Springer Online Journal Archives 1860-2000
Chemistry and Pharmacology
Abstract Purpose. It is a common preconception that supercoiledplasmid DNA is more desirable for the transfection of cells that the relaxedform of the plasmid. This notion has led to the recommendation that aspecification for the minimum amount of plasmid in the supercoiled formshould exist in a gene therapy product. We have tested this notion byexamining the effects of the degree of supercoiling on cationiclipid-mediated gene transfer in vitro and in vivo. Methods. An ion-exchange high performance liquidchromatography (HPLC) method was developed to accurately quantitatethe relative amounts of supercoiled DNA in purified plasmid. A sample of thepurified plasmid was fully relaxed using topoisomerase. Next, the ability ofvarious levels of supercoiled plasmid to transfect mammalian cells wasmeasured. Results. This study suggests that there is no relationbetween the degree of supercoiling and lipofection efficiency. Subsequenttransfection using several different lipofection agents, different celltypes, and an in vivo model support these results. Conclusions. In considering a specification for the amountof supercoiled plasmid in a gene therapy product, it must be noted that therelaxed forms of the plasmid are no less efficient at gene delivery than thesupercoiled forms.
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