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-β1 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.