High levels of human glucocerebrosidase activity in macrophages of long-term reconstituted mice after retroviral infection of hematopoietic stem cells.
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Gaucher disease is a leading candidate for somatic gene therapy using bone marrow (BM) cells as target tissue. Towards this end, we have constructed a retroviral vector (LG) in which the human glucocerebrosidase (GC) cDNA is driven by the Moloney murine leukemia virus (MoMLV) long terminal repeat (LTR). Day 12 to 14 colony-forming unit-spleen progenitor cells were infected by the LG virus with a 100% efficiency, and GC messenger RNA (mRNA) and protein were detected in the progeny of these cells. Tissues from long-term reconstituted mice analyzed 8 months posttransplantation with LG-infected BM contained the intact provirus at greater than 1 copy per cell, indicating effective infection of hematopoietic stem cells. Human GC mRNA generated by the viral LTR was detected in macrophages as well as other hematopoietic cells. Enzyme activity was increased fivefold and twofold in macrophages from BM and spleen, respectively, and could be precipitated with an antibody specific for human GC. Immunohistochemical analysis detected the human GC protein in 81% of the macrophages from five recipient mice. These data indicate that, after transduction of hematopoietic stem cells, the LG vector is capable of directing expression of human GC in the majority of macrophages from long-term reconstituted mice and producing enzyme levels comparable with endogenous mouse activity, suggesting that this virus may be useful in the treatment of Gaucher disease. (+info)
Analyses of variant acid beta-glucosidases: effects of Gaucher disease mutations.
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Acid beta-glucosidase (GCase) is a 497-amino acid, membrane-associated lysosomal exo-beta-glucosidase whose defective activity leads to the Gaucher disease phenotypes. To move toward a structure/function map for disease mutations, 52 selected single amino acid substitutions were introduced into GCase, expressed in an insect cell system, purified, and characterized for basic kinetic, stability, and activator response properties. The variant GCases from Gaucher disease patients and selected variant GCases from the mouse had decreased relative k(cat) and differential effects on active site binding and/or attachment of mechanism-based covalent (conduritol B epoxide) or reversible (deoxynojirimycin derivatives) inhibitors. A defect in negatively charged phospholipid activation was present in the majority of variant GCases but was increased in two, N370S and V394L. Deficits in saposin C enhancement of k(cat) were present in variant GCases involving residues 48-122, whereas approximately 2-fold increases were obtained with the L264I GCase. About 50% of variant GCases each had wild-type or increased sensitivity to in vitro cathepsin D digestion. Mapping of these properties onto the crystal structures of GCase indicated wide dispersion of functional properties that can affect catalytic function and stability. Site-directed mutagenesis of cysteine residues showed that the disulfide bonds, Cys(4)-Cys(16) and Cys(18)-Cys(23), and a free Cys(342) were essential for activity; the free Cys(126) and Cys(248) were not. Relative k(cat) was highly sensitive to a His substitution at Arg(496) but not at Arg(495). These studies and high phylogenetic conservation indicate localized and general structural effects of Gaucher disease mutations that were not obvious from the nature of the amino acid substitution, including those predicted to be nondisruptive (e.g. Val --> Leu). These results provide initial studies for the engineering of variant GCases and, potentially, molecular chaperones for therapeutic use. (+info)
Gaucher disease-associated glucocerebrosidases show mutation-dependent chemical chaperoning profiles.
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Gaucher disease is a lysosomal storage disorder caused by deficient glucocerebrosidase activity. We have previously shown that the cellular activity of the most common Gaucher disease-associated glucocerebrosidase variant, N370S, is increased when patient-derived cells are cultured with the chemical chaperone N-nonyl-deoxynojirimycin. Chemical chaperones stabilize proteins against misfolding, enabling their trafficking from the endoplasmic reticulum. Herein, the generality of this therapeutic strategy is evaluated with other glucocerebrosidase variants and with additional candidate chemical chaperones. Improved chemical chaperones are identified for N370S glucocerebrosidase. Moreover, we demonstrate that G202R, a glucocerebrosidase variant that is known to be retained in the endoplasmic reticulum, is also amenable to chemical chaperoning. The L444P variant is not chaperoned by any of the active site-directed molecules tested, likely because this mutation destabilizes a domain distinct from the catalytic domain. (+info)
Topical liver x receptor activators accelerate postnatal acidification of stratum corneum and improve function in the neonate.
