Effect of Multiple Gene Transfers of Insulinlike Growth Factor I Complementary DNA Gene Constructs in Rats After Thermal Injury FREE

INSULINLIKE growth factor I (IGF-I) has been shown to be an effective anabolic therapeutic approach in the treatment of the trauma-induced catabolic response.^{1 - 5} Insulinlike growth factor I improves metabolism, muscle protein synthesis, and gut and immune function after thermal injury.^{1 - 2 ,6 - 7} Furthermore, it mediates the actions of growth hormone in the hypermetabolic state by attenuating lean body mass loss, the compromised immune response, and the acute-phase response.^{2 ,8 - 12} A key determinant of patient outcome after a thermal injury is the posttraumatic wound healing process.^{3 ,8 ,13} Insulinlike growth factor I has been shown to improve wound healing by stimulating collagen formation and the mitogenicity of fibroblasts and keratinocytes.^{4 - 5} There are, however, adverse effects, such as hypoglycemia, electrolyte imbalance, changes in mental status, edema, and cardiac arrest, that limit the clinical use of IGF-I.^{14 - 15} The pathophysiologic cause of these adverse effects is most likely due to a supraphysiological dose of free IGF-I required for biological efficacy.^{14 - 15} Therefore, it is of interest to develop forms of IGF-I administration that deliver the biologically efficient amount of IGF-I without adverse effects.

A new form that delivers small amounts of IGF-I to target areas, thus reducing adverse effects, is IGF-I gene therapy.^{16 - 17} Gene therapy in general is an approach to the treatment of various clinical disorders.^{18 - 20} Of major importance for successful gene therapy, and thus gene transfer, is the selection of the appropriate vector.^{18 - 20} Viruses, adenoviruses in particular, have been preferred vectors for gene transfer.^{18 - 20} However, viral infection–associated toxicity, immunologic compromise, and possible mutagenic or carcinogenic effects make this system potentially dangerous.^{18 - 20} Cholesterol-containing cationic liposomes represent a nonviral carrier with the ability to interact with all cell membranes.¹⁸ The incorporation of a cytomegalovirus (CMV) promoter driving the complementary (c) DNA constructs increased the levels of transgenic expression to be comparable with those achieved with adenoviral constructs.^{18 - 20} Cationic liposomes are also useful in the treatment of many forms of trauma, exerting anti-inflammatory activity by inhibiting nitric oxide, interleukin 1β, and tumor necrosis factor α synthesis, to attenuate the hypermetabolic response.^{21 - 24}

We have shown that a weekly subcutaneous injection of cholesterol-containing cationic liposomes encapsulating the cDNA coding for the IGF-I gene driven by a CMV promoter is an efficient and safe therapeutic approach to increase IGF-I protein skin concentrations with subsequent improvements in wound healing rates.¹⁶ We have further shown that increased IGF-I protein expression was limited to a small area around the injection site.¹⁶ This restriction of liposomal migration was most likely due to interactions between positive surface charges on cationic liposomes and contiguous negatively charged outer cell membranes, which limited liposomal migration, and thus, transfection.²⁵ The purpose of this study was to determine whether multiple injections of IGF-I cDNA constructs via cholesterol-containing cationic liposomes increase efficacy compared with single injections. Efficacy was determined by measuring rates of transfection, IGF-I protein expression, and rates of wound repair.

Twenty-four adult male Sprague-Dawley rats (350-375 g) were placed in wire-bottom cages and housed in a temperature-controlled room with a 12-hour light-dark cycle. The animals were acclimated to their environment for 7 days prior to the start of the study. All rats received equal amounts of a liquid diet (Sustacal; Mead Johnson Nutritionals, Evansville, Ind) and water ad libitum throughout the study. While under general anesthesia and analgesia, each rat received a 60% total body surface area full-thickness scald burn as described by Herndon et al.²⁶ Immediately after the thermal injury, rats were resuscitated with Ringer solution (50 mL/kg of body weight) and then randomly divided into 2 groups to receive weekly subcutaneous injections of liposomes (10 µL in 180 µL of saline) containing 2.2 µg of an IGF-I cDNA construct and 0.2 µg of the reporter gene β-galactosidase, Lac Z gene cDNA construct driven by a CMV promoter (n=12) at 1 injection site on the edge of the burn wound, or weekly subcutaneous injections of liposomes (10 µL in 180 µL of saline) containing 2.2 µg of CMV-driven IGF-I cDNA construct and 0.2 µg of the reporter gene for β-galactosidase, Lac Z gene cDNA (n=12), at 2 injection sites on the edge of the burn wound.

The injection scheme is depicted in Figure 1. The IGF-I cDNA construct consisted of a CMV-driven IGF-I cDNA plasmid prepared at the University of Texas Medical Branch Sealy Center for Molecular Science Recombinant DNA Core Facility, Galveston. The IGF-I cDNA was kindly supplied by P. Rotwein, PhD, National Institutes of Health, Bethesda, Md. The liposomes were formulated from 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyl ethyl ammonium bromide and cholesterol suspended in membrane-filtered water (Life Technologies, Rockville, Md). This reagent interacts spontaneously with IGF-I cDNA to form the lipid cDNA complex. Immediately after the thermal injury, each rat received 0.2 mL of the solutions injected at either 1 site, 1 cm from the wound margin, or at 2 sites distal to each other (Figure 1). This was repeated once a week for 4 weeks. Lipoplexes had to be prepared fresh every week prior to injections.

