Gene therapy is of increasing interest to the clinical dermatologist. Originally, gene therapy was introduced as a means by which genes that were absent or defective in heritable disorders could be delivered or repaired. This form of gene therapy is currently being studied in detail, and many studies have shown promising results, both in animal models in vivo and in cell cultures in vitro. These applications are primarily relevant to diseases that have well-characterized genetic defects; such genetic skin diseases are shown in Table 1. The applications of gene therapy are not limited to the correction of genetic diseases, however. Other applications include suicide genes for cancer therapy, DNA vaccination, and genetic pharmacology, among others (Figure 1).

Although gene therapy has traditionally been thought to be most applicable to single-gene disorders arising from known genetic defects, most applications of gene therapy currently in testing are directed against various forms of cancers or infectious diseases. Gene therapy is becoming a standard experimental approach for treating cancer patients who have not responded to conventional therapies. One particularly promising gene-therapy approach for treatment of cancer involves suicide-gene therapy.<sup>23 Freeman S.M., Ramesh R.M., Marrogi A.J. Immune system in suicide-gene therapy Lancet 1997 ;  349 : 2  [cross-ref]

Cliccare qui per andare alla sezione Riferimenti</sup> In this approach, the tumor cells are modified by insertion of a suicide gene, such as herpes simplex virus thymidine kinase gene (HSV-TK), which can be expressed in tumor cells with cell-type–specific promoters. By phosphorylation, the HSV-TK gene product is able to convert a pro-drug, such as ganciclovir (GCV), into a highly toxic form of the drug that disrupts DNA replication and results in the death of the cells containing the HSV-TK transgene. Ganciclovir by itself is virtually nontoxic at therapeutic concentrations in cells not infected with HSV-TK, and therefore is nontoxic to normal tissues.

An important observation relating to applicability of the HSV-TK gene to tumor ablation relates to a phenomenon known as the bystander effect, in which nearby unmodified tumor cells are also killed when the neighboring HSV-TK–modified cells are exposed to GCV. This phenomenon can be explained by the fact that a small amount of the toxic phosphorylated GCV metabolites from the HSV-TK gene–modified cells can diffuse to nearby unmodified tumor cells. The bystander effect predicts that only a fraction of the total tumor-cell population needs to be modified by suicide genes to result in a significant reduction of the tumor burden by ablation of the cancer cells.

Genetic vaccination is an exciting new way to immunize individuals by introducing DNA into skin, leading to expression of the foreign antigen, which then elicits an immune reaction.<sup>21 Falo L.D. Targeting the skin for genetic immunization Proc Assoc Am Physicians 1999 ;  111 : 211

Cliccare qui per andare alla sezione Riferimenti</sup> The specific conditions currently being targeted for genetic vaccination include infections by various viruses, such as influenza or papilloma viruses, and various parasites. Intramuscular injection of DNA has been studied in great detail as a means to accomplish DNA vaccination. A convenient way to introduce DNA in the skin is by biolistic particles (i.e., a gene gun), a technique which is discussed later. The advantages of DNA vaccination include improved efficiency and lower cost than, for example, the use of recombinant proteins for immunization. Intracutaneous DNA vaccination takes advantage of the antigen-presenting capabilities of Langerhans cells to elicit a T-cell mediated immune reaction.<sup>35 Klinman D.M., Conover J., Leiden J.M. , e al. Safe and effective regulation of hematocrit by gene gun administration of an erythropoietin-encoding DNA plasmid Hum Gene Ther 1999 ;  10 : 659

Cliccare qui per andare alla sezione Riferimenti</sup>

A particularly promising application of gene therapy relates to genetic pharmacology.<sup>25 Greenhalgh D.A., Rothnagel J.A., Roop D.R. Epidermis: An attractive target tissue for gene therapy J Invest Dermatol 1994 ;  103 : 63S

Cliccare qui per andare alla sezione Riferimenti, 26 Hart I.R., Vile R.G. Targeted therapy for malignant melanoma Curr Opin Oncol 1994 ;  6 : 221  [cross-ref]

Cliccare qui per andare alla sezione Riferimenti</sup> In this approach, DNA encoding various therapeutically beneficial genes can be administered to tissues in a manner such that expression of these genes results in pharmacologic improvement of patients. Methods of administering such genes include, for example, intramuscular injections, introduction of the genes to the skin by gene gun delivery, or the grafting of genetically modified keratinocytes into the skin.<sup>41 Krueger G.G., Morgan J.R., Petersen M.J. Biologic aspects of expression of stably integrated transgenes in cells of the skin in vitro and in vivo Proc Assoc Am Physicians 1999 ;  111 : 198

Cliccare qui per andare alla sezione Riferimenti</sup> Examples of some applications of genetic pharmacology are the production of various clotting factors in hematologic disorders and the use of erythropoietin-encoding DNA for enhanced red blood cell production in patients with chronic anemias.<sup>20 Fakharzadeh S., Sarkar R., Zhang Y. , e al. Correction of factor VIII deficiency in a murine model for hemophilia A through targeting factor VIII transgene expression to epidermis J Invest Dermatol 1998 ;  110 : 503

Cliccare qui per andare alla sezione Riferimenti, 35 Klinman D.M., Conover J., Leiden J.M. , e al. Safe and effective regulation of hematocrit by gene gun administration of an erythropoietin-encoding DNA plasmid Hum Gene Ther 1999 ;  10 : 659

Cliccare qui per andare alla sezione Riferimenti</sup> Similarly, expression of various hormones and growth factors can modify pathologic conditions.

The mode of delivery is determined, in part, by the need to maintain the therapeutic levels of the transgene expression. For example, intramuscular injection of erythropoietin complementary DNA (cDNA) elicits a rise in hematocrit that persists over 8 months, but the levels can reach the 75% to 90% range, clearly an undesirable situation. In contrast, monthly gene gun applications of the gene to the skin of mice elicited a stable increase in hematocrit, and the level of erythrocyte production was controlled by adjusting the dose and frequency of the transgene application.<sup>35 Klinman D.M., Conover J., Leiden J.M. , e al. Safe and effective regulation of hematocrit by gene gun administration of an erythropoietin-encoding DNA plasmid Hum Gene Ther 1999 ;  10 : 659

Cliccare qui per andare alla sezione Riferimenti</sup> The results of this study also indicated that both stationary cells (i.e., keratinocytes) and migratory cells (presumably, Langerhans' cells) were transfected and that they secreted biologically active erythropoietin. Thus, the gene gun administration of plasmid DNA appears to be safe and provides additional strategies for achieving the regulated secretion of an exogenous gene product.