Abstract

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 1OO 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. The 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. Once we have these bacteria, we gatehr them in a capsule designed for target delivery in the duodenum and the jejunum and retain Iron Coli in this area to give it enough time to produce enterobactins. The iron is then chelated 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 in case it was allowed by the national agency of drug safety. The majority (56%) answered "yes". Additionally, by the use of a Ffault tree, we screened all the possible major consequence of this kind of bacteria in the intestine. We figured out that most of the danger is caused by the bacteria staying permanently in the intestine. This is why we favored a strategy based on the bacteria staying temporarily in the intestine. 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 iron in the duodenum and jejunum to prevent the absorption. Based on an ODE model, they came to the conclusion that the iron absorption can be divided by two by considering the enterobactins being instantly produced and the flush alsting 42 seconds. This reduction is especially advantageous for working patients. Blood-lettings are time-consuming and reducing its frequency is a huge benefit for the patients.

Now that we knew the project was being accepted by the patient and the strategy being viable, we focused on the biology. Our strain, Iron Coli, has the ability to sense iron, reverse its behaviour and produce enterobactins in case of high iron concentrations. The sensor is defined after we screened all the promoter regions that we knew were under the control of variations of iron. 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 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. This justifies the need of an inverter where we reverse this behaviour by replacing the downstream by a 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