Retrovirally mediated gene transfer in a skin equivalent model of chronic wounds

Given that treatment for chronic wounds is unsatisfactory, it is likely that gene therapy may be tested as a therapeutic modality in this difficult clinical problem. Actively proliferating cells in wounds are also a good target for retroviral transduction, an increasingly useful method for gene therapy. However, it is unclear how gene therapy may best be used in chronic wounds, and experimental models are urgently needed to study and manipulate gene transfer in the context of chronic wounds. In this report, partial- and full-thickness wounds were made in vitro in a human living skin equivalent (LSE) consisting of fully differentiated keratinocytes layered over a collagen matrix seeded with fibroblasts. To mimic a chronic wound situation, we used tissue culture conditions which, as in a chronic wound, allowed fibroblast but not keratinocyte proliferation or migration. The wounded LSE was then placed over a transduced cell line (PA317) which produced a replication defective retrovirus containing as a histological marker the bacterial beta galactosidase gene. Using this close and direct exposure to the virus-producing cell line, distinct staining for beta-galactosidase was observed in partial-thickness wounds, and was limited to fibroblasts away from the upper site of injury and immediately overlying the retrovirus-producing cell monolayer. Expression of beta-galactosidase was uniformly present at the wound edges and along the base of the entire partial thickness wound. These studies demonstrate that, in in vivo conditions mimicking a chronic wound, an intimate apposition of the injured LSE with the virus-producing cell line is needed for gene transfer. Using this in vitro model system, gene transfer protocols may be optimized prior to beginning in vivo studies in chronic wounds.