For wound healing, DNA shot from guns is better, faster and cheaperthan recombinant growth factor applied topically to the lesion. So saidElof Eriksson, chief of plastic surgery at Brigham and Women'sHospital, Boston."DNA is inexpensive and easy to multiply," Eriksson told BioWorldToday, "compared to human epidermal growth factor (hEGF) producedby recombinant technology."The Boston plastic surgeon has a paper pending publication in theProceedings of the National Academy of Sciences (PNAS) titled "Invivo transfer and expression of a human epidermal growth factor geneaccelerates wound repair." He expects it to appear "within a couple ofmonths." (Keratinocytes are cells of the living epidermis.)Meanwhile, yesterday's PNAS reports Eriksson's prior results:"Genetically modified keratinocytes transplanted to woundsreconstitute the epidermis." It describes transplanting into woundssuspensions of single skin cells carrying b-galactosidase and humangrowth hormone genes.Eriksson and his co-authors excised 170 full-thickness experimentalwounds, each 1.5 cm square by more than 0.1 cm deep, from the backsof 13 anesthetized pigs. "Porcine and human skin are similar inmorphology, and cell turnover time," his paper explains. He pointedout that the skin surface area of an average human body, 1.7 squaremeters, "is thousands of times larger" than one of his experimentallesions.Three-Dimensional, See-Through Wound DressingThe team covered each open site with a patented "liquid woundchamber," consisting of a shallow, flared vinyl device sealed to theshaved pig skin at its lower rim with medical adhesive. "This chamberis really nothing but an incubator attached to the skin," Eriksson said."Through its flexible transparent top, we administer media, transgeniccells and other components to regulate the wound-healingmechanisms." What's more, "the chamber provides a significanttherapeutic benefit over gauze dressings."He added that "I look upon this preliminary published work only asproving that one can indeed deliver a gene, in this case one thatencodes epidermal growth factor, to keratinocytes in vivo, getexpression and see a biologic effect."The groups' pending paper describes bombarding the wounds withexpression plasmids carrying supercoiled DNA encoding hEGF coatedonto gold microbeads, one to three microns in diameter.This particle-mediated gene transfer, Eriksson said, was constructed bytwo of the paper's co-authors, William Swain and Michael Macklin, ofAgracetus, Inc., Middleton, Wisc. Agracetus, he pointed out, "makes aliving using particle-transfer to enhance the yield of crop plants, andrender them herbicide- and pesticide-resistant."Gene-coated gold beads were spread and attached to an 18-mm-squaresheet of mylar. Electric-arc discharge propelled this open-facesandwich against a 100-mesh stainless-steel screen. It stopped themylar but allowed the DNA-coated gold projectiles to hurtle throughand penetrate the target cells capable of regeneration in the wound beds-- in this experiment, shallow, partial-thickness excisions."When we moved to particle-mediation from retroviral gene transferstrategies," Eriksson recalled, "we saw a hundred-fold increase inyield, and found, for the first time, that we could transfect keratinocytesin vivo, something we had never been able to do by retroviral transfer."Unlike his previous retroviral vector, which had only limited crampedspace for the gene, "With particle mediation, we are using plasmidswith much larger space for coding sequences, so we can not only insertseveral genes but also add promoter and enhancer segments."Following acceleration of genes into the porcine wounds, these werecapped with vinyl chambers, and wound fluids sampled daily."Analysis of the wound fluid for hEGF and total protein, an indicatorof reformation of the epidermal barrier," the pending paper reports,"showed that wounds bombarded with the hEGF-plasmid exhibited, 24hours later, a 190-fold increase in EGF concentration, and healed 20percent (2.1 days) earlier than the controls." Growth factorconcentration in wound fluid "decreased sharply over the first five tosix days following gene transfer. Plasmid DNA persisted in the woundbeds for 30 days or more."These findings," the paper concluded, "demonstrate the possibleutility of in vivo gene transfer to enhance epidermal repair.Theoretically. . . chronic non-healing ulcers. . . as well as malignantand non-malignant epidermal diseases might be treated with particle-mediated gene therapy.""We do not have any clinical trials in the making right now," Erikssonsaid, "but we designed the system with the idea of going into clinicaltrials." Meanwhile, he has used the chamber in treating some plastic-surgery patients, "but not including gene therapy."Without the chamber for quickly assaying gene expression in woundfluid, "the laborious alternative would be to use biopsies, and having todo a Western blot to analyze the protein."He observed, "You could potentially apply similar strategies fordelivering genes encoding keratinocytes that are systematically useful.For example, vaccine delivery technologies, where instead ofadministering the antigen to the skin, as is current practice, transfer inthe gene that encodes the particular antigen."Eriksson is first inventor of the liquid wound chamber (U.S. patent No.5,152,757). So far, it's not licensed, he said, "but we are thinking abouttalking to some prospective licensees. We've been holding on to it,because we wanted to learn a little more about the system, but I thinkwe are now ready for that."Cell biologist Robert Diegelman directs the wound-healing center atthe Medical College of Virginia in Richmond. He is intimatelyacquainted with Eriksson's latest work."The nice thing about what Dr. Eriksson and his group have been ableto do," Diegelman told BioWorld Today, "is get around some of theproblems we have now with topical drug delivery. That is, wheregrowth factors and cytokines are placed on top of a wound, and thesematerials come in contact with a pretty hostile environment."Diegelman added, "He is trying to get around that problem byreprogramming the cells that are normally there in the wound, andallowing them to express these genetic signals in a more naturalmanner."It's a big breakthrough," Diegelman said, "that he's been able to dothis, and get sustained increased production of epidermal growthfactor."As for human trials, the Virginia wound-healing specialist cautioned,"Obviously, we need to be careful that these cells don't becomeabnormally controlled, or lose normal regulation. When you startputting in growth factors, this could put the cells into a repetitive cycleof proliferation, perhaps. You don't want to start causing tumors bydoing this." n
-- David N. Leff Science Editor
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