Disease characteristics, effectiveness, and safety of vestronidase alfa for the treatment of patients with mucopolysaccharidosis VII in a novel, longitudinal, multicenter disease monitoring program.

BACKGROUND: Mucopolysaccharidosis VII (MPS VII) is an ultra-rare, autosomal recessive, debilitating, progressive lysosomal storage disease caused by reduced activity of ß-glucuronidase (GUS) enzyme. Vestronidase alfa (recombinant human GUS) intravenous enzyme replacement therapy is an approved treatment for patients with MPS VII. METHODS: This disease monitoring program (DMP) is an ongoing, multicenter observational study collecting standardized real-world data from patients with MPS VII (N ≈ 50 planned) treated with vestronidase alfa or any other management approach. Data are monitored and recorded in compliance with Good Clinical Practice guidelines and planned interim analyses of captured data are performed annually. Here we summarize the safety and efficacy outcomes as of 17 November 2022. RESULTS: As of the data cutoff date, 35 patients were enrolled: 28 in the Treated Group and seven in the Untreated Group. Mean (SD) age at MPS VII diagnosis was 4.5 (4.0) years (range, 0.0 to 12.4 years), and mean (SD) age at DMP enrollment was 13.9 (11.1) years (range, 1.5 to 50.2 years). Ten patients (29%) had a history of nonimmune hydrops fetalis. In the 23 patients who initiated treatment prior to DMP enrollment, substantial changes in mean excretion from initial baseline to DMP enrollment were observed for the three urinary glycosaminoglycans (uGAGs): dermatan sulfate (DS), -84%; chondroitin sulfate (CS), -55%; heparan sulfate (HS), -42%. Also in this group, mean reduction from initial baseline to months 6, 12, and 24 were maintained for uGAG DS (-84%, -87%, -89%, respectively), CS (-70%, -71%, -76%, respectively), and HS (+ 3%, -32%, and - 41%, respectively). All adverse events (AEs) were consistent with the known vestronidase alfa safety profile. No patients discontinued vestronidase alfa. One patient died. CONCLUSIONS: To date, the DMP has collected invaluable MPS VII disease characteristic data. The benefit-risk profile of vestronidase alfa remains unchanged and favorable for its use in the treatment of pediatric and adult patients with MPS VII. Reductions in DS and CS uGAG demonstrate effectiveness of vestronidase alfa to Month 24. Enrollment is ongoing.

Circulatory C-type natriuretic peptide reduces mucopolysaccharidosis-associated craniofacial hypoplasia in vivo.

Skeletal alterations in the head and neck region, such as midfacial hypoplasia, foramen magnum stenosis and spinal canal stenosis, are commonly observed in patients with mucopolysaccharidosis (MPS). However, enzyme replacement therapy (ERT), one of the major treatment approaches for MPS, shows limited efficacy for skeletal conditions. In this study, we analysed the craniofacial morphology of mice with MPS type VII, and investigated the underlying mechanisms promoting jaw deformities in these animals. Furthermore, we investigated the effects of C-type natriuretic peptide (CNP), a potent endochondral ossification promoter, on growth impairment of the craniofacial region in MPS VII mice when administered alone or in combination with ERT. MPS VII mice exhibited midfacial hypoplasia caused by impaired endochondral ossification, and histological analysis revealed increased number of swelling cells in the resting zone of the spheno-occipital synchondrosis (SOS), an important growth centre for craniomaxillofacial skeletogenesis. We crossed MPS VII mice with transgenic mice in which CNP was expressed in the liver under the control of the human serum amyloid-P component promoter, resulting in elevated levels of circulatory CNP. The maxillofacial morphological abnormalities associated with MPS VII were ameliorated by CNP expression, and further prevented by a combination of CNP and ERT. Histological analysis showed that ERT decreased the swelling cell number, and CNP treatment increased the width of the proliferative and hypertrophic zones of the SOS. Furthermore, the foramen magnum and spinal stenoses observed in MPS VII mice were significantly alleviated by CNP and ERT combination. These results demonstrate the therapeutic potential of CNP, which can be used to enhance ERT outcome for MPS VII-associated head and neck abnormalities.

