Gene therapy in wound healing

Gene therapy in wound healing

Surg Clin N Am 83 (2003) 597–616 Gene therapy in wound healing Nicola C. Petrie, MB, ChB, MRCS, Feng Yao, PhD, Elof Eriksson, MD, PhD* Laboratory of ...

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Surg Clin N Am 83 (2003) 597–616

Gene therapy in wound healing Nicola C. Petrie, MB, ChB, MRCS, Feng Yao, PhD, Elof Eriksson, MD, PhD* Laboratory of Wound Repair and Gene Transfer, Division of Plastic Surgery, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115, USA

The first approved human gene therapy trial started on September 14, 1990. Researchers at the US National Institutes of Health removed white blood cells from the body of 4-year-old Ashanti DeSilva, born with severe combined immunodeficiency syndrome (SCID), a rare genetic disease. Her cells were cultivated in vitro, the missing gene was inserted, and the genetically modified blood cells were infused back into her bloodstream. The therapy showed promising results as evidenced by Ashanti’s reduced incidence of common colds and general improvement in health, even permitting her to attend school [1]. These encouraging findings laid the path that subsequent researchers were to follow, and as of September 2001, 596 completed, ongoing, or pending trials have been identified worldwide. The United States has funded most trials (78.7%), with the United Kingdom (7.2%), France (2.5%), Canada (1.8%), and Germany (1.7%) also contributing [2]. Gene therapy is a new and emerging technology that employs the process of manipulating genes, the biologic unit of heredity [3], and in essence involves the insertion of a desired gene (termed the transgene) into the recipient’s cells. In this respect, the Human Genome Project (HGP) [4] has played a crucial role. The HGP initially was envisioned in the 1980s and came to fruition with the collaboration between the National Institutes of Health (NIH) [5] and the Department of Energy [6] in the 1990s. The goal of the HGP is to identify and sequence the estimated 30,000 genes that comprise the human genome [7]. When considering the study of gene therapy, it is essential to make the distinction between therapies on somatic cells versus therapies on germ cells (gamete cells, which contain half the normal number of chromosomes and subsequently are capable of reproduction). The essential difference lies in the

* Corresponding author. E-mail address: [email protected] (E. Eriksson). 0039-6109/03/$ - see front matter Ó 2003, Elsevier Inc. All rights reserved. doi:10.1016/S0039-6109(02)00194-9

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fact that somatic gene therapy involves manipulating genes from an individual and is not inherited, and germline therapy involves manipulation of genes before the formation of an individual and is inherited. Ethical, religious, legal, and other issues are raised that ultimately may affect the acceptance of germline gene therapy by society. At present, this research has been approved only in a few countries, and the treatment has been approved only for nonhuman use. Gene therapy initially was envisaged as having its greatest influence on diseases that are caused by single gene defects and as such, by the end of 1993, had been approved for research in SCID, familial hypercholesterolemia, cystic fibrosis, and Gaucher’s disease [8]. The realization shortly followed, however, that its applications were much more vast, and theories regarding these applications took off. The completion of the HGP is due in 2003 in a timely fashion to coincide with the 50th anniversary of Watson and Crick’s description of the fundamental structure of DNA [9]. With the complete library of human DNA subsequently in place, the opportunities for gene therapy in all surgical specialties will be endless [3]. Rationale for the application of gene therapy to wound healing It is estimated that each year in the United States more than 5 million patients experience the debilitating consequences of chronic nonhealing wounds [10]. In addition, there are a significant number of acute wounds, which each day become complicated by dehiscence, infection, or hypertrophy. The physical, financial, social, and psychological consequences that such wounds inflict on individuals and on society provide the rationale for searching for improved methods of treating problematic wounds. The physiologic process of wound healing has been described as a cascade—a snowballing sequence of events, which act and interact with the ultimate goal of achieving the complex, precise purpose of wound closure. The initial injury-induced influx of platelets to the wound site is followed rapidly by their degranulation and release of many mediators, which propel the rapidly evolving cascade. The intricacies of the process of wound healing have been described accurately and extensively elsewhere [11,12], and it is sufficient, for this purpose, to think of the process as consisting of two essential players—cellular products and the cells themselves—that interact through autocrine, paracrine, and endocrine mechanisms. The breakdown of these communication pathways flips the physiologic fuse box to break the wound healing circuit. Underlying diseases such as ischemia, infection, diabetes, and venous stasis are examples of circuit breakers [13]. Only by examining the process closely can clinicians gain insight into the numerous areas amenable to targeting by gene therapy. Not only is an understanding of healing necessary for the application of gene therapy, but

