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

DNA-RNA寡核苷酸治疗先天性血栓性疾病的初步评价

• 批准号: 11671009
• 负责人: TSUJI Hajime
• 金额: $ 2.18万
• 依托单位: KYOTO PREFECTURAL UNIVERSITY OF MEDICINE
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671009/
• 关键词: OLIGONECLEOTIDE; THROMBOSIS; GENE; ANTITHROMBIN

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

抗凝血酶 (AT) 是凝血酶的主要血浆抑制剂，并且是丝氨酸蛋白酶抑制剂 (serpin) 超家族的成员。它主要由肝细胞合成，其基因（13.5 kb）定位于染色体1q（23.1-23.9），由7个外显子和6个内含子组成。遗传性 AT 缺陷是公认的静脉血栓栓塞发生的危险因素，现已证明静脉血栓栓塞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基因治疗的最有效策略。 Kmiec等人于1996年提出了一种利用嵌合RNA/DNA寡核苷酸(RDO)进行定点诱变的新颖而独特的方法，后来Steer等人于1998年证明该方法可有效地在体内引入因子IX诱变，表明其作为治疗先天性血友病和由点突变引起的血栓性疾病的新工具的可能性。本研究的目的是证实这种新方法在人类 AT 基因中引入靶向核苷酸交换以产生 AT 缺陷模型的有效性。含有与人AT基因25个碱基互补的序列的嵌合2'-O-甲基化-RNA/DNA寡核苷酸被构建为含有5390C至T、5393A至T或7431C至G取代以引入错义突变的双链体。使用人肝癌细胞系HuH-7作为靶向细胞，通过RT-PCR确认其中AT mRNA的表达。用SuperFect转染试剂TM (SFTR；Qiagen Co.，德国)进行寡核苷酸的转染。转染效率通过共聚焦显微镜确认的 FITC 标记的全 DNA 寡核苷酸的核和细胞摄取来确定。分别通过 PCR-RFLP 和使用 SEQ4x4 个人测序系统（Amersham Pharmacia Biotech，英国）的直接测序来筛选和确认成功的诱变。结果，SFTR的转染效率在60%至80%之间，并且可以在体外将所有三种类型的诱变引入HuH-7细胞中的人AT基因中。尽管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，但目前的结果表明 RDO 基因疗法可能成为先天性 AT 缺陷患者的替代治疗方法。较少的