VASCULAR INDUCIBLE NITRIC-OXIDE SYNTHASE GENE-THERAPY - REQUIREMENT FOR GUANOSINE TRIPHOSPHATE CYCLOHYDROLASE-I

Background, Human inducible nitric oxide synthase (iNOS) gene transfer inhibits myointimal hyperplasia in vitro. However, unstimulated vascu lar smooth muscle cells (SIS IC) do not synthesize tetrahydrobiopterin (BH4), an essential cofactor for iNOS, which may be an obstacle to su ccessful vascular iNOS gene therapy. We investigated the capacity of g ene transfer of guanosine triphosphate (GTP) cyclohydrolase I (GTPCH), the rate-limiting enzyme for BH4 biosynthesis, to supply cofactor for iNOS activity. Methods, A human GTPCH expression plasmid (pCIS-GTPCH) was transfected into rat aortic SMC (RAOSMC) and BH4-deficient NIH3T3 cells engineered to stably express human iNOS (3T3-iNOS). GTPCH activ ity and intracellular biopterins were assessed as a measure of success ful transfection, and the capacity of GTPCH to reconstitute iNOS activ ity was used to determine whether BH4 was made available to the iNOS p rotein. Results, The pCIS-GTPCH-transfected 3T3 cells had demonstrable GTPCH activity as compared with control cells (169.3 +/- 6.6 pmol/hr/ mg versus 0, p < 0.001). Intracellular biopterin levels were also incr eased in transfected 3T3 and SMC (60.6 +/- 2.6 and 101.7 +/- 28.3 pmol /mg, respectively, versus less than 4 in control cells). GTPCH reconst ituted near-maximal iNOS activity in 3T3-iNOS cells despite a gene tra nsfer efficiency of less than 1%. GTPCH and iNOS enzymes did not have to coexist in the same cell for the synthesized BH4 to support iNOS ac tivity. Conclusions, GTPCH gene transfer reconstitutes iNOS activity i n BH4-deficient cells despite poor transfer efficiency. GTPCH can deli ver a cofactor to targeted cells even if it is synthesized in neighbor ing cells, and may be a means to concurrently deliver BH4 with iNOS in vivo.