Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 104, Issue 14, Pages 6007-6012Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0608616104
Keywords
ataxia-telangiectasia mutated; mutation-based treatment
Categories
Funding
- NIAID NIH HHS [AI 067769, U19 AI067769] Funding Source: Medline
- NINDS NIH HHS [NS 35322, R01 NS035322] Funding Source: Medline
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We used antisense morpholino oligonucleoticles (AMOS) to redirect and restore normal splicing of three prototypic splicing mutations in the ataxia-telangiectasia mutated (ATM) gene. Two of the mutations activated cryptic 5' or 3' splice sites within exonic regions; the third mutation activated a downstream 5' splice site leading to pseudo-exon inclusion of a portion of intron 28. AMOs were targeted to aberrant splice sites created by the mutations; this effectively restored normal ATM splicing at the mRNA level and led to the translation of full-length, functional ATM protein for at least 84 h in the three cell lines examined, as demonstrated by immunoblotting, ionizing irradiation-induced autophosphorylation of ATM, and trans-activation of ATM substrates. Ionizing irradiation-induced cytotoxicity was markedly abrogated after AMID exposure. The ex vivo data strongly suggest that the disease-causing molecular pathogenesis of such prototypic mutations is not the amino acid change of the protein but the mutated DNA code itself, which alters splicing. Such prototypic splicing mutations may be correctable in vivo by systemic administration of AMOs and may provide an approach to customized, mutation-based treatment for ataxia-telangiectasia and other genetic disorders.
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