Gene therapy in wound healing - Nature

Gene Therapy (2007) 14, 1–10 & 2007 Nature Publishing Group All rights reserved 0969-7128/07 $30.00 www.nature.com/gt

REVIEW

Gene therapy in wound healing: present status and future directions LK Branski1, CT Pereira1, DN Herndon and MG Jeschke Department of Surgery, The University of Texas Medical Branch and Shriners Hospitals for Children, Galveston, TX, USA

Gene therapy was traditionally considered a treatment modality for patients with congenital defects of key metabolic functions or late-stage malignancies. The realization that gene therapy applications were much vaster has opened up endless opportunities for therapeutic genetic manipulations, especially in the skin and external wounds. Cutaneous wound healing is a complicated, multistep process with numerous mediators that act in a network of activation and inhibition processes. Gene delivery in this environment poses a particular challenge. Numerous models of gene

delivery have been developed, including naked DNA application, viral transfection, high-pressure injection, liposomal delivery, and more. Of the various methods for gene transfer, cationic cholesterol-containing liposomal constructs are emerging as a method with great potential for non-viral gene transfer in the wound. This article aims to review the research on gene therapy in wound healing and possible future directions in this exciting field. Gene Therapy (2007) 14, 1–10. doi:10.1038/sj.gt.3302837; published online 24 August 2006

Keywords: gene transfer; liposomes; wound healing; growth factors

Introduction Gene therapy is defined as the insertion of a desired gene (transgene) into recipient cells. It was traditionally considered a treatment modality for patients with lifethreatening congenital defects of key metabolic functions or patients with late-stage malignancies. The human genome project has provided us with the complete library of human DNA. This, and the realization that gene therapy applications were much more vast, has opened up endless opportunities for gene therapy in all medical and surgical specialties.1 The skin is an ideal candidate for genetic manipulations. It is easily accessible, rendering it easy to monitor for adverse reactions and easy to transfect. The epidermis has a high turnover – an ideal situation for most gene transfer methods. The predominant cells of the skin, that is fibroblasts and keratinocytes, are easily harvested and cultured, allowing for testing in vitro and for use of skin cells as vehicles in gene transfer. This has lead to an increasing interest in the use of gene therapy in skin diseases, especially wound healing.2 Wounds lead to a loss of integrity of the skin and, if severe enough, may cause major disabilities or even death. Delayed healing, as seen in the elderly, and impaired function due to excessive scarring, as seen after a burn injury, can be a major cause of morbidity for many patients. In the United States alone, over 2 million people suffer from burns and approximately 7 million suffer Correspondence: Dr MG Jeschke, Shriners Hospitals for Children, 815 Market Street, Galveston, TX 77550, USA. E-mail: [email protected] 1 These two authors contributed equally to the present work. Received 30 December 2005; revised 30 May 2006; accepted 20 June 2006; published online 24 August 2006

from chronic wounds caused by diabetes, pressure, venous diseases, and arterial diseases.3 The primary goals of wound care are rapid closure and a functional, aesthetically satisfactory, scar. Recent advances in cellular and molecular biology have greatly expanded our understanding of the processes involved in wound repair and tissue regeneration. Wound healing is a dynamic, interactive, and complex process involving soluble mediators, blood cells, extracellular matrix, and parenchymal cells.4 Wound repair processes follow a specific time sequence and can be categorized into three broad overlapping phases: inflammation, tissue formation, and tissue remodeling. Transient gene therapy is of particular interest in wound healing since a transient increase in strategic growth factors is required only until wound closure is achieved. This review article aims to discuss the emerging techniques in gene transfer and their use in wound healing.

The biology of wound healing Wound repair is a cascade of events that follows injury and consists of three phases: inflammation, tissue formation, and tissue remodeling.5,6 In the first phase, within minutes after injury, neutrophils, monocytes, and lymphocytes invade the wound site. Designed to defend the wound from invading microorganisms, they secrete proteinases and reactive oxygen species and clean up cell debris by phagocytosis. Additionally, the first phase of wound repair is crucial for the secretion of growth factors and cytokines – macrophages, activated from monocytes, function as the hub of early wound healing with the