Alfavestronidase no tratamento de mucopolissacaridose tipo VII / Alfavestronidase in the treatment of type VII mucopolysaccharidosis

Introdução: A mucopolissacaridose VII é um distúrbio de armazenamento lisossomal de origem genética causado pelo funcionamento inadequado da enzima beta-glicuronidase que desempenha papel essencial na degradação de glicosaminoglicanos específicos constituintes do tecido conjuntivo, especialmente sulfato de condroitina (SC), sulfato de dermatano (SD) e sulfato de heparano (SH). Do ponto de vista fisiopatológico, há um acúmulo desses substratos em tecidos e órgãos causando quadros clínicos variáveis com uma ampla gama de manifestações que envolvem anormalidades no sistema musculoesquelético, cardiovascular, respiratório e neurológico. A história natural da doença é pouco conhecida, mas os indivíduos podem apresentar deformidades esqueléticas, dificuldade de locomoção, problemas de coordenação motora global e fina, doenças valvares cardíacas e cardiomiopatias, dificuldades respiratórias, limitações cognitivas e retardo mental. PERGUNTA: "a alfavestronidase apresenta eficácia e segurança na terapia enzimática substitutiva (TES) para o tratamento de mucopolissacaridose VII (Síndrome de Sly)?". TECNOLOGIA: alfavestronidase1 (Mepsevii®). EVIDÊNCIAS CIENTÍFICAS: A terapia de reposição enzimática com alfavestronidase foi relacionada a uma diminuição rápida e sustentada de glicosaminoglicanos na urina, o que, do ponto de vista farmacodinâmico, demonstra mecanismo de ação compatível com a fisiopatologia da doença. A eficácia do tratamento foi avaliada por meio de índice de respondedor multidomínios combinando seis desfechos clínicos relevantes no contexto de tratamento da doença. A variação média no índice de respondedor clínico multidomínio foi de +0,5 (DP ±0,8) após 24 semanas (p=0,0527), com diferença estatística entre as avaliações na linha de base e após 24 semanas (interindividual). A variação média no índice de respondedor clínico multidomínio incluindo escore total de fadiga foi de +0,8 (DP ±0,94) (p=0,01) após 24 semanas. Essas variações positivas demonstram uma melhora global nos seis domínios avaliados. De forma mais específica, a eficácia da terapia de reposição enzimática se demonstra por aumento clinicamente significativo e progressivo na distância percorrida em teste de 6 metros de caminhada, melhoria da função pulmonar expressa por aumento de 21% em teste de capacidade vital forçada em um dos participantes e diminuição da frequência de uso de suporte respiratório em alguns dos participantes dos estudos. Em alguns dos participantes dos estudos observou-se melhora clinicamente significativa em escores de fadiga, com relatos de aumento de frequência escolar e de normalização de hábitos alimentares. AVALIAÇÃO ECONÔMICA: O demandante apresentou um estudo de custo por desfecho em que apresenta as razões entre as variações médias no índice de respondedor clínico multidomínio (+0,5) e no índice de respondedor clínico multidomínio incluindo escore total de fadiga de (+0,8) após 24 semanas de tratamento e o custo do tratamento com alfavestronidase. Não foram apresentados modelos econômicos, assim como não foram considerados os impactos da utilização de alfavestronidase em desfechos e nos custos de saúde considerando a história natural da mucopolissacaridose tipo VII. Assim foram obtidas razões de R$ 3.141.796,90 por unidade de benefício terapêutico ganho e de R$ 1.963.623,06 por unidade de benefício terapêutico ganho incluindo modificação em escore de fadiga. Entretanto como o demandante não apresenta modelo econômico não fica claro se essas razões representariam as de custo-efetividade incremental. CONSIDERAÇÃO FINAL: Considerando-se a característica progressiva da doença, demonstrou-se a eficácia da terapia de reposição enzimática com alfavestronidase pela melhoria clinicamente significativa e progressiva (>23 metros e 10% de incremento em relação à linha de base) registrada para alguns dos participantes dos estudos no teste de caminhada de 6 minutos, pela melhoria na função pulmonar de alguns participantes, incluindo uma melhora de 21% no teste de capacidade vital forçada e também caso de diminuição da frequência de utilização suporte respiratório após 164 semanas de tratamento. Alguns dos participantes dos estudos apresentaram uma melhoria na coordenação motora global em relação à linha de base. Do ponto de