Vestronidase alfa: Recombinant human ß-glucuronidase as an enzyme replacement therapy for MPS VII.

Mucopolysaccharidosis VII (MPS VII) is a rare lysosomal storage disease characterized by a deficiency in the enzyme ß-glucuronidase that has previously been successfully treated in a mouse model with enzyme replacement therapy. Here, we present the generation of a novel, highly sialylated version of recombinant human ß-glucuronidase (rhGUS), vestronidase alfa, that has high uptake, resulting in an improved enzyme replacement therapy for the treatment of patients with MPS VII. In vitro, vestronidase alfa has 10-fold more sialic acid per mole of rhGUS monomer than a prior rhGUS version (referred to as GUS 43/44) and demonstrated very high affinity at ~1 nM half maximal uptake in human MPS VII fibroblasts. Vestronidase alfa has a longer enzymatic half-life after uptake into fibroblasts compared with other enzymes used as replacement therapy for MPS (40 days vs 3 to 4 days, respectively). In pharmacokinetic and tissue distribution experiments in Sprague-Dawley rats, intravenous administration of vestronidase alfa resulted in higher serum rhGUS levels and enhanced ß-glucuronidase activity distributed to target tissues. Weekly intravenous injections of vestronidase alfa (0.1 mg/kg to 20 mg/kg) in a murine model of MPS VII demonstrated efficient enzyme delivery to all tissues, including bone and brain, as well as reduced lysosomal storage of glycosaminoglycans (GAGs) in a dose-dependent manner, resulting in increased survival after 8 weeks of treatment. Vestronidase alfa was well-tolerated and demonstrated no toxicity at concentrations that reached 5-times the proposed clinical dose. In a first-in-human phase 1/2 clinical trial, a dose-dependent reduction in urine GAG levels was sustained over 38 weeks of treatment with vestronidase alfa. Together, these results support the therapeutic potential of vestronidase alfa as an enzyme replacement therapy for patients with MPS VII.

Biochemical evidence for superior correction of neuronal storage by chemically modified enzyme in murine mucopolysaccharidosis VII.

Enzyme replacement therapy has been used successfully in many lysosomal storage diseases. However, correction of brain storage has been limited by the inability of infused enzyme to cross the blood-brain barrier (BBB). We recently reported that PerT-GUS, a form of ß-glucuronidase (GUS) chemically modified to eliminate its uptake and clearance by carbohydrate-dependent receptors, crossed the BBB and cleared neuronal storage in an immunotolerant model of murine mucopolysaccharidosis (MPS) type VII. In this respect, the chemically modified enzyme was superior to native ß-glucuronidase. Chemically modified enzyme was also delivered more effectively to heart, kidney, and muscle. However, liver and spleen, which express high levels of carbohydrate receptors, received nearly fourfold lower levels of PerT-GUS compared with native GUS. A recent report on PerT-treated sulfamidase in murine MPS IIIA confirmed enhanced delivery to other tissues but failed to observe clearance of storage in neurons. To confirm and extend our original observations, we compared the efficacy of 12 weekly i.v. infusions of PerT-GUS versus native GUS on (i) delivery of enzyme to brain; (ii) improvement in histopathology; and (iii) correction of secondary elevations of other lysosomal enzymes. Such correction is a recognized biomarker for correction of neuronal storage. PerT-GUS was superior to native GUS in all three categories. These results provide additional evidence that long-circulating enzyme, chemically modified to escape carbohydrate-mediated clearance, may offer advantages in treating MPS VII. The relevance of this approach to treat other lysosomal storage diseases that affect brain awaits confirmation.

Assessment of bone dysplasia by micro-CT and glycosaminoglycan levels in mouse models for mucopolysaccharidosis type I, IIIA, IVA, and VII.