Gene therapy in wound healing LK Branski et al

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expression of colony-stimulating factor 1, tumor necrosis factor a, and platelet-derived growth factor. These and other cytokines and chemoatractants moderate the transition between the initial phase of inflammation and the tissue repair within the first 2 days after injury. The second phase of wound healing, which takes place 2–7 days after injury, starts with the migration of keratinocytes at the wound edge. Fibroblasts and endothelial cells migrate into the clot, deposit extracellular matrix and form the so-called granulation tissue. In the third phase, the wound remodeling, fibroblasts have transformed into myofibroblasts that mediate wound contraction, and collagen has been deposited in abundance. The keratinocytes have closed the wound surface with a neoepidermis. This third phase of wound healing shows a transition from granulation tissue to scar tissue, a continued deposition of collagen and constant remodeling of the scar that lasts for months.5,7 The complicated interactions of wound healing are mediated by molecules that interact as mediators and receptors and play a pivotal role in linking the steps of wound healing. These proteins, known as growth factors, were discovered in the 1970s. They directly regulate many of the processes that are essential for wound healing. Growth factors are synthesized and secreted by many types of cells involved in tissue repair including platelets, inflammatory cells, fibroblasts, epithelial cells, and vascular endothelial cells.4,8,9 A deficiency of growth factors leads to impaired wound healing and is caused by numerous factors. Decreased production and secretion, as for example, in diabetic ulcers, impairs wound healing directly.10 Loss of growth factors owing to macromolecular leakage of fibrogen, a-macroglobulin, and albumin has been shown in venous stasis ulcers, diabetic ulcers, and burns.9,11,12 In a review on growth factors by Werner and Grose,5 the current knowledge on expression and function of endogenous growth factors in

Table 1

different phases of wound healing is summarized. There is a need for high levels of growth factors and cytokines as key regulators during the healing process, but abnormal growth factor expression may lead to impaired wound healing or abnormal scarring, such as scar hypertrophy or keloid formation.

Therapeutical application of growth factors Exogenous application of growth factors has been extensively used in chronic wounds where growth factors are deficient.13,14 Hence, increasing the concentration of strategic growth factors in the wound at precise time points represents a promising therapeutic approach. Despite such clear rationale, suggesting the probability of enhancing wound healing trajectories with topical growth factor application, clinical success has been limited. To date, only one drug using recombinant growth factors has received approval by the US Food and Drug Administration: recombinant human plateletderived growth factor BB. However, this growth factor has only been approved for use in diabetic foot ulcers.12 There are certain reasons why growth factors are limited in clinical use. It is well described how various growth factors affect different processes of the wound-healing scheme (Table 1), and that wound healing consists of a complex and variable interaction between cells, soluble cytokines, blood elements, and extracellular matrix.4,15 The application of a single growth factor might have only a transient effect in the enhancement of wound healing. Recombinant growth factors have been tried as means for increasing local concentrations of growth factors.14 However, they are much more expensive and labor intensive to produce compared with plasmids or vectors used for gene transfer. Proteolytic enzymes in the wound

Important growth factors involved in various phases of wound healing and their functions

Growth Factor

Source

Target

Effect

EGF family EGF TGF-a HB-EGF

Platelets Macrophages, epidermal cells Macrophages

Epidermal, mesenchymal Epidermal, mesenchymal Epidermal, mesenchymal

Pleiotropic motility/proliferation Pleiotropic motility/proliferation Pleiotropic motility/proliferation

FGF family b-FGF KGF (FGF-7)

Macrophages, endothelial cells Fibroblasts

Fibroblasts Epidermal

Wound vascularization Epidermal motility/proliferation

TGF-b family TGFb1–b2 TGF-b3

Platelets, macrophages Macrophages

Keratinocytes, fibroblasts Epidermal

Fibrosis, tensile strength Antiscarring

Other PDGF IGF-I VEGF IL-1a/b CSF HGF Activin

Platelets, macrophages, epidermal cells Plasma, platelets Macrophages, epidermal cells Neutrophils Multiple cells Multiple cells Fibroblasts, epidermal cells

Fibroblasts, macrophage Endothelial, fibroblasts

Chemotactic, matrix production Mitogen, activation Angiogenesis Pleiotropic growth factor expression Activation, granulation formation Activation fibroblasts, matrix production Unknown

