To GzmB or not to GzmB Preventing Tissue Damage from Chronic Inflammation TAS_Taipei 2015
Erythematosus Transplant Irritable bowel disease Hypersensitivity ASTHMA Crohn's disease rejection Aneurysm PSORIASIS Kawasaki Discoid Systemic Allograft Scleroderma disease lupus vasculopathy lupus CHRONIC CHRONIC Photoaging Sjogren's Tendinopathies WOUND syndrome Myositis INFLAMMATION Pulmonary Neonatal sarcoidosis Stevens-Johnson erythematosus RHEUMATOID lupus syndrome Myasthenia Toxic Multiple ARTHRITIS DIABETES gravis epidermal sclerosis Atherosclerosis necrolysis Pneumonitis
Swelling Redness INFLAMMATION? Pain Heat
Chronic Inflammation Inflammation wound
1. Chronic inflammation => diseases 2. Prevent damage from chronic inflammation
↑ INFLAMMATION ↑ RANZYME B
What is Granzyme B (GzmB)? Overexpressed during • inflammation Immune Cell Target Cell Serine Protease; cleaves • other proteins Enters and kills harmful • cells (apoptosis)
Granzyme B cleaves ECM Proteins Extracellular Matrix (ECM) • Collection of extracellular molecules • Elastin, Collagen, and Decorin Provides Stability, Elasticity and Scaffolding of tissues
Granzyme B cleaves ECM Proteins Chronic inflammation: Prolonged GzmB activity • More ECM proteins cleaved • Leads to TISSUE DAMAGE - Skin fragility - Joint destruction
What Does Granzyme B Do? Extracellular Intracellular
Chronic Health More GzmB Inflammation Conditions
Project Goal Our project aims to prevent tissue damage from chronic inflammation by limiting GzmB activity in the ECM without affecting its intracellular functions.
EXPERIMENTAL PROTOTYPE Prevent tissue damage from Topical drug delivery • • chronic inflammation Limit GzmB activity in the ECM • POLICY AND PRACTICES Research and Change • Outreach and Entertainment •
Experimental Device Design Mutation allows Secrete proteins Regulate when inhibition of out of bacteria device turns on extracellular GzmB
Granzyme B Inhibitor HUMAN MOUSE NO Extracellular Inhibitor Extracellular Inhibitor
Granzyme B Inhibitor (ACT3m) Goal: Inhibit Extracellular GzmB • ACT: human extracellular protein • 3 mutations allow ACT to bind to GzmB
Granzyme B Inhibitor (ACT3m) Goal: Inhibit Extracellular GzmB Mouse GzmB extracellular Inhibitor Human ACT Wildtype Human ACT3m: Extracellular Inhibitor • ACT: human extracellular protein • 3 mutations allow ACT to bind to GzmB
Motor Protein (YebF) Goal: Send GzmB inhibitor out of the cell YebF secretes attached proteins out of E. coli
Temperature-Sensitive Promoter Goal: Control activation of device Test: What temperature activates the TempSens promoter? How long does it take to activate?
