Clinical and basic scientific researchi toward gene therapy in progressive muscular dystrophies

进行性肌营养不良症基因治疗的临床和基础科学研究

• 批准号: 07670906
• 负责人: SAITO Kayoko
• 金额: $ 1.6万
• 依托单位: Tokyo Women's Medical College
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07670906/
• 关键词: progressive muscular dystrophy; Duchenne muscular dystrophy; Fukuyama muscular dystrophy; somatic mosaicism; gene therapy; cationic liposomes; cultured myoblasts; 体細胞モザイク; Duchenne型筋ジストロフィー; 転写産物; 遺伝子産物; Sequence; 骨格筋細胞培養; アデノウイルスベクター

We have conducted the molecular genetic analysis and clinical application for genotype-phenotype correlation in progressive muscular dystrophies, especially Duchenne muscular dystrophy (DMD) and Fukuyama congenital muscular dystrophy (FCMD). For the purpose of obtaining complinentary primary genetic defects in the DMD gene by introducing copies of recombinant gene constructs into nuscle cells ex vivo, the potential use of cationic liposomes as physical gene delivery systems for cultured human dystrophic skeletal muscles was examined, from the basic scientific point of view. Various cationic liposome formulations, i.e.cellfectin, DMRIE-C,lipofectin, lipofectAMINE and lipofectAMINE PLUS,were examined for their use in plasmid DNA (beta-gal reporter gene) transfection. For transgene expression, muscle cultures were stained or were applied to detect beta-gal activities by the ELISA method. These in vitro studies indicated that lipofectAMINE PLUS showed high efficiency. In addition, myoblast non-clonal cultures were more efficient than clonal cultures. This is the first successful introduction of foreign genes into primary cultures of human dystrophic myoblasts by means of cationic liposomes.

我们对进行性肌营养不良症，特别是Duchenne型肌营养不良症（DMD）和Fukuyama型先天性肌营养不良症（FCMD）的基因型-表型相关性进行了分子遗传学分析和临床应用。为了通过将重组基因构建体的拷贝引入离体的神经节细胞中来获得DMD基因中的互补的原发性遗传缺陷，从基础科学的观点出发，检查了阳离子脂质体作为用于培养的人营养不良骨骼肌的物理基因递送系统的潜在用途。检查了各种阳离子脂质体制剂，即cellfectin、DMRIE-C、lipofectin、lipofectAMINE和lipofectAMINE PLUS在质粒DNA（β-gal报告基因）转染中的用途。对于转基因表达，对肌肉培养物进行染色或通过ELISA方法检测β-gal活性。这些体外研究表明，lipofectAMINE PLUS显示出高效率。此外，成肌细胞非克隆培养比克隆培养更有效。这是第一次通过阳离子脂质体成功地将外源基因导入人营养不良成肌细胞原代培养物中。

项目成果

K.Saito et al.: "Prenatal diagnosis in eight Fukuyama type congenital muscular dystrophy families by haplotype analysis using the the new markers closest to the gene." Am J Med Genet. (in press).

K.Saito 等人：“使用最接近该基因的新标记，通过单倍型分析对八个福山型先天性肌营养不良症家族进行产前诊断。”

斎藤加代子: "福山型先天性筋ジストロフィー研究最近の進歩" 脳と発達. 27. 447-454 (1995)

Kayoko Saito：“福山先天性肌营养不良症研究的最新进展”《大脑与发育》27. 447-454 (1995)。

K.Saito, et al.: "Prenatal diagnosis in eight Fukuyama type congenital muscular dystrophy families by haplotype analysis using the new markers closest to the gene" Americal Journal of Medical Genetics. (in press).

K.Saito 等人：“使用最接近基因的新标记通过单倍型分析对八个福山型先天性肌营养不良症家族进行产前诊断”美国医学遗传学杂志。

斎藤 加代子 ら: "カチオニック・リポソームを用いたヒト培養骨格筋細胞への遺伝子導入" 東京女子医科大学雑誌. (投稿中).

Kayoko Saito 等：“使用阳离子脂质体将基因转移到培养的人类骨骼肌细胞”，东京女子医科大学学报（目前正在提交）。

Y.Fukuyama, M.Osawa, K.Saito: "Conegenital muscular dystrophies" Elsevier Science, 432 (1997)

Y.Fukuyama、M.Osawa、K.Saito：“先天性肌营养不良症”Elsevier Science，432（1997）