Macrophages, epidermal Macrophage Unknown Unknown

Abbreviations: CSF, colony-stimulating factor; EGF, epidermal growth factor; FGF, fibroblast growth factor; HGF, hepatocyte growth factor; IGF, iinsulin-like growth factor-I; IL-1, interleukin-1; KGF, keratinocyte growth factor; PDGF-a, -b, platelet-derived growth factor-a, -b; TGF-a, transforming growth factor-a; TGF-b, transforming growth factor-b; VEGF, vascular endothelial growth factor. Gene Therapy


exudates tend to lyse topically applied growth factors. Further, of the active protein that remains on the wound bed after these interactions, the final diffusion into the wound tissue is also predictably low. In a study with topically applied basic fibroblast growth factor and epidermal growth factor, only 1–9% of the applied dose reached a depth of 1–3 mm.16 Recombinant human platelet-derived growth factor BB, the only growth factor in current clinical use, requires a dose of 100 mg/cm2 on alternate days, which is almost 50 times the amount that has been shown to be active in genetic studies.17 The pivotal role of growth factors in the regulation of wound repair has led to the development of multiple molecular genetic approaches that mainly focus on stimulation and enhancement of this protein group. In this review, we therefore discuss the current status of gene transfer research with a focus on growth factor genes.

Gene transfer techniques There are two basic strategies that depend upon the introduction and expression of foreign DNA into host cells: stable transformation for the permanent correction (permanent insertion of DNA, known as gene therapy) and transient transformation (for short-term expression of a gene product, known as gene medicine).18 For successful gene delivery, the selection of an appropriate vector has been shown to be paramount.19 Genes can be delivered to target tissue by either ex vivo or in vivo approaches. Ex vivo techniques rely on the isolation and cultivation of selected cells, their transfection in vitro and subsequent transplantation to a host. This approach is laborious and more expensive than in vivo techniques, in which genes are introduced directly to target tissue without the need for cell culture. This can be achieved using viral or non-viral genes (Table 2).

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Viral gene transfer Gene transfer with viral vectors relies on the natural ability of viruses to carry and express their genes in hosts. These vectors introduce DNA into cells with high efficiency. Viral gene therapy takes advantage of this by deleting structural genes or genes essential for virus replication and replacing them with sequences encoding a gene of interest.20 Gene therapy vectors are being developed by the modification of different types of viruses. They can be classified into two classes following their different strategies for replication, assembling and survival. The non-lytic replication method entails virions, produced from the cellular membrane of an infected cell, leaving the host cell relatively intact. This group includes retroviruses and lentiviruses. The lytic replication method involves the release of virions with the collapse of the host cell after infection. Human adenoviruses, herpes simplex viruses, as well as the adeno-associated viruses, belonging to the parvovirus group, are lytic replicators. Production of viral gene therapy vectors starts with genetic modification of the virus. Deletion of the original viral genes for replication or assembling is followed by insertion of a therapeutic gene.20,21 The ability to reproduce is then returned to the viral vector by using specialized cell lines called ‘packaging cell lines’ that are engineered to replace a function of a deleted viral gene and for the production of recombinant viruses.22 Adenoviruses. Administration of genes via the adenoviral vector has advantages in cell specificity, complexity, and stability. Adenoviruses are double-stranded DNA viruses with a genome size of about 36 kb. They are the most used and best described vector in viral gene therapy. In spite of their limited genome size of 36 kb, they can accommodate up to 34 bp of foreign DNA

Table 2 Other animal experiments in wound gene transfer Target tissue

Gene

Vehicle

Animal model

Transfer

Reference

Re-epithelization

EGF GH IGF-I TGF-b1

Plasmid Retroviral Liposomal Hydrogel

Mouse Pig Rat Mouse

Ex vivo In vivo In vivo In vivo

Rosenthal et al.87 Vogt et al.73 Jeschke et al.88 Lee et al.89

Neovascularization

VEGF VEGF iNOS HGFa VEGF, bFGF, PDGF-B

Liposomal Viral Adenoviral Plasmid Liposomal

Rat Mouse Mouse Rat Rat

In In In In In

Taub et al.90 Mesri et al.91 Yamasaki et al.79 Kunugiza et al.92 Liu et al.93

Granulation tissue

PDGF-A PDGF-A PDGF-B PDGF-B PDGF-B

Retroviral Retroviral Adenoviral Retroviral Adenoviral

Mouse Rat Rabbit Rat Mouse

Ex vivo Ex vivo In vivo Ex vivo In vivo

Eming et al.94 Machens et al.95 Liechty et al.28 Breitbart et al.96 Keswani et al.97

