Overview

Our project focuses on developing a novel treatment for hematological disorders caused by an iron overload, such as hemochromatosis and thalassemia. These autosomal recessive disorders have symptoms including cirrhosis, arthritis, and heart failure, which result from overabsorption of iron from the duodenum. Although these are among the most common heritable diseases, treatment options are limited. Even today patients are mostly treated by frequent bloodletting, which many people cannot support. The aim of our project is to combat these diseases at the source by developing a therapy that prevents the intestinal absorption of iron.

We engineer the Escherichia coli Ferric Uptake Regulation (FUR) system using a genetic inverter so that they produce siderophores (iron chelators) in response of high concentrations of iron. These engineered bacteria are delivered to the patient's intestine by encapsulating them in an ingestible polymer (capsule) that specifically degrades in the duodenum. Once released into the intestine, the bacteria respond to ambient iron by secreting elevated levels of siderophores, thereby chelating the iron to prevent its absorption by the patient.

The Iron Coli Project deals with iron, and more precisely about the most frequent genetic disease related to that ion, hemochromatosis. In our human body, iron is essentially found under hemic structures. For example, iron is bound to hemoglobin inside red blood cells to transport oxygen. Moreover, iron binds to cytochrome and has a role in detoxification in the liver but also in energy production by the mitochondria.
So iron is essential to our human body for many metabolic pathways. In fact, it is finely regulated in our organism and unfortunately, the unbalance of this regulation leads to dramatic consequences. Our human body has a total pool of 4000 mg of iron. Every day, we absorb 1 mg by the upper intestine (duodenum and jejunum) and excrete 1 mg by tissue renewel (intestin, skin, hair, bleeding, etc...). However, a hemochromatosic patient absorbs on average 4 times more iron. On a long term, this patient accumulates iron in many organs which is very toxic in its free form. The only mechanism we have to decrease the blood level of iron is to store it in our tissues. Heart, liver, kidneys, muscles, all these organs start to store the iron, which enhances even more its toxicity. The main complications are chronic insuficiences such as heart, liver and kidney failures, but also intense fatigue and articulary pain.
Hemochromatosis is the most frequent genetic disease in Europe. The homozygous recessive mutation, C282Y, on the HFE protein is present at a rate of 1/300, but only 1 out of 100 have the symptoms for hemochromatosis. This mutation leads to the deregulation of the iron overabsorption from the intestine. The only efficient treatment available is to remove 500 mM of blood at a frequency from once a week to twice a year. This treatment, although efficient, is time-consumming and is sometimes barely tolerated by the patients.

Our project focuses on finding an alternative treatment to these blood-lettings. To do so, we used a natural system found in E. coli where the bacteria produce iron chelators, called enterobactins, in case of iron starvation. Enterobactins are chelating molecules that have an affinity of 10^39 M^-1 for iron. For our Iron Coli Project, we used the natural ability of E. coli to sense the environmental iron and reversed its behaviour to finally produce enterobactins in the presence of high iron concentrations. Then, we would like to put our engineered bacteria in a capsule designed to target the duodenum and jejunum and to release our bacteria in this area. The iron is then chelated by our Iron Coli and prevents the absorption from the intestine to the blood, which is our goal to cure hemochromatosis.

As a first step, it was crucial to ask for the patients opinion about such a bacterial treatment. Through a survey (N = 270), we asked them if they would take this treatment if it was allowed by the national agency of drug safety. A majority (56%) answered "yes". Additionally, by the use of a fault tree, we screened all the possible major consequences of this kind of bacteria in the intestine. We figured out that if our Iron coli stays permanently in the intestine, which means that it would colonize the intestine, it involves too much risks and danger. This is why we favored a strategy based on the bacteria staying temporarily in the intestine. That is what we called the flush strategy, where we want our E.coli to be flushed out of the intestin. This way, it would produce its enterobactins, the iron chelators, in the upper intestine and then die afterwards.

Our team first created a model to predict if Iron Coli can chelate sufficient amount of iron in the duodenum and jejunum to prevent the absorption. Based on an ODE model, they came to the conclusion that the iron absorption of a hemochromatosic patient can be divided by two, considering the enterobactins being instantly produced and the flush lasting 42 seconds. This decrease of the iron absorbed is especially advantageous for working patients as blood-lettings are time-consuming and reducing its frequency would be a huge benefit for the patients.

Now that we knew the project would be accepted by the patients and that our bacterial treatment would be beneficial to them, we focused on the biology side of our project. Our strain, Iron Coli, has the ability to sense iron, reverse its behaviour and produce enterobactins in case of high iron concentrations. To obtain our sensing device, we first screened the genome of E. coli in order to find promoter regions under the control of iron variations. Out of the 15 promoters we screened, only these following were evaluated: FepA, Fes, AceB and YncE. It came out the pAceB was the most sensitive to iron variations and we kept this one for further analysis. In fact, this promoter, as all the others, has the ability to repress the expression of the gene downstream thanks to an interaction with the Ferric Uptake Regulator (FUR) protein. This justifies the need of an inverter where we reverse this behaviour by replacing the downstream gene by the coding sequence of the LacI protein. Indeed, after cotransformation with a reporter plasmid containing pLacO-mRFP, the RFP is expressed in case of high concentrations of iron and repressed when the iron is low. Finally, the last step in the design of Iron oli is to produce these enterobactins. Their maturation requires 6 genes: EntA, EntB, EntC, EntD, EntE and EntF. We tried to solve a second issue by a model: what are the consequence by overproducing the enterobactins inside an E. coli strain? Using a FLux Balance Analysis, we came to the conclusion that the side pathways that use chorismate and isochorismate have a minor effect on the enterobactin production. Moreover, the enterobaction production reaches a plateau and finally, we need to add chorismate in the medium if we want to produce them. However, our third model predicts that these enterobactins are produced only after 5 hours after the iron in the environment is sensed. The cloning of these 6 genes was the hardest step and still, it remained unfinished