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“Plaque Man” – QGEM 2009. Targeted Drug Delivery to Atherosclerotic Plaques. Summary. Atherosclerosis: An Introduction to the Disease Project Design: A Synthetic Biology Approach to Targeted Drug Delivery Binding Inducibility Effectors Results: What did we accomplish? Future Work. - PowerPoint PPT Presentation
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TARGETED DRUG DELIVERY TO ATHEROSCLEROTIC PLAQUES
“Plaque Man” – QGEM 2009
Atherosclerosis: An Introduction to the Disease
Project Design: A Synthetic Biology Approach to Targeted Drug Delivery Binding Inducibility EffectorsResults: What did we accomplish?Future Work
Summary
Atherosclerosis: An Introduction to the Disease
Project Design: A Synthetic Biology Approach to Targeted Drug Delivery Binding Inducibility EffectorsResults: What did we accomplish?Future Work
Summary
Atherosclerosis
Main vascular disease Main cause of
myocardial and cerebral infarction
Statistics on CVD: 31% of all deaths in
Canada (2005) Costs over $22.2 billion
per year 15.4% of total
hospitalizations in Canada Severely atherosclerotic vessel
wall
Endothelium Single cell layer lining all blood vessels Protect, regulate vascular tone, secrete anti-
thrombotic factors, regulate inflammatory response
Atherosclerosis primarily an inflammatory disease, caused by endothelial dysfunction Endothelial dysfunction generally occurs at sites of
arterial bifurcation, due to shear stress
Atherosclerosis
Timeline progression of atherosclerosis
Atherosclerosis
Contents of a plaque include …
Lipid rich core: cholesterol (modified) Necrotic tissue and remodelling cell
types: myocytes, platelet, fibroblasts, myofibroblasts, lymphocytes
Matrix proteins: proteins and cells involved in previous fibrous caps become engulfed as the plaque grows
Atherosclerosis
Extended growth of the plaque can lead to stenosis (narrowing of blood vessel) Can result in angina pectoris
Rupture of the plaque can create blood clots
Creation of a blood clot in the artery
Atherosclerosis
Atherosclerosis: An Introduction to the Disease
Project Design: A Synthetic Biology Approach to Targeted Drug Delivery Binding Inducibility EffectorsResults: What did we accomplish?Future Work
Summary
VCAM1: expressed on inflamed endothelial cell; binds VLA4, normally found on mononuclear leukocytes
E. coli chassis binding to damaged artery wall
Receptor-Ligand Binding
Integrin composed of α (ITGA-4) and β (ITGB-1) subunits
Lpp-OmpARBSpTet TerTEV cut Linker VLA-4
E. Coli VLA-4 Fragment Presentation
Binding Circuit Design
GOAL: localize active substances to the site of infection/damage Effective concentrations
easier to reach Avoid damage to other
parts of the body Since we are using cells,
this means that binding must induce drug secretion
Expression of luminescent proteins dependent on population density.
Quorum Sensing
PCONST LuxIAHL Synthase
PCONST LuxR
This complex between LuxR receptor and AHL goes on to activate pLux, the quorum sensing promoter
Quorum Sensing Schematic
Connected to pLux — induced when the chassis populates the plaque
Three effectors: Serum Amyloid A (SAA) Atrial Natriuretic Peptide (ANP) Heme Oxygenase-1 (HO-1)
Plaque Busting Effectors
SAA is an acute phase protein produced by the liver that affects HDL mediated reverse cholesterol transport
SAA allows cholesterol to be secreted from macrophages
Secrete SAA from bacterial system using a twin-arginine translocation (TAT) pathway
Effectors: Serum Amyloid A
SAATAT
Effectors: Atrial Natriuretic Peptide Essentially taking
advantage of the NO system
Causes vasodilation: Bind endothelial membrane
receptors Activating guanylate cyclase Increases [cGMP]
Natriuretic peptide precursor A (NPPA)
Effectors: ANP Vasodilation can relieve turbulence around
the plaque Can also restore perfusion:
Similar to nitro-glycerine treatment for angina pectoris, and Viagra (sildenafil citrate).
Digital particle imaging (DPI) and Doppler ultrasound (DUS) flow maps for carotid bifurcation models with 70% concentric stenosis (left panels) and 70% eccentric stenosis (right panels) at peak systole
Effectors: ANP
NO pathway has been exploited to treat CVD for over a century
Attempt to display ANP on cell surface Similar to VLA4
ANP Cell Surface Display Construct
Effectors: Heme Oxygenase-1
Heme protein is composed of four subunits {A, B, C, D}, and can be broken down by HO-1 into …
Carbon Monoxide (CO) Biliverdin Fe2+
Chemical structure of heme
Benefits of vasodilation (refer to ANP) Antioxidants exert a cyto-protective
effect: Attempt to produce and then break
down heme from bacterial system:
HemARBSpLux TerHemB HemC
HO-1RBSpConst Ter
HemD
Effectors: HO-1
LuxR/LuxI
RBSpConst Ter
Lpp-OmpARBSpTet TerTEV cut Linker VLA-4
Circuit Design: General Summary
Ter RBS TEV protease
pLux
RBS Effector DNAseTer RBS TEV protease
pLux
RBS Effector TerRBS
Atherosclerosis: An Introduction to the Disease
Project Design: A Synthetic Biology Approach to Targeted Drug Delivery Binding Inducibility EffectorsResults: What did we
accomplish?Future Work
Summary
Identifying heme and heme/HO-1 complex via spectroscopy
Heme - 412nm
Heme/HO-1 - 408nm
HO-1
SDS-PAGE and Western blot analysis of SAA production and secretion by E. coli cells
SAA
Flowchart of PCR stitching for constructing the binding construct
Making VLA-4 Binding Construct
Proposed circuitry for targeted drug delivery to atherosclerotic plaques
3 New BioBrick parts submitted:
Further characterized BBa_I15008 by demonstrating heme/HO-1 complex
Lpp-OmpARBSpConst TerTEV cut Linker ANP
VLA4 ANP
Accomplishments
Finish and sequence binding and SAA constructs
Endothelial Adhesion Assay Test E. coli binding to murine C166
endothelial cells Quantify GFP emission using fluorimeter Concentration of AHL at plaque
ANP activation Test phosphorylation of VASP – marker for
increased cGMP pool Kinetics test for production of heme and
metabolism by HO-1
Future Work
Functionality of Individual Components and Theoretical Systems Approach
Limitations of prokaryotes and specifically E. coli Innate Receptor Recognition – CX3CR1
Fractalkine Immunogenicity
“Cell-Based Therapies” Anticancer therapies – Chemoembolization Addition of retroviral component – Genetic
Disorders
Implications
The Team
Team (left to right): Chris Y., Bryant S., Parthiv A., Kate T., Mike F., James M., Bogdan M., and Harry Z. (not pictured: Chris P. and Jonas G.)
Thanks to our advisors
Faculty Advisors Dr. Peter Greer Dr. Ian Chin-Sang Dr. Virginia Walker Dr. Nancy Martin Dr. Waheed
Sangrar Dr. David LeBrun
Also thanks to: Jun Liu Jeff Boudreau Dr. Don Maurice Dr. Tom Tam Dr. Keith Brunt Dr. J. Christopher
Anderson (UC Berkeley)