vista farmacodinâmico, a diminuição rápida e sustentada de condroitan e dermatan sulfato na urina é uma informação complementar que reforça a atividade da enzima, embora ainda seja necessário estabelecer a correlação desse desfecho bioquímico com desfechos clínicos relevantes para a doença. O medicamento é seguro e as reações anafilactóides registradas em alguns casos podem ser evitadas pelo controle do tempo de infusão e utilização de medicamentos adequados. RECOMENDAÇÃO PRELIMINAR DA CONITEC: Os membros do plenário presentes na 85ª reunião ordinária, no dia 04 de fevereiro de 2020, indicaram que o tema seja submetido à consulta pública com recomendação preliminar de não incorporação ao SUS de alfavestronidase para mucopolissacaridose tipo VII. Considerou-se que o tempo de mercado e a experiência clínica com o medicamento ainda são incipientes e o impacto orçamentário associado à terapia de reposição enzimática com alfavestronidase é relevante da ordem de 3 milhões de reais por paciente por ano. CONSULTA PÚBLICA: Por meio da consulta pública número 03 de 2020, realizada entre os dias 21/02/2020 e 17/03/2020, foram recebidas 83 contribuições, sendo 13 pelo formulário para contribuições técnico- científicas e 70 pelo formulário para contribuições sobre experiência ou opinião. Na grande maioria das contribuições declarou-se discordância com a recomendação preliminar da Conitec. A principal evidência submetida foi o estudo observacional de extensão de Harmatz e colaboradores (2020), ainda não publicado no momento da elaboração desse parecer. No estudo, em que se avalia como desfecho primário a segurança, acompanha-se grupo de 12 pacientes por 3 anos demonstrando-se que o efeito da terapia de reposição enzimática com alfavestronidase na diminuição da excreção urinária de glicosaminoglicanos é mantida durante o período de acompanhamento. Ao final do estudo havia resultados disponíveis para quatro dos doze indivíduos que iniciaram o acompanhamento. Parte dos pacientes também apresentou melhora sustentada no índice de respondedor multidomínios, utilizado na avaliação da evolução clínica da doença. Em relação ao estudo econômico reforça-se que não se submeteu estudo de custo-efetividade ou outro modelo constante nas Diretrizes Metodológicas do Ministério da Saúde, mas apenas um estuo de custo por desfecho. No tocante à análise de impacto orçamentário pleiteou-se que vários aspectos metodológicos estariam superestimados tais como peso médio dos pacientes, taxa de absorção da tecnologia pelo SUS e quantitativo de pacientes, sugerindo-se que o impacto orçamentário projetado estaria cerca de 50% superestimado. As contribuições de experiência e opinião foram convergentes com as técnicas reforçando-se que o peso médio e o quantitativo de pacientes no Brasil elegíveis para receber o tratamento estaria superestimado. RECOMENDAÇÃO FINAL DA CONITEC: A Conitec em sua 88ª reunião ordinária que ocorreu em 07 de julho de 2020 considerou haver argumentos suficientes, recebidos e avaliados após a consulta pública, para modificar a recomendação inicial desfavorável à incorporação de alfavestronidase emitindo recomendação final favorável à incorporação do medicamento. Esses argumentos dizem respeito à demonstração da segurança e efetividade em longo prazo da terapia de reposição enzimática com alfavestronidase, semelhante às já avaliadas para outras mucopolissacaridoses. Considerou também a plausibilidade da diminuição do impacto orçamentário em função de estudos e opiniões de especialistas a respeito do peso médio e número de pacientes no Brasil que seriam menores do que os inicialmente utilizados no dossiê inicial submetido pela empresa. Dessa forma, o plenário da Conitec recomendou favoravelmente a incorporação de alfavestronidase para tratamento de mucopolissacaridose tipo VII mediante avaliação da efetividade e reapresentação para a Comissão após três anos e elaboração de Protocolo Clínico e Diretrizes Terapêuticas. Foi assinado o registro de deliberação número 532. DECISÃO: incorporar a alfavestronidase para o tratamento de mucopolissacaridose tipo VII, condicionado ao monitoramento dos resultados, à reavaliação pela Conitec após três anos de uso e à elaboração de Protocolo Clínico do Ministério da Saúde, no âmbito do Sistema Único de Saúde - SUS, conforme Portaria nº 26, publicada no Diário Oficial da União nº 153, seção 1, página 40, em 11 de agosto de 2020.

