Helix-B Technologies: CRISPR-Cas Genetic Testing to Combat Preeclampsia in Developing Countries
Preeclampsia Increases the Risk of Complications in Pregnancy
Preeclampsia is a serious pregnancy complication characterized by high blood pressure and protein in the urine.
The early signs of preeclampsia can be subtle. Common symptoms include persistent high blood pressure, proteinuria, sudden weight gain, swelling in the hands, feet and face, persistent headaches, blurred vision or other visual disturbances, and pain in the upper abdomen. Because of these symptoms, preeclampsia can go undiagnosed; weight gain or high blood pressure mistaken for normal pregnancy changes.
Preeclampsia can progress quickly and lead to life-threatening complications during pregnancy or birth for mother and child if not properly managed. For the mother, the condition can escalate to eclampsia and seizures. It can also cause HELLP syndrome (hemolysis, elevated liver enzymes, and low platelet count), which indicates severe liver and blood clotting issues. Difficulty clotting can lead to bleeding complications and hemorrhage. Studies have shown that the risk of post-partum hemorrhage, or excessive blood loss after birth, is increased among women with preeclampsia.
Developing Countries Boast a Mortality Rate of Preeclampsia 9 Times Higher Than in Developed Countries
Preeclampsia, among other complications, is more prominent in developing countries, ranging from 1.8% to 16.7% of all pregnancies compared to 0.4% in developed countries.
Furthermore, preeclampsia is associated with only a slightly increased risk of maternal death, ranging from 0%-1.8% in developed countries. This is because their hospitals are equipped with high-end medical infrastructure.
However, the mortality rate associated with preeclampsia is as high as 15% in developing countries. This is because, in these regions, mothers do not have access to the basic laboratory equipment necessary for preeclampsia diagnosis.
In developing countries, their mortality rate is over 9 times higher than in developing countries.
Moreover, there is a significant blood and resource shortage, exacerbating issues related to treating possible complications.
In Sub-Saharan Africa, more than 40,500 mothers die from preeclampsia yearly.
Imagine if it was possible to save these lives with something as simple as a pregnancy test.
But instead of pregnancy, this test can tell you if you are likely to get preeclampsia, a condition associated with being the second leading cause of maternal mortality in the region.
Prenatal Care Ensures the Safety of Mother and Baby
Prenatal care is one of the most critical aspects of ensuring a healthy pregnancy and a safe delivery. This practice ensures the safety and well-being of both mother and baby and plays a key role in identifying and addressing potential complications, including preeclampsia before they become fatal.
Prenatal care involves regular check-ups with healthcare providers, during which expectant mothers undergo a variety of tests and screenings to track their health and the development of their babies.
In regards to preeclampsia, after 20 weeks of gestation, healthcare providers look for high blood pressure to rule out the disorder. According to the Mayo Clinic, high blood pressure in pregnancy is defined as a systolic pressure above 140 mm Hg or a diastolic pressure above 90 mm Hg. In addition to high blood pressure, they look for at least one of the following: proteinuria (protein in your urine), low platelet count, elevated liver enzymes, pulmonary edema (fluid in the lungs), headaches or vision disturbances.
Healthcare providers may also order additional tests such as blood tests, urine tests, fetal ultrasounds, biomarker tests or nonstress tests. However, these require laboratory equipment, well-trained personnel and infrastructure.
So what if you don’t have access to prenatal care or specialized equipment for preeclampsia diagnostics?
Developed Countries Lack Accessibility to Preeclampsia Testing
In smaller clinics or more rural areas, they do not have access to the basic laboratory equipment needed to perform blood tests or biomarker assays. In addition to a lack of infrastructure, areas like Sub-Saharan Africa are suffering from a blood and resource shortage.
In the unfortunate case where a mother with undiagnosed preeclampsia develops a post-partum complication such as uterine hemorrhage, doctors only have a matter of a few minutes to give a blood transfusion. However, it can be difficult to have the necessary blood on hand.
The detection of preeclampsia is vital to be prepared for complications. In regions like sub-Saharan Africa where resources such as blood are limited, accessibility to testing poses a problem.
So what if we knew who was more likely to get preeclampsia?
