Endometriosis is painful — but the cure isn’t out of reach. (Our idea to solve the issue)
Using DNA methylation via CRISPRoff to repress the over-expression of the genes encoding for SF1 and ERβ production. (Mehr Bains)
The Problem | Endometriosis, a long-term chronic disease-causing infertility and debilitating pain affecting daily activities, affects 190 million women of reproducible age globally, and 1 in 10 women in the UK. With no cure, treatment involves managing symptoms using hormone therapy or invasive surgery, which can cause unwanted menopausal side effects and developed resistance to treatment.
The Mission| Create breakthrough epigenetic therapy to cure and alleviate the mental and physical suffering caused by endometriosis.
The Vision | A world without endometriosis.
Our Values |
📌 We never stop innovating.
📌 We have high standards for impact.
📌 We use the first principles to target the problem.
📌 We do the unconventional.
📌 We silence the disease, not the conversation about it.
The Problem of Endometriosis
Endometriosis is a long-term chronic disease-causing infertility and debilitating pain that affects women of reproducible age. It occurs when endometrial-like cells grow outside of the uterus and form lesions. It is responsible for pelvic pain and other symptoms that cause interference in everyday activities, which negatively impacts the quality of life for many women. Currently, it affects 190 million women globally, with 1 in 10 women in the UK having the condition. Although it is a highly prevalent issue, endometriosis has no cure and its treatment involves only managing symptoms by using unreliable hormone therapy or invasive surgery. The unlucky few must suffer the next 4–12 years, and without the correct treatment, the issue can rapidly progress.
The current major medical treatment aside from pain medication is focused on the hormonal alteration of the menstrual cycle with a major goal to produce a pseudo-pregnancy, pseudo-menopause, or chronic anovulation, creating an acyclic, hypo-estrogenic environment. This is achieved either by blocking ovarian estrogen secretion (GnRH agonists or antagonists), by inducing pseudopregnancy (progestins), or by locally inhibiting estrogenic stimulation of the ectopic endometrium. However, patients can become resistant to hormone therapies over time or symptoms may reoccur if the medicine isn’t taken. As well, this can cause unwanted menopausal symptoms.
The other treatment modalities include medical therapy, surgical intervention, or a combination of both. The recurrence risk after surgery is high: 7–30% of patients reported recurrences three years after laparoscopic surgery and increases to 40–50% five years after treatment. In fact, recurrent symptoms occur in about 10% of women even after treatments are performed.
Additionally, 80% or more people have not heard of endometriosis, creating stigma and a lack of awareness about the disease, even though it is so prevalent. This also leads to a long diagnostic delay which contributes to the severity and progression of the disease. Another issue is that pain is often dismissed as being normal and not a result of a disease. The symptoms can often be confused as being due to menstruation and in some cases, patients aren’t believed when they say that they’re experiencing abnormal pain. Any solution addressing endometriosis will need to tackle this component of the issue as well.
Therefore, this is a call for a novel innovative solution to truly cure endometriosis while eliminating the side effects, that ENDOCURE plans to bring to the industry.
Epigenetic Regulation Via Methylation
Cells are able to change the regulation levels of specific genes in their genomes, through a process known as methylation. Methylation involves adding a methyl (CH3) group that blocks the gene from being read by the cell. As a result, the gene is silenced, as it won’t be transcribed, translated, or turned into a protein. The process causes the DNA and histones to pack closely together which prevents transcription factors from binding to the DNA and the gene isn’t expressed. However, when this process goes wrong, diseases can occur, one of which is endometriosis.
What is CRISPRoff?
In 2021, a study by Nunez et al. was published discussing a new CRISPR variant called CRISPRoff. Contrary to the traditional CRISPR complex which breaks the double strands of DNA and leaves permanent changes to the cell genome, CRISPRoff gives us the ability to silence genes and essentially stop them from affecting the body, by working on an epigenetic level instead of at a genetic level. CRISPRoff also comes with an “undo button” in the form of CRISPR on, which can reverse the changes made by CRISPRoff. In short, CRISPRoff is a recently discovered epigenetic tool that makes reversible and heritable changes in gene expression, without causing double-stranded breaks in DNA, therefore making it a much safer treatment than the CRISPR Cas9 system.
The first component of the CRISPRoff system is a dCas9 protein. This is a mutated form of Cas9 which is labelled dead because it cannot make double-stranded breaks in DNA but it’s still programmable using a guide RNA (sgRNA), which can guide it to the complement DNA sequence at the desired target site. The next component is KRAB, which can influence what proteins are recruited near the gene. Another component of this technology is Dnmt3A and 3L which are DNA methyltransferase proteins that can methylate DNA and basically alter the epigenome.
