Ashwin Amurthur, Peter Qiao, Michael Magaraci, Avin Veerakumar
How can Synthetic Biology Improve Medicine?
Current Medical Therapies Specific Effects Non-specific effects Parameters: 1. Targeting Specificity 2. Dosage Control Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
How can we use Synthetic By Engineering Bacteria to Biology to improve Targeting act as the Therapeutic Specificity and Dose Control?
Cancer As a Case Study Current Cancer Therapies Targeting Specificity Spatial – Radiation Therapy or Cellular – mAb, Chemotherapy Dose Control Passive Diffusion Dose Scheduling Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
Cancer As a Case Study Current Cancer Therapies Proposed Bacterial Therapy Targeting Specificity Targeting Specificity Spatial – Radiation Therapy Combined Spatial or and Cellular – mAb, Chemotherapy Cellular Targeting Dose Control Dose Control Passive Diffusion Active Diffusion Dose Scheduling Tunable Transgene Expression System Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
Tumor Cell Cytoplasm Proposed System Video Cell Membrane HER2 Credit to Autodesk for providing us with Maya! Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
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 pDawn-mCherry Dark (D) (L) pDawn-His-ClyA Dark pDawn-His-ClyA Light pDawn-mCherry Light Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
Spatial control of cell lysis Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
Characterizing ClyA-mediated lysis of SKBR3 cancer cells ClyA Cytotoxicity in SKBR3 cells ClyA Cytotoxicity in HEK293T cells (High HER2 Level Expression) (Basal HER2 Level Expression) **** p<0.0001 Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
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: BBa_K811000- K811002 • INPNC BioBricks: BBa_K811003- K811004 • INPNC-MCS: BBa_K811005 • General surface display vector for any iGEM team • Only one ligation needed! • Regional “Best BioBrick, INPNC-MCS Platform Engineered” Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
Human Practices
VerifiGEM User Interface
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
Perception Barriers to Bacterial Therapeutics Perception Barriers • Negative portrayal of E. coli in the media • Public unfamiliar with synthetic biology Public perception of E. coli is negative Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
Perception Barriers: 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
Biological Barriers to Bacterial Therapeutics Biological Barriers • E. coli produces compounds that are immunogenic • Lab strains are poor candidates for use in the human body Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
Addressing the Biological Barrier • E. coli Nissle 1917 – Nonpathogenic – Used as a probiotic supplement in Canada http://www.npr.org/blogs/thesalt/2012/07/09/156381323/confusion-at-the-yogurt-aisle-time-for- and Europe probiotics-101 – Low immunogenicity 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 bacteria 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
Accomplishments A Novel, Modular Platform for a Targeted Bacterial Therapeutic Light-Activated Drug Delivery Surface Display and Targeting Created an easily adapted surface Functionalized bacteria as a light- display BioBrick activated drug delivery platform First to display DARPin on the Demonstrated light-dependent surface of E. coli hemolysis in a spatially controlled Showed HER-2 dependent binding manner of bacteria to human cells Human Practices VerifiGEM Analyzed barriers to bacterial therapeutics Nissle 1917 – future chassis for bacterial therapeutics Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
Future Directions Optimization of our Platform Increase Therapeutic Viability Use other wavelengths of light (such Port the entire system into Nissle as red) with more clinical relevance 1917 Decrease non-specific binding of our Test our system in a mouse model in engineered therapeutic through vitro modification of E. coli surface Light-Based Drug Surface Display & Human Introduction Conclusion Delivery Targeting Practices
Recommend
More recommend
