Gene Therapy for Cutaneous Wound Repair

Introduction The biology of normal wound healing is well characterized and has been the subject of several recent reviews.[1–3] Wound healing proceeds via an ordered cascade of events that is mediated by specific growth factors, growth factor receptors, and cytokines.[4–6] Normal cutaneous wound healing has three phases: inflammation, proliferation, and remodeling (maturation). In chronic wounds, such as lower-extremity neuropathic ulcers occurring in patients with diabetes, the wound healing response is impaired. This impairment is due in part to a deficiency of endogenous growth factors (e.g., platelet-derived growth factor [pdgf], epidermal growth factor [EGF], fibroblast growth factor-2 [FGF-2], transforming growth factor-beta [TGF-b]) and growth factor receptors.[7,8] The deficiency of growth factors in chronic wounds has been linked to elevated levels of matrix metalloproteinases and other neutrophil proteases.[9,10] The finding of decreased levels of growth factors in nonhealing wounds gave rise to the obvious approach of treating chronic wounds with growth factor proteins. To date, numerous in-vivo studies have been performed with a variety of growth factor proteins (e.g., PDGF, TGF-b, FGF, EGF, keratinocyte growth factor [KGF], vascular endothelial growth factor [VEGF], and insulin-like growth factor [IGF]) in a variety of wound healing models.[4,11] The preclinical studies were very encouraging, demonstrating significant enhancements of wound repair, and biological responses consistent with the known functions of the specific growth factors.[11] In contrast, the clinical success of growth factor proteins has been disappointing.[11–13] To date, only recombinant human platelet-derived growth factor-BB (rhPDGF-BB) has been commercialized for the treatment of diabetic ulcers, and even it yields only modest improvements in healing.[5,14] The disappointing clinical experience with growth factors has been attributed to their short half-lives, degradation by wound proteases, and failure to maintain local protein levels above the therapeutic threshold.[9,10] Consequently, clinical efficacy requires high and frequent dosing, which is prohibitively expensive. In light of these limitations, gene therapy has attracted significant attention as an alternative, cost-effective approach for cutaneous wound therapy.[12] While achieving long-term expression of a therapeutic gene remains a challenge for gene replacement strategies, only transient gene expression is required for wound repair. Tissue repair cells (i.e., fibroblasts, endothelial cells, inflammatory cells) are the target cells for deoxyribonucleic acid (DNA) uptake. It is speculated that gene therapy will enable production and persistence of growth factors throughout the inflammatory and proliferative phases, yielding improved healing responses compared with protein-based therapy. As the wound remodels, the transfected repair cells will die and transgene expression will cease. In other words, expression of the therapeutic protein will persist only as long as it is needed to promote wound repair. The two general approaches for gene transfer are ex-vivo and in-vivo DNA delivery. In ex-vivo gene transfer, isolated cells are genetically modified in vitro and then transplanted back into the host. Genetically modified keratinocytes expressing growth factors have been effective in animal models of wound healing.[15,16] While promising, the extensive ex-vivo manipulations and expenses associated this approach have been significant hurdles to clinical development. For in-vivo gene transfer, the genetic material is delivered directly to the target tissue, thus avoiding the manipulations of ex-vivo gene delivery. For cutaneous wound healing applications, the ready accessibility of the target tissue makes in-vivo gene transfer the logical and preferred approach. The methods by which DNA can be delivered in-vivo fall into one of three categories: biologic, chemical, and physical. The biologic method employs derivatives of naturally occurring viruses and exploits the highly efficient natural mechanisms by which these viruses enter cells, transport their DNA payloads to the nucleus, and use the cell’s machinery to activate viral gene expression. Four classes of virus have received the most attention as potential gene therapy vectors: retroviruses (including lentivirus), adeno-associated viruses (AAV), adenoviruses (Ad), and herpes simplex virus type 1 (HSV-1) (Table 1). For gene therapy vectors, regions of the wild-type viral genome required for replication are deleted, and the transgene of interest (and associated regulatory regions) are inserted. Of course, viral replication is required for vector manufacture, and therefore the missing viral functions must be provided in trans during production. This generally is accomplished by manufacturing the virus in a packaging cell line that has been genetically engineered to express the missing viral proteins or by co-infection of cells with a helper virus. Both approaches have associated risks. Recombination can occur between the viral genome and packaging cell DNA, creating a replication competent viral contaminant, and great care must be taken to remove all helper virus from vector preparations. Retroviruses Retroviruses are enveloped viruses with a 7 to 11kb single-stranded ribonucleic acid (RNA) genome. Different subclasses of retrovirus can be identified based on their genomic complexity.[17] Oncoretroviruses are the simplest retroviruses, consisting of three genes: the gag gene, which encodes core proteins; the pol gene, which encodes reverse transcriptase; and the env gene, which encodes the viral envelope protein.[18] Oncoretroviruses are exemplified by Murine Leukemia Virus (MLV) and infect only dividing cells. For use as gene therapy vectors, the gag, pol, and env genes are removed and replaced with the therapeutic transgene. Retroviruses are limited in the size of transgene that can be inserted and still allow for productive packaging (