Engineering Cellular Nanorobots
Robots extend our capabilities Mars rover Operates in place that is difficult to reach Navigates difficult terrain
Can we make microscopic robots that treat disease? Body is challenging environment Diseased areas difficult to identify, reach and treat (esp. cancer)
Can we make microscopic robots that treat disease? Body is challenging environment Diseased areas difficult to identify, reach and treat (esp. cancer) POSSIBLE SOLUTION: Cellular robots CHEMOTAXIS: neutrophil chasing bacteria
White blood cells are like robots Signaling Movement Receptors pathways (actin cytoskeleton) SENSOR PROCESSING FUNCTION OUR GOAL: Can we rearrange the functional modules in a cell to create cellular “robots” with new behaviors?
White blood cells are like robots Signaling Movement Receptors pathways (actin cytoskeleton) SENSOR PROCESSING FUNCTION OUR GOAL: Can we rearrange the functional modules in a cell to create cellular “robots” with new behaviors?
OUR TEAM • Partnership between UCSF and Lincoln High School • 7 high school students from Lincoln HS • 2 undergraduate (iGEM alumni) • 2 international students (Slovenia and China) • 1 middle-school teacher (iGEM guest)
How can High School students develop a research project? 1. Team – selected from Lincoln HS’s advanced biotechnology class (2 yrs) 2. Pre-meetings – start after-school prep meetings in the spring; read and discuss papers
3. Bootcamp -- intensive 2 week program; basics of cell motility; cell culture technique 4. Brainstorming - hold two-day team challenge event to develop ideas, goals, plans 5. Execution - hit the lab!
OUR CHALLENGES 1) Navigation: Engineer our cells to chemotax to new signals Link additional receptors to motility Tune sensitivity of receptors 2) Speed: Tune the speed of chemotaxis Build brakes and accelerators 3) Payload: Make cells deliver a cargo Tether beads to cells
NAVIGATION Reprogram cells to move to new targets Approach: Insert new sensors new native chemoattractant receptor receptors new input engineered cells
NAVIGATION Chemotaxis Sensors: GPCRs MOST chemotaxis sensors are G-Protein Coupled Receptors (GPCRs). What are GPCRs? 7 Transmembrane proteins – intracellular loops signal to the inside of the cell Involved in sensing hundreds of different signals: smell, taste, hormone and neuronal signals, etc. (Could these GPCRs mediate chemotaxis?)
NAVIGATION Tested 23 new GPCRs for chemotaxis new signal cells signal signal Cells + Control new sensor Cells
NAVIGATION Tested 23 new GPCRs for chemotaxis new signal cells signal signal Cells + Control new sensor Cells
NAVIGATION 6 of 23 tested GPCRs show chemotaxis GPCR ligand ligand type OPRD1 enkephalin opioid peptide OPRL1 nociceptin neuropeptide MTNR1A melatonin hormone neurotransmitte M4 acetylcholine r neurotransmitte M3/2 chimera acetylcholine r ** Some ligands involved in disease ** HTR1A serotonin hormone CONCLUSION: We can modify our cells to migrate to new signals by inserting new sensors.
NAVIGATION Expanding the range of signals 3 classes of GPCRs M3 M2 M3/2 chimera (based on downstream X signaling pathway) Gq-coupled GPCR can be All GPCRs that work in our converted into a chemotaxis assay fall into one class receptor! (Gi) Significance: Cells could potentially migrate toward a wider range of signals.
NAVIGATION Tuning sensor sensitivity Approach: Attach modules that regulate receptor recycling (trafficking to and from the membrane) less sensitive more sensitive CONCLUSION: Altering receptor recycling can increase or decrease sensitivity.
NAVIGATION Conclusions 1) We can introduce receptors that will result in chemotaxis to new signals (Gi-coupled receptors) 2) We can connect more signals to chemotaxis machinery using chimeras (convert Gq Gi) 3) We can tune the sensitivity of cells to signals by linking receptors to different recycling modules
Engineering SPEED PROCESSING SPEED
SPEED Concept Overview • Movement is regulated by a specific membrane lipid: PIP3 • PIP3 is made from PIP2 after receptor is activated • PIP3 activates actin engine PIP3 PIP2 actin polymerization
SPEED Concept Overview BUT to get directional movement, distribution is POLARIZED • PIP3 at FRONT • PIP2 at BACK Feedback loops are important for clearly defining FRONT vs. BACK PIP3 activates conversion of PIP2 to PIP3 PIP2 activates conversion of PIP3 to PIP2 PIP3 + Polarized Feedback Loops cell PIP2 + Feedback loops are important for cell polarity
SPEED Reengineering PIP3 polarity Approach: Construct synthetic feedback loops by fusing localization with catalytic domains PIP2 PIP3 PIP3 binding PIP3 binding domain domain PIP3 genera>ng PIP2 genera>ng - + enzyme enzyme positive loop (stronger polarity) negative loop (weaker polarity) ACCELERATOR BRAKE
SPEED Creating a Brake: one example + = wild-type negative feedback loop PTEN ( PIP2 binding ) fused to RasC - const. active ( PIP3 generating ) speed: 5.9 µ m/min speed: 3.5 µ m/min
SPEED Creating a Brake: one example + = wild-type negative feedback loop PTEN ( PIP2 binding ) fused to RasC - const. active ( PIP3 generating ) speed: 5.9 µ m/min speed: 3.5 µ m/min
SPEED Conclusions • we can regulate cell speed by introducing synthetic feedback loops - created 7 brakes and 1 potential accelerator FUTURE DIRECTIONS : make it inducible (i.e. can we use other signals to control when the brake is applied – like a stoplight signal )
PAYLOAD What can we do with these engineered cells? Can we have them carry a payload?
PAYLOAD Goal: Deliver therapies or imaging agents Proof-of-concept: Make cells carry fluorescent beads ConA
PAYLOAD Goal: Deliver therapies or imaging agents Proof-of-concept: Make cells carry fluorescent beads
PAYLOAD Goal: Deliver therapies or imaging agents Proof-of-concept: Make cells carry fluorescent beads CONCLUSION: Cells can deliver a payload
VISION: Example of Possible Application TARGET: Carcinoid tumors In gut and lungs; often malignant Small and very hard to find Secrete high levels of serotonin (neuroactive hormone detected by Gi- coupled GPCR) Serotoni n therapeutic agent cellular robot
SUMMARY We were able to… • Engineer cells to NAVIGATE to new GPCR coupled signals • Tune INPUT SENSITIVITY by linking different recycling modules to receptors • Control SPEED by modifying polarization feedback circuits • Make cells carry a PAYLOAD of beads Progress towards a cellular robot platform for diverse therapeutic functions Submitted >200 parts to the registry
The FUTURE? Cells programmed to search the body for specific targets Neutrophils converging on sites of infection in live mouse. Peters et al., Science 2008
ACKOWLEDGMENTS UCSF SPONSORS Buddies Benjamin Rhau Oliver Hoeller Raquel Gomes Aynur Tasdemir Jason Park Delquin Gong Bethany Simmons Andrew Houk Arthur Millius David Pincus Saber Khan Advisors Wendell Lim Orion Weiner James Onuffer
Brake Example: PTEN-RasCDa 7 6 5 * um/min 4 Spee 3 d 2 1 0 wildtype RasCda PTEN PTEN-RasCda * p<0.0001
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