EUROPEAN TISSUE REPAIR SOCIETY

GENE THERAPY II

GENE DELIVERY OF PDGF FOR WOUND HEALING THERAPY

GENE Therapy is an approach to modifying host cells to express a therapeutic protein by transfer of genetic material through a variety of techniques. Early studies of candidate disease states amenable to such therapy focused on congenital conditions involving the permanent mutation of a gene. However, gene therapy techniques may also be of benefit in temporary conditions, such as impaired healing, where the shortage or lack of a specific protein contributes to the aetiology of the problem. Platelet-derived growth factor (PDGF) is one of the first cytokines released in response to injury and is therefore an obvious candidate for development as a therapeutic agent. Advances in cloning technology have enabled expression of a recombinant PDGF, which has subsequently been applied as an exogenous treatment in a number of animal models. Despite the inefficiency of exogenous growth factor delivery, these studies demonstrated proof of concept that PDGF was able to influence wound healing favourably and provided a platform from which to launch strategies to deliver PDGF more efficiently by gene therapy.

Background

The existence of a platelet-derived mitogen, which would later become known as PDGF, was first suspected in 1974 following the observations that serum prepared from platelet- free plasma displayed significantly less mitogenic potency than serum prepared from whole blood.^1,2 The responsible factor was later purified in several laboratories and shown to be a glycoprotein of 27,000–31,000Da composed of two peptide chains linked by disulphide bonds.^3-6 These initial studies established the existence of two different chains termed A and B that associated to form three isoforms – homodimers AA and BB and the heterodimer AB. More recently two further isoforms have been described existing as homodimers of two new chains termed C⁷ and D,^8,9 PDGF is normally sequestered within the alpha granules of circulating platelets.^10,11 When the platelet is induced to degranulate, for example, as a result of contact with subendothelial surfaces exposed as a consequence of injury to the vascular endothelial cells PDGF is released into the serum and therefore into the wound microenvironment. ^1,2,12,13 Once released, PDGF has been shown to exert its biological effects through interaction with specific, high affinity receptors^14–18 demonstrated by purification studies to be protein tyrosine kinase receptors.^19–21 Cross competition studies have revealed the existence of two different PDGF receptor classes^2,23 which are activated by the formation of receptor dimers following the binding of one subunit of PDGF to each recep-tor.^20,24,25 The type A PDGF receptor binds both the PDGF-A and PDGF-B chains (and therefore PDGF dimers AA, AB and BB), whereas the B type receptor binds only the PDGF-B chain (and therefore only the BB dimer). The diverse locations at which this receptor has been demonstrated and also the numerous cell types which have been shown to respond to PDGF in culture highlight the many varied functions of the PDGF protein.^{1,15,17,26-30} Within the context of wound healing, PDGF has a number of roles. Platelet aggregation and degranulation are the initial cellular events in response to tissue injury and mediate the localized delivery of PDGF to the wound bed. The presence of PDGF then initiates a chemotactic cascade attracting fibroblasts, macrophages, neutrophils and smooth muscle cells.^31–37 The initial supply of PDGF from platelets is rapidly exhausted following the formation of a fibrin plug at the site of endothelial damage. However, since PDGF is also produced by fibroblasts, ³⁸ macrophages,^39,40 smooth and skeletal muscle cells^41–43 and endothelial cells.^44–47 the initial chemotaxis induced by PDGF derived from platelets initiates a positive feedback loop which stimulates continued expression of PDGF from alternate sources. Other functions of PDGF include the stimulation of the proliferation of fibroblasts,48 smooth muscle cells and endothelial cells.^{1,2,26,29,49,50} In addition, it induces the formation of a provisional extracellular matrix both directly, through the production of collagenase⁵¹ and indirectly by stimulating the expression of Transforming Growth Factor β1(TGFβ1) which has been shown to upregulate the synthesis of fibronectin and procollagen I.^52,53

