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

(1)dystrophin功能分析：为了确定dystrophin的功能意义，我们首先尝试用基因打靶的方法分别建立肌型和脑型dystrophin缺陷小鼠模型。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有2到3只肌型肌营养不良基因敲除小鼠的报道。因此，我们开始致力于培育脑型dystrophin基因敲除小鼠。(2)肌肉和脑型dystrophin基因的发育和组织特异性调控机制：为了研究肌型dystrophin基因在小鼠的发育和组织特异性调控机制，我们制备了携带肌型dystrophin启动子900bp基因组片段的转基因小鼠，并将其融合到细菌LacZ基因的编码区。研究发现，19只小鼠携带了转基因。其中6行显示LacZ在右心表达。TH…在8.5dP.C.(日间交配)的胚胎右心房和右室心肌中检测到较多的e报告基因表达。结果表明，900bp的基因组片段含有右心表达的调控元件。因此，可以认为，在小鼠肌型肌营养不良蛋白启动子中，骨骼肌和左侧热量的调节区与右心的调节区是不同的。至于脑型，我们观察到小鼠脑型dystrophin基因的2.1kb 5‘片段包含其在大脑皮层表达所需的调控元件，但在海马区不包含。(3)DMD基因治疗的初步研究：我们的最终目标是通过使用反义寡核苷酸控制RNA剪接(外显子跳跃)，将dystrophin基因(DMD)的框架外(Out Of Frame)转化为框架内(BMD)基因。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MyoD基因转导的正常成肌细胞和DMD成肌细胞分化成肌管中dystrophin表达的系统；2)利用微型dystrophin基因构建的体外剪接系统。较少

(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