Scar modulation

TGF-b TGF-b HGF FGF

Antisense Plasmid Adenoviral Plasmid

Mouse Rat Rabbit Mouse

In In In In

Choi et al.98 Benn et al.99 Ha et al.100 Sun et al.66

a

vivo vivo vivo vivo vivo

vivo vivo vivo vivo

With prostacyclin synthase (PGIS) – gene. Gene Therapy


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(third-generation adenoviral vectors or ‘gutless vectors’). They can be obtained in vitro at very high concentrations (1010–1011 viral particles/ml) and show the ability to transduce both dividing and non-dividing target cells.20,23 Adenoviruses as carriers were shown to exert a 95% efficiency in infecting skin cells in vitro24 and safe in regard to infection rates in a skin-transfection model.25 Integration of viral genome into the host genome does not occur; therefore, the expression of foreign-delivered genes is only temporal.26,27 One group has reported good results in the treatment of chronic wounds with the adenoviral vector. Liechty et al.28 transferred platelet-derived growth factor B to chronic wounds in the ischemic rabbit ear model via the adenoviral vector and found that these ischemic wounds, treated with only one administration of viral gene, re-epithelized significantly quicker than the nonischemic control wounds. A limitation of the applicability is an inflammatory response and negative effects on wound healing that occur after administration of the control adenovirus. The conclusion was drawn that adenoviral application alone may lead to inflammatory response and impaired wound healing.28 The cause of this inflammatory response is probably due to the capsid protein of the virus.29

Adeno-associated viruses. This single-stranded, nonenveloped DNA virus of 4.5 kb size causes a latent infection of human cells. Most of the adeno-associated viruses are non-pathogenic to humans. Its main advantages are the high stability and safety, and no risk for insertional mutagenesis; the heat-inactivation resistance; and broad tissue tropism. Adeno-associated viral vectors can accommodate up to 4–5 kb of DNA insert and need certain helper viruses to transfect their target.20 Adeno-associated viral vectors have been studied intensively and are currently used in phase 1 clinical trials to treat cystic fibrosis and a-antitrypsin deficiency in adults.30,31 Deodato et al.32 and Galeano et al.33 developed a model for external gene delivery of vascular endothelial growth factor A by the serotype 2 of adenoassociated viruses. Wound healing in the rat showed significant acceleration in 6–10 days after surgery (measurements of wound area reduction) and a wellstructured granulation and greater vascularization after 18 days in the treatment group compared to the control.32 The group confirmed these findings in a rat burn model, using the same gene and vector and demonstrating an increased epithelium regeneration and neo-angiogenesis.33 However, the authors attributed the increased vascularization to the excellent tropism of the vector to the skeletal muscle layer underlying the skin in rodents. This thin muscle layer is not available in humans and generally causes wounds in the rat to heal faster, indicating that this vector might not be as potent when used in humans. New approaches with the use of adeno-associated viral vectors indicate that different serotypes might influence the targeting abilities and transduction rates for specific targets as endothelial cells, leading to improved expression of the genes.34 Furthermore, it has been shown in transfection experiments in keratinocytes, using adeno-associated virus 2 expressing green fluorescent protein, that a treatment of keratinocytes with proteasome inhibitor MG132 resulted in a transient Gene Therapy

augmentation of green fluorescent protein expression in up to 37%.35 Up to now, no further in vivo studies with the use of adeno-associated viral vectors have been performed.

