Antisense RNA Sequences Modulating the Ataxin-1 Message: Molecular Model of Gene Therapy for Spinocerebellar Ataxia Type 1, a Dominant-Acting Unstable Trinucleotide Repeat Disease

• Published in: Cell transplantation 2008-01, Vol.17 (7), p.723-734
• Main Authors: Gao, Youxin, Zu, Tao, Low, Walter C., Orr, Harry T., Mcivor, R. Scott
• Format: Article
• Language: English
• Subjects: Animals; Ataxin-1; Ataxins; Base Sequence; CHO Cells; Cricetinae; Cricetulus; Genetic Therapy; Humans; Introns; Molecular Sequence Data; Nerve Tissue Proteins - genetics; Nerve Tissue Proteins - metabolism; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Nucleic Acid Conformation; RNA - chemistry; RNA - genetics; RNA - metabolism; RNA Splicing; RNA, Antisense - genetics; RNA, Antisense - metabolism; Spinocerebellar Ataxias - genetics; Spinocerebellar Ataxias - metabolism; Spinocerebellar Ataxias - therapy; Transfection; Trinucleotide Repeats
• ISSN: 0963-6897; 1555-3892
• DOI: 10.3727/096368908786516729

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.