Repair of thalassemic human b-globin mRNA in mammalian cells by antisense oligonucleotides ( RNA splicing y gene therapy )

In one form of b-thalassemia, a genetic blood disorder, a mutation in intron 2 of the b-globin gene (IVS2654) causes aberrant splicing of b-globin pre-mRNA and, consequently, b-globin deficiency. Treatment of mammalian cells stably expressing the IVS2-654 human b-globin gene with antisense oligonucleotides targeted at the aberrant splice sites restored correct splicing in a dose-dependent fashion, generating correct human b-globin mRNA and polypeptide. Both products persisted for up to 72 hr posttreatment. The oligonucleotides modified splicing by a true antisense mechanism without overt unspecific effects on cell growth and splicing of other pre-mRNAs. This novel approach in which antisense oligonucleotides are used to restore rather than to down-regulate the activity of the target gene is applicable to other splicing mutants and is of potential clinical interest. b-Thalassemia, a genetic blood disorder, affects a large number of people in the Mediterranean basin, Middle East, South East Asia, and Africa. Close to 100 thalassemic mutations causing defective b-globin gene expression and b-globin deficiency have been identified, but no more than 10 mutations are responsible for '90% of cases worldwide (1). Of the frequently occurring mutations, the ones that cause aberrant splicing of intron 1 of the human b-globin gene are predominant in South Eastern Europe, Cyprus, Lebanon (mutation IVS1-110), India, Malaysia, and Indonesia (IVS1-5). Additional splicing mutations in intron 1 (IVS1-6) as well as in intron 2 of the b-globin gene (IVS2-745) are also common in the above countries, while IVS2-654 is frequent among b-thalassemia patients in China and Thailand (1–8). All of these mutations activate aberrant splice sites and change the splicing pathway even though the correct splice sites remain potentially functional. We hypothesized that blocking of the aberrant splice sites or other sequence elements involved in splicing with antisense oligonucleotides may force the splicing machinery to reselect the correct splice sites and induce the formation of b-globin mRNA and polypeptide, hence restoring