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Latest revision as of 22:32, 21 October 2009


The QGEM project centres on the treatment of atherosclerosis by targeted drug delivery from E.coli. The project is broken down into three major components. The first, and the main, component is the cell membrane protein that allows E.coli to bind to the site of atherosclerotic plaque. The second component is the inducible effector system that produces factors to treat the plaque. The last component is the terminator system that detaches E.coli from the plaque and inhibits proliferation of E.coli in the blood stream.

Part One: Binding System

Part Two: Effector System

Part Three: Cleavage and Termination

Part One: Binding System

In order to target E.coli cells to atherosclerotic plaques, we selected the VLA-4/VCAM-1 binding system. VCAM-1 (vascular cell adhesion molecule-1) is a specific marker that is commonly found at sites of damaged endothelium, such as the site of an atherosclerotic plaque. VLA-4 (Integrin α4β1 or very late antigen-4) is normally expressed on leukocyte membranes, and it directs the leukocytes to damaged sites in the vascular system. Studies have shown that the ITGA-4 chain of VLA-4 antigen can sufficient bind to VCAM-1.

Our goal is to express a fragment of ITGA-4 on the surface of the E.coli plasma membrane. To do this, we have modified the attachment system previously explored by NYMU-Taipei team 2008. Below is a schematic diagram of the binding construct.

          Pconst – RBS – LppOmpA – linker - TEV cleavage site X2 – linker – VLA-4 – STOP 

The Lpp (Lipoprotein signal peptide) and OmpA (Outer membrane protein A) fusion results in presentation of the protein at the outer membrane of E.coli. Two cleavage sites for Tobacco-Etch Virus (TEV) protease are inserted between two linker regions. The TEV protease is a part of the terminator system that detaches E.coli from the endothelial cells. The length of the linker sequence between TEV cut site and VLA-4 can be adjusted in order to optimize binding efficiency.

Part Two: Effector System

Ideally, our effector system should only begin to produce the effectors once a threshold density of E.coli cells is reached at the plaque site. Since the system is in a prokaryotic chassis, signal transduction via the VLA-4/VCAM binding is not a realistic approach. Instead, we choose to employ the highly characterized LuxI/LuxR quorum sensing system in bacteria. Briefly, the effector proteins to be released at the site of plaque are placed under the control of pLux promoter, activated by a threshold concentration of AHL. AHL is constitutively produced by our cells and it will reach the threshold concentration once a sufficient amount of E.coli cells is bound to the plaque.

In terms of treatment options we explored a number of routes; however, the following three proved to be the most feasible and advantageous choices. While we are not medical professionals and have not addressed the concept of dosage, the effectors we have chosen are such that mild overdoses would not be of great concern (i.e. beneficial effects far outweigh negative effects). We have also chosen these effectors for having minimal side effects.

1. Serum Amyloid A

SAA converts cholesterol stored in plaques into a form more accessible by High Density Lipoprotein (HDL), which is the body’s natural mechanism for returning cholesterol to the liver for packaging, metabolism and/or excretion. It is our hope that this effector, when released specifically at the site of atherosclerosis, will induce plaque regression.

However, the clinical effectiveness of SAA is still in doubt for several reasons:

    * Not all atherosclerotic plaques are associated with cholesterol.
    * The body can naturally produce SAA at an astoundingly high rate, given the correct signals.
    * We are unsure if the molecule will reach the area of the plaque it is effective in.
    * We are unsure if the body produces enough HDL to clear atherosclerotic plaques.

2. Heme Oxygenase 1 (HO-1)

The HO-1 effector system involves the production of heme and HO-1. HO-1 catalyzes the degradation of heme into carbon monoxide (CO), biliverdin (BV), and free iron (Fe++), all of which have therapeutically beneficial effects on atherosclerotic plaques. CO acts as a local vasodilator, which may minimize chances of plaque rupture, as well as retard plaque growth. BV inhibits the proliferation of vascular smooth muscle cells, which has been shown to lead to stenosis of blood vessels. Fe++ induces the production of ferritin, which has an antioxidant effect that may protect the surrounding tissues from free radical attack and reduce the chance of plaque rupture.

3. Atrial Natriuretic Peptide (ANP)

ANP activates membrane-bound guanylate cyclase (GCA), which increases the level of intracellular cGMP, a signalling molecule that mediates vasodilation. The effect of ANP is similar to that of carbon monoxide. cGMP also possess an anti-clotting effect, although it is unlikely that we can take advantage of this since platelets do not have membrane-bound guanylate cyclase.

Part Three: Cleavage and Termination

After our chassis has been bound to the site of the plaque for a period of time, it should begin to produce DNases and proteases. The DNases will function to sheer the bacterial genome, making it unable to proliferate in the blood; this functions as a safe-guard against bacteremia. The proteases serve to detach the chassis from the plaque site, so as to avoid a build-up of dead cell debris in an already inflamed area.

Last Updated: October 20, 2009 by Fr3P