What is Endometriosis?
According to the World Health Organization, 1 in 10 women in their reproductive age suffer from endometriosis, a disease which causes chronic pain, making it 190 million people worldwide (1).
Endometriosis is a medical condition in which tissue similar to the lining of the uterus (endometrium) grows outside the uterus, as illustrated in Figure 1. This tissue can grow on other organs in the pelvic region, such as the ovaries, fallopian tubes, the outer surface of the uterus, and even beyond the pelvic organs (1). Symptoms of endometriosis include pain before, during, and after menstruation, pain during intercourse, heavy and irregular periods, and infertility (1).
Despite the severity of these symptoms, endometriosis is often overlooked on an individual level. Many women feel that something is wrong but frequently dismiss their symptoms as insignificant, largely due to societal normalization of these symptoms as “just painful periods” (2).
There is no cure, but treatment options focus on managing symptoms. These may include pain relief medications, hormonal therapy (to slow the growth of endometrial tissue), and surgical removal of the tissue in more severe cases (1).
Treatments for endometriosis aim to relieve symptoms, reduce the growth of endometrial-like tissue, and improve quality of life (3). The choice of treatment depends on the severity of the condition, the person's symptoms, and whether they wish to preserve fertility.
Our Project
EndoERαSE aims to create a minimally invasive treatment for endometriosis. This is accomplished by using a plasmid to induce apoptosis in endometriotic tissue while leaving the endometrial tissue unharmed.
The project was inspired by previous iGEM projects working with endometriosis, such as LactoBack, ENDOSeek, and UteRus. Each of these projects decided to tackle the issue in different ways.
Our team found several issues with preexisting treatment methods, including laparoscopy - which is both a treatment and diagnostic tool, hormone treatment - such as birth control -, and painkillers. While laparoscopy has been proven to be able to reduce pain in patients suffering from endometriosis, it is an invasive surgery that has a 36% chance of requiring further surgery (4). Birth control has also been linked to several side effects such as anxiety, irregular periods, and weight gain (5). Lastly, there is evidence which suggests that painkillers might be ineffective against pains resulting from the disease (9).
On our journey to creating a minimally invasive and non-hormonal treatment, we found studies showing that estrogen receptor β (ERβ) is upregulated by more than 100 times (7) in endometriotic cells while estrogen receptor α (ERα) was downregulated (8). This is visualized in Figure 2. Both receptors are regulated by the hormone estrogen (7). When estrogen binds to these receptors, they form a complex of dimerized receptors and estrogen. They then relocate into the nucleus, where they bind to ERβ and ERα promoters, activating the transcription of certain genes.
Experimental Design
Our experimental design will be based on the alteration of activities of the estrogen promoters. We will construct a plasmid, which contains two synthetic parts: one is the promoter for CD47, followed by apoptosis regulator BCL2 associated X (BAX), while the other part is the BRCA1 promoter, followed by apoptosis regulator B-cell lymphoma/leukemia-2 (BCL2). This design can be seen in Figure 3. The promoter for CD47 is activated by Erβ (10) and the BAX gene induces apoptosis (11). The BRCA1 promoter is activated by ERα (12) and BCL2 inhibits BAX, and therefore, apoptosis (11).
The plasmid will be transformed into E. coli DH5α to generate multiple copies. The plasmids will then be transiently transfected into 12Z Human Endometrial-Like Cells, as well as Human Endometrial Stromal Cells (HESC). Upon transfection of the designed plasmid, the transcription of BAX is activated in response to the high ERβ promoter activity in the endometrial-like cells. The activity of ERβ in healthy endometrial cells should be lower than in the sick cells, due to differences in concentration. This should ideally result in no or very little transcription of BAX. Next, we will measure and compare the level of apoptosis in endometrial-like cells and HESC cells transfected with the plasmid. This will be done by applying apoptosis fluorescent markers and tracking the development by fluorescence readings using a microplate reader, flow cytometry, and microscopic analysis. To verify the efficiency of the promoters, we will generate another plasmid, containing the CD47 and BRCA1 promoters regulating a gene each, encoding Red Fluorescent Protein (DsRed1) and Green Fluorescence Protein (GFP), respectively. The promoter activity of this plasmid will then be measured by flow cytometry post-transfection.
Goal
Our aim is to establish a condition in which increased expression of ERβ, alongside reduced expression of ERα, triggers activation of the ERβ promoter, resulting in the transcription of the BAX gene from the plasmid. This leads to the production of BAX protein, which is part of the apoptosis pathway (13). Our hypothesis is that increased expression of BAX will induce apoptosis in endometrial-like cells, resulting in cell death, as shown in Figure 4. This has been previously observed in HeLa cells (14). On the other hand, if the concentration of ERα is upregulated and the concentration of ERβ is downregulated, which is the case in healthy cells compared to endometrial-like cells (8), the cell will transcribe the BCL2 gene, which is regulated by the ERα promoter. Ideally, this will selectively protect healthy cells from undergoing apoptosis, as shown in Figure 5.
Future Perspective
The ERβ and the ERα receptors are present in other cells, such as cells in adipose tissue, the colon, and the bladder (15). If our approach is to be considered for future treatment, the design and the off-target effects on other cells and tissues will need to be thoroughly evaluated. The approach for our treatment does not have extensive research, but a study was performed to investigate the DNA integration of the pcDNA3.1+PA plasmid in reproductive organs in mice. This plasmid is the one we used as a backbone. The study concluded that the plasmid did not integrate into the host's genome (16). Therefore, if our treatment is to be used in the future, it should not alter the host genome. The treatment is also a minimally invasive and non-hormonal option, unlike existing treatments.
