EUROPEAN TISSUE REPAIR SOCIETY

GENE THERAPY I

MECHANISTIC INSIGHTS AND POSSIBLE CLINICAL APPLICABILITY OF NON-VIRAL LIPOSOMAL GENE THERAPY

THE most important function of the skin is to serve as a protective barrier against external influences, such as the environment.¹ The loss of dermal integrity may lead to loss of fluid and energy and to an increase in permeability, which results in an increased risk of bacterial translocation and hence the development of infection or even sepsis. Wound healing is therefore critical for morbidity and mortality in patients with acute and chronic wounds. Dermal and epidermal regeneration is a dynamic interactive and complex process involving soluble mediators, blood cells, extracellular matrix, and parenchymal cells.1 In general wound healing is divided into three phases: inflammation, tissue formation and tissue remodelling. During these phases different cell types synthesize and release mediators that modulate and stimulate wound healing. These mediators encompass chemokines and growth factors. Growth factors have been shown to influence epidermal and dermal regeneration.^2,3 However, the clinical use of recombinant growth factors has not been as beneficial as once expected, because of the complexity and the variability of the interactions between cells, soluble cytokines, blood elements, extracellular matrix, proteases and decreased concentration of receptors for the growth factors.⁴ New therapeutic strategies are needed to improve the efficacy of growth-factor application and consecutive epidermal and dermal regeneration. One of these strategies is the use of non-viral liposomal gene transfer.

Skin gene transfer represents a relatively new approach with great potential because of the accessibility and the possibility of monitoring the modified area.5 For successful gene delivery the selection of an appropriate vector has been shown to be paramount.^6,7 Viruses have been used as delivery vectors due to the specificity with which they can bind to and infect cells. However, viral infection-associated toxicity, immunological compromise, and possible mutagenic or carcinogenic effects make this approach potentially dangerous.^6,7 Non-viral gene therapy has several advantages over viral gene therapy; non-viral delivery systems are easy, simple, direct, inexpensive and don’t require ex-vivo manipulation.⁸ Another advantage is that non-viral genes can be administered repeatedly without causing an immune response or tachyphylaxia, which is in contrast to viral gene transfer.⁸ Disadvantages of non-viral gene transfer are that it results in a transient gene expression, inability to deliver genes selectively to specific cells and variability in the level of gene expression. However, given the characteristics of non-viral gene therapy, it would seem that this approach could be improved and applied clinically in the future. Despite the possibilities of applying non-viral gene therapy to the skin, little is known or understood about the mechanisms of dermal gene transfer, bio-distribution properties, systemic transfection or deposition, cellular uptake and, whether the translated protein is biologically effective. The aims of our studies were to gain mechanistic insights and to determine the possible clinical applicability of non-viral liposomal gene therapy.

Background

Liposomes can be applied either by topical administration or by direct injection of liposomal gene constructs.^5,9,10 Alexander et al. demonstrated that topical application of liposomal constructs containing the Lac Z-gene in shaved 4-week-old mice resulted in a transfection and expression in the epidermis, dermis and hair follicle.⁵ Expression was seen at 6 hrs post-application and persisted at high levels 48 hrs post-application. Seven days after application the expression was reduced but detectable.⁵ Topical administration of liposomal complexes, however, is not as effective as injection with regard to cell transfection and to the level of expression.⁸ This is due to the stratum corneum, which is the protective layer of the epidermis. Only a few studies have been performed to determine the efficiency of non-viral liposomal gene transfer for the delivery of growth factors.^9–11 Cationic liposomal complexes appeared to be suitable for use in acute and chronic wounds, because liposomes attenuated the pro-inflammatory hypermetabolism and the acute phase response post trauma.¹² While the reasons for the beneficial effects associated with liposomes are not fully understood, they may be due to the direct effect of the liposomal lipid moieties on damaged cell membranes. Alternatively they may lead to an enhancement of the uptake of extracellular nutrients and the in situ encapsulation and protection of endogenous growth factors and cytokines which are elaborated locally as part of the hypermetabolic response which is triggered by acute phase proteins and cytokines.

