RETRACTED: Gene therapy and wound healing

RETRACTED: Gene therapy and wound healing

Clinics in Dermatology (2007) 25, 79 – 92 Gene therapy and wound healing TE D Sabine A. Eming, MDa, Thomas Krieg, MDa, Jeffrey M. Davidson, PhDb,c,...

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Clinics in Dermatology (2007) 25, 79 – 92

Gene therapy and wound healing

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Sabine A. Eming, MDa, Thomas Krieg, MDa, Jeffrey M. Davidson, PhDb,c,* a

Department of Dermatology, University of Cologne, D-50937 Cologne, Germany Department of Pathology, Vanderbilt University School of Medicine, Nashville, TN 37232-2561, USA c Medical Research Service, VA Tennessee Valley Healthcare System, Nashville, TN 37212-2637, USA b

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Abstract Wound repair involves the sequential interaction of various cell types, extracellular matrix molecules, and soluble mediators. During the past 10 years, much new information on signals controlling wound cell behavior has emerged. This knowledge has led to a number of novel therapeutic strategies. In particular, the local delivery of pluripotent growth factor molecules to the injured tissue has been intensively investigated over the past decade. Limited success of clinical trails indicates that a crucial aspect of the growth factor wound healing strategy is the effective delivery of these polypeptides to the wound site. A molecular approach in which genetically modified cells synthesize and deliver the desired growth factor in regulated fashion has been used to overcome the limitations associated with the (topical) application of recombinant growth factor proteins. We have summarized the molecular and cellular basis of repair mechanisms and their failure, and we give an overview of techniques and studies applied to gene transfer in tissue repair. D 2007 Elsevier Inc. All rights reserved.

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Rationale for the application of gene therapy in tissue repair

Tissue injury produces a dynamic, interactive response that involves the complex, overlapping interaction of various cell types, extracellular matrix molecules, and soluble mediators.1,2 Skin has a large potential for efficient and functional repair; however, in utero cutaneous repair is a regenerative process, which declines in late gestation as inflammation and consequent scar formation alter the character of repair. Cutaneous scar tissue lacks a normal extracellular matrix arrangement, and the epidermis covering the scar fails to develop appendages such as hair and

* Corresponding author. Department of Pathology C-3321MCN, Vanderbilt University School of Medicine Nashville, TN 37232-2561, USA. Tel.: +1 615 327 4751x5488; fax: +1 615 327 5393. E-mail address: [email protected] (J.M. Davidson). 0738-081X/$ – see front matter D 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.clindermatol.2006.09.011

glands. Therefore, postnatal wound healing substitutes repair for regeneration. Other soft tissues, including cartilage, ligament, tendon, muscle, and nerve tissue, also possess inherently limited capacity for repair, and injury will frequently lead to loss of organ function. Recent advances in cell biology have identified a variety of molecules, specifically growth factors and their receptors, which are critically involved in directing cell functions during soft tissue organogenesis. Interestingly, expression and functional relevance of most of these growth factor-receptor systems have also been demonstrated in the postnatal repair response of various tissues.3 In addition, experimental studies indicate that the topical application of growth factors is a promising therapeutic tool for modifying the repair microenvironment. Those tissues with inherently poor or pathologically altered healing potential would thus profit from therapeutic growth factor interventions that augment the healing capacity while minimizing inflammation and its consequent scar formation. The limited success of clinical

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extracellular matrix molecules, cell-matrix communication is mediated by different classes of cell membrane receptors including the family of integrins.10,11 Together, extracellular matrix molecules and cytokines provide chemotactic cues to recruit and activate tissue specific resident and nonresident inflammatory cells that enter the wound site. Infiltrating neutrophils, monocyte/macrophages, and lymphocytes are predominantly involved in defense functions and the initiation of granulation tissue formation through the synthesis of potent proteases (elastase, cathepsin G, proteinase 3, uPA, MMP-8, and MMP-9), reactive oxygen species, and cytokines. During granulation tissue formation the cellularity increases and invading mesenchymal cells release soluble mediators, which stimulate keratinocyte migration and proliferation to achieve reepithelialization.12,13 Conversely, activated keratinocytes release soluble mediators, which are crucial for the angiogenesis and fibroplasias that characterize granulation tissue formation. When the integrity of the tissue defect is restored, inflammation resolves, and granulation tissue regresses, scar tissue forms, and the wound enters the remodeling phase. During this last phase, after several months, a balance is reached between synthesis of new components of the scar matrix and their degradation by proteases.

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trials with peptide growth factors underscore an important aspect of the growth factor tissue repair paradigm: the effective delivery of these polypeptides to the tissue site.4,5 The wound site is biologically and biochemically complex and, in situations such as the chronic or infected wound, a hostile environment for repair. Current drug delivery strategies suffer from the inherent loss of drug activity due to the combined effects of physical inhibition and biological degradation. This may account for the limited success of clinical studies. Hence, a critical issue to address has been the development of a strategy aimed at optimizing the delivery of growth factors to maximize their therapeutic efficacy. A molecular genetic approach in which genetically modified cells synthesize and deliver the desired growth factor in a time-regulated and locally restricted manner to the wound site would be a powerful means to overcome the limitations associated with the (topical) application of recombinant growth factor proteins. In contrast to many differentiated cell phenotypes, stem cells are potentially permanent residents of the wound site, and transducing them will therefore probably have the most lasting therapeutic effect. In addition, the function of genetically modified cells might be strengthened by implanting them in biomaterial scaffolds that promote cell adhesion, proliferation, migration, and differentiation and that provide the basis for recreating a regenerative rather than a reparative wound environment.6,7 Therefore, as stem cell biology progresses, stem cells might emerge as an exciting target for gene transfer in tissue repair.8

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Mechanisms of physiologic and pathologic wound healing Cell-cell and cell-matrix interactions

