Tomatsu Shunji, Montaño Adriana M, Gutierrez Monica, Grubb Jeffrey H, Oikawa Hirotaka, Dung Vu Chi, Ohashi Amiko, Nishioka Tatsuo, Yamada Masamichi, Yamada Mana, Tosaka Yasuhiro, Trandafirescu Georgeta G, Orii Tadao
Department of Pediatrics, Saint Louis University, Pediatric Research Institute, St Louis, MO 63110-2586, USA.
Mol Genet Metab. 2007 May;91(1):69-78. doi: 10.1016/j.ymgme.2007.01.004. Epub 2007 Mar 2.
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.
IVA型黏多糖贮积症（MPS IVA）是一种常染色体隐性疾病，由N-乙酰半乳糖胺-6-硫酸酯硫酸酯酶（GALNS）缺乏引起。本研究的目的是建立过表达重组人GALNS（rhGALNS）的中国仓鼠卵巢（CHO）细胞，并使用MPS IVA基因敲除小鼠（Galns(-/-)）评估纯化酶的药代动力学和组织分布。通过两步亲和层析法从培养基中纯化CHO细胞衍生的rhGALNS。将rhGALNS以250U/g体重的单剂量静脉注射给3个月大的Galns(-/-)小鼠。在基线和长达240分钟时通过测定GALNS活性检查处理后的小鼠，以评估酶从血液循环中的清除情况。在注入酶4小时后处死小鼠，以研究酶在组织中的分布。rhGALNS纯化了1317倍，产率为71%。该酶被Galns(-/-)软骨细胞摄取（150U/mg/15小时）。摄取被6-磷酸甘露糖抑制。酶活性从循环中消失，半衰期为2.9分钟。注入酶后，该酶被多个组织摄取并检测到（肝脏中占总注入酶的40.7%）。将荧光标记的酶单次注入MPS IVA小鼠24小时后，生物分布模式显示标记酶保留在骨骼、骨髓、肝脏、脾脏、肾脏和心脏中的量。总之，我们已经表明磷酸化的rhGALNS被递送到包括骨骼在内的多个组织，并且它在Galns(-/-)软骨细胞中具有生物活性，这意味着潜在的酶替代治疗。
