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

我们现在计划用反义寡核苷酸（抗病 ODN，31 聚体）对杜氏肌营养不良症患者进行基因治疗，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来获得。灵敏度高、可靠性好；高效液相色谱法（HPLC）和实时定量PCR方法。 1．反相HPLC法（LC-10A（岛津公司）有限公司））从0.1 M三乙胺（pH 8.0）：CH_3CN（55：45→95：5）和ODS柱（SG300（资生堂有限公司））的流动相开始，并且该方法具有高重现性。在0.5-100 μg/ml范围内具有高线性度(r^2>0.993)。高Tm反义ODN的重复性和线性度均较低(r<0.02)，通过改变PCR方法，两者均得到改善(0.05-10μM，r=0.747)。2．研究了不同储存条件下反义ODN在注射液中的变化，4℃、-80℃或室温下的反义ODN在盐水中的溶解度几乎相同。 4℃以下避光条件下可稳定保存5个月（含量大于90%），而盐水中的反义ODN浓度则因光照而降低，在其他培养基中检测到两种降解产物。 、Tris-EDTA缓冲液和5%葡萄糖，反义ODN在遮光下24小时几乎稳定。 3．血浆中反义ODN浓度的测定，超过90%。当添加与反义ODN序列互补的ODN时，血浆中的回收率为10μg/ml，与苯酚提取法相比，该方法被认为是血浆中反义ODN的限量值为0.5μg/ml。简单、易行、灵敏度高。我们建立了简单、高灵敏度的反义ODN分析方法，可用于监测反义基因治疗中注射液和血浆浓度的稳定性。