Reversal of aberrant splicing of beta-thalassemia allele by antisense RNA in vitro and in vivo

Objective To investigate the reversal of aberrant splicing of beta-thalasse mia allele (IVS-2-654 C-->T, beta(654)) by antisense RNA in vitro and in vi vo. Methods The vector expressing antisense RNA which targeted against the aber rant splice sites of beta(654) pre-mRNA was constructed in pcDNA3, and then used to repair the defective splicing of the mutant pre-mRNA in an in vitr o transcription and splicing system, as well as in HeLa beta(654) cells and cultured beta(654) erythroid cells by lipid-mediated DNA-transfection meth od. The effect of the antisense RNA was identified by RT-PCR mediated mRNA quantitative assay as well as globin chain microbiosynthesis. Results The antisense RNA decreased the aberrant splicing product and resto red the correct splicing pattern in vitro and in vivo efficiently. In the i n vitro transcription and splicing system, the revel of normally spliced mR NA [beta/(beta + beta* )] increased from 0.25 to 0.60. In cultured Hela bet a(654) cells, the revel of beta/(beta + beta* ) increased from 0.07 to 0.43 on the 15th day after transfection. In cultured beta(654) erythroid cells, the level of mRNA [beta/(beta+ beta*)] increased from 0.19 to 0.58 on the 8th day after transfection in beta(654)/beta(654) erythroid cells, from 0. 02 to 0.38 in beta(654)/beta(41-42) erythroid cells, and from 0. 45 to 0. 8 3 in beta(654)/beta A erythroid cells, respectively. Correspondingly, the r atios of globin chain (beta/alpha) biosynthesis increased from 0.16 to 0.52 on the 8th day after transfection in beta(654)/beta(654) erythroid cells, 0. 05 to 0.36 in beta(654)/beta(41-42) erythroid cells, and 0.42 to 0.81 in beta(654)/beta A erythroid cells, respectively. The splicing pattern did n ot show significant changes as compared to the untreated, as well as to the control antisense fragment. Conclusions Antisense RNA transcribed from theexpression vector described h ere could efficiently suppress the aberrant splicing pattern of beta(654) m utant mRNA and restore the correct splicing pathway in vitro and in vivo, l eading to the improvement of globin chain biosynthesis in thalassemic cells . Our antisense strategy provides an alternative approach to the gene thera py of beta-thalassemia.