UC Berkeley

Currently, many protein-based biologics on the market are limited to targeting molecules in the serum, on the cell surface, or within endocytic vesicles. However, targets inside cells have not been explored to a great extent because proteins have low cell permeability. To perform their intended functions in cells, proteins must escape from the endosomes, which is a prerequisite since most proteins are taken up into cells through endocytosis. While many strategies have been developed to promote intracellular protein delivery, they suffer from low delivery efficiency, rely on degradation-prone molecular scaffolds, operate via undefined mechanisms, and are evaluated using indirect or amplified assays. This work aims to address the challenges that hinder efficient protein delivery by better exploiting and understanding the concept of endosomal escape.Chapters 2 and 3 highlight the potential of the cell-permeant mini-protein ZF5.3 as a delivery vehicle that facilitates efficient endosomal escape of two therapeutically relevant proteins. Chapter 2 describes the ZF5.3-assisted delivery of a transcriptional regulator methyl-CpG-binding-protein 2 (MeCP2) implicated in Rett syndrome. The ZF5.3-MeCP2 conjugate reached the nucleus at therapeutically relevant concentrations and remained stable and active in the nucleus. Chapter 3 presents preliminary experiments that demonstrate the improved cytosolic delivery of a RAS-binding monobody inhibitor, NS1, using ZF5.3. Chapters 4 and 5 detail the design of two assays that can effectively evaluate protein delivery to the nucleus and cytosol in a high-throughput manner. The intact nuclear flow cytometry (INF) assay, discussed in Chapter 4, allows for the direct and non-amplified measurement of fluorescently tagged molecules in the nucleus. The results showed a strong linear correlation with results obtained using a quantitative technique – fluorescence correlation spectroscopy. Chapter 5 describes initial proof-of-concept experiments towards the development of a cell-based assay named Signal If and only if the Protein delivered is Intact (SIPI) that detects the cytosolic delivery of intact cargos. The development of such robust high-throughput assays can facilitate the understanding of the structure-activity relationship that governs intracellular access for cell-penetrating moieties. Lastly, we seek to understand the endosomal escape mechanism of ZF5.3 conjugated proteins in Chapters 2 and 6. Chapter 6 reports that ZF5.3 conjugated proteins with a lower Tm reached the cytosol at a higher concentration, suggesting protein unfolding plays a role in endosomal escape. Additionally, siRNA knockdown experiments showed that a functional homotypic fusion and protein sorting (HOPS) complex was crucial for the high delivery efficiency of ZF5.3-protein conjugates. These results support the hypothesis that ZF5.3 protein conjugates escape the endosomes through unfolding, followed by translocation through a portal that HOPS creates. Together, we envision the results reported in this work will contribute to the future development of better delivery strategies for proteins and other biologic-based therapeutics.