CRISPR on, on the other hand, contains DNA demethylase which can remove methyl groups and turn the gene “on” as well as transactivator domains that can promote gene expression even further.
The reason why CRISPRoff is safer than CRISPR Cas9 is that CRISPR Cas9 involves making a double-stranded break (DSB) in DNA and the DSB is repaired using the NHEJ or HDR method. The NHEJ method involves putting the ends of DNA together where the break is made, which results in insertion or deletion mutations. The HDR method introduces a donor template into the cell which edits the DNA sequence as the DNA is repaired. In general, DSBs are dangerous for a cell and this system depends on the cell’s ability to repair the DSB properly, which makes the end result somewhat unpredictable.
The epigenetic changes made by CRISPRoff have also been shown to be heritable and able to be retained even through cell division and differentiation. CRISPRoff combines 3L + Dnmt3A and KRAB to be more effective in retaining epigenetic changes than each of them individually. The paper also indicated that even when CRISPRoff was transiently expressed to silence the H2B gene, the gene silencing effect was still retained in over 90% of cells, 10 days after transfection.
CRISPRoff also has the ability to silence many genes, even those without CpG islands. Using a sgRNA library designed to target more than 20,000 genes, their research shows that it can silence most genes in the human genome.
ENDOCURE: Using CRISPRoff to Silence the NR5A1 and ESR2 Genes Encoding For SF1 and ERβ
We propose epigenetic therapy to cure endometriosis by using CRISPRoff to repress the over-expression of NR5A1 and ESR2 genes. Our hypothesis is that silencing these genes, which are correlated with SF1 and ERβ production respectively, will prevent local estrogen and aromatase enzyme production, which have been determined to drive the progression of endometriosis through increased inflammation, resistance to endometriotic cell death and cell proliferation.
We have determined our hypothesis based on a study done by Bulun et al., 2015 that determined the over-expression of SF1 by 12,000X in endometriotic cells is due to the promoter region being unmethylated, therefore allowing unchecked high levels of expression that is not found in normal cells. SF1 has been shown to be a transcriptional regulator that is involved in moderating the cascade that produces estrogen from cholesterol, where a part of this cascade includes a gene encoding for aromatase. As well, we derive our hypothesis from the study demonstrating that the promoter for ERβ is hypo-methylated, leading to 140X increase in expression, where ERβ further targets signalling proteins such as RERG that oversee the action of estrogen in contributing to the pathology of the disease.
Another study done by Nasu et. al, 2011, examines the aberrant DNA methylation that contributes to endometriosis and identifies the hypo-methylation of a CpG island on the ERβ promoter contributes to the aberrant expression of ERβ in endometriotic cells. As well, they determine that the SF1 promoter is hypo-methylated in endometriotic cells, which contributes to the upregulation of aromatase.
We propose the use of CRISPRoff to prevent the DSBs (double-stranded breaks) and the possibility of mutations that arises from random DNA repair when using the CRISPR Cas9 complex, as well as preventing the menopausal side effects of using aromatase inhibitors and hormone therapies as treatment.
Pre-clinical Study
To better understand CRISPRoff and its feasibility for curing endometriosis, we aim to conduct a pre-clinical study to answer the following questions:
- Will the silenced NR5A1 and ESR2 genes stop the progression of endometriosis in the body?
- Should the genes encoding for SF1 and ERβ both be treated with CRISPRoff and repressed, or only one or the other is needed to cure symptoms? Which one is more effective in treating endometriosis?
- How many doses of CRISPRoff are needed to treat cells and in what intervals to maximize the retention of the epigenetic changes made, in future cell generations?
We aim to use murine models for the pre-clinical study, in 3 rounds of experiment.
Round 1:
- Menstruation is induced and endometrium tissue is injected into 3 cohorts of mice, to induce endometriotic lesions outside the uterus.
- Cohort 1 is treated with CRISPRoff containing a sgRNA targeting NR5A1, cohort 2 receives CRISPRoff containing a sgRNA targeting ESR2, and cohort 3 is treated with both CRISPRoff treatments targeting NR5A1 and ESR2. The sgRNA guides CRISPRoff to endometriotic cells and AAV, a viral vector, is used to deliver in vivo to endometriotic lesions outside the uterus.
