Neonatal combination therapy improves some of the clinical manifestations in the Mucopolysaccharidosis type I murine model.

Mucopolysaccharidosis type I (MPS-I), a lysosomal storage disorder caused by a deficiency of alpha-L-iduronidase enzyme, results in the progressive accumulation of glycosaminoglycans and consequent multiorgan dysfunction. Despite the effectiveness of hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT) in correcting clinical manifestations related to visceral organs, complete improvement of musculoskeletal and neurocognitive defects remains an unmet challenge and provides an impact on patients' quality of life. We tested the therapeutic efficacy of combining HSCT and ERT in the neonatal period. Using a mouse model of MPS-I, we demonstrated that the combination therapy improved clinical manifestations in organs usually refractory to current treatment. Moreover, combination with HSCT prevented the production of anti-IDUA antibodies that negatively impact ERT efficacy. The added benefits of combining both treatments also resulted in a reduction of skeletal anomalies and a trend towards decreased neuroinflammation and metabolic abnormalities. As currently there are limited therapeutic options for MPS-I patients, our findings suggest that the combination of HSCT and ERT during the neonatal period may provide a further step forward in the treatment of this rare disease.

Glycosaminoglycan fragments as a measure of disease burden in the mucopolysaccharidosis type I mouse.

Glycosaminoglycan (GAG) catabolism involves endo-hydrolysis of polysaccharides followed by the sequential removal of the non-reducing end residue from the resulting oligosaccharides by exo-enzymes. In the inherited metabolic disorder, mucopolysaccharidosis type I (MPS I), a deficiency in the exo-enzyme, α-l-iduronidase, prevents removal of α-l-iduronic acid residues from the non-reducing end of the GAGs, heparan sulphate (HS) and dermatan sulphate (DS). The excretion of partially degraded HS and DS in urine of MPS I patients has long been recognized, but the question of whether they do indeed reflect GAG load in a particular tissue has not been addressed - an important issue in the context of biomarkers for assessment of disease burden in MPS I. Therefore, we measured specific low molecular weight HS and DS oligosaccharides with terminal α-l-iduronic acid residues, in the brain, liver, kidney, serum and urine, and correlated these findings with total GAG in the MPS I mouse model. Six oligosaccharides were identified in the urine, ranging from di- to pentasaccharides. Of these, five were observed in the kidney, four in the liver and brain, with the three most abundant in urine also seen in serum. These oligosaccharides accounted for just 0.1-2% of total GAG, with a disaccharide showing the best correlation with total GAG. The oligosaccharides and total GAG were most abundant in the liver, with the least observed in the brain. The concentration of oligosaccharides as a percentage of total GAG in urine was similar to that observed in the kidney, and both revealed a similar ratio of HS:DS, suggesting that the oligosaccharide storage pattern in urine is a reflection of that in the kidney. Serum, liver and brain had a similar ratio of HS:DS, which was lower to that seen in the urine and kidney. The distribution of oligosaccharides when ranked from most to least abundant, was also the same between serum, liver and brain suggesting that serum more closely reflects the oligosaccharides of the brain and liver and may therefore be a more informative measurement of disease burden than urine. The accumulation of HS and DS oligosaccharides was observed in the brain as early as one month of age, with the disaccharide showing a continuous increase with age. This demonstrates the progressive nature of the disease and as such this disaccharide could prove to be a useful biomarker to measure disease burden in MPS I.

RTB lectin-mediated delivery of lysosomal α-l-iduronidase mitigates disease manifestations systemically including the central nervous system.

Mucopolysaccharidosis type I (MPS I) is a lysosomal disease resulting from deficiency in the α-L-iduronidase (IDUA) hydrolase and subsequent accumulation of glycosaminoglycan (GAG). Clinically, enzyme replacement therapy (ERT) with IDUA achieves negligible neurological benefits presumably due to blood-brain-barrier (BBB) limitations. To investigate the plant lectin ricin B chain (RTB) as a novel carrier for enzyme delivery to the brain, an IDUA:RTB fusion protein (IDUAL), produced in N. benthamiana leaves, was tested in a murine model of Hurler syndrome (MPS I). Affect mice (n=3 for each group) were intravenously injected with a single dose of IDUAL (0.58, 2 or 5.8mgIDUAequivalents/kg) and analyzed after 24h. IDUA activities in liver, kidney and spleen increased significantly, and liver GAG levels were significantly reduced in all three groups. Plasma IDUA levels for all treated groups were high at 1h after injection and decreased by 95% at 4h, indicating efficient distribution into tissues. For long-term evaluations, IDUAL (0.58 or 2mg/kg, 8 weekly injections) was intravenously injected into MPS I mice (n=12 for each group). Thirteen days after the 8th injection, significant IDUA activity was detected in the liver and spleen. GAG levels in tissues including the brain cortex and cerebellum were significantly reduced in treated animals. Treated MPS I mice also showed significant improvement in neurocognitive testing. ELISA results showed that while there was a significant antibody response against IDUAL and plant-derived IDUA, there was no significant antibody response to RTB. No major toxicity or adverse events were observed. Together, these results showed that infusion of IDUAL allowed for significant IDUA levels and GAG reduction in the brain and subsequent neurological benefits. This RTB-mediated delivery may have significant implications for therapeutic protein delivery impacting a broad spectrum of lysosomal, and potentially neurological diseases.

