Gene transfer in vitro and in vivo by cationic lipids is not significantly affected by levels of supercoiling of a reporter plasmid.
OBJECTIVE
It is a common preconception that supercoiled plasmid DNA is more desirable for the transfection of cells that the relaxed form of the plasmid. This notion has led to the recommendation that a specification for the minimum amount of plasmid in the supercoiled form should exist in a gene therapy product. We have tested this notion by examining the effects of the degree of supercoiling on cationic lipid-mediated gene transfer in vitro and in vivo.


METHODS
An ion-exchange high performance liquid chromatography (HPLC) method was developed to accurately quantitate the relative amounts of supercoiled DNA in purified plasmid. A sample of the purified plasmid was fully relaxed using topoisomerase. Next, the ability of various levels of supercoiled plasmid to transfect mammalian cells was measured.


RESULTS
This study suggests that there is no relation between the degree of supercoiling and lipofection efficiency. Subsequent transfection using several different lipofection agents, different cell types, and an in vivo model support these results.


CONCLUSIONS
In considering a specification for the amount of supercoiled plasmid in a gene therapy product, it must be noted that the relaxed forms of the plasmid are no less efficient at gene delivery than the supercoiled forms.
Bergan
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Targeted Genetics Corporation, Seattle, Washington 98110, USA.
Galbraith
T
T
Sloane
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DL
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004278:DNA, Superhelical / Q000008:administration & dosage / Q000737:chemistry* / Q000235:genetics; D005822:Genetic Vectors / Q000737:chemistry*; D008055:Lipids / Q000737:chemistry*; D051379:Mice; D008810:Mice, Inbred C57BL; D010957:Plasmids / Q000008:administration & dosage* / Q000235:genetics; D014162:Transfection
D004278:DNA, Superhelical; D008055:Lipids; D004264:DNA Topoisomerases, Type I