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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.
AuthorsYouxin Gao, Tao Zu, Walter C Low, Harry T Orr, R Scott McIvor
JournalCell transplantation (Cell Transplant) Vol. 17 Issue 7 Pg. 723-34 ( 2008) ISSN: 0963-6897 [Print] United States
PMID19044200 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)
Chemical References
  • ATXN1 protein, human
  • Ataxin-1
  • Ataxins
  • Nerve Tissue Proteins
  • Nuclear Proteins
  • RNA, Antisense
  • RNA
Topics
  • Animals
  • Ataxin-1
  • Ataxins
  • Base Sequence
  • CHO Cells
  • Cricetinae
  • Cricetulus
  • Genetic Therapy
  • Humans
  • Introns
  • Molecular Sequence Data
  • Nerve Tissue Proteins (genetics, metabolism)
  • Nuclear Proteins (genetics, metabolism)
  • Nucleic Acid Conformation
  • RNA (chemistry, genetics, metabolism)
  • RNA Splicing
  • RNA, Antisense (genetics, metabolism)
  • Spinocerebellar Ataxias (genetics, metabolism, therapy)
  • Transfection
  • Trinucleotide Repeats

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