PRELIMINARY EVALUATION OF THE TREATMENT OF CONGENITAL THROMBOTIC DISORDERS BY DNA-RNA OLIGOMUCLEOTIDE

抗凝血酶（AT）是血浆中凝血酶的主要抑制剂，是丝氨酸蛋白酶抑制剂（serpin）超家族的一员。它主要由肝细胞合成，其基因（13.5 kb）被分配到染色体1q（23.1-23.9）上，由7个外显子和6个内含子组成。遗传性AT缺乏症是静脉血栓栓塞的一个公认的危险因素，现在已被证明是由一系列遗传缺陷引起的。为了从根本上治愈疾病，通过引入同源重组来靶向纠正疾病相关突变被认为是最有效的基因治疗策略。Kmiec等人于1996年提出了一种利用嵌合RNA/DNA寡核苷酸（chimeric RNA/DNA oligonucletide， RDO）进行位点定向诱变的新颖独特方法，1998年Steer等人证实了该方法在引入体内因子IX诱变中是有效的，这表明它有可能成为治疗先天性血友病和其他由点突变引起的更多血源性疾病的新工具。本研究的目的是证实这种新方法在引入人类AT基因靶向核苷酸交换以产生AT缺乏症模型中的有效性。构建了一个与人类AT基因25个碱基互补的2′- o -甲基化rna /DNA嵌合寡核苷酸，其双链结构可能包含5390C到T、5393A到T或7431C到G的取代，从而引入错义突变。以人肝癌细胞株HuH-7为靶细胞，通过RT-PCR证实了AT mRNA的表达。用superect转染试剂^<TM> （SFTR; Qiagen Co.，德国）转染寡核苷酸。转染效率是通过细胞核和细胞摄取fitc标记的全dna寡核苷酸来确定的，共聚焦显微镜证实。成功诱变分别通过PCR-RFLP和SEQ4x4个人测序系统（Amersham Pharmacia Biotech， UK）进行筛选和直接测序。因此，SFTR转染效率在60 - 80%之间，可以在体外将这三种类型的诱变引入HuH-7细胞中的人AT基因。虽然还需要进一步阐明其在体内纠正遗传性AT基因突变的有效性，但目前的结果表明，rdo基因治疗可能是先天性AT缺乏患者的一种替代治疗方法。少

Antithrombin (AT) is the major plasma inhibitor of thrombin, and is a member of the serine proteinase inhibitor (serpin) superfamily. It is mainly synthesized by the hepatocytes, and its gene (13.5 kb) has been assigned to chromosome 1q (23.1-23.9), consisting of 7 exons and 6 introns. Inherited AT deficiency is a well-recognized risk factor for the development of venous thromboembolism, which has now been shown to be caused by a spectrum of genetic defects. In order to fundamentally cure the disease, targeted correction of the disease related mutation by introducing homologous recombination is considered to be the most effective strategy for gene therapy. A novel and unique method of site-directed mutagenesis using chimeric RNA/DNA oligonucleotide (RDO) was introduced by Kmiec et al in 1996, which was later proven to be effective in introducing mutagenesis in the factor IX in vivo by Steer et al in 1998, suggesting its possibility as a novel tool to treat congenital hemophilic and thr … More ombogenic disease arising from a point mutation. The aim of this study is to confirm the effectiveness of this novel method to the introduction of targeted nucleotide exchange in human AT gene to produce a model of AT deficiency. A chimeric 2'-O-methylated-RNA/DNA oligonucleotide containing sequences complementary to 25 bases of the human AT gene was constructed as a duplex containing either 5390C to T, 5393A to T or 7431C to G substitution to introduce missense mutation. As a targeting cell, human hepatoma cell line HuH-7 was used, within which the expression of AT mRNA was confirmed by RT-PCR.Transfection of the oligonucleotide was performed with SuperFect Transfection Reagent^<TM> (SFTR ; Qiagen Co., Germany). The transfection efficiency was determined by nuclear and cellular up-take of FITC-labeled all-DNA oligonucleotide confirmed with confocal microscopy. Successful mutagenesis were screened and confirmed by PCR-RFLP and direct-sequencing using SEQ4x4 personal sequencing system (Amersham Pharmacia Biotech, UK), respectively. As a result, transfection efficiency with SFTR was between 60 and 80% and it was possible to introduce all three types of mutagenesis into human AT gene in HuH-7 cells in vitro. Although it is necessary to further elucidate its effectiveness in correcting inherited AT gene mutation in vivo, the present results suggest that the RDO-gene-therapy could be an alternative treatment for patients with congenital AT deficiency. Less