Figure 3.

LNP uptake differentially affects ASO delivery and trafficking in primary CNS cells

(A) Representative z-projection IF images of cortical neurons, microglia, astrocytes, and OPCs treated for 3 days with 5 μM TA2 or 100 nM TA2-LNPs (EC90-95) showing ASO localization (green) with endolysosomal markers EEA1, LAMP1, and GRASP1 (red). Scale bars, 10 μm and 5 μm (inset). (B–E) Mean Mander’s coefficients quantifying the fraction of overlap/colocalization between TA2 and EEA1, LAMP1, or GRASP1 after 3 days of gymnosis (solid) or LNP delivery (checked) in cortical neurons (B), microglia (C), astrocytes (D), or OPCs (E). ASO signal within and on the membrane was analyzed from single-plane manually thresholded images. Unpaired two-tailed Mann-Whitney test, ∗∗∗∗p < 0.0001. (F) Representative 2D deconvolved IF images showing ASO colocalization with LAMP1 in cortical neurons (left) and microglia (right) after 3 days of gymnosis or LNP delivery. Scale bars, 2 μm. (G) Internalized TA2 concentrations in cortical neurons and microglia after gymnosis or ASO-LNP delivery as determined by MOL-PCR. Cells were treated with 100 nM final ASO concentration for 24 h, mean ± SEM are shown (n = 3). Unpaired two-tailed Student’s t test, ∗∗∗p < 0.001.