Ashwin Amurthur, Peter Qiao, Michael Magaraci, Avin Veerakumar How - - PowerPoint PPT Presentation

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Ashwin Amurthur, Peter Qiao, Michael Magaraci, Avin Veerakumar How - - PowerPoint PPT Presentation

Dec 23, 2023 255 likes •685 views

Ashwin Amurthur, Peter Qiao, Michael Magaraci, Avin Veerakumar How can Synthetic Biology Improve Medicine? Current Therapies Maximizing Specific Effects Minimizing Non- Specific Effects Leads to two main optimization parameters

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SLIDE 1

Ashwin Amurthur, Peter Qiao, Michael Magaraci, Avin Veerakumar

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SLIDE 2

How can Synthetic Biology Improve Medicine?

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SLIDE 3

Current Therapies

  • Maximizing Specific

Effects

  • Minimizing Non-

Specific Effects

  • Leads to two main
  • ptimization

parameters

– Targeting Specificity – Dosage Control

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SLIDE 4

Cancer As a Case Study

Current Cancer Therapies

  • Significant Off Target Effects

– Chemotherapy – Radiation Therapy

  • Dosage Control

– Passive Diffusion – Difficulty reaching tumors – Not optimized

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SLIDE 5

Cancer As a Case Study

Current Cancer Therapies

  • Significant Off Target Effects

– Chemotherapy – Radiation Therapy

  • Dosage Control

– Passive Diffusion – Difficulty reaching tumors – Not optimized

Proposed Bacterial Therapies

  • Specifically target cancer

cell lines

– Strains that actively seek hypoxic environments – Engineered cell-surface receptors allow for specific binding

  • Induced cytotoxicity

– Systems to regulate gene expression – Cancer-specific therapeutics

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Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

Achieving Light-Activated Cell Lysis

Goal is to demonstrate that:

  • YF1/FixJ (pDawn) blue light sensor can be activated for

downstream transgene expression

  • YF1/FixJ blue light sensor allows for light dependent lysis
  • f mammalian cells
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SLIDE 9

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

YF1/FixJ BL Sensor Allows For Light- Dependent Transgene Expression

0h 1h 2h 3h 4h 6h 8h 22h

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Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

Cytolysin A (ClyA)

(Wallace et. al, 2000)

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SLIDE 11

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

Light-triggered lysis of mammalian cells by pDawn-ClyA bacteria

(D) (L)

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SLIDE 12

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

ClyA hemolysis can be characterized through dose-response

ClyA Cytotoxicity Assay – SKBR3 1.26 * 105 HER2 receptors per cell (High HER2 expressing) ClyA Cytotoxicity Assay – HEK293T 4.23 * 103 HER2 receptors per cell (Low HER2 expressing)

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Spatial control of cell Lysis

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Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

Targeting Cancer Cells

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Ice Nucleation Protein, NC Domain

  • Outer membrane protein

Discovered in P. syringae

  • Promotes ice crystallization
  • Can remove internal repeats

and display proteins on the surface of E. coli

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

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SLIDE 16

Displaying DARPin H10-2-G3

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

DARPin H10-2-G3

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Assaying Display of DARPin H10-2-G3

  • HA tag allowed detection of surface proteins

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

INPNC-HA (+IPTG) INPNC-HA (-IPTG)

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SLIDE 18

DARPin was Successfully Displayed!

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

DARPin-HA (-IPTG) DARPin-HA (+IPTG)

INPNC-DARPin-HA (-IPTG) INPNC-DARPin-HA (+IPTG)

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SLIDE 19

Can our Bacteria Bind to Cancer Cells?

  • SKBR3 Cells are derived

from breast tumors

  • Overexpress HER2

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

DAPI, HER2

Breast Tumor Tissue1 Our Cultured SKBR3 Cells

  • 1. Y. Xiao, X. Gao, G. Gannot, M.R. Emmert-Buck, S. Srivastava,

P.D. Wagner, M.A.Amos and P.E. Barker. Quantitation of HER2 and telomerase biomarkers in solid tumors with IgY antibodies and nanocrystal detection. International Journal of Cancer

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DARPin-displaying Bacteria Bind to SKBR3 Cells Preferentially

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

HEK293T (Basal HER2) SKBR3 (High HER2)

DAPI HER2 eGFP

+E. coli (+IPTG) +E. coli (-IPTG)

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DARPin-displaying Bacteria Bind to SKBR3 Cells Preferentially

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

DAPI, HER2, eGFP

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Submitted BioBricks

  • ClyA BioBricks
  • INPNC: BBa_K811003
  • INPNC-HA: BBa_K811004
  • INPNC-MCS: BBa_K811005
  • General surface display

vector for any iGEM team

  • Only one ligation needed!