Insulin-like growth factor-I (IGF-I) has been shown to improve hypermetabolism and the acute-phase-response after a thermal injury.^13,14 Treatment with IGF-I has been shown to accelerate wound healing by stimulating collagen synthesis and the mitogenicity of fibroblasts and keratinocytes.¹⁵ Several adverse side effects have limited the use of IGF-I in the treatment of burns, most probably due to the supraphysiological doses of free IGF-I needed for the desired therapeutic effects.¹⁶ We therefore specifically wanted to deliver smaller amounts of IGF-I which would be as efficient as the administration of IGF-I protein, but without side effects. We chose non-viral over viral gene transfer, because the immune system in burn patients is massively compromised. In collaboration with the NIH (Dr. P. Rotwein), we designed an IGF-I vector and in several subsequent studies, starting in vitro with 3T3- Fibroblasts, we determined mechanisms and biodistribution of non-viral gene transfer after injecting the gene complexes into the skin. After injecting liposomal cDNA constructs transfection was detected in myofibroblasts, endothelial cells, and macrophages, including multinucleate giant cells. All these cells are known to be proliferative. Other groups described difficulties in transfection rates with the use of liposomes; in an acute wound we reached transfection rates between 70–90% (Figure 1). For other pathophysiological states it is therefore important to improve transfection by modification of the liposomal constructs. For gene expression to occur,DNA plasmids enter the cell and its nucleus. Ribosomes transcribe the nuclear cDNA into mRNA, which is then transported to the rough endoplasmic reticulum where it is translated into protein. Our observations that mRNA for IGF-I was only detected in the skin of transfected rats is consistent with increases in skin IGF-I protein concentrations. Our data indicate that cDNA encoding for b-galactosidase and IGF-I was transcribed and translated into b-galactosidase and IGF-I protein in the skin. To evaluate whether the synthesized IGF-I has physiologic effects we measured wound healing, in terms of re-epithelialisation, and skin cell proliferation.¹⁷ In several studies we found that animals receiving the IGF-I cDNA had an accelerated rate of re-epithelialisation/ wound healing when compared to multiple other treatments, such as naked IGF-I protein, IGF-I protein encapsulated in liposomes, liposomes encoding the Lac Z gene, or normal saline (Figure 2).

Figure 1: Representative photomicrograph of the wound edge taken 33 days after burn and 5 days after the last injection, histochemically stained for b-galactosidase. The skin was injected with liposomes containing the IGF-I and Lac Z gene complexes. The reaction product for b-galactosidase is present within several layers of the skin at the wound edge. Magnification 80x.

Figure 2:Area of wound re-epithelialisation measured by planimetry. Rats receiving multiple injections of encapsulated IGF-I cDNA constructs had the highest percent of re-epithelialisation throughout the study period compared to the vehicle or saline groups. Comparison of multiple with single injections showed that increasing the number of injection sites enhanced the degree of reepithelialisation by nearly 20%. * IGF-I cDNA multiple vs. saline, liposomes and IGF-I single injection. ** IGF-I single injection vs. vehicle and saline (p<.05). Data presented as means±SEM.

Figure 3:Re-epithelialisation in rats treated with liposomes containing the cDNA for KGF or liposomes alone. Animals receiving liposomes containing the KGF cDNA demonstrated an accelerated re-epithelialisation by 170% when compared to controls. Significant difference between rats receiving KGF vs. vehicle, p<.05. Data presented as means±SEM.

Figure 4: Skin cell proliferation determined by Ki67 and apoptosis by TUNEL. A) Rats receiving KGF had an increased skin cell proliferation and a decreased cell apoptosis, thus an improved net-balance compared with rats receiving liposomes. * Significant difference between rats receiving the KGF gene complex vs. vehicle, p<.05. Data presented as means±SEM. B-C) Representative histological section of dermal tissue at the wound edge in rats receiving liposomes or the KGF cDNA construct. Dark staining (redbrown) was considered positive for proliferating skin cells. Mainly basal cells were found to be positive, thus undergoing proliferation. Magnification 200x.

