|Budget Amount *help
¥3,400,000 (Direct Cost : ¥3,400,000)
Fiscal Year 2002 : ¥1,700,000 (Direct Cost : ¥1,700,000)
Fiscal Year 2001 : ¥1,700,000 (Direct Cost : ¥1,700,000)
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
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