Testing Smad7-based biologics for treating chronic wounds

描述(由申请人提供)：与各种疾病(如糖尿病)和急性创伤(如肥厚性疤痕)的异常愈合相关的慢性皮肤创伤是主要的医疗负担，需要基于科学发现的治疗干预措施。我们以前的研究表明，TGFb信号拮抗剂Smad7可以加速皮肤伤口的愈合。我们已经开发出一种Smad7融合蛋白，它包含人Smad7与HIV-1Tat蛋白转导结构域(PTD)的融合。TAT-Smad7蛋白与细胞接触后可以迅速穿透细胞。我们的初步数据显示，皮肤伤口局部应用TAT-Smad7可以加速野生型和糖尿病(db/db)小鼠的愈合。我们的初步数据还表明，含有Smad7的N端258aa(TAT-N-Smad7)或Smad7的C端259-426aa(TAT-C-Smad7)的TAT融合蛋白可能具有TAT-Smad7的全部或部分作用。这项第一阶段的STTR应用建议比较全长Smad7(TAT-Smad7)、N端Smad7(TAT-N-Smad7)和C端Smad7(TAT-C-Smad7)在体外和体内闭合伤口的融合蛋白效果，并建立定量方法来控制它们的生物活性，以便将来用于商业用途。AIM 1将生产全长TAT-Smad7， TAT-N-Smad7和TAT-C-Smad7，并确定角质形成细胞迁移和增殖的定量是否可用于不同批次的TAT-Smad7及其截断衍生物的质量控制。培养的人角质形成细胞将用这些蛋白质处理，并通过活细胞成像来量化它们对角质形成细胞增殖和迁移的影响。我们还将对核pSmad2和NFkB p50进行染色，以确定它们的生物学效应是否与阻断TGFb和NFkB信号有关，如全长TAT-Smad7中所见。目的2比较TAT-Smad7及其截短衍生物在体内创面愈合中的作用。我们将在正常和db/db小鼠的切除伤口上测试3种TAT-Smad7变体，以比较愈合过程中伤口闭合和再上皮化率以及伤口重塑过程中的纤维化反应。TUR提出的研究将缩小领先的基于Smad7的TAT融合蛋白(S)，以进一步发展为局部应用的治疗性生物制剂来治疗皮肤伤口。完成这一应用程序将使我们准备好通过第二阶段应用程序执行与这些生物制品的体内配方和长期毒性相关的IND使能研究，以及使用 GMP级重组蛋白。

DESCRIPTION (provided by applicant): Chronic skin wounds associated with various diseases (e.g., diabetes) and aberrant healing from acute wounding (e.g., hypertrophic scarring) is a major health care burden, which need scientific discovery-based therapeutic interventions. Our previous studies show that Smad7, a TGFb signaling antagonist, accelerates skin wound healing. We have developed a Smad7 fusion protein that contains the human Smad7 fused to the HIV-1 Tat protein transduction domain (PTD). The Tat-Smad7 protein can rapidly penetrate cells upon contact. Our preliminary data revealed that topical Tat-Smad7 application to skin wounds accelerated healing in wildtype and diabetes (db/db) mice. Our preliminary data also suggest that the Tat fusion protein containing either the N-terminal 258aa of Smad7 (Tat-N-Smad7) or the C-terminal 259-426aa of Smad7 (Tat-C-Smad7) could have full or partial effects of Tat-Smad7. This Phase I STTR application proposes to compare fusion protein efficacies of full length Smad7 (Tat-Smad7), N-terminal Smad7 (Tat-N-Smad7) and C-terminal Smad7 (Tat-C-Smad7) on wound closure in vitro and in vivo, and establish quantification methods for quality control of their bioactivities for future commercial use. Aim 1 will produce full length Tat-Smad7, Tat-N-Smad7 and Tat-C-Smad7, and determine if quantification of keratinocyte migration and proliferation can be used for quality control among different batches of Tat-Smad7 and its truncated derivatives. Cultured human keratinocytes will be treated with these proteins and quantify their effects on keratinocytes proliferation and migration via live-cell imaging. We will also stain nuclear pSmad2 and NFkB p50 to determine if their biological effects are associated with blocking TGFb and NFkB signaling as seen in full length Tat-Smad7. Aim 2 will compare the efficacies of Tat-Smad7 and its truncated derivatives on wound healing in vivo. We will test the 3 Tat-Smad7 variants on excisional wounds in normal and db/db mice to compare the rates of wound closure and re-epithelialization during healing and fibrotic response during wound remodeling. Tur proposed studies will narrow the lead Smad7-based Tat fusion protein(s) to further develop into topically applied therapeutic biologics to treat skin wounds. Completing this application will prepare us to perform IND-enabling studies via a Phase II application related to formulation and long-term toxicities of these biologics in vivo, as well as pig wound studies using GMP grade recombinant proteins.