Mucopolysaccharidoses (MPS) are a group of lysosomal storage diseases caused by mutations in lysosomal enzymes involved in degradation of glycosaminoglycans (GAGs). Patients with MPS grow poorly and become physically disabled due to systemic bone disease. While many of the major skeletal effects in mouse models for MPS have been described, no detailed analysis that compares GAGs levels and characteristics of bone by micro-CT has been done. The aims of this study were to assess severity of bone dysplasia among four MPS mouse models (MPS I, IIIA, IVA and VII), to determine the relationship between severity of bone dysplasia and serum keratan sulfate (KS) and heparan sulfate (HS) levels in those models, and to explore the mechanism of KS elevation in MPS I, IIIA, and VII mouse models. Clinically, MPS VII mice had the most severe bone pathology; however, MPS I and IVA mice also showed skeletal pathology. MPS I and VII mice showed severe bone dysplasia, higher bone mineral density, narrowed spinal canal, and shorter sclerotic bones by micro-CT and radiographs. Serum KS and HS levels were elevated in MPS I, IIIA, and VII mice. Severity of skeletal disease displayed by micro-CT, radiographs and histopathology correlated with the level of KS elevation. We showed that elevated HS levels in MPS mouse models could inhibit N-acetylgalactosamine-6-sulfate sulfatase enzyme. These studies suggest that KS could be released from chondrocytes affected by accumulation of other GAGs and that KS could be useful as a biomarker for severity of bone dysplasia in MPS disorders.

Long circulating enzyme replacement therapy rescues bone pathology in mucopolysaccharidosis VII murine model.

Mucopolysaccharidosis (MPS) type VII is a lysosomal storage disease caused by deficiency of the lysosomal enzyme ß-glucuronidase (GUS), leading to accumulation of glycosaminoglycans (GAGs). Enzyme replacement therapy (ERT) effectively clears GAG storage in the viscera. Recent studies showed that a chemically modified form of GUS (PerT-GUS), which escaped clearance by mannose 6-phosphate and mannose receptors and showed prolonged circulation, reduced CNS storage more effectively than native GUS. Clearance of storage in bone has been limited due to the avascularity of the growth plate. To evaluate the effectiveness of long-circulating PerT-GUS in reducing the skeletal pathology, we treated MPS VII mice for 12 weeks beginning at 5 weeks of age with PerT-GUS or native GUS and used micro-CT, radiographs, and quantitative histopathological analysis for assessment of bones. Micro-CT findings showed PerT-GUS treated mice had a significantly lower BMD. Histopathological analysis also showed reduced storage material and a more organized growth plate in PerT-GUS treated mice compared with native GUS treated mice. Long term treatment with PerT-GUS from birth up to 57 weeks also significantly improved bone lesions demonstrated by micro-CT, radiographs and quantitative histopathological assay. In conclusion, long-circulating PerT-GUS provides a significant impact to rescue of bone lesions and CNS involvement.

Identification of N-glycans displaying mannose-6-phosphate and their site of attachment on therapeutic enzymes for lysosomal storage disorder treatment.

Characterization of mono- and bis-mannose-6-phosphate (M6P) bearing oligosaccharides present on acid hydrolase enzymes poses a considerable analytical challenge. In the current paper, we investigated the use of UPLC profiling on a 1.7 µm HILIC phase and capillary electrophoresis with laser induced fluorescence detection (CE-LIF) combined with exoglycosidase digestion and weak anion exchange fractionation for the characterization of M6P bearing glycans on recombinant ß-glucuronidase expressed in Chinese Hamster Ovary (CHO) cells. Using this multidimensional approach a number of peaks were observed to resist digestion, suggesting the presence and blocking activity of the M6P tag. To investigate further, mixed oxide affinity purification on a combined TiO(2)/ZrO(2) resin facilitated the selective enrichment of oligosaccharides bearing mono- or diphospho-esters that corresponded to those peaks previously identified to resist exoglycosidase digestion. Alkaline phosphatase digestion identified Man(6)GlcNAc(2) and Man(7)GlcNAc(2) glycans as the primary carriers of the M6P tag. Site-specific glycoproteomic analysis revealed that Man(7)GlcNAc(2)-M6P oligosaccharides were present at asparagine 272 and 420, while asparagine 631 displayed Man(6)GlcNAc(2)-M6P. The analytical strategy applied herein represents a novel yet simple approach for the qualitative and semiquantitative structural characterization of M6P containing oligosaccharides on therapeutic enzymes.