Retroviruses. Retroviral vectors are single-stranded RNA viruses that replicate through a DNA intermediate. They can be designed as highly tissue specific and safe of recombination after transfection.20 They are largely used in clinical trials, as more than one-third of gene therapy studies are based on retroviral gene transfer.36 Retroviruses are limited by low transfection efficiency, a relative particle instability and an inability to transduce non-dividing cells. The latter limitation is not of great concern for wound healing models, as the target cells are in the proliferating phase, and has partly been overcome by vectors that are constructed using lentiviruses and have surface glycoproteins replaced by genes from other viral genome, expanding the spectrum of target cells and making non-dividing cells a feasible target for retroviral vectors.37 Morgan et al.38 used retrovirus-mediated gene transfer in athymic mice with recombinant human growth hormone grafted on an epithelial sheet. The authors found that these cultured keratinocytes reconstituted an epidermis that was similar in appearance to that resulting from normal cells, but from which human growth hormone could be extracted. Since then, however, no other studies have shown significant acceleration of wound healing using retroviral vectors. Non-viral gene transfer Non-viral gene therapy in wound healing has several advantages over viral gene therapy. There is no additional inflammation and infection risk, most methods are simple and inexpensive, and do not require ex vivo manipulation.39 The transient expression can be used to an advantage in wound healing where stable expression is a distinct disadvantage once the wound has healed. However, non-viral gene transfer methods tend to be non-specific with regards to the cells being transfected and are variable in the level of gene expression. Non-viral gene transfer methods can be divided into two main groups: (a) naked DNA transfection and (b) liposomal transfection. They are described in detail below. (a) Naked DNA transfection. There are several different methods to transfer naked DNA to the skin, such as direct injection, topical application, gene gun, microseeding, and electroporation. Direct injection. Hengge et al.40 first described direct injection in 1995, where he injected naked DNA coding for interleukin 8 and found a significant recruitment of dermal neutrophils post transfer. However, the injection of naked DNA into the skin has been proven to be inefficient at transferring genes. Owing to the fragility of the naked DNA constructs in the extracellular environment and its relatively large size and electric charge, naked DNA is not likely to penetrate the cells.39 The topical application of naked LacZ DNA to the skin has been shown to rapidly penetrate the skin and b-galactosidase was mainly expressed in the dermis, epidermis, and follicular keratinocytes.40,41 The stratum corneum, which is the outer protective layer of the


epidermis, is hydrophobic and it is difficult to penetrate this layer being a negatively charged cDNA. Studies to deliver DNA coding for growth factors to show efficacy of this technique have not been performed.

Gene gun and microseeding. Particle-mediated gene transfer (commonly known as gene gun technology) uses physical force to penetrate the cellular membrane. Goldor tungsten-coated microparticles (1–5 mm in diameter) carry the gene of interest, mostly in the form of a circular DNA plasmid. A ballistic device then propels these DNA-coated particles into skin cells.42 Gene transfer is mostly transient (stable gene transfer only occurs in a 10 3 to 10 4 ratio) and peaks between the first and the third day after injecting the particles into the skin.43,44 It has been hypothesized that depending on the amount of force that is applied, individual epidermal cell layers can be reached. Advantages for the use of the gene gun are its wide range of application for many different genes at the same time. Furthermore, it can be used to transfect many different forms of tissue.45 Recent in vivo rat studies showed that gene gun particle-mediated transfection of different plateletderived growth factor isomers significantly improved wound healing by increasing tensile strength after wounding.42 An in vivo transfection with gold particles with the gene for epithelial growth factor in porcine partial-thickness wounds demonstrated an acceleration of wound healing and an increased rate of re-epithelization.46 Different depth of transfection has been reported: one study showed that the gene gun primarily delivers particles no further than the epidermis.44 Therefore, transfection rate is relatively low and ranges up to 10% of epidermis cells. However, Dileo et al.47 showed that using a modified gene gun to deliver plasmids with higher discharge pressures resulted in higher levels of gene expression in epidermis and subdermal tissues than observed with conventional guns. Gene expression in skin and muscle reached its peak level at 24 h after application and remained measurable for at least 1 week. The tissue damage was described as insignificant.47 Studies by the Eriksson group introduced a new method, the micro-seeding technique.48 Micro-seeding is a method that delivers naked DNA directly into target cells by solid microneedles mounted on a modified tattooing machine. Like the gene gun, it also delivers a variety of DNA into the healing wound. Physiological and supraphysiological levels of the transferred DNA can be reached for 1–2 weeks;48 however, transfection is only achieved in the superficial layers of skin with minimal if any deep transfection. Electroporation and gene transfer. Electroporation or electric stimulation means the application of an electric field to tissue. The technique of electroporation has been successfully used to accelerate the closure of diabetic and chronic wounds.49,50 Two recent publications from 2004 describe the synergistic use of electroporation in combination with tissue growth factor b1 – cDNA or keratinocyte growth factor – plasmid DNA.51,52 In the first study, tissue growth factor b1 – cDNA was injected in full-thickness excisional wounds in diabetic mice. In the group that received both electroporation and gene transfer, the wound bed showed an increased re-epithelization rate and an increase in collagen synthesis and angiogenesis.

