Targeted Drug Delivery to Atherosclerotic Plaques

1. “Plaque Man” – QGEM 2009 Targeted Drug Delivery to Atherosclerotic Plaques

2. 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

3. 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

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. Atherosclerosis • Endothelium • Single cell layer lining all blood vessels • Protect, regulate vascular tone, secrete anti-thrombotic factors, regulate inflammatory response • Atherosclerosis primarily aninflammatorydisease, caused by endothelial dysfunction • Endothelial dysfunction generally occurs at sites of arterial bifurcation, due to shear stress

6. Atherosclerosis Timeline progression of atherosclerosis

7. 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

8. Atherosclerosis Creation of a blood clot in the artery • 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

9. 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

10. Receptor-Ligand Binding • VCAM1: expressed on inflamed endothelial cell; binds VLA4, normally found on mononuclear leukocytes • Integrin composed ofα (ITGA-4) and β (ITGB-1) subunits E. coli chassis binding to damaged artery wall

11. Binding Circuit Design TEV cut Linker VLA-4 RBS Lpp-OmpA Ter pTet E. Coli VLA-4 Fragment Presentation

12. Quorum Sensing • 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.

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

14. Plaque Busting Effectors • 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)

15. Effectors: Serum Amyloid A SAA TAT 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

16. Effectors: Atrial Natriuretic Peptide • Essentially taking advantage of the NO system • Causes vasodilation: • Bind endothelial membrane receptors • Activating guanylatecyclase • 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 todisplay ANPon 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 • Fe2+ Chemical structure of heme

20. Effectors: HO-1 pLux RBS HemA HemB HemC Ter pConst RBS Ter HO-1 HemD Benefits of vasodilation (refer to ANP) Antioxidants exert a cyto-protective effect: Attempt to produce and then break downheme from bacterial system:

21. Circuit Design: General Summary TEV cut Linker VLA-4 pTet RBS Lpp-OmpA Ter pLux pLux RBS RBS Effector Effector Ter Ter RBS RBS TEV protease TEV protease DNAse Ter RBS LuxR/LuxI pConst RBS Ter

22. 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

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

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

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

26. Accomplishments TEV cut Linker ANP pConst RBS Lpp-OmpA Ter ANP VLA4 Proposed circuitry for targeted drug delivery to atherosclerotic plaques 3 New BioBrick parts submitted: Further characterized BBa_I15008 by demonstrating heme/HO-1 complex

27. Future Work • 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

28. Implications • 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

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)