Targeted drug delivery to atherosclerotic plaques Team (alphabetical) : Amin P., Freeman F., MacLeod J,. Momciu B., Palmer C., Shum B., Turner K., Yan C., Zhou H.
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Team (alphabetical): Amin P., Freeman F., MacLeod J,. Momciu B., Palmer C., Shum B., Turner K., Yan C., Zhou H.
Faculty: LeBrun D., Walker V., Sangrar W., Martin N., Chin-Sang I., Greer P.Submitted October 2009 by Queen’s University, Kingston, Ontario, Canada
Results and Discussion
In addition to designing all proposed circuits and submitting the binding construct, VLA-4, and ANP Biobricks to the Parts Registry (see Parts Submitted section), the following positive results were obtained:
After targeting the chassis to the site of the plaque, several chosen effectors should be released. As such, binding must induce drug secretion. In order to achieve this, use of the quorum sensing system was proposed (see Figure 5).
Atherosclerosis is associated with the buildup of plaques in the vascular walls. Currently, treatment for atherosclerosis involves preventative measures and surgical removal of plaque, angioplasty, and stent placement. We sought to develop an E. coli chassis delivering anti-atherosclerotic substances to the site of plaque in vasculature. Inflamed endothelial cells express VCAM-1, a receptor that normally binds to the leukocyte antigen VLA-4. We attempted to express a VLA-4 fragment in E. coli, in order to selectively attach the cells to plaques. In vitro binding test uses inflamed murine endothelial cells, which express VCAM-1. Our bacterial chassis also carries several inducible “effector” systems which, upon binding, release substances that facilitate plaque stabilization and regression including heme oxygenase-1, serum amyloid A and atrial natriuretic peptide. Expression of HO-1 in E. coli has been confirmed using spectroscopy and testing for SAA secretion yielded negative results possibly due to defective ligation. This is subject to further testing along with ANP-induced gene expression in endothelial cells. The chassis system offers the benefits of maximizing treatment through localization and minimizing impact to other areas of the body through targeted drug delivery.
Figure 7: Results of a wavelength scan between 350 and 550nm of a culture expressing I716390 (heme construct), showing strong peaks at 412nm.
Figure 5: Conceptual flowchart of quorum sensing inducibility.
A complex between AHL and the LuxR receptor will form and go on to activate pLux, the quorum sensing promoter. Once pLux is activated (see Figure 4), the effectors could be produced and released.
Several effectors were chosen based on their proven anti-atherosclerotic effects:
Serum amyloid A (SAA) has been shown to allow cholesterol to be secreted from foam cells and taken up by high-density lipoprotein (HDL) by inhibiting the enzymes ACAT and CEH. This may increase the chances of foam cells becoming mobile and thus may reduce the size of the plaque. In addition, since HDL’s role is to remove cholesterol from the blood stream and bring it to the liver for excretion, SAA promotes the body’s natural way of removing cholesterol.
Atrial natriuetic peptide (ANP) is known to bind to membrane receptors on endothelial cells and activate guanylate cyclase, which is part of the nitric oxide (NO) pathway. NO has been exploited to treat cardiovascular disease for over a century. It is a vasodilator, which increases blood flow and relieves turbulence around the plaque. However, NO cannot be synthesized by prokaryotes. By targeting an intermediate in the NO pathway, the vasodilating effects may still be observed, and the prokaryotic chassis would still be sufficient.
Heme is a synthetic group comparable to hemoglobin which carries iron in a porphyrin ring. It is degraded by heme oxygenase-1 (HO-1), which produces carbon monoxide (CO), biliverdin (BV), and free iron (Fe++). All three of these degradation products have an ameliorating effect on plaques, including vasodilation, antioxidant effects, and inhibition of muscle cell proliferation, which can reduce the formation of the fibrous cap. In order to achieve these effects, this effector system attempts to produce heme and inducibly break it down with HO-1. In addition, HO-1 is an anti-inflammatory enzyme which is produced in the blood stream in response to oxidative stress, which means that this effector also promotes the natural resolution of atherosclerotic plaques.