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also an understanding that the relationship is symbiotic in that information gained from genetic studies themselves is positive feedback to increase understanding of wound healing further. Gene therapy in wound healing holds significant promise as a tool for understanding the role of Growth Factors (GF) in wound healing and as a therapeutic strategy [10]. Although gene therapy initially was focused on the correction of inherited diseases for which no therapeutic approaches were available [14], the technique can be applied equally to the local, temporary treatment of acquired diseases, including impaired wound healing and tissue repair [15]. As of September 2001, only 12.6% of clinical trials in gene therapy were for monogenic diseases, with most (63%) addressing cancer treatment [2]. The realization of the difficulty of achieving permanent expression of the transgene focused attention in other areas. Most gene delivery techniques enable only transient transgene expression, and although this is a detrimental quality limiting their use for the treatment of genetic disease, it is a quality that lends itself perfectly to the treatment of temporary conditions, such as wounds, which require only a transient pulse of gene product. This transient expression also may be desirable in view of reduced long-term risks [16]. Recombinant growth factors—the forerunners to wound healing gene therapy Studies performed by Cooper and others [17,18] and supported by the findings of Pierce and colleagues [19] reported that the levels of some growth factors, including platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), endothelial growth factor (EGF), and transforming growth factor-b (TFG-b), were reduced markedly in wound fluid from chronic pressure ulcers compared with acute wounds. The suggestion that a deficiency of such cytokines was responsible for delayed healing was supported further by the finding that increased levels of PDGF were detected in patients with chronic ulcers that eventually healed [20]. Hypotheses to explain the reduced levels of growth factors include degradation by increased levels of wound matrix metalloproteinases [13], trapping of cytokines within the fibrin cuffs surrounding capillaries in venous stasis ulcers [21], and degradation by proteases of bacterial origin present in chronic wounds [22]. Later studies by Trengove and colleagues [23] mainly refuted these earlier results, however. The development of recombinant growth factors prompted by these findings was obvious, and since then many animal studies have been performed that show how the application of topical cytokines can improve the tissue repair response [24]. Cytokines tested by topical application include PDGF, TFG-b, EGF, bFGF, granulocyte-macrophage colonystimulating factor [25], insulin-like growth factor-1, human growth hormone (hGH), and keratocyte growth factor-2, most of which have been reviewed in detail by Robson [26]. Although their effects initially were promising,

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recombinant growth factors soon were found to be limited by their short half-life, inactivation by wound proteases, and lack of an optimal delivery system, resulting in the need for high initial doses and daily application incurring high costs [22] and potential toxicity. It was this shortfalling that led to the application of gene therapy for wound healing. It was reasonable to suppose that by delivering the DNA or gene corresponding to the same recombinant protein directly into the target tissue, one might bypass the aforementioned limitations. Skin as a target for gene therapy The skin is an attractive target for gene therapy for many reasons. The predominant cells of the skin, fibroblasts and keratinocytes [27], are harvested easily, and protocols exist for their successful culture. This situation not only enables skin cells to be tested in vitro, but also highlights their availability for use as vehicles in ex vivo protocols (see later) because such cells readily can be transplanted back to a host. The superficial location of the skin enables it to be monitored easily for any adverse effects or reactions and renders it accessible to direct DNA transfer by many techniques, including injection, microseeding, and topical application, avoiding unnecessary systemic delivery. The infusion of a transgene was responsible for gaining adverse publicity for gene therapy after the death, on September 17, 1999, of an 18-year-old man enrolled in a phase I gene therapy clinical trial for the treatment of an inherited liver disease, ornithine transcarbamoylase deficiency. The cause of death was thought to be due to an overwhelming systemic immune reaction 4 days after administration of a high dose of the engineered adenovirus [28]. The lack of target specificity, which is characteristic of systemic delivery, is an additional incentive to favor topical delivery. Rather than employing systemic delivery to target the skin, it has been suggested that local delivery may be able to target systemic conditions through the secretion of products from skin cells into the circulation [16]. Techniques for the transfer of genetic material The techniques employed for gene therapy have evolved over more than two decades and provide a varied armamentarium for the modern-day researcher. Although there have been shifting trends in some methodologies, others have stood the test of time. It is likely that several delivery systems will find a niche, either as dictated by biologic needs or because the expense of developing a system to replace an existing functional method would be prohibitive. The techniques in common use today in the laboratory and in clinical practice are described here. The common goals of all gene delivery systems are severalfold: First, they must be able to incorporate reliably a therapeutic gene by a reproducible

