A study of pathogenesis, and analysis for clinical and a gene therapy in Duchenne muscular dystrophy.

杜氏肌营养不良症的发病机制研究、临床及基因治疗分析。

• 批准号: 06454302
• 负责人: MIIKE Teruhisa
• 金额: $ 3.84万
• 依托单位: KUMAMOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06454302/
• 关键词: Dystrophin; Gene targeting; Transgenic mouse; Promoter; Regulatory element; lacZ; Splicing mechanism; Gene therapy; ジストロフィン(dy); 標的遺伝子組み換え法; 心筋特異的dyプロモーター; 拡張型心筋症; 神経成長因子

(1) Analysis of dystrophin function :To determine the functional significane of dystrophin, first we have been trying to produce a muscle type and a brain type dystrophin defective model mouse using gene targeting method, respectively. In spite of our efforts, this project has not been succeeded yet. On the other hand 2 to 3 muscle type dystrophin knock out mice have been reported already, in America and Japan last year. So we started to concentrate to produce a brain type dystrophin knock out mouse.(2) Mechanism of developmental and tissue specific regulation of muscle and brain type systrophin :In order to study the mechanism of developmental and tissuespecitic regulation of muscle type dystrophin gene in mice, we generated transgenic mice carrying the 900bp genomic fragment from muscle type dystrophin promoter, fused to the coding region of the bacterial lacZ gene. Ti was found that nineteen mice carried the transgene. Of which six lines showed lacZ expression in the right heart. Th … More e reporter gene expression was detected in presumptive right atria and ventricular myocardium of embryo at 8.5 d.p.c.(dayspost coitus). The results show that the 900bp genomic fragment contain the regulatory element for expression in right heart. Therefore, it is possible to consider that the regulatory regions of skeletal muscle, and also left heat are distinct from the that of right heart within the mouse muscle type dystrophin promoter. As to the brain type, we observed that 2.1kb 5'fragment of the mouse brain type dystrophin gene contains the regulatory element required for its expression in the cerebral cortex, but not in the hippocampus.(3) Preliminary study for DMD gene therapy :Our final goal is to convert out of frame of dystrophin gene (DMD) into in frame (BMD) dystrophin gene by controlling RNA splicing (exon skipping) using antisense oligonucleotide. As a preliminary study we are trying to establish two systems to evaluate effect of the gene therapy : 1) Comparing system of dystrophin expression in myotube differentiated from myoblast which was originated in MyoD gene transducted fibroblast from normal and DMD,and 2) System of in-vitro splicing using mini dystrophin gene construct. Less

(1)抗肌萎缩蛋白功能分析：为了确定抗肌萎缩蛋白的功能意义，我们首先尝试使用基因打靶方法分别产生肌肉型和脑型抗肌萎缩蛋白缺陷模型小鼠。尽管我们努力了，这个项目还没有成功。另一方面，去年在美国和日本已经报道了2至3只肌肉型肌营养不良蛋白敲除小鼠。所以我们开始集中精力生产一种脑型肌营养不良蛋白基因敲除小鼠。(2)肌型和脑型肌萎缩蛋白的发育和组织特异性调控机制：为了研究肌型肌萎缩蛋白基因在小鼠中的发育和组织特异性调控机制，我们构建了携带肌型肌萎缩蛋白启动子900 bp基因组片段的转基因小鼠，该基因片段与细菌lacZ基因的编码区融合。发现19只小鼠携带转基因。其中6条细胞系显示lacZ在右心表达。日 ...更多信息 在8.5d.p.c.的胚胎右心房和心室心肌中检测到报告基因的表达。（dayspost coitus）.结果表明，900 bp的基因组片段含有右心表达调控元件。因此，有可能认为骨骼肌的调节区域以及左心室与右心室在小鼠肌肉型肌营养不良蛋白启动子内的调节区域不同。在脑型方面，我们观察到小鼠脑型dystrophin基因的2.1kb 5 '端片段含有其在大脑皮层表达所需的调控元件，而在海马中没有。(3)DMD基因治疗的初步研究：我们的最终目标是利用反义寡核苷酸控制RNA剪接（外显子跳跃），将DMD基因的读框外（out-frame）转化为读框内（in-frame）。作为初步研究，我们试图建立两个系统来评估基因治疗的效果：1）比较来自正常和DMD的成肌细胞的MyoD基因转导的成肌细胞分化的肌管中dystrophin表达的系统; 2）使用微型dystrophin基因构建体的体外剪接系统。少

项目成果

Uchino,et al.: "Dystrophin and dystrophin-related protein in the central nervous system of normal and and Duchenne muscular dystrophy." Acta Neuropathol. 87. 129-134 (1994)

Uchino 等人：“正常和杜氏肌营养不良症中枢神经系统中的肌营养不良蛋白和肌营养不良蛋白相关蛋白。”

S Kimura: "A 900bp genomic fragment from mouse dystrophin promoter region directs lacZ reporter expression only to the right heart of transgenic mice." (submitted).

S Kimura：“来自小鼠肌营养不良蛋白启动子区域的 900bp 基因组片段将 lacZ 报告基因的表达仅引导至转基因小鼠的右心。”

Makoto Uchino, Teruhisa Miike, Hiroshi Iwashita, Eiichiro Uyama, Kowashi Yoshioka, Shigeto Sugino, Masayuki Ando: "PCR and immunoblot analyzes of dystrophin in Beker muscular dystrophy." J Neurol Sci. 124. 225-229 (1994)

Makoto Uchino、Teruhisa Miike、Hiroshi Iwashita、Eiichiro Uyama、Kowashi Yoshioka、Shigeto Sugino、Masayuki Ando：“Beker 肌营养不良症中肌营养不良蛋白的 PCR 和免疫印迹分析。”

Shigemi Kimura, Kuniya Abe, Misao Suzuki, Kowashi Yoshioka, Masakatsu Ogawa, Tadashi Kaname, Teruhisa Miike, Ken-ichi Yamamura: "A 900bp genomic fragment from mouse dystrophin promoter region directs lacZ reporter expression only to the right heart of tra

Shigemi Kimura、Kuniya Abe、Misao Suzuki、Kowashi Yoshioka、Masakatsu Okawa、Tadashi Kaname、Teruhisa Miike、Ken-ichi Yamamura：“来自小鼠肌营养不良蛋白启动子区域的 900bp 基因组片段将 lacZ 报告基因表达仅引导至 tra 的右心。

A Tomoda: "Two patients with distal muscular dystrophy and autonomic nerve dysfunctin." Brain Dev. 16. 65-70 (1994)

A Tomoda：“两名患有远端肌营养不良症和自主神经功能障碍的患者。”