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| | == The "SWITCH" Project == | | == The "SWITCH" Project == |
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| - | ===Proteins in medical treatments=== | + | ===In a nutshell=== |
| - | In the past decade, proteins have gained increasing attention as therapeutic agents. Among them, monoclonal antibodies have gained a particular importance, since they present several advantages compared to standard drugs. Reduced risk of non-specific action, longer therapeutic effect and smaller risk of toxicity upon elimination are just several advantages monoclonal antibodies possess when used as drugs.
| + | Our project is all about implementing a light activated genetic switches into mammalian cells. The production of complex proteins that require proper folding is becoming more and more commonplace in the pharmaceutical industry among others. Mammalian cells are an ideal environment to make these proteins but getting them produced at the right time and at the right rate requires an expression system keyed to a specific signal. Until now these systems have mostly been limited to chemical signals which have many drawbacks. By using a light activated expression system, we hope to get mammalian cells to respond to a light signal with gene expression. |
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| - | ===Problems with synthesis=== | + | ===A new expression system: LovTAP fusion protein=== |
| - | Despite all of their advantages, monoclonal antibodies are sometimes challenging to produce. The majority of them are manufactured by recombinant cells that are cultured in huge volumes (called “bioreactors”). In order to achieve a reasonable price for the final product, the antibody needs to be produced at a high concentration. Unfortunately, many antibodies are toxic to the producing cells at high concentrations, which means they get killed before a sufficient concentration of final product is reached.
| + | Our main goal this summer was to implement a previously untested expression system into our mammalian cells. The LovTAP-VP16 fusion protein we used was based on a previously used fusion protein, LovTAP, which binds to DNA at a specific site once it is activated with blue light. By attaching an activating domain to the protein that is know to work in mammalian cells we hoped to get it to bind DNA after exposure to light and promote expression of a gene next to its binding site. |
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| - | So far, this problem has been solved by using cells that are designed to produce the desired protein only when they uptake and sense a particular small molecule. With this technique, cells begin by growing normally and start to produce the desired protein only when the small molecule signal is present in sufficient concentration to trigger the production of the desired product at an optimal concentration, maintaining a balance between protein production and cell health. Unfortunately, this method has a couple of disadvantages. First, the “switch” molecule is often difficult and costly to remove. Second, redesigning cells so that they react to the “switch” molecule can perturb many biological pathways, which in turn decreases the efficiency of antibody production by disrupting normal cell growth and proliferation.
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| - | ===Cool solution===
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| - | We thought that the best solution for this problem would be a simple switch, that would activate the synthesis of a therapeutic protein in cells just by shining a specific wavelength of light on the cell culture. Our hope was that this method would have a minimal effect on cellular pathways and reduce the effect on cell health. This way the cells would grow happily in darkness and start producing the toxic protein only when light is supplied. With such a switch, there would be nothing to remove from the cell culture, allowing recombinant proteins to be produced more economically than by supplying large amounts of a small molecule trigger.
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| - | ===How are we going to do it: LovTAP protein===
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| - | A really nice way to realize our idea would be to use the LovTAP protein. When illuminated with blue light, LovTAP changes configuration and acts as a negative regulator of gene expression in bacteria, enabling the ability to switch on and off gene activity. Unfortunately, LovTAP does nothing in the mammalian cells, which are the chemical factories used for the synthesis of antibodies. We've decided to overcome this problem by attaching a VP16 viral promoter domain to the LovTAP protein, so that it will become a powerful positive regulator of gene expression in mammalian cells. If we transect the LovTAP-VP16 construct along with the gene of the protein we would like to synthesize (monoclonal antibodies) under the control of LovTAP, we could use it as the perfect light “switch” to control gene expression. It will activate protein production when mammalian cells are exposed to blue light and minimally disturb other cellular pathways.
