Work

14. CRISPR/Cas9 Edited Dendritic Cells Accelerate Wound Healing By Targeting Wound Fibroblasts Via Growth Factor Signaling

Year: 2024

Type: article

Abstract: Purpose: Cell-based therapies have shown secretory, immunomodulatory, and regenerative effects on chronic wounds. CRISPR/Cas9 gene editing is a powerful tool to enhance the efficacy of cell-based therapies for wound healing. Recently CRISPR-mediated knockout of Ndrg2 in dendritic cells (DCs) has been used to generate a highly potent immunotherapy to accelerate healing in diabetic and non-diabetic wounds. However, how genetically engineered cells interact with local cell types of the wound healing environment to accelerate healing has been incompletely understood. Methods: Ndrg2 knockout in bone marrow derived DCs was performed using Cas9/sgRNA-ribonucleoproteins and electroporation. Splinted excisional wounds in wild-type mice were treated with collagen hydrogels seeded with genetically edited DCs, control DCs which had undergone electroporation only, or blank hydrogels. To investigate how genetically edited DCs interact with different native cell types during wound healing and affect their transcriptional signatures, we performed single-cell RNA sequencing of wound tissue treated with edited DCs, control DCs, or blank hydrogels. To study intercellular communication networks between the transplanted DCs and local cells of the wound, ligand-receptor interactions were analyzed using the CellChat algorithm. Results: The highest number of differentially expressed genes (DEGs) between the groups was found in wound fibroblasts, indicating that edited DCs had the strongest impact on fibroblast gene expression within the wound bed. Several wound healing related genes were among the DEGs in fibroblasts, such as Ngfr, Lgals1, Plod2, and Ppib. CellChat analysis demonstrated that edited DCs showed a stronger signaling activity within their wound environment compared to control DCs. Edited DCs communicated with wound fibroblasts mostly via growth factor pathways, such as the vascular endothelial growth factor (VEGF), the insulin-like growth factor (IGF) and platelet-derived growth factor (PDGF) pathways, which stimulate cell proliferation and angiogenesis. In addition, edited DCs showed an upregulation of Spp1 (osteopontin) signaling, which is a critical driver of early wound repair in diabetic wounds. Conclusion: Our data indicates a strong therapeutic response of resident cells of the wound bed to secreted factors from genetically edited DCs, thus providing a molecular basis for the observed accelerated wound closure in response to edited cell therapies. Since clinical-grade manufacturing of autologous DCs for adoptive cell transfer has already been established in oncology, genetically edited DCs have a high potential for successful clinical translation as a cell-based therapy for patients suffering from complex wounds that are refractive to standard treatments.

Source: Plastic & Reconstructive Surgery Global Open

DOI: 10.1097/01.gox.0001015148.77646.c2