Extended Data Fig. 3: Pharmacokinetics of Chol-HDO in CNS and plasma.
a, Time course of ASO distribution detected by anti-PS antibody staining in the cortex at 1, 24, and 48 hours after a single IV injection of 50 mg/kg Chol-HDO with LNA wings. The scale bar is 20 μm. b, Immunohistochemistry staining of anti-PS antibody in cerebral cortex, the choroid plexus and ependymal cells that line the lateral ventricle in the mouse brain 3 days after a single IV injection of 50 mg/kg single-stranded ASO or Chol-HDO with LNA wings. The ASO signals were observed in the choroid plexus (arrow) and ependymal cells (arrowhead) from mice treated with both ASO and Chol-HDO (upper panel). In contrast, the ASO signals were detected in neuron and glia in cortex from mouse treated with Chol-HDO, but not with ASO (lower panel). The scale bar is 50 μm. Lower panel show the higher magnification image of inset in upper panel. c, Ratiometric FRET imaging to localize separated cRNA and intact Chol-HDO with a Cy5-labeled ASO and a Cy3-labeled cRNA sense strand in layer I and II of the somatosensory cortex (upper panels) and cerebellum (lower panels). When the duplex is intact, the Cy3 transfers energy to Cy5 resulting in the gain of a unique FRET signal (ex540/em680). Far left heat map shows relative distribution of separated cRNA (red) and intact Chol-HDO (purple/blue/green). The majority of Chol-HDO localized at the pial basement membrane is intact and local separation is observed inside of neural cells (inset shows the neuron with close-colocalization of intact duplex and separated cRNA indicating separation of the duplex), vascular endothelial cells and ganglionic (Purkinje cell) layer of the cerebellum. The scale bar is 50 μm in the upper panel or 100 μm in the lower panel. d, Time course of ASO concentration in the plasma and cortex after a single IV injection of 50 mg/kg Chol-HDO containing ASO with LNA wings (3–4/group) on 1, 3, 7, 14, 21, 28, 42 or 56 days. As for plasma, the ASO concentration was also measured at 1, 3, 7, 14, 21, or 28 days after a single IV injection of 50 mg/kg ASO with LNA wings alone (3–4/groups). e, Comparison of Malat1 RNA levels after IV injection of 4 total dosing of 6.25 mg/kg (twice a week) and a single doing of 25 mg/kg Chol-HDO with LNA wings in different brain regions. (4/group; *P < 0.05; ** P < 0.001). f, Correlation of Malat1 RNA levels and ASO concentration in kidney after 1, 2, or 4 total IV dosing (1 dose/week) of 50 mg/kg of ASO or Chol-HDO with LNA wings (2–4/group).