Animals were killed by decapitation 5 days after the last injection (33 days after thermal injury). Blood was collected into serum and plasma separators and spun at 1000g for 15 minutes. Supernatant and pellet were separated and stored at −73°C. Liver and 3 dorsal skin samples, defined as biopsy specimen 1, 2, or 3 (Figure 1), were harvested, snap frozen in liquid nitrogen, and stored at −73°C for analysis.

Transfection was determined by measuring the presence of β-galactosidase. The presence of β-galactosidase protein was detected by chemiluminescent reporter gene assay (Galacto-light Plus; Tropix Inc, Bedford, Mass) in skin. Samples were prepared as follows²⁷ : 100-mg tissue was homogenized in 200 µL of lysis buffer (40 mmol/L Tris, pH 7.5, 1 mmol/L EDTA, 150 mmol/L sodium chloride) for approximately 30 seconds. Samples were centrifuged at 12,000g for 3 minutes. The supernatant was removed and the volume was measured and stored on ice. The residual pellet was rinsed with 200 µL of lysis buffer and microcentrifuged. The assay followed manufacturers directions using 96-well plates.

Insulinlike growth factor I protein concentrations were measured by radioimmunoassay in the 3 skin biopsy specimens. Proteins were extracted by pulverizing approximately 40 mg of tissue under liquid nitrogen, adding an extraction buffer (phosphate-buffered saline 0.25 mL phenylmethylsolfonyl fluoride, 50 mg of leupeptin, 100 mg of aprotinin, and 50 mg of antipain) in a volume of 1:7 (7 mL of buffer per gram of tissue), and homogenizing the mixture. Samples were frozen overnight at −80°C. After thawing, 50 µL of the homogenate was added to 150 µL of extraction solution and centrifuged at 13,500 rpm for 5 minutes, 100 µL of supernatant was added to 400 µL of neutralization solution, and the radioimmunoassay was performed as described in the kit guidelines (Diagnostic System Laboratories, Webster, Tex).

Biological efficacy of the delivery system was defined by measuring body weights, protein content in serum and liver, and wound healing, defined as reepithelization rate. Body weights were measured at the same time each week. Protein content in serum was measured using a nephelometer (Behring, Deerfield, Ill). Liver protein concentration was determined by protein assay (Bio Rad, Hercules, Calif) based on the method by Bradford.²⁸ Wound healing was determined as follows. The wound eschar was left intact for the first 28 days and then removed by gentle traction, caution being taken not to disturb or destroy the healing edge along the periphery. After removing the eschar, the animals were placed on a standard surface and the wound area traced onto acetate sheets along the well-demarcated reepithelized and nonburned interface and the leading edge of the neoepithelium. The areas of these tracings were calculated by computerized planimetry (Sigma Scan and Sigma Plot software; Jandel, San Rafael, Calif).

Studies were reviewed and approved for humane animal treatment by the Animal Care and Use Committee of the University of Texas Medical Branch, assuring that all animals received humane care according to the criteria outlined in the guide for the care and use of laboratory animals published by the National Institutes of Health, Bethesda, Md. Statistical comparisons were made by analysis of variance and t test with the Bonferroni correction. Data are expressed as mean±SEM. Significance was accepted at P<.05.

All rats in each group survived the 60% total body surface area scald burn and drug injections with no evidence of any adverse effects. Total body weight increased almost 2% per week for the first 4 weeks postinjury in animals transfected with single and multiple injections of liposomes–IGF-I cDNA construct. There were no differences between the 2 groups for changes in body weights. Rats receiving multiple injections of the IGF-I cDNA construct had higher serum protein levels (single injection, 52±0.5 g/L vs multiple injections, 55±0.6 g/L) and total liver protein (single injection, 82.0±0.3 mg/mL vs multiple injections, 91.0±3.8 mg/mL) compared with single injection–treated animals (P<.05). After the eschar was removed, the percent area of burn wound reepithelization was significantly larger 33 days after burn in rats receiving the multiple injections of IGF-I cDNA compared with rats receiving the single injection, 38%±2% vs 31%±2%, respectively (P<.05; Figure 2).

Transfection, determined by chemiluminescent reporter gene assay to detect β-galactosidase, was increased around the wound perimeter in animals receiving multiple injections of liposome-encapsulated Lac Z gene cDNA and IGF-I cDNA constructs when compared with single injections, P<.05 (Figure 3).

Skin concentrations of IGF-I protein decreased from skin biopsy point 1 to point 3 in rats receiving the single injection of IGF-I cDNA construct along the wound edge (Figure 4, left). Animals receiving multiple injections of the cDNA construct showed consistently elevated IGF-I protein concentrations along the wound edge (Figure 4, right).

