A Directed Evolution Approach to Affinity-Based Protein Delivery

项目摘要 组织再生是一个动态的，仔细协调的过程，其中许多蛋白质和细胞群 参加由慢性疾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亲和性水凝胶 其可以提供多种治疗性蛋白质的分阶段递送以增强组织修复。我们已经开发 一种“自下而上”的蛋白质递送模块化方法，其中小蛋白质之间的特异性亲和力相互作用 结构域（即结合配偶体）和治疗性蛋白质被整合到生物材料中， 可预测地控制多种蛋白质的释放。我们使用酵母表面展示的定向进化 文库以鉴定对蛋白质具有高特异性和中等亲和力的结合配偶体， 在不同的时间尺度上释放。我们希望我们的方法能更准确地概括自然， 在愈合级联过程中交错呈现多种蛋白质，提供必要的组合 来激活修复过程的关键阶段。在目标1中，我们将发展用于治疗的结合伴侣。 酵母表面展示蛋白质。我们将对每种治疗药物的特异性抗体进行分类， 蛋白质以产生大量多样性的正交的蛋白质-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性质，从而获得所需的蛋白质释放曲线。我们会进食 将这些信息转化为COMSOL生物转运模型，以预测体外和体内的蛋白质递送。在目标3中， 将合成含有结合配偶体的透明质酸水凝胶，以研究多种 蛋白质在体外和体内。我们的建模结果将为生物材料的设计提供信息， 释放曲线不同的多种蛋白质。将通过以下方法评估水凝胶的体外蛋白质释放： ELISA法植入水凝胶内的荧光标记蛋白质的体内保留将使用 大鼠的纵向活体荧光成像。最终，我们期望实现对 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 亲和相互作用我们希望我们的多功能生物材料平台可以应用于精确递送 广泛的蛋白质，并将使系统的研究所需的蛋白质呈递的时间， 组织愈合这项工作将奠定基础，培养临床相关的战略，刺激强大的 多器官系统的组织修复。

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.