A Genome-wide CRISPR screen unveils the endosomal maturation protein WDR91 as a promoter of productive ASO activity in melanoma

Abstract

Antisense oligonucleotides (ASOs) belong to promising therapeutic molecules for the treatment of neurologic, muscular and metabolic disorders. Ten ASOs have been approved so far and many of them are in advanced clinical development covering a dozen therapeutic areas including cancer. Yet, their mechanisms of internalization, cell trafficking and action of these molecules remain poorly understood. Moreover, with an estimated 1% of ASO reaching the correct cellular compartment following systemic delivery, the majority of targeted diseases requires recurrent injections of ASOs. A deeper understanding of these mechanisms would guide the improvement of their efficiency and thus, reduce the amount of delivered ASOs and their potential side-effects. Here, we performed a genome-wide CRISPR knockout screen and identified several proteins which could significantly impact ASO potency in a melanoma cell line. From these candidates, we validated WD Repeat Domain 91 (WDR91) as a modulator whose depletion strongly inhibits ASO activity in melanoma. WDR91 is known as a regulator of endosomal maturation, suggesting that ASOs productive release in cytosol could mainly occur during early endosome maturation or from the late endosomes as supported by other studies. We show that this effect is also observed in another cell line with a different assay, ASO chemistry and mode of action, which could highlight a general mechanism that positively impacts ASOs trafficking in cells.