Spinocerebellar ataxia type 1 (
SCA1) is a dominant inherited disease caused by expanded trinucleotide repeats resulting in an increased
polyglutamine tract in the gene product. As a potential therapeutic approach for
SCA1, we tested antisense RNAs targeting two regions of the
ataxin-1 message. Single-stranded regions around the translational initiation site and the intron 8
splice donor site of the
ataxin-1 message were identified by computer-assisted
RNA secondary structure prediction. Plasmids were generated to contain a 254-bp antisense sequence spanning the translation initiation site (pLasBDini) or a 317-bp sequence spanning the intron 8
splice donor site (pLasBDei) of the
ataxin-1 message. These plasmids were transfected into Chinese hamster ovary cells engineered to express either expanded or unexpanded
ataxin-1 message and
protein. Reduced levels of mutant
ataxin-1 message (82 CAG repeats), wild-type
ataxin-1 message (30 CAG repeats), and
ataxin-1 protein were observed by Northern and Western blot analyses in pLasBDini-transfected clones. pLasBDei-transfected 293 cells exhibited a shift in
ataxin-1 message to a size several kilobases longer than that of the natural message.
Reverse transcriptase/polymerase chain reaction assays demonstrated the retention of message spanning the intron 8 splice acceptor and the inability to amplify sequences between exons 8 and 9, implying that normal splicing of intron 8 had been interrupted. We conclude that antisense RNAs were effective in reducing or modifying
ataxin-1 messages in transfected cells, and may be an effective genetic strategy for
therapy of
SCA1 and similar dominant-acting
neurological disorders.