Correction of prototypic ATM splicing mutations and aberrant ATM function with antisense morpholino oligonucleotides.
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.
Du
Liutao
L
Department of Pathology and Laboratory Medicine, The David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA. ldu@mednet.ucla.edu
Pollard
Julianne M
JM
Gatti
Richard A
RA
D001260:Ataxia Telangiectasia / Q000235:genetics*; D016923:Cell Death / Q000528:radiation effects; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004790:Enzyme Induction / Q000187:drug effects; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes; D005720:Gamma Rays; D006801:Humans; D007700:Kinetics; D008957:Models, Genetic; D009154:Mutation; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D011494:Protein Kinases / Q000032:analysis / Q000096:biosynthesis*; D012326:RNA Splicing / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction
D005456:Fluorescent Dyes; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D011494:Protein Kinases; D016650:Fluorescein-5-isothiocyanate