Engineering Cellular Nanorobots Robots extend our capabilities - - PowerPoint PPT Presentation

Engineering Cellular Nanorobots

Mars rover

• Operates in place that

is difficult to reach

• Navigates difficult

terrain

Robots extend our capabilities


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

Receptors
 Signaling pathways
 Movement (actin cytoskeleton)


OUR GOAL: Can we rearrange the functional modules in a cell to create cellular “robots” with new behaviors?

SENSOR
 PROCESSING
 FUNCTION


White blood cells are like robots

Receptors
 Signaling pathways
 Movement (actin cytoskeleton)


OUR GOAL: Can we rearrange the functional modules in a cell to create cellular “robots” with new behaviors?

SENSOR
 PROCESSING
 FUNCTION


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? 


• 2. Pre-meetings – start after-school prep

meetings in the spring; read and discuss papers

• 1. Team – selected from Lincoln HS’s

advanced biotechnology class (2 yrs)

• 4. Brainstorming - hold two-day team

challenge event to develop ideas, goals, plans

• 3. Bootcamp -- intensive 2 week program;

basics of cell motility; cell culture technique

• 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


engineered cells

Approach: Insert new sensors

new input

NAVIGATION Reprogram cells to move to new targets


new receptor

native chemoattractant receptors

• 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

Chemotaxis Sensors: GPCRs


NAVIGATION

Tested 23 new GPCRs for chemotaxis

Control Cells Cells + new sensor

signal signal

new signal cells

NAVIGATION

Tested 23 new GPCRs for chemotaxis

Control Cells Cells + new sensor

signal signal

new signal cells

NAVIGATION

6 of 23 tested GPCRs show chemotaxis

CONCLUSION: We can modify our cells to migrate to new signals by inserting new sensors.


GPCR ligand ligand type OPRD1 enkephalin

• pioid peptide

OPRL1 nociceptin neuropeptide MTNR1A melatonin hormone M4 acetylcholine neurotransmitte r M3/2 chimera acetylcholine neurotransmitte r HTR1A serotonin hormone ** Some ligands involved in disease **

NAVIGATION

Expanding the range of signals

3 classes of GPCRs (based on downstream signaling pathway)

Gq-coupled GPCR can be converted into a chemotaxis receptor!

Significance: Cells could potentially migrate toward a wider range of signals.

All GPCRs that work in our assay fall into one class (Gi)

X

M3 M2 M3/2 chimera

NAVIGATION

Tuning sensor sensitivity

less sensitive more sensitive

CONCLUSION: Altering receptor recycling can increase or decrease sensitivity.

Approach:
Attach modules that regulate receptor recycling (trafficking to and from the membrane)

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


• Movement is regulated by a specific membrane lipid: PIP3
• PIP3 is made from PIP2 after receptor is activated
• PIP3 activates actin engine

actin polymerization PIP2 PIP3

SPEED

Concept Overview


Feedback loops are important for cell polarity

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 PIP2 PIP3 + +

Feedback Loops Polarized cell

SPEED

Concept Overview


positive loop (stronger polarity)

PIP2

negative loop (weaker polarity)

ACCELERATOR BRAKE PIP3

PIP3
binding
 domain
 PIP3
genera>ng 
 enzyme
 PIP3
binding
 domain
 PIP2
genera>ng 
 enzyme


+

• SPEED

Reengineering PIP3 polarity

Approach: Construct synthetic feedback loops by fusing localization with catalytic domains

+ = wild-type negative feedback loop

PTEN (PIP2 binding) fused to RasC - const. active (PIP3 generating)

speed: 5.9 µm/min speed: 3.5 µm/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 µm/min speed: 3.5 µm/min

SPEED

Creating a Brake: one example

• 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)

SPEED

Conclusions

Can we have them carry a payload?

PAYLOAD What can we do with these engineered cells?

Proof-of-concept: Make cells carry fluorescent beads

PAYLOAD Goal: Deliver therapies or imaging agents

ConA

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


PAYLOAD Goal: Deliver therapies or imaging agents

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)

cellular robot therapeutic agent

Serotoni n

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

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

SPONSORS UCSF

1 2 3 4 5 6 7 wildtype RasCda PTEN PTEN-RasCda um/min

Brake Example: PTEN-RasCDa

* * p<0.0001

Spee d