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.
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.
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.
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.
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:
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:
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.
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.
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.
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.
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.
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.
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.