Evaluation of the analytical methods of antisense oligonueleotide applicable to the antisense therapy

适用于反义治疗的反义寡核苷酸分析方法评价

• 批准号: 13672386
• 负责人: OKUMURA Katsuhiko
• 金额: $ 2.18万
• 依托单位: KOBE UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13672386/
• 关键词: muscular dystrophy; gene therapy; antisense; blood concentration; HPLC; real time quantitative PCR

We are now planning to perform the gene therapy with antisense oligonueleotide (antisease ODN, 31-mer) for duchenne muscular dystrophy patients. For the monitoring of this antisense ODN concentrations in injection mediums and/or in plasma, two analytical methods were developed to obtain the high sensitivity and the good reliability ; the high performance liquid chromatography (HPLC) and the real time quantitative PCR methods.1. The reverse phase HPLC method (LC-10A (Shimazu Co. Ltd.)) was consisted from the mobile phase of 0.1 M triethylamine (pH 8.0) : CH_3CN (55 : 45→95 : 5) and ODS column (SG300 (Shiseido Co, Ltd.)). And this method works with high reproducibility at the range of 0.5-100 μg/ml with high linearity (r^2>0.993). For the real time quantitative PCR method, the reproducibility and linearity of antisense ODN with high Tm was much low (r<O.02). By the change of the PCR process, both of them were improved (0.05-lOμM, r=0.747).2. By HPLC method, stability of antisense ODN in … More injection was studied under several storage conditions ; shading or lighting, and 4℃, -80℃, or room temperature. Antisense ODN solved in saline was almost stable for 5 months under shading condition at less than 4℃ (more than 90% contents). In contrast, antisense ODN concentrations in saline were decreased by the light exposure, and two degradation products were detected. In other mediums ; saline, distilled water, Tris-EDTA buffer, and 5% glucose, antisense ODN were almost stable until 24 hrs under shading.3. In the measurement of antisense ODN concentrations in plasma, more than 90% recoveries were obtained at the 10 μg/ml in plasma, when additioning with ODNs complemental to the sequence of antisense ODN. The limited value of antisense ODN in plasma was 0.5 μg/ml. Compared with phenol extraction method, this method was considered to be simple, easy and high sensitive.We established the antisense ODN analytical methods with simple and high sensitivity, which might be applicable to monitor it's stability in injections and plasma concentration for the antisense gene therapy. Less

我们现在计划对杜氏肌营养不良症患者进行反义寡核苷酸（抗病ODN，31-mer）基因治疗。为了监测注射介质和/或血浆中的反义ODN浓度，开发了两种分析方法以获得高灵敏度和良好的可靠性；高效液相色谱法(HPLC)和实时定量PCR方法。 1．反相HPLC方法(LC-10A(Shimazu Co. Ltd.))由流动相0.1M三乙胺(pH 8.0):CH_3CN(55:45→95:5)和ODS柱(SG300(Shiseido Co., Ltd.))组成。该方法在0.5-100 μg/ml范围内重现性好，线性度高（r^2>0.993）。对于实时定量PCR方法，高Tm反义ODN的重现性和线性度很低(r<O.02)。通过改变PCR流程，两者均得到改善(0.05-10μM，r＝0.747)。2．采用HPLC法研究了反义ODN注射液在多种储存条件下的稳定性；遮光或光照，4℃、-80℃或室温。反义ODN在生理盐水中溶解，在4℃以下遮光条件下5个月基本稳定（含量90%以上）。相反，盐水中的反义 ODN 浓度因光照而降低，并检测到两种降解产物。在其他媒介中；生理盐水、蒸馏水、Tris-EDTA缓冲液、5%葡萄糖、反义ODN在遮光下24小时几乎稳定。3．在测定血浆中反义ODN浓度时，当添加与反义ODN序列互补的ODN时，血浆中10μg/ml的回收率超过90％。血浆中反义ODN的限量值为0.5μg/ml。与苯酚提取法相比，该方法简单易行、灵敏度高。我们建立了简单、灵敏度高的反义ODN分析方法，可用于反义基因治疗中监测其注射稳定性和血药浓度。较少的