DNA transfection of macaque and murine respiratory tissue is greatly enhanced by use of a nuclease inhibitor.
BACKGROUND
Nuclease activity present within respiratory tissues contributes to the rapid clearance of injected DNA and therefore may reduce the transfection activity of directly injected transgenes. Most gene transfer technologies transduce or transfect murine tissues more efficiently than corresponding primate tissues. Therefore, it is prudent to assess the utility of novel gene transfer strategies in both rodent and primate models before proceeding with further development.


METHODS
This study analyzed the effects of ATA (a nuclease inhibitor) on the direct transfection of macaque and murine lung tissue; compared the levels of DNase activity in murine, primate, and human lung fluids; and tested the inhibitory activity of ATA on the DNase activity present in these samples. Fluorescent microspheres were used to detect areas of transfection in lung.


RESULTS
Intratracheal administration of a nuclease inhibitor (ATA) with naked DNA (0.5 microg ATA/g body weight) enhanced direct transfection efficacy in macaque lung by over 86-fold and by over 54-fold in mouse lung. Hematoxylin and eosin staining showed no apparent tissue toxicity. Moreover, macaque, human, and mouse lung fluids were found to possess similar levels of DNase activity and this activity was inhibited by similar concentrations of ATA. The authors also successfully pioneered the use of carboxylate-modified microsphere tracers to identify areas of transfection and/or treatment.


CONCLUSIONS
This work provides evidence that using direct nuclease inhibitors will enhance lung transfection and that nuclease activity is present in all lung fluids tested, which can be inhibited by the use of direct DNase inhibitors.
Gene Delivery Alliance, Inc., Rockville, MD 20850, USA.
Jill
J
Glasspool-Malone
Peter R
PR
Steenland
Ruth J
RJ
McDonald
Rigoberto A
RA
Sanchez
Tammara L
TL
Watts
Joseph
J
Zabner
Robert W
RW
Malone
D000818:Animals; D001312:Aurintricarboxylic Acid / Q000494:pharmacology*; D001992:Bronchoalveolar Lavage Fluid; D004247:DNA / Q000008:administration & dosage* / Q000235:genetics; D003851:Deoxyribonucleases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008168:Lung / Q000201:enzymology / Q000378:metabolism*; D008251:Macaca; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008863:Microspheres; D014162:Transfection
D004791:Enzyme Inhibitors; D001312:Aurintricarboxylic Acid; D004247:DNA; D003851:Deoxyribonucleases