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 – Targeting Specificity – Dosage Control
Cancer As a Case Study Current Cancer Therapies • Significant Off Target Effects – Chemotherapy – Radiation Therapy • Dosage Control – Passive Diffusion – Difficulty reaching tumors – Not optimized
Cancer As a Case Study Current Cancer Therapies Proposed Bacterial Therapies • Significant Off Target Effects • Specifically target cancer cell lines – Chemotherapy – Radiation Therapy – Strains that actively seek hypoxic environments • Dosage Control – Engineered cell-surface – Passive Diffusion receptors allow for specific – Difficulty reaching tumors binding – Not optimized • Induced cytotoxicity – Systems to regulate gene expression – Cancer-specific therapeutics
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 of mammalian cells Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
YF1/FixJ BL Sensor Allows For Light- Dependent Transgene Expression 0h 1h 2h 3h 4h 6h 8h 22h Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
Cytolysin A (ClyA) (Wallace et. al, 2000) Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
Light-triggered lysis of mammalian cells by pDawn-ClyA bacteria (D) (L) Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
ClyA hemolysis can be characterized through dose-response ClyA Cytotoxicity Assay – HEK293T ClyA Cytotoxicity Assay – SKBR3 4.23 * 10 3 HER2 receptors per cell 1.26 * 10 5 HER2 receptors per cell (Low HER2 expressing) (High HER2 expressing) Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
Spatial control of cell Lysis
Targeting Cancer Cells Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
I ce N ucleation P rotein, 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 Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
Displaying DARPin H10-2-G3 DARPin H10-2-G3 Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
Assaying Display of DARPin H10-2-G3 • HA tag allowed detection of surface proteins INPNC-HA (-IPTG) INPNC-HA (+IPTG) Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
DARPin was Successfully Displayed! DARPin-HA (-IPTG) INPNC-DARPin-HA (-IPTG) INPNC-DARPin-HA (+IPTG) DARPin-HA (+IPTG) Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
Can our Bacteria Bind to Cancer Cells? • SKBR3 Cells are derived DAPI, HER2 from breast tumors • Overexpress HER2 Breast Tumor Tissue 1 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 Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
DARPin-displaying Bacteria Bind to SKBR3 Cells Preferentially +E. coli (+IPTG) + E. coli (-IPTG) HEK293T (Basal HER2) DAPI HER2 eGFP SKBR3 (High HER2) Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
DARPin-displaying Bacteria Bind to SKBR3 Cells Preferentially DAPI, HER2, eGFP Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
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! Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
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 Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
Human Practices
Looking Back on Eight Years of iGEM: Where are We Now? • Many people have tried to apply synthetic biology to treat disease. – http://2008.igem.org/Team:MIT Over 75 Health/Medicine teams since 2009 – Many, many papers http://2009.igem.org/Team:Stanford since 1995! http://2011.igem.org/Team:Missouri_Miners • Where are they now? • Why? Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
Biological and Perception Barriers to Bacterial Therapeutics Perception Barriers Biological Barriers • Negative portrayal • E. coli produces of E. coli in the compounds that are media immunogenic • Public unfamiliar • Lab strains are poor with synthetic candidates for use in biology the human body Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
The Hype Cycle Linden, A. (2003). Understanding gartner’s hype cycles . Conshohocken: Gartner. Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
Education & Outreach • Presentation/Q&A session with high school students • Clark Park Science Discovery Day Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
Solving the Biological and Perception Problems • E. coli Nissle 1917 – Nonpathogenic – Used as a probiotic supplement in Canada and Europe http://www.npr.org/blogs/thesalt/2012/07/09/156381323/confusion-at-the-yogurt-aisle-time-for- probiotics-101 – Low immunogenicity – Generally Regarded As Safe by the FDA http://www.msnbc.msn.com/id/27454348/ns/health- http://sitemaker.umich.edu/huffnagl diet_and_nutrition/t/probiotics-more-hype- e.lab/_the_probiotics_revolution___ help/#.UHd2ScXA-f4 book_ Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
The pDawn Expression System is Functional in Nissle 1917 • Chemically +H 2 O +1000 μ g (Neg. Control) pDawn-his-ClyA competent Nissle 1917 achieved light controlled clyA hemolysis pDawn-his-clyA pDawn-his-clyA pDawn-mCherry pDawn-mCherry Dark Light Light Dark Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
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 Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
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 Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
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 Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
Acknowledgements • Dr. Casim A. Sarkar • Dr. Jordan Miller • Dr. Mark Goulian • Najaf Shah • Daphne Ng • Sevile Mannickarottu • Henry Ma • Nikita Singh • Karsticum Computing Inc. Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
Sponsors
Questions and Answers
Backup Slides
Testing the System with mCherry Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
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 Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
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