Matrix-Immobilized Growth Factor Gene Therapy Enhances Tissue Repair

Author(s): 
Dan-Ling Gu, MD;1 Thanh Nguyen, BS;2 M. Laurie Phillips, PhD;3 Lois A. Chandler, PhD;4 Barbara Sosnowski, PhD4

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

Microscopic evaluation of granulation tissue in the trichrome-stained wound sections confirmed these gross observations and demonstrated that AdPDGF-B/collagen induced wound healing in a dose-responsive manner (Figure 2). In general, neotissues present in AdPDGF-B/collagen-treated wounds were thick and highly cellular as a result of the robust infiltration and proliferation of fibroblasts, microvascular endothelial cells, and monocytes in the exogenous collagen matrix, particularly in the mid- and high-dose treated wounds (Figure 2, E–J). By contrast, the authors observed minimal healing in the control- and low-dose treated wounds (Figure 2, A–D). Granulation tissue in these wounds was sparse, and the extent of cellular influx and proliferation at the wound margins and in the wound bed was minimal compared to the three higher dose AdPDGF-B/collagen-treated wounds.

Consistent with the authors’ qualitative assessment of tissue repair, measurements of granulation tissue formation by quantitative morphometric analysis clearly revealed a dose-related effect to AdPDGF-B/collagen therapy on the diabetic wounds (Table 1).
Treatment with AdPDGF-B/collagen resulted in a clear dose-related effect on healing of full-thickness dermal wounds in diabetic mice. In this model of impaired wound healing, no treatment effect was observed at 1.2x108 PN of AdPDGF-B per cm2 of wound surface, and the maximum effective dose was observed at 1.2x1010 PN/cm2. No additional benefit was achieved by increasing the dose to 3.6x1010 PN/cm2. The results demonstrate that in the diabetic mouse model, a single administration of the maximum effective dose of AdPDGF-B/collagen improves wound healing nearly three-fold as determined by the amount of granulation tissue formed in the wound.

AdPDGF-B/collagen in a Full-Thickness Dermal Wound Model in Rabbits

In clinical practice, because of the intractable nature of human diabetic foot ulcers, it is anticipated that repeated applications of AdPDGF-B/collagen may be needed to achieve complete closure of wounds. To determine whether repeated treatment alters the tissue distribution, safety, and efficacy of AdPDGF-B/collagen, the authors developed a large (>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

Compared to both the control- and the low-dose AdPDGF-B/collagen-treated wounds, granulation tissues at Day 23 in the mid- and high-dose AdPDGF-B/collagen-treated wounds were macroscopically thicker, with a visibly greater number of fibroblasts, neovasculature, and
monocytes.

In many cases, spatially distinct layers of granulation tissue differing in degree of maturity occupied the mid- and high-dose wound beds, suggestive of the progressive stages (i.e., inflammation, tissue formation, tissue remodeling) of tissue repair anticipated following sequential weekly treatments (Figure 3). Neotissues at the wound base, which coincide with the first treatment, were more mature and remodeled, judging by the presence of cross-linked collagen fibrils organized horizontally through this area and the reduction in tissue cellularity. The center layers of the wound bed were occupied by an increasing number of proliferating cells, comprising nascent granulation tissue arising from the second and third treatments. The final (4th) application of AdPDGF-B/collagen was distinguishable at the surface of the wound as a layer of predominantly nonintegrated bovine collagen infiltrated by fibroblasts and inflammatory cells, characteristic of the early phase of wound healing. By contrast, granulation tissues in the control- and low-dose AdPDGF-B/collagen-treated wounds at this time point were sparse and loosely scattered throughout the wound space.

Complete wound closure (reepithelization) was achieved in the majority of the wounds in all AdPDGF-B treatment groups by Day 36 (data not shown), indicating that AdPDGF-B/collagen therapy does not impede reepithelization in this wound healing model.

The rabbit full-thickness excisional model was also used to evaluate the safety, biodistribution, and immunogenicity profiles of AdPDGF-B/collagen following a single administration and multiple administrations over a four-week period. In brief, repeat administrations of AdPDGF-B/collagen was found to be safe, well tolerated, and predominately localized to the wound bed with vector dissemination limited to the nearest draining lymph nodes. In addition, AdPDGF-B/collagen treatment induced significant tissue repair, even in the presence of pre-existing anti-Ad immune response (manuscript submitted).

Discussion

The in-vivo data generated using the rat PVA sponge model indicate that a single application of AdPDGF-B in collagen leads to sustained (6–8 days), local production of PDGF-BB and subsequent rapid tissue formation including cellular influx and proliferation, ECM deposition, and tissue modeling. Fibroblasts and inflammatory cells could be seen invading the collagen, and these cells produced human PDGF-BB protein as early as 24 hours after treatment (manuscript submitted). The repair cells present within the developing neotissue, such as mononuclear leukocytes, endothelial cells, and fibroblasts, were shown to be responsible for both transgene mRNA and PDGF-BB protein production.

