1. Overview

According to the data from WHO, cardiovascular diseases are the main leading cause of death globally. Cyclic guanosine monophosphate (cGMP) is a critical second messenger molecule.It can transduce nitric-oxide and natriuretic-peptide-coupled signaling and remit the myocardial hypertrophy by relaxing the 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 would provide the proof of principle for future gene therapy.

Soluble guanylate cyclase (sGC) is an enzyme that synthesize cGMP from GTP. We up-regulate sGC by overexpressing its α and β subunits  in a mammalian cell line. However, elevated levels of cGMP leads to the feed-back expression of PDE5a, a cGMP-specific phosphodiesteras that degrades cGMP. Thus, we further modified the pathway by silencing the PDE5a. To achieve controllable up-regulation of cGMP level in the cell, we designed a hypoxia-inducible operon, HRE, as a switch to up regulate cGMP only in hypoxia situation.

2. Background

From the data of WHO in 2012, it’s obvious that ischemic heart disease was the main leading cause of death in the world over the past decade. During this period, the figure of ischemic heart disease kept on rising and it rose to 13.2% in 2012. A large number of patients suffered from ischemic heart disease every year. The increasing number of patients also left a heavy burden to government and society. In order to change this serious situation, something must be done to stop the rapid growth tendency of ischemic heart disease. Therefore, this summer we tried to do something to protect against this disease.

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,