Developing VDEPT for DT-diaphorase (NQO1) using an AAV vector plasmid.
OBJECTIVE
One enzyme/prodrug combination that has the potential to be used in virally directed enzyme/prodrug therapy (VDEPT) is the obligate 2-electron reducing enzyme, DT-diaphorase (NQO1), with bioreductive agents such as EO9. The present studies were undertaken to determine if this enzyme, as well as the reporter molecule, green fluorescent protein (GFP), could be expressed from a single dicistronic unit under control of the CMV promoter in an adeno-associated virus (AAV) background.


METHODS
The human ovarian tumor cell line, SAU, and the mouse sarcoma cell line, KHT/iv, were studied due to their low level of NQO1 expression. These cells were transfected with pTRUF3-NQO1 using a liposome-mediated protocol.


RESULTS
The results indicate that this construct has the ability to increase the total protein level of NQO1 by 66-fold in SAU and 102-fold in KHT/iv after 24 h. Furthermore, the level of NQO1 activity in SAU increased from undetectable levels to approximately 200 nmol/min/mg, and the NQO1 activity in KHT/iv increased approximately 10-fold following transfection. Expression of the GFP reporter was readily detectable in both cell types using FACS analysis.


CONCLUSIONS
Taken together, these results indicate that this proviral AAV vector plasmid will allow for the production of a recombinant AAV, which can coordinately express the enzyme NQO1 and the GFP reporter for use in vivo in VDEPT studies with various bioreductive agents which are substrates for NQO1.
Warrington
K H
KH
Department of Pharmacology and Experimental Therapeutics, University of Florida, Gainesville 32610, USA.
Teschendorf
C
C
Cao
L
L
Muzyczka
N
N
Siemann
D W
DW
D000818:Animals; D000229:Dependovirus; D005260:Female; D005822:Genetic Vectors / Q000627:therapeutic use*; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics / Q000378:metabolism*; D010051:Ovarian Neoplasms / Q000378:metabolism; D012513:Sarcoma, Experimental / Q000378:metabolism; D014162:Transfection / Q000379:methods*; D014407:Tumor Cells, Cultured
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D016660:NAD(P)H Dehydrogenase (Quinone)