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)