Understanding the mechanism by which certain peptides and peptide mimetics escape from endosomes into the cytosol is essential for the design of next-generation peptide- and protein-based therapeutics. We previously reported that small protein mimetics containing a discrete penta-arg motif escaped from endosomes with unprecedented efficiency. Penta-arg proteins access the cytosol with efficiencies that are at least 3-fold higher than those observed for the widely used cationic sequences derived from HIV Tat or octaarginine. Here, we describe a high-content imaging-based, genome-wide, RNA interference (RNAi) screen and used it to identify genes that up- and down-regulate the endosomal release of penta-arg proteins. Candidate genes were validated using both flow cytometry (FC - to assess total uptake) and fluorescence correlation spectroscopy (FCS - to precisely and accurately quantify endosomal release). This combined approach distinguishes genes that control overall uptake from those that specifically modulate endosomal escape, and have provided key insights into the molecular machinery controlling the release of various canonical and novel peptide mimetics from endosomes.

What machinery controls the release of peptide mimetics from endosomes? A genome-wide analysis

253rd National Meeting (2017)

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