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Project Description

Introducing Project Lactoback

In our team, almost everyone knows someone suffering from endometriosis. Therefore, this project tackles an issue so very personal but also global, as it affects at least one in ten women worldwide.

Despite how common it is, this condition has no cure or satisfactory treatments; there is too little awareness of the debilitating nature of this disease, despite the high proportion of people affected by it in society.

We believe women deserve a timely diagnosis and appropriate treatment.
We believe women shouldn't find it normal to be in pain.

We want to address this problem from an often-overlooked perspective -
the relationship of this disease with the vaginal microbiome.


Endometriosis: The Problem

In endometriosis, some of the cells normally found inside the uterus are also found outside the uterus, where they undergo a monthly cycle, as if they were in the uterus, and shed. This leads to progressively worsening internal inflammation, scarring, cysts and extreme pain. Currently, the main therapies consist of hormonal therapies and operations, both of which are very invasive. One of the difficulties to develop a good therapy is that we don't know what causes the disease.

A lot of current research approaches endometriosis similarly to cancer, since, like cancer cells, endometriosis cells are cells that are found in the wrong place (they form ectopic tissue). However, one avenue of treatment that has received little attention so far is the role of the microbiome in endometriosis. There are multiple indications that a woman's microbiome - that of the vaginal and gut - are important in the development of endometriosis.

In fact, an imbalanced “unhealthy” vaginal microbiome (with what is known as vaginal dysbiosis) is often associated with endometriosis. While recent research suggests that a “healthy” vaginal microbiome is dominated by a single bacterial species, such as the probiotic bacterium Lactobacillus crispatus, this dominance is decreased in the “unhealthy” microbiome.

Thus, our therapy aims to restore the healthy state of the vaginal microbiome by promoting the growth of Lactobacillus crispatus, one of the most frequent dominant species, using a probiotic helper bacterium.

The Vaginal Microbiome

Recently, research has shown that a healthy vaiginal microbiome is not very diverse, but dominated by a few bacteria, with one bacterial species often dominating over 90%. However, while the vagina itself is not diverse, the composition of the bacterial populations in the vagina diverges strongly across different women and especially ethnicities, as Ravel et al. found in a 2011 study of the vaginal microbiome on reproductive-age women.

For a long time, scientists thought that the microflora in a healthy vagina was always dominated by bacteria of the Lactobacillus genus. In fact, strictly anaerobic populations can also dominate the microbiome, and even within the Lactobacillus genus, there are great differences in vaginal composition between women. While 45.4% of white women have L. crispatus as the dominant species, 42.7% of Asian women have L. iners. Thus, a specific vaginal microbiome composition might be fine in one person but cause issues in another.

However, one thing remained the same across all healthy vaginal microbiomes: all these bacterial communities included bacteria that produce lactic acid. By lowering the pH of the vagina, the production of this lactic acid ensures that the vaginal mucosa does not get colonised by pathogens. It has also been shown to affect the immune system of the host by eliciting the secretion of important anti-inflammatory mediators from vaginal epithelial cells. Indeed, Lactobacillus also produces lactic acid and other antimicrobial substances. Thus, the vaginal microbiome is crucial in preventing several urogenital diseases such as bacterial vaginosis, STDs, UTIs etc.

Endometriosis and Vaginal Dysbiosis

Although scientists are still not sure whether dysbiosis (in the gut or in the female reproductive tract) causes endometriosis or vice versa, this might be an important factor contributing to the pathogenesis of endometriosis. By disrupting normal immune function, dysbiosis leads to elevated levels of proinflammatory cytokines, a compromised immunosurveillance system and altered immune cell profiles. Over time, this dysfunctional immunoregulation can progress into a state of chronic inflammation, creating an environment favourable to increased adhesions and angiogenesis, both of which promote the formation of endometrial lesions as mentioned above.

Thus,re-establishing a healthy vaginal microbiome might be a key component in not only reducing endometriosis associated pain but also in preventing the formation of new endometrial lesions, slowing down the overall progression of the disease.


Our Approach: A live bacterial therapy

After antibiotics, there is a second avenue of treatment for vaginal dysbiosis, which we plan to improve on with our project: the use of probiotics - living, “healthy” bacteria which get administered either orally or directly into the vagina. Previous studies suggest that introducing Lactobacilli can help restore a healthy Lactobacillus-rich vaginal microbiome faster, and that vaginal administration of Lactobacillus crispatus especially - one of the Lactobacillus species dominating in many healthy women - reduced the likelihood of vaginal dysbiosis returned...