TempSens Promoter Activates at 37 ℃ 30 ℃ 32 ℃ 37 ℃ 35 ℃ Normal Light Blue Light
TempSens Promoter Activates after ~3h 0h 1h 2h 3h 4h 5h Positive Control TempSens + GFPgen Negative Control
Full GzmB Inhibitor Device Biuret test: Protein Gel: DNA Gel : Protein products successfully Possible protein products Constructed full device secreted
Modeling Complete GzmB inhibition = BAD Dr. Phillip I. Bird GOAL: Bring GzmB levels down to normal
ACT3m Inhibition of GzmB GOAL: Bring GzmB levels down to normal
Calculator GOAL: Bring GzmB levels down to normal Different levels of GzmB Normal levels of GzmB
Experimental Summary Inhibitor Device: ACT3m to inhibit extracelullar GzmB - YebF to transport ACT3m out of bacteria - pTempSens to control expression - Modeling Created an inhibitor concentration - calculator
EXPERIMENTAL PROTOTYPE Prevent tissue damage from Topical drug delivery • • chronic inflammation Limit GzmB activity in the ECM • POLICY AND PRACTICES Research and Change • Outreach and Entertainment •
Prototype GOAL: Deliver GzmB inhibitor
Prototype >50% of People Have ✓ No bacteria in body TOPICAL “Mixed Feelings” ✓ Non-invasive or TREATMENT “Would avoid GMOs ✓ Easy to apply at all costs”
Prototype Vision Localized application: Bandage General application: Cream • Wounds • Psoriasis • Inflamed sites
Bandage Activation by Heat Pack • Measured 100 individuals • Skin temperatures below activation temperature • We maintain control
Localized Application: Bandage Only deliver GzmB inhibitor - Prevent bacteria from entering body - with semi-permeable membrane E.coli is 0.5 µm x 2 µm - Membrane 0.2 µm pore size -
Testing Semi-Permeable Membrane Can BACTERIA pass through? Top LB layer with bacteria Semi-permeable membrane sandwiched in between ? Check bottom LB layer for bacteria
Testing Semi-Permeable Membrane Can BACTERIA pass through? Positive Control (Medical Gauze) Top Layer Membrane Bottom Layer 0.2 µ m Membrane No Bacteria
Testing Semi-Permeable Membrane Can PROTEINS pass through? Before C B A After A B C C A B
Building Bandage Prototype Prototype on Agar Taken Apart Bottom Agar Prototype Removed = Bacteria
General Application: Cream
General Application: Cream Cream contains extracted proteins Delivering proteins through skin • TD1 (peptide) • Transfersomes (specialized liposomes) • TPM (form of vitamin E) • Microneedles
EXPERIMENTAL PROTOTYPE Prevent tissue damage from Topical drug delivery • • chronic inflammation Limit GzmB activity in the ECM • POLICY AND PRACTICES Research and Change • Outreach and Entertainment •
Policy and Practice Outreach and Entertainment • Raise awareness about synthetic biology Research and Change • Public and expert feedback • Brief congress on changing regulations
Outreach Spread Awareness iGEM Club Spring Fair Teaching Classes
Outreach Educational Development Research Ethics Panel Speaker Series Symposium
Outreach Project Sharing NYMU NCTU Mingdao
NYMU Partnership
Entertainment Pipet King Spotify Playlist
Research Public Opinion GzmB Medical TAS community Dr. Phillip I. Bird Dr. Stephanie Hsieh
Research Policy Questionnaire Dr. Sarah R. Carter J. Craig Venter Institute Former White House analyst
Change “We did not develop this medicine for Indians…we developed it for western patients who could afford it.” Marijn Dekkers, Bayer CEO
Change POLICY BRIEF Changing tax incentives to redistribute money from marketing to R&D
EXPERIMENTAL PROTOTYPE Prevent tissue damage from Topical drug delivery • • chronic inflammation Limiting GzmB activity in the • ECM POLICY AND PRACTICES Research and Change • Outreach and Entertainment •
Achievements • 15 new biobricks added to the registry, 3 submitted • Designed, built & characterized GzmB inhibitor device • Designed & built Safety Device • Improved documentation for Temperature Sensitive promoter (characterization of BBa_K608351, iGEM11_Freiburg) • Created a calculator for amount of treatment (see wiki) • Created Policy Brief for pharmaceutical and biopharmaceutical change • Experimentally validated a functional bandage prototype • Collaboration with NYMU_Taipei on modeling, experiments & prototype
Attributions + Acknowledgments • Dr. Sharon Hennessy • Dr. Richard Hartzell • Friends of TAS • Dr. Sarah Carter • Dr. Stephanie Hsieh • Dr. Phillip Bird • NYMU_Taipei iGEM Team
Thank You for Listening!
Safety Device Goal: Kill bacteria if they escape into the environment Degrades bacterial DAP Acid represses promoter cell wall DAP Acid
Excess GzmB decreases skin quality
Recommend
More recommend
