BMN 250, a fusion of lysosomal alpha-N-acetylglucosaminidase with IGF2, exhibits different patterns of cellular uptake into critical cell types of Sanfilippo syndrome B disease pathogenesis.

Sanfilippo syndrome type B (Sanfilippo B; Mucopolysaccharidosis type IIIB) occurs due to genetic deficiency of lysosomal alpha-N-acetylglucosaminidase (NAGLU) and subsequent lysosomal accumulation of heparan sulfate (HS), which coincides with devastating neurodegenerative disease. Because NAGLU expressed in Chinese hamster ovary cells is not mannose-6-phosphorylated, we developed an insulin-like growth factor 2 (IGF2)-tagged NAGLU molecule (BMN 250; tralesinidase alfa) that binds avidly to the IGF2 / cation-independent mannose 6-phosphate receptor (CI-MPR) for glycosylation independent lysosomal targeting. BMN 250 is currently being developed as an investigational enzyme replacement therapy for Sanfilippo B. Here we distinguish two cellular uptake mechanisms by which BMN 250 is targeted to lysosomes. In normal rodent-derived neurons and astrocytes, the majority of BMN250 uptake over 24 hours reaches saturation, which can be competitively inhibited with IGF2, suggestive of CI-MPR-mediated uptake. Kuptake, defined as the concentration of enzyme at half-maximal uptake, is 5 nM and 3 nM in neurons and astrocytes, with a maximal uptake capacity (Vmax) corresponding to 764 nmol/hr/mg and 5380 nmol/hr/mg, respectively. Similar to neurons and astrocytes, BMN 250 uptake in Sanfilippo B patient fibroblasts is predominantly CI-MPR-mediated, resulting in augmentation of NAGLU activity with doses of enzyme that fall well below the Kuptake (5 nM), which are sufficient to prevent HS accumulation. In contrast, uptake of the untagged recombinant human NAGLU (rhNAGLU) enzyme in neurons, astrocytes and fibroblasts is negligible at the same doses tested. In microglia, receptor-independent uptake, defined as enzyme uptake resistant to competition with excess IGF2, results in appreciable lysosomal delivery of BMN 250 and rhNAGLU (Vmax = 12,336 nmol/hr/mg and 5469 nmol/hr/mg, respectively). These results suggest that while receptor-independent mechanisms exist for lysosomal targeting of rhNAGLU in microglia, BMN 250, by its IGF2 tag moiety, confers increased CI-MPR-mediated lysosomal targeting to neurons and astrocytes, two additional critical cell types of Sanfilippo B disease pathogenesis.

Mannitol-facilitated CNS entry of rAAV2 vector significantly delayed the neurological disease progression in MPS IIIB mice.

The presence of the blood-brain barrier (BBB) presents the most critical challenge in therapeutic development for mucopolysaccharidosis (MPS) IIIB, a lysosomal storage disease with severe neurological manifestation, because of alpha-N-acetylglucosaminidase (NaGlu) deficiency. Earlier, we showed a global central nervous system (CNS) transduction in mice by mannitol-facilitated entry of intravenous (IV)-delivered recombinant adeno-associated viral serotype 2 (rAAV2) vector. In this study, we optimized the approach and showed that the maximal transduction in the CNS occurred when the rAAV2 vector was IV injected at 8 min after mannitol administration, and was approximately 10-fold more efficient than IV delivery of the vector at 5 or 10 min after mannitol infusion. Using this optimal (8 min) regimen, a single IV infusion of rAAV2-CMV-hNaGlu vector is therapeutically beneficial for treating the CNS disease of MPS IIIB in adult mice, with significantly extended survival, improved behavioral performance, and reduction of brain lysosomal storage pathology. The therapeutic benefit correlated with maximal delivery to the CNS, but not peripheral tissues. This milestone data shows the first effective gene delivery across the BBB to treat CNS disease. The critical timing of vector delivery and mannitol infusion highlights the important contribution of this pretreatment to successful intervention, and the long history of safe use of mannitol in patients bodes well for its application in CNS gene therapy.