...and yet, vaginal dysbiosis still returns.

This is where we think synthetic biology is a viable solution: it allows us to create living medications which can “patrol” within your body, acting as soon as your disease recurs. Our probiotic would be genetically engineered to support Lactobacillus every time vaginal dysbiosis started to return. Furthermore, since we are already introducing live bacteria as probiotics, it is not such a stretch to think that someday we could use live bacteria that have been genetically modified to act as better medications.

Compared to traditional probiotics, which simply add an arbitrary amount of bacteria, our treatment establishes a self-regulating vaginal microbiome that helps sustain a healthy equilibrium. Having this therapy, which could patrol the body and act on demand, would be beneficial in multiple ways:

  • It would save a lot of money. Optimally, you would only ingest the medication once, instead of taking the normal probiotic medication repeatedly. Your total cost would be around 30 Euro instead of hundreds or thousands over your lifetime.
  • It would act before you have to go to the doctor - before you get full-blown vaginal dysbiosis.
  • By acting early, it would prevent not just vaginal dysbiosis but the effects of vaginal dysbiosis , which may include the onset or worsening of endometriosis (“may” because scientists have not found out yet whether endometriosis causes dysbiosis or dysbiosis causes endometriosis). Either way, it remains a useful treatment.

The Disadvantages of Antibiotics

Antibiotics have several disadvantages. Firstly, they do not kill bacteria very specifically, meaning that they also wipe out the healthy bacteria residing inside your body [a.]. This also means that they're unlikely to only influence the microbiome you're trying to help, but may also disrupt other microbiomes in the body, like that of the gut, which - unlike the vagina - is quite diverse when healthy. Furthermore, several of the pathogenic strains involved in dysbiosis are now resistant to metronidazole and clindamycin [b.], two of the four antibiotics mentioned as treatment options for vaginal dysbiosis by Mayo Clinic [c.]. Even more importantly, antibiotics have a high recurrence rate: within 3-12 months, the dysbiosis often returns [c.]. Our therapy, on the other hand would act against vaginal dysbiosis over a long time, by acting whenever vaginal dysbiosis threatens to return.

With our therapy, you would not get to the point where you had to go to the doctor for vaginal dysbiosis - to the point where the imbalance could already be having other effects in the body - such as the onset or worsening of endometriosis.

Human Practices: Making the Invisible Visible

Endometriosis is a unique disease. A long history of gender and social inequalities lead to the current situation in which patients still suffer from lack of proper treatment and social acceptance. Some may see a topic such as this as a minefield, but we saw it as an opportunity to expand our vision beyond the lab bench and create a project that truly addresses the issues felt by those affected by endometriosis. We elaborate further on these issues in the interview and survey section.

Our vision of integrated human practices is based on three pillars, all of which interact with and affect each other:

  • Product Development: Adapting our project to the deeds of our stakeholders and planning for future implementation.
  • Surveys & Interviews: Listening to patients and experts and understanding their problems and needs so our work is as helpful as possible.
  • Awareness: Connecting with the public and contributing our part to solving the social issues.

Our project was constantly adapting to feedback and new information. LactoBack has turned into something quite different than we originally envisioned, and that is a good thing. We are proud of the awareness we created in our community and are prepared to further plan the future of LactoBack. We understand the needs of patients and the medical community and are more informed about the process of bringing new medication from the lab to the pharmacy shelves.

Check out the Human Practices Page to see how we engaged with the public to further our project.

Dry Lab: Setting Targets, Going Beyond

The dry lab team helps streamline the project by reducing the wet lab's experimental load and by simulating and predicting how our system would perform long term.

Working Hand-in-Hand With the Wet Lab

We help with the selection of gene targets for flux redirection via flux balance analysis (FBA) - a genome-scale model of reactions and metabolites present in the cell - to determine the most useful combinations of genes to conditionally knock down in the wet lab. The results of our simulations are then used to engineer dCas9 sgRNA-based parts.
Throughout the project, we also verify our model predictions and effects in vitro using mass spectrometry-assisted metabolomics as well as various metabolite assays.


