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Iduronidase (IDUA)-deficient mice accumulate glycosaminoglycans in cells and tissues and exhibit many of the same neuropathological symptoms of patients suffering from Mucopolysaccharidosis I. Intravenous enzyme-replacement therapy forMucopolysaccharidosis I ameliorates glycosaminoglycan storage and many of the somatic aspects of the disease but fails to treat neurological symptoms due to poor transport across the blood-brain barrier. In this study, we examined the delivery of IDUA conjugated to guanidinoneomycin (GNeo), a molecular transporter. GNeo-IDUA and IDUA injected intravenously resulted in reduced hepatic glycosaminoglycan accumulation but had no effect in the brain due to fast clearance from the circulation. In contrast, intranasally administered GNeo-IDUA entered the brain rapidly. Repetitive intranasal treatment with GNeo-IDUA reduced glycosaminoglycan storage, lysosome size and number, and neurodegenerative astrogliosis in the olfactory bulb and primary somatosensory cortex, whereas IDUA was less effective. The enhanced efficacy of GNeo-IDUA was not the result of increased nose-to-brain delivery or enzyme stability, but rather due to more efficient uptake into neurons and astrocytes. GNeo conjugation also enhanced glycosaminoglycan clearance by intranasally delivered sulfamidase to the brain of sulfamidase-deficient mice, a model of Mucopolysaccharidosis IIIA. These findings suggest the general utility of the guanidinoglycoside-based delivery system for restoring missing lysosomal enzymes in the brain.

Therapeutic development and monitoring require demonstration of effects on disease phenotype. However, due to the complexity of measuring clinically-relevant effects in rare multisystem diseases, robust biomarkers are essential. For the mucopolysaccharidoses (MPS), the measurement of glycosaminoglycan levels is relevant as glycosaminoglycan accumulation is the primary event that occurs due to reduced lysosomal enzyme activity. Traditional dye-based assays that measure total glycosaminoglycan levels have a high background, due to a normal, baseline glycosaminoglycan content in unaffected individuals. An assay that selectively detects the disease-specific non-reducing ends of heparan sulfate glycosaminoglycans that remain undegraded due to deficiency of a specific enzyme in the catabolic pathway avoids the normal background, increasing sensitivity and specificity. We evaluated glycosaminoglycan content by dye-based and non-reducing end methods using urine, serum, and cerebrospinal fluid from MPS I human samples before and after treatment with intravenous recombinant human alpha-l-iduronidase. We found that both urine total glycosaminoglycans and serum heparan sulfate derived non-reducing end levels were markedly decreased compared to baseline after 26 weeks and 52 weeks of therapy, with a significantly greater percentage reduction in serum non-reducing end (89.8% at 26 weeks and 81.3% at 52 weeks) compared to urine total glycosaminoglycans (68.3% at 26 weeks and 62.4% at 52 weeks, p

Before the availability of an enzyme replacement therapy (ERT) for mucopolysaccharidosis type II (MPS II), patients were treated by bone marrow transplantation (BMT). However, the effectiveness of BMT for MPS II was equivocal, particularly at addressing the CNS manifestations. To study this further, we subjected a murine model of MPS II to BMT and evaluated the effect at correcting the biochemical and pathological aberrations in the viscera and CNS. Our results indicated that BMT reduced the accumulation of glycosaminoglycans (GAGs) in a variety of visceral organs, but not in the CNS. With the availability of an approved ERT for MPS II, we investigated and compared the relative merits of the two strategies either as a mono or combination therapy. We showed that the combination of BMT and ERT was additive at reducing tissue levels of GAGs in the heart, kidney and lung. Moreover, ERT conferred greater efficacy if the immunological response against the infused recombinant enzyme was low. Finally, we showed that pathologic GAGs might potentially represent a sensitive biomarker to monitor the therapeutic efficacy of therapies for MPS II.

Mucopolysaccharidosis type IIIB (MPS IIIB) or Sanfilippo Syndrome type B is a lysosomal storage disease resulting from the deficiency of N-acetyl glucosaminidase (NAGLU) activity. We previously showed that intracranial adeno-associated virus (AAV)-based gene therapy results in partial improvements of several aspects of the disease. In an attempt to further correct the disease, MPS IIIB mice were treated at 2-4 days of age with intracranial AAV2/5-NAGLU (IC-AAV), intravenous lentiviral-NAGLU (IV-LENTI) or the combination of both (BOTH). The BOTH group had the most complete biochemical and histological improvements of any treatment group. Compared with untreated MPS IIIB animals, all treatments resulted in significant improvements in motor function (rotarod) and hearing (auditory-evoked brainstem response). In addition, each treatment group had a significantly increased median life span compared with the untreated group (322 days). The combination arm had the greatest increase (612 days), followed by IC-AAV (463 days) and IV-LENTI (358 days). Finally, the BOTH group had nearly normal circadian rhythm measures with improvement in time to activity onset. In summary, targeting both the systemic and central nervous system disease of MPS IIIB early in life appears to be the most efficacious approach for this inherited metabolic disorder.