Our Solution: CRISPR-Cas Diagnostics for Genetic Predisposition
FLT1: Gene of Interest
Since the early 1960s, studies have found a genetic link to preeclampsia. Chelsey et al. revealed a familial aggregation of preeclampsia and eclampsia and increased rates of preeclampsia in pregnancies of sisters, daughters and granddaughters.
Previous studies have searched for variants in the renin-angiotensin system, coagulation factors, oxidative stress pathways, dyslipidemia and immunoregulatory components (HLA region). While these studies laid the groundwork, the large genome-wide association study (GWAS) has allowed advances in identifying the genetic basis of various common diseases.
McGinnis and colleagues identified specific genetic variants in the fetal genome near the Fms-like tyrosine kinase 1 (FLT1) gene on chromosome 13 associated with the risk of preeclampsia (see below). They determined that multiple causal variants at the fetal FLT1 locus contribute to disease risk, offering a clearer understanding of the genetic basis of preeclampsia.
Researchers found three distinct single nucleotide polymorphisms (SNPs): rs4769613 (53% frequency), rs12050029 (14% frequency) and rs149427560 (6% frequency). The former two SNPs are located in the placental enhancer region near FLT1 and thus impact the expression of the FLT1 gene. Multiple causal variants at the fetal FLT1 locus contribute to disease risk.
Combining DETECTR and Lateral Flow Design
Our goal is to lower maternal mortality rates in regions like Sub-Saharan Africa by providing accessible genetic testing for the FLT1 variants, for better management of preeclampsia and improved resource allocation.
Lateral Flow Assays — Accessibility improved
Lateral flow assays (LFA) are low-cost, rapid and portable detection often used for detecting complex mixtures in a sample in fields such as biomedicine, agriculture and environmental sciences. Results can be displayed within 5–30 minutes and development costs and ease of production make it a low-cost, simple and rapid detection method.
When you think of a lateral flow, you might want to imagine a pregnancy test or the COVID-19 rapid detection PCR tests with the control and testing line.
These tests have overlapping membranes glued to a hard backing for better handling and stability. This makes it a greater portability and a more compact method of testing.
The mechanism behind LFAs is relatively simple. A few drops of the liquid sample or extract are released onto the absorbent sample pad at one end of the strip. Within this pad, buffers and surfactants are present to ensure the binding of the sample to the capture reagents. The sample then moves through the conjugate release pad through the detection zone. The detection zone is a porous membrane with specific biological components immobilized in lines. Their job is to catch the analyte (= substance being measured). The control line indicates that the liquid successfully moved through the detection zone while the test line will catch the analyte if it is present in the sample.
DETECTR — Harnessing CRISPR-Cas12a To Power our LFAs
In 2018, Chen et al. developed DNA Endonuclease-Targeted CRISPR Trans Report, more commonly known as DETECTR.
DETECTR is a CRISPR-based diagnostic method that uses the Cas12a protein and a single guide RNA to recognize and cleave specific dsDNA sequences. When the sgRNA-Cas12 complex encounters its target, it binds to it and activates the protein leading to specific cleavage and collateral cleavage.
Specific cleavage occurs when the Cas12 cuts the target DNA. Collateral cleavage is Cas12’s unique property. It indiscriminately cleaves single-stranded DNA (ssDNA) in its vicinity. This collateral cleavage is used for signal generation: the Cas12 cleaves DNA reporters with a quenched fluorophore to indicate that the target DNA has been located.
Specific cleavage is scissors in the hands of a concentrated adult, while collateral cleavage occurs when a cut-happy toddler gets their hands on the scissors and cuts everything on sight.
Now All Together…
At Helix-B technologies, we want to combine the accessibility and ease of use of lateral flow assays with the specificity of CRISPR-Cas nucleic acid detection.
DETECTR is what will allow the LFA to function.
Here’s how we would design a lateral flow test using DETECTR (CRISPR-Cas12a) to detect the genetic variants located near the FLT1 fetal locus (SNP rs4769613.
Included in the kit: lyophilized (freeze-dried) Cas12a protein, guide RNA (gRNA), amplification reagents, the physical lateral flow test and a small test tube.
- Cas12a, primers and gRNA: Design primers for the amplification of the target region. Design a guide RNA that targets the region encompassing the SNP rs4769613.