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Wound repair is a dynamic, interactive response to tissue injury that involves a complex interaction and cross talk of various cell types, extracellular matrix molecules, soluble mediators, and cytokines.1 Knowledge of the signals temporally triggering and controlling wound healing is fundamental for our understanding of tissue repair. The healing response has been divided into 4 phases: hemostasis, inflammation, granulation tissue formation, and remodeling.1,2,9 Although individual soft tissues have their intrinsic complexities in repair, the sequence of these biological events is similar for all tissues. Immediately after injury, limited proteolysis activates the clotting cascade to initiate hemostasis, consisting of the polymerization and deposition of fibrin (clot formation) as well as platelet degranulation. Additional serum- and cellderived extracellular matrix molecules including fibronectin, vitronectin, and thrombospondin accumulate at the wound site. This complex network provides a provisional matrix through which cells can migrate during the repair process. It also serves as a reservoir of cytokines and growth factors that are released from activated platelets. As cells adhere to

Growth factors in tissue repair

In addition to the importance of cell-cell and cell-matrix interactions, a complex network of cytokines, which include proinflammatory cytokines, growth factors, and chemokines, controls all stages of the repair process. Experimental studies have helped to elucidate the function of individual cytokines and their receptors. Specifically, the development of transgenic and knockout mouse technologies has provided new insight into the function of many different cytokine and growth factor genes.3,14 After injury, the release of several pro-inflammatory cytokines (interleukin 1a, interleukin b, tumor necrosis factor a, interferon) regulates the inflammatory phase and activates additional mediator systems, such as growth factors and chemokines.15 The role of chemokines in tissue repair is currently being explored, whereas the function of growth factors has been intensively studied.3,16 Growth factors predominantly control wound- and tissue-specific cellular activities through signal transduction cascades that direct cell division, migration, differentiation, and the synthesis of extracellular matrix molecules.3 During granulation tissue formation, macrophages provide an important source of growth factors necessary to stimulate fibroplasia and angiogenesis (transforming growth factor a, transforming growth factor b, basic fibroblast growth factor, plateletderived growth factor [PDGF], members of the vascular endothelial growth factor [VEGF] family) and therefore have been considered essential for repair.17 This dogma is now being challenged.18 In particular, several fibroblast-derived growth factors have been shown to be crucial in directing keratinocyte function during reepithelialization, including keratinocyte growth factor (KGF), hepatocyte growth factor

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Mechanisms of impaired healing

Factors deleterious in tissue repair

Prolonged inflammation Disturbed balance proteases/protease inhibitors Reactive oxygen species Inactivation of growth factors/matrix molecules Bacterial toxins and proteases Inhibitory molecules (eg, soluble growth factor receptors)

Studies point to the synthesis of factors within the chronic wound environment that could have deleterious effects on the repair process. For example, high-level expression of soluble growth factor receptors can inhibit the activity of mediators essential for wound repair.32 Recent data furthermore show that bacterial factors might exert detrimental effects on cell functions crucial in wound repair, including toxins and proteases.33-36 In summary, these data highlight the need to identify deleterious factors that contribute to the hostile chronic wound environment to further develop current treatment modalities. Potentially, identified factors could provide novel therapeutic targets for gene therapy.

Techniques for transfer of genetic material

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The well-defined chronology of cellular events is critical for repair. The combination of certain systemic (vessel disease, diabetes mellitus, age, therapeutic interventions, genetic predisposition) and local factors (Table 1) leads to a disturbance of the delicate balance of cell, matrix, and growth factor interactions. Clinically, these situations often appear as chronic, nonhealing ulcers or excessive scarring observed in hypertrophic scars or keloids. A detailed pathobiologic understanding of the hostile wound environment will be crucial for the development of any efficient local therapeutic approach. Local mechanisms underlying nonhealing cutaneous wounds are however only partly characterized at the cellular and molecular level. Efforts to define the highly aggressive wound environment of chronic wounds promise further improvement of current treatment modalities. Characteristically, chronic leg ulcers, the most common form of nonhealing chronic wounds in Western countries, fail to progress through the normal pattern of wound repair, but instead remain in a state of chronic inflammation characterized by abundant neutrophil and macrophage infiltration. Exuberant inflammation interferes with all subsequent wound healing processes and manifests itself as inadequate mesenchymal cell chemotaxis and proliferation, angiogenesis, epithelialization, wound contraction, collagen synthesis, and remodeling. There is increasing evidence that the persisting infiltration of neutrophils and macrophages plays a major role in the generation of a protease-rich and prooxidant hostile microenvironment.21-23 Recent experimental and several comprehensive clinical studies demonstrated that the proteolytic activity of matrix metalloproteinases and serine proteinases is significantly up-regulated in the exudate of chronic wounds,24-27 thus contributing to the degradation of extracellular matrix molecules,24,25 growth factors, and their receptors.28-30 In addition, proteolytic degradation products generated by high protease activity in the chronic wound environment may exert inhibitory effects on cell function.31

Table 1

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(HGF), and insulin-like growth factor (IGF).19 Conversely, keratinocytes are a rich source for growth factors mediating autocrine and paracrine cell functions, for example, PDGF, transforming growth factor b, and VEGF. Because of these pivotal properties of growth factors, the hypothesis that they might accelerate the process of wound repair has been tested and confirmed in a wide variety of experimental animal models.3 Despite intense efforts in more than a decade, the clinical promise of growth factors in tissue injury application has yet to be achieved.4,5 The only clinical success has been PDGF-BB.20 A major concern of growth factor therapy in tissue repair is the effective delivery of these polypeptides into the wound site that promotes healing of the injured tissue. Introduction of growth factor genes into cells participating in the healing response might be an ideal mechanism for drug delivery by local, autologous expression.

Gene delivery systems can be classified as either viral or nonviral. Both approaches have been extensively investigated in various models of repair and have been shown to deliver transgenes effectively into the wound site. Several recent articles provide greater detail about the diverse gene transfer technologies37-41. Therefore, we will concentrate on a survey of the distinct advantages and disadvantages of these technologies applied to gene transfer in soft tissue repair (Table 2).

