Team:Slovenia

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Challenge

Biological drugs are very effective and are increasingly used to treat different diseases. Often, due to their systemic administration, adverse effects are observed. Additionally high cost of biopharmaceutials imposes a great burden on health systems. We aimed to develop a safe and cost-effective biological delivery system for biopharmaceuticals, which would increase the quality of patients' lives. This system would increase compliance to the therapy, minimize the number of required invasive procedures, introduce more effective multistage therapy while the local administration will reduce the side-effects.

We addressed this challenge by implementing microencapsulated engineered mammalian cells that can be regulated from the outside to produce different therapeutics and comprise safety mechanisms.

The switch 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...	Safety 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...	Implementation 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...
Modeling 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...	Society 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...	Perspectives 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. Read more...

Abstract for non-scientists

We designed a new type of therapy where we modify human cells, pack them into small capsules and introduce into the diseased tissue. In order to make this therapy useful we invented new type of switches that will allow the medical doctors to turn on or off production of different biological drug in the patient. At the end of the therapy the capsule will be degraded and cells that produced drugs will be destroyed. For the first applications we selected therapy of hepatitis C, where we induce production of drug that has antiviral activity and at the later stage a drug that helps regeneration of liver. For the therapy of heart infarction we designed cells that suppress inflammation and promote formation of new blood vessels around the affected tissue. In our experiments we demonstrated function of new devices that have to be integrated into final therapy. Students also made mathematical model of switch and on the distribution of drugs throughout body that should decrease the side effects of therapy.

Outcome

Achievements in technical details:

this is the first experimental implementation of a bistable toggle switch based on noncooperative DNA-binding elements and the first bistable switch based on designed DNA-binding elements,
we designed and tested bistable toggle switch for mammalian cells based on orthogonal TAL-repressors and activators,
modeling demonstrated improved robustness of a switch based on a positive feedback loop with respect to leaky transcription,
introduced three safety mechanisms into microencapsulated mammalian cells:
- escape tag for cell elimination by natural killer cells,
- secretory alginate lyase for degradation of alginate microcapsules,
- induction of apoptosis of therapeutic cells by the introduction of a prodrug,
introduced IFN-alpha/HGH effector pair for the therapy of hepatitis C to inactivate the virus and promote liver regeneration in the later stage,
introduced anakinra/VEGF-PDGF-B for therapy of ischaemia to suppress inflammation and promote angiogenesis in the later stage,
deposited 89 BioBricks,
improved an existing BioBrick.

Project sponsors