The long-term safety and efficacy of vestronidase alfa, rhGUS enzyme replacement therapy, in subjects with mucopolysaccharidosis VII.

Vestronidase alfa (recombinant human beta-glucuronidase) is an enzyme replacement therapy (ERT) for Mucopolysaccharidosis (MPS) VII, a highly heterogeneous, ultra-rare disease. Twelve subjects, ages 8-25 years, completed a Phase 3, randomized, placebo-controlled, blind-start, single crossover study (UX003-CL301; NCT02377921), receiving 24-48 weeks of vestronidase alfa 4 mg/kg IV. All 12 subjects completed the blind-start study, which showed significantly reduced urinary glycosaminoglycans (GAG) and clinical improvement in a multi-domain responder index, and enrolled in a long-term, open-label, extension study (UX003-CL202; NCT02432144). Here, we report the final results of the extension study, up to an additional 144 weeks after completion of the blind-start study. Three subjects (25%) completed all 144 weeks of study, eight subjects (67%) ended study participation before Week 144 to switch to commercially available vestronidase alfa, and one subject discontinued due to non-compliance after receiving one infusion of vestronidase alfa in the extension study. The safety profile of vestronidase alfa in the extension study was consistent with observations in the preceding blind-start study, with most adverse events mild to moderate in severity. There were no treatment or study discontinuations due to AEs and no noteworthy changes in a standard safety chemistry panel. Out of the eleven subjects who tested positive for anti-drug antibodies at any time during the blind-start or extension study, including the baseline assessment in the blind-start study, seven subjects tested positive for neutralizing antibodies and all seven continued to demonstrate a reduction in urinary GAG levels. There was no association between antibody formation and infusion associated reactions. Subjects receiving continuous vestronidase alfa treatment showed a sustained urinary GAG reduction and clinical response evaluated using a multi-domain responder index that includes assessments in pulmonary function, motor function, range of motion, mobility, and visual acuity. Reduction in fatigue was also maintained in the overall population. As ERT is not expected to cross the blood brain barrier, limiting the impact on neurological signs of disease, and not all subjects presented with neurological symptoms, outcomes related to central nervous system pathology are not focused on in this report. Results from this study show the long-term safety and durability of clinical efficacy in subjects with MPS VII with long-term vestronidase alfa treatment.

Vestronidase Alfa: A Review in Mucopolysaccharidosis VII.

Mucopolysaccharidosis VII is an extremely rare, autosomal recessive lysosomal storage disorder characterized by a deficiency of ß-glucuronidase activity, resulting in partial degradation and accumulation of GAGs in numerous tissues throughout the body, with consequent cellular damage and organ dysfunction. Enzyme replacement therapy (ERT) with intravenous vestronidase alfa (Mepsevii™), a recombinant form of human ß-glucuronidase, is the first disease-specific therapy approved for the treatment of mucopolysaccharidosis VII in pediatric and adult patients. In the pivotal, blind start, phase 3 trial, 24 weeks of vestronidase alfa therapy significantly reduced urinary GAG (uGAG) excretion in patients with mucopolysaccharidosis VII. Based on a Multi-Domain Responder Index (MDRI; comprises six clinically important morbidity domains, with prespecified minimally important differences for each domain), most evaluable patients experienced an improvement in ≥ 1 domain during the 24-week primary assessment period (overall positive mean change of 0.5 domains). The clinical benefits of vestronidase alfa were sustained during longer-term treatment, as was the reduction in uGAG excretion. Vestronidase alfa has a manageable tolerability profile, with most adverse reactions of mild to moderate severity. Given the lack of treatment options and the clinical benefits it provides, intravenous vestronidase alfa is an important emerging ERT for patients with mucopolysaccharidosis VII.

Pharmacokinetic and Pharmacodynamic Modeling to Optimize the Dose of Vestronidase Alfa, an Enzyme Replacement Therapy for Treatment of Patients with Mucopolysaccharidosis Type VII: Results from Three Trials.

