Restoration of correct splicing in thalassemic pre-mRNA by antisense oligonucleotides.
Antisense 2'-O-methylribooligonucleotides were targeted against specific sequence elements in mutated human beta-globin pre-mRNAs to restore correct splicing of these RNAs in vitro. The following mutations of the beta-globin gene, A-->G at nt 110 of the first intron (beta 110), T-->G at nt 705 and C-->T at nt 654 of the second intron (IVS2(705) and IVS2(654), respectively), which led to aberrant splicing of the corresponding pre-mRNAs, were previously identified as the underlying causes of beta-thalassemia. Aberrant splicing of beta 110 pre-mRNA was efficiently reversed by an oligonucleotide targeted against the branch point sequence in the first intron of the pre-mRNA but not by an oligonucleotide targeted against the aberrant 3' splice site. In both IVS2(705) and IVS2(654) pre-mRNAs, correct splicing was restored by oligonucleotides targeted against the aberrant 5' splice sites created by the mutations in the second intron or against a cryptic 3' splice site located upstream and activated in the mutated background. These experiments represent an approach in which antisense oligonucleotides are used to restore the function of a defective gene and not, as usual, to down-regulate the expression of an undesirable gene.
Dominski
Z
Z
Department of Pharmacology, University of North Carolina, Chapel Hill 27599.
Kole
R
R
D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D005914:Globins / Q000096:biosynthesis / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D007438:Introns / Q000187:drug effects; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D012322:RNA Precursors / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism*; D012326:RNA Splicing; D015183:Restriction Mapping; D017086:beta-Thalassemia / Q000235:genetics* / Q000378:metabolism*
D016376:Oligonucleotides, Antisense; D012322:RNA Precursors; D005914:Globins