Tina Rozaklis

Newborn Screening for Lysosomal Storage Disorders: Clinical Evaluation of a Two-Tier Strategy

Objective. To evaluate the use of protein markers using immune-quantification assays and of metabolite markers using tandem mass spectrometry for the identification, at birth, of individuals who have a lysosomal storage disorder. Methods. A retrospective analysis was conducted of Guthrie cards that were collected from newborns in Denmark during the period 1982–1997. Patients whose lysosomal storage disorder (LSD; 47 representing 12 disorders) was diagnosed in Denmark during the period 1982–1997 were selected, and their Guthrie cards were retrieved from storage. Control cards (227) were retrieved from the same period. Additional control cards (273) were collected from the South Australian Screening Centre (Australia). Results. From 2 protein and 94 metabolite markers, 15 were selected and evaluated for their use in the identification of LSDs. Glycosphingolipid and oligosaccharide markers showed 100% sensitivity and specificity for the identification of Fabry disease, α-mannosidosis, mucopolysaccharidosis (MPS) IVA, MPS IIIA, Tay-Sachs disease, and I-cell disease. Lower sensitivities were observed for Gaucher disease and sialidosis. No useful markers were identified for Krabbe disease, MPS II, Pompe disease, and Sandhoff disease. The protein markers LAMP-1 and saposin C were not able to differentiate individuals who had an LSD from the control population. Conclusions. Newborn screening for selected LSDs is possible with current technology. However, additional development is required to provide a broad coverage of disorders in a single, viable program.

Examination of intravenous and intra-CSF protein delivery for treatment of neurological disease

Mucopolysaccharidosis type IIIA is a neurodegenerative lysosomal storage disorder characterized by progressive loss of learned skills, sleep disturbance and behavioural problems. Absent or greatly reduced activity of sulphamidase, a lysosomal protein, results in intracellular accumulation of heparan sulphate. Subsequent neuroinflammation and neurodegeneration typify this and many other lysosomal storage disorders. We propose that intra‐cerebrospinal fluid protein delivery represents a potential therapeutic avenue for treatment of this and other neurodegenerative conditions; however, technical restraints restrict examination of its use prior to adulthood in mice. We have used a naturally‐occurring Mucopolysaccharidosis type IIIA mouse model to determine the effectiveness of combining intravenous protein replacement (1 mg/kg) from birth to 6 weeks of age with intra‐cerebrospinal fluid sulphamidase delivery (100 μg, fortnightly from 6 weeks) on behaviour, the level of heparan sulphate‐oligosaccharide storage and other neuropathology. Mice receiving combination treatment exhibited similar clinical improvement and reduction in heparan sulphate storage to those only receiving intra‐cerebrospinal fluid enzyme. Reductions in micro‐ and astrogliosis and delayed development of ubiquitin‐positive lesions were seen in both groups. A third group of intravenous‐only treated mice did not exhibit clinical or neuropathological improvements. Intra‐cerebrospinal fluid injection of sulphamidase effectively, but dose‐dependently, treats neurological pathology in Mucopolysaccharidosis type IIIA, even when treatment begins in mice with established disease.

Disease-Specific Markers for the Mucopolysaccharidoses

Unprecedented demands are now placed on clinicians for early diagnosis as we enter into an era of advancing treatment opportunities for the mucopolysaccharidoses (MPS). Biochemical monitoring of any therapeutic avenue will also be prerequisite. To this end, we aimed to identify a range of urinary oligosaccharides that could be used to identify and characterize patients with MPS. We analyzed 94 urine samples from 68 patients with MPS and 26 control individuals for oligosaccharides derived from glycosaminoglycans using electrospray ionization-tandem mass spectrometry. The oligosaccharide profile for each patient group was compared with that of the control group. The Mann-Whitney U test was used to measure the difference between each patient group and the controls for each analyte. Urine samples from patients before and at successive times after bone marrow transplantation were also evaluated. A number of oligosaccharides were identified in the urine of each MPS subtype, and for each of these, specific oligosaccharide profiles were formulated. These profiles enabled the identification of all 68 patients and their subtypes with the exception of MPS IIIB and IIIC. Selected oligosaccharides were used to assess three individuals after a bone marrow transplant, and, in each case, a substantial reduction in the level of diagnostic oligosaccharides, posttransplantation, was observed. The identification and measurement of glycosaminoglycan-derived oligosaccharides in urine provides a sensitive and specific screen for the early identification of individuals with MPS. The resulting oligosaccharide profiles not only characterize subtype but also provide a disease-specific fingerprint for the biochemical monitoring of current and proposed therapies.

Mucopolysaccharidosis IIIA (Sanfilippo syndrome) in a New Zealand Huntaway dog with ataxia.