INTRODUCTION: Mucopolysaccharidosis type VII (MPS VII, Sly Syndrome) is a progressive, debilitating, ultra-rare lysosomal storage disorder caused by the deficiency of ß-glucuronidase (GUS), an enzyme required for breakdown of glycosaminoglycans (GAGs). Vestronidase alfa, a recombinant human GUS, is an enzyme replacement therapy approved in the US and EU for the treatment of MPS VII. METHODS: The pharmacokinetics (PK) and pharmacodynamics (PD) of vestronidase alfa were evaluated in 23 adult and pediatric subjects with MPS VII enrolled in phase I-III clinical trials to optimize the clinical dosing regimen of vestronidase alfa. The serum concentration-time profiles were adequately described by a two-compartment population PK model incorporating subjects' body weight as the only significant covariate. RESULTS: Model-based simulations predicted a substantially decreased time duration of serum exposures exceeding the level of Kuptake (the in vitro determined vestronidase alfa concentration corresponding to 50% maximum rate of cellular uptake) for 4 or 8 mg/kg once every 4 weeks dosing, compared with 4 mg/kg once every other week (QOW) dosing by intravenous infusion, suggesting that given the same total monthly dose, the QOW dosing frequency should result in more efficient delivery to the GUS-deficient tissue cells, and therefore superior treatment efficacy. A standard inhibitory maximal effect model reasonably explained the observed pharmacological PD responses of reduction in urinary GAGs from pretreatment baseline, which appeared to have reached the plateau of maximal effect at the 4 mg/kg QOW dose. CONCLUSION: The modeling results, together with the clinical evidence of safety and efficacy, supported the recommended 4 mg/kg QOW dosing regimen of vestronidase alfa for pediatric and adult patients with MPS VII. CLINICAL TRIAL REGISTRATION: NCT01856218, NCT02418455, NCT02230566.

Neuromuscular degeneration and locomotor deficit in a Drosophila model of mucopolysaccharidosis VII is attenuated by treatment with resveratrol.

Mucopolysaccharidosis VII (MPS VII) is a recessively inherited lysosomal storage disorder caused by ß-glucuronidase enzyme deficiency. The disease is characterized by widespread accumulation of non-degraded or partially degraded glycosaminoglycans, leading to cellular and multiple tissue dysfunctions. The patients exhibit diverse clinical symptoms, and eventually succumb to premature death. The only possible remedy is the recently approved enzyme replacement therapy, which is an expensive, invasive and lifelong treatment procedure. Small-molecule therapeutics for MPS VII have so far remained elusive primarily due to lack of molecular insights into the disease pathogenesis and unavailability of a suitable animal model that can be used for rapid drug screening. To address these issues, we developed a Drosophila model of MPS VII by knocking out the CG2135 gene, the fly ß-glucuronidase orthologue. The CG2135-/- fly recapitulated cardinal features of MPS VII, such as reduced lifespan, progressive motor impairment and neuropathological abnormalities. Loss of dopaminergic neurons and muscle degeneration due to extensive apoptosis was implicated as the basis of locomotor deficit in this fly. Such hitherto unknown mechanistic links have considerably advanced our understanding of the MPS VII pathophysiology and warrant leveraging this genetically tractable model for deeper enquiry about the disease progression. We were also prompted to test whether phenotypic abnormalities in the CG2135-/- fly can be attenuated by resveratrol, a natural polyphenol with potential health benefits. Indeed, resveratrol treatment significantly ameliorated neuromuscular pathology and restored normal motor function in the CG2135-/- fly. This intriguing finding merits further preclinical studies for developing an alternative therapy for MPS VII.This article has an associated First Person interview with the first author of the paper.

First human treatment with investigational rhGUS enzyme replacement therapy in an advanced stage MPS VII patient.

Mucopolysaccharidosis type VII (MPS VII, Sly syndrome) is a very rare lysosomal storage disease caused by a deficiency of the enzyme ß-glucuronidase (GUS), which is required for the degradation of three glycosaminoglycans (GAGs): dermatan sulfate, heparan sulfate, and chondroitin sulfate. Progressive accumulation of these GAGs in lysosomes leads to increasing dysfunction in numerous tissues and organs. Enzyme replacement therapy (ERT) has been used successfully for other MPS disorders, but there is no approved treatment for MPS VII. Here we describe the first human treatment with recombinant human GUS (rhGUS), an investigational therapy for MPS VII, in a 12-year old boy with advanced stage MPS VII. Despite a tracheostomy, nocturnal continuous positive airway pressure, and oxygen therapy, significant pulmonary restriction and obstruction led to oxygen dependence and end-tidal carbon dioxide (ETCO2) levels in the 60-80mmHg range, eventually approaching respiratory failure (ETCO2 of 100mmHg) and the need for full-time ventilation. Since no additional medical measures could improve his function, we implemented experimental ERT by infusing rhGUS at 2mg/kg over 4h every 2 weeks for 24 weeks. Safety was evaluated by standard assessments and observance for any infusion associated reactions (IARs). Urinary GAG (uGAG) levels, pulmonary function, oxygen dependence, CO2 levels, cardiac valve function, liver and spleen size, and growth velocity were assessed to evaluate response to therapy. rhGUS infusions were well tolerated. No serious adverse events (SAEs) or IARs were observed. After initiation of rhGUS infusions, the patient's uGAG excretion decreased by more than 50%. Liver and spleen size were reduced within 2 weeks of the first infusion and reached normal size by 24 weeks. Pulmonary function appeared to improve during the course of treatment based on reduced changes in ETCO2 after off-ventilator challenges and a reduced oxygen requirement. The patient regained the ability to eat orally, gained weight, and his energy and activity levels increased. Over 24 weeks, treatment with every-other-week infusions of rhGUS was well tolerated with no SAEs, IARs, or hypersensitivity reactions and was associated with measurable improvement in objective clinical measures and quality of life.

