Syn-R-bodies: hijacking a bacterial pH-responsive polymer as a modular platform for the delivery of biological therapeutics
Engineering cells directly in vivo represents a transformative opportunity in modern medicine. One method Nature uses to deliver proteins into the cytoplasm of cells is R-bodies, natural protein polymers that undergo a reversible change from a ~ 0.5 µm cylinder to a rigid helical tube ~10 µm that is hypothesized to release toxins from organelles into the cytoplasm of cells. Here, we develop synthetic R-bodies (Syn-R-bodies), in which genetic code expansion and bioorthogonal click chemistry are used to convert R-bodies into a modular, programmable platform for introducing targeting moieties and therapeutic payloads for the delivery large biomolecule payloads to the cytoplasm of cells. First, we introduce bio-orthogonal click handles into a tunable subset of R-body monomers using genetic code expansion. Then, post-assembly installation of a bi-functional conjugation arm with competitive reactivities functionalizes Syn-R-bodies with both receptor-targeting ligands and diverse payloads, demonstrating the modularity of the system. Modified Syn-R-bodies retain the characteristic reversible, pH-triggered contract–extend transition and exhibit morphologies comparable to unmodified polymers by SEM, confocal microscopy, and flow cytometry. Using receptor targeting to the cation-independent mannose 6-phosphate receptor (CI-MPR) or transferrin receptor (TfR), Syn-R-bodies drive productive receptor-mediated uptake and access to the cytosolic space. Together, Syn-R-bodies provide a modular, mechanically actuated platform for cytosolic delivery of large biomolecules that addresses a core limitation of existing non- viral in vivo cell engineering technologies.