A self-inactivating gamma-retroviral vector reduces manifestations of mucopolysaccharidosis I in mice.

Mucopolysaccharidosis I (MPS I) is a lysosomal storage disease due to deficiency in alpha-L-iduronidase (IDUA) that results in accumulation of glycosaminoglycans (GAGs) throughout the body, causing numerous clinical defects. Intravenous administration of a gamma-retroviral vector (gamma-RV) with an intact long terminal repeat (LTR) reduced the clinical manifestations of MPS I, but could cause insertional mutagenesis. Although self-inactivating (SIN) gamma-RVs in which the enhancer and promoter elements in the viral LTR are absent after transduction reduces this risk, such vectors could be less effective. This report demonstrates that intravenous (i.v.) injection of a SIN gamma-RV expressing canine IDUA from the liver-specific human alpha(1)-antitrypsin promoter into adult or newborn MPS I mice completely prevents biochemical abnormalities in several organs, and improved bone disease, vision, hearing, and aorta to a similar extent as was seen with administration of the LTR-intact vector to adults. Improvements were less profound than when using an LTR-intact gamma-RV in newborns, which likely reflects a lower level of transduction and expression for the SIN vector-transduced mice, and might be overcome by using a higher dose of SIN vector. A SIN gamma-RV vector ameliorates clinical manifestations of MPS I in mice and should be safer than an LTR-intact gamma-RV.

Structural study on mutant alpha-L-iduronidases: insight into mucopolysaccharidosis type I.

To elucidate the basis of mucopolysaccharidosis type I (MPS I), we constructed structural models of mutant alpha-L: -iduronidases (IDUAs) resulting from 33 amino acid substitutions that lead to MPS I (17 severe, eight intermediate, and eight attenuated). Then, we examined the structural changes in the enzyme protein by calculating the number of atoms affected and determined the root-mean-square distance (RMSD) and the solvent-accessible surface area (ASA). In the severe MPS I group, the number of atoms influenced by a mutation and the average RMSD value were larger than those in the attenuated group, and the residues associated with the mutations identified in the severe group tended to be less solvent accessible than those in the attenuated group. The clinically intermediate phenotype group exhibited intermediate values for the numbers of atoms affected, RMSD, and ASA between those in the severe group and those in the attenuated group. The results indicated that large structural changes had occurred in the core region in the severe MPS I group and small ones on the molecular surface in the attenuated MPS I group. Color imaging revealed the distributions and degrees of the structural changes caused by representative mutations for MPS I. Thus, structural analysis is useful for elucidating the basis of MPS I. As there was a difference in IDUA structural change between the severe MPS I group and the attenuated one, except for a couple of mutations, structural analysis can help predict the clinical outcome of the disease.

Human alpha-L-iduronidase. Catalytic properties and an integrated role in the lysosomal degradation of heparan sulphate.

The kinetic parameters (Km and kcat) of human liver alpha-L-iduronidase were determined with a variety of heparin-derived disaccharide and tetrasaccharide substrates. More structurally complex substrates, in which several aspects of the aglycone structure of the natural substrates heparin and heparan sulphate were maintained, were hydrolysed with catalytic efficiencies up to 255 times that observed for the simplest disaccharide substrate to be hydrolysed. The major aglycone structure that influenced both substrate binding and enzyme activity was the presence of a C-6 sulphate ester on the residue adjacent to the iduronic acid residue being hydrolysed. Sulphate ions and a number of substrate and product analogues were potent inhibitors of enzyme activity. Human liver alpha-L-iduronidase activity towards 4-methylumbelliferyl alpha-L-iduronide at pH 4.8 had two Km values of 37 microM and 1.92 mM with corresponding kcat. values of 299 and 650 mol of product formed/min per mol of enzyme respectively, which may explain the wide range of Km values previously reported for alpha-L-iduronidase activity toward its substrate. Skin fibroblast alpha-L-iduronidase activity towards the heparin-derived oligosaccharides was influenced by the same substrate aglycone structural features as was observed for the human liver enzyme. A comparison was made of the effect of substrate aglycone structure upon catalytic activities of the enzymes which act to degrade the highly sulphated regions of heparan sulphate. A model was proposed whereby the substrate is directed from alpha-L-iduronidase to subsequent enzyme activities to ensure the efficient degradation of heparan sulphate.