Plasmid Gene Delivery to Human Keratinocytes Through a Fibrin-Mediated Transfection System

Abstract

We have developed a matrix-mediated transfection system to deliver plasmids to human keratinocytes. The matrix is a soluble, self-hardening fibrin matrix (Tissucol), Baxter) that has been used clinically. Recently it has been shown that full thickness burn wounds can be successfully treated with a keratinocyte fibrin glue suspension. Further, it has been demonstrated that hEGF transfected cells accelerate wound healing. In this study, we inoculated the matrix with the hEGF expression plasmid and resuspended the matrix with either cultured or noncultured human keratinocytes. We obtained successful transfection rates of these cells (up to a 100-fold increase compared to controls containing no EGF expression plasmid) in vitro. After transplantation to full thickness wounds on athymic mice we were able to show a 180-fold increase in EGF concentration compared to controls, which persisted over the entire 7-day monitored period, decreasing from 180 to 20 pg/mL at day seven. This unique approach indicates the possible utility to combine a matrix for cell transplantation with a transfection system to release therapeutic proteins in vitro and in vivo.

Vascularization of the Dermal Support Enhances Wound Re-Epithelialization by In Situ Delivery of Epidermal Keratinocytes

“…Plasmid DNA [42][43][44][45] and recombinant viruses 46,47 have also been incorporated into fibrin hydrogels by our group and others, and they may be also used in this setting to promote vascularization. For the most part, genetic modification in this setting is local to the cells that populate the dermis during the granulation tissue phase of wound healing, for example, macrophages, fibroblasts, and endothelial cells.…”

A Fibrin Glue Composition as Carrier for Nucleic Acid Vectors

“…Several research groups have demonstrated the utility of fibrin glues for in situ immobilizing nonviral (17)(18)(19)(20)(21) and viral gene vector suspensions, mostly adenovirus or carrier materials for adenovirus (22)(23)(24)(25)(26)(27)(28)(29), at target sites and in some cases have provided evidence of therapeutic benefit of fibrin matrix mediated gene delivery in models of wound healing, induction of angiogenesis or bone healing. Hence we were interested in the feasibility of generating a lyophilized fibrinogen component comprising COPROGs which was to be readily applicable by the user in complete analogy to fibrin glue which is widely used as a tissue sealant in clinical surgery.…”

Tissue engineering of cultured skin substitutes

“…DeLuca and coworkers tried to characterize the potential stem cell-like behaviour of basal keratinocytes into holoclones and meroclones [119], proposing that the proliferative compartment of stratified squamous epithelia consists of stem and transient amplifying (TA) keratinocytes. These authors were also [7,8,57,87] able to demonstrate that epithelial cells from the corneal limbus have the capacity to reform a corneal epithelium with translucency after a culture period and retransplantation into the recipient eyes of patients [111]. They described that conjunctival keratinocytes with high proliferative capacity give rise to goblet cells at least twice in their life and, more importantly, at rather precise times of their life history, namely at 45-50 cell doublings and at ~15 cell doublings before senescence.…”

“…When resurfacing third degree burns or chronic wounds the lack of dermis remains a clinical challenge. The development of dermal analogues or substitutes or combinations of keratinocytes and dermal substitutes or matrix cells such as fibroblasts [5,8,34,63,81,96,101,133,143,147] aims towards this problem.…”

“…Gene therapy in combination with keratinocyte culture techniques lends itself as an obvious tool to enhance performance of cultured skin substitutes, to accelerate cell growth during the in vitro and in vivo process and to address difficult to heal wounds [3,8,57], [7,8,16,57,87,138].…”