1. Overview

According to data from the World Health Organization (WHO), cardiovascular diseases are the leading causes of death globally. Cyclic guanosine monophosphate (cGMP) is a critical second messenger molecule. One of its many complex functions is to transduce nitric-oxide and natriuretic-peptide-coupled signaling and remit myocardial dysfunctions by relaxing blood vessels. This summer, we tried to use synthetic biology to modify the cGMP metabolic pathway in a human cell line. We hope that our work could provide basis for a future gene therapy using synthetic biology.

Increased understanding of the molecular events operating in the NO-sGC-cGMP axis has stimulated therapeutic targeting of the pathway in human diseases. Soluble guanylate cyclase (sGC) is an enzyme that synthesizes cGMP from GTP and is found as a heterodimer of α and β subunits. BBa_K1720000 and BBa_K1720001 contain the full coding sequences of the α and β subunits of human sGC, respectively. Co-expression of the two parts increases sGC activity and cGMP synthesis in the cell. However, elevated cellular level of cGMP may lead to feed-back expression of PDE5A, a phosphodiesterase that degrades cGMP. Creation of novel circuit that consists of U6 promoter followed by PDE5A shRNA may enhance the function of our parts. To achieve controllable up-regulation of cGMP level in the cell, we also designed a hypoxia-inducible operon, HRE, as a switch to up-regulate cGMP only in hypoxia situation.

2. Background

Ischemic and hypertensive heart disease were the leading causes of death in the world over the past decade. Over 7.4 million people were dead of these diseases in 2012 and the number is increasing. Patients and their families suffer from the diseases. It also left a heavy burden to government and society. This summer, we tried to solve the problem with our knowledge in synthetic biology.

The discovery of Nitric oxide (NO) as an endogenous signaling molecule and as a mediator of the cardiovascular effects was acknowledged by the Nobel Prize in Physiology/Medicine in 1998. NO activates sGC by binding to its prosthetic heme group and thereby catalyzing the synthesis of cGMP. As a critical second messenger molecule, cGMP regulates many vital homeostatic mechanisms, i.e. endothelial cell permeability, vascular smooth muscle contractility and cardiomyocyte hypertrophy.

Understanding of the NO-sGC-cGMP pathway has initiated significant translational interest, but this was almost exclusively embodied by the use of NO donors and PDE5 inhibitors. NO donors are not ideal for chronic treatment of heart diseases because of partial patient response, development of tolerance over time and short retention in the plasma. The use of PDE inhibitors is also likely to be limited as the efficacy of such molecules is dependent on endogenous cGMP generation. We therefore thought that it may be beneficial to ‘think outside the box’ to target the NO-sGC-cGMP axis using molecular medicines.

3. Project

3.1 Over Expression of sGC

Dysfunction of NO signaling is related to many pathological disorders, such as myocardial hypertrophy, arterial hypertension, pulmonary hypertension, heart failure, atherosclerosis and restenosis. Soluble guanylate cyclase (sGC) is a critical enzyme in NO-sGC-cGMP pathway, which catalyzes the production of cGMP from GTP. In this project, we aimed to up-regulate the concentration of cGMP by the overexpression of sGC to enhance the function of NO signaling pathway. SGC is a heterodimeric protein, contains two subunits: α and β. Both α and β subunits have a few different isoforms. We overexpressed the α3 (GUCY1A3) and β3 (GUCY1B3) isoforms because they are the most abundant forms in the cardiovascular system.

Crystal structure of the heterodimeric catalytic domain of wild-type human soluble guanylate cyclase.

(Sources:http://www.rcsb.org/pdb/pv/pv.do?pdbid=4NI2&bionumber=1# )

3.2 Silence the PDE5A

cGMP is catabolized to 5’GMP by specific members of the phosphodiesterase (PDE) superfamily. Our goal to increase cGMP synthesis by overexpressing sGC was achieved, but this may also lead to increased cGMP catabolism through the up-regulation of PDE5A. Thus, we created novel circuit consisting U6 promoter followed by PDE5A shRNA to enhance the effects of sGC overexpression.

3.3 On-Off: Hypoxia-Inducible Promoter

Absolutely,the level of cGMP will be up regulated when we enhance cGMP synthesis and block its degradation at the same time.If the devices “overexpress sGC” and “silence PDE5A” work in myocardium cells overtime,it may lead several bad effects we don’t expect.To achieve controllable up-regulation of cGMP level in the cell, we also need a switch to up regulate cGMP only in ischemia or hypoxia situation.

We found that in mammalian cells, there is already a hypoxia-inducible system. The hypoxia-inducible factor (HIF) activates transcription via binding to highly variable hypoxia-responsive elements (HREs) that are composite regulatory elements comprising the conserved HIF-binding site (HBS) with an A/GCGTG core sequence and a highly variable flanking sequence. Space between HBSs, distance from the core promoter, and orientation of HBSs are all connected with hypoxia-responsive activity. Following these rules, we design a HRE sequence and inserted it into the CMV promoter, ahead of the TATA box. We name the reconstructed CMV promoter as hypoxia-inducible promoter. To examine its function, we test an original CMV promoter submitted by the team Freiburg in 2012 as negative control,BBa_k747096.Thus, we improved the characterization of a previously existing basic part.