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. 2023 May 9;32:773–793. doi: 10.1016/j.omtn.2023.05.005

Figure 2.

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.