Sunday, October 10, 2004

Feasibility of Adenoviral Gene Delivery in Unburned and Burned Skin

Tobias Hirsch, MS, Dominik Mittler, MS, Frank Jacobsen, PhD, Mathias Schulte, MS, Marcus Lehnhardt, MD, Daniel Druecke, MD, Heinz-Herbert Homann, MD, PhD, Hans-Ulrich Steinau, MD, PhD, and Lars Steinstraesser, MD.

Introduction: The exogenously enhanced transient gene expression of growth factors or antimicrobial Peptides in wounds is an attractive alternative to the application of expensive synthetically manufactured proteins. The aim of this study was to evaluate in vitro and in vivo adenoviral gene transfer in a burn wound Methods: Primary human keratinocytes and HaCaT cells were transfected with different MOI (1, 10, 100, or 1000) and analyzed at 12, 24, 48 hours and 5 days. Sprague Dawley rats were randomized and burned (20% BSA) on their flanks by immersing for 20s into a 60C water bath using an apertured isolation mould. 48 hours later Adenovirus 2 x 108 virus particles (Ad5-CMV-LacZ) were intradermally and subcutaneously injected into both flanks. Transfection efficiency was measured in burned and unburned skin homogenates two and seven days after adenovirus application. Transfected cells were localized with X-Gal staining or in formalin-fixed sections using Immunhistochemistry. Quantitative Analysis was performed by luminometric Assays. Results: The in vitro Transfection showed a stable and high-efficient expression rate in both cell types and over all timepoints. As of a MOI of 10 we obtained significant transfection rates at all time points (p< 0.05 vs. negative Control). MOI 1000 reached the highest transfection rate in both cell types. Adenoviral induced reporter gene expression was significantly higher (p= 0,004) in burned skin compared to unburned skin. Gene expression rate increases from two to seven days in burned as well as in unburned skin. Discussion: This study demonstrates a capable adenoviral transfection efficacy in burns, which is stable for at least seven days. Application of adenovirus vectors in wounds may offer a promising drug delivery method to promote wound healing locally for potential clinical use.