Team:Montpellier/Description
PROJECT DESCRIPTION
KARMA : Katch And ReMove Antigens
Our project aims to develop a new molecular tool “KARMA”, capable of cleaving a specifically targeted protein in a complex environment. This idea was first inspired by the DNA editing tool: CRISPR/Cas9.
Since the role of many proteins in pathologies is known and well-characterized, it would be very interesting to have handy a programmable tool capable of cleaving these targets in a specific way. With the CRISPR tool, researchers can target a specific region of the genome in order to cut it. So we asked ourselves how can we come up with such a programmable tool to cleave proteins
KARMA anatomy...
KARMA is a tool designed to degrade specific proteins, which means that if we’re dealing with a mix of different proteins, only the targeted one will be degraded. The specificity of the reaction is achieved by the fusion of two components: a VHH (=Nanobody) and a protease (=a protein that degrades proteins). The fusion of these two components is called KARMA.
“For this tool we will first need a homing missile, that will specifically seek for the protein of interest and bind to it, leading the molecular scissors.Antibodies seemed to be the best solution, but an entire antibody is complicated to produce experimentally because complete antibodies require certain posttranslational modifications and maturations that take place in eukaryotic systems. In comparison, VHHs (nanobodies) have the advantage of being much smaller ( > 15kDa) and have affinities in the range we were looking for to increase the specificity of our protease. In addition, VHHs can be easily expressed in prokaryotic systems.
“The second part of the KARMA tool is the “scissors” that will cut the protein of interest. In principle, we should use an aspecific protease, such as trypsin (an endopeptidase). Thus the endopeptidase would have the ability to degrade a protein by cleaving it several times. Importantly, to decrease non-specific degradation, the protease activity should be attenuated (by mutations for instance). Addition of the specificity module (VHH) will increase the binding efficiency on specific targets, and thus specifically restore the degradation activity on these targets. ”
“In order to combine the two fractions of our tool, we had to think of a way to attach them by taking into account the steric clutter that each molecule can have on the other. That’s why we thought it would be better to use a flexible linker to space our two parts rather than directly fusing the protease and the VHH together. The linker is therefore composed of an amino acid sequence (GGGGS)n. In our tool, this sequence is repeated up to 6 times in order to vary the size of the linker.
State of the art:
In a pathology when a protein such as an enzyme, for example, is newly identified and plays an important role in the development of the disease, it becomes a therapeutic target that we aim to inhibit, destroy or cleave. Currently, the most common strategy is to use inhibitors: molecules that are capable of interacting with the protein and block its action by steric obstruction of its site of action, or by changing its conformation.
The development of molecular inhibitors is a long and complex process. Developing these molecules requires both structural bioinformatics and threedimensional model of proteins/organic molecule who could potentially inhibit them. Then you need to carry out a molecular bank screening to identify the right inhibitory molecule, it is difficult and very long. Here’s an example [1]
Recently, a new chemical approach has been developed to cleave intracellular protein targets known to interact in various pathologies. PROTACS (Proteolysis Targeting Chimera) is composed of 2 active domains: a ligand capable of binding specifically to its target and a molecule capable of ubiquitinating the target, thus triggering the recruitment of the proteasome. This results in the degradation of the target by proteolysis.
But there are some caveats with this technique, some cells don’t have the best working proteasome ever, ubiquitination doesn’t always lead to degradation by the proteasome and this technique can only be used in cells.
With our tool we tried to combine the best of those two strategies. Specificity and degradation. KARMA is composed of a protease for cleaving activity and an antibody for specific targeting. It has the advantage of exploiting a repertoire that has long been underestimated and yet is growing rapidly, proteases. Their therapeutic potential has long been ignored despite their clinical applications in recent decades [2] In the living world there are all kinds of proteases capable of degrading all kinds of proteins. KARMA will also use another extremely rich repertoire that has been growing rapidly in recent times, the antibodies in therapeutic treatments. The usefulness of antibodies in therapy has been widely demonstrated, there is to date a wide variety of antibodies capable of specifically targeting a large number of targets. In addition, the theoretical diversity of antibodies is about 5.1013, i. e. an infinite number of combinations for an infinite amount of targets.
KARMA's strength resides in being a modular tool that draws on two extremely rich directories, proteases and antibodies, to adapt to a wide range of applications.