Mucopolysaccharidosis type VII: A powerful experimental system and therapeutic challenge.

Mucopolysaccharidosis type VII (MPSVII) is an inborn error of metabolism caused by a deficiency in the lysosomal enzyme B-glucuronidase (GUSB). As such, MPSVII is one of a larger class of inherited diseases referred to as lysosomal storage diseases (LSD). (1) The absence of GUSB activity leads to the progressive accumulation of undegraded glycosaminoglycans (GAGs) in many tissues of the body. Mucopolysaccharidosis VII has a complex clinical phenotype, including skeletal dysplasia, hepatosplenomegally, sensory deficits, cognitive impairment, and premature death. Although the natural history of the human disease is not precisely defined, small and large animal models of MPSVII have played a major role in our understanding of the disease process and towards effective treatments. The mouse model of MPSVII is a particularly powerful system due to its similarity to the human disease and the ability to generate large numbers of genetically defined animals. It has been shown in the murine model of MPSVII that recombinant enzyme replacement therapy (ERT) can ameliorate most of the clinical signs of disease if initiated during the neonatal period. Progenitor cell transplantation (hematopoietic, neuronal, mesenchymal) can correct many of the pathological signs of disease in MPSVII mice. Viral-mediated gene therapy has also been shown to decrease the severity of the disease in both the murine and canine models of MPSVII. Although pre-clinical experiments have shown that a number of approaches can effectively treat MPSVII, translation of those therapies into the clinic has lagged behind other LSDs. This is due in large part to the ultra-rare nature of MPSVII. Encouragingly, a clinical trial of ERT for MPSVII has recently been initiated. It will be interesting to determine if the positive pre-clinical data gathered in animal models of MPSVII translate to affected children. This clinical trial may also establish a paradigm for the treatment of other ultra-rare disorders.

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.

Storage correction in cells of patients suffering from mucopolysaccharidoses types IIIA and VII after treatment with genistein and other isoflavones.

Mucopolysaccharidoses are autosomal and recessive lysosomal storage disorders caused by the deficiency of a lysosomal enzyme involved in glycosaminoglycan catabolism. The Sanfilippo type A disease (MPS III A) results from sulfamidase deficiency, which leads to accumulation of heparan sulfate, whereas Sly disease (MPS VII) results from beta-glucuronidase deficiency, leading to accumulation of heparan, dermatan, and chondroitin sulfates. These syndromes are characterized by severe central nervous system degeneration, resulting in progressive mental retardation, and fatality occurs in severely affected children. To date, no effective treatment is available except for bone marrow transplantation in specific cases. Recently, the use of genistein, an isoflavone that inhibits glycosaminoglycans synthesis, has been tested as substrate reduction therapy for neuronopathic forms of these diseases.We tested five natural analogs to genistein in human fibroblasts from both Sanfilippo A and Sly patients. Four molecules were as efficient as genistein in decreasing glycosaminoglycan accumulation. Moreover, a combination of several isoflavones was more efficient than one single isoflavone, suggesting a synergistic effect. These preliminary data may offer new perspectives for treating Sly and Sanfilippo A diseases and could be relevant to other neurological forms of mucopolysaccharidoses.