Viral techniques Viruses are natural vehicles for gene delivery. The overall technological strategy is to generate a replication-defective particle by replacing some or all viral genes with the gene of interest.39,42,43 The viral proteins necessary for efficient cell entry and gene delivery that were previously encoded by the removed genes are supplied by packaging cell lines. Viruses, in general, efficiently transduce cells and in some cases permanently integrate into the host cell’s genome. The choice of viral type depends to some extent on whether permanent or transient expression of the gene product is desirable. Retroviruses, adenoviruses, and adeno-associated viruses (AAVs) are the most commonly used viral gene delivery systems in general, as well as in soft tissue repair applications.39

Recombinant retrovirus Recombinant retroviruses have been one of the most successful methods for gene transfer and clinical applications. The molecular biology of these viruses is well understood, and the manipulation of these viral genomes

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Viral genome Virus titer (pfu/mL) Packing size (bases) Integration Nondividing cells Duration of expression Transduction efficiency Safety concerns

Retrovirus/lentivirus Adenovirus

AAV

HSV-1

RNA 106-107

dsDNA 1010-1012

ssDNA 105-107

dsDNA 109-1011

8300

36,000

4700

152,000

Unlimited

Yes—random No/yes

No—episomal Yes

Yes—chromosome 19 Yes

No—episomal Yes

No—episomal

Long

Transient

Long

Short

Short

High (in vitro)

High (in vitro/in vivo)

Insertional mutagenesis

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Table 2

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Inflammation/cytotoxicity Insertional mutagenesis Inflammation/cytotoxicity Low toxicity

dsDNA indicates double-stranded DNA; ssDNA, single-stranded DNA.

Low

Low

for in vivo instability of gene expression is unclear: methylation or deletion of the proviral DNA is a possibility.50 Advances in the construction of packaging cell lines and the development of sensitive screening methods for replication competent virus made the risk of generating replication-competent virus essentially impossible.50 Nonetheless, recent reports highlight the risk of vector-induced target cell transformation in human gene therapy trials.51,52 In tissue repair applications this risk could be reduced by choosing allogeneic cells as target for transgene expression. For example, epithelial sheets of cultured allogeneic cells have been successfully used for the treatment of several skin defects.53 Unlike autografts, grafts of allogeneic cultured cells are not permanently integrated and are thought to persist less than 2 weeks.54 Allogeneic epidermal sheet grafts that have been genetically modified by retrovirus, thus, could serve as efficient, transient, and safe delivery systems for factors promoting cutaneous tissue repair.

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is straightforward and controllable. The major advantages of retroviral-mediated gene transfer are that recombinant retroviruses are capable of transferring genes to a wide range of different cell types including normal diploid cells, the genes are stably integrated without rearrangements into the chromosomal DNA at relatively high frequency, and retroviruses are capable of simultaneously transferring genes to large numbers of cells at high efficiencies. One disadvantage of retroviral-mediated gene transfer is that only a gene of limited size (b6 kilobase [kb]) can be packaged and transferred. Although the producer cell lines produce virus in relatively low titers (105-107/mL), recent progress has been reported on methods to concentrate viral particles.44 Integration of the transferred gene, furthermore, is dependent on cell division, which significantly limits the types of cells and tissues that can be modified. Naldini et al45 were the first to describe the construction of a retroviral vector based on a lentivirus, which was able to integrate into the genome of nonproliferating cells. This finding has therefore overcome one of the major obstacles in retroviral-mediated gene transfer because one of the major targets in genetic skin diseases are epidermal stem cells, which are believed to divide and cycle slowly. Kuhn et al have however recently presented surprising data, which indicate that lentiviral vectors are not necessarily superior to retroviral vectors at introducing genes into keratinocyte progenitor cells during in vitro culture.46 Khavari’s group recently reported on the development of the lentivectorbased technology by introducing elements to control transgene expression and rendering it more suitable for cutaneous gene transfer.47 An additional area in need of development is stability of the levels of gene expression in vivo of retrovirally transduced cells. Although long-term gene expression has been reported in some systems, including fibroblasts and muscle cells, transient expression in retrovirally modified cells has also been described.48,49 The basis

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plasmid DNA (liposomes)

Recombinant adenovirus To date, adenoviral vectors have demonstrated the highest gene transfer efficiency in vivo.55 Unlike retroviruses, adenoviruses can be easily concentrated to high viral titers and can infect dividing as well as nondividing cells, with high efficiency. This is a major advantage for some applications, including gene transfer to the lung where cell division is infrequent. The major reservations for the use of adenoviral vectors has been the cytotoxicity of viral proteins and the host cellular immune response to the adenoviral proteins of the virus particle that results in local inflammation and destruction of transduced cells. The host immune response also limits repeated administrations of the vector, which might be needed because recombinant adenoviruses rarely integrate into the host genome. Currently, strategies are being developed to limit the immune response by the use of bgutted vectorsQ56,57; however, inflammation induced by the adenoviral capsid protein itself

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Recombinant AAV

Herpes simplex virus type 1

Gene transfer of plasmid DNA

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Adeno-associated viruses (AAVs) are small nonenveloped viruses, which are not known to cause any human disease.40 They require adenovirus or herpesvirus helper functions for replication. The AAV can transduce a wide range of dividing and nondividing cells, but its main advantage is that the wild-type virus integrates reliably into a specific position on chromosome 19, which reduces the probability of inadvertent activation of a protooncogene.40,60 In AAV vectors that have had the viral recombination function deleted, integration in the host genome occurs randomly as sites of double-stranded DNA breaks. Adeno-associated virus serotypes can have tissueselective trophism. The vector is currently used only in few clinical gene therapy trials, demonstrating its restriction in its use.60 Disadvantages of AAVs are that the vector packages only small transgenes (up to 4.7 kb) and the preparation of the recombinant vector is frequently contaminated with wild-type adenovirus that must be separated or inactivated. The integration mechanism, furthermore, is not as precise as retroviral vectors and generally results in tandem repeats of the transgene being inserted into the host chromosome.41 Immune responses can occur with AAV administration, and many individuals have prior immunity.

