Gene Therapy for Sly disease using AAV vector.

使用 AAV 载体对 Sly 病进行基因治疗。

• 批准号: 11670790
• 负责人: OHASHI Touya
• 金额: $ 2.24万
• 依托单位: The Jikei University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670790/
• 关键词: Gene Therapy; AAV; MPS VII; Sly disease; MPS VII

We generated adeno-associate vector which express human beta-glucuronidase. Genzyme generously provided us AAV plasmid vector and helper virus. Using this system, full length human beta-glucuronidase cDNA was inserted to AAV expression plasmid, pNT C3CMV.Resultant plasmid was cotransfected with helper virus, p5rep-CMVcap to 293 cells. At the same time we infect temperature sensitive adenovirus, Ad5ts149 to 293 cells. After 48 hours culture at 39℃, the cells and medium was collected and recombinant virus was purified using sulfated sepharose column chromatography. We infected this purified virus to the skin fibroblasts cultivated from MPS VII mice. The activity of beta-glucuronidase increased in the infected fibroblasts. We also looked at transdution efficiency by histochemical staining of beta-glucuronidase activity in cells. More than 60% cells expressed human beta-glucuronidase. This observation suggest that our AAV vector efficiently infect Cosl cells in vitro. As an next step, we injected this virus to muscle of MPS VII mice. The enzymatic : activity increased in serum up to 20 % of normal serum. We are currently investigating the improvement of pathology of various organs from MPS VII.

我们生成了腺体 - 辅助载体使用该系统，将人β-葡萄糖酶cDNA插入AAV Express Plasmid，PNT C3CMV。旋转质粒是cotransive cotransive selpransed with Helper withper with helper withper with helper witus with helper witus wit p5Rep-cmvcap at at A in Same温度敏感的腺病毒，AD5TS149至293个细胞，收集细胞和培养基，并使用过度柱色谱纯化重组病毒。在感染的成纤维细胞中，葡萄糖醛酸酶增加了β-葡萄糖醛酸糖酶活性的组织化学效率MPS VII小鼠的肌肉。

项目成果

Eto Y.,Ohashi T.: "Gene therapy/cell therapy for lysosomal storage disease."J Inherited Metabo Dis. 23(3). 293-8 (2000)

Eto Y.，Ohashi T.：“溶酶体贮积病的基因疗法/细胞疗法。”J Inherited Metabo Dis。

Watabe K., Ohashi T., Sakamoto T., Kawazoe Y., Takeshima T., Oyanagi K., Inoue K., Eto Y., and Kim S.U.: "Rescue of lesioned adult rat spinal motoneurons by adenoviral gene transfer of glial cell line-derived neurotrophic factor."Journal of Neuroscience R

Watabe K.、Ohashi T.、Sakamoto T.、Kawazoe Y.、Takeshima T.、Oyanagi K.、Inoue K.、Eto Y. 和 Kim S.U.：“通过胶质细胞腺病毒基因转移来拯救受损的成年大鼠脊髓运动神经元

Tsujino S., Kanazawa N., Ohashi T., Eto Y., Saito T., Kira J., Yamada T.: "Three novel mutations (G27E, insAAC, R179X) in the ORNT1 gene of Japanese patients with HHH syndrome."Arch of Neuro. 147(5). 625-31 (2000)

Tsujino S.、Kanazawa N.、Ohashi T.、Eto Y.、Saito T.、Kira J.、Yamada T.：“日本 HHH 综合征患者 ORNT1 基因中的三种新突变（G27E、insAAC、R179X）。

Yokoo T., Ohashi T., Eto Y., et al.: "Prophyaxis of Antibody-Induced Acute Glomerulonephritis・・・"Hum Gene Ther. 10. 2673-2678 (1999)

Yokoo T.、Ohashi T.、Eto Y. 等人：“抗体诱发的急性肾小球肾炎的预防……”Hum Gene Ther。

Ohashi T.,Yokoo T.,Iizuka S.: "Eduction of Lysosomal storage in Murine Mucoplysaccharidosis Type VII by Transplantation of Normal and Genetically Modified Macrophages."Blood. 95(11). 3631-3 (2000)

Ohashi T.、Yokoo T.、Iizuka S.：“通过移植正常和转基因巨噬细胞来排出 VII 型小鼠粘多糖贮积症中的溶酶体储存。”血液。