Preclinical Evaluation of Inducible Nitric Oxide Synthase Lipoplex Gene Therapy for Inhibition of Stent-Induced Vascular Neointimal Lesion Formation

Abstract

Several reports have established the concept of nitric oxide synthase (NOS) gene transfer for inhibiting smooth muscle cell (SMC) proliferation after vascular injury. To minimize potential risks associated with viral gene transfer, we developed a liposome-based gene transfer approach employing inducible NOS (iNOS) overexpression for inhibition of stent-induced neointimal lesion formation. Therapeutic lipoplexes were transferred to femoral or coronary arteries of Goettingen minipigs, using the Infiltrator local drug delivery device. Efficiency of local iNOS lipoplex transfer was analyzed by iNOS-specific immunohistochemistry. NO-mediated inhibition of stent-induced neointimal lesion formation was analyzed by intravascular ultrasound (IVUS) and computerized morphometry. Gene transfer efficiency increased dose dependently to a maximum of 44.3 ± 4.2% iNOS-positive vessel area (dose, 2 μg of iNOS lipoplex). Proliferating cell nuclear antigen (PCNA) expression of medial SMCs (immunohistochemistry) was inhibited significantly by transfer of 2 μg of iNOS lipoplexes (111 ± 27 cells [iNOS] versus 481 ± 67 cells [control; PCNA-positive medial cells]). IVUS analysis demonstrated that local transfer of iNOS lipoplexes resulted in a significant reduction of femoral in-stent plaque area (control, 40.85 ± 6.37 mm²; iNOS, 24.69 ± 1.8 mm²; p = 0.03). Coronary in-stent lesion formation was reduced by about 45% as determined by histologic morphometry (control, 4.0 ± 0.29; iNOS, 2.2 ± 0.30; p < 0.01). In conclusion, this study demonstrates that local intramural delivery of iNOS lipoplexes can exert therapeutic effects in inhibiting stent-induced neointimal lesion formation. Together with the nonviral character of this gene therapy approach, these findings may have important impact on the transition of NOS-based gene therapy to clinical practice.