Chemically modified beta-glucuronidase crosses blood-brain barrier and clears neuronal storage in murine mucopolysaccharidosis VII.

Enzyme replacement therapy has been used successfully in many lysosomal storage diseases. However, correction of brain storage has been limited by the inability of infused enzyme to cross the blood-brain barrier. The newborn mouse is an exception because recombinant enzyme is delivered to neonatal brain after mannose 6-phosphate receptor-mediated transcytosis. Access to this route is very limited after 2 weeks of age. Recently, several studies showed that multiple infusions of high doses of enzyme partially cleared storage in adult brain. These results raised the question of whether correction of brain storage by repeated high doses of enzyme depends on mannose 6-phosphate receptor-mediated uptake or whether enzyme gains access to brain storage by another route when brain capillaries are exposed to prolonged, high levels of circulating enzyme. To address this question, we used an enzyme whose carbohydrate-dependent receptor-mediated uptake was inactivated by chemical modification. Treatment of human beta-glucuronidase (GUS) with sodium metaperiodate followed by sodium borohydride reduction (PerT-GUS) eliminated uptake by mannose 6-phosphate and mannose receptors in cultured cells and dramatically slowed its plasma clearance from a t(1/2) of <10 min to 18 h. Surprisingly, PerT-GUS infused weekly for 12 weeks was more effective in clearing central nervous system storage than native GUS at the same dose. In fact, PerT-GUS resulted in almost complete reversal of storage in neocortical and hippocampal neurons. This enhanced correction of neuronal storage by long-circulating enzyme, which targets no known receptor, suggests a delivery system across the blood-brain barrier that might be exploited therapeutically.

Infused Fc-tagged beta-glucuronidase crosses the placenta and produces clearance of storage in utero in mucopolysaccharidosis VII mice.

Glycosaminoglycan storage begins in prenatal life in patients with mucopolysaccharidosis (MPS). In fact, prenatal hydrops is a common manifestation of MPS VII because of beta-glucuronidase (GUS) deficiency. One way to address prenatal storage might be to deliver the missing enzyme across the placenta into the fetal circulation. Maternal IgG is transported across the placenta by the neonatal Fc receptor (FcRn), which recognizes the Fc domain of IgG and mediates transcytosis from maternal to fetal circulation. We hypothesized that we could exploit this process to deliver corrective enzyme to the fetus. To test this hypothesis, the C-terminal fusion protein, GUS-Fc, was compared with native, untagged, recombinant GUS for clearance from the maternal circulation, delivery to the fetus, and reduction of lysosomal storage in offspring of MPS VII mice. We observed that GUS-Fc, infused into pregnant mothers on embryonic days 17 and 18, was transported across the placenta. Similarly infused untagged GUS was not delivered to the fetus. GUS-Fc plasma enzyme activity in newborn MPS VII mice was 1,000 times that seen after administration of untagged GUS and approximately 100 times that of untreated WT newborns. Reduced lysosomal storage in heart valves, liver, and spleen provided evidence that in utero enzyme replacement therapy with GUS-Fc targeted sites of storage in the MPS VII fetus. We hypothesize that this noninvasive approach could deliver the missing lysosomal enzyme to a fetus with any lysosomal storage disease. It might also provide a method for inducing immune tolerance to the missing enzyme or another foreign protein.

Mutations and polymorphisms in GUSB gene in mucopolysaccharidosis VII (Sly Syndrome).

