Additionally, we synthesised a "dual activity" conjugate with ispenisib conjugated to the NCL1 gapmer, harnessing both RNase H-mediated degradation and lysosome trafficking. Synthesis was carried out as above, from a 5′-terminally amine-modified gapmer (SI Figure ). This dual activity NCL1 gapmer LyTON still outperformed the NCL1 gapmer with 40.7% less transcript at 100 nM (Figure , SI Figure ). While the NCL1 gapmer LyTON appeared to have performed better than the NCL1 2'-OMe LyTON, this difference was not significant (SI Figure ) This indicated that we may have reached a plateau in gene silencing efficiency, likely based on the affinity of the ASO sequence to bind its target mRNA. We measured little to no significant differences in the toxicity profiles of the unconjugated and ispinesib-conjugated oligonucleotides, assessed by Cell Titer Glo (SI Figure ) and RNA transcript levels of key housekeeping genes (SI Figure ). This lines up with our initial expectations, as our treatments are in the sub-micromolar range, well below concentrations that result in toxicity associated with kinesin spindle protein (Eg5) inhibition (the canonical target of ispinesib). These results demonstrated that we could achieve significant reduction of target mRNA with NCL1 2'-OMe LyTON treatment, which could not be attributed to standard RNase H activity. Next, we sought to confirm whether the mechanism of mRNA degradation involved the lysosome, as expected. Bafilomycin, a wellcharacterised inhibitor of lysosome activity , was used (Figure ). Bafilomycin inhibits vacuolar V-ATPase and prevents lysosomal acidification -disrupting basal lysosomal flux. If the NCL1 2'-OMe LyTON was trafficking the target mRNA to the lysosome, treatment with bafilomycin would result in loss of knockdown activity (Figure ). First, we treated the HEK293T cells with bafilomycin for 24h and confirmed the inhibition of lysosomal activity through an increase in LC3-II levels (SI Figure ). Cell toxicity was observed with 100 nM bafilomycin, therefore, the lower 10 nM concentration (without any cell toxicity effects) was chosen for further studies with the ASOs (SI Figure ). The knockdown of NCL1 transcript levels was then measured upon transfection with the NCL1 gapmer, NCL1 gapmer LyTON, NCL1 2'-OMe ASO, and NCL1 2'-OMe LyTON in the presence and absence of bafilomycin (Figure ). As expected, the bafilomycin treatment did not affect the activity of the NCL1 gapmer or NCL1 2'-OMe ASO. However, the enhanced mRNA knockdown activity of both the NCL1 2'-OMe and NCL1 gapmer LyTONs were completely lost in the presence of bafilomycin, showing comparable activity to the parent unmodified NCL1 2'-OMe ASO or NCL1 gapmer, respectively. This confirmed that LyTONs relied upon lysosomal degradation for activity, and upon inhibition of normal lysosomal flux only acted as a conventional ASOs. After establishing the chemistry and mechanism of action of LyTONs, we decided to target an exciting therapeutically-relevant gene of interest. For this, we chose the MEN1 gene that encodes for the protein, Menin -which plays a particularly significant role in MLL-rearranged leukemias. Menin acts as a scaffold for oncogenic MLL fusion proteins resulting in the transcriptional activation programs for leukemic cell survival and growth . Menin also has a key role in KRAS-driven cancers . Inhibition of Menin has been shown to disrupt such cancer-sustenance pathways, leading to reduced proliferation and increased apoptosis . Smallmolecule Menin inhibitors have shown early clinical promise, however, recent clinical studies have found emerging resistance and patient relapse due to somatic mutations in MEN1 . The structural mutations in Menin occur within a key region of the inhibitor-binding pocket essential for stabilizing Menin inhibitor binding but not required for interaction with MLL. These small molecule drug resistance challenges, which manifest at the protein level, can potentially be circumvented by targeting MEN1 mRNA using ASOs.