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In neonatal rat stratum corneum (SC), pH declines from pH 6.8 at birth to adult levels (pH 5.0-5.5) over 5-6 d. Liver X receptor (LXR) activators stimulate keratinocyte differentiation, improve permeability barrier homeostasis, and accelerate the in utero development of the SC. In this manuscript we determined the effect of LXR activators on SC acidification in the neonatal period and whether these activators correct the functional abnormalities in permeability barrier homeostasis and SC integrity/cohesion. Formation of the acid SC-buffer system was accelerated by topically applying the LXR activator, 22(R)-hydroxycholesterol, and non-oxysterol activators of LXR, TO-901317, and GW-3965. A sterol which does not activate LXR had no effect. LXR activation increased secretory phospholipase A(2) (sPLA(2)) activity and conversely, inhibition of sPLA(2) activity prevented the LXR induced increase in SC acidification, suggesting that increasing sPLA(2) accounts in part, for the LXR stimulation of acidification. LXR activation resulted in an improvement in permeability barrier homeostasis, associated with an increased maturation of lamellar membranes attributable to an increased beta-glucocerebrosidase activity. SC integrity cohesion also normalized in LXR-activator-treated animals and was associated with an increase in corneodesmosomes and in desmoglein 1 expression. These results demonstrate that LXR activators stimulate the formation of an acidic SC and improve both permeability barrier homeostasis and SC integrity/cohesion. (+info)
Short-term effect of miglustat in every day clinical use in treatment-naive or previously treated patients with type 1 Gaucher's disease.
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In a prospective, open-label study, 25 patients with mild-to-moderate type 1 Gaucher's disease (GD1) were treated with miglustat (Zavesca), an oral glucosylceramide synthase inhibitor, over 12 months. Of the 25 patients, 10 were therapy-naive and 15 had previously received enzyme replacement therapy (ERT). Clinical status, blood parameters, biomarkers, and organomegaly were assessed at baseline at 6 months and at 12 months. At 6 months the previously untreated patients showed a mean increase in hemoglobin of 0.77 g/dL, platelet counts improved or remaining stable, chitotriosidase and CCL18 decreased. These results were similar to those observed in 40 Spanish GD1 patients on ERT. Bone marrow infiltration cleared at 12 months. In the previously treated group, clinical and hematologic parameters and biomarkers were maintained/ improved at 12 months. Miglustat was well tolerated. The efficacy of miglustat treatment after 6 months was comparable to that of ERT. (+info)
Reexamination of the cysteine residues in glucocerebrosidase.
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Glucocerebrosidase, the deficient enzyme in Gaucher disease, catalyzes the cleavage of the beta-glycosidic linkage of glucosylceramide. A previous study on the enzyme identified three disulfide bridges and a single sulfhydryl [Lee, Y., Kinoshita, H., Radke, G., Weiler, S., Barranger, J.A. and Tomich, J.M. (1995) Position of the sulfhydryl group and the disulfide bonds of human glucocerebrosidase. J. Protein Chem. 14(3), 127-137] but recent publication of the X-ray structure identifies only two disulfide bridges with three free sulfhydryls [Dvir, H., Harel, M., McCarthy, A.A., Toker, L., Silman, I., Futerman, A.H. and Sussman, J.L. (2003) X-ray structure of human acid-beta-glucosidase, the defective enzyme in Gaucher disease. EMBO. 4(7), 704-709]. Using chemical modifications, acid cleavage and enzymatic digestion methods, we report that three free sulfhydryls exist and that the remaining four cysteines form two disulfide bonds located within the first 25 amino-terminal residues, supporting the X-ray structure. (+info)
The clinical effectiveness and cost-effectiveness of enzyme replacement therapy for Gaucher's disease: a systematic review.