Wound healing is of major importance to the survival and clinical outcome of burn patients.^{8 ,13} Somatic gene therapy is a potentially useful strategy for the delivery of growth factors to accelerate wound healing. Insulinlike growth factor I enhances wound healing through stimulation of collagen formation and mitogenicity of fibroblasts and keratinocytes by binding to their receptors.^{4 - 5} Despite the advantages of IGF-I therapy after trauma, adverse effects limited the clinical use of IGF-I.^{14 - 15} We have previously demonstrated that a new form of IGF-I administration, subcutaneous injection of liposomes encapsulating the cDNA coding for IGF-I, increased skin IGF-I concentrations along with improved wound healing without detectable adverse effects.^{16 - 17} We further showed that liposomal migration, and thus areas of transfection, was restricted to the areas immediate to the injection site.¹⁶ Therefore, we hypothesized that multiple injections of lipoplexes containing the cDNA for IGF-I enhance transfection with increases in IGF-I concentration around the wound edge, which accelerates the wound healing process.

After the subcutaneous injection of the IGF-I cDNA and the reporter Lac Z gene construct, we determined transfection in dermal cells. The major mechanism by which effective transfection occurs is most likely localized endocytosis.^{23 ,29 - 30} DNA plasmids enter the cell and the cell nucleus and the released cDNA is then taken up by the nucleus.²⁰ How the cDNA is taken up by the nucleus is currently unknown.²⁰ The ribosomes then transcribe the IGF-I cDNA into mRNA, which is transported to the rough endoplasmic reticulum, where the messenger RNA is translated into the IGF-I protein. This transient increase in the local expression of IGF-I protein is most likely to cause a concurrent stimulation of insulinlike growth factor binding protein-3 protein synthesis and locally increased levels of the biological active complex IGF-I/IGFBP-3, without any concomitant supraphysiological increases in circulating levels of free IGF-I protein, and therefore no deleterious adverse effects.^{14 - 15}

In agreement with previous results, we demonstrated in this study that transfection is restricted to a perimeter near the sites of injection.¹⁶ The restricted nature of the expression of IGF-I protein was most likely due to the local character of the interactions between the positive surface charges on cationic liposomes and negatively charged outer cell membranes, which restricted liposomal migration.²⁵ This restriction of liposomal migration leads to restrained transfection and protein expression. As we demonstrated in this study, liposomal migration, transfection, and protein expression can all be increased by multiple injections. Rats receiving multiple injections of IGF-I cDNA demonstrated high transfection rates around the wound edge, with concurrent increased IGF-I protein expression. Rats receiving 1 injection of IGF-I cDNA demonstrated a gradient of transfection and protein expression, with a high density around the injection site and no detectable transfection as well as protein expression distal from the injection site. This finding is clinically relevant because the liposomes encapsulating the gene should be applied at well-defined distances from the wound to provide optimal transfection and protein expression.

A physiological response to the increased IGF-I skin concentration was accelerated wound healing and improved total protein concentrations in serum and liver. Given that the subcutaneous injection of IGF-I cDNA construct is localized, we suggest that the physiological effects are due to improved wound healing and cell recovery after injury and not from increased circulating levels of IGF-I protein.¹⁶ The advantages of early wound closure, demonstrated in several clinical studies,^{31 - 32} include a diminished hypermetabolic burn response and a decrease in inflammatory mediators, such as interleukin 1, interleukin 6, interleukin 8, and tumor necrosis factor α.^{31 - 32} Furthermore, we have shown that IGF-I protein decreases proinflammatory cytokines interleukin 1β and tumor necrosis factor α expression after thermal injury (M.G.J., unpublished data, 1999). Therefore, IGF-I may exert its systemic beneficial effect through the enhancement of re-epithelialization and/or the decrease of the proinflammatory response in the skin, which is one of the major sources of cytokine synthesis and release after burn.^{33 - 34}

In this study, we showed that multiple subcutaneous injections of the IGF-I cDNA increased the number of transfected cells and protein expression compared with a single injection. The physiological responses to increases in skin IGF-I were an enhancement in wound healing, with subsequently systemic improvements to the hypermetabolic response. From these findings, we conclude that multiple injections of cholesterol-containing cationic liposomes encapsulating an expression plasmid vector for IGF-I cDNA given to rats with a 60% total body surface area thermal injury were effective in increasing IGF-I skin protein concentrations without adverse effects. Thus, treatment with multiple injections of the IGF-I cDNA construct may be an effective therapeutic approach to improve clinical outcomes after thermal injury.

This study was supported by the Clayton Foundation for Research, Houston, and the Shriners Hospital for Children, Galveston, Tex.

We thank Anne S. Burke, BS, Meelie A. DebRoy, MD, and Jyoti Rai, MD, for their technical assistance.

Corresponding author: Robert E. Barrow, PhD, Department of Surgery, Shriners Hospital for Children, 815 Market St, Galveston, TX 77550 (e-mail: Rbarrow@sbi.utmb.edu).