MODULATION OF IN-VITRO SPLICING OF THE UPSTREAM INTRON BY MODIFYING AN INTRA-EXON SEQUENCE WHICH IS DELETED FROM THE DYSTROPHIN GENE IN DYSTROPHIN KOBE

Molecular analysis of dystrophin Kobe showed that exon 19 of the dystr ophin gene bearing 52-bp deletion was skipped during splicing, althoug h the known consensus sequences at the 5' and 3' splice sites of exon 19 were maintained (Matsuo, M., T. Masumura, H. Nishio, T. Nakajima, Y . Kitoh, T. Takumi, J. Koga, and H. Nakamura. 1991. J. Clin. Invest. 8 7:2127-2131). These data suggest that the deleted sequence of exon 19 may function as a cis-acting element for exact splicing for the upstre am and downstream introns. To investigate this potential role of exon 19, an in vitro splicing system using artificial dystrophin mRNA precu rsors (pre-mRNAs) was established. Pre-mRNA containing exon 18, trunca ted intron 18, and exon 19 was spliced precisely in vitro, whereas spl icing of intron 18 was almost completely abolished when the wild-type exon 19 was replaced by the dystrophin Kobe exon 19. Splicing of intro n 18 was not fully reactivated when dystrophin Kobe exon 19 was restor ed to nearly normal length by inserting other sequences into the delet ed site. These results suggest that the presence of the exon 19 sequen ce which is lost in dystrophin Kobe is more critical for splicing of i ntron 18 than the length of the exon 19 sequence. Characteristically, the efficiency of splicing of this intron seemed to correlate with the presence of polypurine tracks within the downstream exon 19. Moreover , an antisense 31-mer 2'-O-methyl ribonucleotide complementary to the 5' half of the deleted sequence in dystrophin Kobe exon 19 inhibited s plicing of wild-type pre-mRNA in a dose- and time-dependent manner. Th is first in vitro evidence that dystrophin pre-mRNA splicing can be mo dulated by an antisense oligonucleotide raises the possibility of a ne w therapeutic approach for Duchenne muscular dystrophy.