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Design of Tumor-Killing Bacteria

Design of Tumor-Killing Bacteria. J. Christopher Anderson Adam P. Arkin and Christopher A. Voigt Labs U.C. Berkeley and UCSF. Celebrities of the Prokaryotic World. Applications. Euprymna scolopes and symbiotic Vibrio fischeri. Agrobacterium infection.

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Design of Tumor-Killing Bacteria

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  1. Design of Tumor-Killing Bacteria J. Christopher Anderson Adam P. Arkin and Christopher A. Voigt Labs U.C. Berkeley and UCSF

  2. Celebrities of the Prokaryotic World Applications Euprymna scolopes and symbiotic Vibrio fischeri Agrobacterium infection Fruiting Body formation by Myxococcus xanthus Microbial cellulose production by Acetobacter xylinum Erythromycin biosynthesis by Saccharopolyspora erythraea Oil bioremediation by Acinetobacter sp.

  3. Specialty Chemicals Therapeutics Bioremediation Agents Biopolymers Materials • Compactnanoscalesensor, controller, actuator packages • Highly engineerable • They are self-assembling and self-replicating • Derived from cheap, renewable resources Biosensors

  4. Applications What is Synthetic Biology? Ground-up Genetic or Cellular Engineering, we add DNA sequences into well-characterized model organisms to understand biological processes or create useful organisms • Putting the Engineering back in Genetic Engineering • Focus on regulation and multi-gene circuits or networks • New, whole-cell technologies • Synthesis methodology and exploiting 1Mb of memory

  5. Standardization of Parts and the Biobrick Language Systems Basic Parts Promoters Ribosome Binding Sites Open Reading Frames Terminators Devices A GFP Producing Device tetR RBS GFP Ter. Ter.

  6. The Parts Registry Processors http://parts.mit.edu

  7. Electronic Signal Carrier Devices Systems Sensors Processors Actuators

  8. Transcriptional Signal Carrier Devices Systems Reporter Genes Signal Integrators Biosynthetic Genes Digital Switches Virulence Genes Sensors Processors Actuators

  9. Systems Very Smart Drugs Really smart drugs

  10. History of Bacterial Therapeutics Applications Digestive Disorders Bladder Cancer Hair Removal/Restoration Nail Fungus GI Parasites Autoimmune Diseases Viral Infections Arteriosclerosis Solid Tumors

  11. Treating Cancer with Bacteria Applications • Cancer Immunotherapy • Localization to and killing of cancer cells

  12. Treating Cancer with Bacteria Applications 20 min 2 days Vibrio cholerae Clostridium Bifidobacterium Listeria monocytogenes Bordetella pertussis Escherichia coli DH5a Salmonella (Dang et al., 2001; Low et al., 1999; Yu et al., 2004) • Why isn’t cancer cured? • Bacteria accumulate in necrotic regions • No intimate association, no regression • Poor localization in human trials • Not a general therapeutic platform • Why growth in tumors? • Reduced immune surveillance • Preferential anaerobic growth • Increased nutrient concentrations • Differential growth rates • Differential clearance rates Is there any good news? Systemic infections can be safely administered Bacteria can access all areas of solid tumors

  13. A Simple Model of Systemic Infection Applications Model for bacterial growth, complement mediated killing, phagocytosis, transport between the bloodstream, organs, and tumors Bacteria in the bloodstream Bacteria in organs Bacteria in tumors

  14. A Simple Model of Systemic Infection Applications Growth in blood

  15. A Simple Model of Systemic Infection Applications Phagocytosis mediated killing

  16. A Simple Model of Systemic Infection Applications Complement mediated killing

  17. A Simple Model of Systemic Infection Applications Transport to and from other tissues

  18. A Simple Model of Systemic Infection Applications Transport to and from tumors

  19. A Simple Model of Systemic Infection Applications Phagocytosis in other tissue

  20. A Simple Model of Systemic Infection Applications Phagocytosis in tumor

  21. A Simple Model of Systemic Infection Applications

  22. Model Analysis of Localization Applications For a given set of parameters, where are the bacteria after 24 hrs? • Most parameter sets result in clearance • Tumor localization is robust to initial dosage and growth rates • Highly sensitive to clearance and transfer rates

  23. Adding Specific Invasion Allows Robust Localization Applications

  24. How to Achieve Robust Localization Applications • Efficient and specific invasion of target cell population • Slow growth rates • Slow clearance rates • Rapid transfer

  25. Therapeutic Bacteria in the Bloodstream

  26. Pancreas

  27. Bloodborne Targets Leukemia HIV-infected Cells Autoimmune Disease Arterial Plaques

  28. Solid Tumor

  29. Bacteria in the bloodstream Uncontrolled bacterial growth Sepsis Innate Immune responses Complement-mediated lysis Phagocytosis

  30. Really smart drugs Growth Rate Control Chassis Blocking High-Affinity Iron Transport

  31. Really smart drugs Growth Rate Control Chassis Blocking Diaminopimelic Acid Biosynthesis

  32. Really smart drugs Advanced Growth Control Chassis Growth-suppression circuits You et. al. Nature. 2004 rE. coli Church and Coworkers

  33. Systems The Target System Really smart drugs

  34. Sensing the Tumor Microenvironment Sensors Gullino, 1975 Salmon, 2003

  35. Environmental Signal Integration Processors

  36. Systems The Target System Really smart drugs

  37. Serum and Complement Resistance K-Capsule O-Antigen Periplasm Jann et al., 1990. Actuators Really smart drugs • Increase serum half-life from <5 min to 4 hours • K1 and K92 are robust to animal host • Poorly antigenic • Function additively

  38. Environmental Restriction of Capsule Expression Actuators

  39. Systems The Target System Really smart drugs

  40. Controlled Invasion of Mammalian Cells Actuators

  41. Environmentally-Controlled Invasion of Cancer Cells Actuators FdhInv - Anaerobic Induction AraInv - Arabinose Induction TetInv - Constitutive Anaerobic Arabinose Normal

  42. Systems The Target System Really smart drugs

  43. First Generation Modular AND Gate Processors

  44. Modularity of Inputs and Outputs [Sal] LuxR PluxR Psal AND [Mg] AND PhoPQ PhoPQ GFP Invasin PmgrB PmgrB NahR [Mg] Processors [AI-1] 4000 1 10-1 3000 10-2 Fluorescence (au) Fraction Invasive 2000 10-3 1000 10-4 10-5 0 100 M AHL - + - + 30 mM Mg - - + + 100 g/ml Sal - + - + 30 mM Mg - - + +

  45. Quantitative Analysis of Signal Integration Processors

  46. Saturation Mutagenesis of Ribosome Binding Sites Processors RBS Start CAAGGAATTAACCATG NNNGGAATTAACCRTG Biobrick RBS Family

  47. Second Generation Signal Integrators Processors

  48. Second Generation Signal Integrators High IN Low OUT Processors

  49. Systems The Target System Really smart drugs

  50. Intracellular Fate of Invasive E. coli Class I MHC Display Class II MHC Display Lysosome Plasmid Protein Vacuole Vacuole (Grillot-Courvalin, 1998) Vessicle (Gentschev, 1995) Devices

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