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.

Optical Imaging for Stem Cell Differentiation to Neuronal Lineage

“…Optical reporter systems driven by the cardiac troponin T promoter (pcTnT) or the granzyme B promoter (pGB) were also based on a similar TSTA strategy. These systems exhibited heart-specific expression or highly amplified reporter activity during T cell activation [27,28]. Figure 2 illustrates significantly stronger bioluminescence signals in pcTnT-eBid-TSTA-mediated cardiac reporter gene expression 28 days after intramyocardial injection compared to the pCMV-fluc-and pcTnT-fluc-mediated gene reporters.…”

“…The TSTA system generated greater luciferase signal than the constitutive promoter-driven luciferase system (pCMV-fluc). Reprinted with permission from [27] Two days after induction of neuronal differentiation by treatment with retinoic acid, a significantly reduced Gaussia luciferase signal was observed (yellow arrow). In vivo normalization was conducted using the cotransfection method with the firefly luciferase gene under the control of cytomegalovirus (CMV) promoter (CMV/Fluc) to track the survival of the implanted P19 cells using CMV/Fluc vector.…”

Noninvasive Imaging of Hypoxia-Inducible Factor-1α Gene Therapy for Myocardial Ischemia

“…pcDNA3.1( + ) (Invitrogen, Carlsbad, CA), pCEP4/HIF1alpha (ATCC, Johns Hopkins Special Collection, Manassas, VA), pIRES-EGFP (Clontech, MountainView, CA), pCMVhRL (Promega, Madison, WI), pBCVP2-G5L (Zhang et al, 2002), pcTnT-bid-TSTA (Chen et al, 2010), and p5X-HRE-fluc (Huang et al, 2008) were used as starting vectors for the construction of other vectors. An intermediate vector containing a cytomegalovirus (CMV) promoter driving the expression of two tandem repeats of herpes simplex VP16 transcriptional activator (VP2) was constructed by PCR amplifying VP2 from pBCVP2-G5L, followed by inserting it into the BamHI/XhoI sites of pcDNA3.1(+) downstream of CMV.…”

“…Cell culture and plasmid transfection HL-1 mouse cardiomyocytes were cultured in Claycomb Medium ( JRH Biosciences, Lenexa, KS) as previously described (Chen et al, 2010). Using lipofectamine 2000 (1 ll/ 1 lg total DNA; Invitrogen), cells were cotransfected with increasing doses of pcTnT-HIF-1a-VP2-TSTA-fluc (0-1 lg), a fixed dose of p5X-HRE-hRluc (1 lg), as well as pCMV-b-gal (0.4 lg) and pcDNA3.1( + ) (0-1 lg) to correct for transfection efficiency and total DNA, respectively.…”

“…Recently, we have developed a novel bidirectional two-step transcriptional amplification (TSTA) vector strategy, which can achieve high levels of balanced, correlated transgene expression, even when a weak tissue-specific promoter is used for transcriptional regulation (Ray et al, 2008). Such a vector strategy maximizes both therapeutic efficacy and the sensitivity for indirect gene imaging, and has the potential to mediate transcriptionally targeted gene therapy as previously described (Chen et al, 2010).…”