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

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SLIDE 23

Summary

  • First light-activated

drug delivery in bacteria

  • First display of a

DARPin on cell surface

  • DARPin targeted

cancer cells in vitro

  • Created general

Surface Display platform

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

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SLIDE 24

Human Practices

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SLIDE 26
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SLIDE 27
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SLIDE 28 http://2009.igem.org/Team:Stanford
  • Many people have tried

to apply synthetic biology to treat disease. – Over 75 Health/Medicine teams since 2009 – Many, many papers since 1995!

  • Where are they now?
  • Why?

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

Looking Back on Eight Years of iGEM: Where are We Now?

http://2008.igem.org/Team:MIT http://2011.igem.org/Team:Missouri_Miners
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Biological and Perception Barriers to Bacterial Therapeutics

Biological Barriers

  • E. coli produces

compounds that are immunogenic

  • Lab strains are poor

candidates for use in the human body Perception Barriers

  • Negative portrayal
  • f E. coli in the

media

  • Public unfamiliar

with synthetic biology

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

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The Hype Cycle

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion Linden, A. (2003). Understanding gartner’s hype cycles. Conshohocken: Gartner.

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Education & Outreach

  • Presentation/Q&A

session with high school students

  • Clark Park Science

Discovery Day

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

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Solving the Biological and Perception Problems

  • E. coli Nissle 1917

– Nonpathogenic – Used as a probiotic supplement in Canada and Europe – Low immunogenicity – Generally Regarded As Safe by the FDA

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

http://www.npr.org/blogs/thesalt/2012/07/09/156381323/confusion-at-the-yogurt-aisle-time-for- probiotics-101 http://www.msnbc.msn.com/id/27454348/ns/health- diet_and_nutrition/t/probiotics-more-hype- help/#.UHd2ScXA-f4 http://sitemaker.umich.edu/huffnagl e.lab/_the_probiotics_revolution___ book_
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The pDawn Expression System is Functional in Nissle 1917

  • Chemically

competent Nissle 1917 achieved light controlled clyA hemolysis

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

+1000 μg pDawn-his-ClyA +H2O (Neg. Control)

pDawn-mCherry Light pDawn-mCherry Dark pDawn-his-clyA Dark pDawn-his-clyA Light

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Recommendation for Future H&M iGEM Teams

  • When designing a project, keep clinical

endpoints in mind

  • Utilize strains of bacterial like Nissle 1917

– Scientific benefits – Perception benefits

  • Use outreach events as an opportunity to

smooth out the hype curve

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

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Project Summary

  • Light dependent clyA expression & hemolysis
  • INPNC based display of proteins

– Easily adapted for surface display of any protein

  • HER-2 dependent binding of bacteria to human cells
  • Modular binding & cell lysis systems

– Can be repurposed – Interchangable parts

  • A low cost way of improving the reliability of biobricks
  • A careful consideration of the adoption of bacterial

therapeutics

– Nissle 1917

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

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Future Directions

  • Use other wavelengths of light
  • Port the entire system into Nissle 1917
  • Integrate our system into E. coli. genome
  • Test our system in a mouse model

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

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SLIDE 37

Acknowledgements

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

  • Dr. Casim A. Sarkar
  • Dr. Jordan Miller
  • Dr. Mark Goulian
  • Najaf Shah
  • Daphne Ng
  • Sevile Mannickarottu
  • Henry Ma
  • Nikita Singh
  • Karsticum Computing Inc.
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SLIDE 38

Sponsors

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SLIDE 39

Questions and Answers

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SLIDE 40

Backup Slides

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SLIDE 41

Testing the System with mCherry

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

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Testing the System with mCherry

  • Fused INPNC to mCherry

with 12aa GS Linker

  • Sonicated INPNC-

mCherry expressing E. coli and separated lysate and membrane fraction

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion