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“Plaque Man” – QGEM 2009.  Atherosclerosis: An Introduction to the Disease  Project Design: A Synthetic Biology Approach to Targeted Drug Delivery 

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Presentation on theme: "“Plaque Man” – QGEM 2009.  Atherosclerosis: An Introduction to the Disease  Project Design: A Synthetic Biology Approach to Targeted Drug Delivery "— Presentation transcript:

1 “Plaque Man” – QGEM 2009

2  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 Summary

3  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 Summary

4 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

5  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

6 Timeline progression of atherosclerosis Atherosclerosis

7  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

8  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

9  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 Summary

10  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

11 Lpp-OmpARBS pTet Ter TEV cutLinkerVLA-4 E. Coli VLA-4 Fragment Presentation Binding Circuit Design

12  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

13 P CONST LuxI AHL Synthase P CONST LuxR This complex between LuxR receptor and AHL goes on to activate pLux, the quorum sensing promoter Quorum Sensing Schematic

14  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

15  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 SAA TAT

16 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)

17 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

18 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

19 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  Fe 2+ Chemical structure of heme

20  Benefits of vasodilation (refer to ANP)  Antioxidants exert a cyto-protective effect:  Attempt to produce and then break down heme from bacterial system: HemARBSpLuxTerHemBHemC HO-1 RBSpConstTer HemD Effectors: HO-1

21 LuxR/ LuxI RBSpConstTer Lpp-OmpARBSpTetTer TEV cutLinkerVLA-4 Circuit Design: General Summary TerRBSTEV protease pLuxRBSEffectorDNAseTerRBSTEV protease pLuxRBSEffectorTer RBS

22  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 Summary

23  Identifying heme and heme/HO-1 complex via spectroscopy Heme - 412nm Heme/HO-1 - 408nm HO-1

24 SDS-PAGE and Western blot analysis of SAA production and secretion by E. coli cells SAA

25 Flowchart of PCR stitching for constructing the binding construct Making VLA-4 Binding Construct

26  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-OmpARBSpConstTer TEV cutLinkerANP VLA4 ANP Accomplishments

27  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

28  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

29 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.)

30 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)

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