lower, recent studies indicate that effective gene transfer approaches in tissue repair exert functional alterations at lowlevel expression of the transgene. Nonviral gene transfer systems have the advantage to deliver genes to target cells without the potential for recombination with wild-type viruses and possible cellular damage due to persistence or repeated exposure to the viral vectors.65 These synthetic systems are also easier to manufacture on a large scale because they typically use plasmid constructs that can be grown with existing fermentation technology. Direct plasmid application, lipofection, and receptor-mediated delivery vectors are the most promising nonviral systems. There are many other nonviral transfection techniques that are too inefficient for clinical use (ie, electroporation, laborious microinjection of DNA). Recently, Ortiz-Urda et al66 presented a novel and elegant approach to stably transfect epidermal progenitor cells by a nonviral approach. The authors successfully applied a Streptomyces phage AC31 integrasebased system to insert the large human COL7A1 gene into keratinocytes from patients who have from recessive dystrophic epidermolysis bullosa. This approach may prove very useful for the nonviral delivery of large genes.

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cannot be completely prevented.58,59 Loss of the vector DNA from the gene-modified cells results in transient gene expression and limits long-term gene expression, which make this the vector of choice for short-term gene expression in targets such as the wound site.

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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 HSV1 can infect and lyse other nonneuronal epithelial cells. Because of its large genome size, up to 30 to 50 kb transgene can be packaged into recombinant HSV-1 vectors. At present, 2 major classes of HSV-1 vectors have been developed, namely, replication-defective viruses and replication conditional mutants.61,62 The ability of HSV and these mutant recombinants to establish life-long latent infections however raises concerns about the use of these vectors in humans. To ensure its safety, an anti–HSV-1 –specific HSV-1 recombinant virus capable of inhibiting its own replication and the replication of parenteral wild-type virus has been constructed.63 In addition, development has been made to create HSV-1 amplicon vectors essentially free of HSV- 1 helper virus, which might be a promising genetic vehicle for in vivo gene delivery.64

Nonviral techniques There have been rapid advances in the development of more physiologic means (nonviral) to introduce genes into target cells. Although nonviral gene transfer efficiencies are

In recent years, significant progress has been made in augmenting the efficiency of plasmid DNA transfer into wounds. These technologies include the direct injection of plasmid DNA into the target tissue, particle-mediated gene transfer, microseeding, and electroporation. One of the most practical methods to deliver the DNA to the target tissue is the direct injection of DNA into the tissue. This approach was first investigated by Hengge and coworkers67 who demonstrated that DNA directly injected into the skin can be taken up by keratinocytes and its transient expression can induce a biological response. The efficacy of this approach has now been supported by reports of other groups.68-70 A more sophisticated technique for the delivery of naked DNA has been described by Ciernik et al71 and Eriksson et al72 and is named puncture-mediated gene transfer or microseeding, respectively. These techniques deliver the DNA directly to target cells by multiple perforations using oscillating solid microneedles. The authors state that transgene expression by microseeding is more efficient than delivering DNA by a single dermal injection. Another physical method for introducing DNA into cells is the so-called particle bombardment. This technology is based on the acceleration of DNA-coated microprojectiles using high-pressure helium as a propellant.73-79 By varying the propellant pressure the DNA-loaded particles can be targeted to individual epidermal cell layers; optimal gene expression is achieved when most of the gold particles are present in the basal epidermal cell layer.

Liposomes Lipofection has been used with a variety of cell types in vitro as well as tissues in vivo.80,81 The main advantages of

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Fig. 1 Gene transfer technologies applied in tissue repair. In the case of in vitro gene transfer, target cells are isolated from a small biopsy and cultured. Once a sufficient cell number is obtained, cells are incubated with the viral or nonviral vector containing the therapeutic gene of interest; cells can be selected to ensure a high number of transfected cells; transgenic cells can be incorporated in biodegradable matrix (biological, synthetic); and genetically engineered grafts can then be transplanted to the donor. In contrast, for the in vivo gene transfer approach, plasmid DNA or virus, with or without a delivery vehicle, is injected directly into the desired organ.

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liposomes are that there are few constraints to the size of the gene that can be delivered. Liposomes are relatively nontoxic and can be applied repeatedly. A drawback of the method is the low frequency of stable transfection. The ability to target these liposome vectors to specific cells is also limited. Recent efforts to target liposomes to specific cell types have focused on the incorporation of a variety of ligands into the liposome-DNA complex.82

Nanoparticles

More complex delivery vehicles with nanometer dimensions can be formulated from both organic and inorganic constituents. These formulations can incorporate complex functions, including controlled release, targeting, and encapsulation. Both viral and nonviral DNA can be incorporated in an active form for use as parenteral, local, or inhalant delivery of expression vectors.83-85

Gene therapy in soft tissue repair

Gene transfer delivery technologies applied to soft tissue repair Different delivery technologies for gene transfer applied in tissue repair have been investigated and have been successfully applied to ex vivo and in vivo gene therapy (Fig. 1).37,38,86 The ex vivo approach permits the introduction

of genetic material directly into a particular cell type by isolating involved cells from the patient, genetically manipulating these cells in culture, and then transplanting them back into the donor. Thus far, differentiated parenchymal cells of diverse tissues have been intensively investigated as target cells for gene transfer. As culture conditions for nondifferentiated progenitor cells advance, however, those cells might emerge as an additional exciting target for gene transfer.8 The ex vivo approach, furthermore, allows to enhance the function of gene-modified cells by combining modified cells with biomaterials that support cell function before transplantation.6,7 In vivo gene therapy obviates the need for proper cell culture and transplantation because the genes are delivered directly into the target tissue. This method simply requires that the DNA vector harboring the encoding sequence be inserted into host cells in vivo. This straightforward approach, besides being simple, is especially relevant to tissues where cells are difficult or impossible to culture and/or transplant, such as in the nervous system. Although there are clear advantages to this in vivo approach, it is sometimes difficult to target genes to specific cells of a particular tissue in vivo. In fact, recent studies highlight the risk that intradermally applied plasmid DNA can be transported by CD11b+ cells beyond regional lymph nodes to distant organs87; furthermore, cellular uptake and transport of the transgene were amplified by inflammation in this model.