Mucopolysaccharidosis VII (MPS VII; Sly syndrome) is an autosomal recessive disorder caused by a deficiency of beta-glucuronidase (GUS, EC 3.2.1.31; GUSB). GUS is required to degrade glycosaminoglycans (GAGs), including heparan sulfate (HS), dermatan sulfate (DS), and chondroitin-4,6-sulfate (CS). Accumulation of undegraded GAGs in lysosomes of affected tissues leads to mental retardation, short stature, hepatosplenomegaly, bone dysplasia, and hydrops fetalis. We summarize information on the 49 unique, disease-causing mutations determined so far in the GUS gene, including nine novel mutations (eight missense and one splice-site). This heterogeneity in GUS gene mutations contributes to the extensive clinical variability among patients with MPS VII. One pseudodeficiency allele, one polymorphism causing an amino acid change, and one silent variant in the coding region are also described. Among the 103 analyzed mutant alleles, missense mutations accounted for 78.6%; nonsense mutations, 12.6%; deletions, 5.8%; and splice-site mutations, 2.9%. Transitional mutations at CpG dinucleotides made up 40.8% of all the described mutations. The five most frequent mutations (accounting for 44/103 alleles) were exonic point mutations, p.L176F, p.R357X, p.P408S, p.P415L, and p.A619 V. Genotype/phenotype correlation was attempted by correlating the effects of certain missense mutations or enzyme activity and stability within phenotypes. These were in turn correlated with the location of the mutation in the tertiary structure of GUS. A total of seven murine, one feline, and one canine model of MPS VII have been characterized for phenotype and genotype.

Mannose 6-phosphate receptor-mediated transport of sulfamidase across the blood-brain barrier in the newborn mouse.

Mucopolysaccharidosis type IIIA (MPS IIIA), which is a lysosomal storage disorder (LSD) caused by inherited deficiency of sulfamidase, is characterized by severe, progressive central nervous system (CNS) dysfunction. Enzyme replacement therapy (ERT) to treat CNS storage is challenging, because the access of enzymes to the brain is restricted by the blood-brain barrier (BBB). In a prior study, we found that phosphorylated beta-glucuronidase (P-GUS) could be transcytosed across the BBB in newborn mice by the mannose 6-phosphate (M6P) receptor. In order to determine whether sulfamidase can utilize this pathway, we examined brain influx and the specificity of uptake of sulfamidase after intravenous (i.v.) injection in 2-day-old and 8-week-old mice. [(131)I]Sulfamidase was transported across the BBB in neonates at rates higher than that of simultaneously injected [(125)I]albumin. In contrast, the transport of [(131)I]sulfamidase was negligible in 8-week-old mice, thereby showing that the BBB transport mechanism is developmentally downregulated. Capillary depletion revealed that 83.7% of the [(131)I]sulfamidase taken up by the brain was in the parenchyma, demonstrating transfer across the capillary wall. The uptake of [(131)I]sulfamidase into the brain was significantly reduced by co-injections of M6P and P-GUS. That is, the transport of sulfamidase into the brain parenchyma in early postnatal life is mediated by the M6P receptor, which is shared with P-GUS and is likely accessible to other M6P-containing lysosomal enzymes.

Acidic amino acid tag enhances response to enzyme replacement in mucopolysaccharidosis type VII mice.

We have tested an acidic oligopeptide-based targeting system for delivery of enzymes to tissues, especially bone and brain, in a murine mucopolysaccharidosis type VII (MPS VII) model. This strategy is based upon tagging a short peptide consisting of acidic amino acids (AAA) to N terminus of human beta-glucuronidase (GUS). The pharmacokinetics, biodistribution, and the pathological effect on MPS VII mouse after 12 weekly infusions were determined for recombinant human untagged and tagged GUS. The tagged GUS was taken up by MPS VII fibroblasts in a mannose 6-phosphate receptor-dependent manner. Intravenously injected AAA-tagged enzyme had five times more prolonged blood clearance compared with the untagged enzyme. The tagged enzyme was delivered effectively to bone, bone marrow, and brain in MPS VII mice and was effective in reversing the storage pathology. The storage in osteoblasts was cleared similarly with both enzyme types. However, cartilage showed a little response to any of the enzymes. The tagged enzyme reduced storage in cortical neurons, hippocampus, and glia cells. A highly sensitive method of tandem mass spectrometry on serum indicated that the concentration of serum dermatan sulfate and heparan sulfate in mice treated with the tagged enzyme decreased more than the untagged enzyme. These preclinical studies suggest that this AAA-based targeting system may enhance enzyme-replacement therapy.

