Multivalent Conjugates for Enhanced Bioactivity of Growth Factor Based Therapies

描述(申请人提供)：2010年，超过875,000名美国糖尿病患者被诊断为下肢溃疡。这些伤口未能愈合，每年导致73500多人截肢。这些伤口的一个共同特征是创面床血管不足，导致缺血、感染和坏死。除糖尿病溃疡外，冠状动脉、骨科、牙科和肌肉组织再生过程中的有限血管形成也是常见的糖尿病并发症。包括Sonic Hedgehog(Shh)在内的几种生长因子可以通过促进伤口新生血管来促进伤口愈合，但它们很快就会从伤口环境中清除出来，并受到蛋白质分解消化的影响。因此，生长因子在体内的定位生物活性持续时间短，限制了其作为临床治疗手段的应用。我们已经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的稳定性，以促进它们作为生物疗法的临床转化。利用这种方法，我们将Shh连接到透明质酸(HYA)的直链上，通过改变Shh：HYA的比例，我们可以调节它激活Shh途径的能力。将Shh连接到大分子上也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，多价共轭分子 Shh(MvShh)将增强和维持Shh诱导的基因表达，促进糖尿病伤口愈合过程中的新生血管。在具体目标1中，我们将使用从db/db小鼠获得的真皮成纤维细胞来鉴定体外产生最大途径激活的mvShh配方，db/db小鼠是一种表现出伤口愈合受损和血管生成基因表达减少的糖尿病模型动物。在特定的目标2中，我们将把mvShh结合物的生物活性与它们加速db/db小鼠全层切除伤口愈合的能力联系起来。最后，在特定的目标3中，我们将使用相同的db/db创伤愈合模型来研究Shh的多价呈现如何在体内增强Shh诱导的血管生成基因的表达和血管形成。通过检验我们的假设，我们将评估mvShh结合物作为糖尿病溃疡的治疗方法。Shh在血管生成信号中多价结合的一般机制也可能扩展到其他各种微血管疾病。同样，这项研究将建立多价结合的理论基础，将其作为基于蛋白质的疗法的使能战略，这些疗法需要局部递送和持续的生物活性。 公共卫生意义：2010年，超过875,000名美国糖尿病患者被诊断为慢性下肢溃疡，这种糖尿病并发症每年产生超过300亿美元的相关医疗费用。我们正在开发一种先进的治疗方法，通过鼓励新生血管和改善损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.