Application for antibiotic resistance:
We are currently losing a biological war with antibiotic resistance. This war began in 1928 when Alexander Fleming first isolated an antibiotic that he called penicillin. He observed that this molecule prevents the development of bacteria. This molecule was then used for its therapeutic potential to fight infections. It took a few years, until 1940 to be able to produce enough of it and introduce it clinically for the first time in 1943. Its release was a therapeutic breakthrough... but not for long, only 3 years later, the first cases of resistance to it appeared. In only 3 years, bacteria gained the ability to resist to the molecule. Since then, antibiotic resistance has continued to increase with every single antibiotic that was found, and scientific research has been a real race against time in order to fight these resistant germs. Unfortunately, the development of new therapeutic molecules takes an average of 15 years while the average time for bacteria to resist slowly decreases.
Bacteria exposed to antibiotics evolve and develop defense mechanisms that makes them no longer sensitive to their effects. It is such a huge problem the World Health Organization predicts that, by 2050 more than 10 million people worldwide, will die due to antibiotics resistance. Making it the first cause of death, way ahead of cancer.
Resistance to beta-lactam antibiotics
Penicillin is a beta-lactam antibiotic (a class of antibiotics that have a beta-lactam nucleus). Bacteria that have acquired resistance to beta-lactam antibiotics are able to produce an enzyme that can break down this class of antibiotics before they reach their target..
KARMA against beta-lactamases
In Montpellier we wanted to develop a new approach to fight this resistance mechanism. Currently, one of the methods to fight against beta-lactamase is to develop molecules inhibiting the enzyme, which takes a long time... we wanted to come up with another approach, by degrading the beta-lactamase with our molecular tool. Since beta-lactamase is an enzyme and therefore a protein, we thought that a non-specific protease may be able to cleave beta-lactamase. To be able to use this enzymatic degradation approach, we had to address the specificity problem of a non-specific enzyme, the solution was the antibody fused to our protease.
Advantage and innovation
With our tool, we will not only inhibit beta-lactamase, we will degrade it ! This has several advantages over the usual strategy used to develop inhibitory molecules. The development of specific inhibitors is very long (an average of 10 to 15 years) and relatively complicated. An inhibitory molecule operates under the principle of a "key-lock", these molecules (the key) go inside a specific pocket (lock) of the enzyme decreasing or completely inhibiting the activity of the enzyme. But bacteria evolve very quickly, they are able to slightly modify their pockets so that the keys can no longer fit in. Some emerging beta-lactamase compounds have shown resistance to all inhibitory molecules marketed in the pharmaceutical market. (See Beta-lactamase NDM-1).
Compared to classical inhibitors, the use of a non-specific protease to cleave beta-lactamase is very interesting to solve the resistance issue: even upon mutation, beta-lactamase remains a protein composed of amino acids and will therefore always be sensitive to the activity of an aspecific protease such as trypsin. One might argue that the protein may mutate to escape VHH recognition but it’s faster to obtain multiple VHH against a target than molecules.
A strong proof of concept...
The idea of fusing a protease to a VHH in order to achieve specific protein degradation may look good on paper, but what if our theory does not work ?
Moving on directly to a concrete application like beta-lactamase degradation would have been too complicated to characterize for a first try. So we decided to first carry out a solid proof of concept, which requires:
a well-characterized protease: we chose the TEV protease, which has a well-known consensus cleavage site. Also, its optimal temperature is 30°C and we hoped that its activity would be attenuated at 37°C.
a degradation reporter: we chose to monitor fluorescence increase as a readout for degradation. So we used a fusion between sfGFP and a degradation tag (SSRA) and added a TEV cleavage site in between.
a specificity module: we fused a VHH against GFP to the TEV. Sequence analysis suggested that it should bind to sfGFP.
a negative control: we made similar degradation reporters with RFP instead of sfGFP. As the VHH does not bind to RFP, these constructs are used as negative controls and mimic the non-specific proteins present in the vicinity of the target proteins and KARMA.
KARMA for proof of concept :
For the specificity module, we used an anti-GFP VHH. We were unable to find an anti-sfGFP VHH, however, by analyzing the epitope of the anti-GFP VHH we were able to see that it should bind to the sfGFP (7 amino acids out of 9).