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method, and second, they must be able to deliver the transgene in a way that is taken up by cells [29]. The cells themselves must express the transgene product in a desirable amount, for the optimal period of time, and in the appropriate target location. The various systems can be categorized suitably into biologic, chemical, and physical techniques, although some overlap does occur. The efficiency of each system can be measured by incorporating a marker or reporter gene or by directly measuring the expression of the target gene. The b-galactosidase (Lac-Z) gene of Escherichia coli enables the identification of transfected cells by a simple enzymatic reaction, which, on staining with its substrate, X-Gal, turns cells successfully transfected with the Lac-Z transgene blue [30]. Biologic (viral) techniques Viruses are obligate intracellular parasites, designed through the course of evolution to infect cells, often with great specificity and efficiency (Fig. 1 and Table 1). Although many viruses have been developed for use in gene therapy, interest has centered on four types: retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses (AAVs), and herpes simplex virus (HSV) type 1. The principles behind creating a recombinant viral vector involve deleting essential parts of the viral genome to render the virus replication deficient and subsequently inserting the gene of interest. The genes deleted vary according to the viral vector being used, and the transgene inserted is limited by the packaging capacity of that same vector. Although the endogenous viral promoter may be able to drive the expression of the inserted gene, this process usually is optimized by cloning the target gene under the control of a more powerful promoter sequence,

Fig. 1. Gene therapy by transduction of a viral vector. Uptake of the vector occurs either by endocytosis of a nonenveloped virus (A) or by fusion of the viral envelope with the cell membrane for enveloped viral particles (B). When internalized, the virus transports the transgene to the cell nucleus (C), where it is transcribed into mRNA (D). The mRNAs subsequently are transported into the cytoplasm (E), where they are translated (F) into the final transgene product (G).

Enveloped

Linear ss RNA

Insertion into host genome

NonSs DNA 5 kb Dividing and enveloped nondividing Enveloped Linear ds 152 kb Dividing and DNA nondividing

36 kb Dividing and nondividing

7

High (1010 PFU/mL)

Yes (specific for Low (106–107 the wild type) CFU/mL) No, remains High (109–1011 episomal PFU/mL)

No, remains episomal

6

High (10 –10 CFU/mL)

Yield

High

High

High

High

Potential insertional mutagenesis

Disadvantages

Possible formation of replication competent virus Short Only transient expression possible Immunogenicity (limiting repeat administration) Long Potential insertional mutagenesis Varies—long Toxicity to target in neuronal cells (except cells but short neuronal cells) in others

Long

Transfection Duration of efficiency expression

Abbreviations: AAV, adeno-associated virus; FU, colony-forming units; HSV, herpes simplex virus; PFU, plaque-forming unit.

HSV

AAV

Infecting capacity

8 kb Dividing cells Yes, random only but large range

Size

Genome

Form

Adenovirus NonLinear ds (firstenveloped DNA generation vectors)

