Team:Slovenia

We designed a new type of bistable toggle switch for mammalian cells based on designed DNA-binding proteins, which would allow the simultaneous introduction of several orthogonal switches and construction of complex logic devices. We discovered that the classical toggle switch topology was ineffective since TAL effectors bind noncooperatively as monomers. We solved this problem by designing a switch comprising a pair of mutual repressors (TAL-KRAB) coupled with a pair of activators (TAL-VP16) that form a positive feedback loop. This arrangement resulted in experimental confirmation of bistability in mammalian cells that can be regulated by small molecule inducers. Read more...

We tailored the benefits of microencapsulated engineered cells by designing safety mechanisms to degrade the alginate capsules at the end of therapy, terminate therapeutic cells by induction of apoptosis and introduction of an escape killing tag that marks potential escaped cells to destruction by the host natural killer cells. Read more...

We implemented the effector therapeutics for therapy of hepatitis C and ischaemic heart disease, by introducing five different therapeutic proteins that could, in agreement with our pharmacokinetic models, reduce the side effects and improve the efficiency of the therapy. Switching between production of effectors with antiviral or anti-inflammatory effect and tissue regeneration could be regulated by the physician by delivery of small molecule inducers from the outside. Read more...

Exhaustive modeling demonstrated that the classical toggle switch is not stable without cooperativity while it confirmed the improved robustness of the switch that included two positive feedback loops. This topology does not require cooperativity since the nonlinearity is introduced by positive feedback loop. Pharmacokinetic model of the local delivery of therapeutics by microencapsulated cells predicted that this system has reduced systemic side effects. Read more...

Different aspects of the project as well as medical applications of synthetic biology were discussed with a wide range of stakeholders, including medical professionals, patients, regulators, general public and scientists that will support introduction of this technology into clinical use. We involved a network of high school students into the dissemination.Read more...

• the safety device including termination, escape and degradation component will allow implementation for different therapeutic purposes,
• stable cell lines comprising switch with the selected therapeutic effectors and safety mechanisms will be established,
• we plan to initiate in vivo tests first on ischaemia and wound healing,
• the designed orthogonal TAL-transcriptional factor logics will allow introduction multistability from several parallel switches and other complex logical devices.

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