摘要：

In vivo cutaneous gene therapy is possible with the use of liposome mediated transfection in keratinocytes and fibroblasts. Liposomes were prepared with the encapsulation of DNA within lipids, dimethyl, dioctadecyl-ammonium bromide (DDAB) and dioleyl (C18:1(cis)-9), L-alpha-phosphatidylethanolamine (DOPE) by sonication. These were used for in vitro transfection of human foreskin keratinocytes and fibroblasts with CMVluciferase. Luciferase activities were measured by chemiluminescence and activities were expressed as relative light units per milligram of protein (RLU/mg). Activities to the level of 10(super)8 RLU/mg was obtained for the transfection of normal human foreskin keratinocytes and 10(super)7 RLU/mg for fibroblasts. Optimal activities to 10(super)9 RLU/mg were obtained in normal foreskin keratinocytes using DDAB:DOPE at a ratio of 1:3. Compared with other lipophilic agents, the following activities were obtained in foreskin keratinocytes immortalized with the human papilloma viral E6 and E7 proteins (sonicated liposomes-0.3 10(super)6 RLU/mg, cellfectin-1.3 10(super)6 RLU/mg, lipofectin-33.5 10(super)6 RLU/mg). Although lipofectin appears to yield the highest activities, this agent has been reported in literature to be unstable, yielding a toxic metabolite. Toxicity studies revealed that the sonicated lipids (DDAB:DOPE) have little or no effect on the survival of keratinocytes up to 100 microgram/ml. Therefore, we used sonicated lipids for intradermal injection of CMVluciferase into human foreskin explants which gave activities up to 10(sup)3 RLU/mg. When injected subcutaneously into severe combined immunodeficient (SCID) mice, activities to 10(super)6 RLU/mg were obtained. We are experimenting with SCID mice engrafted with human keratinocytes and fibroblasts.

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