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Diagnostic Survey of Public Showed Preference for Healthcare

Chagas Disease – Our Real World Problem

Chagas Disease – Our Real World Problem

“Chagas disease, caused by the protozoan Trypanosoma cruzi, is responsible for a greater disease burden than any other parasitic disease in the New World”

Immunocompromised

Coinfection with HIV Infants

Variable efficiency

Evolution of surface antigens Differences between strains

Limitations in Diagnostics

Prof Yves Carlier, expert in Infectious Diseases (Université Libre de Bruxelles) Provided us with useful insights into Chagas disease throughout our project

Would screening all infants impact epidemiology? Would our diagnostic be a viable investment? Can our project make a real difference?

Investigating the Feasibility of Our Diagnostics

Years Population

Total infected without diagnostic Total infected with diagnostic

Epidemiological Model Shows a Congenital Chagas Diagnostic is Viable

>130,000 fewer infected individuals $61 mil in healthcare costs saved annually 37,000 DALYs per year eliminated

Epidemiological Model Shows a Congenital Chagas Diagnostic is Viable

Years Population

Total infected without diagnostic Total infected with diagnostic

Prof Mike Bonsall, Professor of Mathematical Biology (University of Oxford) Helped us gain a better understanding of the principles of disease modelling, and equipped us with the skills to create our own epidemiological model for Chagas disease

CIRCUIT OUTPUT INPUT

Canonical Diagnostic Circuitry

Protease Detection is an Ideal Opportunity for a Platform Diagnostic

INPUT

Cruzipain

Protease Detection is an Ideal Opportunity for a Platform Diagnostic

INPUT

Chagas Disease African Sleeping Sickness Malaria Schistosomiasis Toxoplasmosis

(Cruzipain)

CIRCUIT OUTPUT Cruzipain

INPUT

Canonical Diagnostic Circuitry

Blood Clotting Assay is Most Appropriate for our Diagnostic

OUTPUT

Clotted Blood

Negative

Blood Clotting Assay is Most Appropriate for our Diagnostic

OUTPUT

Positive

Non- clotted Blood Clotted Blood

Negative

Blood Clotting Assay is Most Appropriate for our Diagnostic

OUTPUT

CIRCUIT

INPUT :

Cruzipain Hirudin

OUTPUT

Canonical Diagnostic Circuitry

CIRCUIT

Canonical Diagnostic Circuitry

Lower risk of contamination No need for impractical cell culture Freeze-dried powder eliminates need for cold chain

CIRCUIT

Prof Keith Pardee, pioneer in cell-free technologies (University of Toronto) “[Freeze-dried cell-free] systems … could alleviate both the restrictions of live-cell biosynthesis and cold-chain distribution requirements” – Keith Pardee

Cell-free Overcomes Conventional Synbio Problems

CIRCUIT

Canonical Diagnostic Circuitry

INPUT

Cruzipain

OUTPUT

Hirudin

DNA-Based System Protein-Based System CIRCUIT We Propose Two Novel Cell-Free Protease Detection Systems

TetR dimer

Redesigned System Produced TEV Protease for Amplification

ATC

Hirudin

Dimerisation Domain Cruzipain-cleavable linker DNA-binding domain

Initial Design Produced Hirudin Directly

Redesigned System Produced TEV Protease for Amplification

Hirudin

Transcription and Translation of Hirudin is Insufficient to Prevent Blood Coagulation

Time (min) [Hirudin] (µM)

1.3 µM threshold of hirudin needed to stop blood coagulation Time taken to produce sufficient hirudin to stop clotting Time taken for blood to clot

Redesigned System Produced TEV Protease for Amplification

TEV Protease TEV Protease

Modified Model Showed Amplification Increased Hirudin Production

Time (min) [Hirudin] (µM)

1.3 µM threshold of hirudin needed to stop blood coagulation Time taken to produce sufficient hirudin to stop clotting

Time taken for blood to clot

With amplification Without amplification

pTet-eYFP Was Designed as Proof-of- Concept for DNA Added to System

eYFP (TEV Proxy) RBS Tet Operator

Strong RBS Increases Hirudin Production Rate

Time (min) [Hirudin] (µM)

Concentration of hirudin needed to stop blood coagulation

Stronger RBS Strong RBS Weak RBS Weaker RBS

pTet-eYFP is repressed by TetR

Repression of pTet-eYFP can be Relieved by Addition of ATC

0. 3750. 7500. 11250. 15000. 18750.

