A Directed Evolution Approach to Affinity-Based Protein Delivery

项目总结 组织再生是一个动态的、精心协调的过程，在这个过程中，许多蛋白质和细胞群体 参与进来。由慢性病或严重损伤引起的愈合级联中断很容易损害 组织再生，导致无法愈合的损伤。我们的长期目标是设计基于亲和力的水凝胶 它可以提供多种治疗性蛋白质的阶段性输送，以增强组织修复。我们已经开发出 一种“自下而上”的模块化蛋白质传递方法，在这种方法中，小蛋白之间的特定亲和力相互作用 结构域(即结合伙伴)和治疗性蛋白被整合到生物材料中，以独立和 可以预测地控制多种蛋白质的释放。我们正在使用酵母表面展示的定向进化 寻找与蛋白质具有高度特异性和中等亲和力的结合伙伴的文库，以使蛋白质能够 在不同的时间尺度上释放。我们预计，我们的方法将更准确地概括自然、 在愈合级联过程中，多种蛋白质交错呈现，提供必要的组合 以激活修复过程的关键阶段。在目标1中，我们将进化出用于治疗的结合伙伴 利用酵母菌表面展示蛋白质。我们将描述每种疗法特有的一类亲和抗体。 蛋白质产生大量多样性的正交性、蛋白质-亲和体亲和相互作用。在目标2中，我们将使用 统计建模，以优化生物材料的属性，以实现所需的蛋白质释放曲线。我们会喂饱你 将这些信息输入到COMSOL生物运输模型中，以预测蛋白质在体外和体内的输送。在目标3中，我们 将合成含有结合伙伴的透明质酸水凝胶以研究多个 体外和体内的蛋白质。我们的建模结果将为实现交付的生物材料的设计提供信息 具有不同释放谱的多个蛋白质。水凝胶的体外蛋白质释放将通过以下方式进行评估 是ELISA.植入的水凝胶中的荧光标记蛋白的体内保留率将使用 大鼠纵向活体荧光成像。最终，我们希望实现对 通过正交蛋白质材料释放与组织修复相关的多种治疗性蛋白质 亲和力相互作用。我们期待我们的多功能生物材料平台可以应用于精确交付 广泛的蛋白质，并将使系统研究所需的蛋白质呈递时间 组织愈合。这项工作将为培养临床相关的刺激健壮的策略奠定基础。 多器官系统中的组织修复。

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.