Genetic engineering of IgG-glucuronidase fusion proteins.

beta-Glucuronidase (GUSB) is a lysosomal enzyme that could be developed as a brain therapy for Type VII Mucopolysaccharidosis. However, GUSB does not cross the blood-brain barrier (BBB). To enable BBB transport of the enzyme, human GUSB was re-engineered as a fusion protein with the chimeric monoclonal antibody (MAb) to the human insulin receptor (HIR). The HIRMAb crosses the BBB on the endogenous insulin receptor, and acts as a molecular Trojan horse to ferry into brain the GUSB. The 611 amino acid GUSB was fused to either the carboxyl or amino terminus of the heavy chain of the HIRMAb. This study illustrates the differential retention of functionality of IgG-enzyme fusion proteins depending on how the fusion protein is engineered.

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.

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.

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.

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.

Glycosylation-independent targeting enhances enzyme delivery to lysosomes and decreases storage in mucopolysaccharidosis type VII mice.

Enzyme-replacement therapy is an established means of treating lysosomal storage diseases. Infused therapeutic enzymes are targeted to lysosomes of affected cells by interactions with cell-surface receptors that recognize carbohydrate moieties, such as mannose and mannose 6-phosphate, on the enzymes. We have tested an alternative, peptide-based targeting system for delivery of enzymes to lysosomes in a murine mucopolysaccharidosis type VII (MPS VII) model. This strategy depends on the interaction of a fragment of insulin-like growth factor II (IGF-II), with the IGF-II binding site on the bifunctional, IGF-II cation-independent mannose 6-phosphate receptor. A chimeric protein containing a portion of mature human IGF-II fused to the C terminus of human beta-glucuronidase was taken up by MPS VII fibroblasts in a mannose 6-phosphate-independent manner, and its uptake was inhibited by the addition of IGF-II. Furthermore, the tagged enzyme was delivered effectively to clinically significant tissues in MPS VII mice and was effective in reversing the storage pathology. The tagged enzyme was able to reduce storage in glomerular podocytes and osteoblasts at a dose at which untagged enzyme was much less effective. This peptide-based, glycosylation-independent lysosomal targeting system may enhance enzyme-replacement therapy for certain human lysosomal storage diseases.

Numerous transcriptional alterations in liver persist after short-term enzyme-replacement therapy in a murine model of mucopolysaccharidosis type VII.

The lysosomal storage disease MPS VII (mucopolysaccharidosis type VII) is caused by a deficiency in beta-glucuronidase activity, and results in the accumulation of partially degraded glycosaminoglycans in many cell types. Although MPS VII is a simple monogenetic disorder, the clinical presentation is complex and incompletely understood. ERT (enzyme replacement therapy) is relatively effective at improving the clinical course of the disease; however, some pathologies persist. In order to clarify the molecular events contributing to the disease phenotype and how ERT might impact upon them, we analysed liver tissue from untreated and treated MPS VII mice at both 2 and 5 months of age using biochemical assays and microarray analysis. Overall, as the disease progresses, more genes have altered expression and, at either age, numerous transcriptional changes in multiple pathways appear to be refractory to therapy. With respect to the primary site of disease, both transcriptional and post-transcriptional mechanisms are involved in the regulation of lysosomal enzymes and other lysosome-associated proteins. Many of the changes observed in both lysosome-associated mRNAs and proteins are normalized by enzyme replacement. In addition, gene expression changes in seemingly unrelated pathways may account for the complex metabolic phenotype of the MPS VII mouse. In particular, beta-glucuronidase deficiency appears to induce physiological malnutrition in MPS VII mice. Malnutrition may account for the pronounced adipose storage deficiency observed in this animal. Studying the molecular response to lysosomal storage, especially those changes recalcitrant to therapy, has revealed additional targets that may improve the efficacy of existing therapies.

Selective neurodegeneration in murine mucopolysaccharidosis VII is progressive and reversible.

The mucopolysaccharidoses are caused by inherited deficiencies of lysosomal enzymes involved in the degradative pathway of glycosaminoglycans. Lysosomal storage leads to cellular and organ dysfunction, including mental retardation. Storage lesions are found throughout the diseased brain, but little is known about the cellular and molecular mechanisms that underlie brain dysfunction. In the mouse model of mucopolysaccharidosis VII, we found that specific regions of the brain are vulnerable to neurodegeneration, characterized by the presence of ubiquitin inclusions, neurofilament inclusions, and reactive astrogliosis. The pathological lesions were found predominantly in the hippocampus and cerebral cortex, and they increased progressively with age. Treatment with a recombinant viral vector to correct the enzymatic defect quantitatively reversed the neurodegenerative lesions in targeted regions to normal levels.