iGEM 2012 iGEM 2012 Cambridge, MA, 3. - 5. 11. 2012 Amsterdam, 6. – 7. 10. 2012
Standard therapy with Biological drugs Advantages • Specific • Safe OUR SURVEY OF HEPATITIS C PATIENTS • Effective None 12% Drawbacks Strong 37% • Systemic application • Side effects Mild 51% Adverse effects of interferon therapy
Opportunities of SB in medical therapy 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
The idea MICROENCAPSULATION ADVANTAGES IN SITU PRODUCTION • Local therapeutic OF BIOLOGICAL DRUGS concentrations EXTERNAL REGULATION • Less systemic side effects SAFE TERMINATION
Safety mechanisms CAPSULE TERMINATION ESCAPE DEGRADATION TAG TAG HSV- Capsule THYMIDINE MICA degradation KINASE NKG2D ALGINATE GANCICLOVIR LYASE NK cell Design of safety mechanisms: Urban and Anja
Safety mechanisms CAPSULE Alginate lyase DEGRADATION Sphingobacterium multivorum Heat-denatured enzyme alginate lyase Capsule degradation ALGINATE LYASE Cloning, encapsulation, microscopy:Urban
Safety mechanisms CAPSULE TERMINATION DEGRADATION TAG HSV- Capsule THYMIDINE degradation KINASE ALGINATE Blue – cell nuclei GANCICLOVIR Pink – dead cells LYASE mGMK:TK30 was obtained from Freiburg 2010 BioBrick Cloning, cell experiments: Anja
Safety mechanisms ESCAPE TAG MICA NKG2D Natural killer cell 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 VS. Local Production Pharmacokinetic modelling: Maja with the help of Boštjan
Pharmacokinetics: ischaemic heart disesase Systemic application: 100 mg/day Switch-IT local production: 30 μg/day Pharmacokinetic modelling: Maja with the help of Boštjan
Consulting stakeholders PHYSICIANS PATIENTS SCIENTISTS JOURNALISTS PUBLIC REGULATORS
Advice of medical experts infectologistt traumatologistt cardiologistt Hepatitis C Ischaemic heart disease Wound healing Medical need Definitely Yes, microcapsule delivery into the liver Feasibility artery, heart muscle, wound tissue Improvements Add therapeutics for tissue regeneration High level of control is a must Meetings: all students and some advisors
Improved idea MICROENCAPSULATION EXTERNAL REGULATION SWITCH between the production from one to another drug Advanced therapies with different drug combinations
Regulation requires multiple switches Classic toggle switch … in mammalian cells Gardner et al., 2000 Kramer et al., 2004 How can we design multiple orthogonal switches ? Use designed DNA binding domains!
TAL effectors
Designed TAL repressors and activators Over 90% repression TAL KRAB 0 Control TALA:KRAB Over 1500-fold activation TAL VP16 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 80% of cells express both reporters overlay mCitrine mNeptune
Modeling Improved modeling approaches. Algorithmic model: Allows for explicit modeling of transcriptional regulators competing for binding sites
Modeling Improved modeling approaches. Experimental model: makes use of obtained experimental data to predict the switch behavior Parameter derivation f (x) = … e -kx … Function fitting to determine fold repression pCMV_TALA:KRAB [ng] / pCMV_fLUC [ng] Modelling the switches: Dušan, Martin
Mutual repressor switch simulation Conclusions : cooperativity coefficient above ~ 2.0 Minimal or no promoter leakage required for bistability TAL effector binding is expected Concentration [nM] to be non-cooperative … Cooperativity = 2.0 Cooperativity = 1.0 Time [h] Modelling the switches: Dušan, Martin
The positive feedback loop switch Theory: introduce additional regulatory loops in the system. Can any protein act as an activator and repressor at the same time? Macía , Widder and Solé , 2009
The positive feedback loop switch … we could use a repressor and an activator, competing for the same binding site.
The positive feedback loop switch Cloning of bistable positive feedback loop switch: Boštjan, Fedja, Zala
The positive feedback loop switch Optimal ratio repressor plasmids : activator plasmids Relative level [%] 3:1 Time [h] 1. Exhibits bistable behaviour without assuming cooperativity. http://2012.igem.org/Team:Slovenia/ Interactive 2. More robust with respect to promoter leakage.
How does it work?
How does it work? Erythromycin
How does it work? Erythromycin
Proof of bistability! Induced Induced Nonstimulated reporter 1 reporter 2 cells BFP Citrine Flow citometry experiments: Miha, Urban, Anja, Lucija
Inducer 1 mCitrine BFP BFP CITRINE Inducer 1 Confocal microscopy : Uroš
Stable state 1 mCitrine BFP BFP CITRINE Confocal microscopy : Uroš
Inducer 2 mCitrine BFP BFP CITRINE Inducer 2 Confocal microscopy : Uroš
Stable state 2 mCitrine BFP BFP CITRINE Confocal microscopy : Uroš
Does it switch? BFP BFP mCitrine mCitrine Switching monitored by the activity of SEAP and firefly luciferase. Confocal microscopy SEAP and fLuc experiments:Anja, Lucija
Improved Switch IT therapy HEPATITIS C ISCHAEMIC HEART DISEASE IFN- α HGF Antiviral action Liver regeneration ANAKINRA PDGF VEGF Antiinflammatory Promotion of action angiogenesis HCV Healthy HEPATITIS C infected liver VIRUS liver
Impact of orthogonal switches on synthetic biology We can prepare hundreds of TALs in few days. 3 switches • Introduction of numerous orthogonal switches into cells to define complex states 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 with only 10 switches count to 1000
Contribution to Registry and Attributions 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.
Achievements New type of bistable orthogonal switch based on monomeric designed DNA-binding protein domains Safety mechanisms Therapeutic implementations of biological drug production and delivery device Consultation with stakeholders
Our goal: defense against disease Students: Advisers: Urban Bezeljak Rok Gaber Anja Golob Tina Lebar Lucija Kadunc Jan Lonzarić Dušan Vučko Anže Smole Martin Stražar Roman Jerala Boštjan Pirš Mojca Benčina Miha Jerala Vida Forstnerič Uroš Zupančič Alja Oblak Maja Somrak Miha Mraz Zala Lužnik Miha Moškon Fedja Pavlovec Andreja Majerle
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