Design rules for efficient endosomal escape

The inefficient translocation of proteins across biological membranes limits their application as therapeutic compounds and research tools. In most cases, translocation involves two steps: uptake into the endocytic pathway and endosomal escape. Certain charged or amphiphilic molecules promote protein uptake but few enable efficient endosomal escape. One exception is ZF5.3, a mini-protein that exploits natural endosomal maturation machinery to translocate across endosomal membranes. Although certain ZF5.3-protein conjugates are delivered efficiently into the cytosol or nucleus, overall delivery efficiency varies widely with no obvious design rules. Here we evaluate the role of protein size and thermal stability in the ability to efficiently escape endosomes when attached to ZF5.3. Using fluorescence correlation spectroscopy, a singlemolecule technique that provides a precise measure of intra-cytosolic protein concentration, we demonstrate that delivery efficiency depends on both size and the ease with which a protein unfolds. Regardless of size and pI, low-Tm cargos of ZF5.3 (including intrinsically disordered domains) bias its endosomal escape route toward a high-efficiency pathway that requires the homotypic fusion and protein sorting (HOPS) complex. Small protein domains are delivered with moderate efficiency through the same HOPS portal even if the Tm is high. These findings imply a novel protein- and/or lipid-dependent pathway out of endosomes that is exploited by ZF5.3 and provide clear guidance for the selection or design of optimally deliverable therapeutic cargo. Significance Statement The results described in this paper provide new insights into how protein delivery works and how it can be best utilized in the future. Although intracellular protein delivery has been studied for decades, this paper describes the first interrogation of why certain protein cargos are privileged for efficient endosomal escape. These results represent a fundamental advance in the long-awaited goal of efficient protein delivery and provide design rules to overcome one of the most significant challenges for the future of biotechnology. ### Competing Interest Statement M.Z. and A.S. have submitted a provisional patent application related to this work.