title: A titratable two-step transcriptional amplification strategy for targeted gene therapy based on ligand-induced intramolecular folding of a mutant human estrogen receptor.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
authors: Department of Medicine, Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford University, Stanford, CA, USA.
authors: Ian Y
authors: IY
authors: Chen
authors: Ramasamy
authors: R
authors: Paulmurugan
authors: Carsten H
authors: CH
authors: Nielsen
authors: David S
authors: DS
authors: Wang
authors: Vinca
authors: V
authors: Chow
authors: Robert C
authors: RC
authors: Robbins
authors: Sanjiv S
authors: SS
authors: Gambhir
mesh_terms: D000818:Animals; D015153:Blotting, Western; D003952:Diagnostic Imaging; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008099:Liver / Q000378:metabolism; D008163:Luminescent Measurements; D051379:Mice; D050505:Mutant Proteins / Q000737:chemistry* / Q000378:metabolism*; D009206:Myocardium / Q000473:pathology; D041681:NIH 3T3 Cells; D009928:Organ Specificity / Q000235:genetics; D010957:Plasmids / Q000378:metabolism; D017510:Protein Folding; D011960:Receptors, Estrogen / Q000737:chemistry* / Q000378:metabolism*; D015203:Reproducibility of Results; D014158:Transcription, Genetic
chemical_list: D008024:Ligands; D050505:Mutant Proteins; D011960:Receptors, Estrogen
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