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

Cell Transplantation

Article

Walter C. Low¹  Youxin Gao²  Tao Zu²  R. Scott Mcivor³  Harry T. Orr³ 

[1] Department of Neurosurgery, University of Minnesota, Minneapolis, MN, USA;Institute of Human Genetics, Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, USA;Institute of Human Genetics, Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, USA;Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN, USA;

关键词: Cerebellum;    Ataxia;    Spinocerebellar ataxia type 1;    Gene therapy;   

DOI  :  10.3727/096368908786516729

 received in 2008-05-08, accepted in 2008-07-05,  发布年份 2008

来源: Sage Journals

【 摘 要 】

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.

【 授权许可】

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© 2008 Cognizant Comm. Corp.