Phage display selection of a peptide DNase II inhibitor that enhances gene delivery.
BACKGROUND
Nuclease activity is thought to be a significant barrier to effective gene delivery employing synthetic vectors. In particular, the lysosomal DNase, DNase II, has significant access to plasmid DNA, when the protective condensing agent has been shed. Here, we present the identification of a peptide DNase II inhibitor, enabling enhanced levels of gene delivery.


METHODS
A DNase II inhibitor was identified by phage display from a cyclic, random 12-amino acid library. Activity was assayed by inhibition of DNase II degradation of DNA. Transfection enhancement levels were measured over a range of DNA doses with CV-1 and MDBK cell types using PEI and cationic lipoplexes as vectors.


RESULTS
We postulated that a DNase II inhibitor would enhance transfection by enabling a larger fraction of plasmid DNA to traffic through the cell and enter the nucleus. Peptides based on the selected sequence (SLRLLQWFLWAC) [ID2] were shown to inhibit DNase II with an observed KI,app of 0.2-2 microM. Lipoplex-mediated transfection in vitro was found to be enhanced by ID2-3 across the entire range of plasmid DNA doses examined (0.10-3.0 microg/mL). Transfection with PEI/DNA complexes was found to be specifically enhanced in the presence of ID2 peptides, with a saturable DNA-dose curve as would be expected for a competitive inhibitor. Transfection enhancements as high as 270-fold were found in the presence of ID2-3.


CONCLUSIONS
A novel peptide DNase II inhibitor has been used to increase transfection. The level of enhancement was found to be significant in multiple cell types with multiple synthetic vectors.
Sperinde
J J
JJ
Department of Biopharmaceutical Sciences, School of Pharmacy, University of California at San Francisco, 94143-0446, USA.
Choi
S J
SJ
Szoka
F C
FC
D000595:Amino Acid Sequence; D000818:Animals; D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D017931:DNA Primers; D004706:Endodeoxyribonucleases / Q000037:antagonists & inhibitors*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D018014:Gene Transfer Techniques; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*
D017931:DNA Primers; D004791:Enzyme Inhibitors; D010455:Peptides; D004706:Endodeoxyribonucleases; C019329:deoxyribonuclease II