Recombinant Therapy

The rationale for using PDGF to accelerate healing is based not only on in-vitro observations but also on data from a number of other studies. High concentrations of PDGF have been found in media conditioned by human osteosarcoma, ⁵⁴ melanoma⁵⁵ and glioma cell lines.⁵⁶ In addition, the B chain of PDGF is highly homologous (92%) to v-sis, the oncogene of simian sarcoma virus,57 an acutely transforming retrovirus isolated from a fibrosarcoma of a pet woolly monkey.⁵⁸ These facts support the role of PDGF as a powerful mitogen. Furthermore, it has been suggested that chronic wounds may be growth-factor deficient, consistent with reported observations that the extracellular environment of chronic wounds is highly proteolytic.^59-61 Actively-healing wounds have been shown to express abundant quantities of PDGF whereas other studies suggest it to be lacking in chronic non-healing wounds.^62,63 Beer and colleagues were able to demonstrate reduced levels of PDGFA, PDGFβ receptor and PDGFβ receptor in the skin of genetically diabetic mice.⁶⁴ The apparent association between low levels of PDGF and chronic wounds prompted an interest in the pharmacological application of exogenous proteins, which subsequently demonstrated the vulnerary effects of recombinant PDGF isoforms in both acute and impaired-healing wounds in a number of animal models. Pierce and colleagues⁶⁵ reported that the delivery of 5- 20μg rPDGF-B to rat incisional wounds was able to increase the breaking strength and the inflammatory cell influx. Lepisto et al⁶⁶ subcutaneously implanted hollow cylindrical cellulose sponges in rats, which were injected daily with 0, 5, 50 or 500ng of PDGF-AA or PDGF-BB and demonstrated a dose-dependent acceleration of granulation tissue formation in response to PDGF-BB, which was less obvious than with PDGF-AA. In a model of impaired healing, Mustoe et al⁶⁷ demonstrated that a single application of 2–10μg rPDGF-B increased wound breaking strength and cellularity in incisional wounds created in surface-irradiated rats.These results were supported by findings in an alternate model of impaired healing where daily application of 1–10μg rPDGF-BB to full-thickness wounds in genetically diabetic mice significantly enhanced the number of fibroblasts and capillaries in the wound bed in addition to accelerating the rate of wound closure.⁶⁸ Data generated from animal experiments demonstrating the advantageous effects of recombinant PDGF-B on the healing of acute and chronic wounds was instrumental in progression to a number of clinical trials69 and to the development of Regranex™, a commercially available PDGFBB analogue. Recombinant proteins provide possibly the easiest and most rapid method of delivering a therapeutic protein to a target tissue. However, they suffer from a number of shortcomings. Their synthetic manufacture lacks the complicated processes necessary for post-translational modification, a process that may or may not alter their biological properties. Secondly, they have a very short halflife, due, in part to their rapid degradation by wound proteases. Poor bioavailability from the vehicle employed may also hamper the protein’s function. It is these issues, and others, which contribute to the necessity of delivering high doses at frequent intervals, thereby making recombinant therapy an expensive and labour-intensive option.

