Toward vocal fold scar mitigation through CRISPR/Cas-9-mediated gene editing of fibroblasts - ABSTRACT Vocal fold (VF) scarring negatively impacts both voice quality and quality of life, and effective treatments are currently lacking. VF fibroblasts (VFF) play a crucial role in VF scarring, as they are involved in wound healing and the production and remodeling of the VF lamina propria extracellular matrix in response to injury. Recent advances in understanding VFF gene expression have identified a signaling pathway that may contribute to the scar-like phenotype of differentiated VFF, specifically myofibroblasts. This research proposal aims to identify gene targets that could prevent the differentiation of VFF into myofibroblasts, thereby reducing the overproduction of fibrous proteins and excessive cell proliferation that contribute to scarring. The central hypothesis of this proposal is that inhibiting TGF-β signaling pathways will reduce VFF differentiation and proliferation, leading to a gene expression profile and cellular behavior more characteristic of undifferentiated VFF, with a concomitant reduction in fibrosis. We plan to quantify the effects of gene editing in VFF using an in vitro model and perform functional testing to address specific hypotheses. Specifically, we will target two pathways within the TGF-β signaling cascade: SMAD-dependent pathway and PI3K/AKT SMAD-independent pathway. CRISPR/Cas9 technology will be used to knock out specific genes involved in these pathways. After gene editing, we will assess the cellular response using assays for proliferation, differentiation, and collagen contraction, and perform bulk RNA sequencing to examine gene expression changes. This research is the first to apply CRISPR/Cas9 technology to edit VFF, providing genetic control over VFF differentiation in the context of scarring. The proposed study will shed light on the role of these specific genes and pathways in myofibroblast function. Given that no reliable, minimally invasive treatments for VF scarring currently exist, this proposal may reveal novel targets for further investigation and potential therapies for VF scar remediation.
