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.
