A synthetic cGMP-sensitive gene switch providing Viagra (R)-controlled gene expression in mammalian cells and mice

摘要

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 phosphocliesterases (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 Rhodospirillurn centenum to the Herpes simplex-derived transactivation domain VP16, we created a novel synthetic mammalian cGMP-sensing transcription factor (GTA) that activates synthetic promoters (P-GTA) 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/P-GTA-driven transgene expression was induced by B-type natriuretic peptide (BNP; Nesiritide (R)) 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 GTAJP(GTA)-driven transgene expression, which was suppressed by en expression of PDEs (PDE-3A, PDE-5A and PDE-9A) and was re triggered by the corresponding PDE inhibitor drugs (Pletal (R), Perfan (R), Primacor (R) (PDE-3A), Viagra (R), Levitra (R), Cialis (R) (PDE-5A) and BAY73-6691 (PDE-9A)). Mice implanted with microencapsulated designer cells co expressing the GTA/P-GTA device with NPR-A and PDE-5A showed control of blood SEAP levels through administration of sildenafil (Viagra (R)). 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. (C) 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.