RESTORATION OF CORRECT SPLICING IN THALASSEMIC PREMESSENGER RNA BY ANTISENSE OLIGONUCLEOTIDES

Antisense 2'-O-methylribooligonucleotides were targeted against specif ic sequence elements in mutated human beta-globin pre-mRNAs to restore correct splicing of these RNAs in vitro. The following mutations of t he beta-globin gene, A --> G at nt 110 of the first intron (beta110), 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 beta110 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 olig onucleotide targeted against the aberrant 3' splice site. In both IVS2 (705) and IVS2(654) pre-mRNAs, correct splicing was restored by oligon udeotides targeted against the aberrant 5' splice sites created by the mutations in the second intron or against a cryptic 3' splice site lo cated upstream and activated in the mutated background. These experime nts represent an approach in which antisense oligonucleotides are used to restore the function of a defective gene and not, as usual, to dow n-regulate the expression of an undesirable gene.