Figure 3.

Single dose screen of 5-9-5 2′-O-methoxyethyl (MOE) and cEt antisense oligonucleotides (ASOs). 300 µg of ASO was delivered by intracerebroventricular (ICV) bolus injection to the right lateral ventricle of 2–3-month-old Hu97/18 mice. Four weeks later, brains were collected and sectioned in a 2 mm coronal rodent brain matrix. The first section containing mostly olfactory bulb was discarded. The second section, containing anterior cortex and striatum, was used for HTT quantitation by allelic separation immunoblotting. The remaining posterior portion of the brain was used for immunohistochemical evaluation of ASO distribution and tolerability. (a) Example western blots showing wt and muHTT protein. ASOs in orange contain only MOE modifications. ASOs in blue contain cEt modifications. (b) Quantitation of HTT protein in both hemispheres of 4 animals. Density of HTT bands was normalized to calnexin loading control and then expressed as a percentage of the same allele (either wtHTT or muHTT) from brain lysates of PBS injected animals on the same membrane. Error bars are SEM. *P < 0.05, **P < 0.01, ***P < 0.001 difference between wt and muHTT by Bonferroni post hoc analysis following two-way analysis of variance. (c) Example immunohistochemistry demonstrating the range of astrogliosis (GFAP reactivity) observed with screened ASOs, as a measure of tolerability. Scale bars are 250 µm for 5× image and 100 µm for 20× image.