Electrotransfer of Plasmid Vector DNA into Muscle
Wolff et al. (1990) first reported that plasmid DNA injected into skeletal muscle is taken up by muscle cells and the genes in the plasmid are expressed for more than two months thereafter, although the transfected DNA does not usually undergo chromosomal integration (Wolff et al., 1991, 1992). However, the relatively low expression levels attained by this method have hampered its applications for uses other than as a DNA vaccine (Davis et al., 1995). There are a number of reports analyzing the conditions that affect the efficiency of gene transfer by intramuscular DNA injection and assessing the fine structures of expression plasmid vectors that may affect expression levels (Davis et al., 1993; Liang et al., 1996; Norman et al., 1997). Furthermore, various attempts were done to improve the efficiency of gene transfer by intramus cular DNA injection. Consequently, regenerating muscle was shown to produce 80-fold or more protein than did normal muscle, following injection of an expression plas-mid. Muscle regeneration was induced by treatment with cardiotoxin or bupivacaine (Wells, 1993; Vitadello et al., 1994). We previously demonstrated that by combining a strong promoter and bupivacaine pretreatment intramuscular injection of an IL-5 expression plasmid results in IL-5 production in muscle at a level sufficient to induce marked proliferation of eosinophils in the bone marrow and eosinophil infiltration of various organs (Tokui et al., 1997). It was also reported that a single intramuscular injection of an erythropoietin expression plasmid produced physiologically significant elevations in serum erythropoietin levels and increased hematocrits in adult mice (Tripathy et al., 1996). Hematocrits in these animals remained elevated at >60% for at least 90 days after a single injection. However, improvements to this method have not been sufficient to extend its applications including clinical use.
Electroporation has long been used for DNA transfection of cells in vitro and it implies a physical process which exposes cells to a brief, high voltage that induces temporary poration to the cell membrane, allowing the influx of large molecules such as plasmid DNA. The entry of plasmid directly into cytoplasm may bypass the endosome7#x2014;lysosome pathway, reducing the degree of DNA degradation. Its initial application in vivo was to transfer DNA into liver cells (Heller et al., 1996) and chick embryos (Muramatsu et al., 1997). We tried in vivo electroporation in skeletal muscle of mice by low voltage rectangular pulses, which had been successfully applied in chick embryos in ovo (Muramatsu et al., 1997; Funahashi et al., 1999). By applying electroporation, naked plasmid DNA can be delivered several hundred-fold more efficiently to muscle cells in vivo (Aihara & Miyazaki, 1998; Mir et al., 1999; Rizzuto et al., 1999). The resulting expression levels are considered sufficient to warrant further investigation of this method for human gene therapy for various diseases (Maruyama et al., 2000).
KeywordsGene Transfer Expression Plasmid Electric Pulse Tibialis Anterior Muscle Direct Gene Transfer
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