Dataset Information

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

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.

SUBMITTER: Chen IY

PROVIDER: S-EPMC2900530 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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

Chen I Y IY Gheysens O O Ray S S Wang Q Q Padmanabhan P P Paulmurugan R R Loening A M AM Rodriguez-Porcel M M Willmann J K JK Sheikh A Y AY Nielsen C H CH Hoyt G G Contag C H CH Robbins R C RC Biswal S S Wu J C JC Gambhir S S SS

Gene therapy 20100318 7

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 biolumin ...[more]

PMID: 20237511

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Project description:BackgroundNumerous self-gated cardiac imaging techniques have been reported in the literature. Most can track either cardiac or respiratory motion, and many incur some overhead to imaging data acquisition. We previously described a Cartesian cine imaging technique, pseudo-projection motion tracking with golden-step phase encoding, capable of tracking both cardiac and respiratory motion at no cost to imaging data acquisition. In this work, we describe improvements to the technique by dramatically reducing its vulnerability to eddy current and flow artifacts and demonstrating its effectiveness in expanded cardiovascular applications.MethodsAs with our previous golden-step technique, the Cartesian phase encodes over time were arranged based on the integer golden step, and readouts near ky?=?0 (pseudo-projections) were used to derive motion. In this work, however, the readouts were divided into equal and consecutive temporal segments, within which the readouts were sorted according to ky. The sorting reduces the phase encode jump between consecutive readouts while maintaining the pseudo-randomness of ky to sample both cardiac and respiratory motion without comprising the ability to retrospectively set the temporal resolution of the original technique. On human volunteers, free-breathing, electrocardiographic (ECG)-free cine scans were acquired for all slices of the short axis stack and the 4-chamber view of the long axis. Retrospectively, cardiac motion and respiratory motion were automatically extracted from the pseudo-projections to guide cine reconstruction. The resultant image quality in terms of sharpness and cardiac functional metrics was compared against breath-hold ECG-gated reference cines.ResultsWith sorting, motion tracking of both cardiac and respiratory motion was effective for all slices orientations imaged, and artifact occurrence due to eddy current and flow was efficiently eliminated. The image sharpness derived from the self-gated cines was found to be comparable to the reference cines (mean difference less than 0.05?mm-?1 for short-axis images and 0.075?mm-?1 for long-axis images), and the functional metrics (mean difference?<?4?ml) were found not to be statistically different from those from the reference.ConclusionsThis technique dramatically reduced the eddy current and flow artifacts while preserving the ability of cost-free motion tracking and the flexibility of choosing arbitrary navigator zone width, number of cardiac phases, and duration of scanning. With the restriction of the artifacts removed, the Cartesian golden-step cine imaging can now be applied to cardiac imaging slices of more diverse orientation and anatomy at greater reliability.

Dataset Information

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

Publications

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

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