Research for gene therapy of α-glucosidase deficiency using adenoviral vector

腺病毒载体治疗α-葡萄糖苷酶缺乏症的基因治疗研究

• 批准号: 09670687
• 负责人: TSUJINO Seiichi
• 金额: $ 2.3万
• 依托单位: National Institute of Neuroscience
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670687/
• 关键词: α-glucosidase deficiency; adenoviral vector; gene therapy; knockout mouse; quail; α-グルコシダーゼノックアウトマウス

α-glucsidase deficiency (acid maltase deficiency: AMD) causes a lysosomal glycogenosis inherited as an autosomal recessive trait. The infantile type of AMD (Pompe disease) leads to early death due to severe dysfunction of cardiac and respiratory muscles and no effective therapy is available. Replication-defective adenovirus vectors offer a promising tool for in vivo gene delivery and gene therapy. We constructed a recombinant adenovirus containing the human α-glucosidase cDNA downstream of the CAG promoter, composed of modified chicken β-actin promoter and CMV-IE enhancer (AxCANAM). We used two animal models to estimate the effects of AxCANAM; naturally generated Japanese quail with AMD and α-glucosidase knockout (AM-KO) mice.First, we used Japanese quail with AMD. When cultured fibroblasts from AMD quails were infected with AxCANAM, α-glucosidase activity in the cells increased in proportion to multiplicity of infection (MOI). When AxCANAM was injected into unilateral superficial pect … More oral muscle of AMD quails, PAS staining showed that glycogenosomes disappeared and stainability of acid phosphatase was reduced in the injected area as compared to the contralateral muscle of the same birds. Biochemically, α-glucosidase activity increased and glycogen content decreased in the injected muscle. Western blot analysis showed that AMD quail muscle injected with AxCANAM expressed human α-glucosidase protein processed to active forms. These results suggest that the human α-glucosidase cDNA transferred by an adenovirus vector was sufficiently expressed, leading to a marked reduction of the glycogen accumulation in the skeletal muscle of AMD quails.Second, we used knockout mice, which was generated with exon 6 of α-glucosidase gene deleted. When AxCANAM was injected into cardiac cavity of AM-KO mice, α-glucosidase activity increased and glycogen content decreased in skeletal muscle, heart, and liver. Stainability of PAS and acid phosphatase was reduced in these tissues. After the injection, α-glucosidase was expressed mainly in liver, but it was thought that the precurcer protein was presumably secreted into blood, uptaked by peripheral tissues, such as muscle, and processed to active α-glucosidase in lysosomes, thereby leading to systemic effects. Less

α-葡萄糖苷酶缺乏症（酸性麦芽糖酶缺乏症：AMD）导致溶酶体糖原累积症，作为常染色体隐性遗传性状遗传。婴儿型AMD（庞贝氏症）由于心脏和呼吸肌的严重功能障碍而导致早期死亡，并且没有有效的治疗方法。复制缺陷型腺病毒载体为体内基因递送和基因治疗提供了一种有前途的工具。我们构建了含有CAG启动子下游的人α-葡萄糖苷酶cDNA的重组腺病毒，该重组腺病毒由修饰的鸡β-actin启动子和CMV IE增强子（AxCANAM）组成。我们使用两种动物模型来评估AxCANAM的作用;自然产生的患有AMD的日本鹌鹑和α-葡萄糖苷酶敲除（AM-KO）小鼠。当培养的AMD鹌鹑成纤维细胞被AxCANAM感染时，细胞中的α-葡萄糖苷酶活性与感染复数（MOI）成比例地增加。AxCANAM注入一侧浅表部时， ...更多信息 PAS染色显示，与对侧相比，注射区糖原小体消失，酸性磷酸酶活性降低。生物化学上，注射肌肉中α-葡萄糖苷酶活性增加，糖原含量减少。Western blot分析显示，AxCANAM诱导AMD鹌鹑肌肉表达人α-葡萄糖苷酶蛋白，并将其加工成活性形式。这些结果表明，腺病毒载体介导的人α-葡萄糖苷酶cDNA在AMD鹌鹑骨骼肌中得到了有效表达，并显著降低了骨骼肌中糖原的积累。当AxCANAM注射到AM-KO小鼠的心腔内时，骨骼肌、心脏和肝脏中的α-葡萄糖苷酶活性增加，糖原含量减少。PAS和酸性磷酸酶活性降低。注射后，α-葡萄糖苷酶主要在肝脏中表达，但据认为，促凝蛋白可能分泌到血液中，被外周组织（如肌肉）摄取，并在溶酶体中加工成活性α-葡萄糖苷酶，从而导致全身效应。少

项目成果

Tsujino,S.,et al.: "Adenovirus-mediated transfer of human acid maltase gene reduces glycogen accumulation in skeletal muscle of Japanese quail with acid maltase deficiency" Human Gene Therapy. 9. 1609-1616 (1998)

Tsujino,S.,et al.：“腺病毒介导的人类酸性麦芽糖酶基因转移减少了酸性麦芽糖酶缺乏的日本鹌鹑骨骼肌中糖原的积累”人类基因疗法。

Tsujino et al.: "Adenovirus-mediated transfer of human acid maltase gene reduces glycogen accumulation in skeletal muscle of Japanese quail with acid maltase deficiency."Hum Gen Therapy. 9. 1609-1616 (1998)

Tsujino 等人：“腺病毒介导的人类酸性麦芽糖酶基因转移减少了酸性麦芽糖酶缺乏的日本鹌鹑骨骼肌中糖原的积累。”Hum Gen 疗法。

Tsujino, S., et al.: "Adenovirus-mediated trnasfer of human acid maltase gene rednces glycogen aconmulation in skefetal muslle of Japanese guail with acid maltase deficieng"Human Gene Therapy. 9. 1609-1616 (1998)

Tsujino，S.，等人：“腺病毒介导的人酸性麦芽糖酶基因转移减少了患有酸性麦芽糖酶缺陷的日本番石榴骨骼肌中的糖原聚集”人类基因疗法。

辻野精一: "筋糖原病(遺伝性糖代謝異常性ミオパチー)"神経研究の進歩. (印刷中). (2000)

Seiichi Tsujino：“肌糖原病（遗传性葡萄糖代谢障碍性肌病）”神经学研究进展（2000 年出版）。