DEVELOPING VDEPT FOR DT-DIAPHORASE (NQO1) USING AN AAV VECTOR PLASMID

Purpose: One enzyme/prodrug combination that has the potential to be u sed in virally directed enzyme/prodrug therapy (VDEPT)is the obligate 2-electron reducing enzyme, DT-diaphorase (NQO1), with bioreductive ag ents such as EO9, The present studies were undertaken to determine if this enzyme, as well as the reporter molecule, green fluorescent prote in (GFP), could be expressed from a single dicistronic unit under cont rol of the CMV promoter in an adeno-associated virus (AAV) background, Methods: The human ovarian tumor cell line, SAU, and the mouse sarcom a cell line, KHT/iv, were studied due to their low level of NQO1 expre ssion. These cells were transfected with pTRUF3-NQO1 using a liposome- mediated protocol. Results: The results indicate that this construct h as 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 NQ O1 activity in SAU increased from undetectable levels to similar to 20 0 nmol/min/mg, and the NQO1 activity in KHT/iv increased similar to 10 -fold following transfection, Expression of the GFP reporter was readi ly detectable in both cell types using FAGS analysis. Conclusions: Tak en together, these results indicate that this proviral AAV vector plas mid will allow for the production of a recombinant AAV, which can coor dinately express the enzyme NQO1 and the GFP reporter for use in vivo in VDEPT studies with various bioreductive agents which are substrates for NQO1, (C) 1998 Elsevier Science Inc.