Efficient Uptake of Recombinant α-Galactosidase A Produced with a Gene-Manipulated Yeast by Fabry Mice Kidneys

Department of Analytical Biochemistry, Meiji Pharmaceutical University, Tokyo, Japan.
Molecular Medicine (Impact Factor: 4.51). 05/2012; 18(1):76-82. DOI: 10.2119/molmed.2011.00248
Source: PubMed


To economically produce recombinant human α-galactosidase A (GLA) with a cell culture system that does not require bovine serum, we chose methylotrophic yeast cells with the OCH1 gene, which encodes α-1,6-mannosyltransferase, deleted and over-expressing the Mnn4p (MNN4) gene, which encodes a positive regulator of mannosylphosphate transferase, as a host cell line. The enzyme (yr-hGLA) produced with the gene-manipulated yeast cells has almost the same enzymological parameters as those of the recombinant human GLA produced with cultured human fibroblasts (agalsidase alfa), which is currently used for enzyme replacement therapy for Fabry disease. However, the basic structures of their sugar chains are quite different. yr-hGLA has a high content of phosphorylated N-glycans and is well incorporated into the kidneys, the main target organ in Fabry disease, where it cleaves the accumulated glycosphingolipids. A glycoprotein production system involving this gene-manipulated yeast cell line will be useful for the development of a new enzyme replacement therapy for Fabry disease.

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Available from: Tadayasu Togawa, Oct 05, 2015
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    • "Under plasma (37 °C) conditions PRX-102, agalsidase beta and agalsidase alfa were spiked into human plasma to a final concentration of 1 μg/mL, and incubated at 37 °C for 0, 15, 30, 45, and 60 min. Following the exposure to plasma, the samples were analyzed for residual enzymatic activity (compared to t = 0), utilizing 4-methylumbelliferyl-α-D-galactopyranoside (4- MU-Gal, Sigma M-7633) as an artificial substrate for α-Gal-A [26] [27] [28]. "
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    ABSTRACT: Fabry disease is an X-linked recessive disorder caused by the loss of function of the lysosomal enzyme α-Galactosidase-A. Although two enzyme replacement therapies (ERTs) are commercially available, they may not effectively reverse some of the Fabry pathology. PRX-102 is a novel enzyme for the therapy of Fabry disease expressed in a BY2 Tobacco cell culture. PRX-102 is chemically modified, resulting in a cross-linked homo-dimer. We have characterized the in-vitro and in-vivo properties of PRX-102 and compared the results with the two commercially produced α-Galactosidase-A enzymes. Results show that PRX-102 has prolonged in-vitro stability in plasma, after 1h incubation it retains 30% activity compared with complete inactivation of the commercial enzymes. Under lysosomal-like conditions PRX-102 maintains over 80% activity following 10days of incubation, while commercial enzymes become inactive after 2days. Pharmacokinetic profile of PRX-102 measured in male Fabry mice shows a 10 fold increase in t1/2 in mice (581min) compared to approved drugs. The enzyme has significantly different kinetic parameters to the alternative ERTs available (p-value<0.05, one way ANOVA), although these differences do not indicate any significant biochemical variations. PRX-102 is uptaken to primary human Fabry fibroblasts. The repeat administration of the enzyme to Fabry mice caused significant reduction (p-value<0.05) of Gb3 in various tissues (the measured residual content was 64% in kidney, liver was cleaned, 23% in heart, 5.7% in skin and 16.2% in spleen). PRX-102 has a relatively simple glycosylation pattern, characteristic to plants, having mainly tri-mannose structures with the addition of either α(1-3)-linked fucose or β(1-2)-linked xylose, or both, in addition to various high mannose structures, while agalsidase beta has a mixture of sialylated glycans in addition to high mannose structures. This study concludes that PRX-102 is equivalent in functionality to the current ERTs available, with superior stability and prolonged circulatory half-life. Therefore we propose that PRX-102 is a promising alternative for treatment of Fabry disease.
    Molecular Genetics and Metabolism 08/2014; 114(2). DOI:10.1016/j.ymgme.2014.08.002 · 2.63 Impact Factor
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    ABSTRACT: The prevalence rate for Fabry disease is conventionally considered to be 1 case in 40,000; however, due to increased screening accuracy, reports now suggest that prevalence is 1 case in 1,500 among male children, and it is likely that the clinical importance of the condition will increase in the future. In dialysis patients to date, prevalence rates are between 0.16 and 1.2 %. Globotriaosylsphingosine (Lyso-GL-3), which is a substrate of α-galactosidase A (α-Gal A), has surfaced as a new biomarker, and is also effective in the determination and monitoring of the effects of enzyme replacement therapy. In terms of genetic abnormalities, the E66Q mutation has recently become a topic of discussion, and although doubts have been expressed over whether or not it is the gene responsible for Fabry disease, there is still a strong possibility that it is a functional genetic polymorphism. At present, the standard treatment for Fabry disease is enzyme replacement therapy, and in order to overcome the problems involved with this, a method of producing recombinant human α-Gal A using methanol-assimilating yeast, and chemical or medicinal chaperone treatment are of current interest. Migalastat hydrochloride is known as a pharmacological chaperone, but is currently in Phase III global clinical trials. Adding saposin B to modified α-N-acetyl galactosaminidase is also under consideration as a treatment method.
    Clinical and Experimental Nephrology 11/2013; 18(2). DOI:10.1007/s10157-013-0897-2 · 2.02 Impact Factor
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    ABSTRACT: Fabry disease is an X-linked lysosomal storage disorder with an estimated incidence of 1 in 40 000 males. In 1973, Johnson and Brady showed that the infusion of purified alpha-galactosidase A (aGalA), derived from human placenta, rapidly cleared globotriaosylceramide (GL-3) from systemic circulation in patients with Fabry disease, however, technical complications prevented further development of enzyme replacement therapy (ERT) for Fabry disease until 2001 when recombinant aGalA became available for therapeutic use. ERT has limited effect in patients with cerebrovascular involvement, as the recombinant enzymes cannot penetrate the blood–brain barrier. Clinically, the development of an immune response is anticipated in a number of patients treated with any recombinant human proteins and is suggested to be more common, especially when the native protein is deficient or absent as is the case with many male patients with Fabry disease.
    Enzyme Technologies, 12/2013: pages 321-334; , ISBN: 9780470286265
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