Marked enhancement of direct respiratory tissue transfection by aurintricarboxylic acid

Simple, nontoxic, and pharmaceutically defined methods for genetic modifica tion of respiratory tissues may enable development of a variety of molecula r medicines. Clinical applications for such medicines include treatment of inborn errors of metabolism, interventions for asthma and iatrogenic pulmon ary fibrosis, and disease prophylaxis via mucosal polynucleotide vaccinatio n. "Free," "direct," or "naked" plasmid adminstration is a simple, apparent ly safe, and pharmaceutically defined gene delivery method. Murine, macaque , and clinical human studies have demonstrated transfection of respiratory tissues after direct application of free plasmid. The aim of this study was to develop a simple and safe alternative to respiratory tissue transductio n, and specifically to provide a theoretical framework for developing a cat egory of adjuvants, nuclease inhibitors, that augment the transfection acti vity of free plasmid. Plasmid employing the human CMV IE promoter/enhancer to drive expression of the Photinus pyralis luciferase reporter protein was administered intratracheally into mouse lung with or without the nuclease inhibitor aurintricarboxylic acid (ATA). Lavage samples and tissue extracts were used to demonstrate inhibition of lung nuclease activity. ATA dose es calation studies were performed using lung homogenate assays to characteriz e transfection. Potential toxicity was assessed histologically. The data in dicate that nucleases present in respiratory fluids accelerate clearance of biologically active plasmid from lung, that intratracheal coadministration of ATA together with plasmid reduces extracellular DNA clearance, and that this treatment results in marked enhancement of reporter protein expressio n. The effective dose for ATA enhancement of direct lung transfection was 0 .5 mu g/g mouse weight, and the LD50 was approximately 6 mu g/g. These find ings provide a theoretical and practical foundation for further development of an alternative gene delivery system: free plasmid-based respiratory tra nsfection technology.