In another series of studies we asked the question as to whether only IGF-I gene transfer is possible or whether another growth factor could be effectively administered using this method. Keratinocyte growth factor (KGF) stimulates epithelial cell differentiation and proliferation, which are of major importance for wound healing. In an animal study we could show that KGF cDNA gene transfer improved epidermal regeneration by 170% by exhibiting the most rapid area and linear wound re-epithelialization (Figure 3).¹⁸ KGF improved epidermal cell net balance by increasing skin cell proliferation and decreasing skin cell apoptosis (Figure 4). Dermal regeneration was further improved in KGF cDNA treated animals by an increased collagen deposition and better morphology. KGF cDNA increased neo-vascularisation and concomitant VEGF concentrations when compared with vehicle. KGF cDNA gene transfer not only stimulated epithelial cells, but also mesenchymal cells through increases in IGF-I concentration. ¹⁸

References

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2. Clark RAF, ed. The molecular and cellular biology of wound repair. 2nd ed. New York: Plenum Press, 1996.
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4. Wlaschek M, Achterberg V, and Meyer-Ingold W. Protease inhibitors protect growth factor activity in chronic wounds. Br J Dermatol 1997; 137: 646–56.
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8. Vogel JC. Nonviral skin gene therapy. Human Gene Therapy 2000; 11: 2253–2259.
9. Jeschke MG, Barrow RE, Hawkins HK, Yang K, Hayes R et al. IGF-I gene transfer in thermal trauma. Gene Therapy 1999; 6: 1015–1020.
10. Jeschke MG, Barrow RE, Hawkins HK, Chrysopoulo MT, Perez-Polo JR et al. Impact of multiple injections of an IGF-I gene construct after thermal injury. Arch Surg 1999; 134: 1137–1141.
11. Sun L, Xu L, Chang H, Henry FA, and Nielsen TB. Transfection with aFGF cDNA improves wound healing. J Invest Dermatol 1997; 108: 313–318.
12. Jeschke MG, Barrow RE, Perez-Polo JR, and Herndon DN. Cholesterol-containing cationic liposomes attenuate type I acute phase proteins and pro-inflammatory cytokines in thermally injured rats. Arch Surg 1999; 134: 1098–1102.
13. Meyer NA, Barrow RE, and Herndon DN. Combined insulin-like growth factor-1 and growth hormone improves weight loss and wound healing in burned rats. J Trauma 1996; 31: 1008–1012.
14. Jeschke MG, Herndon DN, and Barrow RE. Insulinlike growth factor-I plus insulin-like growth factor binding protein-3 affects the hepatic acute phase response and hepatic morphology in thermally injured rats. Ann Surg 2000; 231: 408–416.
15. Martin P. Wound healing-aiming for perfect skin regeneration. Science, 1997; 276: 75–81.
16. Jabri N, Schalch DS, Schwartz SL, Fischer JS, Kipnes MS et al. Adverse effects of recombinant human insulin-like growth factor-I in obese insulin-resistant type II diabetic patients. Diabetes 1994; 43: 369–74.
17. Jeschke MG, Barrow RE, Hawkins HK, Tao Z, Perez-Polo JR et al. Biodistribution and feasibility of non-viral IGF–I gene transfers in thermally injured skin. Lab Invest 2000; 80: 151–158.
18. Jeschke MG, Richter G, Hofstätter F, Perez-Polo JR, Herndon DN et al. Non-viral, liposomal Keratinocyte Growth Factor (KGF) cDNA Gene Transfer improves dermal and epidermal regeneration through stimulation of epithelial and mesenchymal factors. Gene Therapy 2002; 9: 1065–1074

Corresponding author:
Priv. Doz. Dr. med. Marc G Jeschke
Klinik und Poliklinik für Chirurgie
Klinikum der Universität Regensburg
Franz-Joseph-Strauss Allee 11
93053 Regensburg, Germany
Tel: +49 (0)941 944 6801 Fax: +49 (0)941 944 6802
e-mail: Mcjeschke@hotmail.com