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OBJECTIVES: The aim of this review is to determine the clinical effectiveness and cost-effectiveness of enzyme replacement therapy (ERT) in the treatment of symptomatic Gaucher's disease. DATA SOURCES: Major electronic databases were searched from their inception to August 2003; and updated from January 2003 to July/August 2004. REVIEW METHODS: Databases were searched for studies that met the criteria and selected data were extracted and evaluated. Studies were assessed for their relevance to the UK context and the review objective. The bibliographic databases were also searched to identify existing cost studies, economic evaluations and models. A Markov decision model was constructed based on patients moving between states defined by the modified Severity Score Index (SSI). Most of the parameters were derived from the published literature. ERT was assumed to restore patients to full health in the base case. RESULTS: Sixty-three studies were included, all suggestive of benefit with ERT. However, the way in which the effects translate into patient well-being and survival or the need for services and resources has not been reliably estimated. Quality of life improvements with ERT have been reported. Nonetheless, studies based on the Short Form 36 (SF-36) indicate that patients treated with ERT continue to have reduced health-related quality of life (HRQoL) compared with the general population. No study attached utility values to quality of life measures for ERT-treated patients. Thirty-one studies relevant to the natural history of the disease were found. Sixteen looked at multiple clinical characteristics of a cohort of patients with type I Gaucher's disease. There was considerable within-study and between-study heterogeneity, but all showed that Gaucher's disease was a progressive condition. Some suggested that the disease may become more indolent in adulthood; however, studies were discrepant on this point. Most disease is diagnosed in adulthood, although about one-quarter presented in childhood, these patients having the most severe symptoms and greatest rate of progression. Modelling of natural history was undertaken using the five papers that reported the SSI for each patient, along with patient-level data on age, age at diagnosis, splenectomy status and genotype, to address the question of whether disease stabilises in adulthood and the degree of correlation between phenotype and genotype. Analysis of the available data suggested that disease progression is likely to slow markedly in adulthood and that genotype is a useful predictor of clinical expression of the disease. Five studies looked at quality of life. Data on this topic were also obtained from the registries. The evidence suggests that the vast majority of the clinical characteristics of type I Gaucher's disease have little impact on subjective HRQoL and that therefore for the majority of people with type I Gaucher's disease this may not be a severe condition. Bone and skeletal symptoms contribute most to the morbidity of the disease and can lead to severe pain and immobility. The mean cost per patient treated was approximately pounds sterling 86,000 per annum in England and Wales. The cost per patient varied considerably by dose. Four existing economic evaluations were found, all of which calculated a very high cost per quality-adjusted life-year (QALY). Using the Markov decision model, ERT was assumed to restore patients to full health in the base case. The estimated incremental cost per QALY [incremental cost-effectiveness ratio (ICER)] in the base case ranged from pounds sterling 380,000 to pounds sterling 476,000 per QALY, depending on genotype. Univariate sensitivity analyses examined ERT not restoring full health, more severe disease progression in the untreated cohort, and only treating the most severely affected patients. These produced ICERs of approximately pounds sterling 1.4 million, pounds sterling 296,000 and pounds sterling 275,000 per QALY, respectively. The base-case unit cost of the drug is pounds sterling 2.975. The unit cost would have had to be reduced ten-fold, to pounds sterling 0.30, to obtain an ICER of pounds sterling 30,000 per QALY. At a unit cost of pounds sterling 1 the ICER would be pounds sterling 120,000 per QALY. CONCLUSIONS: Although ERT for treating the 'average' Gaucher's disease patient exceeds the normal upper threshold for cost-effectiveness seen in NHS policy decisions by over ten-fold, some argue that since orphan drug legislation encouraged the manufacture of Cerezyme, and Gaucher's disease can be defined as an orphan disease, the NHS has little option but to provide it, despite its great expense. More information is required before the generalisability of the findings can be determined. Although data from the UK have been used wherever possible, these were very thin indeed. Nonetheless, even large errors in estimates of the distribution of genotype, genotype--phenotype associations, effectiveness and numbers of patients will not reduce the ICER to anywhere near the upper level of treatments usually considered cost-effective. Further research could help to clarify the many uncertainties that exist. However, although doing so will be of clinical interest, it is questionable whether, within the current pricing environment, such research would have any substantive impact on policy decisions. It is highly improbable that, whatever the findings of such research, the ICER could be brought down by the orders of magnitude required to make ERT an efficient use of health service resources. (The possible exception to this would be investigating the most efficient alternative treatment strategies for using ERT in a paediatric population only.) Moreover, if under equity considerations for orphan diseases the NHS feels it is important to provide this drug, regardless of its cost-effectiveness, then refining the precision of the ICER estimate also becomes superfluous. (+info)
Conditional expression of human acid beta-glucosidase improves the visceral phenotype in a Gaucher disease mouse model.
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The reversibility and regression of histological and biochemical findings in a mouse model of Gaucher disease (4L/PS-NA) was evaluated using a liver-enriched activator protein promoter control of a tetracycline-controlled transcriptional activation-responsive human acid beta-glucosidase (hGCase) transgenic system. 4L/PS-NA has the acid beta-glucosidase (GCase) V394L/V394L (4L) point mutation combined with hypomorphic ( approximately 6% wild-type) expression of the mouse prosaposin transgene (PS-NA). The hGCase/4L/PS-NA had exclusive liver expression of hGCase controlled by doxycycline (DOX). In the absence of DOX, hGCase was secreted from liver at levels of approximately 120 microg/ml serum with only approximately 8% of full activity, following exposure to pH 7.4 in serum. The hGCase activity and protein were detected in cells of the liver (massive), lung, and spleen, but not the brain. The visceral tissue storage cells and glucosylceramide (GC) accumulation in hGCase/4L/PS-NA were decreased from that in 4L/PS-NA mice. Turning off hGCase expression with dietary DOX led to reaccumulation of storage cells and of GC in liver, lung, and spleen, and macrophage activation in those tissues. This study demonstrates that conditionally expressed hGCase supplemented the existing mutant mouse GCase to control visceral substrate accumulation in vivo. (+info)