Development of enzyme replacement therapy for lysosomal diseases using yeast expression system.

使用酵母表达系统开发溶酶体疾病的酶替代疗法。

• 批准号: 15591149
• 负责人: SAKURABA Hitoshi
• 金额: $ 2.24万
• 依托单位: Tokyo Metropolitan Organization for Medical Research
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15591149/
• 关键词: lysosomal disease; Fabry disease; α-galactosidase; enxyme replacement therapy; yeast; マンノース-6-リン酸; アルファ-ガラクトシダーゼ; 疾患モデルマウス; 構造; ポンペ病; α-グルコシダーゼ

Efforts have been made to develop therapies for lysosomal diseases including Fabry disease (α-galactosidase deficiency). We have produced a recombinant α-galactosidase with engineered N-linked sugar chains facilitating uptake and transport to lysosomes in a Saccharomyces cerevisiae mutant. We improved the production and purification procedures, allowing us to obtain a large amount of highly purified enzyme protein with mannose-6-phosphate residues at the non-reducing ends of sugar chains. The products were incorporated into cultured fibroblasts derived from a patient with Fabry disease via mannose-6-phosphate receptors. The ceramide trihexoside (CTH) accumulated in lysosomes was cleaved dose-dependently, and the disappearance of deposited CTH was maintained for at least 7 days after administration. We next examined the effect of the recombinant α-galactosidase on Fabry mice. Repeated intravascular administration of the enzyme led to successful degradation of CTH accumulated in the liver, kidneys, heart, and spleen. As the culture of yeast cells is easy and economical, and does not require fetal calf serum, the recombinant α-galactosidase produced in yeast cells is highly promising as an enzyme source for enzyme replacement therapy in Fabry disease.

已经努力开发包括法布里病(α-半乳糖苷酶缺乏症)在内的溶酶体疾病的治疗方法。我们已经在酿酒酵母突变株中生产了重组的α-半乳糖苷酶，该酶具有N-连接的糖链，便于摄取和转运到溶酶体。我们改进了生产和纯化工艺，获得了大量高纯度的酶蛋白，其中甘露糖-6-磷酸残基位于糖链的非还原末端。这些产物通过甘露糖-6-磷酸受体被整合到来自一名法布里病患者的培养成纤维细胞中。溶酶体中积累的神经酰胺三己糖苷(CTH)呈剂量依赖性地被切割，沉积的CTH在给药后至少7天内消失。接下来，我们研究了重组α-半乳糖苷酶对法布里小鼠的影响。反复血管内给药可成功降解积聚在肝脏、肾脏、心脏和脾中的CTH。由于酵母细胞的培养简单、经济，且不需要胎牛血清，在酵母细胞中生产的重组α-半乳糖苷酶作为酶替代治疗Fabry病的酶来源是非常有前途的。

项目成果

Three dimensional structural studies of α-N-acetylgalactosaminidase (α-NAGA) in α-NAGA deficiency (Kanzaki desease) : Different gene mutations cause peculiar structural changes in α-NAGAs resulting in different substrate specificities and clinical phenoty

α-N-乙酰半乳糖胺酶（α-NAGA）在 α-NAGA 缺乏症（神崎病）中的三维结构研究：不同的基因突变导致 α-NAGA 发生特殊的结构变化，从而导致不同的底物特异性和临床表型

• 发表时间: 2005
• 期刊: J.Dermatol.Sci. 37
• 作者: Kanekura;T.;et al.
• 通讯作者: et al.

Clinical, biochemical, and cytochemical studies on a Japanese Salla disease case associated with a renal disorder.

对与肾脏疾病相关的日本 Salla 病病例进行临床、生化和细胞化学研究。

• 发表时间: 2004
• 期刊: J.Hum.Genet 49
• 作者: Ishiwari;K. et al.
• 通讯作者: K. et al.

Matsuzawa, F., et al.: "Structural basis of the GM2 gangliosidosis B variant."J.Hum.Genet.. 48. 582-589 (2003)

Matsuzawa, F., et al.：“GM2 神经节苷脂沉积症 B 变体的结构基础。”J.Hum.Genet.. 48. 582-589 (2003)

Sakuraba, H., et al.: "Structural and immunocytochemical studies on α-N-acetylgalactosaminidase deficiency (Schindler/Kanzaki disease)."J.Hum.Genet.. 49. 1-8 (2004)

Sakuraba, H., et al.：“α-N-乙酰氨基半乳糖苷酶缺乏症（Schindler/Kanzaki 病）的结构和免疫细胞化学研究。J.Hum.Genet.. 49. 1-8 (2004)”

An asymptomatic heterozygous female with Fabry disease.

患有法布里病的无症状杂合女性。

• 发表时间: 2005
• 期刊: J.Nippon Med.Sch. 72
• 作者: Inagaki S;et al.
• 通讯作者: et al.