Current treatment

Current treatments of IBD aim to relieve acute flare-ups or at maintaining remission (this means that medication aims to avoid the occurence of a flare-up). This is done with a combination of 5-aminosalicylic acid therapy and steroids, or TNF-alpha antibodies. Other types of medications that are sometimes used are immunomodulators and antibiotics^[8].

Even the most advanced forms of treatment only have a 50% success rate and lots of side effects ^[9]. They also affect the immune system. The side effects of current therapies can be blamed on the systemic method of administration of these drugs^[10]. It is common for patients to get lung infections as a result of the medications they use, since they cause weakening of the immune system^[11]. Furthermore, other common side effects are headaches, abdominal pain, diarrhea, nausea, vomiting, muscle or joint pain, loss of appetite, and more. Working on alternative approaches is therefore necessary to minimize patient discomfort and hospitalization time, and to improve the success rate^[7].

The financial burden of IBD on the health care system is also significant. Average costs of treatment in the United States amount to 26 thousand dollars per patient per year. Furthermore, the costs of treatment are highly correlated to the course of the disease. Patients with worsening of their disease sometimes need expensive hospitalization time and surgeries^[12]. This means that it is extremely important to have ways to keep patients in remission so that no additional interventions are needed.

Please click on the dropdown menus below to learn more about current treatments on the market.

Our solution

Treating IBD is not yet possible without many side effects and a lot of money. To solve these problems, team iGEM TU-Eindhoven genetically engineer E. coli to turn them into a probiotic. This resulted in the development of cELPro. As shown in Figure 5, the bacteria were inserted with two plasmids, one of which produces Elastin-Like Polypeptides (ELPs) that form an intracellular hydrogel, and the other Interleukin 10 (IL-10). ELPs are synthetic biopolymers that have an affinity for each other based on hydrophobic interactions^[20]. To increase the affinity even further, they were equipped with complementary Leucine zipper cross-linkers attached at their ends. The hydrogel forms in a temperature-dependent manner based on these interactions^[21]. The goal of the hydrogel is to make sure that the bacteria stop dividing, which makes them safer to ingest. They are not able to grow out of control anymore once the gel is formed, but they can still produce the therapeutic IL-10. This makes the release of IL-10 controlled and provides a great alternative to current treatment options.

The bacteria are freeze-dried and put in a pill that the patient takes regularly when they experience a disease flare-up (Figure 6). The pill has a coating that can withstand the acidic environment of the stomach. The bacteria reach the gut alive, containing the hydrogel, where it produces IL-10 to locally treat IBD. IL-10 is an immunoregulatory cytokine that has the potential as an anti-inflammatory biological therapy for IBD. It has been used in research for treatment of IBD with promising results, but since it was often administered systemically, a high concentration was needed, causing many side effects^[22]. By administering IL-10 locally with bacteria, these problems are tackled. In earlier research, it has been tried before to use bacterial strains that secrete IL-10 to treat IBD in mice with promising results^[23]. Additionally, there has even been a promising study in human subjects who experienced almost no side effects^[24]. However, biocontainment remains a challenge and often requires many steps to make sure the bacteria do not grow out of control outside of the patient. Solutions with transgenic bacteria have been tested before, but the route to make these bacteria is very labor-intensive^[25]. This problem will be solved by our technology, since it will be easier to develop and more scalable. The sections below will explain the parts of our idea further.

Elastin-like polypeptides (ELPs)

ELPs are protein polymers which are derived from human tropo-elastin. One of their key features is that they exhibit a phase separation behaviour that is often reversible. Samples remain soluble below a certain transition temperature (T_t) but form micelles or aggregates above T_t (Figure 7). For ELPs many applications have already been found in purification, sensing, activation, and nano assembly. Furthermore, they are non-immunogenic, can be substrates for proteolytic biodegradation, and can be decorated with pharmacologically active peptides, proteins, and small molecules^{20}. This makes them very interesting for therapeutic applications. Furthermore, recombinant synthesis allows precise control over ELP properties and weight, which results in protein polymers with uniform physicochemical properties that can be used in the design of multifunctional biologics^[20].

