction of urinary glycosaminoglycan concentration in all cats indicated the ability of rh4S to metabolize stored substrates regardless of the presence of circulating antibodies. Similarly, lysosomal storage in reticuloendothelial cells and fibroblasts of kidney interstistium, dura and skin was reduced in all cats irrespective of their antibody titre although cats with elevated titre had less beneficial effect on cardiovascular tissues (aorta smooth muscle cells, heart valve fibroblasts). Overall improvement in the disease condition (at physical, neurological, and skeletal levels) was most pronounced for cats treated from birth compared with cats treated at a later age.