Indirect imaging of cardiac-specific transgene expression using a bidirectional two-step transcriptional amplification strategy

• Published in: Gene therapy Vol. 17; no. 7; pp. 827 - 838
• Main Authors: Chen, I Y, Gheysens, O, Ray, S, Wang, Q, Padmanabhan, P, Paulmurugan, R, Loening, A M, Rodriguez-Porcel, M, Willmann, J K, Sheikh, A Y, Nielsen, C H, Hoyt, G, Contag, C H, Robbins, R C, Biswal, S, Wu, J C, Gambhir, S S
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.07.2010; Nature Publishing Group
• Subjects: 631/1647/2300/1514; 631/1647/245/2222; 631/443/592/75; 631/61/201; Anesthesia. Intensive care medicine. Transfusions. Cell therapy and gene therapy; Animals; Applied cell therapy and gene therapy; Biological and medical sciences; Bioluminescence; Biomedical and Life Sciences; Biomedicine; Biotechnology; Calcium-binding protein; Cardiovascular disease; Care and treatment; Cell Biology; Cell Line; Cell lines; Female; Fundamental and applied biological sciences. Psychology; Gene Amplification; Gene Expression; Gene Targeting; Gene Therapy; Gene Transfer Techniques; Genes, Reporter; Genetic aspects; Genetic Vectors; Health aspects; Health. Pharmaceutical industry; Heart; Heart diseases; Hepatocytes; Human Genetics; Industrial applications and implications. Economical aspects; Kinases; Liver; Liver - metabolism; Luciferases, Firefly - genetics; Luciferases, Renilla - genetics; Medical sciences; Mice; Mice, Inbred BALB C; Molecular and cellular biology; Molecular genetics; Myocardium; Myocardium - metabolism; Nanotechnology; original-article; Plasmids; Promoter Regions, Genetic; Renilla; Reporter gene; Rodents; Scientific imaging; Transcription; Transcription, Genetic; Transcription. Transcription factor. Splicing. Rna processing; Transfusions. Complications. Transfusion reactions. Cell and gene therapy; Transgenes; Troponin; Troponin - genetics; Troponin T; United States; two-step transcriptional amplification; bioluminescence imaging; plasmid vector; cardiac specificity; bidirectional construct; reporter gene expression; Heart; Bioluminescence; Transcription; Gene expression; Plasmid; Gene amplification; Specificity; Reporter gene; Imaging; Strategy; Circulatory system; Gene therapy; Vector
• ISSN: 0969-7128; 1476-5462; 1476-5462
• DOI: 10.1038/gt.2010.30

Transcriptional targeting for cardiac gene therapy is limited by the relatively weak activity of most cardiac-specific promoters. We have developed a bidirectional plasmid vector, which uses a two-step transcriptional amplification (TSTA) strategy to enhance the expression of two optical reporter genes, firefly luciferase (fluc) and Renilla luciferase (hrluc), driven by the cardiac troponin T (cTnT) promoter. The vector was characterized in vitro and in living mice using luminometry and bioluminescence imaging to assess its ability to mediate strong, correlated reporter gene expression in a cardiac cell line and the myocardium, while minimizing expression in non-cardiac cell lines and the liver. In vitro , the TSTA system significantly enhanced cTnT-mediated reporter gene expression with moderate preservation of cardiac specificity. After intramyocardial and hydrodynamic tail vein delivery of an hrluc-enhanced variant of the vector, long-term fluc expression was observed in the heart, but not in the liver. In both the cardiac cell line and the myocardium, fluc expression correlated well with hrluc expression. These results show the vector's ability to effectively amplify and couple transgene expression in a cardiac-specific manner. Further replacement of either reporter gene with a therapeutic gene should allow non-invasive imaging of targeted gene therapy in living subjects.