- Mice are tested using Western blotting and qPCR to quantitatively evaluate the mRNA and protein expression levels from the NR5A1 and ESR2 genes of SR1 and ERβ respectively, after CRISPRoff treatment. DNA methylation activity at the promoter regions for the genes is also measured using bisulphite sequencing.
Round 2:
- The cured endometriotic cells from round 1 of mice will be transferred to this round, to another 3 cohorts of mice that have not been injected with endometriotic cells, to determine if the mice over-express SR1 and ERβ or not.
Round 3:
The experiment in round 1 is repeated to determine the following:
- Whether CRISPRoff does again demonstrate that the NR5A1 and ESR2 genes are silenced
- The number of doses of CRISPRoff that are required
- The time interval in which the doses should be administered to ensure epigenetic changes are retained in cell memory
We will be moving to larger animals for pre-clinical testing if this study is proven to be successful.
One challenge to acknowledge in carrying out this trial is choosing the correct viral vector to insert CRISPRoff into the murine model to target the desired tissue. We aim to test segmenting the CRISPRoff treatment over 3 AAVs before insertion since VLPs have been shown to not work for inserting CRISPRoff.
Stages For FDA Approval
Phase 1: These trials usually enroll 20 to 100 healthy volunteers or mice with the condition being studied, and last 1–2 years. This phase measures safety by testing for any adverse side effects of the treatment, but not necessarily how effective the drug or device is.
Phase 2: Around 70% of potential new drugs enter Phase 2, which continues to measure safety, while also looking at how effective the treatment is and carefully investigating its side effects. Phase 2 trials recruit up to several hundred patients with the condition to take part. This phase typically lasts several months to two years.
Phase 3: Just 33% of drugs make it to Phase 3, which tests the potential treatment in the largest number of people. This phase measures both safety and effectiveness with many volunteers, sometimes thousands. Phase 3 trials last from one to four years.
FDA approval: After Phase 3, a pharmaceutical company may submit a New Drug Application (NDA) or a biologics license application (BLA) for the treatment to the Food and Drug Administration (FDA). The FDA then reviews results from all stages of the trial to determine whether it will approve the drug and allow the pharmaceutical company to begin marketing it to the public.
Phase 4: This phase is often called “Post-Approval Research and Monitoring.” After a new treatment is approved by the FDA, the pharmaceutical or device company may want to continue monitoring patients to learn more about the treatment’s longer-term effects, while comparing it against other already-approved options. It may take time for long-term side effects to appear, making this an important phase.
Cost Breakdown
The average cost of phase 1, 2, and 3 clinical trials across therapeutic areas is around $3.1 million, $14.6 million, and $17.5 million respectively. Pivotal (phase 3) studies for new drugs approved by the Food and Drug Administration (FDA) of the United States cost a median of $41,117 per patient. FDA approval and phase 4 will cost $2.0 million and $6.8 million respectively. Hence, the total cost of development for EndoCure will be approximately $44 million.
The Outcome
The cost of current surgery methods to treat endometriosis ranges from $14,564.73 (vaginal hysterectomy) to $26,002. One study estimated that the annual cost of endometriosis was $5200 per patient, with lost productivity and lost leisure time costs being 78%.
The treatment cost for endometriosis using ENDOCURE is $2 million per person, based on our research. Although expensive, the cost will rapidly decrease as the industry expands and the costs for genetic and epigenetic therapy decrease. To begin our rollout, we start in developed countries as they possess the infrastructure and abilities to implement the process. We aim to target the UK for launching the treatment and cure 675,000 patients, and then take the treatment global and provide a cure for as many women affected as possible, as the costs for CRISPRoff treatment decrease with market growth. As well, more people will get access to our solution when awareness of endometriosis and its symptoms increases.
The benefits of such a technology are priceless for the well-being of women and are a leap towards advancing femtech. Beyond treatment, we’d like to develop an early detection test for endometriosis using biomarkers, and begin a campaign to raise awareness and reduce stigma through conversation and education.
The Opportunity
The femtech global market size was $40.2 billion in 2020 and is projected to grow with a CAGR of 13.3% to $75.1 billion by 2025. The endometriosis market in the 5EU (France, Germany, Italy, Spain and the UK) is expected to grow with a CAGR of 12.5% (higher than the US or Japan) from 2020–2030. Europe has 25% of all femtech startups, and the UK specifically has 10%. Europe also has a prominent endometriosis market position as their healthcare sector continues to grow and more $ is put into the industry.
