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.

Figure 1

Diagrams of experimental and control plasmid vectors. Abbreviations: cTnT, rat cardiac troponin T promoter; fluc, firefly luciferase gene; PA, SV40 poly(A) tail; hrluc, synthetic Renilla luciferase gene; CMVenh, cytomegalovirus enhancer; TSTA, two-step transcriptional amplification; Gal4–VP2, fusion gene combining yeast Gal4 and two tandem repeats of herpes simplex virus VP16; 8xGal4bs, 8 tandem repeats of Gal4-binding site; E4, adenovirus E4 minimal promoter; hrluc/M185V, gene expressing the mutant RLuc (RLuc/M185V) with enhanced light output; CMV, cytomegalovirus promoter.

Figure 2

In vitro characterizations of the TSTA-based strategies in terms of transcriptional amplification, cardiac specificity, and correlated reporter gene expression. Cardiac (HL-1) and non-cardiac cells were separately transfected with control (pNull-fluc, pNull-hrluc), one-step (pcTnT-fluc, pcTnT-hrluc), CMVenh-based (pCMVenh-cTnT-fluc), unidirectional TSTA (pcTnT-TSTA-fluc), bidirectional TSTA (pcTnT-Bid-TSTA), and reference (pCMV-fluc, pCMV-hrluc) vectors, with each vector co-delivered with a normalization vector (pCMV-β-gal). The cell lysates were assayed 24 h later for FLuc, RLuc, and β-GAL activities. The FLuc and RLuc activities were normalized to total protein, corrected for transfection efficiency (β-GAL activity), and, respectively, expressed as a percentage of FLuc activity of pCMV-fluc and RLuc activity of pCMV-hrluc. (a) The FLuc activity is shown for all fluc-containing vectors except for pcTnT-Bid-TSTA, whose FLuc (black solid bar) and RLuc (gray solid bar) activities are shown in (b) and compared against those of fluc- and hrluc-containing control, one-step, and reference vectors. *P< 0.005 compared with pNull-fluc; ^P< 0.001 compared with pCMVenh-cTnT-fluc; **P< 0.001 compared with pcTnT-fluc; ^^P< 0.002 compared with pcTnT-hrluc. (c) The cardiac-specificity index (CSI), derived from FLuc activity, is shown for each vector except for pNull-fluc, whose CSI could not be fairly assessed mathematically because of its near-zero expression in both cardiac and non-cardiac cell lines. *P< 0.04 compared with pCMV-fluc; ^P< 0.03 compared with pcTnT-fluc; ^†P< 0.01 compared with pCMVenh-cTnT-fluc. (d) HL-1 cells transfected with increasing doses of pcTnT-Bid-TSTA were assayed 24 h later for both FLuc and RLuc activities, which were normalized by total protein and plotted here against each other for each plasmid dose used. For all figures, the error bars represent s.e.m. for triplicate determinations.

Figure 3

Bioluminescence imaging of pcTnT-eBid-TSTA-mediated cardiac reporter gene expression in living mice. Serial BLI was performed on four mouse cohorts, which had separately undergone intramyocardial co-injections of (1) pCMV-fluc+pCMV-β-gal, (2) pcTnT-fluc+pCMV-β-gal, (3) pcTnT-eBid-TSTA+pCMV-β-gal, or (4) pNull-fluc+pCMV-β-gal. (a) The BLI images of three representative mice that received pCMV-fluc (left), pcTnT-fluc (middle), and pcTnT-eBid-TSTA (right), respectively, are shown for only days 2, 16, and 28. Each image is displayed on a rainbow scale in units of photons s⁻¹ cm⁻² sr⁻¹) and overlaid onto a grayscale reference image of the corresponding mice. (b) The average heart signal on the post-operative days indicated was corrected for transfection efficiency [β-GAL activity in units of absorbance (Abs)] and plotted for each mouse cohort: pCMV-fluc (solid black diamond), pcTnT-fluc (light gray square), pcTnT-eBid-TSTA (dark gray triangle), and pNull-fluc (empty circle). Note that the signal is displayed on a log scale. The error bars represent s.e.m. for five mice. When considering the entire study period, *P< 0.012 between pcTnT-eBid-TSTA and pcTnT-fluc; ^P< 0.007 between pcTnT-eBid-TSTA and pNull-fluc.

Figure 4

Bioluminescence imaging of pcTnT-eBid-TSTA-mediated hepatic reporter gene expression in living mice. Serial BLI was performed on four mouse cohorts, which had separately undergone hydrodynamic tail vein co-injections of (1) pCMV-fluc+pCMV-β-gal, (2) pcTnT-fluc+pCMV-β-gal, (3) pcTnT-eBid-TSTA+pCMV-β-gal, or (4) pNull-fluc+pCMV-β-gal. (a) The BLI images of three representative mice, one from each of the groups that received pCMV-fluc (left), pcTnT-fluc (middle), and pcTnT-eBid-TSTA (right), are shown for days 2, 16, and 28. Each image is displayed on a rainbow scale in units of photons s⁻¹ cm⁻² sr⁻¹ and overlaid onto a grayscale reference image of the corresponding mouse. (b) The average hepatic signal on the post-operative days shown was normalized for transfection efficiency (β-GAL activity) and plotted for the different mouse groups: pCMV-fluc (solid black diamond), pcTnT-fluc (light gray square), pcTnT-eBid-TSTA (dark gray triangle), and pNull-fluc (empty circle). Note that the signal is displayed on a log scale. The error bars represent s.e.m. for five mice. *P< 0.0001 between pcTnT-eBid-TSTA (or pCMV-fluc) and pcTnT-fluc (or pNull-fluc) before day 12. ^P< 0.0001 between pCMV-fluc and all other vectors after day 12.

Figure 5

Correlated cardiac reporter gene expression from pcTnT-eBid-TSTA after intramyocardial vector delivery. (a) Two representative mice were intramyocardially injected with either pcTnT-eBid-TSTA (experimental) or pNull-fluc (control) and scanned for both hrluc/M185V and fluc expression 2 days later using BLI. Both RLuc/M185V (left two images) and FLuc (right two images) images (rainbow scale and grayscale for luminescence and reference images, respectively) show a robust heart signal (red arrow) for the experimental pcTnT-eBid-TSTA mouse and minimal to no signal for the pNull-fluc control mouse. Note that for the dose of coelenterazine used in this study (50 µg), a background hepatic signal (orange arrows) can always be seen on the RLuc/M185V images of both control and experimental mice. (b) Serial BLI of the entire mouse cohort injected with cTnT-eBid-TSTA revealed the kinetics of hrluc/M185V and fluc expression over 28 days. The average RLuc/M185V- and FLuc-mediated signals are displayed on a log scale. The error bars represent s.e.m. for five mice. (c) The FLuc signal at each time point is plotted against the corresponding RLuc/M185V signal. All signal measures are expressed in units of photons s⁻¹ cm⁻² sr⁻¹.