HFE association with transferrin receptor 2 increases cellular uptake of transferrin-bound iron.

Mutations in either HFE or transferrin receptor 2 (TfR2) cause decreased expression of the iron regulatory hormone hepcidin and hemochromatosis. HFE and TfR2 were recently discovered to form a stable complex at the cell membrane when co-expressed in heterologous cell lines. We analyzed the functional consequences of the co-expression of these proteins using transfected TRVb cells, a Chinese hamster ovary derived cell line without endogenous HFE or transferrin receptor. The co-expression of TfR2 in TRVb cells expressing HFE led to accelerated HFE biosynthesis and late-Golgi maturation, suggesting interaction prior to cell surface localization. The co-expression of HFE in cells expressing TfR2 led to increased affinity for diferric transferrin, increased transferrin-dependent iron uptake, and relative resistance to iron chelation. These observations indicate that HFE influences the functional properties of TfR2, and suggests a model in which the interaction of these proteins might influence signal transduction to hepcidin.

Expression of membrane-bound carbonic anhydrases IV, IX, and XIV in the mouse heart.

Expression of membrane-bound carbonic anhydrases (CAs) of CA IV, CA IX, CA XII, and CA XIV has been investigated in the mouse heart. Western blots using microsomal membranes of wild-type hearts demonstrate a 39-, 43-, and 54-kDa band representing CA IV, CA IX, and CA XIV, respectively, but CA XII could not be detected. Expression of CA IX in the CA IV/CA XIV knockout animals was further confirmed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Cardiac cells were immunostained using anti-CA/FITC and anti-alpha-actinin/TRITC, as well as anti-CA/FITC and anti-SERCA2/TRITC. Subcellular CA localization was investigated by confocal laser scanning microscopy. CA localization in the sarcolemmal (SL) membrane was examined by double immunostaining using anti-CA/FITC and anti-MCT-1/TRITC. CAs showed a distinct distribution pattern in the sarcoplasmic reticulum (SR) membrane. CA XIV is predominantly localized in the longitudinal SR, whereas CA IX is mainly expressed in the terminal SR/t-tubular region. CA IV is present in both SR regions, whereas CA XII is not found in the SR. In the SL membrane, only CA IV and CA XIV are present. We conclude that CA IV and CA XIV are associated with the SR as well as with the SL membrane, CA IX is located in the terminal SR/t-tubular region, and CA XII is not present in the mouse heart. Therefore, the unique subcellular localization of CA IX and CA XIV in cardiac myocytes suggests different functions of both enzymes in excitation-contraction coupling.

Pharmacologic manipulation of lysosomal enzyme transport across the blood-brain barrier.

The adult blood-brain barrier, unlike the neonatal blood-brain barrier, does not transport lysosomal enzymes into brain, making enzyme replacement therapy ineffective in treating the central nervous system symptoms of lysosomal storage diseases. However, enzyme transport can be re-induced with alpha-adrenergics. Here, we examined agents that are known to alter the blood-brain barrier transport of large molecules or to induce lysosomal enzyme transport across the blood-brain barrier ((±)epinephrine, insulin, retinoic acid, and lipopolysaccharide) in 2-week-old and adult mice. In 2-week-old adolescent mice, all these pharmacologic agents increased brain and heart uptake of phosphorylated human ß-glucuronidase. In 8-week-old adult mice, manipulations with (±)epinephrine, insulin, and retinoic acid were significantly effective on uptake by brain and heart. The increased uptake of phosphorylated human ß-glucuronidase was inhibited by mannose 6-phosphate for the agents (±)epinephrine and retinoic acid and by L-NG-nitroarginine methyl ester for the agent lipopolysaccharide in neonatal and adult mice. An in situ brain perfusion study revealed that retinoic acid directly modulated the transport of phosphorylated human ß-glucuronidase across the blood-brain barrier. The present study indicates that there are multiple opportunities to at least transiently induce phosphorylated human ß-glucuronidase transport at the adult blood-brain barrier.

