[Repair of the splicing defect of thalassemic allele beta IVS-2-654 C-->T in cultured human erythroid cells by antisense RNA].

OBJECTIVE

To investigate the repair of the splicing defect of thalassemic allele beta IVS-2-654 C-->T (beta 654) in cultured human adult erythroid cells (hAE) by antisense RNA A2.

METHODS

Vectors expressing antisense RNA fragments A2 which targeted against the aberrant splice sites of beta 654 pre-mRNA were introduced into hAE by lipid-mediated DNA-transfection method. Quantitation of beta-globin mRNA by RT-PCR and micro-biosynthesis of globin chain assay were used to measure the expression of globin genes in hAE from day 0 to day 8 after transfection. Meanwhile, hAE transfected with random antisense sequence was used as control.

RESULTS

Antisense RNA A2 treatment restored the correct splicing of beta 654 pre-mRNA. The proportions of correctly spliced beta-globin mRNA (beta/beta + beta*) increased from 0.024 (before transfection) to 0.380 (8th day after transfection) in hAE obtained from individual with beta 654/ beta 41-42 mutations and, from 0.396 to 0.883 in hAE obtained from individual with beta 654/beta A genotype. Correspondently, microglobin biosynthesis of chain revealed that the ratio of beta/alpha increased from 0.052 to 0.359 in the former and, from 0.624 to 0.820 in the later case, respectively. There was no difference with or without antisense RNA A2 treatment on hAE obtained from normal controls.

CONCLUSIONS

Antisense RNA A2 treatment could restore efficiently the correct splicing of beta 654 pre-mRNA and, consequently improve the imbalance of globin chains in thalassemic cells. This method is of potential clinical interest in gene therapy of the disease.