Abstract Aim: To investigate the nature of a progressive ataxia in a New Zealand Huntaway dog. Methods: The affected dog was examined clinically before being humanely killed and necropsied. Selected tissues were submitted to light and electron microscopy and to biochemical analyses. Results: The histological lesions were interpreted as indicative of one of the forms of mucopolysaccharidosis type-III (dMPS-III), a lysosomal storage disease. Biochemically there was a deficiency of heparan sulphamidase. All the heparan sulphate chains had non-reducing-end glucosamine-N-sulphate residues. Conclusion: The disease is dMPS-IIIA (Sanfilippo syndrome). An autosomal recessive mode of inheritance can be provisionally assumed from the nature of this disease in other species.

Impact of high-dose, chemically modified sulfamidase on pathology in a murine model of MPS IIIA

Mucopolysaccharidosis type IIIA (MPS IIIA) is a neurodegenerative lysosomal storage disorder that results from a deficiency of sulfamidase (N-sulfoglucosamine sulfohydrolase), with consequential accumulation of its substrate, partially degraded heparan sulfate. Conventional doses (e.g. 1mg/kg) of intravenously delivered recombinant human sulfamidase (rhSGSH) do not improve neuropathology in MPS IIIA mice due to an inability to traverse the blood-brain barrier; however high-dose treatment or administration of enzyme that has been chemically modified to remove mannose-6-phosphate glycans has been shown to reduce neuropathology in related animal models. We have combined these approaches to evaluate the ability of 1, 5, 10 or 20mg/kg of similarly chemically modified or unmodified rhSGSH to reduce neuropathology following repeated intravenous delivery to adult MPS IIIA mice. rhSGSH was detected in brain homogenates from mice treated with all doses of modified rhSGSH and those receiving the two higher doses of unmodified rhSGSH, albeit at significantly lower levels. Immunohistochemically, rhSGSH visualized in the brain was localized to the endothelium, meninges and choroid plexus, with no convincing punctate intra-neuronal staining seen. This presumably underlies the failure of the treatment to reduce the relative level of a heparan sulfate-derived oligosaccharide (GlcNS-UA), or secondarily stored substrates that accumulate in MPS IIIA brain cells. However, modification of rhSGSH significantly increased its effectiveness in degrading GlcNS-UA in non-CNS tissues, potentially as a result of its reduced plasma clearance. If this observation is generally applicable, chemical modification may permit the use of significantly lower doses of lysosomal enzymes in patients currently receiving intravenous enzyme replacement therapy.

Allogeneic stem cell transplantation does not improve neurological deficits in mucopolysaccharidosis type IIIA mice

Mucopolysaccharidosis type IIIA (MPS IIIA) is a neurodegenerative metabolic disorder caused by mutations in the N-sulfoglucosamine sulfohydrolase gene with resultant accumulation of partially degraded heparan sulfate (HS). Whilst allogeneic bone marrow transplantation (BMT) is indicated for several lysosomal storage disorders featuring neurodegeneration, its use in MPS III is highly controversial. Published evidence suggests that BMT does not improve cognitive function in MPS III patients. Despite this, patients continue to be transplanted in some centers. We therefore sought to determine the clinical effectiveness of BMT in a murine model of MPS IIIA. Pre-symptomatic young adult mice pre-conditioned with total body irradiation generated complete and stable donor-type chimerism. Whilst HS-derived disaccharides were reduced by up to 27% in the brain parenchyma, this was insufficient to decrease secondary cholesterol and GM3 ganglioside storage or permit clinical improvement. These results suggest that BMT is ineffective in its unmodified form and should not be considered as a treatment for MPS IIIA children.

Helper-dependent canine adenovirus vector-mediated transgene expression in a neurodegenerative lysosomal storage disorder.

Mucopolysaccharidosis type IIIA (MPS-IIIA) is a severe neurodegenerative lysosomal storage disorder caused by a deficiency of N-sulfoglucosamine sulfohydrolase (SGSH) activity with subsequent accumulation of partially-degraded heparan sulfate and other glycolipids. In this study, we have evaluated a gene therapy approach using a helper-dependent canine adenovirus vector that expresses human SGSH as a means of delivering sustained transgene expression to the brain. Initial testing in a mixed neural cell culture model demonstrated that the vector could significantly increase SGSH activity in transduced cells, resulting in near-normalization of heparan sulfate-derived fragments. While administration of vector by direct injection into the brain of adult MPS-IIIA mice enabled transgene expression for at least 8.5 months post-treatment, it was only in discrete areas of brain. Heparan sulfate storage was reduced in some regions following treatment, however there was no improvement in secondary neuropathological changes. These data demonstrate that helper-dependent canine adenovirus vectors are capable of neural transduction and mediate long-term transgene expression, but increased SGSH expression throughout the brain is likely to be required in order to effectively treat all aspects of the MPS-IIIA phenotype.

Delivery of therapeutic protein for prevention of neurodegenerative changes: comparison of different CSF-delivery methods.