For the protease, we chose to work with the TEV protease which is a very specific protease, (consensus : ENLYFQ/S). Since TEV is a widely used protease in biology, it was easier for us to work with a very well characterized and safe protease so that we could control our experiment.
Reporter gene:
To perform our proof of concept, we used a well-known reporter gene system by fusing a degradation tag (ssrA) [3] to sfGFP. The ssrA tag is a small peptide sequence that is targeted to an endogenous E. coli recycling machine called ClpXP. Adding a ssrA tag to a protein significantly reduces its half-life. sfGFP-ssrA expressed in a bacterium will, therefore, produce less fluorescence than sfGFP. In order to evaluate the enzymatic activity of our tool by fluorescence restoration, we added a consensus cleavage sequence of the TEV protease between the sfGFP and the ssrA tag (sfGFP-TEVcs-ssrA). Thus when the TEV protease performs its cleavage activity it separates the proteolysis tag and prevents ClpXP from cleaving the sfGFP. The stronger the protease activity, the higher the fluorescence.
Education and public engagements
Although the scientific quality of a project has a very important place in the iGEM competition, this does not reflect the whole spirit of this competition. Science must be accessible to all and it is extremely important in an iGEM project to communicate and raise public awareness of the different themes addressed. Our project is above all a story, an adventure that we must tell. In order to get closer to the general public on projects and life sciences in general, many iGEM teams choose to organize various scientific conferences or events in the universities where they study. However, the spectators of these events are often already largely aware of the sciences field. People who live in more isolated places living in remote villages, for example, are only marginally affected by these types of events, which take place mainly in large cities, where universities and major cultural centres are located. In order to overcome this problem, we have chosen this year to popularize science through a somewhat innovative means, a caravan, which gives us the necessary mobility to reach this public.
To this end, thanks to the collaboration of one of our university teachers, Boris Chenaud, who agreed to lend us his Cara-Science. It is a small caravan with a purpose and the opportunity to chat face to face with a science explorer.
Thus Le Cara-Tour was born and we travelled through town and village where the carascience could stop in various places (market places, main squares, festival, park etc...) in order to be able to meet directly with the public so that they can discuss with us about our project and science in general. Thanks to the BiologyEcology Department of the Faculty of Sciences of Montpellier, scientific equipment (microscope, magnifying glass, etc.) has been provided in the cara-science to carry out experiments for adults and children in order to stimulate scientific curiosity.
We also participated in the Science Festival with our own stand where we welcome the public to explain synthetic biology through an exhibition on the comparison of superhero superpower with some bacteria that may possess identical powers. Various activities have also been carried out for the younger ones such as bacterial painting, or the creation of DNA sculptures.
Collaboration & Integrated Human Practices
One of the medal criteria of the competition is based on team collaboration. We attached particular importance to this challenge because we also thought that in a society it is very important to be able to work between teams regardless of the issues. We are therefore all very sensitive to the ecological issues of our society and we wish during our iGEM adventure to have an ecological part concerning our project. Thanks to the iGEM Nantes team, which organized an art exhibition on synthetic biology, we invited them to exhibit a work of art that we provided to them in order to raise public awareness of the impact of the research activity from an environmental point of view.
We had therefore collected for 3 weeks the various consumable waste materials necessary for our experiments, which we had previously cleaned and disinfected, then painted them and finally made another work of art. During this collaboration and the design of our work of art, we carried out various surveys on the different waste treatments of the laboratories on behalf of the iGEM teams and laboratories around us, in order to produce some statistics to realize the environmental impact that science can have and perhaps reflect on potential new solutions.
Reference :
[1] (Doman et al., J. Med. Chem., 45, 2002, 2213-2221 (Pharmacia Corp.)): Out of a bank of 400,000 molecules tested, only 85 molecules were found to be active (0.021%). Then on the 85 molecules, there is no guarantee that they can be used clinically (toxicity, cross-reactivity etc.)
[2] "Craik, Charles S., Michael J. Page, and Edwin L. Madison. 2011. "Proteases as Therapeutics". Biochemical Journal 435(1): 1-16."
[3] : Guiziou, S., Sauveplane, V., Chang, H.-J., Clerté, C., Declerck, N., Jules, M., & Bonnet, J. (2016). A part toolbox to tune genetic expression in Bacillus subtilis. Nucleic Acids Research, gkw624.