Tuesday, October 10, 2006 - 2:00 PM

Ex-Vivo Gene Transduction of Microvascular Free Flaps with Antimicrobial Peptides to Treat Chronic Infections

Shadi Ghali, MD, Kirit Bhatt, MD, Marlese Patricia Dempsey, MD, Eric Chang, Sunil Singh, BA, Yubin Shi, PhD, and Gurtner C. Geoffrey, MD.

Purpose: Recalcitrant infections often require microsurgical free tissue transfer to fill soft tissue and osseous defects. Despite the transfer of vascularized tissue, there is still a 30-40% failure rate due to infection. Antimicrobial peptides are a ubiquitous group of natural molecules with minimal pathogenic resistance. This study examines the utility of using free flaps in a therapeutic as well as reconstructive capacity to deliver the human antimicrobial peptide LL37 ex-vivo.

Methods: Fasciocutaneous flaps were raised in adult fischer rats and transfected ex-vivo with adenovirus-LacZ reporter gene (4.6x109PFU) or adenovirus-LL37 (4.4x109 PFU) +/- vascular endothelial growth factor (VEGF) to improve transduction efficiency. Flaps and periflap tissue were analyzed at days 3,7,14 and 24 for transgene expression by ELISA and/or immunohistochemistry. Distal organs were also analyzed as a measure of toxicity. A reproducible catheter infection model with bioluminescent Staphylococcus Aureus (~5x108 cfu/ml) was also established to determine the biological efficacy of ex-vivo transduction of these flaps with the therapeutic adenovirus-LL37. Non-invasive bioluminescent photon emissions were recorded and final bacterial counts were analyzed at day 7.

Results: Maximal -galactosidase expression from the adenovirus-LacZ reporter gene was demonstrated immunohistochemically and by ELISA (62ng/ml) at day 7. LL37 expression at day 7 was demonstrated by ELISA (70ng/ml). The addition of VEGF resulted in a 50% increase in transduction efficiency (104ng/ml) and a 30% increase in peri-flap transgene expression. The spleen and liver showed minimal protein expression. Flaps transduced with adeno-LL37 demonstrated superior bacterial clearance compared to controls (~4x105 cfu/ml vs ~3x107 cfu/ml).

Conclusions: We have demonstrated successful transduction of the reporter gene, LacZ as well as the therapeutic gene product, LL37 using ex-vivo gene delivery methods. The gene product was expressed maximally at day 7 and was concentrated in the flap and peri-flap tissues, where recalcitrant infection commonly occurs. Transduction efficiency was improved with the addition of VEGF and biological efficacy was demonstrated in a rat infection model. We believe that the ex-vivo gene transduction of microvascular free flaps with therapeutic genes is feasible, efficacious and safe and has enormous potential in the field of reconstructive plastic surgery.

View Synopsis (.doc format, 92.0 kb)