|Budget Amount *help
¥2,600,000 (Direct Cost : ¥2,600,000)
Fiscal Year 1997 : ¥1,100,000 (Direct Cost : ¥1,100,000)
Fiscal Year 1996 : ¥1,500,000 (Direct Cost : ¥1,500,000)
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.