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

抗凝血酶(AT)是凝血酶的主要血浆抑制物，是丝氨酸蛋白酶抑制物(SERPIN)超家族成员。它主要由肝细胞合成，其基因全长13.5kb，位于染色体1q(23.1-23.9)，由7个外显子和6个内含子组成。遗传性血管紧张素转换酶缺乏症是公认的静脉血栓栓塞症的危险因素，现已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年提出了一种新颖而独特的利用嵌合RNADNA寡核苷酸进行定点突变的方法，该方法后来被Steer等人于1998年在体内有效地引入了对凝血因子IX的突变，表明它可能成为治疗先天性血友病和Thr…的一种新工具。更多的基因疾病是由点突变引起的。本研究的目的是证实这种新的方法在人类AT基因中引入靶向核苷酸交换以制造AT缺乏症模型的有效性。构建了含有人AT基因25个碱基互补序列的嵌合2‘-O-甲基化RNA/DNA寡核苷酸，构建了含有5390C-T、5393A-T或7431C-G替换的双链，以引入错义突变。以人肝癌细胞系HH-7为靶细胞，RT-PCR法证实AT基因的表达，用SuperFect转基因试剂(SuperFect转基因试剂)进行寡核苷酸转导。通过共聚焦显微镜证实FITC标记的All-DNA寡核苷酸的核摄取和细胞摄取来确定转染率。利用SEQ4x4个人测序系统(amersham Pharmacia Biotech，UK)，分别用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)和直接测序法对诱变成功进行筛选和确认。结果表明，SFTR的转染率在60%~80%之间，三种诱变方法均有可能在体外将人AT基因导入HH-7细胞中。尽管RDO基因治疗在体内纠正遗传性AT基因突变的有效性尚需进一步阐明，但本研究结果提示RDO基因治疗可作为先天性AT缺乏症患者的一种替代治疗方法。较少

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