A Directed Evolution Approach to Affinity-Based Protein Delivery

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PROJECT SUMMARY Tissue regeneration is a dynamic, carefully coordinated process in which many proteins and cell populations participate. Disruptions in the healing cascade caused by chronic disease or severe injury can easily impair tissue regeneration, resulting in injuries that do not heal. Our long-term goal is to design affinity-based hydrogels that can provide phased delivery of multiple therapeutic proteins to enhance tissue repair. We have developed a “bottom-up” modular approach to protein delivery, in which specific affinity interactions between small protein domains (i.e. binding partners) and therapeutic proteins are integrated into biomaterials to independently and predictably control the release of multiple proteins. We are using directed evolution of yeast surface display libraries to identify binding partners with high specificity and moderate affinities for proteins, to enable protein release over different timescales. We expect that our approach will more accurately recapitulate the natural, staggered presentation of multiple proteins during the healing cascade, providing the necessary combinations of proteins to activate key phases of the repair process. In Aim 1, we will evolve binding partners for therapeutic proteins using yeast surface display. We will characterize an assortment of affibodies specific to each therapeutic protein to generate a large diversity of orthogonal, protein-affibody affinity interactions. In Aim 2, we will use statistical modeling to optimize biomaterial properties to achieve desired protein release profiles. We will feed this information into COMSOL bio-transport models to predict protein delivery in vitro and in vivo. In Aim 3, we will synthesize hyaluronic acid hydrogels containing binding partners to investigate tunable co-delivery of multiple proteins in vitro and in vivo. Our modeling results will inform the design of biomaterials that enable the delivery of multiple proteins with distinct release profiles. In vitro protein release from hydrogels will be evaluated via ELISA. In vivo retention of fluorescently labeled proteins within implanted hydrogels will be evaluated using longitudinal live fluorescence imaging of rats. Ultimately, we expect to achieve independent control over the release of a wide range of therapeutic proteins relevant to tissue repair through orthogonal protein-material affinity interactions. We expect that our versatile biomaterial platform can be applied to the precise delivery of a broad range of proteins and will enable systematic investigation of the timing of protein presentation required for tissue healing. This work will lay the foundation to cultivate clinically-relevant strategies for stimulating robust tissue repair in multiple organ systems.