Retrovirus

Structure

Table 1 Comparison of commonly employed viral vectors for gene therapy

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such as the cytomegalovirus (CMV) promoter. Because the recombinant virus, in its construction, is rendered replication deficient, additional genes necessary for its replication must be provided in trans in a packaging cell line, to enable the subsequent multiplication, following which the yield of recombinant virus should be purified. Retrovirus Retrovirus is an enveloped virus containing a single-stranded RNA molecule of approximately 8 kb. The genome contains at least three genes, the gag gene (encoding core proteins), the pol gene (encoding reverse transcriptase), and the env gene (encoding viral envelope protein) [31]. After infection, the viral genome is reverse transcribed into double-stranded DNA, which integrates into the host genome. Lentiviruses are a subclass of retroviruses, which are considerably more complex and able to infect proliferating and nonproliferating cells. Retroviruses first were introduced as vectors in the 1980s [32] and were appealing in their ability to integrate into the host cell’s genome [33]. This method of transduction offers the possibility of long-term transgene expression, and it is inactivated by substances present in human serum [34], reducing its potential systemic toxicity. These characteristics make retroviruses the most commonly employed vectors in current gene therapy clinical trials, accounting for 36% of all vectors used [2]. Disadvantages of the retroviral vector include the difficulty of generating high titers [31], the loss of infectivity after attempts at its purification and concentration based on the labile nature of the virus [35], and the requirement that the target cells should be dividing for retroviral integration and expression of viral genes. Two additional complications of retroviral vectors limit their use. One is the theoretical risk of inducing neoplastic transformation in the target cell—an event termed insertional mutagenesis—which could occur should the viral genome integrate in proximity to a cellular proto-oncogene, driving its production, or by disrupting a tumor-suppressor gene. The other complication is the risk of generating a replication-competent virus, as evidenced by several outbreaks of wild-type virus from recombinant virus– producing cell lines that have been reported [35]. Retroviral vector–mediated gene transfer to the skin first was reported by Morgan and coworkers [36], who modified keratinocytes to secrete biologically active hGH. The effect that hGH had with respect to wound healing was investigated further by transplanting the transduced cultured keratinocytes into porcine full-thickness wounds. No acceleration of healing was observed, although increased levels of hGH were shown [37]. Retroviral vectors also have a potential role in vascular surgery and have been used in animal studies to overexpress thrombolytic genes, such as tissue plasminogen activator and urokinase, in endothelial cells [38]. Advances aim at improving the specificity of the vector by altering glycoproteins in the viral envelope and manipulating the target cell range [39].

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Adenovirus Adenovirus is a nonenveloped virus containing a linear double-stranded DNA of 35 kb and existing as greater than 40 serotypes. Adenoviral vectors classically comprise of serotypes 2 and 5 [33] and are the vector of choice in 28% of current gene therapy clinical trials [2]. Adenoviruses are internalized by receptor-mediated endocytosis, and their genomes are transported to the nucleus where they are replicated episomally, that is, without integration into the host genome. As a result, there is no risk of insertional mutagenesis. The viral genome is organized into several early (E1 through E4) and late (L1 through L5) transcriptional regions depending on whether they are expressed before or after viral replication [31]. The early units seem to have regulatory functions, whereas the late units code for structural proteins. First-generation recombinant adenoviral vectors are characterized by the deletion of either the E1 or E3 gene. Advantages of adenoviral vectors include their ability to transduce nondividing cells of all types at high efficiency, without integration into the host cell’s genome; their production in high titers [33]; and their relatively large packaging capacity. Disadvantages include their relatively short duration of transgene expression and the inflammatory response elicited. Evidence suggests that the inflammatory reaction generated could be detrimental in the wound-healing scenario [40], although conflicting reports hypothesize that by augmenting inflammation wound healing could be enhanced [10]. Sylvester and colleagues [41] examined the effect of Ad Lac-Z on the healing of excisional wounds of human skin transplanted onto SCID mice. An increase in inflammatory response was seen (consisting of polymorphonuclear neutrophils and mononuclear cells) that was present in adenoviral and control wounds but was more marked with the adenovirus, although no detrimental effect on wound healing was seen [41]. Pameijer and coworkers [42] investigated the effect of repeated administration of an adenoviral vector. Their results showed an elevated titer of antibody after the second injection, which did not seem to affect transgene expression, and although beneficial effects were not as marked, they still were statistically significant [10]. Within the scope of wound healing research, adenoviral transfer of PDGF-BB by intradermal injection into ischemic rabbit excisional wounds has been shown to enhance wound reepithelialization [40]. The production of gutless vectors, which contain no viral coding sequences, has rendered these vectors less immunogenic [43]. This situation may reduce potential toxicity, significantly prolong transgene expression, and preserve the biologic potency of the vector even after multiple readministrations [10]. Although the death in a gene therapy clinical trial was attributed to an adenoviral vector, the dose given had been large (1013 particles) and administered into the liver, possibly accessing the systemic circulation. Local site-directed gene therapy used in wound healing studies with modest doses has been well tolerated in preclinical animal toxicity