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| | ===Another approach: Melanopsin=== | | ===Another approach: Melanopsin=== |
| - | An another nice tool to realize a light-induced switch would be to use a light receptor that already exists in mammalian cells, such as melanopsin. This way, the receptor will sense the light and transmit a chemical signal to the rest of the cell in order to activate the production of the desired protein. Even though this switch is more complex then the previous one and will affect several pathways, it still eliminates the need for an activating small molecule. In addition, it might be more efficient then the LovTAP-VP16 based switch, since it uses a protein that already exists in mammalian cells, and not a fusion protein.
| + | Our team also worked with another light activated expression system that has already been proven to work. Martin Fusseneger et al. have designed a pathway that makes extensive use of gene expression pathways already present in the cell. By adding a light activated receptor on the cell membrane they promote the release of calcium into the cytoplasm upon exposure to light. This activates the native response to an increased calcium concentration and promoter proteins sensitive to calcium will signal for the expression of genes with specific promoter sites. By introducing a new gene with a calcium sensitive promoter next to it, we can use light to induce its expression. |
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| | ===CHO cells & Co=== | | ===CHO cells & Co=== |
| - | In order to check the validity of our ideas, we attempted to implement both light-induced switches in CHO cells.
| + | The light activated expression systems we tried out would be extremely convenient for production of proteins in an industrial context and CHO (chinese hamster ovary) cells are the workhorses of industrial protein production. For our project we transfected these cells with each expression system and conducted a battery of tests. Along with this cell line we also chose to try them out on HEK (human embryonic kidney) cells since the melanopsin system had already been proven to work in them and we wanted data we could compare to our untested LovTAP-VP16 system. |
| - | For the LovTAP-VP16 switch, we've transfected CHO cells with LovTAP-VP16 encoding plasmid along with a reporter plasmid containing a protein that fluoresces in red (dsRed). We can tell that the switch functions correctly if after illumination with blue light we can detect a substantial increase of red fluorescence in the cells.
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| - | For the melanopsin-based switch, we will transfect a plasmid expressing a natural light receptor (melanopsin), along with a reporter that would synthesize green fluorescent protein in response to the signaling pathway that is naturally triggered by melanopsin. We will know the switch functions correctly if we can detect a substantial increase in green fluorescence in cell culture after illumination with blue light. | + | |
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| - | ===The Problem===
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| - | Producing complex therapeutic proteins requires biosynthesis in mammalian cells. Such proteins can sometimes have a certain level of toxicity for the cells and limit their productivity if they are produced constantly and are accumulating. To avoid this, the pharmaceutical industry use 'rewired' cells that synthesize toxic proteins only when a special molecule is added to the bioreactor. This solution has two disadvantages. First, cell rewiring affects several pathways and decreases cell productivity. Second, the 'special' molecule will mix with the final product and a purification will be needed to get rid of it.
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| - | The pharmaceutical industry needs an easier way to induce the production of a specific compound in mammalian cell.
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| - | This is where our "SWITCH" project steps in.
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| - | ===The Simple Switch===
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| - | We want to design a light-sensitive switch, so that the product will only be synthesized by the cells in presence of blue light, allowing them to grow happily in the dark.
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| - | The switch is based on an already existing chimeric protein, LovTAP. This protein was originally intended to act as a light-induced repressor in bacteria. The EPFL 2009 iGEM team proposed to fuse it with VP16, a viral activator, in order to convert it into a light-induced activator in mammalian cells. This way, it will be enough to add a LovTAP binding site in front of the gene of interest and a LovTAP encoding gene to induce protein expression without using any additional chemical or disturbing too many pathways.
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| - | This year we will try to realize this idea by transfecting CHO (Chinese hamster ovary) cells with two plasmids: one coding for the LovTAP-VP16 fusion protein and another with a read-out ( red fluorescence ) protein preceded by a binding site for LovTAP-VP16. If everything goes as expected, LovTAP-VP16 will only bind the plasmid and activate the production of read-out when exposed to light.
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| - | ===The Complex Switch===
| + | In each different cell line we tested a transfection containing an protein designed to et off expression in response to light and a target gene or readout. Instead of producing therapeutic proteins right off the bat we chose to express easily detectable fluorescent proteins so that we could compare the efficiency of both systems in turning on a particular gene. |
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| - | In addition to LovTAP switch, we will be realizing a more complex, melanopsin-based light switch, as described by Fussenegger et al. In this switch, light-sensitive melanopsin triggers a cascade involving calcium ion channels that eventually leads to the transcription of the gene of interest. The switch involves more complex signalling cascades, but it also uses only one natural protein and might function better then the simple switch. Fussenegger's team did this using HEK (human embryo kidney) cells, and we will also try to make it work on CHO cells, so that we can determine which switch works better in the CHO cells.
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