Gene Therapy - Present Status of the Technology

Gene therapy differs from recombinant therapy in that it delivers the DNA or mRNA template for the protein rather than the protein itself. In this way, protein expression can be prolonged and many of the problems associated with recombinant therapy overcome. The skin is an ideal candidate for gene therapy techniques for a number of reasons. Its superficial location allows topical delivery of the transgene, thereby obviating the need for systemic delivery and all the risks which accompany it. The therapy can be precisely localised and readily monitored and furthermore, the transient, evolving nature of a healing wound means that only temporary expression of the therapeutic gene is needed, thereby reducing the risks associated with long-term therapy. A number of techniques for delivering PDGF and other growth factors to wounds have been described and applied. Each of these techniques has specific advantages and disadvantages. Direct injection of naked DNA into the superficial dermis has been used successfully to express the β-Galactosidase reporter gene in porcine and human skin and less efficiently in mouse skin.⁷⁰ This simple technique has been modified, in an attempt to retain the DNA vector and its transgene product within the wound, by incorporating the vector into a biodegradable matrix – an approach termed Gene Activated Matrix (GAM). Tyrone et al⁷¹ delivered collagen lattices embedded with 1 mg PDGF-A or PDGF-B naked plasmid DNA into excisional wounds in an ischaemic rabbit ear model and were able to demonstrate an increase in new granulation tissue formation and rate of re-epithelialisation. This technique, first described by Fang and co-workers,⁷² relies on cells migrating into the collagen matrix, taking up plasmid DNA and expressing the protein product. Enhancement of granulation tissue formation and vascularisation has also been observed following subcutaneous implantation of a matrix containing a plasmid encoding PDGF-BB.⁷³ Manipulation of the composition of the chosen GAM offers the added advantage of providing control over its permissiveness for cellular infiltration and vector retention. However, the local therapeutic dose of transgene achieved is low, difficult to quantify accurately and is only transient in nature. Particle-mediated gene therapy uses a device, termed ‘gene gun’, which propels microprojectiles (e.g., gold, tungsten) coated with DNA into specific tissues. Transfection rates of this technique have been estimated in monolayer culture to be 3–15%⁷⁴ and are transient in nature. Other limitations include selective targeting and a cumbersome methodology although it does allow the delivery of multiple genes and/or large DNA molecules due to the high loading capacity of the microprojectiles. Our laboratory was the first to demonstrate the beneficial effect which delivery of growth-factor plasmid DNA by this technique exerted on wound healing.⁷⁵ Eming and colleagues⁷⁶ used a particle-bombardment technique to deliver PDGF-AA or PDGF-BB cDNA to rat skin immediately prior to creating full thickness incisional wounds. They were able to demonstrate a significant increase in wound breaking strength using either isoform, although the effect was maximal with PDGF-AA which led to a 3.5- fold increase in wound breaking strength at day 7 compared with controls. We have recently performed ex-vivo PDGF-A transfections of porcine fibroblasts and demonstrated that, when transplanted back to their autologous donor, these cells are capable of expressing 20-fold higher PDGF levels than controls and that they enhance reepithelialisation over and above that induced by fibroblasts alone (unpublished data). The above gene-therapy strategies demonstrate an ability to deliver successfully the PDGF transgene to skin, and in some cases to confer a beneficial effect on wound healing outcome. However, low rates of transfection and the transient nature of transgene expression pose major limitations to these approaches.

