Matrix-Immobilized Growth Factor Gene Therapy Enhances Tissue Repair

Disclosure: This work was supported in part by a NIH research grant 1R43AR46154. Introduction The natural process of wound healing proceeds through an orderly sequence of events mediated by specific growth factors. In chronic wounds, such as diabetic ulcers, pressure ulcers, and venous stasis ulcers, this process is impaired. This is in part due to a deficiency in endogenous growth factors, which have the primary role of stimulating cell migration, proliferation, and extracellular matrix deposition. Significant effort has been put into development of growth factor proteins as wound healing therapeutics. Despite encouraging pre-clinical studies evaluating a wide variety of growth factors, minimal clinical success has been achieved. Only one growth factor, platelet-derived growth factor-BB (PDGF-BB), has been approved by the Food and Drug Administration for the treatment of diabetic ulcers.[1,2] The failure of protein growth factors largely is due to inadequate delivery and persistence of the protein at the treatment site. In the last several years, gene therapy has been evaluated as an alternative approach for tissue repair. According to Chandler and Sosnowski,[3] there are a wide variety of gene therapy methods being evaluated for wound repair including ex-vivo delivery to keratinocytes, particle-mediated delivery of plasmid DNA, and direct injection. These approaches all have low gene transfer efficiencies and thus have not progressed to clinical studies. To circumvent the current limitation of gene therapy for wound repair, the authors have developed a novel gene therapy strategy called gene activated matrix (GAM). GAM consists of a gene encoding a growth factor immobilized within a biocompatible matrix, which is topically applied directly to the wound. Using this approach, the DNA vector as well as the transgene product is retained within the defect site. The matrix 1) immobilizes the DNA vector, thus increasing its persistence and availability at the wound site and 2) serves as a scaffold that is conducive for in-growth, proliferation, and transduction of repair fibroblasts. The tissue repair cells originating in the healthy tissue surrounding the wound site migrate into the matrix and then take-up and express the therapeutic transgene, resulting in improved healing. In-vitro studies using an adenoviral vector encoding PDGF-B (AdPDGF-B) formulated with a bovine Type I collagen gel have shown significant association of the vector with the matrix resulting in enhanced retention at the wound site.[4,5] The authors developed a gene therapy strategy for dermal wound repair using AdPDGF-B and a collagen gel. Stimulation of granulation tissue formation has been shown in multiple in-vivo models. While these animal models are poor surrogates for human healing deficits, they do provide important information on the biological activity and mechanism of action of the growth factor GAMs. The wound healing activity of AdPDGF-B formulated with a collagen matrix (final concentration 2.6%) is reviewed in three different models of dermal repair: the polyvinyl alcohol (PVA) sponge model, the diabetic db/db mouse model, and a rabbit full-thickness excisional wound model. In each model, AdPDGF-B/collagen delivery resulted in accelerated and augmented tissue repair. AdPDGF-B/Collagen in a PVA Sponge Implant The PVA sponge model is an established model of de-novo tissue generation.[6–8] PVA sponges are implanted subcutaneously in rats, allowing for infiltration by tissue repair cells including inflammatory and stromal cells. The granulation tissue that is produced is remodeled over time giving rise to mature granulation tissue similar to that found in healing dermal wounds. When AdPDGF-B formulated in collagen is injected into the center of an implanted sponge, it induces granulation tissue formation in a dose-response manner.[5] An adenoviral vector encoding the reporter gene luciferase does not result in significant quantities of granulation tissue. Using this model, the kinetics of granulation tissue formation was also evaluated. Histological analysis (Masson’s trichrome) revealed that by Day 4 posttreatment, void spaces were filled by cellular and vascularized tissue. At later time points (Day 6 and Day 8) arterioles and venules can be seen,[4] indicative of continuing vascular development. In the same model, Doukas, et al.,[5] directly compared gene therapy with protein delivery. A single application of AdPDGF-B/collagen resulted in a three-fold increase in the percentage of sponge filled by granulation tissue compared to collagen alone. When the PDGF-BB protein was administered as a single 100mg dose, the tissue formation response was significantly lower. In fact, it took three injections of PDGF-BB protein (10mg every other day) in order to achieve responses comparable to a single administration of the AdPDGF-B gene. These data show the ability of AdPDGF-B/collagen to rapidly induce tissue formation and supports the hypothesis that gene delivery with in-situ growth factor production translates into greater histogenesis than that seen with growth factor proteins. AdPDGF-B/Collagen in the Diabetic db/db Mouse Model The db/db mouse exhibits clinically relevant characteristics (i.e., obesity, insulin resistance, hyperinsulinemia, and severe hyperglycemia) of human adult onset diabetes with a concomitant delay in wound healing.[9–11] Complications seen in people with diabetes, such as peripheral neuropathy, microvascular lesions, basement membrane thickening, glomerular filtration abnormalities, and immunodeficiency, have also been observed in the db/db mouse. Because of these similar characteristics this model has been extensively used for a wide variety of dermal repair investigations.[11–14] To evaluate the wound healing response of AdPDGF-B/collagen in this model, two 8mm diameter, full-thickness, circular wounds were surgically created through the dermis on the back of each mouse. Treatments consisted of AdPDGF-B formulated with bovine Type I collagen to a final concentration of 2.6-percent collagen (AdPDGF-B/collagen) or an equal volume of buffer in 2.6 percent collagen as control (control/collagen). Wounds were treated topically with control/collagen or AdPDGF-B/collagen at adenoviral doses of 1.2x108, 1.2x109, 1.2x1010, or 3.6x1010 virus particles (PN) per square centimeter (cm2) of wound surface. Ten days post-treatment, wounds were harvested, processed for histology, and quantitatively evaluated for granulation tissue formation (Masson’s trichrome) by morphometric analyses. Granulation tissue was defined as neotissue consisting of new blood vessels, fibroblasts, macrophages, and loose connective tissue. All measured areas of granulation tissue deposition were limited to the original wound margins as indicated by the surgical excision sites from the dermis to the panniculus carnosus (Figure 1). Within the wound space, areas not classified as granulation tissue (and therefore excluded from morphometric analyses) included areas of new epithelium, fibrin clots, hematomas, edema fluid (if present), residual bovine collagen from the treatment, and areas that were predominantly occupied by inflammatory cells (e.g., leukocytes). At sacrifice, all animals had elevated serum glucose levels of 300 to 1000mg/dL (normal 6cm2) full-thickness, non-ischemic, dermal wound model in rabbits that can accommodate repeated treatments with AdPDGF-B/collagen. Animals received either collagen alone or three different dose levels of AdPDGF-B/collagen (1.0 x 109 PN, 1.1 x 1010 PN and 1.2 x 1011 PN/cm2). Wounds were treated four times at 1-week intervals (Day 1, 8, 15, 22). Morphometric analysis was performed on tissue samples taken at Day 23 and Day 36. Following four weekly treatments with AdPDGF-B/collagen a clear, dose-dependent effect on granulation tissue development in the wound bed was observed. Microscopic evaluation of wounds on Day 23 clearly showed that repeat administration of AdPDGF-B/collagen significantly improved dermal healing as determined by the amount (cross-sectional area) of granulation tissue formed within the wound bed (Table 2). At the low vector dose of 1.0 x 109 PN/cm2 of wound surface, AdPDGF-B/collagen enhanced granulation tissue deposition by 36 percent relative to the control/collagen treatments; however, this difference was not statistically significant. At both mid- (1.1 x 1010 PN/cm2) and high- (1.2 x 1011 PN/cm2) AdPDGF-B/collagen doses, dermal healing improved significantly by 73 percent (p