A calcium-sensitive promoter construct for gene therapy

Abstract

Targeting diseased cells is a challenging issue in both pharmacological and biological therapeutics. Gene therapy is emerging as a novel approach for treating rare diseases and for illnesses for which there is no other alternative. An important limitation of gene therapy has been the off-target effects and therefore efforts have been focused on increasing the specificity of gene transfer to the targeted organ. Here, we describe a promoter containing six nuclear factor of activated T cells (NFAT) consensus sequences, which is as efficient as the cytomegalovirus (CMV) promoter to drive expression in vascular smooth muscle cells both in vitro and in vivo. In contrast to the CMV promoter it is activated in a Ca2+ -dependent manner after endoplasmic reticulum depletion and allows the transgene expression only in proliferative/diseased cells. Overexpression of sarco/ endoplasmic reticulum (SR/ER) Ca2 + ATPase 2a under the control of this NFAT promoter inhibits restenosis after angioplasty in rats. In conclusion, this promoter may be useful for gene therapy in vascular proliferative diseases and other diseases involving upregulation of the NFAT pathway.

Expression of sarco (endo) plasmic reticulum calcium ATPase (SERCA) system in normal mouse cardiovascular tissues, heart failure and atherosclerosis

“…Several observations support the evidence that the calcium response shape appears to be dependent on SERCA2a expression: 1) blocking SERCA activity also strongly inhibits the Ca 2+ oscillations, demonstrating that they are caused by release of Ca 2+ from the SR [36–38]; 2) trans-differentiation of VSMC towards synthetic phenotype is associated with a loss of both Ca 2+ oscillations and SERCA2a expression (this paper and [39, 40]; 3) SERCA2a gene transfer to synthetic cultured VSMC modifies the agonist-induced calcium transient from steady-state to oscillatory mode [41]. Importantly, restoring SERCA2a expression by gene transfer in synthetic cultured VSMC also inhibits Ca 2+ dependent activation of transcription factor NFAT required for proliferation and migration of VSMC [30, 41, 42]. …”

The Current and Future Landscape of SERCA Gene Therapy for Heart Failure: A Clinical Perspective

“…97 In vitro SERCA2a gene transfer inhibits proliferation and migration of these cells secondary to inhibition of NFAT. [98][99][100] It is plausible that coronary artery disease may be another future target for SERCA gene therapy. Furthermore, AV fistulas created for renal dialysis commonly fail and vascular smooth muscle cell proliferation can contribute to this.…”

Dominant negative Ras attenuates pathological ventricular remodeling in pressure overload cardiac hypertrophy

“…The active mutant Ras Val12 (a substitution of glycine for valine, G12V, in the 12 th amino-acid) was used as a positive control for DN-Ras [5]. Ad-βGal, encoding β-galactosidase under CMV promoter followed by target gene-IRES-reporter gene (a green fluorescent protein (GFP)) construct translated as two independent proteins [19] was used as an additional control; Ad.NFAT-Luc virus, carrying the luciferase reporter gene controlled by NFAT- responsive promoter [20] was used to measure NFAT activation. Ad-VIVIT, encoding NFAT competing peptide VIVIT and GFP under CMV promoter[21, 22] was used as an additional control for NFAT-reporter experiments.…”