Gene therapy and wound healing

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Wound repair: candidate tissues for gene therapy Skin

The skin is a uniquely attractive tissue site for development of new genetic therapeutic approaches both for its accessibility as well as for the large number of diseases that are amenable to cutaneous gene transfer. Among these opportunities are inherited skin disorders, metabolic disorders, tumor disease, and wound repair.37,38,103,104 For in vitro and in vivo gene transfer strategies, skin cells are easily accessible. Conditions under which epidermal and dermal cells are isolated, rapidly grown, and can be repeatedly subcultured in vitro have been described and make gene manipulations of these cells ex vitro possible. Transplantation procedures for both cell types are well established so that gene-modified cells can be successfully transplanted back to the recipient, either as monocultures or as skin equivalents. Our group was one of the first who investigated the application of genetically modified keratinocytes for the local synthesis and sustained delivery of growth factors to manipulate the healing environment. In these studies, we used retroviral-mediated gene transfer to introduce genes encoding different PDGF isoforms or IGF-1 into cultured human keratinocytes. Genetically modified keratinocytes were shown to synthesize and secrete high levels of transgenes in vitro. When modified cells were transplanted as an epithelial sheet to athymic mice, PDGF-A–expressing cells promoted the development of a granulation tissue in the adjacent dermal tissue.105 IGF-1–expressing keratinocytes increased the proliferation of modified cells, demonstrating that genetic modification can be used to modify the autocrine control of keratinocyte proliferation.106 These data demonstrated the feasibility of genetically modifying the cells of a skin substitute to secrete high levels of growth factors and the ability of this genetically modified skin substitute to affect epidermal and dermal cell function in vivo.

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Genetic approaches to tissue repair are based on the efficient delivery of the vector or the modified cells to the wound site. Recent studies indicate that the choice of the vehicle in which the vector or the transgenic cells are incorporated can be critical for the efficiency of gene delivery. The transgene can be delivered as a solution or as part of a scaffold matrix of natural or synthetic origin. Specifically, porous biomaterial scaffolds have been shown to contribute to gene transfer efficiency by providing the surface on which cells and DNA vectors interact.88 Recently, the potency of different biodegradable carriers to serve as in vivo gene delivery medium has been investigated. Type I collagen (lyophilized sponge, paste, and gel), hyaluronan, and alginate can be used as matrix carriers of plasmid DNA, as well as synthetic materials such as polylactide-co-glycolide and carboxymethylcellulose.88 These gene delivery systems are referred to as geneactivated matrix (GAM). Gene-activated matrix has been found not only to optimize in vivo vector delivery, but also to limit gene transfer to those cells that actively invade the artificial scaffold. 89-91 It is anticipated that a GAM formulation could also support the migration of wound repair cells into the structural matrix, where they encounter the viral vector and subsequently produce the therapeutic protein within the local wound environment. In preclinical in vivo models, direct GAM plasmid gene transfer to repair cells in skin, in bone, tendon and ligament, heart and skeletal muscle, and cranial nerve has been reported.88 Clinical trials are underway with a GAM-formulated adenoviral PDGF for diabetic foot ulcers.92

has been cleared of heterologous gene sequences, thus minimizing immunogenicity.99 Additional improvement has been achieved by modifying the inducible systems to function with non-immunosuppressive drug analogues.100 Alternatively, an approach to select gene-modified cells in vivo, specifically keratinocytes, has recently been described. In these studies, Pfutzner et al101 demonstrated a sustained and enhanced transgene expression in vivo by topical colchicine selection of keratinocytes transduced with the multidrug resistance gene (MDR1). This system might be useful to enhance both the duration and expression level of a desired therapeutic gene. Along these lines, although certainly not as precise as a regulated gene expression system, the GAM can also be considered as a gene-targeting device in that only those cells that contact or invade the GAM during the repair process will be transfected.102

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Vehicle for vector transfer

Regulation of transgene expression

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A successful gene therapy strategy relies not only on the efficient delivery of the transgene into the target cell, but also requires maximum control over localization and duration of transgene expression. One strategy to control transgene activity is achieved by inserting tissue-specific or inducible promoters into recombinant vectors. Using this approach, Jaakkola et al93 were able to direct adenovirusmediated transgene expression to cutaneous wound edge keratinocytes. Multiple regulatory systems have been developed furthermore, including the tetracycline-inducible system,94,95 the ecdysone (an insect steroid)–inducible system,96 the RU486/antiprogestin mifepristone–inducible system,97,98 and the rapamycin-inducible system.99 The common feature of these systems lies in their use of a minimal mammalian promoter, which by itself exhibits little basal transcriptional activity, fused to its corresponding cis-acting DNA binding elements, and a hybrid transactivator, whose trans-activating activity is regulated by a pharmacologic molecule. One concern of these heterologous, regulated gene expression systems is that some of the regulatory factors may elicit an undesirable immune reaction. A humanized system, using rapamycin, reportedly

