Characterizing and exploiting the endocytic pathway for macromulecular delivery

Sammanfattning: Macromolecular drugs with cytosolic or nuclear targets often exhibit low therapeutic activity due, at least in part, to their inability to escape endosomal compartments following cellular internalization. There exist a range of strategies to address this inefficient delivery by attempting to bolster the endosomal escape of the therapeutic cargo. Many of these delivery strategies apply to a broad range of molecular species, including proteins and nucleic acids. Two strategies of particular interest involve the use of extracellular vesicles (EVs) or endosomolytic small molecule compounds (SMCs). EVs are nanoscale, membrane-bound particles produced by all cell types and present in all bodily fluids. As a biological nanoparticulate species, EVs are inherently capable of delivering the material they contain to cells. Further, EVs can be modified through recombinant protein-based engineering strategies which can bestow a range of functional utilities such as fusogenicity, preferential cargo loading, and molecular targeting. However, the use of EVs as a scalable therapeutic modality is hampered by an inability to reliably mass-produce a homogenous population of these nanoparticles in vitro. SMCs, on the other hand, are easily synthesized at scale and can function in a stochastic manner dependent on an appropriate co-dosing strategy with their complementary therapeutic cargo. However, the mechanisms underlying SMC-mediated macromolecular delivery can be difficult to elucidate due to a lack of high-resolution characterization techniques. In this thesis, two issues - one underpinning each strategy - are investigated. First, the effects of culture media composition on the production of proteinloaded EVs in vitro are explored, with the ultimate aim of increasing EV output while characterizing the cellular biology driving the EV production. Certain serum components can differentially affect EV biogenesis by influencing ceramide-dependent EV biogenesis. In the second project, a functional screen of a novel family of SMCs is conducted to identify several chemical analogs in this family that demonstrate endosomolytic activity. Thereafter, superresolution and real-time microscopic assays are employed to determine the mechanism and consequence of the novel compounds during their co-treatment with a splice-switching oligonucleotide (SSO). SSOs are clinically relevant small-RNA therapeutics that alter the production of splice variants for a given genetic transcript. The novel SMCs bolster SSO activity by disrupting the structure of endosomes in a manner dependent on the acidification of the endosomal compartments, suggesting the SMCs display a buffering capacity at certain concentrations. The findings herein strengthen the potential of each delivery strategy as a therapeutically relevant approach to functionally delivering macromolecular cargo to cells.