To test whether we could use the ATTEC-warhead ispinesib for lysosomal trafficking and subsequent lysosomal degradation of target mRNA (Figure ), we initially opted to attach it to an ASO with a fully 2'-O-Methyl (2'-OMe) sugar modified-backbone. This chemistry is known to be RNase H-inactive, and would allow us to assess the Ispinesib-ASO without interference from RNase H-mediated degradation. We chose a previously validated ASO sequence designed to target cytoplasmic NCL1 mRNA 25 (Figure ). A 5'-dibenzocyclooctyne (DBCO) NCL1 ASO was synthesised by reacting a 5′-terminally modified-amine 2'-OMe ASO with excess DBCO-Nhydroxysuccinimide (NHS) ester. We then used strain-promoted azide-alkyne click (SPAAC) chemistry to couple commercially available ispinesib-azide to the 5'-DBCO 2'-OMe ASO (Figure ), achieving >90% yields for all bioconjugation reactions performed and >95% purity post HPLC purification (SI Figure ).