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could be detected in tissue and wound fluid collected from transfected wound sites. The authors state that transgene expression by microseeding is more efficient than delivering DNA by a single dermal injection. Another physical method, which has been investigated for introducing DNA into wounded tissue, is the particle bombardment. We used this technique to evaluate the expression of different PDGF isoforms on wound healing in a rat incisional wound healing model.116 Transgene expression was readily detected up to day 3 after transfection, using reverse transcriptase–polymerase chain reaction analysis, but expression of the transgene fell to detection limits by day 5. To examine the biological effect of transient local PDGF expression we determined the wound breaking strength 7 and 14 days after a single administration of recombinant DNA to the skin. On average, we obtained 75% to 100% increase in mechanical strength of incisions for up to 2 weeks after transfection. These findings indicate that particle-mediated gene transfer is a promising tool for the delivery of growth factor DNA to the skin and can significantly augment the wound healing response; these findings were supported by several groups investigating this technique. Benn et al117 demonstrated that overexpression of transforming growth factor b by particle bombardment resulted in a significant increase in tensile strength of healing rat tissue. Another group demonstrated that the in vivo transfection of porcine partial-thickness wounds with a vector expressing EGF increased the rate of reepithelialization and shortened the time of wound closure.112 Overall, these studies demonstrate that particlemediated transfection using appropriate expression vectors might be a simple and effective means of enhancing the rate of wound repair in an animal model. Recently, several groups reported DNA delivery in vivo from polymer coatings, microspheres, and synthetic matrices.89,91 In the study of Shea and coworkers,91 plasmid DNA was entrapped within porous polymer matrices of polylactide-co-glycolide. After immersion in water, DNAwas slowly released from the matrix for as long as 30 days, and plasmid was shown to be biologically active. When transplanted into the subcutaneous tissue of rats, a large number of transfected cells were observed in the implant periphery. Matrices carrying the gene for PDGF resulted 2 and 4 weeks after transplantation in a significant increase in the thickness of granulation tissue and the number of blood vessels. This study demonstrated that a gene-activated matrix can serve for the sustained gene delivery and is sufficient to affect tissue formation. In addition, in vivo liposome-mediated gene transfer of KGF or IGF-1 has been shown to be effective in augmenting the healing response.118 Adenoviral vectors have been investigated for gene transfer in animal wound healing models. Most evidence suggests that adequate rates of nitric oxide production are essential for normal wound healing. Inducible nitric oxide synthase (iNOS) synthesizes nitric oxide in a sustained manner, and iNOS-knockout mice show a significant delay in repair. Yamasaki and coworkers119 demonstrated the

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In addition to transplanting keratinocytes as epidermal sheets, efforts have concentrated on developing a composite graft of cultured keratinocytes seeded onto a dermal analog so that both epidermal and dermal components are supplied. Although the long-term outcome of these composite grafts is comparable to native skin autografts for the treatment of full-thickness burns, the composite grafts are more fragile, slower to revascularize, and show a decreased percentage of initial engraftment resulting in an increased incidence of regrafting.107 The molecular and cellular events that control the take of a graft are complex and unclear, but certainly involve the actions of both cells of the wound bed as well as of the graft. Genetically modified cells that overexpress potent growth factors could enhance the performance of composite skin grafts. To test this hypothesis we seeded genetically modified keratinocytes, which express the marker gene lacZ or PDGF, on acellular human dermis and transplanted these genemodified composite grafts to athymic mice.108 As indicated by lacZ expression, numerous gene-modified cells were present 1 week after transplantation. Grafts overexpressing PDGF showed an increase in fibrovascular ingrowth in the dermal template and graft contraction was reduced. These data indicate that genetic modification of cells combined with biomaterials can expand the potency of gene-modified cells and biomaterials. Dermal fibroblasts have also been genetically modified to express various genes relevant to angiogenesis and/or fibroblast cell function, including VEGF,109 PDGF-B,110 and PDGF-A.111 In these studies, predominantly viral vectors were chosen to modify cells in vitro and to transplant modified cells to the recipient either by injection or by seeding the cells onto a dermal matrix. Both transplantation approaches resulted in active transgene expression in vivo, and serum levels of the corresponding proteins could be temporarily detected. These studies demonstrate the potential of autologous implants of genetically modified fibroblasts as an approach to alter cell function critical in tissue repair. Alternative to the in vitro gene transfer strategy, several in vivo approaches have been investigated.67,70,72,80,81, 108,112-115 One of the most practical methods to deliver the DNA to the skin is the direct injection of DNA. This approach was first described by Hengge; injection of the gene encoding interleukin 8, a potent chemoattractant for neutrophils, resulted in significant dermal neutrophil recruitment.67 This study demonstrated that DNA directly injected into the skin can be taken up by keratinocytes and its transient expression can induce a biological response. A more sophisticated technique for the delivery of naked DNA has recently described by Ciernik et al and Eriksson et al and is named puncture-mediated gene transfer or microseeding, respectively.71,72 Partial-thickness wounds were gene modified with an epidermal growth factor (EGF) expression construct using this technique in a pig model. Two days after transfection, significant levels of EGF protein

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delivery to the injured site via gene transfer might thus be an effective approach to augment nerve repair. Gene transfer to the peripheral nerve system, however, poses an exceptional challenge because the peripheral nerve system contains a wide range of cell types, many of which are postmitotic. Therefore, specifically viral vector systems, including adenovirus, AAV, and herpes simplex virus, have been investigated and demonstrated as a potential for direct gene transfer to motor neurons and Schwann cells.123,124 Schwann cells close to injured axons might be ideally suited as cellular stage for the local production of recombinant proteins, which influence regenerative and reparative processes.125,126 Recently, in vivo adenoviral gene delivery of glial cell line–derived neurotrophic factor to the injured site has demonstrated to prevent the degeneration of motor neurons with peripheral nerve injury127; transgene expression was localized predominantly to motor neurons. These preclinical data indicate that delivery of therapeutic proteins might be augmented via gene transfer also in nerve injury. Currently, a combined phase I/II clinical trial is investigating the safety and bioactivity of intramuscular gene transfer using hVEGF165 plasmid DNA in diabetic neuropathy associated with/without peripheral artery disease (www.ClinicalTrials.gov identifier: NCT00056290).

