Engineered Tandem Thymosin Peptide Promotes Corneal Wound Healing.

PURPOSE: Thymosin beta-4 (TB4) is a small peptide upregulated in injured tissues, playing roles in cell migration, angiogenesis, inflammation, and oxidative stress. Studies show TB4 significantly promotes corneal wound healing after injury, leading to a clinical trial (RGN-259), with full US Food and Drug Administration approval still pending. Current limitations to TB4-based therapies are a short half-life and high peptide synthesis costs, limiting large-scale applications. Here, we engineered a tandem thymosin beta-4 (tTB4) peptide with improved therapeutic potential and scalability for corneal wound repair. METHODS: tTB4 was produced by fusing two TB4 monomers into a single polypeptide, creating dual G-actin binding domains. Binding activity was investigated using structural, in vitro, and in vivo studies. tTB4 and TB4 effects on cell viability and migration were assessed in human telomerase-immortalized corneal epithelial (hTCEpi) cells. Their efficacy in promoting corneal wound healing was tested in a murine alkali-induced corneal injury model. RESULTS: Structural modeling revealed tTB4 has a mechanistic advantage in enhancing actin polymerization over TB4, as it can simultaneously bind and sequester two G-actin molecules, facilitating filament formation. In vitro, tTB4 promoted increased hTCEpi viability and migration compared to TB4. In vivo, tTB4 promoted corneal wound healing and reduced scarring with greater efficacy than TB4. CONCLUSIONS: The engineered tTB4 exhibited superior bioactivity and enhanced corneal wound-healing efficacy over TB4. Given that tTB4 can be produced by bacterial fermentation, synthesis is simplified and more cost-effective than TB4. Together, our data demonstrate the potential of tandem peptide engineering to improve regenerative peptide pharmacokinetics and therapeutic performance.