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studies and a single small phase I trial in humans [44]. These reassuring findings have enabled the vectors to be approved for use in two ongoing clinical trials investigating the effect of Ad-PDGF-B in the treatment of chronic venous stasis ulcers and chronic lower extremity neuropathic diabetic ulcers [45]. Adeno-associated virus AAV is a nonpathogenic human parvovirus dependent on a helper virus (usually adenovirus) to proliferate. Its genome consists of a 5-kb singlestranded DNA molecule comprising two genes, rep (encoding proteins that control viral replication and integration into the host genome) and cap (encoding capsid structural proteins). Although five identified serotypes have been described, AAV 2 is employed most commonly [33]. AAV vectors are capable of infecting dividing and nondividing cells, and the wild-type virus integrates reliably at a specific point on chromosome 19q, eliminating unpredictable insertional mutagenesis [46]. The vector currently is employed only in 2% of gene therapy clinical trials, showing the restrictions in its use owing to the cumbersome methodology and a low yield when purified from any contaminating virus. Herpes simplex virus type 1 HSV 1 is a human neurotropic virus and is used primarily as a vector for gene transfer to the nervous system, although the wild-type HSV 1 can infect and lyse other nonneuronal cell types, such as those of the skin [47]. Its genome consists of a linear double-stranded DNA of 152 kb containing more than 80 genes, half of which are not essential for growth in cell culture. Currently two main types of vectors exist—replication-defective viruses and replication conditional mutants [48]. In view of concerns regarding the safety of HSV 1, Yao and Eriksson [49] constructed a novel anti–HSV 1 recombinant virus capable of inhibiting its own replication and the replication of parental wild-type virus. An additional development has been that of the HSV 1 amplicon vector, which comprises a plasmid, consisting of a transgene and the HSV 1 origin of replication, and packaging sequence, packaged in a HSV 1 virion. Because it expresses no viral proteins, it exhibits little toxicity and low antigenicity, while maintaining a packaging capacity of more than 22 kb [48]. It is speculated that such developments would generate increasing interest in the use of HSV 1 vectors in future gene therapy clinical trials, in which currently they represent only 0.5% [2]. Chemical methods Cationic liposomes Numerous chemical methods of gene transfer have been used in the past, such as calcium phosphate [50], but the only one of interest at present seems to be cationic liposomes. These are lipid bilayers that are rendered cationic (positively charged) and associate in a noncovalent fashion with negatively

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charged DNA to form liposome-DNA complexes. The rationale for coating nucleic acid with lipids is to allow highly negatively charged nucleic acid molecules to traverse the plasma membrane of the target cell [51]. Lipofection is the term used to refer to gene transfer by this mechanism and involves the addition of the complexes to the target cells, following which the molecules adsorb to the cell membrane and deliver the bound DNA. The advantages of lipofection include repeated application, lack of toxicity, and ability to carry large amounts of DNA. Although currently used in 13% of all gene therapy clinical trials [2], lipofection is limited by a low rate of stable integration into the target cell and a lack of specificity [31]. Physical methods Direct injection using a hypodermic needle Direct injection is a simple technique currently employed as the delivery method of choice in 9% of all gene therapy clinical trials. It involves the injection of naked plasmid DNA solution directly to the target tissue to enable transgene expression [52]. It is a desirable mode of transfection in that it is versatile and can be used for plasmid DNA solution and liposomeDNA complexes, and it has a lower incidence of effects exhibited by some viral vectors, such as elicitation of an adverse immune response and insertional mutagenesis [53]. The challenge when using this technique is to overcome the low levels of transfection seen [54]. Hengge and colleagues [53] investigated the dissemination of transgene after the injection of a large dose of marker plasmid to establish the safety of the technique. Most organs were found to contain the plasmid DNA transiently for several days, whereas integration into the host genome was not detected. These investigators concluded that skin gene therapy with naked plasmid DNA can be considered safe because of the rapid biodegradation of plasmid DNA and the exclusive and transient expression of foreign genes. Meuli and coworkers [55] showed that direct intradermal injection of multiple plasmid DNAs into wound sites consistently results in site-specific transgene expression, which is targeted to dermal and subdermal layers and a variety of cell types. Microseeding Microseeding is a technique for in vivo gene transfer whereby the plasmid DNA solution of choice is delivered directly to the target cells of the skin by a set of oscillating solid microneedles driven by a modified tattooing device [56]. The advantages of the technique are that because no special preparation is required, recombinant viral vectors also can be delivered efficiently; it does not deposit any foreign material as occurs with particlemediated gene transfer; and it has a much higher transfection efficiency compared with single injection, possibly as a result of DNA delivery by smaller, finer solid microneedles [48].