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reversal of impaired wound repair in iNOS-deficient mice by the topical adenoviral-mediated iNOS gene transfer. A single, topical application of an adenoviral vector expressing the iNOS gene improved wound healing in the knockout mice. Expression of the mRNA transgene was present up to 10 days after transfection, suggesting that adenoviralmediated gene transfer is applicable to deliver potent mediators with wound healing properties. Liechty et al120 investigated the topical application of an adenoviral vector expressing PDGF-B in an ischemic impaired rabbit wound healing model. In these studies, control animals received an adenoviral vector expressing the marker gene lacZ. In support of the studies from Yamasaki, these studies also demonstrated that adenoviral vectors could efficiently deliver genes to a wound. Liechty however reported that mock-transfected control wounds showed a significant defect in reepithelialization, which might be due to an inflammatory response to the adenovirus or a direct cytotoxic effect of the adenovirus. Recently, Doukas et al90 demonstrated that in vivo adenoviral-mediated gene transfer could be augmented by formulating the adenoviral vector in a collagen matrix before subcutaneous implantation. These studies demonstrated that in addition to plasmid DNA, immobilizing matrices enable the controlled delivery of adenoviral vectors. Currently, a phase I clinical trial has evaluated the safety and potential clinical utility of topical applications of GAM501, a gene for PDGF-B contained within an E1-deleted adenoviral vector and formulated in a bovine type I collagen gel (www.ClinicalTrials.gov identifier: NCT00065663). Similarly, a phase I trial with PDGF-B adenovirus is underway in a population of patients with venous ulcer (www.ClinicalTrials.gov identifier: NCT00000431). Hepatocyte growth factor has wound healing properties.121,122 A phase II clinical trial is currently investigating the overall safety and clinical outcome of a HGF plasmid vector gene therapy approach in peripheral vascular disease; evaluation includes reduction of amputation and mortality, wound healing, rest-pain reduction, and improvement in subject’s ability to function without adverse consequences on quality of life (www.ClinicalTrials.gov identifier: NCT00189540).

Nerve tissue

Current treatments of peripheral nerve injury often result in only partial recovery. Recent advances in the understanding of molecular pathways and their physiologic role demonstrate that several proteins, including neurotrophic factors, intrinsic neuronal growth cone proteins, and celladhesion molecules, play an important role in the development, maintenance, and regeneration of peripheral nerve. In vitro analysis suggested that application of these proteins might be used to promote neuroregeneration. Animal experiments, however, were rather disappointing in this respect, possibly because of poor protein delivery to the injured site. Also in nerve injury, local growth factor

Candidate genes for tissue repair: a perspective

Genes other than those coding for growth factors have been investigated, to manipulate the healing response, and could be interesting candidates in tissue repair. As outlined above, a major pathogenic factor of the chronic nonhealing wound environment is a high level of wound proteases.24 - 27,29,30 Administration of recombinant tissue inhibitor of metalloproteinase 2, a metalloproteinase inhibitor, promoted tissue repair in a rat model.128 Along these lines, our group generated a novel protease-resistant VEGF165 molecule, which is characterized by an increased stability in the protease-rich microenvironment of the chronic wound.28,129 This VEGF mutant is expected to exert superior angiogenic properties at the chronic wound site. Furthermore, gene transfer of pro-inflammatory cytokines involved in regulating the innate immune response has been suggested to augment the antibacterial properties of grafts.130 Alternatively, transcription factors might be considered as gene target in wound repair. These factors have the potential of activating multiple targets and pathways. Hypoxia-inducible factor 1 activates the transcription of hypoxia-inducible genes by binding to a hypoxic response element in the gene promoter.131 Genes that are regulated by hypoxia-inducible factor 1 include VEGF and iNOS; overexpression of hypoxia-inducible factor 1 can activate multiple members of the VEGF family, inducing revascularization and enhancing wound repair.132 In cartilage repair, transcription factors such as Sox-9, L-Sox5, and

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The outlook for the development of better methods for treating and repairing tissue defects using gene-based strategies appears particularly bright. Numerous experimental investigations have demonstrated the feasibility of delivery and expression of gene products to various cell types involved in soft tissue repair. Overexpression of certain gene products by viral and nonviral methods can stimulate a number of cell types toward desired differentiation or biosynthesis pathways. Certain transgenes appear to enhance tissue repair in some animal models, suggesting that the strategy is feasible and efficacious. In contrast to genetic diseases, it is most likely that expression of a specific transgene product is required only for a limited

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Sox-6 that promote chondrogenesis and maintenance of the chondrocyte phenotype present additional mediators that may be useful cartilage injury.133 The transcription factor HoxD3 has significant effects in diabetic mouse wound closure when administered as a plasmid in a methylcellulose disk,134,135 and more recent findings with HoxA3 in the same model show enhanced cell migration and angiogenesis.136 The transcriptional co-factor, cardiac ankyrin repeat protein, furthermore, exerts potent effects on endothelial migration and wound neovascularization in several animal models.137

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Fig. 2 Gene delivery to cells and tissues expands the range of targets. Traditional wound therapy with biomacromolecules such as growth factors relies on the interaction of the signal with a cell surface receptor, which then transmits its signal to the nucleus to activate gene expression. Gene expression profiling has shown that surface receptors, intracellular signal pathways (transducers), and factors that directly interact with the genetic machinery (transcription factors) are also modulated during the healing process. These intracellular signals are prime candidates for gene delivery methods.

Recently, signal transduction molecules, such as Smads, have been shown to be key regulators of inflammation in cutaneous repair and chondrocyte differentiation. Modifying Smad activity could thus augment the healing response in cutaneous as well as cartilage repair.138,139 Because these later regulator molecules function intracellularly and cannot be delivered to cells in soluble form, gene transfer is perhaps the only avenue through which they might be directly applied for medical use (Fig. 2). In addition, growth factor genes have been modified to render them more suitable for gene transfer. For example, the use of FGF-2, a pleiotropic mitogen in different organ systems, in gene transfer approaches has been limited because FGF-2 lacks a classic secretion signal peptide. Sohn et al140 generated a novel, active secreted form of FGF-2, which potentially offers a suitable molecule to be investigated for gene transfer applications.

Fig. 3

Diverse research areas and applications of gene therapy in biotechnology.

