Construction of antisense RNA expression vectors and correction of splicing defect in human beta-globin gene (IVS-2-654 C-->T mutant) in HeLa cells.

The antisense fragments, which were available in in vitro system, were cloned into the mammalian expression vector pcDNA3, and were transfected into H654 cells, a mammalian cell line stably expressing the thalassaemic (IVS-2-654 C-->T) human beta-globin gene. In these transfected cells, the level of correctly spliced beta-globin mRNA in total beta-globin mRNA (beta/(beta + beta*)) was improved from 0.07 (0 d) to 0.22 (3 d), and this effect persisted for up to 15 d post transfection. All the results demonstrated that antisense RNAs were able to be transcribed from the antisense fragment expression vectors stably and effectively suppressed aberrant splicing pattern of the mutated beta-globin gene (IVS-2-654 C-->T) and restored correct splicing pathway. This work provided a novel approach with potential clinical significance to gene therapy of this kind of splicing mutants including beta-thalassaemia (IVS-2-654 C-->T) by antisense RNAs.