Indirect imaging of cardiac-specific transgene expression using a bidirectional two-step transcriptional amplification strategy
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Indirect imaging of cardiac-specific transgene expression using a bidirectional two-step transcriptional amplification strategy. / 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, Carsten Haagen; Hoyt, G; Contag, C H; Robbins, R C; Biswal, S; Wu, J C; Gambhir, S S.
In: Gene Therapy (Basingstoke), Vol. 17, No. 7, 2010, p. 827-38.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Indirect imaging of cardiac-specific transgene expression using a bidirectional two-step transcriptional amplification strategy
AU - Chen, I Y
AU - Gheysens, O
AU - Ray, S
AU - Wang, Q
AU - Padmanabhan, P
AU - Paulmurugan, R
AU - Loening, A M
AU - Rodriguez-Porcel, M
AU - Willmann, J K
AU - Sheikh, A Y
AU - Nielsen, Carsten Haagen
AU - Hoyt, G
AU - Contag, C H
AU - Robbins, R C
AU - Biswal, S
AU - Wu, J C
AU - Gambhir, S S
PY - 2010
Y1 - 2010
N2 - 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.
AB - 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.
KW - Animals
KW - Cell Line
KW - Female
KW - Gene Amplification
KW - Gene Targeting
KW - Gene Transfer Techniques
KW - Genes, Reporter
KW - Genetic Vectors
KW - Liver
KW - Luciferases, Firefly
KW - Luciferases, Renilla
KW - Mice
KW - Mice, Inbred BALB C
KW - Myocardium
KW - Plasmids
KW - Promoter Regions, Genetic
KW - Transcription, Genetic
KW - Transgenes
KW - Troponin
U2 - 10.1038/gt.2010.30
DO - 10.1038/gt.2010.30
M3 - Journal article
C2 - 20237511
VL - 17
SP - 827
EP - 838
JO - Gene Therapy
JF - Gene Therapy
SN - 0969-7128
IS - 7
ER -
ID: 32982715