Team:Slovenia

From 2012.igem.org

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 an advanced and safe method of in situ production of biological drugs to increase the quality of patients' lives. This system should increase compliance to therapy, decreas the number of required invasive drug administrations 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 effective, reliable, safe and potentially more cost-effective.

The switch Switches are the basic regulatory elements through which the state of cells can be controlled. We designed a new type of a universal bistable toggle switch for mammalian cells based on designed DNA-binding proteins. Its properties enable simultaneous introduction of several orthogonal switches and construction of complex logic devices. We found that a classic toggle switch topology is ineffective if based on TAL effectors. Our modeling explaines that the reason for this is noncooperative binding of monomeric TAL effectors to DNA. We solved this problem by introducing nonlinearity through the addition of the addition of positive feedback loops consisting of pairs of TAL activators and repressors competing for binding. This arrangement resulted, both theoretically and experimentally in mammalian cells, in functional switch that can be regulated by small molecule inducers. Read more...	Safety In order to enable safe application of engineered cells in the microencapsulation based therapy we designed three safety mechanisms to degrade the alginate capsules at the end of the therapy, terminate the therapeutic cells by induction of apoptosis and to tag any escaped cells for elimination by the host's natural killer cells. Read more...	Implementation In consultations with medical experts we tailored our therapeutic devices based on implanted microencapsulated engineered cells to the treatments of hepatitis C and ischaemic heart disease by in situ production of therapeutic protein effectors whose efficiency has already been demonstrated. In agreement with our pharmacokinetic models, this strategy could reduce side effects and improve efficiency of these therapies. Switching between production of effectors with antiviral or anti-inflammatory and tissue regenerative effect could be regulated by administrating a small molecule inducer. Read more...
Modeling Modeling was used to simulate and improve the properties of the switch and the pharmacokinetic distribution of drugs in the tissue, which is required for an effective therapy and decreased side effects. We introduced improved methods of switch simulations such as a quantitative parameter derivation and algorithmic/mixed simulation that can capture mixed regulator binding to operators. We also included into the wiki a server for online switch simulation. 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. Exhaustive modeling of the switch demonstrated that the classic genetic toggle switch arrangement is not stable without cooperativity, but it confirmed functionality and improved robustness of the our switch design with two positive feedback loops. This topology does not require cooperativity since nonlinearity is introduced by the positive feedback loop. Read more...	Society Different aspects of general medical applications of synthetic biology and our project specifically were discussed with a wide range of stakeholders, including medical professionals, patients, experts on the law and ethics of GMO use and release, scientists, the media and the general public that will all have to participate in a successful introduction of synthetic biology applications to clinical use. We attempted to organize a network of Slovenian high schools to share the excitement of synthetic biology with younger generations and to demonstrate its application in medicine and other fields. Read more...	Perspectives We anticipate that designed DNA-binding element-based transcriptional factor logic will play a very important role in the development of synthetic biology, TAL-based switches allow simultaneous introduction of multiple switches to adopt multiple cellular states with numerous medical and other applications , The safety mechanisms, including the inducible leak-free termination (prodrug), escape detection and capsule degradation components will allow implementation for different therapeutic purposes, for an effective application stable cell lines containing integrated switches with selected therapeutic effectors and safety mechanisms will have to be established, most likely by the use of human artificial chromosomes, we plan to *initiate in vivo* experiments first on ischaemia and wound healing**. Read more...

Abstract in plain english

Biological drugs are being used ever more often as advanced drugs for the treatment of numerous diseases, due to their more specific mode of action. In current therapies the biological drugs are usually distributed more or less throughout the whole body, although each 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 was 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 cells from spreading throughout the body and protect them from destruction by cells of the host immune system. We constructed a device that allows implanted cells to produce different types of drugs while switching between those production states can be controlled from the outside by a physician, depending on the stage of the disease. We designed our device specifically for the therapy of hepatitis C or heart attack. Against hepatitis C the engineered cells produce a protein with antiviral activity, whose biological activity we have tested and confirmed. After the state of the cells is switched, a protein that improves liver regeneration would be produced. For the therapy after a heart attack we designed cells to suppress local inflammation and promote formation of new blood vessels only 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, effective and applicable in the real world for the therapy of different types of diseases.

Achievements in technical details

We present the first experimental implementation of a bistable toggle switch in mammalian cells based on noncooperative DNA-binding proteins as well as the first demonstration of a 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 IL-1 receptor antagonist (anakinra) and vascular endothelial growth factor/platelet-derived growth factor BB as effectors for therapy of ischaemia to suppress inflammation followed by the local promotion of 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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