igem 2012 amsterdam, 6. 7. 10. 20122012.igem.org/files/presentation/slovenia.pdf · theory:...

iGEM 2012

Amsterdam, 6. – 7. 10. 2012

Advantages

• Specific

• Safe

• Effective

Biological drugs

Drawbacks

• High cost

• Invasive systemic application

• Side effects

None 12%

Mild 51%

Strong 37%

Adverse effects of interferon therapy

OUR SURVEY

The ideal therapy:

• Deliver the drug where it is needed

• Combine different drugs in the correct temporal order

• Completely safe

• Affordable

Opportunities of SB in medical therapy

MICROENCAPSULATION

EXTERNAL REGULATION

IN SITU PRODUCTION OF BIOLOGICAL DRUG COMBINATIONS

SAFE TERMINATION

The idea

ADVANTAGES

• Advanced therapies with different drug combinations

• Local therapeutic concentrations • Less systemic side effects

Termination or therapy Combinations of

therapeutics

Single cell

Design of multiple switches

Gardner et al., 2000 Kramer et al., 2004

Classic toggle switch … in mammalian cells

How can we design multiple orthogonal switches ?

Use designed DNA binding domains !

TAL effectors

Designed TAL repressors and activators

TAL KRAB

TAL VP16

Over 90% repression

Over 1500-fold activation

0 Control TALA:KRAB

Mutual repressor switch

mNeptune mCitrine

overlay

80% of cells express both reporters

Modeling

Two conventional modelling approaches: Deterministic model (ODE) Stochastic model (Chemical reactions)

Two improved approaches: Quantitative model Algorithmic model

pCMV_TALA:KRAB [ng] / pCMV_fLUC [ng]



Modeling




Function fitting to determine fold repression

f(x) = … e-kx …

Modeling


Mutual repressor switch simulation

Analysis of bistability: Minimal promoter leakage Cooperativity coefficient above ~2.0

Time [h]

Co

nc

en

tratio

n [n

M]

Cooperativity = 2.0

Cooperativity = 1.0

The positive feedback loop switch

Macía, Widder and Solé, 2009

Theory: introduce additional regulatory loops in the system.

Can any protein act as an activator and repressor at the same time?

…we could use a repressor and an activator,

competing for the same binding site.


Exhibits bistable behaviour without assuming cooperativity.

More robust in respect to promoter leakage.

Time [h]

Co

nc

en

tra

tio

n [

nM

] The positive feedback loop switch

Erythromycin

How does it work?

Nonstimulated cells

Proof of bistability! B

FP

Citrine

Induced reporter 1

Induced reporter 2

Inducer 1

mCitrine BFP

BF

P

CITRINE

Inducer 1

mCitrine BFP

BF

P

CITRINE

Stable state 1

Inducer 2

mCitrine BFP

BF

P

CITRINE

Inducer 2

mCitrine BFP

BF

P

CITRINE

Stable state 2

Designed positive feedback loop switch

• We have designed and experimentally tested a bistable switch based on designed DNA binding elements.

• Bistable switch based on orthogonal DNA binding elements allows introduction of complex logic responsive to the signals from within the organism or from the outside and could be used as scalable biological memory.

• Simulation demonstrated high robustness even with monomeric binding elements.

Safety

TERMINATION TAG

GANCICLOVIR

HSV- THYMIDINE KINASE

CAPSULE DEGRADATION

ALGINATE LYASE

Capsule degradation

ESCAPE TAG

MICA

NKG2D

NK cell

CAPSULE DEGRADATION

ALGINATE LYASE

Capsule degradation

Capsule degradation

Sphingobacterium multivorum alginate lyase

Heat-denatured enzyme

Alginate lyase

Termination tag

TERMINATION TAG

GANCICLOVIR

HSV- THYMIDINE KINASE

CAPSULE DEGRADATION

ALGINATE LYASE

Capsule degradation

mGMK:TK30 was obtained from Freiburg 2010 BioBrick

Blue – cell nuclei Pink – dead cells

Escape tag

ESCAPE TAG

MICA

NKG2D

Natural killer cell

Do cells in a microcapsule stay viable?