Localization of carbonic anhydrase XII to the basolateral membrane of H+-secreting cells of mouse and rat kidney.

Membrane-associated carbonic anhydrase (CA) has a crucial role in renal HCO(3)(-) absorption. CA activity has been localized to both luminal and basolateral membranes of the tubule epithelial cells. CA XII is a transmembrane isoenzyme that has been demonstrated in the basolateral plasma membrane of human renal, intestinal, and reproductive epithelia. The present study was designed to demonstrate the distribution of CA XII expression in the rodent kidney. A new polyclonal antibody to recombinant mouse CA XII was used in both Western blotting and immunohistochemistry. Western blotting analysis revealed a 40-45-kD polypeptide in CA XII-expressing CHO cells and isolated membranes of mouse and rat kidney. Immunofluorescence staining localized CA XII in the basolateral plasma membranes of S1 and S2 proximal tubule segments. Abundant basolateral staining of CA XII was seen in a subpopulation of cells in both cortical and medullary collecting ducts. Double immunofluorescence staining identified these cells as H(+)-secreting type A intercalated cells. The localization of CA XII in the peritubular space of proximal tubules suggests that it may play a role in renal HCO(3)(-) absorption, whereas the function of CA XII in the type A intercalated cells needs further investigation.

New strategies for enzyme replacement therapy for lysosomal storage diseases.

Enzyme replacement therapy is an established means of treating lysosomal storage diseases. Infused enzymes are normally targeted to the lysosomes of affected cells by interactions with cell-surface receptors that recognize carbohydrate moieties such as mannose and mannose 6-phosphate on the enzymes. Therefore, we have investigated alternative strategies to deliver the lysosomal enzyme beta-glucuronidase in the enzyme-deficient mucopolysaccharidosis type VII mouse model. Here we summarize our recent efforts to use nontraditional ways to deliver beta-glucuronidase. First, we used a chimeric protein of the insulin-like growth factor II (IGF-II) fused to beta-glucuronidase to deliver enzyme via the IGF-II binding site on the bifunctional IGF-II/mannose 6-phosphate receptor. Second, we used the 11-amino-acid human immunodeficiency virus (HIV) Tat domain fused to beta-glucuronidase to mediate uptake by absorptive endocytosis. Interaction with heparan sulfate on the cell surface internalizes and delivers the Tat-tagged enzyme to the lysosome via plasma membrane recycling. Third, we created a chimeric beta-glucuronidase fused to the Fc portion of human immunoglobulin G (IgG) Fc, which was transported by the neonatal Fc receptor from the maternal circulation across the placenta to sites of storage in fetal tissues. Finally, periodate treatment was used to eliminate interaction with carbohydrate receptors, creating an enzyme with increased plasma half-life, resulting in transport across the blood-brain barrier and clearance of storage in neurons. These strategies for delivering lysosomal enzymes could also be used to target nonlysosomal proteins or enzymes identified for bioremediation of other conditions.

Epinephrine enhances lysosomal enzyme delivery across the blood brain barrier by up-regulation of the mannose 6-phosphate receptor.

