Dominski Z, Kole R
Department of Pharmacology, University of North Carolina, Chapel Hill 27599.
Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8673-7. doi: 10.1073/pnas.90.18.8673.
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.
反义2'-O-甲基核糖寡核苷酸靶向突变的人β-珠蛋白前体mRNA中的特定序列元件，以在体外恢复这些RNA的正确剪接。先前已确定β-珠蛋白基因的以下突变，即第一内含子第110位核苷酸处的A→G（β110）、第二内含子第705位核苷酸处的T→G和第654位核苷酸处的C→T（分别为IVS2(705)和IVS2(654)），这些突变导致相应前体mRNA的异常剪接，是β地中海贫血的根本原因。针对前体mRNA第一内含子中的分支点序列的寡核苷酸可有效逆转β110前体mRNA的异常剪接，但针对异常3'剪接位点的寡核苷酸则不能。在IVS2(705)和IVS2(654)前体mRNA中，针对第二内含子中突变产生的异常5'剪接位点或针对位于上游且在突变背景下被激活的隐蔽3'剪接位点的寡核苷酸可恢复正确剪接。这些实验代表了一种方法，即使用反义寡核苷酸来恢复缺陷基因的功能，而不是像通常那样下调不良基因的表达。
