How do we see our project?
Our project is higly fundamental. We understand how much examination is to be done before it could enter the real world as an obesity treatment. However, we see many different implemetations of our result, which can help better understand the LEAP-2 role in obesity, or design different bacteria secretion systems. Let's speak a bit about them all!
- Usage of data on LMWP effects on secretion in designing of different secretion crcuits
- Usage in physiology to study the ghrelin - LEAP-2 interactions
- Usage as an intranasal probiotic for obesity treatment (the most distant one)
Cell penetrating peptides, or CPPs are well known for their ability to allow polar molecules pass lipid membranes from the outside. But what would happen, if bacteria tried to secrete a protein with an attached CPP? This question still has no answer, so we had no other option, but to learn it by ourselves. The exact mechanism of CPP function is unknown, so our findings may lead to a new discovery at that field. We are going to try only one CPP - low molecular weight protamine, because CPPs are not the key point of our study, however a secretion assay is important for us.
In the beginning of our project development we have consulted a local physiologist, who specializes on weight regulation mechanisms. She was rather doubtful, about LEAP-2 would even work, as we had expected, because numerous compensatory effects would appear. Nevertheless, LEAP-2 remains on the scope of weight physiologists, and our bacteria could provide an interesting model for further non-invasive investigation of its physiological role, whether as a source of recombinant LEAP-2 for nasal infusions, or even as a mice symbiont.
We understand the gravity of clinical trials, and how much is to be done before we could proceed to just mice tests, not to mention next stages. That's why interim benefits are essential. However, our main objective is to create a symbiotic bacterium for obesity therapy. Our survey with almost 200 answers shows, that more than half of respondents would be interested in such medication, and most of them don't mind a probiotic to be intranasal, thus, there is a request for our project among potential customers. Many people have commented that validation of safety and effectiveness is the most important in drug development.
In vitro model
The article, on which we based our idea of making a fusion protein with the cell-penetrating peptide, LWMP,  states that the mechanism by which their fusion protein travels to the hypothalamus via the olfactory and trigeminal nerves is unclear. It is speculated that endocytosis and macropinocytosis play an important role, however, the intra-tissue penetration mechanisms are “largely unknown” and most likely occur via an extracellular mechanism.
Therefore uncovering the underlying mechanism behind this type of delivery is a point of interest to us, and an interesting area of research in itself. To account for both potential mechanisms we decided to use two simple in vitro models.
The first model would test for paracellular penetration via “leaky” tight junctions and would be a simple ALI monolayer system, similar to the ones we were originally researching . The cell type isn’t crucial, provided the cells in the monolayer form tight junctions. In this model, we would also test the interaction of our protein with mucins in an artificial mucosal layer.
The second model would test for intracellular transport in neurones. We plan to use a microfluidic model to remove the effects of diffusion, similar to the one shown in the image . In the neurones, we would monitor the localisation of LEAP (i.e. in lysosomes, the Golgi apparatus, the ER). We can do this with the help of the Myc-tag found in our protein via immunostaining or replace the Myc-tag with a fluorescent tag. In the case of intracellular mechanisms of transport, we would, ideally, find our protein in synaptic vesicles and demonstrate synaptic transmission.
As a final point, though in vitro models have many advantages, even the most complex ones cannot account for all of the interconnected physiological effects happening in the organism. Our models, for example, do not take into account the effect of gravity - the olfactory epithelium the mucus washes over is “upside-down”. Nor do these models account for the fact that the volume of the olfactory bulb can fluctuate in response to changes in the sense of smell . On ethical grounds, an additional step may be added after our in vitro models and prior to doing any fully-fledged in vivo tests on mice - a model using murine olfactory epithelium slices, for example. In any case, while more complex experiments such as these do fit in our vision of the implementation of our project, they cannot be realistically done in the timeframe of the competition and we consider them to be areas of future research and further development.
Nasal transport in vitro proof summary
- Epithelium permeability examination: use an epithelium layer explant to check if our protein could bypass tight cell junctions
- Neuron uptake examination: use an olfactory neuron culture to test the neuron uptake of our protein and its localization inside the cell
As for the closest after iGEM plans, we are going to complete everything yet uncompleted with the secretion assays. Not everything has gone according to our plans, but so is science. Next step, would be the test of nose-to-brain transfer possibility on neuron and epithelial cell cultures. We will have two separate experiments to make the mode of passage for our protein clear, because it influences the physiological effect greatly.