Figure 4.

ASO-LNPs induce gliosis in mice following i.c.v. injection

(A) TA2-LNP formulations screened for TMEM106b mRNA regulation in vitro. (B and C) TMEM106b mRNA expression in cortical neurons (B) and microglia (C) as determined by qRT-PCR. Cells were treated with 20 nM TA2 (leftmost bar) or TA2-LNP formulations (1–6) for 24 h. Data were normalized to GAPDH- and PBS-treated control cells. Three internal replicates were averaged and the experiments were completed in triplicate. One-way ANOVA with multiple comparisons Dunnett’s test (TA2 control), ∗p < 0.03, ∗∗p < 0.002, ∗∗∗p < 0.0002. (D) Representative cryo-EM images of unloaded, and TA2-loaded GenVoy (formulation 1) and MC3 (formulation 6) LNPs showing the internal structures of the formulations selected for in vivo delivery. Scale bars, 200 nm. (E and F) Particle sizes (E) and polydispersity measurements (F) of unloaded and TA2-loaded LNPs as determined by dynamic light scattering. (G) C57BL/6N mice were i.c.v. injected with saline (n = 6), TA2 (100 or 25 μg, n = 6 per group), TA2-LNPs (25 μg, n = 6 per group), or empty LNPs (n = 3 per group) and analyzed over 14 days. Illustration created using BioRender.com. (H) Acute toxicity 1 h post injection. (I) Percent body weight change normalized to day 0. GenVoy-treated groups showed signs of toxicity and were monitored closely post injection. One mouse in the TA2-GenVoy group did not survive the duration of the experiment. (J) Representative Iba1 IHC images from brain sections obtained 14 days post i.c.v. injection. Scale bars, 500 μm. (K and L) IHC quantification for total (K) and clustered (L) Iba1 signals. For all in vivo statistical analyses, one-way ANOVA with multiple comparisons Dunnett’s test (saline control) was used, ∗p < 0.03, ∗∗p < 0.002, ∗∗∗p < 0.0002, ∗∗∗∗p < 0.0001. Individual group comparisons (empty vs. TA2-loaded LNPs) were analyzed using unpaired two-tailed Student’s t test, ∗p < 0.05. For all panels, mean ± SEM are shown and the 100 μg TA2 dose is represented by bold font.