RETROVIRALLY MEDIATED GENE-TRANSFER IN A SKIN EQUIVALENT MODEL OF CHRONIC WOUNDS

Given that treatment for chronic wounds is unsatisfactory, it is likel y that gene therapy may be tested as a therapeutic modality in this di fficult clinical problem. Actively proliferating cells in wounds are a lso a good target for retroviral transduction, an increasingly useful method for gene therapy. However, it is unclear how gene therapy may b est be used in chronic wounds, and experimental models are urgently ne eded to study and manipulate gene transfer in the context of chronic w ounds. In this report, partial- and full-thickness wounds were made in vitro in a human living skin equivalent (LSE) consisting of fully dif ferentiated keratinocytes layered over a collagen matrix seeded with f ibroblasts. To mimic a chronic wound situation, we used tissue culture conditions which, as in a chronic wound, allowed fibroblast but not k eratinocyte proliferation or migration. The wounded LSE was then place d over a transduced cell line (PA317) which produced a replication def ective retrovirus containing as a histological marker the bacterial be ta galactosidase gene. Using this close and direct exposure to the vir us-producing cell line, distinct staining for beta-galactosidase was o bserved in partial-thickness wounds, and was limited to fibroblasts aw ay from the upper site of injury and immediately overlying the retrovi rus-producing cell monolayer. Expression of beta-galactosidase was uni formly present at the wound edges and along the base of the entire par tial thickness wound. These studies demonstrate that, in in vivo condi tions mimicking a chronic wound, an intimate apposition of the injured LSE with the virus-producing cell line is needed for gene transfer. U sing this in vitro model system, gene transfer protocols may be optimi zed prior to beginning in vivo studies in chronic wounds.