igem 2012 amsterdam, 6. 7. 10. 20122012.igem.org/files/presentation/slovenia.pdf · theory:...
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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 !
Designed TAL repressors and activators
TAL KRAB
TAL VP16
Over 90% repression
Over 1500-fold activation
0 Control TALA:KRAB
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]
Two conventional modelling approaches: Deterministic model (ODE) Stochastic model (Chemical reactions)
Two improved approaches: Quantitative model Algorithmic model
Modeling
pCMV_TALA:KRAB [ng] / pCMV_fLUC [ng]
Two conventional modelling approaches: Deterministic model (ODE) Stochastic model (Chemical reactions)
Two improved approaches: Quantitative model Algorithmic model
Function fitting to determine fold repression
f(x) = … e-kx …
Modeling
pCMV_TALA:KRAB [ng] / pCMV_fLUC [ng]
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.
The positive feedback loop switch
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
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
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.
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
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.
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