Human Mesenchymal Stromal Cells Engineered to Overexpress PDGF-B Using CRISPR/Cas9/rAAV6-based Tools Improve Wound Healing
Nina Kosaric, A.B., Wai Srifa, B.A., Geoffrey C. Gurtner, M.D., Matthew H. Porteus, M.D..
Stanford University, Stanford, CA, USA.
PURPOSE: Mesenchymal Stromal Cells (MSCs) are a promising source for cell therapy due to their pro-regenerative and immunomodulatory effects. These properties can be enhanced by engineering MSCs to overexpress and secrete growth factors favorable to wound healing processes. Here, we aimed to develop a platform for targeted gene insertion in human bone marrow (hBM)-MSCs that utilizes CRISPR/Cas9 nuclease and recombinant adeno-associated virus serotype 6 (rAAV6) to deliver a designed homologous recombination-based repair template. Using this method, we engineered hBM-MSCs to overexpress and secrete soluble platelet derived growth factor B (PDGF-B) and examined the ability of this cell line to promote wound healing.
METHODS: PDGF-B secreting hBM-MSCs (PDGFB-hBM-MSCs) were generated by genome integration of a bi-cistronic PDGFB-2A-GFP expression cassette at the CCR5 locus. To examine the ability of these cells to promote wound healing, we utilized a diabetic murine excisional wound healing model. Wounds were treated with a single, local injection of PDGFB-hBM-MSCs, GFP-hBM-MSCs, or vehicle control (phosphate buffer saline (PBS)) immediately after wounding. The percentage change in wound size over time was used as a measure of the rate of wound healing.
RESULTS: We observed accelerated wound healing kinetics in wounds treated with PDGFB-hBM-MSCs compared to wounds in the other treatment groups as early as day 13 after wounding, and this significant difference in healing rate persisted through 23 days post-wounding. This resulted in significantly earlier wound closure in wounds treated with PDGFB-hBM-MSCs (20.2 days ± 0.68 days) compared with GFP-MSCs (25.4 days ± 0.83 days, p<0.0001) and PBS (26.33 days ± 0.67 days, p<0.0001). These data suggest that PDGF-B plays a critical role in the response to tissue injury during cutaneous wound healing, and that hBM-MSCs can be engineered to overexpress proteins to increase their therapeutic potential.
CONCLUSION: Our engineering platform can be used to integrate a GFP reporter expression cassette into a genomic ‘safe harbor’ locus without changing the defining characteristics of MSCs. Using this platform, we successfully generated hBM-MSCs that overexpress and secrete PDGF-B, a growth factor essential to wound healing. Single, local injection of these cells under excisional wounds of diabetic mice at the time of wounding accelerates wound healing rates and reduces time to complete wound closure significantly when compared to injections of GFP-MSCs or a vehicle control.
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