反义RNA对地中海贫血红系细胞β-珠蛋白mRNA异常剪接的纠正作用

Repair of thalassemic human β globin mRNA in cultured erythroid cells by antisense RNA

目的研究反义ＲＮＡ对β地中海贫血红系细胞ＩＶＳ２６５４Ｃ→Ｔ（β６５４）突变ｍＲＮＡ异常剪接的纠正作用。方法构建特异性针对β６５４ｍＲＮＡ前体异常剪接位点的反义ＲＮＡ表达载体，和不针对上述位点的反义对照载体，转染培养的β６５４红系细胞后，抽提总ＲＮＡ；以ＲＴＰＣＲ法作ｍＲＮＡ定量，检测正常和异常剪接的β珠蛋白ｍＲＮＡ产物的比例［β／（β＋β）］；以珠蛋白肽链体外生物合成分析红系细胞中β和α珠蛋白肽链合成的比例（β／α）。结果在ＲＮＡ水平，β６５４／β６５４纯合子的β／（β＋β）值由０．１９（转染后第０天）上升到０．５８（第８天），β６５４／βＡ杂合子由０．４５（０天）上升到０．８３（第８天）；在珠蛋白肽链水平，β６５４／β６５４细胞的β／α值由０．１６（０天）上升到０．５２（第８天），β６５４／βＡ细胞由０．３９（０天）上升到０．８４（第８天）。反义ＲＮＡ处理βＡ／βＡ红系细胞以及对照ＲＮＡ处理这３类红系细胞，对其ｍＲＮＡ剪接和珠蛋白肽链合成均影响不大。结论特异性反义ＲＮＡ能抑制红系细胞β６５４ｍＲＮＡ前体的异常剪接，部分恢复其正常剪接途径。

Objective To investigate the restoration of correct splicing of β thalassemia allele (IVS2 654C→T,β 654 ) in cultured erythroid cells by antisense RNA.Methods Vectors transcribing an antisense RNA that was targeted against the aberrant splice sites of β 654 pre transcript(pCMVA) and transcribing a control antisense fragment (pCMVC) were constructed. The total RNA was extracted at given time point after transfection, and the effect of antisence RNA was studied by RT PCR mediated mRNA quantitative assay as well as globin chain microbiosynthesis. Results The antisense fragment transcribed from pCMVA effectively improved the β 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 β 654 /β 654 homozygous erythroid cells, and from 0.45(day 0) to 0.83(day 8) in β 654 /β A heterozygous erythroid cells, as determined by the ratio of normally spliced β globin transcript over total β globin transcript. Correspondingly, the ratios of globin chain biosynthesis(β/α) increased from 0.16(day 0) to 0.52( day 8) in β 654 /β 654 erythroid cells, and from 0.39(day 0) to 0.84(day 8) in β 654 /β A erythroid cells. Antisense RNA has no significant effect on the splicing pattern in β A /β A erythroid cells. The splicing pattern in transfected cells with pCMVA showed significant changes compared to that in untransfected cells and in transfected cells with pCMVc. Conclusions The antisense RNA transcribed from the mammalian expression vector pCMVA could efficiently and specifically suppress the aberrant splicing pattern of β 654 mutant pre transcript and restore the correct splicing pathway in vivo, leading to improvement of globin chain biosynthesis in thalassemic cells.