Plastic Surgery Research Council
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PSRC 60th Annual Meeting
Program and Abstracts

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Ex Vivo Free Flap Transfection With Minicircle DNA For Targeted Localized Gene Therapy
Peter A. Than, MD1, Robert R. Rennert, MD1, Christopher R. Davis, BSc, MB, ChB, MRCS1, Shane Morrison, MD1, Tai Le2, Michael W. Findlay, MBBS PhD FRACS FACS1, Geoffrey C. Gurtner, MD FACS1.
1Stanford University School of Medicine, Stanford, CA, USA, 2Stanford University School of Medicine, Menlo Park, CA, USA.

Purpose: Optimism surrounding gene therapy has been tempered by its failure to translate to clinical use. The major barriers relate to achieving efficient, targeted gene delivery without systemic toxicity. To address these issues, our laboratory previously developed a technique for ex vivo transduction of microvascular free flaps using viral vectors to deliver therapeutic proteins to the site of reconstruction. Free flaps, used ubiquitously in reconstructive surgery, have proven to be an excellent platform for targeted gene delivery. We have used these genetically modified “biologic protein pumps” to successfully deliver anti-cancer and anti-microbial agents to sites requiring reconstruction following resection or debridement. Building upon this work, we sought to further improve safety and efficacy by exploring alternative gene delivery vectors. Here we use minicircle DNA (MC DNA), a new generation of vector that is devoid of bacterial backbone sequences. This property makes it smaller, more easily transfected, resistant to in vivo gene silencing, and safer.
Methods: Adipose derived stem cells (ASCs) were harvested from the inguinal fat pad of Sprague Dawley rats and cultured under standard conditions. ASC transfection in vitro was achieved by electropulsation for the delivery of MC DNA encoding either human factor IX, firefly luciferase, or green fluorescent protein (GFP). In vivo studies in mice were performed by injection of MC DNA into the inguinal fat pad. In rats, the superficial inferior epigastric (SIE) flap was harvested and infused with MC DNA through the cannulated artery with the vein clamped. After a dwell period of 1 hour the venous clamp was released, excess MC DNA was flushed from the flap circulation, and re-implantation was performed by microsurgical techniques. Fluorescence microscopy, in vivo bioluminescence imaging, and enzyme linked immunosorbent assay (ELISA) were used to monitor protein expression.
Results: Electroporation of ASCs resulted in 70% transfection efficiency. MC DNA is capable of delivering high levels of transgene expression in vitro for greater than 28 days when assessed by fluorescence microscopy, bioluminescence, and ELISA. Injection into the inguinal fat pad in mice and ex vivo SIE flap transduction in rats yielded high-levels of local transgene expression sustained for greater than 28 days without significant systemic protein release or toxicity when assessed by similar assays.
Conclusions: Transfection of free flaps ex vivo allows localized, targeted gene delivery, avoids systemic toxicity, and is reversible. Utilization of MC DNA, a new generation of vector, provides an improved safety and transfection profile. The high level of local transgene expression without significant systemic expression is advantageous in a number of applications including resection and reconstruction for oncologic or infectious purposes, allowing continuous release of therapeutic proteins directly into the region of interest. This expands the functionality of the flap from a purely reconstructive role to include a therapeutic one.


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