A titratable two-step transcriptional amplification strategy for targeted gene therapy based on ligand-induced intramolecular folding of a mutant human estrogen receptor

TY - JOUR

T1 - A titratable two-step transcriptional amplification strategy for targeted gene therapy based on ligand-induced intramolecular folding of a mutant human estrogen receptor

AU - Chen, Ian Y

AU - Paulmurugan, Ramasamy

AU - Nielsen, Carsten Haagen

AU - Wang, David S

AU - Chow, Vinca

AU - Robbins, Robert C

AU - Gambhir, Sanjiv S

PY - 2014/4

Y1 - 2014/4

N2 - PURPOSE: The efficacy and safety of cardiac gene therapy depend critically on the level and the distribution of therapeutic gene expression following vector administration. We aimed to develop a titratable two-step transcriptional amplification (tTSTA) vector strategy, which allows modulation of transcriptionally targeted gene expression in the myocardium.PROCEDURES: We constructed a tTSTA plasmid vector (pcTnT-tTSTA-fluc), which uses the cardiac troponin T (cTnT) promoter to drive the expression of the recombinant transcriptional activator GAL4-mER(LBD)-VP2, whose ability to transactivate the downstream firefly luciferase reporter gene (fluc) depends on the binding of its mutant estrogen receptor (ER(G521T)) ligand binding domain (LBD) to an ER ligand such as raloxifene. Mice underwent either intramyocardial or hydrodynamic tail vein (HTV) injection of pcTnT-tTSTA-fluc, followed by differential modulation of fluc expression with varying doses of intraperitoneal raloxifene prior to bioluminescence imaging to assess the kinetics of myocardial or hepatic fluc expression.RESULTS: Intramyocardial injection of pcTnT-tTSTA-fluc followed by titration with intraperitoneal raloxifene led to up to tenfold induction of myocardial fluc expression. HTV injection of pcTnT-tTSTA-fluc led to negligible long-term hepatic fluc expression, regardless of the raloxifene dose given.CONCLUSIONS: The tTSTA vector strategy can effectively modulate transgene expression in a tissue-specific manner. Further refinement of this strategy should help maximize the benefit-to-risk ratio of cardiac gene therapy.

AB - PURPOSE: The efficacy and safety of cardiac gene therapy depend critically on the level and the distribution of therapeutic gene expression following vector administration. We aimed to develop a titratable two-step transcriptional amplification (tTSTA) vector strategy, which allows modulation of transcriptionally targeted gene expression in the myocardium.PROCEDURES: We constructed a tTSTA plasmid vector (pcTnT-tTSTA-fluc), which uses the cardiac troponin T (cTnT) promoter to drive the expression of the recombinant transcriptional activator GAL4-mER(LBD)-VP2, whose ability to transactivate the downstream firefly luciferase reporter gene (fluc) depends on the binding of its mutant estrogen receptor (ER(G521T)) ligand binding domain (LBD) to an ER ligand such as raloxifene. Mice underwent either intramyocardial or hydrodynamic tail vein (HTV) injection of pcTnT-tTSTA-fluc, followed by differential modulation of fluc expression with varying doses of intraperitoneal raloxifene prior to bioluminescence imaging to assess the kinetics of myocardial or hepatic fluc expression.RESULTS: Intramyocardial injection of pcTnT-tTSTA-fluc followed by titration with intraperitoneal raloxifene led to up to tenfold induction of myocardial fluc expression. HTV injection of pcTnT-tTSTA-fluc led to negligible long-term hepatic fluc expression, regardless of the raloxifene dose given.CONCLUSIONS: The tTSTA vector strategy can effectively modulate transgene expression in a tissue-specific manner. Further refinement of this strategy should help maximize the benefit-to-risk ratio of cardiac gene therapy.

KW - Animals

KW - Blotting, Western

KW - Diagnostic Imaging

KW - Gene Expression Regulation

KW - Genes, Reporter

KW - Genetic Therapy

KW - Genetic Vectors

KW - Humans

KW - Ligands

KW - Liver

KW - Luminescent Measurements

KW - Mice

KW - Mutant Proteins

KW - Myocardium

KW - NIH 3T3 Cells

KW - Organ Specificity

KW - Plasmids

KW - Protein Folding

KW - Receptors, Estrogen

KW - Reproducibility of Results

KW - Transcription, Genetic

U2 - 10.1007/s11307-013-0673-4

DO - 10.1007/s11307-013-0673-4

M3 - Journal article

C2 - 23955099

SN - 1536-1632

VL - 16

SP - 224

EP - 234

JO - Molecular Imaging and Biology

JF - Molecular Imaging and Biology

IS - 2

ER -