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 -