To GzmB or not to GzmB Preventing Tissue Damage from Chronic - - PowerPoint PPT Presentation

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To GzmB or not to GzmB Preventing Tissue Damage from Chronic - - PowerPoint PPT Presentation

Feb 14, 2023 131 likes •725 views

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

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SLIDE 1

Preventing Tissue Damage from Chronic Inflammation

TAS_Taipei 2015

To GzmB or not to GzmB

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SLIDE 2

PSORIASIS

RHEUMATOID ARTHRITIS

CHRONIC INFLAMMATION

ASTHMA

Photoaging

DIABETES

Tendinopathies

Stevens-Johnson syndrome

Toxic epidermal necrolysis

Discoid lupus

Erythematosus Crohn's disease

Irritable bowel disease

Multiple sclerosis

Sjogren's syndrome Myositis

Systemic lupus

erythematosus Neonatal lupus

Scleroderma

Kawasaki disease

Atherosclerosis

Transplant rejection

Aneurysm

Allograft vasculopathy

Hypersensitivity Myasthenia gravis

Pulmonary sarcoidosis

Pneumonitis

CHRONIC WOUND

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SLIDE 3

Redness

INFLAMMATION?

Swelling Heat Pain

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SLIDE 4

Inflammation Chronic Inflammation

wound

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SLIDE 5
  • 1. Chronic inflammation => diseases
  • 2. Prevent damage from chronic inflammation
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SLIDE 6

INFLAMMATION RANZYME B

↑ ↑

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SLIDE 7

What is Granzyme B (GzmB)?

  • Overexpressed during

inflammation

  • Serine Protease; cleaves
  • ther proteins
  • Enters and kills harmful

cells (apoptosis)

Immune Cell Target Cell

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SLIDE 8

Granzyme B cleaves ECM Proteins

Extracellular Matrix (ECM)

  • Collection of extracellular

molecules

  • Elastin, Collagen, and

Decorin Provides Stability, Elasticity and Scaffolding of tissues

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SLIDE 9

Chronic inflammation:

  • Prolonged GzmB activity
  • More ECM proteins cleaved

Leads to TISSUE DAMAGE

  • Skin fragility
  • Joint destruction

Granzyme B cleaves ECM Proteins

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SLIDE 10

Extracellular Intracellular

What Does Granzyme B Do?

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SLIDE 11
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SLIDE 12

Chronic Inflammation More GzmB Health Conditions

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Project Goal

Our project aims to prevent tissue damage from chronic inflammation by limiting GzmB activity in the ECM without affecting its intracellular functions.

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SLIDE 14

EXPERIMENTAL PROTOTYPE POLICY AND PRACTICES

  • Prevent tissue damage from

chronic inflammation

  • Limit GzmB activity in the ECM
  • Topical drug delivery
  • Research and Change
  • Outreach and Entertainment
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SLIDE 15

Experimental

Device Design

Secrete proteins

  • ut of bacteria

Mutation allows inhibition of extracellular GzmB Regulate when device turns on

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SLIDE 16

MOUSE HUMAN Extracellular Inhibitor

NO

Extracellular Inhibitor

Granzyme B Inhibitor

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SLIDE 17

Granzyme B Inhibitor (ACT3m)

Goal: Inhibit Extracellular GzmB

  • ACT: human extracellular protein
  • 3 mutations allow ACT to bind to GzmB
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SLIDE 18

Mouse GzmB extracellular Inhibitor Human ACT Wildtype Human ACT3m: Extracellular Inhibitor

Granzyme B Inhibitor (ACT3m)

  • ACT: human extracellular protein
  • 3 mutations allow ACT to bind to GzmB

Goal: Inhibit Extracellular GzmB

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SLIDE 19

Motor Protein (YebF)

YebF secretes attached proteins

  • ut of E. coli

Goal: Send GzmB inhibitor out of the cell

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Temperature-Sensitive Promoter

Test: What temperature activates the TempSens promoter? How long does it take to activate? Goal: Control activation of device

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TempSens Promoter Activates at 37℃

Normal Light Blue Light

30 ℃ 32 ℃ 35 ℃ 37 ℃

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SLIDE 22

TempSens Promoter Activates after ~3h

Positive Control TempSens + GFPgen

0h 1h 2h 3h 4h 5h

Negative Control

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Full GzmB Inhibitor Device

DNA Gel: Constructed full device Protein Gel: Possible protein products Biuret test: Protein products successfully secreted

