Owing to the rapid development of in vivo applications for nonviral gene delivery vectors, it is necessary to have a better understanding of how the structure-activity relationships of these lipid-DNA complexes are affected by their environment. Indeed, research in gene therapy first focused on in vitro cell culture studies to determine the mechanisms involved in the delivery of DNA into the cell. New biophysical techniques such as electron microscopy and X-ray diffraction have been developed to discern the structure of the lipid-DNA complex. However, further studies have revealed discrepancies between optimal lipid-DNA formulations for in vitro transfection and for in vivo administration of these vectors. Furthermore, some immune stimulatory effects have been associated with in vivo lipid-DNA administration. This review summarizes the current state of knowledge on in vitro and in vivo lipid-DNA complex transfections. New prospects of vectors for in vivo gene transfer are also discussed.

Electrical stimulation (ES) is a therapeutic treatment for wound healing. Electroporation, a type of ES, is a well-established method for gene delivery. We hypothesize that proper conditions can be found with which both electrical and gene therapies can be additively applied to treat diabetic wound healing. For the studies of transforming growth factor-beta1 (TGF-beta1) local expression and therapeutic effects, full thickness excisional wound model of db/db mice was used, we measured TGF-beta1 cytokine level at 24 h postwounding and examined wounds histologically. Furthermore, wound closure was evaluated by wound-area measurements at each day for 14 d. We found that syringe electrodes are more effective than the conventional caliper electrodes. Furthermore, diabetic skin was more sensitive to the electroporative damage than the normal skin. The optimal condition for diabetic skin was six pulses of 100 V per cm for 20 ms. Under such condition, the healing rate of electrically treated wound was significantly accelerated. Furthermore, when TGF-beta1 gene was delivered by electric pulses, the healing rate was further enhanced. Five to seven days postapplication of intradermal injection of plasmid TGF-beta1 followed by electroporation, the wound bed showed an increased reepithelialization rate, collagen synthesis, and angiogenesis. The data indicates that indeed the electric effect and gene effect work synergistic in the genetically diabetic model.

Although particle-mediated gene transfer technology (gene gun) has been applied for gene transfer to external tissues, the application of this technology to other tissues has met with limited success. Here we report the development of a new design of a gene gun that uses helium discharge to propel DNA-coated gold beads that are suspended in liquid. Higher discharge pressures allow for the delivery of DNA to deeper tissues. Using the new gene gun to deliver a luciferase expression plasmid resulted in higher levels of gene expression in the skin than observed with conventional guns, as well as in subdermal tissues, including subcutaneous tumors. Even when using as little as 125 ng of DNA, gene expression in skin and muscle reached its peak level at 24 hr postbombardment and remained for at least 1 week. The use of a LacZ expression plasmid showed that gene expression was distributed throughout the skin with no observable pathology. The new gene gun was used to deliver a model tumor rejection antigen (a modified human papilloma virus [HPV] E7 gene) to mice. All of the treated animals developed protective immunity against HPV-positive tumors. These results demonstrate that our new design can be used in standard gene gun applications and extends the reach of gene gun technology to tissues that were previously unavailable.