Viruses are natural vehicles for gene delivery and their inherent ability to infect cells and thereby transfer genetic material prompted their exploitation as vectors for gene therapy. The principles underlying the development of a viral vector involve generating a replication-deficient virus by removal of essential genes and replacement of these genes with a foreign DNA sequence encoding the transgene product of choice. Retroviruses, Adenoviruses and Adenoassociated viruses are the most widely tested viral gene delivery systems. Breitbart and colleagues⁷⁷ retrovirally transduced rat dermal fibroblasts to express PDGF-BB and transplanted the cells onto polyglycolic acid (PGA) scaffold matrices prior to implantation into full thickness excisional wounds in rats. The authors were able to demonstrate in-vitro expression of 2ng ml^-1 PDGF-B per million cells per 24 hours and an increase in fibroblast cellularity in PDGF-treated wounds compared with controls. In a similar study Eming et al⁷⁸ transduced human keratinocytes retrovirally to overexpress PDGF-A and achieved a transfection rate of approximately 50%. When seeded onto an acellular dermal matrix and transplanted into full thickness excisional wounds on mice these composite skin substitutes exhibited reduced wound contraction and enhanced re-vascularisation. Two major drawbacks of retroviral vectors, namely their selectivity for infecting only dividing cells and their ability to integrate randomly into chromosomes and so potentially induce cell transformation, have led to the preferred use of adenoviral vectors.⁷⁹ Adenoviruses are able to accommodate the insertion of equivalent amounts of exogenous DNA and are able to infect both dividing and non-dividing cells. Additionally, adenoviruses are able to infect human skin cells at more than 95% efficiency,^80,81 demonstrating stable gene-expression for 2–6 weeks depending on the proliferation rate of cells⁸² and their genome does not integrate into the host chromosomes, but remains episomal. Liechty and co-workers⁸³ achieved adenoviral-mediated overexpression of PDGF-B by injecting 106 or 108 PFU Ad-PDGFB into full-thickness wounds on an ischaemic rabbit ear model. They were able to correct the impairment in wound re-epithelialisation, not only compared with the vehicle-, Ad-LacZ- or 5μg rPDGF-B-treated ischaemic wounds, but also compared with non-ischaemic control wounds. Other effects seen were an increase in granulation tissue formation, extracellular matrix production and angiogenesis compared with controls. Ad-PDGF-B has also been delivered in a collagen pad to porcine full-thickness wounds, where it increased volumes of granulation tissue formation and neo-vascularisation.⁸⁴ Promising results using Adenoviral-mediated PDGF-B gene therapy in animal models has provided the necessary support for the approval of a phase I clinical trial to establish the safety and efficiency of using adenoviral-mediated PDGF-B for the treatment of diabetic insensate foot ulcers.⁸⁵ Despite the many advantages of using adenoviruses, understandable concern remains regarding their safety. A potential for recombination with wild-type viruses, in addition to a propensity to stimulate a host immune response, poses serious practical and ethical issues clouding the use of adenoviruses for gene therapy. Furthermore, although transfection rates achieved can be as high as 95%, limited duration of transgene expression remains an issue, although this may be considered advantageous in a wound healing setting. We have recently established porcine fibroblast stable cell lines, which permanently express either PDGF-A or PDGF-B. Using an ex-vivo gene-therapy model we have transplanted these cells into full-thickness porcine wounds and demonstrated high levels and prolonged expression of each transgene (unpublished data). Our data demonstrate that high levels of PDGF-A or PDGF-B may actually be deleterious to wound healing and suggests that a narrow window exists between the beneficial therapeutic effect and the toxic effect with increasing gene dosage. This highlights the need for developing a gene therapy strategy which not only promotes the healing process, but also provides proper regulatory controls to prevent aberrant wound healing. Cutroneo⁸⁶ has described the use of an anti-sense technology to regulate gene expression. In this, small oligonucleotide sequences complementary to the initiating AUG codon of a target gene hybridize with the 5’ end of the mRNA, causing translation arrest. Disadvantages of this technique include anti-sense instability, and the inability to optimally control gene expression. An alternate technique is to use a genetic switch to control expression of the desired transgene.Yao et al⁸⁷ have developed a tetracycline- regulable system by inserting the tetracycline operator (Tet-O) upstream of a cassette for a transgene sequence. This system, marketed as T-Rex™ (Invitrogen), offers the possibility of regulating transgene expression by greater than 1000-fold in a dose-dependent and reversible manner. In the absence of tetracycline, the tetracycline binding protein (Tet-R) binds to the tetracycline operator and prevents transcription of the transgene. The addition of tetracycline at low concentration binds the Tet-R thereby releasing it from the Tetracycline-operator and allowing transcription to proceed unimpeded. We have recently developed a stable cell line expressing PDGF-B under control of the tetracycline switch and demonstrated regulable control of PDGF-B both in-vitro and in-vivo (unpublished data).

The recent advances in PDGF gene-therapy technology have rekindled interest in this well-characterized growth factor by overcoming problems associated with recombinant technology and the limited expression achieved by earlier gene therapy techniques. The advent of systems allowing accurate regulation of transgene expression will enable us to address previous concerns regarding the safety of gene therapy strategies and will offer exciting possibilities for the clinical application of PDGF gene therapy in the future.

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