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-thalassemia allele (IVS-2-654 C-->T, beta 654) by antisense RNA in vitro and in vivo.

METHODS

The vector expressing antisense RNA which targeted against the aberrant 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 vitro transcription and splicing system, as well as in HeLa beta 654 cells and cultured beta 654 erythroid cells by lipid-mediated DNA-transfection method. 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 restored the correct splicing pattern in vitro and in vivo efficiently. In the in vitro transcription and splicing system, the level of normally spliced mRNA [beta/(beta + beta*)] increased from 0.25 to 0.60. In cultured HeLa beta 654 cells, the level 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 0.83 in beta 654/beta A erythroid cells, respectively. Correspondingly, the ratios 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 not show significant changes as compared to the untreated, as well as to the control antisense fragment.

CONCLUSIONS

Antisense RNA transcribed from the expression vector described here could efficiently suppress the aberrant splicing pattern of beta 654 mutant mRNA and restore the correct splicing pathway in vitro and in vivo, leading to the improvement of globin chain biosynthesis in thalassemic cells. Our antisense strategy provides an alternative approach to the gene therapy of beta-thalassemia.