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SLIDE 24

Modeling

Complete GzmB inhibition = BAD GOAL: Bring GzmB levels down to normal

  • Dr. Phillip I. Bird
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SLIDE 25

ACT3m Inhibition of GzmB

GOAL: Bring GzmB levels down to normal

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SLIDE 26

Calculator

Different levels of GzmB Normal levels of GzmB GOAL: Bring GzmB levels down to normal

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SLIDE 27

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

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SLIDE 28

EXPERIMENTAL PROTOTYPE POLICY AND PRACTICES

  • Prevent tissue damage from

chronic inflammation

  • Limit GzmB activity in the ECM
  • Topical drug delivery
  • Research and Change
  • Outreach and Entertainment
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SLIDE 29

GOAL: Deliver GzmB inhibitor

Prototype

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>50% of People Have “Mixed Feelings”

  • r

“Would avoid GMOs at all costs”

✓ No bacteria in body ✓ Non-invasive ✓ Easy to apply

TOPICAL TREATMENT

Prototype

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Prototype Vision

Localized application: Bandage

  • Wounds
  • Inflamed sites

General application: Cream

  • Psoriasis
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SLIDE 32

Bandage Activation by Heat Pack

  • Measured 100

individuals

  • Skin temperatures

below activation temperature

  • We maintain control
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SLIDE 33

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
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SLIDE 34

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

?

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SLIDE 35

0.2 µm Membrane Positive Control (Medical Gauze) Top Layer Membrane Bottom Layer

Testing Semi-Permeable Membrane

Can BACTERIA pass through? No Bacteria

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Testing Semi-Permeable Membrane

Can PROTEINS pass through? A B C C B A C A B Before After

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Prototype on Agar Taken Apart

Bottom Agar

=

Prototype Removed

Bacteria

Building Bandage Prototype

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General Application: Cream

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SLIDE 39

General Application: Cream

Cream contains extracted proteins Delivering proteins through skin

  • TD1 (peptide)
  • Transfersomes (specialized liposomes)
  • TPM (form of vitamin E)
  • Microneedles
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EXPERIMENTAL PROTOTYPE POLICY AND PRACTICES

  • Prevent tissue damage from

chronic inflammation

  • Limit GzmB activity in the ECM
  • Topical drug delivery
  • Research and Change
  • Outreach and Entertainment
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SLIDE 41

Policy and Practice

Outreach and Entertainment

  • Raise awareness about synthetic biology

Research and Change

  • Public and expert feedback
  • Brief congress on changing regulations
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SLIDE 42

iGEM Club Outreach

Spread Awareness

Spring Fair

Teaching Classes

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SLIDE 43

Outreach

Educational Development

Speaker Series Ethics Panel

Research Symposium

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SLIDE 44

Outreach

Project Sharing

NYMU NCTU

Mingdao

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SLIDE 45

NYMU Partnership

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SLIDE 46

Entertainment

Pipet King Spotify Playlist

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SLIDE 47

Research

Medical

  • Dr. Stephanie Hsieh

TAS community

  • Dr. Phillip I. Bird

Public Opinion GzmB

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SLIDE 48

Research

Policy Questionnaire

  • Dr. Sarah R. Carter
  • J. Craig Venter Institute

Former White House analyst

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SLIDE 49

Change

“We did not develop this medicine for Indians…we developed it for western patients who could afford it.” Marijn Dekkers, Bayer CEO

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SLIDE 50

Change

POLICY BRIEF Changing tax incentives to redistribute money from marketing to R&D

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SLIDE 51

EXPERIMENTAL PROTOTYPE POLICY AND PRACTICES

  • Prevent tissue damage from

chronic inflammation

  • Limiting GzmB activity in the

ECM

  • Topical drug delivery
  • Research and Change
  • Outreach and Entertainment
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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
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SLIDE 53

Attributions + Acknowledgments

  • Dr. Sharon Hennessy
  • Dr. Richard Hartzell
  • Friends of TAS
  • Dr. Sarah Carter
  • Dr. Stephanie Hsieh
  • Dr. Phillip Bird
  • NYMU_Taipei iGEM Team
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SLIDE 54

Thank You for Listening!

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SLIDE 55

Safety Device

Goal: Kill bacteria if they escape into the environment

DAP Acid

Degrades bacterial cell wall

DAP Acid represses promoter

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SLIDE 56
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SLIDE 57
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SLIDE 58

Excess GzmB decreases skin quality