Project Grant R01AR083161

A RELAXIN-LOADED HYDROGEL FOR THE TREATMENT OF HYPERTROPHIC SCARS - PROJECT SUMMARY/ABSTRACT *THIS PROPOSAL DESCRIBES AN INNOVATIVE TREATMENT FOR HYPERTROPHIC SCARS THAT PREVENTS AND TREATS PATHOLOGIC FI- BROGENESIS AFTER DERMAL INJURY. HYPERTROPHIC SCARS AFFECT TENS OF MILLIONS OF PEOPLE EACH YEAR, INCLUDING OVER 50% OF SURGICAL PATIENTS AND 90% OF DEEP PARTIAL THICKNESS AND FULL-THICKNESS BURN INJURY PATENTS. CURRENT NON-SURGICAL TREATMENTS (E.G., LASER/CRYOTHERAPY, CYTOTOXIC DRUGS, CORTICOSTEROIDS) ARE PAINFUL AND LIMITED IN THEIR SCOPE AND EFFICACY, WHILE SURGICAL INTERVENTIONS ARE INVASIVE, EXPENSIVE, AND MAY CAUSE RECURRING HYPERTROPHIC SCAR FOR- MATION. THERE IS CURRENTLY NO OPTIMAL TREATMENT FOR HYPERTROPHIC SCARS, AS THESE OPTIONS DO NOT TARGET THE UNDER- LYING PATHOLOGY OF SCAR DEVELOPMENT. WE PROPOSE THE USE OF HUMAN RELAXIN-2 (RLX-2), AN ENDOGENOUS 6-KDA ANTIFIBROTIC PEPTIDE HORMONE, AS A NOVEL PROTEIN THERAPEUTIC FOR THE TREATMENT OF HYPERTROPHIC SCARS THAT TARGETS THE FIBROTIC BUILD-UP OF EXTRACELLULAR MATRIX (ECM) PROTEINS IN ABNORMAL SCAR DEVELOPMENT. IMPORTANTLY, WE EMPLOY A RLX-2-LOADED IN SITU POLYMERIZING HYDROGEL, WHEREBY RLX-2 IS UNIQUELY LINKED TO THE HYDROGEL AND SUBSE- QUENTLY RELEASED IN ITS NATIVE FORM, TO ACHIEVE SUSTAINED AND LOCALIZED DELIVERY. ADDITIONALLY, WE DESCRIBE DETAILED MECHANISM-OF-ACTION (MOA) STUDIES TO EXAMINE RLX-2 AS A THERAPEUTIC PROTEIN TO REDUCE FIBROSIS IN DERMAL FIBROBLASTS THROUGH: 1) IMPEDING MYOFIBROBLAST DIFFERENTIATION AND ECM DEPOSITION BY INHIBITING THE TGF-B1 PATH- WAY; 2) UPREGULATING MATRIX METALLOPROTEINASES THAT DEGRADE COLLAGEN; AND 3) DOWNREGULATING TISSUE INHIBITORS OF METALLOPROTEINASES. WE HYPOTHESIZE THAT THE SUSTAINED RELEASE OF RLX-2 FROM A LOCALLY APPLIED HYDROGEL TO DEEP DERMAL WOUNDS WILL PROMOTE PROPER WOUND HEALING, REDUCE EXCESSIVE COLLAGEN LEVELS, AND ALLE- VIATE SCAR SIZE AND APPEARANCE (I.E., PREVENT AND TREAT HYPERTROPHIC SCARS). IMPORTANTLY, PRELIMINARY DATA SUPPORT THE PROPOSED STUDIES, WELL-CHARACTERIZED MATERIALS AND RIGOROUS EXPERIMENTAL DESIGNS ARE ESTABLISHED, AND ESSENTIAL CROSS-DISCIPLINARY COLLABORATIONS AND EXPERTISE ARE IN PLACE TO ADDRESS THE HYPOTHESIS. THE SPECIFIC AIMS OF THIS FIVE-YEAR PROPOSAL ARE AS FOLLOWS. AIM 1 DETERMINES THE MOA OF RLX-2 IN VITRO IN A 3D ORGANOTYPIC SKIN MODEL AND IN EX VIVO PATIENT BIOPSY EXPLANTS TO PROVIDE KNOWLEDGE ON THE MOLECULAR SIGNALING BEHIND RLX- 2'S ABILITY TO REDUCE FIBROTIC SCARRING. AIM 2 DEVELOPS A RLX-2-LOADED HYDROGEL FOR LOCALIZED AND SUSTAINED TOPICAL RELEASE OF RLX-2. WE WILL USE OUR NEWLY DISCOVERED METHOD THAT HARNESSES INTEIN CHEMISTRY TO ENABLE SOLUBLE EXPRESSION AND PURIFICATION OF RECOMBINANT RLX-2 FUNCTIONALIZED TO EFFICIENTLY CONJUGATE TO THE HYDROGEL AND THEN RELEASE UNALTERED TO THE SKIN. AIM 3 ASSESSES THE PHARMACOKINETICS, BIODISTRIBUTION, AND EFFICACY OF THE RLX-2- LOADED HYDROGEL IN AN IN VIVO SWINE MODEL OF HYPERTROPHIC SCARRING, ALONG WITH LIVE TISSUE IMAGING METHODS TO EXAMINE RLX-2 EFFECTS ON WOUND HEALING DYNAMICS AND REDUCTION OF FIBROTIC SCARRING. TOGETHER, THESE AIMS WILL DEMONSTRATE THE EFFICACY OF A LOCALLY DELIVERED RLX-2-BASED SYSTEM TO REDUCE ABERRANT SCARRING AND WILL ELUCIDATE THE MOA UNDERLYING THIS ACTIVITY. FURTHERMORE, WE WILL ESTABLISH A UNIQUE PROTEIN PURIFICATION-FUNCTIONALIZATION STRATEGY WHICH WILL BE BROADLY APPLICABLE TO THE SUSTAINED DELIVERY OF OTHER THERAPEUTIC PROTEINS.