ENDOCURE
At ENDOCURE, we envision a world where women have the freedom to live with a healthy and high quality of life. Femtech is an advancing industry that is becoming a global priority as more research is being done on women’s health and we’re excited to take a step to be involved in advancing this field. We believe our idea of using breakthrough epigenetic therapy to cure the physical suffering caused by endometriosis in 10% of all women of reproducible age will be a game-changer.
If this sounds similar to your vision of the future, we’d love to have a chat and see how we can tackle endometriosis together.
This article was written by Mehr Bains, Arjun Mahes, and Aditya Mahes, on behalf of the ENDOCURE team.
We’d also like to thank everyone who provided feedback for this article!
References
Nuñez, J. K., Chen, J., Pommier, G. C., Cogan, J. Z., Replogle, J. M., Adriaens, C., Ramadoss, G. N., Shi, Q., Hung, K. L., Samelson, A. J., Pogson, A. N., Kim, J., Chung, A., Leonetti, M. D., Chang, H. Y., Kampmann, M., Bernstein, B. E., Hovestadt, V., Gilbert, L. A., & Weissman, J. S. (2021). Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing. Cell, S0092–8674(21)00353–6. Advance online publication. https://doi.org/10.1016/j.cell.2021.03.025
Bulun SE, Monsivais D, Kakinuma T, Furukawa Y, Bernardi L, Pavone ME, Dyson M. Molecular biology of endometriosis: from aromatase to genomic abnormalities. Semin Reprod Med. 2015 May;33(3):220–4. doi: 10.1055/s-0035–1554053. Epub 2015 Jun 2. PMID: 26036904.
Nasu, K., Kawano, Y., Tsukamoto, Y., Takano, M., Takai, N., Li, H., Furukawa, Y., Abe, W., Moriyama, M. and Narahara, H. (2011), Aberrant DNA methylation status of endometriosis: Epigenetics as the pathogenesis, biomarker and therapeutic target. Journal of Obstetrics and Gynaecology Research, 37: 683–695. https://doi.org/10.1111/j.1447-0756.2011.01663.xEndometriosis. (2021, March 31). WHO | World Health Organization. Retrieved May 5, 2022, from https://www.who.int/news-room/fact-sheets/detail/endometriosis
Endometriosis Facts and Figures. (n.d.). Endometriosis UK. Retrieved May 5, 2022, from https://www.endometriosis-uk.org/endometriosis-facts-and-figuresKatherine, B., Amelia, P., Jessica, S., Elaine, P., Alicia, S., Nazmin, E., & Richard, B. (2022).
Endometriosis in the Mouse: Challenges and Progress Toward a ‘Best Fit’ Murine Model. Frontiers in Physiology, 12. 10.3389/fphys.2021.806574Dash PK, Kaminski R, Bella R, et al.
Sequential LASER ART and CRISPR Treatments Eliminate HIV-1 in a Subset of Infected Humanized Mice. Nat Commun. 2019;10(1):2753. Published 2019 Jul 2. doi:10.1038/s41467–019–10366-yPeng, S., Cai, J., & Bao, S. (2021).
CMBs carrying PTX and CRISPR/Cas9 targeting <em>C‑erbB‑2</em> plasmids interfere with endometrial cancer cells. Molecular Medicine Reports, 24, 830. https://doi.org/10.3892/mmr.2021.12470FemTech Market Overview. (n.d.).
FemTech Analytics. Retrieved May 5, 2022, from https://www.femtech.health/femtech-market-overviewEndometriosis market projected to grow at a CAGR of 10.6% between 2020 and 2030. (2022, January 6). Pharmaceutical Technology. Retrieved May 5, 2022, from https://www.pharmaceutical-technology.com/uncategorised/endometriosis-market-cagr-2020-2030/
Endometriosis Market To Reach USD 2.42 Billion By 2026 | Reports And Data. (2020, March 5). GlobeNewswire. Retrieved May 5, 2022, from https://www.globenewswire.com/news-release/2020/03/05/1996125/0/en/Endometriosis-Market-To-Reach-USD-2-42-Billion-By-2026-Reports-And-Data.htmlGuildford, A. (2021, March 17).
Endometriosis: Are there any new treatments on the horizon? Medical News Today. Retrieved May 5, 2022, from https://www.medicalnewstoday.com/articles/the-latest-in-endometriosis-research-ways-forward#Existing-treatments
How Much Does a Clinical Trial Cost? (2020, January 2). Sofpromed. Retrieved May 5, 2022, from https://www.sofpromed.com/how-much-does-a-clinical-trial-cost