Figure 2: Diagram of E.coli cells binding to damaged endothelial cells using the VCAM-1/VLA-4 mechanism.
Binding and Detachment Mechanisms
In order to allow for effective drug delivery, the VCAM-1/VLA-4 mechanism was chosen to localize the chassis to the site of inflammation. VCAM-1 is expressed on inflamed endothelial cells, such as those present at the plaque. VLA-4 is an antigen normally expressed on the membrane of leukocytes and is composed of two subunits, ITGA-4 and ITGB-1. A fragment of ITGA-4 is suspected to have binding ability to VCAM-1. This fragment was synthesized and attempts were made to PCR stitch this fragment into the binding circuit (see Figure 3).
Atherosclerosis begins as low-density lipoprotein (LDL) is deposited in the sub-endothelial space. This induces an inflammatory response, attracting macrophages and causing platelet adhesion at the site. Accumulation of cell
debris and foam cells (macrophages which have taken up too much LDL and become immobile) can form a necrotic core at the centre of the lesion, which may lead to calcification (hardening). Additionally, vascular smooth muscle cells may proliferate and form a fibrous cap over the plaque, which can result in stenosis and possible rupturing.
Figure 8: Results of a wavelength scan between 300 and 600nm of a culture expressing I716390 (heme construct) and I15008 (HO-1 construct), showing a strong peaks at ~408nm.
In order to test the HO-1 effector system, Biobricks I716390 (Berkeley 2007) and I15008 (Austin 2004) were expressed by E. coli singularly and together. I716390 has been shown to produce heme and I15008 to produce HO-1. Figures 7 and 8 show the spectroscopic identification of heme and the heme/HO-1 complex, respectively. This demonstrates the degradation of human heme in a prokaryotic system.
Figure 1: A severely atherosclerotic
SDS-PAGE and Western Blot analysis with a polyclonal antibody recognizing whole SAA proteins did not reveal SAA in the culture supernatent and whole cell lysates after a 12 hour incubation. Possible defective ligation of pTet-RBS fragment to the SAA encoding gene may be responsible for these negative results.
Future steps for this project include the completion and sequencing of the binding construct, an endothelial cell adhesion assay, activation tests for ANP, kinetics testing for heme metabolism by HO-1, and testing on the proposed detachment and destruction circuit.
Figure 3: PCR flowchart for constructing the binding circuit.
The binding circuit itself is a modification of the attachment system explored by the NYMU-Taipei iGEM team in 2008, in which the fusion of Lpp and OmpA results in the presentation of the protein on the outer surface of E. coli. In addition to this, the circuit encodes a cleavage site for TEV protease in the ITGA-4 fragment as part of the detachment mechanism (see Parts Submitted section).
The detachment mechanism was proposed to be TEV protease and the kill switch developed by the Berkeley iGEM team in 2007 (see Figure 4). This kill switch can be induced to release an endonuclease which destroys genetic material and prevents the chassis from reproducing. In this way, the ITGA-4 fragment binding to VCAM-1 would be cut by TEV protease, the chassis would be released from the site of the plaque, and the endonuclease would prevent it from proliferating.
The transcription of this detachment mechanism would be controlled by the leaky terminator B0015, which allows RNA polymerase to pass through periodically, with a forward termination efficiency of 0.984. By inserting two of these terminators in a row, the detachment mechanism ideally be activated after a period of time allowing therapeutic delivery of effectors
Release of Effectors
BBa_K214001: Construct to express a fragment of ANP or ITGA-4 on the surface of the E.coli plasma membrane, accomplished by modification of the attachment system previously explored by NYMU-Taipei Team 2008.
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. The cleavage sites for Tobacco-Etch Virus (TEV) protease is positioned before the linker region. 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.
Figure 9: SDS-PAGE and Western blot analysis of SAA expression and secretion of E. coli cells.
Figure 6: The inducement and release of ANP, SAA, and precursors to biliverdin and CO, which work together to break down the plaque and reduce harmful effects such as artery stenosis and attack by oxidizing agents.
BBa_K214003: Interchangeable ITGA-4 Fragment.
Interchangeable ANP component.
Figure 4: Proposed inducible effector and detachment circuit.