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Yao and Eriksson [48] showed that expression of a human EGF transgene in porcine skin was sevenfold higher than transfection by a single intradermal injection and twofold to threefold higher than particle-mediated gene transfer. The technique allows for regulation of gene expression by varying the DNA concentration and the amount of solution delivered to the needles. The efficiency of gene transfer can be enhanced further by delivering the plasmid as a liposome-DNA complex [56]. Particle-mediated transfer (gene gun) Particle-mediated gene transfer using a force to accelerate DNA-coated particles initially was developed by Klein and colleagues [57] in 1987 to deliver genes to plant cells. It employs the approach of bombarding cells and tissues with particles or microparticles coated with DNA. The microparticles are composed of gold or tungsten and measure 1 to 5 lm in diameter. When coated with the DNA of interest, the particles are loaded into a device, known as the gene gun, before being accelerated by a force (either an electrical discharge or high-pressure helium) that drives the particles into the target cell, where the DNA dissociates from the particles and is expressed. Advantages of this technique include its broad spectrum for being able to transfect a wide variety of target tissues in vivo and in vitro, including skin, liver, pancreas, kidney, muscle, and cornea, and the high loading capacity of the microparticles, which permits the introduction of multiple genes. The transfection system is simple, standardized, and reproducible; the DNA/gold preparation is extremely stable with a shelf life of at least 6 months; and the preparation of the delivery system requires only a few hours [15]. Pressure adjustment serves as a control for the depth of penetration as shown by Furth and coworkers [58], who were able to transfect cells 2 cm from the skin surface when a high-pressure jet delivery of 100 to 300 lL was used. Limitations of the technique include a relatively low level of transfection, estimated in monolayer culture as being 3% to 15% [59]; transient transgene expression; delivery of a foreign body; and potentially damaging effects of high pressures. In the context of wound healing, EGF [60], PDGF [61], and TGF-b [62] are among the growth factors that have been delivered by particle-mediated gene transfer, and all were shown to accelerate the wound healing response. Electroporation The principle of electroporation rests on the ability of electrical field pulses to create pores in the cell membrane. Cells suspended in a medium containing plasmid DNA and treated with electrical field pulses take up and express DNA from the medium. Gene therapy techniques The mechanisms developed for circumventing the skin’s defensive systems to deliver nucleic acids to target cells have been outlined previously.

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The techniques by which all of these methods are applied to gene therapy can be categorized into two main disciplines. In vivo gene therapy In vivo gene therapy refers to the transfer of genetic material directly into nonmanipulated target cells of the host (Fig. 2). Ex vivo gene therapy Ex vivo gene therapy involves prior harvesting and cultivation of cells, their transfection or transduction in vitro, and subsequent transplantation to the ultimate host. This technique has been performed using autologous, allogeneic, and xenogeneic cells [36], and although the skin consists of several distinct cell types, each of which can be cultured independently, the ultimate aim of their modification in a wound healing scenario is to affect the property of skin as an intact tissue (Fig. 3). The retrovirus has been used successfully to transduce keratinocytes with PDGF-A (in a mouse model) [63,64] and hGH (in a porcine model) and fibroblasts with human PDGF-B (in rats) [65] and PDGF-A (in mice) [64]. All models reported beneficial effects on wound healing.

Transgene medium Specific mechanisms for DNA delivery and methods in gene therapy to which they can be applied have been described. Another variable to increase further the techniques available to the researcher is the choice of medium for delivery of the transgene. Whether in vivo or ex vivo protocols are followed, the transgene can be delivered as a solution, as part of a scaffold matrix, or incorporated into a skin substitute. Various methods have been tested in an attempt to optimize transgene delivery.

