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Genome Editing of Mouse Fibroblasts by Homologous Recombination for Sustained Secretion of PDGF-B and Augmentation of Wound Healing

Barker, Jenny C. M.D., Ph.D.; Barker, Adam D. M.D.; Bills, Jessica B.S.; Huang, Jiying B.S.; Wight-Carter, Mary D.V.M.; Delgado, Imelda A.A.; Noble, Debby L. B.S.; Huang, Lily J. Ph.D.; Porteus, Matthew H. M.D., Ph.D.; Davis, Kathryn E. Ph.D.

Plastic & Reconstructive Surgery:
doi: 10.1097/PRS.0000000000000427
Experimental: Original Articles
Abstract

Background: Exogenous cytokines, such as platelet-derived growth factor (PDGF)-B, can augment wound healing, but sustained delivery to maintain therapeutic levels remains a problem. “Genome editing” is a new technology in which precise genome modifications are made within cells using engineered site-specific nucleases. Genome editing avoids many of the complications associated with traditional gene therapy and the use of viral vectors, including random integration, imprecise gene expression, and inadvertent oncogene activation.

Methods: This study demonstrates site-specific nuclease-mediated integration of a PDGF-B transgene into a predefined locus within the genome of primary mouse fibroblasts. Engineered fibroblasts were applied to splinted mouse wounds and evaluated after 14 days and 5 months for the retention of engineered fibroblasts, wound healing morphology, angiogenesis, and systemic PDGF-B expression.

Results: The application of engineered PDGF-B–expressing fibroblasts enhanced wound healing compared with controls. Low-level, constitutive expression of PDGF-B was achieved without detectable levels of systemic PDGF-B. The mechanism of improved wound healing is, at least in part, the result of increased wound vascularization, as the wounds treated with PDGF-B fibroblasts had a blood vessel density 2.5 times greater than controls. After 5 months, the engineered fibroblasts persisted in the wound bed. No adverse effects were detected from the application of these fibroblasts after 5 months as assessed by hematoxylin and eosin staining of wounds and by mouse necropsy.

Conclusions: These data support that site-specific genome editing allows for sustained cell-based cytokine delivery. Furthermore, sustained release of PDGF-B increases the speed and quality of wound healing after a single application.

Author Information

Dallas, Texas; and Stanford, Calif.

From the Department of Plastic Surgery, the Medical Scientist Training Program, the Animal Resource Center, Veterinary Pathology, and the Department of Cell Biology, The University of Texas Southwestern Medical Center; the Department of Surgery, Baylor University Medical Center; and the Department of Pediatrics, Stanford University.

Received for publication September 12, 2013; accepted January 28, 2014.

Disclosure: Dr. Davis has grants from Convatec, Thermotek, Unilever, Kensey Nash, Andrew Technologies, TA Sciences, and Innovative Therapies. She is a consultant for Thermotek and Innovative Therapies, Inc. None of the noted financial relationships affected or influenced the research presented in this article. The studies in this article were performed independently of any funds received from these companies. The other authors have no financial interest to disclose.

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Kathryn E. Davis, Ph.D., Department of Plastic Surgery, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, F4.310A, Dallas, Texas 75390-8560, kathryn.davis@utsouthwestern.edu

Matthew H. Porteus, M.D., Ph.D., Department of Pediatrics, Stanford University, Stanford, Calif. 94305, mporteus@stanford.edu

©2014American Society of Plastic Surgeons