The peak of cellular influx and proliferation coincided with an apparent peak in production and secretion of PDGF-BB between 4 and 6 days following treatment. Immunostaining demonstrated that during this time, a reservoir of extra-cellular PDGF-BB accumulated in the collagen matrix. Six days post-treatment, statistically significant, dose-related increases in the percent of sponge interior occupied by granulation tissue of up to 260 percent and increases in protein content (100%) and DNA content (170%) were observed versus collagen alone.5 Eight days following treatment, the neotissue induced by AdPDGF-B/collagen treatment showed signs of maturation, with a reduction in proliferating cells and a remodeling of the extracellular matrix, which consisted of new matrix deposition and neovascularization. Based on this data, a weekly dosing schedule was selected for initial human clinical studies.

The db/db diabetic mouse was the animal model of choice for analysis of a dose-response to AdPDGF-B/collagen as these mice exhibit clinically relevant characteristics of human adult onset diabetes (i.e., obesity, insulin resistance, hyperinsulinemia, and severe hyperglycemia) with a concomitant impairment of wound healing. This animal model is also sensitive to dose effects, and lends itself readily to quantification. In this model, a single application of AdPDGF-B/collagen at a dose of 1.2x108 PN/cm2 did not enhance granulation tissue deposition compared to the control treatments. However, at vector doses of 1.2x109, 1.2x1010, and 3.6x1010 PN/cm2, AdPDGF-B/collagen induced 2.4-, 2.8-, and 2.6-fold increases in granulation tissue formation respectively, over controls (p

Although the db/db mouse exhibits impaired wound healing, the acute, surgical wounds created in this model do not fully replicate the conditions present in nonhealing wounds of diabetic humans (reviewed in Pierce[15]). The chronic inflammatory state, which develops in human diabetes-associated ulcers, shows large amounts of matrix metalloproteinases and a greater ratio of proteases to protease-inhibitors than that seen in db/db mice. More proteases within wounds translates into more destruction of newly forming tissue and shortened half-lives of protein therapeutics placed within the wounds. These observations suggest that higher doses of AdPDGF-B/collagen (per cm2 of wound) and repeated application may be required for effective treatment of human diabetic ulcers.

Although the primary emphasis of the rabbit dermal wound study was safety, the authors also quantified the wound healing response to repeated treatment of AdPDGF-B/collagen. Examination of wounds following four weekly applications indicated that repeated administration of AdPDGF-B/collagen was highly efficacious. When compared to control/collagen treated wounds, AdPDGF-B/collagen induced granulation tissue area increases of 36 percent at 1.0x109 (NS), and of 73 percent at 1.1x1010 (p

Microscopically, wound beds harvested at Day 23 revealed that discrete layers of granulation tissue (with the least mature cellular architecture closest to the wound surface) could be seen, suggestive of progressive stages of tissue repair associated with sequential treatments. The final (fourth) application of AdPDGF-B/collagen was distinguishable at the top of the wound as a non-integrated collagen layer. As was observed in the PVA sponge studies, 24 hours after treatment this collagen was already infiltrated by fibroblasts and inflammatory cells, characteristic of the early phase of wound healing. By contrast, repeated treatments with control/collagen alone resulted in minimal healing as evidenced by the sparse level of cellular infiltrate within the collagen matrix.

The apparent maximal effective dose in this normal wound healing model is between 1.1x1010 and 1.2x1011 PN/cm2 of wound surface area, similar to that observed in the db/db diabetic mouse model. Although there was not a statistically significant difference between the low dose of 1.0x109 PN/cm2 and the collagen/control-treated wounds, this was likely due to the sample size, as a trend toward efficacy is apparent. Thus the no-observed-effect level of AdPDGF-B/collagen in this animal model is below 1.0x109 PN/cm2 of wound surface area, consistent with the dose response determined in the db/db diabetic mouse model.

Together, the nonclinical studies described here demonstrate that AdPDGF-B/collagen treatment is effective in a variety of tissue repair models. In view of the apparent lack of local or systemic toxicity (manuscript submitted) and the efficacy of AdPDGF-B/collagen in promoting wound healing, initiation of human clinical studies for treatment of chronic nonhealing diabetic foot ulcers is justified.

Recently, investigators began a phase I clinical research study utilizing AdPDGF-B/collagen in the treatment of diabetic ulcers. The primary objective of the clinical research study is to evaluate the safety of the topical gel and to determine the maximum tolerated dose. Results of the clinical research study will be reported once the trial has been completed.

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