Figure 2.
LNP delivery increases ASO potency in murine primary CNS cell cultures
(A–C) TMEM106b mRNA expression in cortical neurons (teal), microglia (burgundy), astrocytes (blue), and OPCs (salmon) as assessed by qRT-PCR. (A) Cells were treated with 10 μM NC1 or TA2, and TMEM106b mRNA expression was analyzed after 3 days. (B) Dose-response curves analyzing TMEM106b mRNA expression after 3 days of TA2 treatment. (C) TMEM106b mRNA expression after 0, 4, 8, 16, 24, 48, 72, 144, and 192 h of treatment with 500 nM TA2. (D) Representative z-projection IF images of cells treated with 5 μM TA2 for 3 days (EC90-95). Scale bars, 25 μm. (E–H) TA2 dose-response curves generated in neurons treated for 3 days with 20 μM bicuculline (E), 1 μM TTX (F), 5 mM KCl (G), or 5 μM glutamate (H) and compared with non-treated (NT) cultures (teal). (I and J) TA2 dose-response curves generated in microglia treated for 3 days with 1× LPS (I) or 20 ng/mL IL-4+IL-13 (J) and compared with NT cultures (burgundy). (K) Cells were treated with 2, 4, 20, 100, and 500 nM TA2-LNPs (dashed) and compared with gymnosis controls (solid), showing increased potency with LNP delivery. TMEM106b mRNA expression was analyzed by qRT-PCR after 3 days. (L) Representative z-projection IF images of cells treated with 100 nM TA2-LNPs for 3 days (EC90-95). Scale bars, 25 μm. Gymnosis dose-response curves were generated using 2, 4, 20, 100, 500, 2,500, 5,000, and 10,000 nM TA2. All mRNA expression data were normalized to GAPDH- and PBS-treated control cells. Three internal replicates were averaged and experiments were completed in triplicate, mean ± SEM are shown.