to gzmb or not to gzmb
play

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

Feb 14, 2023 •131 likes •718 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

  1. To GzmB or not to GzmB Preventing Tissue Damage from Chronic Inflammation TAS_Taipei 2015

  2. 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

  3. Swelling Redness INFLAMMATION? Pain Heat

  4. Chronic Inflammation Inflammation wound

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

  6. ↑ INFLAMMATION ↑ RANZYME B

  7. What is Granzyme B (GzmB)? Overexpressed during • inflammation Immune Cell Target Cell Serine Protease; cleaves • other proteins Enters and kills harmful • cells (apoptosis)

  8. Granzyme B cleaves ECM Proteins Extracellular Matrix (ECM) • Collection of extracellular molecules • Elastin, Collagen, and Decorin Provides Stability, Elasticity and Scaffolding of tissues

  9. Granzyme B cleaves ECM Proteins Chronic inflammation: Prolonged GzmB activity • More ECM proteins cleaved • Leads to TISSUE DAMAGE - Skin fragility - Joint destruction

  10. What Does Granzyme B Do? Extracellular Intracellular

  12. Chronic Health More GzmB Inflammation Conditions

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

  14. 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 •

  15. Experimental Device Design Mutation allows Secrete proteins Regulate when inhibition of out of bacteria device turns on extracellular GzmB

  16. Granzyme B Inhibitor HUMAN MOUSE NO Extracellular Inhibitor Extracellular Inhibitor

  17. Granzyme B Inhibitor (ACT3m) Goal: Inhibit Extracellular GzmB • ACT: human extracellular protein • 3 mutations allow ACT to bind to GzmB

  18. 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

  19. Motor Protein (YebF) Goal: Send GzmB inhibitor out of the cell YebF secretes attached proteins out of E. coli

  20. Temperature-Sensitive Promoter Goal: Control activation of device Test: What temperature activates the TempSens promoter? How long does it take to activate?

  21. TempSens Promoter Activates at 37 ℃ 30 ℃ 32 ℃ 37 ℃ 35 ℃ Normal Light Blue Light

  22. TempSens Promoter Activates after ~3h 0h 1h 2h 3h 4h 5h Positive Control TempSens + GFPgen Negative Control

  23. Full GzmB Inhibitor Device Biuret test: Protein Gel: DNA Gel : Protein products successfully Possible protein products Constructed full device secreted

  24. Modeling Complete GzmB inhibition = BAD Dr. Phillip I. Bird GOAL: Bring GzmB levels down to normal

  25. ACT3m Inhibition of GzmB GOAL: Bring GzmB levels down to normal

  26. Calculator GOAL: Bring GzmB levels down to normal Different levels of GzmB Normal levels of GzmB

  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

  28. 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 •

  29. Prototype GOAL: Deliver GzmB inhibitor

  30. 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”

  31. Prototype Vision Localized application: Bandage General application: Cream • Wounds • Psoriasis • Inflamed sites

  32. Bandage Activation by Heat Pack • Measured 100 individuals • Skin temperatures below activation temperature • We maintain control

  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 -

  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

  35. Testing Semi-Permeable Membrane Can BACTERIA pass through? Positive Control (Medical Gauze) Top Layer Membrane Bottom Layer 0.2 µ m Membrane No Bacteria

  36. Testing Semi-Permeable Membrane Can PROTEINS pass through? Before C B A After A B C C A B

  37. Building Bandage Prototype Prototype on Agar Taken Apart Bottom Agar Prototype Removed = Bacteria

  38. General Application: Cream

  39. General Application: Cream Cream contains extracted proteins Delivering proteins through skin • TD1 (peptide) • Transfersomes (specialized liposomes) • TPM (form of vitamin E) • Microneedles

  40. 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 •

  41. Policy and Practice Outreach and Entertainment • Raise awareness about synthetic biology Research and Change • Public and expert feedback • Brief congress on changing regulations

  42. Outreach Spread Awareness iGEM Club Spring Fair Teaching Classes

  43. Outreach Educational Development Research Ethics Panel Speaker Series Symposium

  44. Outreach Project Sharing NYMU NCTU Mingdao

  45. NYMU Partnership

  46. Entertainment Pipet King Spotify Playlist

  47. Research Public Opinion GzmB Medical TAS community Dr. Phillip I. Bird Dr. Stephanie Hsieh

  48. Research Policy Questionnaire Dr. Sarah R. Carter J. Craig Venter Institute Former White House analyst

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

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

  51. 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 •

  52. 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

  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

  54. Thank You for Listening!

  55. Safety Device Goal: Kill bacteria if they escape into the environment Degrades bacterial DAP Acid represses promoter cell wall DAP Acid

  58. Excess GzmB decreases skin quality

Recommend

More recommend