Delivering therapeutic levels of lysosomal enzymes across the blood-brain barrier (BBB) has been a pivotal issue in treating CNS storage diseases, including the mucopolysaccharidoses. An inherited deficiency of beta-glucuronidase (GUS) causes mucopolysaccharidosis type VII that is characterized by increased systemic and CNS storage of glycosaminoglycans. We previously showed that the neonate uses the mannose 6-phosphate (M6P) receptor to transport phosphorylated GUS (P-GUS) across the BBB and that this transporter is lost with maturation. Induction of expression of this BBB transporter would make enzyme replacement therapy in the adult possible. Here, we tested pharmacological manipulation with epinephrine to restore functional transport of P-GUS across the adult BBB. Epinephrine (40 nmol) coinjected i.v. with (131)I-P-GUS induced the transport across the BBB in 8-week-old mice. The brain influx rate of (131)I-P-GUS (0.29 mul/g per min) returned to the level seen in neonates. Capillary depletion showed that 49% of the (131)I-P-GUS in brain was in brain parenchyma. No increases of influx rate or the vascular space for (125)I-albumin, a vascular marker, was observed with epinephrine (40 nmol), showing that enhanced passage was not caused by disruption of the BBB. Brain uptake of (131)I-P-GUS was significantly inhibited by M6P in a dose-dependent manner, whereas epinephrine failed to increase brain uptake of nonphosphorylated GUS. Thus, the effect of epinephrine on the transport of (131)I-P-GUS was ligand specific. These results indicate that epinephrine restores the M6P receptor-mediated functional transport of (131)I-P-GUS across the BBB in adults to levels seen in the neonate.

Characterization and pharmacokinetic study of recombinant human N-acetylgalactosamine-6-sulfate sulfatase.

Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by a deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS). The aims of this study were to establish Chinese hamster ovary (CHO) cells overexpressing recombinant human GALNS (rhGALNS) and to assess pharmacokinetics and tissue distribution of purified enzymes by using MPS IVA knock-out mouse (Galns(-/-)). The CHO-cell derived rhGALNS was purified from the media by a two-step affinity chromatography procedure. The rhGALNS was administered intravenously to 3-month-old Galns(-/-) mice at a single dose of 250U/g of body weight. The treated mice were examined by assaying the GALNS activity at baseline and up to 240min to assess clearance of the enzyme from blood circulation. The mice were sacrificed 4h after infusion of the enzyme to study the enzyme distribution in tissues. The rhGALNS was purified 1317-fold with 71% yield. The enzyme was taken up by Galns(-/-) chondrocytes (150U/mg/15h). The uptake was inhibited by mannose-6-phosphate. The enzyme activity disappeared from circulation with a half-life of 2.9min. After enzyme infusion, the enzyme was taken up and detected in multiple tissues (40.7% of total infused enzymes in liver). Twenty-four hours after a single infusion of the fluorescence-labeled enzymes into MPS IVA mice, biodistribution pattern showed the amount of tagged enzyme retained in bone, bone marrow, liver, spleen, kidney, and heart. In conclusion, we have shown that the phosphorylated rhGALNS is delivered to multiple tissues, including bone, and that it functions bioactively in Galns(-/-) chondrocytes implying a potential enzyme replacement treatment.

Enzyme therapy in mannose receptor-null mucopolysaccharidosis VII mice defines roles for the mannose 6-phosphate and mannose receptors.

Enzyme replacement therapy (ERT) is available for several lysosomal storage diseases. Except for Gaucher disease, for which an enzyme with exposed mannosyl residues targets mannose receptors (MR) on macrophages, ERT targets primarily the mannose 6-phosphate receptor (MPR). Most recombinant lysosomal enzymes contain oligosaccharides with both terminal mannosyl and mannose 6-phosphate residues. Effective MPR-mediated delivery may be compromised by rapid clearance of infused enzyme by the MR on fixed tissue macrophages, especially Kupffer cells. To evaluate the impact of this obstacle to ERT, we introduced the MR-null mutation onto the mucopolysaccharidosis type VII (MPS VII) background and produced doubly deficient MR-/- MPS VII mice. The availability of both MR+/+ and MR-/- mice allowed us to study the effects of eliminating the MR on MR- and MPR-mediated plasma clearance and tissue distribution of infused phosphorylated (P) and nonphosphorylated (NP) forms of human beta-glucuronidase (GUS). In MR+/+ MPS VII mice, the MR clearance system predominated at doses up to 6.4 mg/kg P-GUS. Genetically eliminating the MR slowed plasma clearance of both P- and NP-GUS and enhanced the effectiveness of P-GUS in clearing storage in kidney, bone, and retina. Saturating the MR clearance system by high doses of enzyme also improved targeting to MPR-containing tissues such as muscle, kidney, heart, and hepatocytes. Although ablating the MR clearance system genetically is not practical clinically, blocking the MR-mediated clearance system with high doses of enzyme is feasible. This approach delivers a larger fraction of enzyme to MPR-expressing tissues, thus enhancing the effectiveness of MPR-targeted ERT.

