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剂，是丝氨酸蛋白酶抑制剂（serpin）超家族成员。它主要由肝细胞合成，其基因（13.5kb）定位于染色体1 q（23.1-23.9），由7个外显子和6个内含子组成。遗传性AT缺乏是静脉血栓栓塞症发展的公认危险因素，现在已证明是由一系列遗传缺陷引起的。为了从根本上治愈该疾病，通过引入同源重组靶向校正疾病相关突变被认为是基因治疗的最有效策略。Kmiec等于1996年提出了一种独特的嵌合RNA/DNA寡核苷酸定点突变方法，Steer等于1998年证实了该方法能有效地在体内对因子IX进行诱变，提示其可能成为治疗先天性血友病和Thr基因的新工具。 ...更多信息 一种由点突变引起的遗传性疾病。本研究的目的是证实这种新的方法的有效性，在人类AT基因中引入靶向核苷酸交换，以产生AT缺乏症模型。将含有与人AT基因的25个碱基互补的序列的嵌合2 ′-O-甲基化-RNA/DNA寡核苷酸构建为含有5390 C至T、5393 A至T或7431 C至G取代以引入错义突变的双链体。作为靶细胞，使用人肝癌细胞系HuH-7，其中AT mRNA的表达通过RT-PCR证实。寡核苷酸的转染用SuperFect Transfection Reagent<TM>（SFTR ; Qiagen Co.，德国）。通过共聚焦显微镜证实的FITC标记的全DNA寡核苷酸的核和细胞摄取来确定转染效率。分别通过PCR-RFLP和使用SEQ 4x 4个人测序系统（阿默舍姆Pharmacia Biotech，UK）的直接测序来筛选和确认成功的诱变。结果表明，SFTR的转染效率在60%~ 80%之间，并且有可能在体外将所有三种类型的突变引入HuH-7细胞中的人AT基因中。虽然有必要进一步阐明其在体内纠正遗传性AT基因突变的有效性，但本研究结果表明，RDO基因治疗可能是先天性AT缺乏症患者的替代治疗方法。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