Implementation

Hepatitis C

IFN-α

HGF Biologic activity of IFN-α produced

in therapeutic cells

Ischaemic heart disease

ANAKINRA Antiinflammatory

action

PDGF -BB & VEGF

Promotion of angiogenesis

Biologic activity of anakinra

produced in therapeutic cells

Pharmacokinetic model

Physiologically based, compartmental model.

Key drug processes in the model:

absorption,

distribution,

elimination.

Pharmacokinetics: hepatitis C

The model was used to compare different therapies.

Pharmacokinetics: ischaemic heart disesase

Systemic application: 100 mg/day

Switch-IT: 30 g/day

Systemic application: 100 mg/day Switch-IT: 30 μg/day

Pharmacokinetics: ischaemic heart disesase

SCIENTISTS

PHYSICIANS

PUBLIC JOURNALISTS

REGULATORS

PATIENTS

Human Practice: consulting stakeholders

Do you think it would be beneficial to

develop a therapy with human cells

which could produce biological drugs

inside the diseased tissue and thus

reduce both side effects and the need

for frequent drug application?

Do you think it would be beneficial to develop a therapy with human cells which could produce biological drugs inside the diseased tissue and thus reduce both side effects and the need for frequent drug application?

Encouraging approval of therapy

Would you consume genetically modified food if it had a better taste, looked more appealing or would be healthier?

Physicaly deposited 89 new BioBrick parts, including: • TAL binding domains • Designed repressors and activators • Repeats of TAL binding sites • Reporters • Components of the switch with a positive binding loop • Therapeutic effectors • Safety components • Set of plasmids for cloning with nonstandard sites into BioBrick

standards

Improved an existing BioBrick so that it is functional and was incorporated into the composite part as the functional transcriptional repressor and activator.

Helped other iGEM teams that requested help either in sending parts, participating in surveys, discussions…

Contribution to the iGEM community

Undergraduate student team members contributed ideas for the project, performed all cloning, cell culture and biochemical experiments, cell encapsulation experiments, modeling, performed the survey, discussed project with MDs and regulators.

Conclusions • We designed and experimentally verified the first bistable toggle switch based on monomeric designed DNA-binding protein domains. • This type of switch could be used for complex regulation of mammalian cells and as scalable biological memory. • Mathematical modeling demonstrated improved robustness of a switch based on positive feedback loop.

• We introduced three safety mechanisms for microencapsulated mammalian cells.

• We designed and prepared components for two potential therapeutic implementations of the biological drug production and delivery device.

• We discussed the project with many different stakeholders to improve the acceptance of therapeutic applications of synthetic biology and foster further development.

Our goal: defense against disease

Students: Urban Bezeljak Anja Golob Lucija Kadunc Dušan Vučko Martin Stražar Boštjan Pirš Miha Jerala Uroš Zupančič Maja Somrak Zala Lužnik Fedja Pavlovec

Advisers: Rok Gaber Tina Lebar Jan Lonzarić Anže Smole Roman Jerala Mojca Benčina Vida Forstnerič Alja Oblak Miha Mraz Miha Moškon Andreja Majerle

http://www.uni-lj.si/en/

Achievements • We designed and experimentally verified the first bistable toggle switch based on monomeric designed DNA-binding protein domains. • This type of switch could be used for complex regulation of mammalian cells and as scalable biological memory. • Mathematical modeling demonstrated improved robustness of a switch based on positive feedback loop.

• We introduced three safety mechanisms for microencapsulated mammalian cells.

• We designed and prepared components for two potential therapeutic implementations of the biological drug production and delivery device.

• We discussed the project with many different stakeholders to improve the acceptance of therapeutic applications of synthetic biology and foster further development.

Erythromycin

E:KRAB

PIP:KRAB

Mutual repressor switch

igem 2012 amsterdam, 6. 7. 10. 20122012.igem.org/files/presentation/slovenia.pdf · theory:...

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