Overview
Cervical Cancer accounts for about 8% of the world’s total cancer cases and deaths, 90% of which occur in developing countries where access to adequate healthcare and preventative measures is strikingly scarce. 1 in 50 Indian women develop cervical cancer in their lifetime[1], contributing to one-fourth of the world’s cervical cancer cases and yet, has been largely ignored in public health efforts due to a combination of factors including lack of awareness, social stigma in a historically patriarchal society and inadequate screening methods.
More than 95% of all cases of cervical cancer are caused by persistent HPV infections. HPV (Human Papilloma Virus) is a sexually transmitted virus of which the two highest oncogenic risk types are HPV 16 and 18. Vaccination and screening are the flag bearers for this fight against cervical cancer, as the cancer is treatable if detected in early stages and entirely preventable if detected in pre-cancerous stages.
Most developing countries like India do not have a national level vaccination programme for HPV. Only recently, a vaccine has been introduced in India at much more affordable prices compared to what was previously available. However, these are only effective when administered to youth, ideally before the first sexual intercourse. Further, existing vaccines do not protect against all types of HPV. Hence the answer to the issue lies in a combined effort of vaccination and screening methods.
Another factor plaguing women’s health is the taboo around discussing sexual health, especially in rural areas, which results in a general lack of awareness about safe sex practices and STD testing. Further, existing screening methods are not suitable for the context of developing countries. The most popular existing screening method, the Pap Smear, is an invasive test that requires trained medical professionals, and takes days to produce results. Inhibitions and logistical issues prevent the widespread testing required.
To tackle this problem, we aim to make screening more convenient and accessible for people by developing a rapid, sensitive, and easy-to-use point of care diagnostic kit for the detection of high-risk HPV. The detection mechanism utilizes CRISPR Cas12a technology for detection of HPV DNA as a biomarker. We propose an in-kit DNA extraction process, designed to eliminate the need for any laboratory equipment, involving antibody-based enrichment, capsid degradation and DNA amplification. As opposed to an invasive cervical swab, our method is capable of functioning with a self-administered vaginal swab. Guided by a designed crRNA complementary to the highly conserved HPV16 E7 oncogene, the transcleavage property of CRISPR Cas12 is activated in presence of the target DNA. This allows for cleavage of a reporter molecule in the solution and subsequent visual readout on a lateral flow assay strip. No knowledge of any of these processes would be required to perform the test, due to a simple and clean workflow.
Through our project, Crisprly, we hope to enable large-scale screening and empower women to prevent the life-threatening disease that is cervical cancer. Our efforts attempt to address the lack of appropriate technology and lack of awareness hindering the prevention of an entirely preventable disease.
Inspiration
“Scars may heal, blood counts may normalize, years may pass. But never again will the simple act of waking up to a normal, boring day as a healthy individual be taken for granted, nor go unappreciated.” Cancer is one of the deadliest diseases known to mankind. With its dangerously high mortality rate, cervical cancer is the fourth-most common cause of death among women. Cervical cancer is majorly prevalent in developing countries. According to a report published by the HPV Information Centre (WHO IARC initiative), 483.5 million women in India are at risk for cervical cancer.
On further investigating the issue at the grassroot level, through doctors and health professionals, we discovered certain alarming things like lack of awareness about the Human Papillomavirus (the cause of almost all Cervical cancer cases), sexual health taboo especially among rural women and lack of low-cost, non-invasive screening tests. Early detection and screening can control the incidence of cervical cancer significantly[3] The overwhelming scale, potentially fatal and extremely under-addressed nature of the problem is what led us to tackle it. Using our engineering skills and understanding of biology, we developed a low-cost and easy-to-use HPV detection kit that can be used in low-resource settings without the need of any specialized equipment or trained personnel.
The diagnostic kit could have incredible entrepreneurship potential which enables us to reach out to the government, private hospitals and companies for large-scale implementation that would really bring about a positive change in the status quo. The number of lives that could be undeterred by cervical cancer if detected early and bringing the issue of Sexual Health and HPV Immunisation to the national forefront served as our key motivators as we persevered to come up with a solution that will change millions of lives.
We acknowledged the gravity of the problem at hand and utilised this opportunity to design a solution while also promoting open dialogue on sexual health which is considered a taboo especially in the rural areas through our human practices and public engagement activities. We reached out to a large community of diverse individuals and received good feedback. Furthermore, choosing a topic with less social awareness but a huge societal impact served both as a challenge and a huge opportunity for us, helping us come up with more viable solutions.
Basics of the kit
Human Papillomavirus
Human Papillomavirus or commonly known as HPV is a sexually transmitted virus which globally causes more than 95 % of known cervical cancer. Sexual transmission is majorly through oro-genital, ano-genital, heterosexual and intimate skin-to-skin contact. Infection of HPV is common in both males and females (90%) who are sexually active, with fewer chances of repeated infection in a person’s lifetime. Many cases show no obvious symptoms of HPV infection, and 90% resolve spontaneously within two years. However, a persistent infection can result in warts, lesions, cancer on the genitals, anus or mouth.DNA sequencing data reports close to 200 different types of HPV, of which about half are well characterised.However, not all of these strains are oncogenic. Based on oncogenic potential, HPV types are divided into two groups: low-risk (6, 11, 16, 18, 31, 33, 35, 42, 43, 44, 45, 51, 52, 74) and high-risk (16, 18, 6, 11, 31, 34, 33, 35, 39, 42, 44, 45, 51, 52, 56, 58, 66). HPV 16 and HPV 18 are of particular interest as they are high-risk oncogenic strains that cause most of the cervical cancers and pre-cancerous cervical lesions.
