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.

3. Project

3.1 Over Expression of sGC

Soluble guanylate cyclase (sGC) a critical enzyme in nitric oxide (NO)—soluble guanylate cyclase (sGC)—cyclic guanosine monophosphate (cGMP) pathway. This pathway serves an important physiologic role in vascular tissues,including remission of myocardial hypertrophy. SGC catalyzes GMP to form cGMP ,a second messenger molecule that transduces nitric-oxide and natriuretic-peptide-coupled signaling. Dysfunction of NO signaling results in many pathological disorders, such as myocardial hypertrophy , arterial hypertension, pulmonary hypertension, heart failure, atherosclerosis and restenosis. In our project we aimed to up regulate the concentration of cGMP by the overexpression of sGC to reestablish the function of NO signaling pathway. SGC is a heterodimeric protein, containing 2 subunits: alpha and beta. However, both alpha and beta subunit has a few different isoforms. In our project we overexpressed the alpha3 and beta3 isoforms because they are abundant in 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

Increasing cGMP synthesis by overexpressing sGC is effective, but cGMP catabolism may be also increased. cGMP is catabolized to 5’GMP by specific members of the phosphodiesterase superfamily. The most widely studied cGMP esterase is PDE5A. In 2012,Longhu Li etc. found that Adenoviral short hairpin RNA(shRNA) therapy targeting PDE5a relieves dysfunction following myocardial infarction. Therefore, our project overexpressed sGC and silenced PDE5a with Lentiviral shRNA at the same time.

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.

In mammalian cells,there is a founded hypoxia-inducible systems.The hypoxia-inducible factor (HIF) activates transcription via binding to the highly variable hypoxiaresponsive elements (HREs) which are composite regulatory elements comprising the conserved HIF-binding site (HBS) with an A/GCGTG core sequence and a highly variable flanking sequence.Optimizations of the HBS, spacing between HBSs, the distance from the minimal promoter, and orientation of HBSs are connected with hypoxic activity.Following these factors,we design a HRE sequence,and clone it into CMV promoter,ahead of TATA box.We name the reconstructed CMV promoter as hypoxia-inducible promoter.For describing it well,we test the original CMV promoter as negative control,and this work helps us improve the characterization of a previously existing basic part, BBa_k747096 ,submitted by team Freiburg in 2012 .