Multivalent Conjugates for Enhanced Bioactivity of Growth Factor Based Therapies

描述（由申请人提供）：2010 年，超过 875,000 名美国糖尿病患者被诊断患有下肢溃疡。这些伤口未能闭合每年会导致超过 73,500 例下肢截肢。这些伤口的一个共同特征是伤口床血管化不足，导致缺血、感染和坏死。除了糖尿病性溃疡外，冠状动脉、骨科、牙科和肌肉组织再生过程中的血管化受限也是常见的糖尿病并发症。几种生长因子，包括 Sonic Hedgehog (Shh)，可以通过促进伤口中的新血管形成来增强伤口愈合，但它们会迅速从伤口环境中清除并受到蛋白水解消化。因此，生长因子作为临床疗法的转化因其体内位点特异性生物活性的持续时间短而受到限制。我们开发了多价生长因子结合物，旨在增强生长因子的生物活性和组织水平稳定性，以促进其作为生物疗法的临床转化。使用这种方法，我们将Shh与透明质酸（HyA）的线性链缀合，并且通过改变Shh：HyA的比例，我们可以调节其激活Shh通路的能力。将 Shh 与大分子结合还可以防止其被蛋白水解酶失活，并增强其在靶组织中的分子稳定性。我们的总体假设是多价缀合物 Shh (mvShh) 将增强和维持 Shh 诱导的基因表达，从而促进糖尿病伤口愈合过程中的新生血管形成。在具体目标 1 中，我们将使用从 db/db 小鼠（一种表现出伤口愈合受损和血管生成基因表达减弱的糖尿病模型动物）收获的真皮成纤维细胞来确定可在体外产生最大途径激活的 mvShh 配方。在具体目标 2 中，我们将 mvShh 缀合物的生物活性与其加速 db/db 小鼠全层切除伤口愈合的能力相关联。最后，在具体目标 3 中，我们将使用相同的 db/db 伤口愈合模型来研究 Shh 的多价表达如何增强 Shh 诱导的血管生成基因表达和体内血管形成。通过检验我们的假设，我们将评估 mvShh 缀合物作为糖尿病溃疡的治疗方法。 Shh 多价结合在血管生成信号传导中的一般机制也可以扩展到多种其他微血管疾病。同样，这项研究将为多价结合奠定基础，作为需要局部递送和持续生物活性的基于蛋白质的疗法的启用策略。 公共健康相关性：2010 年，超过 875,000 名美国糖尿病患者被诊断患有慢性下肢溃疡，这种糖尿病并发症每年产生超过 300 亿美元的相关医疗保健费用。我们正在开发一种先进的治疗方法，通过促进新生血管形成和改善受伤部位的血液供应来加速糖尿病伤口的愈合。我们项目的总体目标是评估我们的治疗策略如何增强血管形成的细胞机制，从而开始将其转化为临床治疗。

DESCRIPTION (provided by applicant): In 2010, more than 875,000 Americans with diabetes were diagnosed with a lower extremity ulcer. Failed closure of these wounds results in more than 73,500 lower extremity amputations annually. One common feature to these wounds is inadequate vascularization of the wound bed, leading to ischemia, infection, and necrosis. In addition to diabetic ulcers, limited vascularization during regeneration of coronary, orthopaedic, dental and muscle tissues are frequent diabetic complications. Several growth factors, including Sonic hedgehog (Shh), can enhance wound healing by promoting neovascularization in the wound, but they are rapidly cleared from the wound environment and subject to proteolytic digestion. Thus, the translation of growth factors as clinical therapies has been limited by their short duration of site-specific bioactivity in vivo. We have developed multivalent growth factor conjugates that are designed to enhance the bioactivity and tissue-level stability of growth factors to facilitate their clinical translation as biological therapeutics. Using this approach, w have conjugated Shh to linear chains of hyaluronic acid (HyA), and by varying the ratio of Shh:HyA, we can modulate its ability to activate the Shh pathway. Conjugating Shh to a large macromolecule may also prevent its deactivation by proteolytic enzymes and enhance its molecular stability in the target tissues. Our overall hypothesis is that multivalent conjugates of Shh (mvShh) will enhance and sustain Shh-induced gene expression that promotes neovascularization during diabetic wound healing. In Specific Aim 1, we will identify the mvShh formulations that yield maximal pathway activation in vitro using dermal fibroblasts harvested from db/db mice, a diabetic model animal that exhibits impaired wound healing and diminished angiogenic gene expression. In Specific Aim 2 we will correlate the bioactivity of mvShh conjugates to their ability to accelerate healing of full-thickness excisional wounds in db/db mice. Finally, in Specific Aim 3 we will use the same db/db wound healing model to investigate how multivalent presentation of Shh can enhance Shh-induced expression of angiogenic genes and blood vessel formation in vivo. By testing our hypothesis, we will evaluate mvShh conjugates as a treatment for diabetic ulcers. The general mechanism of multivalent conjugation of Shh in angiogenic signaling may also be extended to a variety of other microvascular disorders. Likewise, this study will build a rationale for multivalent conjugation as an enabling strategy for protein-based therapies that require local delivery and sustained bioactivity. PUBLIC HEALTH RELEVANCE: In 2010, more than 875,000 Americans with diabetes were diagnosed with a chronic lower extremity ulcer, this diabetic complication generates over $30 billion per year in related health care costs. We are developing an advanced therapeutic to accelerate healing in diabetic wounds by encouraging neovascularization and improving blood supply to the site of injury. The overall goal of our project is to evaluate how our treatment strategy can enhance the cellular mechanisms of blood vessel formation, and thus initiating its translation to a clinical therapy.