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<h2>The synthetic biological components that needed to be developed</h2>
<h2>The synthetic biological components that needed to be developed</h2>
<h3>A Universal orthogonal bistable toggle switch for mammalian cells</h3>
<h3>Universal orthogonal bistable toggle switch for mammalian cells</h3>

Revision as of 19:19, 26 September 2012


For this year's Team Slovenia iGEM project we wanted to exploit the advantages of synthetic biology for an alternative biological medical therapy which could overcome some of the difficulties connected to current therapy with biopharmaceuticals.

Each disease and its corresponding therapy is a story of its own, but we sought to design a synthetic biological solution for drug application that could be used to treat different diseases and include safety components that could be useful in different therapeutic applications.

Our solution was to engineer mammalian cells, so that they will produce and deliver biological drugs inside the organism, as if the drugs were produced by the host cells themselves. Within the engineered cells, we planned to incorporate genetic switches allowing controlled production of one or several desired therapeutics, depending on the stage of the disease. The engineered cells are incorporated in microcapsules that may be implanted into the affected tissue to ensure an immune-privileged environment for the therapeutic cells. The semi-permeable capsules allow the flux of nutrients and effector molecules through the capsule’s pores but prevent cell escape and dissemination throughout the body. For the unlikely case of cells escaping capsules, we designed a safety tag ensuring destruction of cells outside microcapsules by the patient's immune system. This form of therapy may be terminated at any given time by an outside signal inducing microcapsule degradation and therapeutic cell apoptosis in which case no traces of the exogenous material should remain in the tissue.

The main features of our proposed therapeutic device:
  1. Production of therapeutic proteins within the organism (no need for purification)
  2. Use of cell microencapsulation to allow application of the same type of engineered cells for all patients to avoid immune response, increase the reliability and predictability of the response and decrease cost
  3. Microencapsulation of cells allows localized release of therapeutic proteins in the affected tissue, which can decrease the systemic side effects
  4. Regulation of production of different therapeutic proteins appropriate for different stages of therapy. Switching between the different states of therapeutic cells is possible by a short pulse of the inducer, that can be applied to patients orally
  5. Introduction of an escape tag for the elimination of cells that may escape from microcapsules through augmented targeting by natural killer cells
  6. At the end of therapy the alginate microcapsule shell is degraded by a secretory alginate lyase followed by induced apoptosis of the therapeutic cells

Figure 1. Microencapsulated engineered cells for the production and delivery of biological therapeutics.
An important advantage of the microencapsulated cell approach is that the therapeutic protein can reach local therapeutic local concentrations in the affected tissue, while maintaining a low systemic concentration, thus avoiding systemic side effects, such as systemic immunosupression. Production of protein therapeutics by cells can also eliminate the need for the repeated invasive administration of therapeutics, which are typically applied by injections. Repeated application of therapeutics into the liver, brain or other complex tissues, where local delivery could be beneficial, is quite difficult. We aimed to develop an implantable cellular device for the local, regulated and safe delivery of therapeutic proteins. The usual obvious solution would be gene therapy, which has, despite its early promises, not delivered many successful clinical applications because of the problems of viral delivery and variable effectiveness due to the random site of integration. Long term survival of encapsulated cells beyond months may be problematic and their efficiency may decrease, therefore we should focus on the therapy of acute conditions.

The desired properties of synthetic cellular devices for the next generation of cell-based therapies
  • Control over the selection of different states of production of therapeutics or their combinations during different stages of therapy should be possible from the outside by a physician
  • The ability to inactivate and destroy cells of the device at the end of the therapy, leaving as little remains as possible and elimination of any cells that might have escaped from microcapsules

The synthetic biological components that needed to be developed

Universal orthogonal bistable toggle switch for mammalian cells

Figure 2. Regulation of the several different states of therapeutic cells based on several orthogonal toggle switches.
The existing genetic switches used in mammalian cells are mainly based on the available prokaryotic transcriptional regulators, whose number is limited and whose properties may differ, hindering balanced switching. Our idea was to investigate the development of genetic switches based on the designed DNA-binding domains, such as TAL effectors that can achieve high orthogonality and support simultaneous introduction of several toggle switches into mammalian cells. Although designed transcription factors have been created based on modular DNA binding proteins, such as zinc fingers and TAL effectors, to our best knowledge there have been no reports of genetic switches based on these types of elements. These devices would be extremely valuable since we could produce numerous orthogonal switches allowing development of complex functions and therapeutic devices.

Safety mechanisms for therapy with microencapsulated cells

For the safe therapeutic implementation of microencapsulated engineered cells in the organism we envisioned that we should be able to cease the therapy without having to physically remove the capsules.

Figure 3. Safety mechanisms that we planned to introduce for the application of microencapsulated engineered mammalian cells in therapy.

Microcapsule degradation - Most of the therapeutic microcapsules are composed of polymerized alginate that is biocompatible but cannot be degraded in mammalian tissues. Alginate lyase harvested from a marine microorganism effectively degrades alginate, so secretion of this enzyme from cells should degrade the capsules and allow their resorption by phagocytic cells.

Termination - We need to be able to inactivate the implanted cells at the end of the therapy or at any other stage if the therapy has to be terminated, regardless of the cause. We introduced a safety mechanism that would enable controled induction of apoptosis of the implanted cells. Therefore we could would inactivate them and enable their resorption without causing inflammation.

Escape tag - ensures that any cell that may escape from a microcapsule is eliminated by the immune system of the host organism. The escaped cells would most likely be recognized as foreign in any case but in order to provide maximal safety measures we decided to introduce an additional tag that would alert the host immune response.

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