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6. Langer R. Biomaterials in drug delivery and tissue engineering: one laboratory’s experience. Acc Chem Res 2000;33:94 - 101. 7. Vacanti JP, Langer R. Tissue engineering: the design and fabrication of living replacement devices for surgical reconstruction and transplantation. Lancet 1999;354(Suppl 1):SI32 - 4. 8. Korbling M, Estrov Z. Adult stem cells for tissue repair—a new therapeutic concept? N Engl J Med 2003;349:570 - 82. 9. Clark RA. Cutaneous tissue repair: basic biologic considerations. I. J Am Acad Dermatol 1985;13:701 - 25. 10. Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell 2002;110:673 - 87. 11. Hynes RO. Integrins: versatility, modulation, and signaling in cell adhesion. Cell 1992;69:11 - 25. 12. Smola H, Stark HJ, Thiekotter G, et al. Dynamics of basement membrane formation by keratinocyte-fibroblast interactions in organotypic skin culture. Exp Cell Res 1998;239:399 - 410. 13. Smola H, Thiekotter G, Fusenig NE. Mutual induction of growth factor gene expression by epidermal-dermal cell interaction. J Cell Biol 1993;122:417 - 29. 14. Scheid A, Meuli M, Gassmann M, et al. Genetically modified mouse models in studies on cutaneous wound healing. Exp Physiol 2000;85:687 - 704. 15. Schroder JM. Cytokine networks in the skin. J Invest Dermatol 1995;105:20S - 4S. 16. Gillitzer R, Goebeler M. Chemokines in cutaneous wound healing. J Leukoc Biol 2001;69:513 - 21. 17. Leibovich SJ, Ross R. The role of the macrophage in wound repair. A study with hydrocortisone and antimacrophage serum. Am J Pathol 1975;78:71 - 100. 18. Martin P, D’Souza D, Martin J, et al. Wound healing in the PU.1 null mouse-tissue repair is not dependent on inflammatory cells. Curr Biol 2003;13:1122 - 8. 19. Werner S, Smola H. Paracrine regulation of keratinocyte proliferation and differentiation. Trends Cell Biol 2001;11:143 - 6. 20. Smiell JM, Wieman TJ, Steed DL, et al. Efficacy and safety of becaplermin (recombinant human platelet-derived growth factor–BB) in patients with nonhealing, lower extremity diabetic ulcers: a combined analysis of four randomized studies. Wound Repair Regen 1999;7:335 - 46. 21. Ashcroft GS, Lei K, Jin W, et al. Secretory leukocyte protease inhibitor mediates non-redundant functions necessary for normal wound healing. Nat Med 2000;6:1147 - 53. 22. Falanga V. Chronic wounds: pathophysiologic and experimental considerations. J Invest Dermatol 1993;100:721 - 5. 23. Wenk J, Foitzik A, Achterberg V, et al. Selective pick-up of increased iron by deferoxamine-coupled cellulose abrogates the iron-driven induction of matrix-degrading metalloproteinase 1 and lipid peroxidation in human dermal fibroblasts in vitro: a new dressing concept. J Invest Dermatol 2001;116:833 - 9. 24. Grinnell F, Ho CH, Wysocki A. Degradation of fibronectin and vitronectin in chronic wound fluid: analysis by cell blotting, immunoblotting, and cell adhesion assays. J Invest Dermatol 1992; 98:410 - 6. 25. Grinnell F, Zhu M. Fibronectin degradation in chronic wounds depends on the relative levels of elastase, alpha1-proteinase inhibitor, and alpha2-macroglobulin. J Invest Dermatol 1996;106:335 - 41. 26. Herouy Y, May AE, Pornschlegel G, et al. Lipodermatosclerosis is characterized by elevated expression and activation of matrix metalloproteinases: implications for venous ulcer formation. J Invest Dermatol 1998;111:822 - 7. 27. Weckroth M, Vaheri A, Lauharanta J, et al. Matrix metalloproteinases, gelatinase and collagenase, in chronic leg ulcers. J Invest Dermatol 1996;106:1119 - 24. 28. Lauer G, Sollberg S, Cole M, et al. Generation of a novel proteolysis resistant vascular endothelial growth factor165 variant by a sitedirected mutation at the plasmin sensitive cleavage site. FEBS Lett 2002;531:309 - 13.

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period, sufficient to adequately stimulate cells toward synthesis of repair. Therefore, current generations of vectors may be sufficient. Key challenges that remain to be addressed before gene transfer in tissue repair can translate into a therapeutic reality is to improve our understanding of molecular and cellular mechanisms of tissue injury that direct healing or nonhealing. An important approach to identify optimal gene targets for intervention will be the careful analysis of the wound environment of healing and nonhealing tissues in humans and the identification of deleterious factors contributing to the microenvironment hostile to repair (Fig. 3). A comprehensive database of gene expression during healing and nonhealing will, furthermore, increase the number of rational therapeutic genes for tissue repair applications. Such a database will be fundamental to appreciate current information provided by the human gene project for tissue repair. Finally, a powerful scientific approach will be provided by the generation of mouse strains with targeted mutations that allows to develop local gene transfer formulations in defined genetic backgrounds. The understanding of the most attractive cellular targets for gene delivery in the injured tissue has also to be deepened however (Fig. 3). In various tissues, differentiated cells have been intensively investigated and successfully used for gene transfer. Stem cells/progenitor cells have however recently emerged as an exciting source to stimulate tissue repair, potentially to direct tissue regeneration. If stem cells could be instructed to differentiate toward a particular lineage and functionally integrate into an injured tissue environment, they could replace cells that have been lost during the disease process. Lastly, given the elective nature of gene therapy applications in soft tissue repair, thorough evaluation of safety issues of the various methods is of particular importance. Many of the cytokines proposed for use in these applications could have detrimental side effects if overexpressed in nontarget organs. For a successful gene transfer approach it is thus fundamental to explore gene transfer methods that will afford maximum control over expression with limited dissemination of vectors, genetically modified cells, or transgene products.

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