Reversal of aberrant splicing of β-thalassaemia allele (IVS-2-654 C→T) by antisense RNA expression vector in cultured human erythroid cells

The antisense fragment targeting the aberrant splice sites of the beta -thalassaemia allele, TVS-2-654 C-->T (beta (654)), pretranscript was cloned into the mammalian expression vector, pcDNA3. The recombinant construct, pCMVA, was then used to repair the defective splicing of the beta (654) mutant pretranscript in cultured beta (654) erythroid cells by the lipofectin-mediated DNA transfection method. The total RNA was extracted at given time points after transfection and the effect of antisense RNA was studied by reverse transcription polymerase chain reaction (RT-PCR)-mediated mRNA quantitative assay, as well as globin chain microbiosynthesis. The antisense fragment transcribed from pCMVA effectively improved the beta (654) splicing pattern in cultured erythroid cells. The level of correctly spliced transcript increased from 0.19 (day 0 after transfection) to 0.58 (day 8) in beta (654)/beta (654) homozygous erythroid cells, and from 0.45 (day 0) to 0.83 (day 8) in beta (654)/beta (A) heterozygous erythroid cells, as determined by the ratio of normally spliced beta -globin transcript over total beta -globin transcript. Correspondingly the ratios of globin chain biosynthesis (beta/alpha) increased from 0.16 (day 0) to 0.52 (day 8) in beta (654)/beta (654) erythroid cells, and from 0.39 (day 0) to 0.84 (day 8) in beta (654)/beta (A) erythroid cells. Antisense RNA had no significant effect on the splicing pattern in beta (A)/beta (A) erythroid cells. The spicing pattern in transfected cells with pCMVA showed significant changes compared with that in untransfected cells and that in transfected cells with the control antisense fragment (human SRY gene sequence), in addition, we did not observe side-effects on cytological features after the introduction of pCMVA, All these results indicated that the antisense RNA transcribed from the mammalian expression vector pCMVA could efficiently and specifically suppress the aberrant splicing pattern of beta (654) mutant pretranscript and restore the correct splicing pathway in vivo, leading to the improvement of globin chain biosynthesis in thalassaemic cells.