Antisense transfer

Transfer 600 p1 of this mixture to another micro tube containing 10 /(I of antisense ODN (10 pmole/p ) complementary to the cleavage site on the mRNA. The remaining 70 to 100 p serves as a control sample ( uncut ) and will be subjected to the following incubations (steps 3 to 6). 

Particles from cationic lipids may also be useful for antisense therapy of skin disease — a nontoxic increase in the oligonucleotide uptake by cultivated keratinocytes and a sebocyte cell line has been reported [66]. Moreover, cationic dendri-mers also efficiently transfer reporter gene DNA to human keratinocytes cultivated in vitro. In the skin of hairless mice, in vivo transfection was possible with complexes, yet reporter gene expression was localized to perifollicular areas. Transfection, however, failed with the naked plasmid. For prolonged contact, biodegradable membranes coated with dendrimer/DNA complexes were used [67]. This hints at a follicular uptake of these complexes and indicates that gene transfection also may be possible with human skin, which has a thicker stratum comeum compared with mouse skin (eight to ten vs. two to three layers [58]). 

Some investigators have used electrical current (electroporation) to improve DNA (or drug) entry into tumor cells with some preliminary success. Liposomes are attractive vehicles for gene delivery, since they can carry plasmid, antisense, or viral DNA. Compared with viral approaches, however, liposomes remain relatively inefficient at facilitating gene transfer, although their safety profile remains more desirable. Some of the attributes and limitations of the nonviral methods are listed in Table 58.1. 

Elomaa, P. et al., Agrobacterium-mtdidXtd transfer of antisense chalcone synthase cDNA to Gerbera hybrida inhibits flower pigmentation. Biotechnology, 11, 508, 1993. 

M Sugano, N Makino, S Sawada, S Otsuka, M Watanabe, H Okamoto, M Kamada, A Mizushima. Effect of antisense oligonucleotides against cholesteryl ester transfer protein on the development of atherosclerosis in cholesterol-fed rabbits. J Biol Chem 273 5033-5036, 1998. 

Xiao, X. et al. (1997). Adeno-associated virus (AAY) vector antisense gene transfer in vivo decreases GABA(A) alphal containing receptors and increases inferior collicular seizure sensitivity. Brain Res. 756(1-2), 76-83. 

Hanna AK, FoxJC, Necklis DG, et al, Antisense basic fibroblast growth factor gene transfer reduces neointimal thickening after arterial injury, J Vase Surg 1997 25(2) 320-325. 

Genetic modification of many dicotyledoneous crops such as potato using gene transfer via A. tumefaciens is, at present, an efficient and reliable technique the application of the antisense route to limit or neutralize the action of undesirable genes has also been applied successfully to potatoes. Conversely, for monocotyledoneous crops such as maize, wheat, and rice, transformation can be accomplished by the much less efficient particle gun technique. This technique has been improved, resulting in rapid progress in the development of maize and later wheat with improved agronomic properties and/or altered starch composition. 

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