Extended Data Fig. 1: Processing mechanism of HDO.
a, Schematic illustration of presumed processing mechanism of HDO in the cell. HDO is composed of a gapmer ASO, duplexed with a cRNA conjugated to delivery ligands. Gapmer ASOs have a central gap-region of DNA flanked by modified nucleotides that enhance affinity for cRNA, such as LNA, cEt or MOE, and whole nucleotides of ASO strand are protected from nucleases by PS modification. In the cRNA strand, the center portion hybridized to the gap portion of ASO remains unmodified for recognition of RNase H, and terminal nucleotides hybridized to both wing portions of ASO are protected from exonucleases by both PS modification and OMe. b, The effect of the cholesterol position and number in cRNA strand of Chol-HDO with LNA wings on Malat1 RNA levels. Cortex was harvested 3 days after one IV dose per week for 4 weeks of PBS or 50 mg/kg Chol-HDO (3–4/group; *P < 0.05; ** P < 0.001). c, Evaluation of Hdac2 expression in the cortex after co-administration of ASO targeting Hdac2 and Chol-HDO containing ASO with cEt wings targeting Malat1 after a single IV injection. Tissues were collected 1 week after dosing. Results show Chol-HDO does not facilitate Hdac2 ASO activity in the CNS indicating the absence of BBB damage by Chol-HDO (3/group). d, ICV administration of ASO targeting Hdac2 with cEt wings shows gene knockdown in the cerebral cortex at 30 or 100 µg doses (2/group), indicating that ASO targeting Hdac2 is effective in the CNS. Abbreviations (for all figures): ASO, antisense oligonucleotide; cEt, 2′, 4′- constrained Ethyl BNA; Chol, cholesterol; Chol-HDO, heteroduplex oligonucleotide conjugated with cholesterol; cRNA, complementary ribonucleic acid; DNA, deoxyribonucleic acid; DHA, Docosahexaenoic acid; Dmpk, dystrophia myotonica protein kinase; Gfap, glial fibrillary acidic protein; Hdac2, histone deacetylase 2; HDO, heteroduplex oligonucleotide; LNA, locked nucleic acid; Malat1, metastasis associated lung adenocarcinoma 1; MOE, 2′-O-Methoxyethyl ribonucleic acid; mRNA, messenger ribonucleic acid; OMe, 2′-O-methyl RNA ; PBS, phosphate-buffered saline; PO phosphodiester; PS phosphorothioate; RNA, ribonucleic acid; s.e.m., standard error of the mean; SOD1, superoxide dismutase 1; Tmax, time to maximal plasma concentration (Tmax); Toc, tocopherol; Toc-HDO, heteroduplex oligonucleotide conjugated with tocopherol.