The general structure of polymeric ELPs is (VPGXG)ⁿ, where the monomeric unit is Val-Pro-Gly-X-Gly, and the "X" denotes a variable amino acid guest residue that impacts the properties of the ELP, such as the T_t. Specifically, the hydrophilicity or hydrophobicity and the presence or absence of a charge on the guest residue affects the Tt. Furthermore, the hydrophobicity of the guest residue can affect the T_t. The "n" denotes the number of monomeric units that the polymer is comprised of^[21]. In general, ELPs are linear below the T_t, but aggregate into spherical clumps, or coacervates, above the T_t^[26].

The TU-Eindhoven 2023 team used these proteins for the formation of the hydrogel in E. coli. The repeating sequences used were (VPGIG)ⁿ and ((VPGAG)3(VPGGG)2)ⁿ which creates a hydrophobic and hydrophilic part in the chain. To make the affinity for the proteins even higher, complementary Leucine zipper domains were also added to the protein. Leucine zippers are amphipathic helices, two complementary zippers are able to dimerize. Figure 8 and 9 show the structure of the designed proteins and their interaction.

Interleukin 10 (IL-10)

Interleukin 10 (IL-10) is an anti-inflammatory cytokine made by most cells of the immune system. It acts via limiting immune responses during infection, allergic reactions, and autoimmunity. IL-10 plays a central role in the prevention of immune-mediated damage to the host. A schematic representation of the function of IL-10 can be seen in Figure 10. However, because of its immunosuppressive effects, too much IL-10 can cause chronic infections^[27]. This is part of the reason that systemic use of IL-10 has not been successful yet in the treatment of IBD. Other reasons include that the concentration in the bowel does not become high enough to see therapeutic effects when systemic administration is used. Furthermore, there might be differences among individuals depending upon disease phenotype/severity. It is also possible that IL-10 is only successful at preventing and not treating the disease. More reasons that IL-10 does not work well when used systemically are that IL-10 alone fails to suppress all the pro-inflammatory mediators involved in chronic inflammation or it might be possible that IL-10’s immunostimulatory effects counterbalance its immunosuppressive properties^[25].

Even though IL-10 has not been proven to be successful in the use of systemic treatments, it is an interesting protein to use in different, more local applications like the solution from the TU-Eindhoven team described above. There have been successes with the use of IL-10 in the treatment of IBD with bacteria before, such as a clinical trial and study in mice using transgenic bacteria that produce IL-10, locally in the bowel^[24][23]. The patients who participated in this study experienced almost no side effects, but they did see a decline in symptoms of their disease. This is why the TU-Eindhoven team focusses on this protein as a therapeutic.

Advantages

By forming the hydrogel, the bacteria become unable to divide. This concept has been tested with an intracelluar PEG polymer-based hydrogel before^[28]. Upon deviding the bacteria were not able to divide anymore. The TU-Eindhoven 2023 team wants to use this approach but with synthetic biology to make a protein-based hydrogel. An advantage of this approach is that all the machinery to make the hydrogel is present in the bacteria, which makes the approach more scalable and affordable. The system requires the addition of the plasmid to the bacteria that encodes for the proteins that form the hydrogel and another one that encodes for the therapeutic protein before they are grown in large quantities. When the hydrogel needs to be formed, all that is needed is to increase the temperature to above the transition temperature of 21 degrees Celcius. The method with the PEG polymer-based gel requires the bacteria to be cultured first before equipping them with the parts that form the hydrogel, which results in more production steps. Furthermore, the production of bacteria is generally easier than the production of complicated antibodies, which makes this solution more scalable compared to current treatment options as well.

Another aspect that makes cELPro more affordable is the fact that it can be administered as a pill. This means that the patient will not have to visit the hospital for injections from trained physicians as often which reduces healthcare costs.

The fact that the bacteria stop dividing makes them safer to consume compared to bacteria that would be dividing, since our bacteria cannot grow out of control or easily mutate. This means that they do not change the microbiome of the patient.

Eventually, the system can be used to create a modular platform to cure a variety of intestinal diseases in a non-invasive way. In conclusion, the treatment is a local, non-invasive, scalable, and affordable alternative to current options for IBD treatment.