- Lateral flow assay: If the SNP is detected (genetic predisposition to preeclampsia), an activated Cas12a protein cleaves the single-stranded DNA reporter molecules, releasing cleaved fluorophore quencher complexes. The reporter molecules encounter the capture zones where they bind to the immobilized antibodies. The accumulation of captured fluorophore at a detection line generates a visible signal indicating whether the SNP has been located in the genome of the mother.
In short, if the mother has the genetic variant making her more likely to get preeclampsia, the lateral flow test will display a line indicating a positive test. If the mother does not have a genetic predisposition to preeclampsia, a different line will appear showing a negative result.
Provided a Positive Result, Healthcare Providers can Tailor Treatment to Manage Possible Preeclampsia
Based on the results obtained from our test, medical professionals will be able to distinguish those with a higher risk for preeclampsia. Although this is not a method of detection, this piece of information will make a difference and is a step towards saving the lives of over 40,000 women in Sub-Saharan Africa and more globally!
- Early Identification and Monitoring: By understanding genetic risks, healthcare providers can identify women at a higher risk for preeclampsia earlier on in their pregnancy. These women can be monitored more closely throughout their pregnancy, which can lead to earlier detection and intervention.
- Personalized Care: Genetic information can help in tailoring specific preventative strategies and treatments. For example, women with a higher genetic risk might benefit from different lifestyle recommendations, medications, or more frequent prenatal visits compared to those with lower risk.
- Preventive Measures: Early identification of genetic predisposition allows for the implementation of preventive measures. This might include dietary changes, supplements like low-dose aspirin, or other medical interventions aimed at reducing the risk of developing preeclampsia.
- Improved Outcomes: By managing and mitigating risks early, the chances of severe complications for both the mother and baby can be reduced. This includes reducing the risk of preterm birth, low birth weight, and other associated complications.
Affordable, Fast, Reliable
The price breakdown of Helix-B Lateral Flow test:
- Lateral flow test: ~ US$2.50 *
- DETECTR: ~ US$15**
Total: ~ US$ 17.50 manufacturing cost
*Lateral flow test can be manufactured for $0.10–3.00 per test. In 2021, reports stated a price ceiling of US$2.50 per test.
**Price estimated off of SHERLOCK estimated device price (Ghouneimy et al.). SHERLOCK is another method of CRISPR-Cas diagnostics using the Cas13 protein to detect RNA sequences. Methods are relatively similar and thus we assume that the price of SHERLOCK greatly resembles DETECTR’s manufacturing price.
The hypothesized manufacturing price of US$17.50 makes this test a reliable and affordable option for genetic testing. However, as developments in biotechnology and DETECTR continue, we hope to see this manufacturing cost decrease to give every woman, no matter which country they live in, access to some form of genetic testing for preeclampsia.
When compared to similar tests including PCR tests ($50–200/test), ELISA tests ($20–100), NGS tests (several hundred to several thousand $)*, rapid tests (up to $50), cfDNA tests (few hundred to several thousands $) *.
In comparison to similar tests, while varying in complexity and infrastructure needed, our solution is viable in terms of cost and accessibility.
*Costs are estimated and the wide range reflects the varying complexity and the number of genetic markers being tested.
Partner Collaboration for Enhanced Distribution
To fulfill our mission of saving mothers in developing countries, we want to partner with several different companies and organizations to get our lateral flow tests in the hands of all expecting mothers.
Our goal is to partner with the National Institutes of Health (NIH) to continue the improvement and efficacy of the project as well as work with the Red Cross to establish distribution in the region (beginning with Sub-Saharan Africa). Additionally, we want to work with the Bill and Melinda Gates Foundation to fund our project and save the lives of countless mothers.
Because of the low cost and large impact of our tests, we also plan on establishing distribution through the government by selling them our tests.
Outcome
At Helix-B we are combining DETECTR and lateral flow tests to get fast and reliable genetic testing in the hands of all expecting mothers to identify possible genetic links to preeclampsia. They can take the test with a simple prick of the finger and their doctor or medical professionals will be well prepared in the case of complications should they have a positive result. This can include having blood or resources on hand to treat hemorrhage.
Proposal by Sophie Qian, Sophie Lassonde and Melissa Guedes.
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