BioMarin Pharmaceutical Inc., Novato, California, United States of America.
PLoS One. 2010 Aug 16;5(8):e12194. doi: 10.1371/journal.pone.0012194.
Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome) is a lysosomal storage disorder caused by deficiency of N-acetylgalactosamine-6-sulfatase (GALNS), an enzyme that degrades keratan sulfate (KS). Currently no therapy for MPS IVA is available. We produced recombinant human (rh)GALNS as a potential enzyme replacement therapy for MPS IVA. Chinese hamster ovary cells stably overexpressing GALNS and sulfatase modifying factor-1 were used to produce active ( approximately 2 U/mg) and pure (>or=97%) rhGALNS. The recombinant enzyme was phosphorylated and was dose-dependently taken up by mannose-6-phosphate receptor (K(uptake) = 2.5 nM), thereby restoring enzyme activity in MPS IVA fibroblasts. In the absence of an animal model with a skeletal phenotype, we established chondrocytes isolated from two MPS IVA patients as a disease model in vitro. MPS IVA chondrocyte GALNS activity was not detectable and the cells exhibited KS storage up to 11-fold higher than unaffected chondrocytes. MPS IVA chondrocytes internalized rhGALNS into lysosomes, resulting in normalization of enzyme activity and decrease in KS storage. rhGALNS treatment also modulated gene expression, increasing expression of chondrogenic genes Collagen II, Collagen X, Aggrecan and Sox9 and decreasing abnormal expression of Collagen I. Intravenous administration of rhGALNS resulted in biodistribution throughout all layers of the heart valve and the entire thickness of the growth plate in wild-type mice. We show that enzyme replacement therapy with recombinant human GALNS results in clearance of keratan sulfate accumulation, and that such treatment ameliorates aberrant gene expression in human chondrocytes in vitro. Penetration of the therapeutic enzyme throughout poorly vascularized, but clinically relevant tissues, including growth plate cartilage and heart valve, as well as macrophages and hepatocytes in wild-type mouse, further supports development of rhGALNS as enzyme replacement therapy for MPS IVA.
黏多糖贮积症 IVA（MPS IVA；Morquio A 综合征）是一种溶酶体贮积病，由 N-乙酰半乳糖胺-6-硫酸酯酶（GALNS）缺乏引起，该酶降解硫酸角质素（KS）。目前尚无治疗 MPS IVA 的方法。我们生产重组人（rh）GALNS 作为 MPS IVA 的潜在酶替代疗法。用稳定过表达 GALNS 和硫酸酯酶修饰因子-1 的中国仓鼠卵巢细胞来生产具有活性（~2 U/mg）和高纯度（>或=97%）的 rhGALNS。重组酶被磷酸化，通过甘露糖-6-磷酸受体（K（摄取）=2.5 nM）进行剂量依赖性摄取，从而恢复 MPS IVA 成纤维细胞中的酶活性。由于缺乏具有骨骼表型的动物模型，我们在体外建立了来自 2 名 MPS IVA 患者的软骨细胞作为疾病模型。MPS IVA 软骨细胞的 GALNS 活性无法检测到，且细胞中 KS 储存量比未受影响的软骨细胞高 11 倍。MPS IVA 软骨细胞将 rhGALNS 内吞到溶酶体中，导致酶活性正常化和 KS 储存减少。rhGALNS 治疗还调节基因表达，增加软骨形成基因 Collagen II、Collagen X、Aggrecan 和 Sox9 的表达，并减少 Collagen I 的异常表达。rhGALNS 静脉给药后，在野生型小鼠的心脏瓣膜的所有层和生长板的整个厚度中均有分布。我们表明，用重组人 GALNS 进行酶替代治疗可清除硫酸角质素的积累，并且这种治疗可改善体外人软骨细胞的异常基因表达。治疗酶在包括生长板软骨和心脏瓣膜在内的、血供较差但临床上相关的组织，以及在野生型小鼠中的巨噬细胞和肝细胞中的渗透，进一步支持 rhGALNS 作为 MPS IVA 的酶替代疗法的开发。
