Cas9-mediated knockout of Ndrg2 enhances the regenerative potential of dendritic cells for wound healing

Abstract

Chronic wounds impose a significant healthcare burden to a broad patient population. Cell-based therapies, while having shown benefits for the treatment of chronic wounds, have not yet achieved widespread adoption into clinical practice. We developed a CRISPR/Cas9 approach to precisely edit murine dendritic cells to enhance their therapeutic potential for healing chronic wounds. Using single-cell RNA sequencing of tolerogenic dendritic cells, we identified N-myc downregulated gene 2 (Ndrg2), which marks a specific population of dendritic cell progenitors, as a promising target for CRISPR knockout. Ndrg2-knockout alters the transcriptomic profile of dendritic cells and preserves an immature cell state with a strong pro-angiogenic and regenerative capacity. We then incorporated our CRISPR-based cell engineering within a therapeutic hydrogel for in vivo cell delivery and developed an effective translational approach for dendritic cell-based immunotherapy that accelerated healing of full-thickness wounds in both non-diabetic and diabetic mouse models. These findings could open the door to future clinical trials using safe gene editing in dendritic cells for treating various types of chronic wounds.

Biomaterials for immunomodulation in wound healing

“…The incorporation of new techniques to enhance cell-based therapies may also help to achieve the translational advancements required for their widespread adoption into clinical practice. For example, gene editing using the CRISPR/Cas9 approach has also recently been used to precisely edit dendritic cells and preserve an immature cell state with strong pro-angiogenic and regenerative capacity—these cells show enhanced therapeutic potential for accelerating the healing of chronic wounds [ 201 ]. Focusing on the role of mechanical forces that shift fibroblasts toward pro-fibrotic phenotypes, leading to myofibroblast differentiation and excessive collagen production, may also prove effective if one can disrupt this mechanical signaling [ 202 ].…”

Unlocking Dendritic Cell-Based Vaccine Efficacy through Genetic Modulation—How Soon Is Now?

“…A concept that seems closer with the development of CRISPR-Cas9-Ribonucleoprotein and macrophage-targeted nano-assembly delivery systems to genetically edit our gene candidates in tumour-associated macrophages in vivo [102]. Such breakthroughs could rapidly expand the application of DC-based therapies beyond cancer to clinical initiatives to fight infectious diseases like HIV [103] or modulate DCs to promote the healing of chronic diabetes ulcers [104]. So, in a bid to answer our original question-How soon is now?-regarding the next generation of DC vaccines capable of generating robust anti-tumour responses: it is close, but important steps remain.…”

Regulatory dendritic cell therapy in organ transplantation

“…Engineering of dendritic cells with lentiviral vectors, the genome-editing tool c lustered r egularly i nterspaced s hort p alindromic r epeats (CRISPR)-Cas9, or by other methods has held increasing appeal. Thus, Cas9-mediated knockout of the v-myc myelocytomatosis viral-related oncogene, n euroblastoma d erived (avian) (N-MYC)- d ownstream- r egulated g ene 2 (Ndrg2) or CD40 ablation preserves dendritic cell immaturity and reduces T-cell activation, which prolongs allograft survival and reduces graft damage, respectively [40 ▪ ,41]. Also, dendritic cell overexpressing immunosuppressive IL-10, with or without a co-encoding immunodominant antigen-derived peptide (DC IL-10 ; DC IL-10/Ag ) have proven effective in establishing homeostasis [42,43 ▪ ].…”