Team:Slovenia

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Revision as of 03:19, 27 September 2012



The Challenge

Biological drugs such as hormones, enzymes, cytokines or antibodies are increasingly used to treat different diseases. Due to systemic administration, these drugs often have adverse effects. Additionally, the high cost of biopharmaceuticals imposes a heavy burden on health systems.

We aimed to use the principles of synthetic biology to develop a safe and cost-effective method of in situ production of biological drugs to increase the quality of patients' lives. This system should increase compliance to therapy, minimize the number of required invasive procedures and enable advanced multistage therapy while local administration could 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. This system should be reliable, cost effective and safe.

The switch

We designed a new type of bistable toggle switch for mammalian cells based on designed DNA-binding proteins that enables simultaneous introduction of several orthogonal switches and construction of complex logic devices. We discovered that a classical toggle switch topology is ineffective if based on TAL effectors, because they bind to DNA non-cooperatively as monomers. We solved this problem by designing a switch comprised of 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 experimentally confirmed bistability in mammalian cells that can be regulated by small molecule inducers. Read more...
Safety

In order to enable safe application of engineered cells for the microencapsulation based therapy we designed safety mechanisms to degrade the alginate capsules at the end of the therapy, terminate the therapeutic cells by induction of apoptosis and introduction of an escape tag that marks any escaped cells for elimination by natural killer cells of the host. Read more...
Implementation

We implemented effector therapeutics for the 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 regenerative effect could be regulated by a physician administrating a small molecule inducer. Read more...
Modeling

Exhaustive mathematical modeling demonstrated that the classical genetic toggle switch arrangement is not stable without cooperativity. At the same time the modeling confirmed improved robustness of the switch when using modular DNA binding element-based transcriptional regulators that formed two positive feedback loops. This topology does not require cooperativity since nonlinearity is introduced by the positive feedback loop. A pharmacokinetic model of the local delivery of therapeutics by microencapsulated cells predicted that this type of drug delivery should have reduced systemic side effects. Read more...
Society

Different aspects of our project as well as medical applications of synthetic biology were discussed with a wide range of stakeholders, including medical professionals, patients, regulators, general public, media and scientists that will have to participate in the successful introduction of synthetic biology applications in clinical use. We engaged a network of high schools to introduce younger generations to synthetic biology and its use in medicine as well as in a broad range of other fields. Read more...
Perspectives
  • The safety device, including the termination, escape prevention and degradation components will allow implementation for different therapeutic purposes,
  • we plan to establish stable cell lines, each containing an integrated switch with the selected therapeutic effectors and safety mechanisms,
  • the designed orthogonal TAL-based transcriptional factor logic will allow construction of tens or hundreds of different switches and achievement of multistable states and other complex logical devices,
  • we plan to initiate in vivo tests on ischaemia and wound healing.

Read more...
Abstract in plain english

Biological drugs have lately been replacing chemical drugs in the treatement of numerous diseases due to their more specific nature. The current route of biological drug administration is usually systemic, which means that the drug is distributed more or less throughout the whole body, although its function should often be restricted to a specific organ or tissue. This can cause serious side effects, requires larger dosages and consequently raises the price of therapy. Our solution to this problem is to implant cells producing biological drugs inside the very tissue where the drug is required. The drug producing cells are safely sealed inside microcapsules, that prevent them from spreading throughout the body and protect them from destruction by cells of the host immune system. We invented a device that allows implanted cells to produce different types of drugs and switching between them can be controlled by a physician depending on the stage of the disease. We designed cells for use in the therapy of hepatitis C, where the engineered cells produce a biological drug that has antiviral activity followed by a drug that improves liver regeneration. For the therapy after a heart attack we designed cells to suppress inflammation and promote formation of new blood vessels around the affected tissue. A physician may initiate self-destruction of the therapeutic cells and capsules by an outside stimulus when the therapy is complete or at any other given time. We believe our system to be safe and effective and applicable to the therapy of different types of diseases.

Achievements in technical details

  • We present the the first experimental implementation of a bistable toggle switch in mammalian cells based on noncooperative DNA-binding proteins as well as the first bistable switch based on designed DNA-binding proteins.
  • A bistable toggle switch based on orthogonal TAL-repressors and activators was designed and tested in mammalian cells.
  • Mathematical modeling demonstrated improved robustness of a switch based on a positive feedback loop with respect to leaky transcription.
  • We introduced three safety mechanisms into microencapsulated mammalian cells:
    • a tag for escaped cells enabling elimination by natural killer cells,
    • a secretory alginate lyase for degradation of alginate microcapsules,
    • apoptosis of therapeutic cells initiated by the addition of a prodrug.
  • We introduced interferon alpha and hepatocyte growth factor as an effector pair for the therapy of hepatitis C to inactivate the virus followed by the promotion of liver regeneration.
  • We introduced anakinra and vascular endothelial growth factor/platelet-derived growth factor BB as effectors for therapy of ischaemia to suppress inflammation followed by angiogenesis.
  • We deposited 89 BioBricks to the Registry and used most of them in functional devices.
  • We improved an existing BioBrick.


Project sponsors

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