iGEM 2012 Amsterdam, 6. – 7. 10. 2012 iGEM 2012 Cambridge, MA, 3. - 5. 11. 2012
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iGEM 2012
Amsterdam, 6. – 7. 10. 2012
iGEM 2012 Cambridge, MA, 3. - 5. 11. 2012
Advantages
• Specific
• Safe
• Effective
Standard therapy with Biological drugs
Drawbacks
• Systemic application
• Side effects
None 12%
Mild 51%
Strong 37%
Adverse effects of interferon therapy
OUR SURVEY OF HEPATITIS C PATIENTS
The ideal therapy:
• Treat the cause and symptoms of the disease
• Efficient, no side effects
• Deliver the drug where it is needed
• Completely safe
• Affordable
Opportunities of SB in medical therapy
MICROENCAPSULATION
EXTERNAL REGULATION
IN SITU PRODUCTION OF BIOLOGICAL DRUGS
SAFE TERMINATION
The idea
ADVANTAGES
• Local therapeutic concentrations
• Less systemic side effects
Safety mechanisms
TERMINATION TAG
GANCICLOVIR
HSV- THYMIDINE KINASE
CAPSULE DEGRADATION
ALGINATE LYASE
Capsule degradation
ESCAPE TAG
MICA
NKG2D
NK cell
Design of safety mechanisms: Urban and Anja
CAPSULE DEGRADATION
ALGINATE LYASE
Capsule degradation
Sphingobacterium multivorum alginate lyase
Heat-denatured enzyme
Alginate lyase
Safety mechanisms
Cloning, encapsulation, microscopy:Urban
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
Safety mechanisms
Cloning, cell experiments: Anja
ESCAPE TAG
MICA
NKG2D
Natural killer cell
Safety mechanisms
Cloning of MIC-A and cell experiments: Anja
Possible implementation
Ischaemic heart disease
ANAKINRA
Antiinflammatory action
Biologic activity of anakinra
produced in therapeutic cells
Cloning of therapeutic proteins: Lucija and Boštjan
Hepatitis C
IFN-α
Biologic activity of IFN-α produced
in therapeutic cells
Cloning of therapeutic proteins: Lucija and Boštjan
Pharmacokinetic model
Physiologically based, compartmental model.
Absorption Distribution Elimination
Used to compare
different therapies.
Pharmacokinetic modelling: Maja with the help of Boštjan
Pharmacokinetics: hepatitis C
Repetitive
injection
Local
Production
VS.
Pharmacokinetic modelling: Maja with the help of Boštjan
Systemic application: 100 mg/day Switch-IT local production: 30 μg/day
Pharmacokinetics: ischaemic heart disesase
Pharmacokinetic modelling: Maja with the help of Boštjan
SCIENTISTS
PHYSICIANS
PUBLIC JOURNALISTS
REGULATORS
PATIENTS
Consulting stakeholders
Advice of medical experts
Medical need
Feasibility
Improvements
Definitely
Yes, microcapsule delivery into the liver artery, heart muscle, wound tissue
Add therapeutics for tissue regeneration High level of control is a must
cardiologistt infectologistt traumatologistt
Ischaemic heart disease Hepatitis C Wound healing
Meetings: all students and some advisors
MICROENCAPSULATION
EXTERNAL REGULATION
SWITCH between the production from one to another drug
Improved idea
Advanced therapies with different drug combinations
Regulation requires 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
TALs were obtained from Addgene (Sander et al., 2011)
Cloning of TAL effectors: Anja, Lucija, Uroš. Testing of them: Anja, Lucija, Uroš, Miha,
Mutual repressor switch
mNeptune mCitrine
overlay
80% of cells express both reporters
Modeling
Improved modeling approaches.
Algorithmic model: Allows for explicit modeling of
transcriptional regulators competing for binding sites
Modeling
Experimental model: makes use of obtained
experimental data to predict the switch
behavior
f(x) = … e-kx …
pCMV_TALA:KRAB [ng] / pCMV_fLUC [ng]
Function fitting
to determine fold
repression
Parameter derivation
Improved modeling approaches.
Modelling the switches: Dušan, Martin
Mutual repressor switch simulation
Conclusions:
cooperativity coefficient above ~2.0
Minimal or no promoter leakage required for
bistability
Co
nc
en
tra
tio
n [
nM
]
Time [h]
Cooperativity = 2.0
Cooperativity = 1.0
TAL effector binding is expected
to be non-cooperative …
Modelling the switches: Dušan, Martin
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
The positive feedback loop switch
Cloning of bistable positive feedback loop switch: Boštjan, Fedja, Zala
Re
lati
ve
le
ve
l [%
]
Time [h]
1. Exhibits bistable behaviour without assuming cooperativity.
2. More robust with respect to promoter leakage.
The positive feedback loop switch
Optimal ratio repressor plasmids : activator plasmids
3:1
http://2012.igem.org/Team:Slovenia/Interactive
How does it work?
Erythromycin
How does it work?
Erythromycin
How does it work?
Nonstimulated cells
Proof of bistability! B
FP
Citrine
Induced reporter 1
Induced reporter 2
Flow citometry experiments: Miha, Urban, Anja, Lucija
Inducer 1
mCitrine BFP
BF
P
CITRINE
Inducer 1
Confocal microscopy : Uroš
mCitrine BFP
BF
P
CITRINE
Stable state 1
Confocal microscopy : Uroš
Inducer 2
mCitrine BFP
BF
P
CITRINE
Inducer 2
Confocal microscopy : Uroš
mCitrine BFP
BF
P
CITRINE
Stable state 2
Confocal microscopy : Uroš
Does it switch?
mCitrine
BFP
mCitrine
BFP
Switching monitored by the activity of SEAP and firefly luciferase.
Confocal microscopy
SEAP and fLuc experiments:Anja, Lucija
HEPATITIS C
IFN-α
Antiviral action
HEPATITIS C
VIRUS
HGF
Liver regeneration
HCV
infected
liver
Healthy
liver
ISCHAEMIC
HEART DISEASE
PDGF VEGF
Promotion of
angiogenesis
ANAKINRA
Antiinflammatory
action
Improved Switch IT therapy
• Introduction of numerous orthogonal switches into cells to define complex states
3 switches
can define 8 cellular states
• Switches are basic elements of memory units. TAL-based switches can be used for scalable biological
memory, to build counters or other logical elements
count to 1000 with only 10 switches
Impact of orthogonal switches on synthetic biology
We can prepare hundreds of TALs in few days.
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 into BioBrick standards using
nonstandard sites
All experimental results and modeling were performed by undergraduates as well as consultations with physicians, patients, regulators.
Advisers provided training, guidance on microscopy, flow cytometry & cell microencapsulation and valuable advice.
Contribution to Registry and Attributions
Achievements
New type of bistable orthogonal switch based on monomeric designed DNA-binding protein domains
Consultation with stakeholders
Therapeutic implementations of biological drug production and delivery device
Safety mechanisms
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
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