The wound healing process itself, however, was not significantly accelerated by the synergistic use of electric stimulation and gene transfer, apart from a transitional effect between the second and fourth day after wounding.51 In a similar study conducted by Marti et al.,52 synergistic electroporation and administration of keratinocyte growth factor DNA showed an accelerated wound healing compared to the control group with no treatment (92 vs 40% area healed), but again no significant improvement in comparison to administration of keratinocyte growth factor DNA alone was observed. The benefit of this concept of electrical stimulation and simultaneous injection of naked DNA remains yet to be proven.

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(b) Liposomal gene therapy. Cationic liposomes are synthetically prepared vesicles with a positively charged surface. When mixed with negatively charged DNA, they form a loose complex and protect the DNA from degradation. The net positive charge of the complex binds readily to negatively charged cell surfaces. For gene expression to occur, DNA plasmids have to enter the cell and its cell nucleus. Possible entry paths are depicted in Figure 1. There are five possible pathways of cell entry: endocytosis, fusion, lipid exchange, adsorption, and direct membrane poration.53,54 However, endocytosis is likely the principal mechanism for transfection. After cellular uptake, the released cDNA is taken up by the nucleus.55 The nuclear membrane represents a barrier to the entry of DNA into the nucleus, which is absent during cell division, suggesting that transfection may be more efficient in rapidly dividing cells.55 Ribosomes transcribe the nuclear cDNA into mRNA, which is then transported to the rough endo-

Figure 1 Possible pathways for entry of liposomes and their subsequent intracellular distribution and translation. Gene Therapy


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plasmic reticulum where it is translated into protein. Following an uptake of these complexes, DNA is discharged into the cytoplasm, transported into a nucleus, and eventually transcribed and translated into a transgenic protein56,57 (Figure 1). When Bangham first discovered liposomes, their in vivo utility was largely met with skepticism owing to low in vivo transfection efficiencies.55 Modification of the standard liposomal structure to a cationic structure improved efficacy of liposomal constructs.56,58 The change from multivalent cationic lipids to monovalent cationic lipids further improved transfection rates.39 The use of a cytomegalovirus promoter in the cDNA constructs used for gene transfer also increased the efficacy and transgenic expression levels to those previously achieved with adenovirus constructs.55,59 As a vector for gene therapy, liposomes have become attractive owing to their non-viral composition, stability, and ability to interact with the cell membrane.55 Cationic liposomes form complexes with the DNA through charge interactions – DNA–liposome complexes bind to the negatively charged cell surface owing to the presence of excess positive charges in the complex. The nature of non-specific interactions results in efficient transfection of many cell types. The flexibility in the design of cationic lipid structures and the variety of methods used in the liposomal preparation broaden their scope for the therapeutic use.43 Further, liposomes can be applied by either topical administration or by direct injection of liposomal gene constructs.41,56 Alexander and Akhurst60 demonstrated in 1995 that topical application of liposomal constructs containing the Lac Z gene in shaved 4-week-old mice resulted in a transfection and expression in the epidermis, dermis, and hair follicle. Expression was seen at 6 h post-application and persisted at high levels 48 h post-application. At 7 days after application, the expression was reduced but detectable.60 Topical administration of liposomal complexes, however, is not as effective in regards to cell transfection and level of expression as injection.39,61 This is due to the stratum corneum, which is the protective layer of the epidermis. Injecting the liposomal gene complexes results in transfection of epidermal cells, predominantly keratinocytes and dermal cells, such as myofibroblasts and inflammatory cells.57 Others and we have shown that transfection is only transient.19,56 The reason for transient transfection and expression is due to degradation and loss of the DNA reservoir from the skin and a lack of stable gene transfection into stem cells, for example, keratinocyte stem cells.19 Thus, cationic liposomes are emerging as a viable technique for gene transfer. Other techniques such as biomaterials,62 calcium phosphate transfection,63 diethylaminoethyl-dextran59 and microbubble-enhanced ultrasound64 have been attempted. These new techniques are promising, but further studies are necessary to define the efficacy and possible clinical applicability.