FLu/Abs(600nm)

0nM ATC 1nM ATC 10nM ATC

Protein-Based Circuitry Overview

Cruzipain

Inactive TEV protease

Prevents Blood Clotting

Active TEV protease

Proof-of-Concept Parts to Investigate Protease Action at Outer Membrane Vesicles

• Simulates activation
• f our system by

Cruzipain

• Can also be used to test

the activation of TEV in

• ur output
• Investigating targeted

delivery to OMVs Use in our diagnostic

Use for future teams

Cleavage by TEV Protease Significantly Increases sfGFP fluorescence

Time (min) Flu/Abs (600nm)

sfGFP no Quencher sfGFP + Quencher

IPTG induction (µM) 500 500

Cleavage by TEV Protease Significantly Increases sfGFP fluorescence

TEV Protease IPTG Time (min) Flu/Abs (600nm)

sfGFP no Quencher sfGFP + Quencher

IPTG induction (µM) 500 500 500 5

sfGFP + Quencher + TEV protease

The 4Es Framework for Applied Design

Clarity Sensitivity & specificity Speed Equipment Presentation Training Materials Transport Delivery Risks Sustainability Disposal

Dr Cristina Alonso-Vega, Expert in Infectious Disease (University of San Simon) Helped develop an understanding of the current political, social and economic landscape in Bolivia that would impact the implantation of our design Dr Piers Millet, Senior Research Fellow at Future of Humanity Institute (University of Oxford) Piers gave us his expert opinion on the current direction that regulation may be moving in; and helped evaluate our cell free report Centre for Health, Law and Emerging Technologies (HeLEX) and Innovation for Science, Innovation and Society (InSIS) We had sustained dialogue about the ethical and social issues related to our project, which heavily influenced our applied design

Our Final Kit

Dr Tempest van Schaik, researcher in Biomedical Engineering (Science Practice) Helped us to understand the importance of cheap and easy prototyping for our kit to maximise efficiency and potential

Prototyped using paper, cardboard, CAD and 3D printing Meets the 4Es framework criteria

At all levels of medical infrastructure Rapid, point-of-care diagnostic Fully contained and cell-free $3.90 – cheaper than current options

Stochastic Modelling Highlighted Effectiveness of System

Time to 1.3µM Amount of Hirudin (min) Probability Density Function True Positives False Positives Time (min) Percentage

Sensitivity > 95% False positives < 10%

Where Do We See Cruzi Going?

CIRCUIT OUTPUT INPUT

Where Do We See Cruzi Going?

INPUT

African Sleeping sickness (Rhodesain) Schistosomiasis

(Cercarial elastase)

Toxoplasmosis (Cathepsin L) Sepsis (LasA)

Advisors

• Dr. George Wadhams
• Dr. Nicolas Delalez
• Professor Antonis Papachristodoulou
• Professor Michael Bonsall
• Harrison Steel
• Associate Professor Maike Bublitz

Sponsors Experts

Professor Cristina Alonso-Vega Professor Michael Laffan Sam Bannon Professor Emilio Malchiodi Professor Mike Bonsall

• Dr. Piers Millett

Professor Jaila Dias Borges Lalwani

• Dr. Michael Morrison

Professor Yves Carlier Eileen Murphy Dr Scott L Diamond Professor Keith Pardee Sarah Dragonetti

• Dr. Ben Riley
• Dr. Darragh Ennis

Tim Ring

• Dr. Matteo Ferla

David Sprent Drs David and Carol Harris Juan Solano Dr Miguel Hernan Vicco Alfons Van Woerkom Professor Matt Higgins HeLEX and InSIS Professor Mark Howarth

Collaborations

Amazona Judd School AQA Unesp Northwestern City of London School McMaster II EPFL TEC CEM

Acknowledgements