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

Gene Therapy volume 17, pages 827–838 (2010)Cite this article

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Abstract

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.

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Acknowledgements

We thank Dr Jarrett Rosenberg for his kind assistance with statistical analysis. This work was supported in part by NHLBI 5R01HL078632 (SSG), NCI ICMIC P50 CA114747 (SSG), NCI SAIRP, American Heart Association Pre-doctoral Fellowship (IYC), Stanford Bio-X Graduate Student Fellowship (IYC), K99-R00 HL88048 (MRP), HL095571 (JCW), and HL093172 (JCW).

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Authors and Affiliations

  1. Departments of Radiology and Bioengineering, Molecular Imaging Program at Stanford (MIPS), Bio-X Program, Stanford University, Stanford, CA, USA

    I Y Chen, O Gheysens, S Ray, R Paulmurugan, A M Loening, J K Willmann, C H Nielsen & S S Gambhir

  2. Departments of Pediatrics, Radiology, and Microbiology and Immunology, Molecular Imaging Program at Stanford (MIPS), Bio-X Program, Stanford University, Stanford, CA, USA

    Q Wang & C H Contag

  3. Department of Radiology, Molecular Imaging Program at Stanford (MIPS), Stanford University, Stanford, CA, USA

    P Padmanabhan & S Biswal

  4. Division of Cardiovascular Diseases, Department of Internal Medicine, Mayo Clinic, Rochester, MN, USA

    M Rodriguez-Porcel

  5. Department of Cardiothoracic Surgery, Stanford University, Stanford, CA, USA

    A Y Sheikh, G Hoyt & R C Robbins

  6. Division of Cardiovascular Medicine, Department of Medicine, Molecular Imaging Program at Stanford (MIPS), Stanford University, Stanford, CA, USA

    J C Wu

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  1. I Y Chen
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  16. J C Wu
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Correspondence to S S Gambhir.

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Chen, I., Gheysens, O., Ray, S. et al. Indirect imaging of cardiac-specific transgene expression using a bidirectional two-step transcriptional amplification strategy. Gene Ther 17, 827–838 (2010). https://doi.org/10.1038/gt.2010.30

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