Growth factor delivery by liposomal transfection. Only few studies have been performed to determine liposomal gene transfer for the delivery of growth factors.42,65,66 Sun et al.66 administered acidic fibroblast growth factor to the skin of diabetic mice with incisional wounds by topical application plus subcutaneous injection. They found that transfection with acidic fibroblast Gene Therapy

growth factor increased wound breaking strength and improved wound quality when compared to controls. The effect of insulin-like growth factor I on epidermal and dermal regeneration after a thermal injury,65 which is characterized by a hypermetabolic response that compromises the immune system,67 has been investigated by Jeschke et al.57,61,65 The burn wound has been shown to be the major source of pro-inflammatory cytokines, which support and enhance the hypermetabolic response.67,68 Cationic liposomal complexes attenuate the pro-inflammatory hypermetabolism and post-traumatic acute phase response,69 treatment with insulin-like growth factor I has been shown to accelerate wound healing by stimulating collagen synthesis and the mitogenicity of fibroblasts and keratinocytes.61 In a preliminary biodistribution study, using thermally injured rat skin, transfection was detected in myofibroblasts, endothelial cells, and macrophages, including multinucleate giant cells. The transfection rate reached in certain areas 70–90%57,65 (Figure 2). The observation that mRNA for insulin-like growth factor I was only detected in the skin of transfected rats is consistent with increases of insulin-like growth factor I concentration in the skin. It has been suggested that the skin may be used as a delivery system for gene products because foreign gene products synthesized in the epidermis have been shown to reach the systemic circulation.45,60,70 The skin was described serving as a bioreactor. Thermally injured rats treated with liposomal insulin-like growth factor I cDNA-constructs had significant less body weight loss and increased protein concentration in muscle, liver, and kidney when compared to treatment with empty liposomes or saline.65 However, no evidence was found that the dermal injection of insulin-like growth factor I cDNA–complexes leads to transfection or increase in b-galactosidase or insulin-like growth factor I expression in blood, liver, spleen, or kidney.57 It seems that injection of liposomal constructs allows for specific targeting of epidermal and dermal cells.57,61

Figure 2 Representative photomicrographs for the histochemical reaction for b-galactosidase of the wound edge taken 5 days after the last injection of liposomes plus LacZ. The reaction product for b-galactosidase is present within several layers of the skin at the wound edge. A granular blue–green reaction product is present within many myofibroblastic and histiocytic cells in the granulation tissue underlying the burn wound. Magnification 40.


Figure 3 Representative photomicrograph of histological staining with Masson Goldner, of an animal receiving gene therapy. The collagen demonstrates an intense staining.

In further studies it was found that animals receiving the insulin-like growth factor I cDNA had an accelerated rate of re-epithelization/wound healing when compared to multiple other treatments, such as the naked protein, insulin-like growth factor I encapsulated in liposomes, liposomes encoding the Lac Z gene, or normal saline.57,65,71 In these experiments, liposomal insulin-like growth factor I cDNA increased the rate of re-epithelization by 15% compared to other experimental settings, with an increase in basal skin cell proliferation. It seems that myofibroblasts, endothelial cells, and macrophages, which were identified to be transfected, produce biologically active insulin-like growth factor I and exert autocrine, paracrine, or endocrine effects61 (Figure 3). Our group subsequently investigated the feasibility of multiple cDNA constructs, using multiple genes (cDNA for keratinocyte growth factor and insulin-like growth factor I) and compared this to the administration of the same growth factors individually.71 Accelerated re-epithelization, increased proliferation, and decreased skin cell apoptosis compared to controls was noted. The re-epithelization in the burn model reached 225% of the untreated control and significantly improved cell survival.71