Fig. 2. Some commonly employed mechanisms for in vivo gene transfer by direct injection (A), transduction using a viral particle solution (B), transfection by particle-mediated gene transfer (gene gun technique) (C), microseeding (D), and lipofection using liposome-DNA complexes (E).

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Fig. 3. Ex vivo gene transfer. Cells first are harvested (A) and grown in culture (B) before genetic modification by many techniques to insert the desired transgene (C). The transfected cells are incorporated into an appropriate medium (D) before their transplantation back to the host (E).

Suspension Delivery as a solution is the simplest technique and can be achieved by injection into the surrounding tissues or topical application of a viral vector solution. This method of delivery can be facilitated by using an enclosed wound chamber, whose purpose is not only to enclose the solution, but also to function as a wet incubator [66]. Gene-activated matrix Transgene delivery through scaffold matrices, termed matrix-enabled gene transfer or gene-activated matrix (GAM), are gaining popularity based on the hypothesis that they increase the length of exposure of the transgene at the target site and are particularly suited to the treatment of wounds of large surface area, in which other modes of delivery would be less practical [67]. Transgenes for growth factors, such as PDGF-A, have been delivered as gene-activated matrices to wounds using plasmid solution [68] and recombinant adenovirus [69]. Evidence exists to support an accelerated healing response after the gene-activated matrix delivery of plasmid PDGFBB when compared with direct injection of the aqueous plasmid [70], and other studies have shown the benefits of biocompatible/biodegradable matrices to deliver therapeutic proteins, including FGF-2 [71], TGF-b, and EGF [72]. Disadvantages include a low transfection efficiency. Skin substitute The technique of incorporating transgene as a skin substitute involves a similar principle to the gene-activated matrix but aims at delivering a composite graft, which better mimics the physiologic function and anatomic architecture of skin.

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From bench to bedside In vitro cell culture has been performed, modified, and improved for many decades and provides the basis for all clinical experimental research by enabling clinicians to learn about cell behavior and develop molecular biology techniques. These cellular models are limited, however, in their lack of representation of the physiologic environment in which cells grow in an in vivo model. It is possible that the complex interactions between dermal fibroblasts and epidermal keratinocytes may not be mimicked sufficiently in vitro. The acceptance of these limitations identifies the need for animal experimentation, although studies engaging animal models must be considered with the understanding that they come from different species that may have different modes and efficiencies of skin cell behavior [16]. Many animal models have been used for wound healing research, including mice [63], rats [65], rabbits [73], and dogs [74]. Each species has advantages and disadvantages based on ease of handling, expense, suitability for specific disease model, genetic similarity between individuals, and resemblance to human skin. For most purposes, the pig has been identified as the animal model that is most representative of human skin, although its use is limited by expense and the technical challenge of experimentation [75]. Just as simple in vitro systems may not mimic the complexity of the real in vivo condition, so too do animal models fall short of accurately representing human patients. This shortcoming requires the additional process of implementing clinical trials that are monitored meticulously and aim, in the first instance, to establish the safety and efficacy of the experimental therapy. Currently there are only a handful of gene therapy clinical trials investigating the application of growth factors to chronic wounds. Margolis and colleagues [45] currently are involved in phase I clinical trials to evaluate the safety of a recombinant adenovirus expressing PDGF-B under the control of the CMV promoter in diabetic foot ulcers and chronic venous ulcers. Other growth factors under investigation include FGF and vascular endothelial growth factor (VEGF) [2].

Ethical issues The use of gene therapy raises numerous ethical issues, not only those pertaining to the HGP, but also issues concerning the running of clinical trials. Pertinent dilemmas raised by the formation of a genetic database include who should have access to such information. Should the data be classified alongside medical records and subject to the same confidentiality laws, or should employers and insurance companies have a right to access? More importantly, is it right to subject patients to information about their possible future disease pattern? Should this also be the case if no current therapy is available for cure? Is it right that the tens of thousands of human

