Extended Data Fig. 2: The distribution and gene knockdown effect of Chol-HDO in multiple CNS regions.
a, Dose response curve showing Malat1 RNA knockdown in the spinal cord after a single IV injection of PBS or Chol-HDO with LNA or cEt wings (Dose for LNA wings, 12.5, 25, 50 or 75 mg/kg, for cEt wings 7, 20 or 50 mg/kg) (4/group). Spinal cord was harvested 3 days after dosing. Malat1 RNA levels in the cortex were normalized to Actin mRNA. Dose dependency was also observed in other brain regions with the cerebellum being the least sensitive to the HDO (data not shown). Time course of Malat1 RNA reductions in the spinal cord on 3, 7, 14, 28, 56, 168 or 252 days after a single IV injection of 50 mg/kg Chol-HDO with LNA wings (4/group; *P < 0.05; ** P < 0.001). b, The effect of 50 mg/kg Toc-HDO and Chol-HDO with LNA wings after a single IV injection on Malat1 RNA expression in various brain regions and spinal cord compared to 50 mg/kg ASO or PBS. Tissues were harvested 3 days after injection (4/group; *P < 0.05; ** P < 0.001). (c/d) In situ hybridization with a Malat1 probe in the cortex (layers I/II–VI) (c) and the anterior horn of spinal cord (d), dosing paradigm as described in Fig. 1c. Each panel (c) shows all cortical layers from the pial surface/layer I (left) to layer VI (right). The dashed lines in (d) indicate the border between white and gray matter, and the area within the dashed line is the anterior horn of spinal cord. The scale bar is 100 μm. Since Malat1 is ubiquitously expressed15, the near complete inhibition of probe signal suggests that RNA expression was reduced in both neurons and glia after Chol-HDO administration. Similar Malat1 RNA reduction was also confirmed in cerebellum, striatum, hippocampus, and brain stem (data not shown). e, Time courses of Malat1 RNA levels in the cortex and spinal cord on 7, 14 or 28 days after single SC injection of 50 mg/kg Chol-HDO with LNA wings (4–6/group; *P < 0.05; ** P < 0.001). f, Dose response curves of Malat1 gene knockdown effect comparing IV and SC injection with dosing of 50 mg/kg Chol-HDO with cEt wings once a week for four weeks (2–3/group). g, The effect of 50 mg/kg Chol-HDO with MOE gapmer ASOs on Malat1 RNA expression levels in different brain regions after a single IV injection of 50 mg/kg. Tissues were harvested 3 days after the last injection (4/group; *P < 0.05). h, The effect of Chol-HDO with cEt gapmer ASOs on Malat1 RNA in brain regions and spinal cord after IV administration of 50 mg/kg Chol-HDO once a week for four weeks. Tissues were harvested 3 days after the last injection (4/group; *P < 0.05; ** P < 0.001). i, Chol-HDO with MOE wings reduces the expression of Gfap, causative gene of Alexander disease, in various brain regions and spinal cord after IV administration of 50 mg/kg Chol-HDO once a week for four weeks. The brain and spinal cords were harvested 3 days after the last injection (4/group; *P < 0.05; ** P < 0.001). There is no clear reduction in GFAP protein in the CNS of mice treated with Chol-HDO by a Quanterix ELISA assay. While this experimental design described above was sufficient for detecting mRNA knockdown, it was too short to evaluate GFAP protein reduction as this protein is known to have a long half-life (~27 days for cortex and ~9 weeks for spinal cord). j, Chol-HDO with LNA wings reduces the expression of SOD1, the causative gene of familial amyotrophic lateral sclerosis, in various brain regions and spinal cord after SC administration of 50 mg/kg Chol-HDO once a week for four weeks (upper panel). The brain and spinal cords were harvested 7 days after the last injection (4–6/group; *P < 0.05). Human SOD1 protein expression from the brain treated with Chol-HDO was also reduced as compared to that from the brain treated with PBS on immunoblotting (lower panel). Immunoblotting and densitometry analysis (3/group; *P < 0.05) showing that Chol-HDO significantly reduced the levels of SOD1 protein applied by 10 μg total protein in brain as compared to the brain treated with PBS (lower panel).
