Project Description

Abstract

The BiG-IF project aims to develop a new treatment for pernicious anaemia (PA), a severe form of vitamin B12 deficiency. In pernicious anaemia, the body has an autoimmune reaction towards the intrinsic factor (IF), a crucial carrier protein that allows vitamin B12 to be taken up in the gut after consumption.

Our idea is to engineer a recombinant IF to which pernicious anaemia autoantibodies cannot bind. This is done through site-directed mutagenesis of the autoantibody binding site, while making sure that the vitamin B12 affinity remains unaffected. This modified protein could then be cheaply produced in bacteria and easily taken in pill form, allowing the patient’s B12 absorption ability to be restored.

Pernicious Anaemia

Vitamin B12 is one of the essential vitamins due to its importance in metabolic effects: It acts as a cofactor in DNA synthesis, in both fatty acid and amino acid metabolism, plays a role in the synthesis of myelin, is essential for the nervous system, and is necessary for the maturation of red blood cells in the bone marrow contributing to the circulatory system. The main sources of vitamin B12 are animal-derived foods, pharmaceutical compounds used in intramuscular and intravenous injection, namely hydroxocobalamin, and supplements in the form of cyanocobalamin.

Pernicious anaemia [1] is an autoimmune condition that prevents the body from absorbing vitamin B12, which is one of the leading causes of vitamin B12 deficiency. An affected individual will have fewer red blood cells carrying oxygen throughout their body. PA can be present for several years before a person with the disease may notice any symptoms. If left untreated, PA can lead to serious medical problems including irreversible damage to the nervous system. Patients with PA are usually prescribed vitamin B12 supplements as treatment.

Prevalence

PA is widespread across all continents and the prevalence of the disease is 0.1% in the general population and up to 2% in ages 60+ [2], depending on the diagnostic criteria. However, the prevalence is probably underestimated as a result of the complexity of the diagnosis. The mean age of patients ranges from 59 to 62 years. PA is more common in people with African or European ancestry than in those with Asian ancestry. The highest prevalence is seen in northern Europeans, especially those in the UK and Scandinavian countries [3].

Why Pernicious Anaemia?

When choosing a project topic, the team had a clear focus on medicine and diagnostics, as these were our main fields of interest. When we came across the condition of pernicious anaemia, we were quickly captured by its relevance. Not only can we potentially contribute to the treatment of a severe disease, but it also is locally relevant due to its prevalence in Scandinavia, as described above.

Furthermore, we came across a research article that described the exact location of the epitope of the protein to which the autoantibodies bind. This paper became foundational to the project, as it was the basis of our plan for where we could modify the IF to avoid the autoimmune response, allowing us to treat PA [4].

Road map

Protein design

To address the challenging task of protein design, we performed thorough modelling to select the best candidates to be tested in the lab. First, we designed our mutants based on the rationale of preserving structure as much as possible. This principle was chosen as the antibody binding region is in close proximity to the B12 binding region [4]. We aimed the mutagenesis to prevent intrinsic factor from binding to autoimmune antibodies while preserving ligand-receptor binding affinity.

Mutant selection

To verify whether the designed mutants would preserve properties of the wild type IF, protein structure predictions were performed [5]. This enabled us to refine the list of tested mutants by filtering out the ones with improperly folded structures.

As a final step of screening for candidates, we performed docking studies to ensure that the binding affinity of engineered protein to vitamin B12 is at least on par with the wild type IF. Docking studies were performed in 2 steps, first making the prediction followed by the energy refinement step [6][7]. As a result, we identified the most promising candidates to be tested in wet lab experiments. We found that most selected mutants have similar or better binding affinity to vitamin B12. These results indicate that our pipeline is adequate and applicable to the research question.

Mutant production

The first step of the process was to clone the IF gene into a vector followed by inserting mutations into the desired regions through site-directed mutagenesis. Starting from single-point mutants, subsequent mutations were added on top of these to sequentially achieve the desired double, triple and quadruple mutants.

Hypothesis testing

HEK 293 cells were transfected with the wild type and mutant variants of IF and the binding of autoantibodies was evaluated using western blot, fluorescence microscopy, and flow cytometry techniques. These experiments enabled us to test our initial hypothesis that mutagenesis in the known epitope of IF prevented the autoantibody binding.

Cherry on top

In addition to extensive work in both dry and wet labs, we developed a simple tool that facilitates protein design based on mutagenesis. Our software, called ProMutor, is a user-friendly web-based tool that would enable others to select protein candidates based on their requirements. ProMutor also has direct access to the well-known protein structure prediction tool called ColabFold. We utilized it for creating our pool of mutants for docking studies and downstream experiments.

Future prospects

Once an effective variant of our engineered IF is found, we would then continue with toxicology and efficacy studies, and assemble our IF with vitamin B12 in a pill as a medical product. We also plan to use bacterial strains instead of mammalian cells to further push down our costs of development and production. The idea behind this plan is to establish a novel drug development company with the settled template we created in our project. We can use this template to tackle problems in all autoimmune diseases in terms of improving treatment and diagnosis. With the results from our iGEM project, we would like to go ahead with much more detailed market analysis, while also furthering our research and bringing this product to fruition.

References