Cambridge, MA, 3. - 5. 11. 2012 Amsterdam, 6. 7. 10. 2012 Standard - - PowerPoint PPT Presentation

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

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

• verlay

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

Concentration [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

Relative level [%] 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!

BFP Citrine

Induced reporter 1 Induced reporter 2

Flow citometry experiments: Miha, Urban, Anja, Lucija

Inducer 1 mCitrine BFP

BFP CITRINE

Inducer 1

Confocal microscopy : Uroš

mCitrine BFP

BFP CITRINE

Stable state 1

Confocal microscopy : Uroš

Inducer 2 mCitrine BFP

BFP CITRINE

Inducer 2

Confocal microscopy : Uroš

mCitrine BFP

BFP 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