A Titratable Two-Step Transcriptional Amplification Strategy for Targeted Gene Therapy Based on Ligand-Induced Intramolecular Folding of a Mutant Human Estrogen Receptor

Abstract

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 (ERG521T) 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.

Hydrodynamic Delivery: Characteristics, Applications, and Technological Advances

“…Liverrelated diseases include liver fibrosis/cirrhosis [149][150][151], nonalcoholic fatty liver disease [152,153], alcoholic liver injury [154,155], portal hypertension [156], fulminant hepatitis [157,158], liver regeneration [159], and acute liver injury [160,161]. Extrahepatic diseases include infectious diseases, such as bacterial [162] and trypanosome [163] infection, cutaneous diseases [117] including atopic skin [164,165], cardiovascular diseases, such as atherosclerosis [166], cardiac injury [167,168], intracranial aneurysm [115], ischemia and hypoxic-ischemic encephalopathy [169][170][171], glomerulonephritis [172], renal injury [173], renal fibrosis [174], and hyperparathyroidism [175], metabolic disorders, such as diabetes mellitus, obesity, and hypertriglyceridemia [83,[176][177][178][179], and inflammatory disorders, such as myocarditis [180,181], pancreatitis [182], autoimmune disease [183], arthritis [184], osteoporosis [185], transplant rejection [59,186], xen...…”

“…Melanoma [24,125,126] Leber congenital amaurosis [141] Atopic skin & cutaneous diseases [114,117,164,165] Sepsis [81] Metastasis (melanoma, breast cancer, RCC * (lungs, liver, kidneys)) [118,119,121] Sickle cell disease [142] Cardiovascular & ischemic diseases [115,[166][167][168][169][170][171] Trypanosome [163] *, renal cell carcinoma Cystathionine β-synthase deficiency [143] Kidney diseases & hyperparathyroidism [172][173][174][175] Fabry disease [144] Diabetes mellitus & obesity [176][177][178][179] α-1 antitrypsin deficiency [69] Hypertriglyceridemia [83] Growth hormone deficiency [145] Inflammatory diseases [180][181][182][183][184] Metachromatic leukodystrophy [146] Osteoporosis [185] Short-chain acyl-CoA dehydrogen. def.…”

Image-Guided Hydrodynamic Gene Delivery: Current Status and Future Directions

“…Persistent gene expression is dependent on the regulatory element in the plasmid, and modifications in this region, such as a combination of albumin promoter and alpha-fetoprotein enhancer introns, can significantly contribute to sustained gene expression [ 11 ]. HGD is also useful for delivering large DNA fragments [ 12 ] of therapeutic genes as tested in small animal models for various disease models including hepatitis, hematological disorders, metabolic disease, infectious disease, muscle disease, and malignant diseases [ 4 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 ]. HGD is also an effective tool in studies of genetic immunization and treatment of viral infection [ 47 , 48 ].…”

Molecular-Genetic Imaging of Cancer

“…In an effort to control further the tissue-specific expression of reporter genes, a titratable TSTA (tTSTA) system has been developed (Chen et al, 2014). tTSTA uses the cardiac troponin T promoter to drive the expression of the Gal4-mER(LBD)-VP2 and fLuc as the reporter.…”