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gene patents pending are granted to researchers or companies? Is a gene any one person’s invention? Should it belong to a company, or should its ownership be by society? Even if the complex ethical and moral issues raised by germline gene therapy and cloning are excluded, there remains a huge area of conflict, and in an attempt to address these concerns, many regulatory bodies have been established to overlook the processes involved and enforce some basic principles. The Recombinant DNA Advisory Committee [76] was one such initiative drawn up by the NIH, and the Food and Drug Administration (FDA) focuses on safety and efficacy of genetically altered products. Regulations were established whereby research proposals went through a review process consisting of a preliminary approval by the home institution’s Institutional Biosafety Committee and Institutional Review Board before a final approval from the Recombinant DNA Advisory Committee. The American Society of Gene Therpy was established in 1996 and has provided a policy for ethical standards involving gene therapy clinical studies. The guiding principle is that the clinical investigators must be able to design and carry out clinical research studies in an objective and unbiased manner, free from conflicts caused by significant financial involvement with the commercial sponsors of the study [77]. In March 2000, as part of ongoing efforts to ensure patient protection in gene therapy trials, the FDA and NIH announced two new initiatives to strengthen further the safeguards for individuals enrolled in clinical studies for gene therapy: the Gene Therapy Clinical Trial Monitoring Plan and the Gene Transfer Safety Symposia [78]. The ultimate aim of all societies concerned with ethical issues is to ensure that clinical trials are conducted maintaining the principles in the constitution of the United Nations Educational, Scientific, and Cultural Organization of the ‘‘dignity, equality and mutual respect of men’’ [79]. Future prospects Gene therapy for wound healing is evolving rapidly, and possibilities for future developments are extensive. Studies showing the reduced expression of receptors during impaired wound healing [18] have prompted speculation that delivery of a growth factor receptor gene could enhance the healing process, a theory investigated by Nanney and colleagues [80] using human EGF receptor cDNA with promising results. The concept of a genetic switch is another exciting development. Regulation of transgene expression in target cells is a crucial and challenging aspect of gene therapy. Many switches have been developed that have been modified to maximize their safety and ease of use [81,82]. Yao and Eriksson [83] described the use of the tet-R system (a genetic switch controlled by a doserelated response to the presence or absence of tetracycline) in vitro and in vivo enabling the manipulation of timing and level of expression of a transgene. Combinations of growth factors or their sequential use may be the future

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answer to accelerating wound healing processes [26], and with the advent of this therapy, these genetic switches will become increasingly important. As discussed earlier, developments in matrix components and tissue engineering technology offer promise for the future. Chandler speculated on the development of slow-release matrices, which may prolong transgene expression [67]. Gene therapy approaches allow for the introduction of enhancers of healing and inhibitors or downregulators [84]. This concept led to the development of epigenetic approaches to wound healing and, specifically, antisense RNA technology. These techniques do not involve the introduction of a DNA sequence that would be used to translate a protein, but rather a sequence that would modify the cell’s ability to express its own endogenous genes. The introduction of antisense cDNA to a growth factor would block the cell from translating that protein [85,86]. Transcription factors also have epigenetic applications for wound healing. Hypoxia inducible factor 1 is a protein that activates the transcription of hypoxia-inducible genes by binding to a hypoxic response element in the gene promoter [87]. Genes that are regulated by hypoxia inducible factor 1 include VEGF; provision of a transcription factor gene could activate multiple members of the VEGF family of growth factors, inducing revascularization and enhancing wound healing [29]. It is important to consider gene therapy applications in other contexts of wound healing, such as for downregulating the exaggerated reaction seen in hypertrophic and keloid scars. Liu and colleagues [88] found that exogenous TGF-b upregulated TGF-b mRNA and a1procollagen in keloid-derived and normal fibroblasts and that blocking TFG-b signaling in keloid cells could downregulate collagen gene expression, serving as a potential therapeutic strategy for keloids. Since the early concepts behind gene therapy, advancing technology, knowledge, and informatics have driven the application forward at an alarming pace. With the completion of the HGP in the near future, the sequence library will be able provide clinicians with DNA templates for a multitude of transgenes. The problem then will be establishing which of the many molecules identified as having potential therapeutic benefit should be the ones of choice. This problem highlights the need for a database of gene expression in wound healing to facilitate further study, as has been emphasized previously [89]. With growth factor genes, protease inhibitor genes, extracellular matrix genes, and genes coding for intracellular regulatory proteins as potential strategies, the therapeutic options are numerous. References [1] Thompson L. The first kids with new genes. Time 1993;141:50. [2] Gene Therapy Clinical Trials. Available at: http://www.wiley.co.uk/wileychi/genmed/ clinical/. Accessed September 2001.

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