Injection of lysosomal enzyme into cisternal or ventricular cerebrospinal fluid (CSF) has been carried out in 11 lysosomal storage disorder models, with each study demonstrating reductions in primary substrate and secondary neuropathological changes, and several reports of improved neurological function. Whilst acute studies in mucopolysaccharidosis (MPS) type II mice revealed that intrathecally-delivered enzyme (into thoraco-lumbar CSF) accesses the brain, the impact of longer-term treatment of affected subjects via this route is unknown. This approach is presently being utilized to treat children with MPS types I, II and III. Our aim was to determine the efficacy of repeated intrathecal injection of recombinant human sulfamidase (rhSGSH) on pathological changes in the MPS IIIA dog brain. The outcomes were compared with those in dogs treated via intra-cisternal or ventricular routes. Control dogs received buffer or no treatment. Significant reductions in primary/secondary substrate levels in brain were observed in dogs treated via all routes, although the extent of the reduction differed regionally. Treatment via all CSF access points resulted in large reductions in microgliosis in superficial cerebral cortex, but only ventricular injection enabled amelioration in deep cerebral cortex. Formation of glutamic acid decarboxylase-positive axonal spheroids in deep cerebellar nuclei was prevented by treatment delivered via any route. Anti-rhSGSH antibodies in the sera of some dogs did not reduce therapeutic efficacy. Our data indicates the capacity of intra-spinal CSF-injected rhSGSH to circulate within CSF-filled spaces, penetrate into brain and mediate a significant reduction in substrate accumulation and secondary pathology in the MPS IIIA dog brain.

Lysosomal sulfate efflux following glycosaminoglycan degradation: measurements in enzyme-supplemented Maroteaux-Lamy syndrome fibroblasts and isolated lysosomes

Studies using lysosomal membrane vesicles have suggested that efflux of the sulfate that results from lysosomal glycosaminoglycan degradation is carrier-mediated. In this study, glycosaminoglycan degradation and sulfate efflux were examined using cultured skin fibroblasts and lysosomes deficient in the lysosomal enzymeN-acetylgalactosamine-4-sulfatase. Such fibroblasts store dermatan sulfate lysosomally, which could be labelled biosynthetically with Na235SO4. The addition of recombinantN-acetylgalactosamine-4-sulfatase to the media of35S labelled fibroblasts degraded up to 82% of the stored dermatan [35S] sulfate over a subsequent 96 h chase and released inorganic [35S] sulfate into the medium. In the presence of 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS), sulfate was reused to a minor extent in newly synthesized proteoglycan. Isolated granules from recombinant enzyme supplemented fibroblasts degraded stored dermatan [35S]sulfate to sulfate which was rapidly released into the medium at a rate that was reduced by the extra-lysosomal presence of the lysosomal sulfate transport inhibitors SITS, Na2SO4 and Na2MoO4. SITS also inhibited dermatan sulfate turnover, although it had no effect on the action of purified recombinant enzymein vitro. These data imply that sulfate clearance occurred concomitantly with dermatan sulfate turnover in the lysosome even at high substrate loading, and that lysosome-derived sulfate, while available, is reutilized minimally in synthetic pathways.

Low-dose, continuous enzyme replacement therapy ameliorates brain pathology in the neurodegenerative lysosomal disorder mucopolysaccharidosis type IIIA.

Repeated replacement of sulphamidase via cerebrospinal fluid injection is an effective treatment for pathological changes in the brain in mice and dogs with the lysosomal storage disorder, mucopolysaccharidosis type IIIA (MPS IIIA). Investigational trials of this approach are underway in children with this condition, however, infusions require attendance at a specialist medical facility. We sought to comprehensively evaluate the effectiveness of sustained‐release (osmotic pump‐delivered) enzyme replacement therapy in murine MPS IIIA as this method, if applied to humans, would require only subcutaneous administration of enzyme once the pump was installed. Six‐week‐old MPS IIIA and unaffected mice were implanted with subcutaneous mini‐osmotic pumps connected to an infusion cannula directed at the right lateral ventricle. Either recombinant human sulphamidase or vehicle were infused over the course of 7 weeks, with pumps replaced part‐way through the experimental period. We observed near‐normalisation of primarily stored substrate (heparan sulphate) in both hemispheres of the MPS IIIA brain and cervical spinal cord, as determined using tandem mass spectrometry. Immunohistochemistry indicated a reduction in secondarily stored GM3 ganglioside and neuroinflammatory markers. A bias towards the infusion side was seen in some, but not all outcomes. The recombinant enzyme appears stable under pump‐like conditions for at least 1 month. Given that infusion pumps are in clinical use in other nervous system disorders, e.g. for treatment of spasticity or brain tumours, this treatment method warrants consideration for testing in large animal models of MPS IIIA and other lysosomal storage disorders that affect the brain.