Cancer Gene Therapy Using Plasmid DNA: Pharmacokinetic Study of DNA Following Injection in Mice

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Abstract
The fate of plasmid DNA complexed with cationic lipids delivered intravenously in mice was evaluated at selected timepoints up to 6 months postinjection. Blood half-life and tissue distribution of plasmid DNA and potential expression in tissues were examined. Southern blot analyses of blood indicated that intact plasmid DNA was rapidly degraded, with a half-life of less than 5 min for intact plasmid, and was no longer detectable at 1 hr postinjection. Southern analyses of tissue demonstrated that intact DNA was differentially retained in the lung, spleen, liver, heart, kidney, marrow, and muscle up to 24 hr postinjection. After 7 days, no intact plasmid DNA was detectable by Southern blot analysis; however, the plasmid was detectable by the polymerase chain reaction (PCR) in all tissues examined at 7 and 28 days postinjection. At 6 months postinjection, femtogram levels of plasmid were detected only in muscle. Immunohistochemical analyses did not detect encoded protein in the tissues harboring residual plasmid at 1 or 7 days postinjection. The present study evaluates the pharmacokinetic half-life and tissue distribution of plasmid DNA following intravenous injection in mice. This study extends the time frame of previous in vivo analyses to 6 months following i.v. injection. Injected mice exhibit no expression of the encoded gene as assayed by immunofluorescence. This represents the first systematic in vivo pharmacokinetic study of intravenously injected DNA complexed with cationic lipids, and is relevant to many gene therapy protocols utilizing direct injection of plasmid DNA plus lipids. The results provide a preliminary basis for the safe initiation of cancer immunotherapy clinical trials in which plasmid DNA is directly injected into tumors.