Volume 3, Issue 1, Pages 8-13 (January 2001) p27–p16 Chimera: A Superior Antiproliferative for the Prevention of Neointimal Hyperplasia  James G. McArthur, Husheng Qian, Dianne Citron, Gautam G. Banik, Lou Lamphere, Jeno Gyuris, Lisa Tsui, Samuel E. George  Molecular Therapy  Volume 3, Issue 1, Pages 8-13 (January 2001) DOI: 10.1006/mthe.2000.0239 Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 1 Structures of the p16, p27, and W9 CDKi. Schematic representations of the CDKi molecules: full-length p16 contains 156 amino acids, full-length p27 contains 198 amino acids. Chimeric p16 and p27 molecule designated W9 was generated by fusing truncated versions of p27 (amino acids 25–93) to near full-length p16 (amino acids 2–156). On the 5’ end of each of the constructs was added an influenza hemagglutinin tag. Molecular Therapy 2001 3, 8-13DOI: (10.1006/mthe.2000.0239) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 2 p16, p27, and W9 protein expression in the balloon-injured carotid artery following AV-CDKi gene transfer. (A) CDKi transgene expression. Gene-modified arteries 3 days following adenoviral infection were sectioned and stained with an anti-HA antibody that recognizes the N-terminal HA-tag on the p16, p27, and W9 proteins. (B) Quantification of CDKi expression was done by image analysis. The levels of transgene expression were 42 ± 11, 33 ± 8, and 41 ± 10 μm2 area of positive expression for W9, p16, and p27, respectively. Molecular Therapy 2001 3, 8-13DOI: (10.1006/mthe.2000.0239) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 3 AV-CDKi inhibits neointimal hyperplasia in the single balloon-injured carotid artery. (A) Three days after balloon injury, vessels were infected with 2 × 1011 viral particles of ΔE1 AV encoding either W9 or lacZ. The vessels were harvested 2 weeks later and analyzed for the extent of neointimal hyperplasia. Sections are shown at low (top) and higher magnification (bottom). Arrows indicate the thickness of the neointima following balloon injury in the sham and gene-modified vessels. (B) Comparison of AV-W9 with AV-p16 and AV-p27. The intima/media ratios for sham-, AV-β-gal-, AV-W9-, Avp27-, and AV-p16-treated vessels are shown. Five measurements of the intima and media of each section were performed on three to five sections from each vessel. Each treatment group included four to five vessels. Molecular Therapy 2001 3, 8-13DOI: (10.1006/mthe.2000.0239) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 4 PCNA gene expression. (Top) The level of PCNA expression was examined in rabbit artery 3 days after gene transfer. Immunohistochemical analyses using an anti-PCNA antibody was counterstained with hematoxylin to visualize nuclei. (Bottom) Quantification of PCNA gene expression. The level of PCNA expression was examined in rabbit artery at both 3 days and 2 weeks after gene transfer. PCNA expression was lower in all AV-CDKi-treated vessels. AV-W9 showed a significant reduction in PCNA expression compared to AV-p16 or AV-p27 alone. The levels of PCNA expression were down in all vessels by 2 weeks after gene transfer, indicating that SMC proliferation that occurs in response to injury was nearly finished. Quantification of PCNA expression was done by cell count. Molecular Therapy 2001 3, 8-13DOI: (10.1006/mthe.2000.0239) Copyright © 2001 American Society for Gene Therapy Terms and Conditions