title: 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.
abstract: 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.
authors.affiliation: Institute of Human Genetics, Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: Youxin
authors.initials: Y
authors.lastname: Gao
authors.forename: Tao
authors.initials: T
authors.lastname: Zu
authors.forename: Walter C
authors.initials: WC
authors.lastname: Low
authors.forename: Harry T
authors.initials: HT
authors.lastname: Orr
authors.forename: R Scott
authors.initials: RS
authors.lastname: McIvor
mesh_terms: D000818:Animals; D000067696:Ataxin-1; D000067528:Ataxins; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D015316:Genetic Therapy; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing; D016372:RNA, Antisense / Q000235:genetics* / Q000378:metabolism; D020754:Spinocerebellar Ataxias / Q000235:genetics* / Q000378:metabolism / Q000628:therapy*; D014162:Transfection; D018911:Trinucleotide Repeats
chemical_list: C000593749:ATXN1 protein, human; D000067696:Ataxin-1; D000067528:Ataxins; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D016372:RNA, Antisense; D012313:RNA