筋ジストロフィーに対するアンチセンスDNAによる化学的治療法の開発

在本研究中，我们对反义寡核苷酸治疗Duchenne肌营养不良症进行了基础性研究。首先，我们建立了C2细胞向肌管诱导分化的实验系统，通过加入寡脱氧核苷酸来评估dystrophin基因的外显子跳跃。通过剪接，我们获得了共识序列的反义S-寡核苷酸。C2细胞在向肌管分化过程中用反义S寡核苷酸处理3d。对dystrophin基因的表达及剪接产物进行分析。加入5‘-和3’-剪接位点的反义S寡核苷酸后，正常剪接产物外显子22-23-24的表达显著降低，并出现外显子22-24条带。为了研究反义寡核苷酸对Ddystrophin基因外显子23的强迫跳跃作用，我们克隆了编码内含子22到内含子23的Dystrophin微基因，构建了哺乳动物正、反义RNA表达载体。将两种载体分别导入C2成肌细胞，G418筛选细胞。稳定的转化子被诱导分化为肌管，RT-PCR分析了dystrophin基因的表达。不幸的是，所有转化子的外显子22 24的表达没有显著变化。另一方面，5‘和3’剪接位点的反义寡核苷酸强制外显子跳跃的结果，提高了dystrophin基因外显子和内含子连接序列的重要性。因此，我们与Kyowa Hakko Kogyo株式会社东京研究实验室的Anazawa博士和Shinkai博士合作，研究了人类dystrophin基因第46号外显子和52号至55号外显子的序列。这些数据是应用人类化疗所必需的。

In this study, we did fundamental studies to develop the chemo-therapeuic treatment with antisense oligodeoxynucleotides against Duchenne muscular dystrophy. First, we developed expermental system using C2 cells which can be induced differentiation to myotube, to evaluate exon-skip of dystrophin gene by the addition of oligodeoxynucleotides. We made antisense S-oligodeoxynucleotides of consensus sequences on RNA splicing. C2 cells were treated with antisense S-oligodeoxynucleotides during the differentiation to myotube for 3 days. The expression and the spliced products of dystrophin gene were analyzed. By the addition of antisense S-oligodeoxynucleotides of5'-and 3'-splice sites, the expression of exons 22+23+24, the normal splicing product, were dramatically reduced and the band of exons 22+24 appeared. This result demonstrated that antisense oligodeoxynucleotides made forced exon-skip of exon 23 in dystrophin gene.To investigate the effect of longer antisense nucleotides, we cloned dystrophin mini-gene which encodes intron 22 to intron 23, and made mammalian expression constructs for sense and antisense RNAs. Both plasmids were transfected to C2 myoblasts and then cells were selected with G418. Stable transformants were induced differentiation to myotube and the expressed dystrophin gene were analyzed by RT-PCR.Unfortunately, there was no significant change in the expression of exons 22+24 in all transformants.On the other hand, our result of the forced exon-skip by antisense oligodeoxynucleotides of 5'-and 3'-splice sites, raise the importance of junctional sequences of exons and introns of dystrophin gene. Therefore, we investigated juntional sequences of exon 46 and exons 52 to 55 of human dystrophin gene in cooperation with Drs. Anazawa and Shinkai, Tokyo Research Laboratories of Kyowa Hakko Kogyo, Co., Ltd.These data are necessary to apply human chemotherapy.