Transcript 48x36 Poster Template

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.
Faculty: LeBrun D., Walker V., Sangrar W., Martin N., Chin-Sang I., Greer P.
Submitted October 2009 by Queen’s University, Kingston, Ontario, Canada
Abstract
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.
Introduction
Atherosclerosis begins as low-density
lipoprotein (LDL) is deposited in the subendothelial 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
Figure 1: A severely atherosclerotic smooth muscle cells may proliferate and form a
fibrous cap over the plaque, which can result in
vessel wall.
stenosis and possible rupturing.
Binding Mechanism
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).
Figure 5: Conceptual flowchart of quorum sensing inducibility.
Figure 2: Diagram of E.coli cells binding to damaged endothelial cells using the
VCAM-1/VLA-4 mechanism.
Several effectors were chosen based on their proven anti-atherosclerotic effects:
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).
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.
Figure 3: PCR flowchart for constructing the binding circuit.
BBa_K214003:
Interchangeable ITGA-4
Fragment.
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).
Figure 4: Proposed inducible effector and detachment circuit.
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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.
Release of Effectors
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:
Figure 7: Results of a wavelength scan between 350 and 550nm of a culture
expressing I716390 (heme construct), showing strong peaks at 412nm.
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.
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 9: SDS-PAGE and Western blot analysis of SAA
expression and secretion of E. coli cells.
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
BBa_K214002:
Interchangeable ANP
component.
Results and Discussion
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.
Binding and Detachment Mechanisms
Parts Submitted
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.
Inducible Effectors
Sponsorship
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.