Team:TU-Eindhoven/T-Cell

iGEM TU Eindhoven

Kill Switch
Kill Switch

To make genetically modified cells or bacteria safe, it is important to include them with safety mechanisms. A common safety mechanism is a kill switch. A kill switch induces the apoptosis pathway of cells. This can be used when genetically modified cells do not fulfil their purpose (anymore), or when they show odd behaviour or any other case in which it could be safer if the genetically modified cells were killed.

One of the most important things about a kill switch is that it can be triggered at a precise moment. Our scaffold protein can help to accomplish that.

Our scaffold protein can play a role in safety mechanisms by working as a kill switch. Our scaffold proteins can function as a small molecule induced kill switch. This kill switch can be created when apoptosis inducers, that need to be in close proximity of each other, are linked to the CT52 protein. Now when CT52 binds on the scaffold, which is only possible in presence of fusicoccin, the apoptosis inducers are in close proximity of each other and can enable the apoptosis pathways. The apoptosis inducer that can be used is caspase-9. If caspase-9 dimerizes, apoptosis pathways will be activated. To make a more efficient kill switch, the tetrameric scaffold protein can be used. Because the tetramer has 4 binding pockets, it is expected that this will result in higher local concentrations compared to the dimeric scaffold protein.

Figure 1: T14-3-3 kill switch on moleculair level

For the kill switch to work, the tetrameric scaffold protein and CT52-caspase-9 need to be built into the DNA of a modified cell. After that the kill switch is present inside the genetically modified cell, but still inactive. When fusicoccin is added locally, in the environment of the cells that need to be killed, this will induce binding of CT52-caspase-9 to the scaffold. This will cause caspase-9 dimerization, which initiates the apoptosis pathways.

We think this could be a useful addition to the safety mechanisms in genetically modified cells. An advantage of this system is that activation depends on the concentration of fusicoccin. Increasing the concentration at specific locations can make the system very specific and precise. Another advantage of this kill switch is that it triggers the apoptosis pathways, this is a clean way to reduce the amount T cells, because no necrosis is involved. Also, the usage of the small molecule fusicoccin is not harmful for cells in the environment of the cell that will be killed, since fusicoccin is not harmful to cells in the concentration in which the kill switch will be used.

There are several applications of this kill switch. It is applicable on almost all modified cells/organisms. One possible application of the kill switch will be elaborately illustrated. Currently, a new cancer therapy is in development. This therapy is called T cell therapy. In this cancer treatment human T cells are extracted from the body, modified so they can recognise cancer cells and placed back in the human body again. Because modification of human cells is highly controversial, a lot of safety measures are needed. See below how our scaffold protein can contribute to this by functioning as a kill switch.

Application T Cell Therapy

Cancer is a term for a group of diseases with the same characteristics. Currently cancer is the second most common cause of death1. Cancer is caused by disturbances in signalling pathways. For example, apoptosis pathways can be blocked.

Cancer can occur in several forms. At this moment, there are several treatments, but none of them has proven to work with enough precision and effectivity. Now, the most important cancer treatments are chemo therapy, surgery and radiation therapy. All of these treatments do not specifically target cancer cells, but a target area is used.

The promising new cancer treatments differ from these treatments because they aim to target cancer cells specifically. For this application scenario the focus will be on one specific treatment: T cell therapy.

The problem of cancer is that cells do not work properly due to mutations. This can for example effect in cells that are not capable of detecting cancer cells. T cell therapy fixes this problem by extracting T cells from the human body, modifying them so they can recognize cancer antigens better and then placing them back in the human body2,3. These modified T cells have a greater ability in recognizing cancer cells and eliminating them than the non-modified T cells.

The big problem with this treatment is that it involves modifying cells of a patient and inserting them back in the patient’s body. Currently this is highly controversial.

Our scaffold protein can play a role as a safety mechanisms that obviously need to be implemented in this treatment, because altering human cells is highly controversial. Our scaffold proteins can contribute to T cell therapy by functioning as a small molecule induced kill switch.

Figure 2: Schematic representation of a modified T cell with T14-3-3 kill switch built in.

When the tetrameric scaffold protein and CT52-caspase-9 are built into the human T cells, these cells are already altered to recognize cancer cells again. When modified T cells show unexpected moving patterns, like amplifying themselves too much or show other irregularities, fusicoccin can be added locally which will induce caspase-9 activation. This will lead to apoptosis of the modified T cell.

We think this could be a useful addition to the safety mechanisms concerning T cell therapy.

References
  • [1]FastStats. (2016). Cdc.gov. Retrieved 31 July 2016, from http://www.cdc.gov/nchs/fastats/leading-causes-of-death.htm
  • [2] Yuhas, A. (2016). Cancer researchers claim 'extraordinary results' using T-cell therapy. the Guardian. Retrieved 31 July 2016, from https://www.theguardian.com/science/2016/feb/15/cancer-extraordinary-results-t-cell-therapy-research-clinical-trials
  • [3] Harpe, M. & Mount, N. (2015). Genetically modified T cells in cancer therapy: opportunities and challenges. Disease Models & Mechanisms, 8(4), 337-350. http://dx.doi.org/10.1242/dmm.018036

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