Figure 6

Cumulative gene silencing effect by repeated administration of Toc-HDO. (A) OAT3 mRNA levels measured by quantitative RT-PCR in brain homogenates after four intravenous injections (at 1-week intervals) of 13-mer ASO, HDO, or Toc-HDO (No. 18) at doses corresponding to 50 mg/kg of ASO. Brains were obtained 72 h after the last injection. Data shown are relative to CD31 mRNA levels and are expressed as mean values ± s.e.m. (N = 3, **P < 0.01). P values were calculated using Student’s two-tailed t-test. (B) Western blot analysis of the OAT3 protein in the microvascular fraction of the brains of mice treated as in (A). β-actin was used as an internal control. P, positive control (kidney sample from a wild-type mouse); N, negative control (kidney sample from an OAT3-knockout mouse). Quantification of the OAT3 band densities are expressed as mean values ± s.e.m. (N = 3, *P < 0.05, **P < 0.01). P values were calculated using Student’s two-tailed t-test. (C) Radioactivity of the ^99mTc-ECD metabolite in the brain measured 2 or 30 min after an intravenous injection of ^99mTc-ECD into mice that were injected four times at 1-week intervals, by up to 72 h before, with PBS (open squares) or 13-mer Toc-HDO (No. 18) at a dose corresponding to 50 mg/kg of ASO (closed squares). Data are expressed as mean values ± s.e.m. (N = 5, *P < 0.05); regression lines show the retention of the ^99mTc-ECD metabolite in the brain. P values were calculated using Student’s two-tailed t-test.