Volume 3, Issue 5, Pages 779-786 (May 2001) Coexpression of Guanylate Kinase with Thymidine Kinase Enhances Prodrug Cell Killing in Vitro and Suppresses Vascular Smooth Muscle Cell Proliferation in Vivo  Levent M. Akyürek, Shriram Nallamshetty, Kazunori Aoki, Hong San, Zhi-Yong Yang, Gary J. Nabel, Elizabeth G. Nabel  Molecular Therapy  Volume 3, Issue 5, Pages 779-786 (May 2001) DOI: 10.1006/mthe.2001.0315 Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 1 Coexpression of GK with TK in NIH 3T3 cells. (A) Schematic representation of the constructs expressing GK with TK. (B) Growth of NIH 3T3 cells transfected with the respective plasmid illustrated in A in the presence of 0.5 μM GCV. A plasmid expression vector lacking any TK gene insert (pAdEF1α-Ø) served as control. Proliferation was measured using a colorimetric assay. Data represent the average of three measurements at OD570–630.*P < 0.01, **P < 0.001. (C) Comparable expression of recombinant TK protein in different plasmid expression vectors. Western blot analysis performed by standard methods using polyclonal rabbit antiserum to HSV-1 TK. (D) CMV and EF1α promoters were directly compared using TKciteGK constructs. Molecular Therapy 2001 3, 779-786DOI: (10.1006/mthe.2001.0315) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 2 (A) Structure of recombinant adenoviral TKciteGK vectors and (B) comparison of the killing activity in primary vascular smc's driven by SM22α, EF1α, or CMV promoters. The E1 and E3 regions of the Ad5Sub360 cosmid adenoviral genome were deleted (ΔE3), rendering the virus replication defective. The AdSM22α-TKciteGK and AdEF1α-TKciteGK vectors encode a TKciteGK gene and bovine growth hormone polyadenylation signal under the transcriptional control of the 441-bp murine SM22α or EF1α promoter, respectively, and the 471-bp human 4F2 transcriptional enhancer. AdCMV-TKciteGK encodes recombinant TKciteGK gene and bovine growth hormone polyadenylation signal under the transcriptional control of CMV immediate-early promoter (CMV E/P) containing the first exon and intron (CMV IE 5'UT). AdΔAE1 adenoviral vector was included as control. Primary vascular smcs were infected with 1000 m.o.i. of either adenoviral vector, and cell proliferation was measured by using a colorimetric assay in the absence or presence of GCV (0.1 μM). Results are given as the mean percentage of three experiments expressing cell viability ± SEM. *P < 0.0001. Molecular Therapy 2001 3, 779-786DOI: (10.1006/mthe.2001.0315) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 3 Comparison of cell killing efficiency between different ACV (A) and GCV (B) doses after infection with TKciteGK adenoviral vectors driven by SM22α, EF1α, or CMV promoters in primary vsmcs. Cells were infected at 1000 m.o.i. Following infection, half of the infected vsmcs were mixed with nontransduced cells the next day and then seeded in 96-well plates and cultured in the presence of ACV or GCV. An MTT calorimetric analysis was performed at day 5. In order to achieve a killing efficiency equivalent to that of GCV, a 5- to 50-fold greater concentration of ACV was required. Molecular Therapy 2001 3, 779-786DOI: (10.1006/mthe.2001.0315) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 4 Effect of GCV on intimal and medial areas in femoral arteries after balloon injury and infection with adenoviral TKciteGK vector. Representative cross sections from iliofemoral arteries of pigs injured and then infected with adenoviral TKciteGK vector and treated with GCV (B) compared with control adenovirus, AdΔE1 (A). Arrowhead indicates internal elastic lamina bordering intimal and medial layers. H&E stain; X200 original magnification. Measurements of I/M area ratios are from arteries infected after a 1-min injury and 3 weeks after infection with AdCMV-TKciteGK vector and treatment with GCV. Data are given as means ± SEM. A statistically significant reduction in I/M area ratios was observed in the AdCMV-TKciteGK/+GCV group compared with AdΔE1/+GCV (*P = 0.0002) (C). Molecular Therapy 2001 3, 779-786DOI: (10.1006/mthe.2001.0315) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 5 Effect of GCV on intimal proliferation in femoral arteries after stent injury and infection with adenoviral TKciteGK vector. Photomicrographs show porcine femoral artery cross sections (original magnification ×20, elastin stain). Following stent deployment, injured arteries were infected with either control adenoviral vector, AdΔE1 (A) or AdCMV-TKciteGK (B) introduced by a double-balloon catheter. Arrowhead indicates internal elastic lamina. Three weeks after stent injury and infection, I/M area ratios were calculated (C). Compared to the control arteries infected with AdΔE1, TKciteGK gene delivery by double-balloon catheter significantly inhibited intimal thickening (*P = 0.006). Data are given as means ± SEM. Molecular Therapy 2001 3, 779-786DOI: (10.1006/mthe.2001.0315) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 6 Analysis of tissues for organ toxicity in pigs infected locally with the AdCMV-hpAP vector. Four days after intraarterial infection of femoral arteries (A and B), organ specimens including liver (C), lung (D), heart (E), and kidney (F) were removed, stained with either H&E or alkaline phosphatase (AP), and analyzed for histological changes. Note an intense AP transgene expression in intimal and upper medial layers (B). No AP transgene expression was observed in major organs (C–F). Local adenoviral vector administration in pigs was not associated with an inflammatory cell infiltration throughout the vessel layers or systemic toxicities by light microscopy. White arrows indicate the internal elastica lamina and black arrows point to arterial structures in organs. Methyl green counterstain (B–E). Original magnification X200. Molecular Therapy 2001 3, 779-786DOI: (10.1006/mthe.2001.0315) Copyright © 2001 American Society for Gene Therapy Terms and Conditions