TY - JOUR
T1 - Correction of prototypic ATM splicing mutations and aberrant ATM function with antisense morpholino oligonucleotides
AU - Du, Liutao
AU - Pollard, Julianne M.
AU - Gatti, Richard A.
PY - 2007/4/3
Y1 - 2007/4/3
N2 - We used antisense morpholino oligonucleotides (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 pseudoexon 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 transactivation of ATM substrates. Ionizing irradiation-induced cytotoxicity was markedly abrogated after AMO 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.
AB - We used antisense morpholino oligonucleotides (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 pseudoexon 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 transactivation of ATM substrates. Ionizing irradiation-induced cytotoxicity was markedly abrogated after AMO 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.
KW - Ataxia-telangiectasia mutated
KW - Mutation-based treatment
UR - http://www.scopus.com/inward/record.url?scp=34347236941&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=34347236941&partnerID=8YFLogxK
U2 - 10.1073/pnas.0608616104
DO - 10.1073/pnas.0608616104
M3 - Article
C2 - 17389389
AN - SCOPUS:34347236941
SN - 0027-8424
VL - 104
SP - 6007
EP - 6012
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 14
ER -