Wet Lab: Targeting an Imbalanced Vaginal Microbiome


We hope to reestablish a healthy microbiome by engineering a self-sustaining helper bacterium population that grows well in dysbiotic - i.e. unhealthy - vaginal conditions. Our helper bacterium would give Lactobacillus crispatus, the dominant bacterium in many women, a competitive edge by conditionally secreting lactic acid, which lowers the vaginal pH and hampers the growth of opportunistic bacteria.


The LactoBack System:

Figure 1: Left: Overview of the engineered E. Coli bacterium: Rhl-QS molecules are depicted in violet. Right: Depiction of a hypothetical timeline from: eubiotic conditions (1), disruption that leads to dysbiosis (2), growth of engineered E. coli bacterium that leads to secretion of lactate and thus lowering of pH (3) and reestablishment of eubiotic conditions (4).

At the start of dysbiosis Lactobacillus crispatus greatly reduces in number. Simultaneously there is a strong increase in pH and opportunistic bacteria, including the engineered helper bacterium take-over. The level of C4-HSL rises with an increasing number of helper bacteria. Once a threshold level is reached, metabolic flux is redirected to produce great quantities of lactic acid. Opportunistic bacteria can no longer compete with Lactobacillus crispatus and a healthy environment is reestablished.


Designing our system

We plan to introduce a quorum sensing system into our helper bacterium that lets it detect its own population density (Fig. 1) so that lactic acid production is only triggered at high densities. This precise live response to the patient's current state, combining monitoring and treatment, is only possible with precise engineering using synthetic biology.

    Lactic acid production - there are two ways to increase the output of a specific metabolite - increasing the efficiency of its production, or cutting off the alternatives. Our approach combines both.
    Lactic acid effects - how much lactic acid our system produces depends on two factors: lactic acid production rate per bacterium and the total lactic acid producing population size. However, lactic acid is harmful to the bacterium itself - therefore, we want to trigger its production only when truly necessary: when the bacterial population reaches a high enough density to make an impact.

While we work on our proof of concept here using E. coli MG1655, in the future, we aim to engineer the functioning into a variety of vaginal bacteria, which would allow for personalised medicine, to reflect the different makeup of the vaginal microbiome in women of different genetic and environmental backgrounds.

Check out the Engineering Page to read more about the steps we took to reach our Bioengineering goals.

References

[1] Alakomi, H.-L. et al. Lactic Acid Permeabilizes Gram-Negative Bacteria by Disrupting the Outer Membrane (2000).

[2] Amabebe, E. & Anumba, D. O. C. The Vaginal Microenvironment: The Physiologic Role of Lactobacilli (2018).

[3] Boris, S. & Barbés, C. Role played by lactobacilli in controlling the population of vaginal pathogens (2000).

[4] Happel, A.-U. et al. Exploring potential of vaginal Lactobacillus isolates from South African women for enhancing treatment for bacterial vaginosis (2020).

[5] Jiang I, Yong P.J., Allaire C. and Bedaiwy M.A. Intricate Connection between the Microbiota and Endometriosis (2021).

[6] Kalia, N., Singh, J. & Kaur, M. Microbiota in vaginal health and pathogenesis of recurrent vulvovaginal infections: a critical review (2020).

[7] O'Hanlon, D. E., Moench, T. R. & Cone, R. A. Vaginal pH and Microbicidal Lactic Acid When Lactobacilli Dominate the Microbiota (2013).

[8] Ravel, J. et al. Vaginal microbiome of reproductive-age women (2011).

[9] Richeux, V. Endometriosis Linked With Genital Microbiome Changes (2022).

[10] Saraf V.S, Sheikh S.A, Ahmad A., Gillevet P.M, Bokhari H., Javed S. Vaginal Microbiome:Normalcy vs Dysbiosis (2021).

[11] Ser, H.-L., Au Yong, S.-J., Shafiee, M. N., Mokhtar, N. M. & Ali, R. A. R. Current Updates on the Role of Microbiome in Endometriosis: A Narrative Review (2023).

[12] Uzuner, C., Mak, J., El-Assaad, F. & Condous, G. The bidirectional relationship between endometriosis and microbiome (2023).

[13] Wang, S. et al. Antimicrobial Compounds Produced by Vaginal Lactobacillus crispatus Are Able to Strongly Inhibit Candida albicans Growth, Hyphal Formation and Regulate Virulence-related Gene Expressions (2017).

[14] . WHO. Endometriosis.