Testing Smad7-based biologics for treating chronic wounds

描述（由申请人提供）：与各种疾病相关的慢性皮肤创伤（例如，糖尿病）和急性创伤的异常愈合（例如，增生性瘢痕）是主要卫生保健负担，需要基于科学发现的治疗干预。我们以前的研究表明，Smad 7，TGF β信号拮抗剂，加速皮肤伤口愈合。我们已经开发了一种Smad 7融合蛋白，其含有与HIV-1达特蛋白转导结构域（PTD）融合的人Smad 7。Tat-Smad 7蛋白在接触时可以迅速穿透细胞。我们的初步数据显示，局部Tat-Smad 7应用于皮肤伤口加速了野生型和糖尿病（db/db）小鼠的愈合。我们的初步数据还表明，含有Smad 7的N-末端258 aa（Tat-N-Smad 7）或Smad 7的C-末端259- 426 aa（Tat-C-Smad 7）的达特融合蛋白可能具有Tat-Smad 7的全部或部分作用。该I期STTR申请提出在体外和体内比较全长Smad 7（Tat-Smad 7）、N-末端Smad 7（Tat-N-Smad 7）和C-末端Smad 7（Tat-C-Smad 7）对伤口闭合的融合蛋白功效，并建立用于未来商业用途的生物活性的质量控制的定量方法。Aim 1将产生全长Tat-Smad 7， Tat-N-Smad 7和Tat-C-Smad 7，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-Smad 7及其截短衍生物之间的质量控制。将用这些蛋白质处理培养的人角质形成细胞，并通过活细胞成像定量其对角质形成细胞增殖和迁移的影响。我们还将染色核pSmad 2和NFkB p50以确定它们的生物学效应是否与阻断TGFb和NFkB信号传导相关，如在全长Tat-Smad 7中所见。目的2将比较Tat-Smad 7及其截短衍生物对体内伤口愈合的功效。我们将在正常和db/db小鼠的切除伤口上测试3种Tat-Smad 7变体，以比较愈合期间伤口闭合和再上皮化的速率以及伤口重塑期间的纤维化反应。Tur提出的研究将缩小基于Smad 7的达特融合蛋白的范围，以进一步开发成局部应用的治疗性生物制剂来治疗皮肤伤口。完成本申请将使我们能够通过与这些生物制剂的体内制剂和长期毒性相关的II期申请进行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.