Actividad Urinaria del N-Acetil Betaglucosaminidasa en el embarazo normal / N-Acetyl Glucosaminidase urinary activity in normal pregnancy

Introducción: Durante el embarazo normal se producen en el riñón una serie de cambios que van a alterar la estructura y función de éste. Existen marcadores bioquímicos precoces de gran sensibilidad que detectan esta disfunción y que son muy útiles en el diagnóstico precoz de las formas complicadas de los trastornos hipertensivos del embarazo. Sin embargo son muy escasos los estudios de seguimiento en el embarazo normal. Uno de estos marcadores es la actividad urinaria del N-acetil beta-glucosaminidasa (NAG), enzima lisosómico de localización fundamentalmente tubular. Casuística y Métodos. Estudiamos un grupo de 64 mujeres sanas no embarazadas y otro de 93 mujeres con embarazo normal que seguimos a las 20 y 30 semanas y en el parto. La determinación de la actividad urinaria de NAG se realizó en orina de 24 horas por el método espectrofotométrico de Horak. Resultados. Encontramos una elevación progresiva del enzima urinario conforme progresa el embarazo, siendo los niveles máximos en el momento del parto. En los primeros trimestres de la gestación la actividad urinaria de NAG se comportó como en las mujeres sanas no embarazadas. Conclusiones. El ascenso en la actividad urinaria de NAG en la última fase de la gestación nos indica que es cuando se producen los mayores cambios de la función tubular condicionados por el aumento de volumen circulante, cambios en la presión oncótica y alteraciones en la reabsorción de glucosa, ácido úrico y aminoácidos

Introduction. During normal pregnancy, kidney suffers a series of changes that modifies its structure and functions. There are very sensitivity early biochemical markers that detect this dysfunction and are very useful in the early diagnosis of complicated ways of hypertensive disorders in pregnancy. However, monitoring studies in normal pregnancy are rare. One of said markers is N-Acetyl-beta-D-glucosaminidase (NAG), a lysosomal enzyme of mainly tubular localization. Casuistry and methods. We studied a group of 64 healthy, non-pregnant women and another with 93 women with a normal pregnancy that were monitored in the gestational weeks 20 and 30 and during the delivery. Determining urinary NAG activity was carried out in 24 hours urine by the chromatographic method of Horak. Results. We found a gradual increase of enzymes in urine as the pregnancy progresses, reaching the highest levels during the delivery. Urinary NAG activity in the last stage of pregnancy shows it is at that time when the highest changes in tubular function take place. Said changes are determined by the increase in circulating volume, the changes in the oncotic pressure, and the disorders in glucose, uric acid, and amino acid reabsorption

Calcitonin stimulates lysosomal enzyme release and uptake in LLC-PK1 cells.

Renal tubular targeted hormones increase urinary excretion of a lysosomal enzyme, N-acetyl-beta-D-glucosaminidase (NAG). To elucidate the mechanism of this event, the calcitonin effect on NAG handling by LLC-PK1 cells was examined. Calcitonin (1 nM to 1 microM), phorbol myristate (10 nM to 1 microM), and ionomycin (1 to 10 microM) promoted NAG release without any increase in lactate dehydrogenase release or any reduction of mitochondrial dehydrogenase activity. Treatment with 100 nM calphostin C or 50 microM KN-93 partially reversed the calcitonin effect on NAG release. Calcitonin promoted secretion of fluorescence ceramide, a reporter of protein transport from Golgi apparatus to cell surface. Calcitonin-stimulated NAG release was partially inhibited by 10 microg/ml brefeldin A, a blocker of protein transport through the Golgi apparatus. Calcitonin accelerated cellular uptake of exogenous NAG, which was inhibited by low temperature, 0.1 mM monodansyl cadaverine (receptor-mediated endocytosis inhibitor), and 10 mM mannose-6-phosphate. Furthermore, calcitonin promoted progression of intracellular membranes stained by a fluorescence membrane marker, styryl pyridinium dye, from cell periphery to perinuclear regions (commonly referred to as recycling vesicles) and increased dye release from preloaded cells. Fluorescence release from the cells pre-loaded with FITC-labeled NAG or albumin was also stimulated by calcitonin. These calcitonin effects on endocytotic and re-exocytotic pathways were inhibited by 100 nM cytochalasin D, 100 nM nocodazole, 0.1 to 1 microM bafilomycin A1, or 0.1 mM monodansyl cadaverine. Increased urinary NAG excretion has been considered to reflect renal tubular damage. However, it was demonstrated here that stimulation of secretory and recycling pathways may be an alternative mechanism for calcitonin-induced enzymuria, which will become a new indicator of renal tubular response to this hormone.