Insulin-like growth factor II peptide fusion enables uptake and lysosomal delivery of α-N-acetylglucosaminidase to mucopolysaccharidosis type IIIB fibroblasts.

Enzyme replacement therapy for MPS IIIB (mucopolysaccharidosis type IIIB; also known as Sanfilippo B syndrome) has been hindered by inadequate mannose 6 phosphorylation and cellular uptake of rhNAGLU (recombinant human α-N-acetylglucosaminidase). We expressed and characterized a modified rhNAGLU fused to the receptor-binding motif of IGF-II (insulin-like growth factor 2) (rhNAGLU-IGF-II) to enhance its ability to enter cells using the cation-independent mannose 6-phosphate receptor, which is also the receptor for IGF-II (at a different binding site). RhNAGLU-IGF-II was stably expressed in CHO (Chinese-hamster ovary) cells, secreted and purified to apparent homogeneity. The Km and pH optimum of the fusion enzyme was similar to those reported for rhNAGLU. Both intracellular uptake and confocal microscopy suggested that MPS IIIB fibroblasts readily take up the fusion enzyme via receptor-mediated endocytosis that was inhibited significantly (P<0.001) by the monomeric IGF-II peptide. Glycosaminoglycan storage was reduced by 60% (P<0.001) to near background levels in MPS IIIB cells after treatment with rhNAGLU-IGF-II, with half-maximal correction at concentrations of 3-12 pM. A similar cellular uptake mechanism via the IGF-II receptor was also demonstrated in two different brain tumour-derived cell lines. Fusion of rhNAGLU to IGF-II enhanced its cellular uptake while maintaining enzymatic activity, supporting its potential as a therapeutic candidate for treating MPS IIIB.

Biochemical characterization of fluorescent-labeled recombinant human alpha-L-iduronidase in vitro.

In vivo tracking of the delivery of therapeutic proteins is a useful tool for preclinical studies. However, many labels are too large to use without disrupting the normal uptake, function, or other properties of the protein. Low-molecular-weight fluorescent labels allow in vivo and ex vivo tracking of the distribution of therapeutic proteins, and should not alter the protein's characteristics. We tested the in vitro properties of fluorescent-labeled recombinant human alpha-l-iduronidase (rhIDU, the enzyme deficient in Hurler syndrome) and compared labeled to unlabeled proteins. Labeled rhIDU retained full enzymatic activity and showed similar kinetics to nonlabeled rhIDU. Uptake of labeled rhIDU into human Hurler fibroblasts, measured by activity assay, was equivalent to unlabeled rhIDU enzyme and showed an uptake constant of 0.72 nM. Labeled rhIDU was also able to enter cells via the mannose 6-phospate receptor pathway and reduce glycosaminoglycan storage in Hurler fibroblasts. Subcellular localization was verified within lysosomes by confocal microscopy. These findings suggest that fluorescent labeling does not significantly interfere with enzymatic activity, stability, or uptake, and validates this method as a way to track exogenously administered enzyme.

Echocardiographic assessment of left ventricular geometry and the mitral valve apparatus in cats with hypertrophic cardiomyopathy.

OBJECTIVES: This retrospective study addressed the general hypothesis that abnormalities of the mitral valve apparatus are common in cats with idiopathic hypertrophic cardiomyopathy (HCM) and contribute to dynamic obstruction of the left ventricular outflow tract (LVOT). ANIMALS, MATERIALS AND METHODS: 106 cats (28 controls and 78 with HCM) had transthoracic two-dimensional and Doppler echocardiography performed with quantification of 33 variables. Three groups of cats (control [Group-1], HCM without obstruction [Group-2], and HCM with obstruction [Group-3]) were identified and compared by analysis of variance, chi(2) analysis, and correlation analysis. RESULTS: Cats in Group-3 had more LV and papillary muscle hypertrophy, increased length of the anterior mitral valve leaflet, and a higher prevalence of false tendons in the LVOT compared to cats in Group-2 (P < or = 0.05). The length of the anterior mitral valve leaflet was correlated to the severity of dynamic obstruction (P < or = 0.05) and the magnitude of LV hypertrophy (P < or = 0.001). Systolic anterior motion of chordae tendineae (CAM) was observed in 16% of control cats and >50% of cats with HCM (P < or = 0.05). CONCLUSIONS: Abnormalities of the mitral valve are common in cats with HCM suggesting a possible role in the pathogenesis of dynamic outflow tract obstruction.