Future directions Gene transfer promises to have great potential in the treatment of wounds. However, out of currently over 300 clinical trials in the field of gene transfer, there are only two trials with growth factors targeting the wound locally: hepatocyte growth factor and platelet-derived growth factor A in chronic wounds.72 As with gene therapy for other indications, the potential of immunological or toxic side effects, and tumor development – in particular with growth factor gene therapies – should be considered in all instances. Several groups demonstrated that liposomes containing DNA sequences for growth factors enhance, accelerate, and improve dermal and epidermal regeneration. The exogenously applied cDNA is transcribed and translated into endogenous protein and exerts its physiologic effects. As the transcription–

translation process is relatively limited to the skin with no systemic transfection, the approach of injecting DNA into the skin is relatively specific. By improving the quality and transfection rates of liposomes, they could be the delivery system of the future for gene therapy. A potential problem of single gene therapy is that increasing the concentration of a single growth factor may not effect all phases of wound healing. A single growth factor cannot counteract all the deficiencies of a burn wound, nor can it control the vicissitudes and complexities of chronic wound healing. Lynch et al.17 demonstrated that the combination of platelet-derived growth factor and insulin-like growth factor I was more effective than either growth factor alone in a partial thickness wound healing model created with the use of a dermatome. Sprugel et al.19 found that a combination of platelet-derived growth factor and basic fibroblast growth factor increased the DNA content of wounds in the rat (implanted wound chamber model) better than any single growth factor. Multiple gene transfections for delivery of multiple growth factors at strategic time points of wound healing (known as sequential growth factor therapy) is therefore the next logical step in augmenting wound healing. Work is in progress in our lab on using liposomal gene therapy transfer in an established porcine wound-healing model. Other labs have also conducted work on porcine models using retroviral technology.73 The concept of a genetic switch is another exciting development where transgenic expression in target cells can be switched ‘on’ or ‘off’ depending on the presence of or absence of a stimulator such as tetracycline.74 Further developments in matrix components and tissue engineering technology offer promise of a slow-release matrix, allowing for prolonged transgenic expression.75 Growth factors are not the only target for gene therapy in wound healing, and some promising new approaches have recently been reported. Transcription (co-) factors such as cardiac ankyrin repeat protein or L-Sox5, Sox6, and Sox9 are active regulators in wound repair. Cardiac ankyrin repeat protein is expressed in skeletal muscle, vessel wall, hair follicle, inflammatory cells, and epidermis of wounds. When delivered to wounds by an adenoviral vector, cardiac ankyrin repeat protein leads to an increase in the vascular component in granulation tissue in the rat model.76 The Sox family are transcription factors that mediate the differentiation of mesenchymal cells into chondrocytes and might present useful targets in the genetic modulation of cartilage repair.77 Another transcription factor, nuclear factor-kB, mediates inflammation on normal cutaneous wound healing. An inhibitor of this transcription factor was administered into the rat using an adenoviral vector, which resulted in increased collagen deposition and nitric oxide levels and accelerated wound healing.78 The direct effect of injection of genes encoding for interleukin 8 has been described by Hengge,40 and Yamasaki et al.79 showed that the adenoviral transfer of inducible nitric oxide synthase leads to improved wound healing in the mouse. Transduction molecules might also represent a target in wound healing therapy. Members of the small mother against decapentaplegic family (SMAD2–4) are regulating factors for fibroblast proliferation, and inhibition of SMAD3 activity has been demonstrated to markedly promote wound healing, as SMAD3

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mediates the role of transforming growth factor-b in inhibiting cell proliferation and inducing cell apoptosis. Cofactors such as c-ski regulate the activity of SMAD and might be accessible for gene therapy.80,81 It is also important to consider gene therapy applications in other contexts of wound healing such as for downregulating exaggerated responses seen in hypertrophic and keloid scars.82 Antisense oligonucleotides have been used to regulate the activity of collagen genes to control fibrosis.83 Biotechnological refinements such as wound chamber technique84 and gene-delivering gel/ matrix products85,86 may help improve the efficacy of gene delivery to wounds. Use of skin as a bioreactor for simultaneous wound healing locally and systemic therapy to reduce hypermetabolism postburn is an as yet unchartered territory and work in this area promises to be exciting with its clinical relevance. Further studies also need to be conducted to accurately detect the growth factor deficiencies/increases in different types of wounds and to elucidate the precise timing of gene expression or downregulation required to augment wound healing and control scar formation.

Acknowledgements We thank Liz Cook, Eileen Figueroa, and Steve Schuenke, Publications Office, Department of Surgery, The University of Texas Medical Branch, Galveston Texas, for their help in the preparation of this manuscript. We acknowledge the Postdoctoral Training in Trauma and Burns (T32-GM08256) and the Clayton Foundation for Research.

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