Intrathecal enzyme replacement therapy is an experimental option to treat central nervous system disease due to lysosomal storage. Previous work shows that MPS I dogs receiving enzyme replacement with recombinant human alpha-L-iduronidase into the cisterna magna showed normal brain glycosaminoglycan (GAG) storage after three or four doses. We analyzed MPS I dogs that received intrathecal enzyme in a previous study using an assay that detects only pathologic GAG (pGAG). To quantify pGAG in MPS I, the assay measures only those GAG which display terminal iduronic acid residues on their non-reducing ends. Mean cortical brain pGAG in six untreated MPS I dogs was 60.9 ± 5.93 pmol per mg wet weight, and was 3.83 ± 2.64 in eight normal or unaffected carrier animals (p

Crystal (Cry) proteins made by the bacterium Bacillus thuringiensis are pore-forming toxins that specifically target insects and nematodes and are used around the world to kill insect pests. To better understand how pore-forming toxins interact with their host, we have screened for Caenorhabditis elegans mutants that resist Cry protein intoxication. We find that Cry toxin resistance involves the loss of two glycosyltransferase genes, bre-2 and bre-4. These glycosyltransferases function in the intestine to confer susceptibility to toxin. Furthermore, they are required for the interaction of active toxin with intestinal cells, suggesting they make an oligosaccharide receptor for toxin. Similarly, the bre-3 resistance gene is also required for toxin interaction with intestinal cells. Cloning of the bre-3 gene indicates it is the C. elegans homologue of the Drosophila egghead (egh) gene. This identification is striking given that the previously identified bre-5 has homology to Drosophila brainiac (brn) and that egh-brn likely function as consecutive glycosyltransferases in Drosophila epithelial cells. We find that, like in Drosophila, bre-3 and bre-5 act in a single pathway in C. elegans. bre-2 and bre-4 are also part of this pathway, thereby extending it. Consistent with its homology to brn, we demonstrate that C. elegans bre-5 rescues the Drosophila brn mutant and that BRE-5 encodes the dominant UDP-GlcNAc:Man GlcNAc transferase activity in C. elegans. Resistance to Cry toxins has uncovered a four component glycosylation pathway that is functionally conserved between nematodes and insects and that provides the basis of the dominant mechanism of resistance in C. elegans.

Treatment for mucopolysaccharidosis type IIIB (MPS IIIB; Sanfilippo syndrome type B) is hampered because recombinant alpha-N-acetylglucosaminidase (NAGLU) contains little/no mannose 6-phosphate. We developed a fusion protein of insulin-like growth factor 2 (IGF2) and NAGLU (rhNAGLU-IGF2). IGF2 is a natural ligand of the mannose-6phosphate receptor, and thus provides glycosylation-independent lysosomal targeting. Purified rhNAGLU-IGF2 is active in biochemical and cell-based assays (see Abstract: Aoyagi-Scharber, et al.). To evaluate rhNAGLU-IGF2 in vivo, adult (16 wk) MPS IIIB mice were catheterized into the left ventricle and given 4 twice-weekly injections of vehicle or rhNAGLU-IGF2 (100 μg). Mice were harvested 1d, 7d, 14d, and 28d after the last injection (n= 4-8 per group). Vehicle-treated heterozygotes were used as controls. Forebrain NAGLU activity ranged from 100x (1d, p b 0.001), 400x (7d, p = 0.001), 30x (14d, p = 0.9), and 130x fold (28d, p = 0.002) in rhNAGLU-IGF2 treated mice vs. vehicle-treated heterozygotes. Hexosaminidase activity was reduced by 26% (1d), 44% (7d), 51% (14d), and 57% (28d) in rhNAGLU-IGF2 treated vs vehicle-treated MPS IIIB mice (p b 0.001). Brain heparan sulfate was reduced from 228 pmol/ 5 μg brain (in vehicle-treated MPS IIIB) to 79.3 (1d), 24.8 (7d), 20.8 (14d), 17.4 (28d) in rhNAGLU-IGF2-treatedMPS IIIBmice (p b 0.001), vs 10.5 in vehicle-treated heterozygotes. In multiple brain regions, robust neuronal uptake was evident by immunostaining with NAGLU antibody and reduced LAMP1 signal with rhNAGLU-IGF2 treatment. In a separate experiment using 8 week-old mice, secondary accumulations were measured 1d after the last injection. Neuronal accumulations of SCMAS, glypican 5, amyloid beta and P-GSK3 beta in themedial entorhinal cortex and of P-tau in the dentate gyrus were reduced to the control level of these proteins. Accumulation of CD68 in activated microglia in the somatosensory cortex was reduced about half-way to control. In summary, we achieved broad distribution of NAGLU and improvement in neuropathology with short-term intraventricular rhNAGLU-IGF2. Glycosylation-independent lysosomal targeting may permit development of superior therapy for Sanfilippo syndrome type B. Support from NIH/NINDS 1R21NS078314-01A1 and BioMarin Pharmaceutical Inc.

Jillian R. Brown , Roger Lawrence , Kanar Al-Mafraji , William C. Lamanna , James R. Beitel , Geert-Jan Boons , Jeffrey D. Esko , Brett E. Crawford , Zacharon Pharmaceuticals, Inc, San Diego, CA, USA, Department of Cellular and Molecular Medicine, Glycobiology Research and Training Center, University of California, San Diego, USA, Complex Carbohydrate Research Center, University of Georgia, Athens, USA