Testing Smad7-based biologics for treating chronic wounds

测试基于 Smad7 的生物制剂治疗慢性伤口

• 批准号: 8779367
• 负责人: Xiao-Jing Wang
• 金额: $ 19.47万
• 依托单位: TAIGA BIOTECHNOLOGIES, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8779367
• 关键词: Acute; Affect; Biological; C-terminal; Cells; Chimeric Proteins; Chronic; Cicatrix; DNA Binding; Data; Diabetes Mellitus; Disease; Drug Formulations; Escherichia coli; Exhibits; Family suidae; Fibrosis; Future; Goals; HIV-1; Healed; Healthcare; Human; Hypertrophic Cicatrix; In Vitro; Infection; Inflammation; Lead; Length; Life; Link; MAP Kinase Gene; Mediating; Methods; Molecular; Mus; N-terminal; NF-kappa B; Non-Insulin-Dependent Diabetes Mellitus; Nuclear; Oral mucous membrane structure; Patients; Peptides; Phase; Physiological; Production; Proteins; Quality Control; Radiation; Recombinant Proteins; Signal Transduction; Skin; Smad7 protein; Small Business Technology Transfer Research; Splint Device; Staining method; Stains; Surgical wound; TNFRSF5 gene; Testing; Therapeutic; Therapeutic Effect; Therapeutic Intervention; Topical application; Toxic effect; Transactivation; United States; Variant; Wound Healing; base; care burden; cell motility; cellular imaging; commercialization; comparative efficacy; cost effective; db/db mouse; healing; in vivo; keratinocyte; migration; novel; oral mucositis; prevent; prophylactic; public health relevance; research study; response; tat Protein; wound

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.

描述（由申请人提供）：与各种疾病（例如，糖尿病）和急性伤口（例如肥厚疤痕）相关的慢性皮肤伤口是一种主要的医疗保健负担，需要基于科学发现的治疗干预措施。我们先前的研究表明，TGFB信号拮抗剂SMAD7加速了皮肤伤口愈合。我们已经开发了一种SMAD7融合蛋白，该蛋白包含与HIV-1 TAT蛋白转导域（PTD）融合的人Smad7。 Tat-Smad7蛋白可以在接触时迅速穿透细胞。我们的初步数据表明，局部TAT-SMAD7在野生型和糖尿病（DB/DB）小鼠中加速皮肤伤口的局部数据。我们的初步数据还表明，含有SMAD7（Tat-N-SMAD7）N末端258AA的TAT融合蛋白或SMAD7（TAT-C-SMAD7）的C末端259-426AA可能具有TAT-SMAD7的全部或部分作用。此阶段I STTR应用建议将全长SMAD7（TAT-SMAD7），N末端SMAD7（TAT-N-SMAD7）和C端SMAD7（TAT-C-SMAD7）（TAT-C-SMAD7）（TAT-N-SMAD7）（TAT-C-SMAD7）的融合蛋白效率进行比较，并在体外和体外封闭中的伤口封闭，并建立质量对未来广告的质量控制的定量方法。 AIM 1将产生全长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在不同批次的Tat-Smad7及其截断衍生物之间进行质量控制。培养的人角质形成细胞将用这些蛋白质治疗，并通过活细胞成像量化其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染色，以确定它们的生物效应是否与全长Tat-Smad7所示的TGFB和NFKB信号传导有关。 AIM 2将比较TAT-SMAD7及其截短的衍生物在体内伤口愈合的效力。我们将测试正常和DB/DB小鼠的杂交伤口上的3个TAT-SMAD7变体，以比较伤口重塑期间愈合过程中的伤口闭合和再上皮反应的速率。 TUR拟议的研究将缩小基于SMAD7的TAT融合蛋白（S），以进一步发展为局部应用的治疗生物制剂，以治疗皮肤伤口。完成此应用将使我们通过与这些生物制剂的配方和长期毒性有关的II期应用程序做好准备 GMP级重组蛋白。