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）相关的慢性皮肤伤口和急性伤口（如肥厚性疤痕）的异常愈合是一个主要的卫生保健负担，需要基于科学发现的治疗干预。我们之前的研究表明Smad7，一种TGFb信号拮抗剂，可以加速皮肤伤口愈合。我们开发了一种Smad7融合蛋白，其中包含融合到HIV-1 Tat蛋白转导结构域（PTD）的人类Smad7。Tat-Smad7蛋白可以在接触细胞后迅速穿透细胞。我们的初步数据显示，局部应用Tat-Smad7皮肤伤口加速野生型和糖尿病小鼠（db/db）的愈合。我们的初步数据还表明，含有Smad7的n -末端258aa （Tat- n -Smad7）或Smad7的c -末端259-426aa （Tat- c -Smad7）的Tat融合蛋白可能具有完全或部分的Tat-Smad7的作用。本I期STTR申请旨在比较全长Smad7 （Tat-Smad7）、n -末端Smad7 （Tat-N-Smad7）和c -末端Smad7 （Tat-C-Smad7）在体外和体内伤口愈合中的融合蛋白效果，并建立定量方法以质量控制其生物活性，以供未来商业应用。Aim 1将产生全长Tat-Smad7，

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.