Reversal of aberrant splicing of beta-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 -tha lassaemia allele, TVS-2-654 C-->T (beta (654)), pretranscript was cloned in to the mammalian expression vector, pcDNA3. The recombinant construct, pCMV A, was then used to repair the defective splicing of the beta (654) mutant pretranscript in cultured beta (654) erythroid cells by the lipofectin-medi ated DNA transfection method. The total RNA was extracted at given time poi nts after transfection and the effect of antisense RNA was studied by rever se transcription polymerase chain reaction (RT-PCR)-mediated mRNA quantitat ive assay, as well as globin chain microbiosynthesis. The antisense fragmen t transcribed from pCMVA effectively improved the beta (654) splicing patte rn in cultured erythroid cells. The level of correctly spliced transcript i ncreased 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 th e 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 splicin g pattern in beta (A)/beta (A) erythroid cells. The spicing pattern in tran sfected cells with pCMVA showed significant changes compared with that in u ntransfected cells and that in transfected cells with the control antisense fragment (human SRY gene sequence), in addition, we did not observe side-e ffects on cytological features after the introduction of pCMVA, All these r esults indicated that the antisense RNA transcribed from the mammalian expr ession vector pCMVA could efficiently and specifically suppress the aberran t splicing pattern of beta (654) mutant pretranscript and restore the corre ct splicing pathway in vivo, leading to the improvement of globin chain bio synthesis in thalassaemic cells.