Enhancement of drug delivery to bone: characterization of human tissue-nonspecific alkaline phosphatase tagged with an acidic oligopeptide.

Hypophosphatasia is caused by deficiency of activity of the tissue-nonspecific alkaline phosphatase (TNSALP), resulting in a defect of bone mineralization. Enzyme replacement therapy (ERT) with partially purified plasma enzyme was attempted but with little clinical improvement. Attaining clinical effectiveness with ERT for hypophosphatasia may require delivering functional TNSALP enzyme to bone. We tagged the C-terminal-anchorless TNSALP enzyme with an acidic oligopeptide (a six or eight residue stretch of L-Asp), and compared the biochemical properties of the purified tagged and untagged enzymes derived from Chinese hamster ovary cell lines. The specific activities of the purified enzymes tagged with the acidic oligopeptide were the same as the untagged enzyme. In vitro affinity experiments showed the tagged enzymes had 30-fold higher affinity for hydroxyapatite than the untagged enzyme. Lectin affinity chromatography for carbohydrate structure showed little difference among the three enzymes. Biodistribution pattern from single infusion of the fluorescence-labeled enzymes into mice showed delayed clearance from the plasma up to 18 h post infusion and the amount of tagged enzyme retained in bone was 4-fold greater than that of the untagged enzyme. In vitro mineralization assays with the bone marrow from a hypophosphatasia patient using each of the three enzymes in the presence of high concentrations of pyrophosphate provided evidence of bone mineralization. These results show the anchorless enzymes tagged with an acidic oligopeptide are delivered efficiently to bone and function bioactively in bone mineralization, at least in vitro. They suggest potential advantages for use of these tagged enzymes in ERT for hypophosphatasia, which should be explored.

Overcoming the blood-brain barrier with high-dose enzyme replacement therapy in murine mucopolysaccharidosis VII.

Enzyme replacement therapy (ERT) effectively reverses storage in several lysosomal storage diseases. However, improvement in brain is limited by the blood-brain barrier except in the newborn period. In this study, we asked whether this barrier could be overcome by higher doses of enzyme than are used in conventional trials. We measured the distribution of recombinant human beta-glucuronidase (hGUS) and reduction in storage by weekly doses of 0.3-40 mg/kg administered i.v. over 1-13 weeks to mucopolysaccharidosis type VII mice immunotolerant to recombinant hGUS. Mice given up to 5 mg/kg enzyme weekly over 3 weeks had moderate reduction in meningeal storage but no change in neo-cortical neurons. Mice given 20-40 mg/kg three times over 1 week showed no reduction in storage in any area of the CNS except the meninges. In contrast, mice receiving 4 mg/kg per week for 13 weeks showed clearance not only in meninges but also in parietal neocortical and hippocampal neurons and glia. Mice given 20 mg/kg once weekly for 4 weeks also had decreased neuronal, glial, and meningeal storage and averaged 2.5% of wild-type hGUS activity in brain. These results indicate that therapeutic enzyme can be delivered across the blood-brain barrier in the adult mucopolysaccharidosis type VII mouse if administered at higher doses than are used in conventional ERT trials and if the larger dose of enzyme is administered over a sufficient period. These results may have important implications for ERT for lysosomal storage diseases with CNS involvement.