Gene Therapy to Fight Infection in Skin Transplants

Abstract

Wound closure can be problematic in patients with massive burns covering a large area of the body and in diabetic patients suffering from chronic ulcers. Insufficient donor sites in severely burned patients results in delayed wound closure and increases the likelihood of infection, which is a major cause of burn wound mortality [1]. Complications in patients wi th diabetic foot ulcers, most commonly resulting from infection, lead to as many as 50,000 amputations annually in the USA [2]. Tissue-engineered skin substitutes have been developed to begin to meet the needs of these patient populations [3]. The most basic skin substitutes are synthetic, acellular materials that act primarily as barriers to fluid loss and microbial invasion. Among the most innovative products are those containing cultured skin cells. Commercially available cellular skin substitutes, such as Apligraf ® , contain allogeneic dermal fibroblasts and epidermal keratinocytes, derived from donated neonatal foreskin, combined with a bovine collagen matrix. Apligraf is indicated for use in the treatment of venous leg ulcers and diabetic foot ulcers, increasing the percentage of wounds healed and decreasing the time required for wound closure [4–6]. However, as it is prepared using allogeneic cells, Apligraf and similar products act as biological bandages. Cells within the graft secrete growth factors and other proteins that facilitate wound healing and provide a matrix for cell migration, but the grafted cells do not persist indefinitely. Multiple applications are often required for complete wound healing. For burn patients, epidermal sheet grafts such as Epicel ® can be prepared with patient-derived keratinocytes for permanent wound coverage [7]. These grafts contain only epidermal cells; thus, they are prone to blistering and scar formation [7]. Cultured skin substitutes (CSS) are a bilayered autologous skin substitute currently in Phase II clinical trials. Comprised of cultured autologous keratinocytes, fibroblasts and biopolymers, CSS are used as an adjunctive treatment for coverage of burn wounds of greater than 50% of the body’s surface area, and have also been used to treat chronic wounds [8–10]. Despite the clinical successes observed with skin substitutes, they exhibit anatomical deficiencies, such as delayed vascularization, when compared with native skin autograft, which increase the risk for destruction due to microbial contamination. The use of topical antimicrobials in combination with skin substitutes can enhance engraftment and facilitate wound healing, but may contribute to the emergence of resistant strains of microorganisms. The development of a bioengineered skin substitute with increased innate resistance to microbial contamination could reduce or eliminate the requirement for exogenous antibiotics. Hypothetically, this could be achieved using a cutaneous gene therapy approach. Gene-encoded antimicrobial peptides, including members of the α- and β-defensin families and cathelicidins, have demonstrated antimicrobial activity against a wide variety of pathogens. Significantly, antimicrobial peptides have been shown to act against microorganisms commonly found in burns and chronic wounds, including multiply-resistant strains. As their mechanism of action is distinct from pharmaceutical antibiotic agents, defensins and related peptides represent a novel approach for treatment or prevention of infection. Antimicrobial peptides are effectors of the innate immune system and represent the body’s first line of defense against infection. Various antimicrobial peptides are expressed in neutrophils and in tissues that serve as interfaces with the environment, such as gut and lung epithelia and skin [11,12]. Rather than relying on specific antigen recognition, as in adaptive immunity, mediators of innate immunity take advantage of shared structural and functional characteristics of microorganisms. Antimicrobial peptides are cationic, allowing them to interact with the anionic lipid bilayer of microbial cell membranes through electrostatic interactions [13]. Following adsorption, the peptides disrupt the integrity of the outer cell membrane; irreversible injury of the target cell can result from entry of the antimicrobial peptide and other normally excluded molecules and leakage of essential cell components. Defensin-related cell death has also been linked to interference with protein synthesis and