Selective photoactivation of proteins in mammalian cells for research and therapy

Protein therapeutics is a fast-growing field with the number of proteins licensed as therapeutics by the U.S. Food and Drug Administration rising every year. Therapeutic proteins often suffer fewer undesired secondary effects than small-molecule drug equivalents due to their unique structure, allowing them to perform more complex functions, and mediate more specific responses inside and outside cells. Furthermore, they are fully degradable by the cellular machinery, eliminating bio-accumulation problems. However, delivery of proteins to the cytosol of a specific population of cells is a considerable challenge, which has so far limited protein therapeutics to extracellular applications. Current tools to deliver proteins inside cells are not specific, and the generic method used to create specific agents, antibody bioconjugation, dramatically increases the chance of inducing an undesired immune response. This thesis focuses on developing, characterising and optimising a novel system to deliver inactive proteins into mammalian cells and recover their activity upon irradiation with a particular wavelength of light. This photo-activation of delivered protein (PADP) system was formed of three main components: an intein to prevent protein activity by disrupting the protein fold, a photocage to control intein activity by light, and a cell-penetrating peptide (CPP) to deliver the protein inside cells. The PADP system was firstly implemented to deliver the red-fluorescent protein mCherry to allow a direct characterisation of the system by fluorescence spectroscopy. The intein-split mCherry construct was studied in vitro to determine the optimal irradiation time, pH and additives for complete photocage cleavage. Several CPPs were attached to native mCherry to determine the best CPP candidate for cytosolic delivery into HeLa cells by flow cytometry and live-cell confocal microscopy. The resulting PADP was then proven to successfully be delivered into HeLa cells and photo-activated to form correctly folded, fluorescent mCherry. The versatility of the PADP concept was then demonstrated by the development of variants to deliver two cytotoxic proteins, saporin and barnase. Cytotoxic proteins are widely studied in cancer research for future use as therapeutics, but current approaches offer no control over protein activity and require antibody bioconjugation. Both PADP constructs selectively killed cells upon light-activation, demonstrating the potential for novel therapeutic proteins. The PADP system is a universal tool that could be potentially implemented in any protein and activated by light with spatiotemporal control

Spatio-temporal control of cell death by selective delivery of photo-activatable proteins

Protein therapeutics offer exquisite selectivity in targeting cellular processes and behaviors, but are rarely used against non-cell surface targets due to their poor cellular uptake. While cell-penetrating peptides can be used to deliver recombinant proteins to the cytosol, it is generally difficult to selectively deliver active proteins to target cells. Here, we report a recombinantly produced, intracellular protein delivery and targeting platform that uses a photocaged intein to regulate the spatio-temporal activation of protein activity in selected cells upon irradiation with light. The platform was successfully demonstrated for two cytotoxic proteins to selectively kill cancer cells after photo-activation of intein splicing. This platform can generically be applied to any protein whose activity can be disrupted by a fused intein, allowing it to underpin a wide variety of future protein therapeutics