title: A synthetic cGMP-sensitive gene switch providing Viagra(®)-controlled gene expression in mammalian cells and mice.
abstract: Cyclic guanosine monophosphate (cGMP) is a universal second messenger that is synthesized from guanosine triphosphate (GTP) by guanylyl cyclases (GCs) and hydrolyzed into guanosine monophosphate (GMP) by phosphodiesterases (PDEs). Small-molecule drugs that induce high cGMP levels in specialized tissues by boosting GC activity or inhibiting PDE activity have become the predominant treatment strategy for a wide range of medical conditions, including congestive heart failure, pulmonary hypertension, atherosclerosis-based claudication and erectile dysfunction. By fusing the cGMP receptor protein (CRP) of Rhodospirillum centenum to the Herpes simplex-derived transactivation domain VP16, we created a novel synthetic mammalian cGMP-sensing transcription factor (GTA) that activates synthetic promoters (PGTA) containing newly identified GTA-specific operator sites in a concentration-dependent manner. In cell lines expressing endogenous natriuretic peptide receptor A (NPR-A) (HeLa), GTA/PGTA-driven transgene expression was induced by B-type natriuretic peptide (BNP; Nesiritide(®)) in a concentration-dependent manner, which activated NPR-A׳s intracellular GC domain and triggered a corresponding cGMP surge. Ectopic expression of NPR-A in NPR-A-negative cell lines (HEK-293T) produced high cGMP levels and mediated maximum GTA/PGTA-driven transgene expression, which was suppressed by co-expression of PDEs (PDE-3A, PDE-5A and PDE-9A) and was re-triggered by the corresponding PDE inhibitor drugs (Pletal(®), Perfan(®), Primacor(®) (PDE-3A), Viagra(®), Levitra(®), Cialis(®) (PDE-5A) and BAY73-6691 (PDE-9A)). Mice implanted with microencapsulated designer cells co-expressing the GTA/PGTA device with NPR-A and PDE-5A showed control of blood SEAP levels through administration of sildenafil (Viagra(®)). Designer cells engineered for PDE inhibitor-modulated transgene expression may provide a cell-based PDE-targeting drug discovery platform and enable drug-adjusted gene- and cell-based therapies.
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH-4058 Basel, Switzerland.
authors.forename: Taeuk
authors.initials: T
authors.lastname: Kim
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH-4058 Basel, Switzerland.
authors.forename: Marc
authors.initials: M
authors.lastname: Folcher
authors.affiliation: Institut Universitaire de Technologie, IUTA, Département Génie Biologique, Villeurbanne Cedex, France.
authors.forename: Ghislaine
authors.initials: G
authors.lastname: Charpin-El Hamri
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH-4058 Basel, Switzerland; Faculty of Science, University of Basel, Mattenstrasse 26, CH-4058 Basel, Switzerland. Electronic address: fussenegger@bsse.ethz.ch.
authors.forename: Martin
authors.initials: M
authors.lastname: Fussenegger
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics; D006152:Cyclic GMP / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D042182:Rhodospirillum centenum / Q000235:genetics*; D000068677:Sildenafil Citrate / Q000494:pharmacology*
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; C022994:cyclic GMP-binding protein; D000068677:Sildenafil Citrate; D006152:Cyclic GMP
keywords: Guanylyl cyclases; Phosphodiesterases; Synthetic gene circuit; cGMP