The oncogenic activity of HPV 16 and 18 is attributed to the viral oncoproteins E6 and E7.[2] These two proteins inhibit tumor-suppressing genes namely p53 and pRb, which results in uncontrolled, rapid multiplication of cells eventually leading to cancer. Our team has selected HPV16 for cancer detection from vaginal load as there is high conservation of the E7 gene. As observed from our bioinformatics analysis, no specific conserved portion in the E7 gene of HPV18 was found. Also, the sequences of E7 in 16 and 18 are significantly different, which ultimately led us to work on HPV16.
Lateral Flow Assays
LFAs or Lateral Flow Assays are the technologies behind the rapid, affordable and portable detection devices that are used most frequently in the field of medical diagnostics due to their potential of providing an instantaneous diagnosis.
They are based on the concept of capillary flow. A liquid sample that may contain the analyte of interest is allowed to flow over a polymeric, usually nitrocellulose strip which contains various molecules that interact with the analyte. This interaction between molecules present on the strip and the analyte is responsible for the appearance of a visual readout that allows us to differentiate between the presence and absence of the analyte in the sample. The liquid sample that may contain the analyte is initially released on the sample pad and flows upto the conjugate release pad which contains the molecules specific to the analyte. Usually, these consist of gold nanoparticles. This mixture of analyte and analyte specific molecules flows up to a porous region of the nitrocellulose strip called the detection zone. Here, depending on the interaction between analyte and molecules a visual readout occurs in the form of a line. A test line appears to indicate the presence of the analyte and a control line appears to ensure appropriate flow of fluid on the strip. The visual readout occurs in the form of colorimetric interactions between a conjugate bound antigen and immobilized antibodies on the strip. The remaining of the fluid flows to the absorbent pad which absorbs all remaining reagents and prevents backflow ensuring that capillary forward flow continues to occur.
Our LFA based device is a nitrocellulose based strip. The analyte consists of a combination of crRNA (guide RNA for CRISPR), LbCas12a enzyme, NEB r.2.1 buffer HPV16 DNA containing E7 region, ssDNA reporter and Milli-Q. The conjugate release pad consists of the following anti-FITC/FAM antibodies and gold nanoparticles which possess the ability to bind with FAM present on our ssDNA reporter. The gold nanoparticles bind to the anti-FITC antibodies which can conjugate to FAM present on the reporter. The test line consists of immobilized IgG anti-rabbit antibodies which can conjugate to the anti-FITC antibodies ,whereas the control line consists of Biotin binding ligand, streptavidin. Depending on the cleavage of ssDNA reporter during trans-cleavage caused by CRISPR/CAS12 reaction, a corresponding visual readout may be observed.
CRISPR
CRISPR technology, derived from the immune system of prokaryotes, is rapidly gaining popularity as the most effective and specific genome editing technology. In bacteria, a combination of proteins works to deactivate bacteriophages by destroying portions of their DNA (protospacers) that they recognise (by standard Watson-Crick base pairing) as incorporated into their own genomes from previous infections. These programmable nucleases, like Cas9, with the desired target guide RNA, are used to find and cause double-strand breaks in DNA sequences.
Cas12a is an endonuclease found in many prokaryotes that recognises a TTTV (where V is any other base) PAM sequence. In genome editing, Cas12a, mostly from Acidaminococcus sp, creates a Double Strand Break (DSB) with a staggered 5′ overhang (unlike the widely used Cas9, which creates a DSB with blunt ends).
One notable feature of the Cas12a enzyme is its unique cleavage behaviour. It exhibits target-activated trans-cleavage ability, thereby efficiently cutting non-specific single-stranded DNA molecules surrounding the Cas system-bound target DNA, apart from cleavage of the target site. This unique characteristic is extremely useful as biosensors for highly specific nucleic acid detection, particularly as early diagnostic tools.[6]
Our project employs CRISPR technology with Cas12a endonuclease to identify our selected biomarker. After binding to the target sequence complementary to the engineered guide RNA, Cas12a will also cleave the DNA probe, FAM-TTATT-Biotin, present in the solution, enabling the reporter to produce visual readouts on the LFA strips to indicate viral infection.
Novelty
1. Low-Resource
The accessibility and convenience of using Crisprly are cornerstones of our project. We aim to design a diagnostic kit without the requirement of trained personnel, or any specialized equipment. It will be extremely easy to use, and all the involved steps can be carried out at room temperature in any setting.
2. Non-Invasive
The accessibility and convenience of using Crisprly are cornerstones of our project. We aim to design a diagnostic kit without the requirement of trained personnel, or any specialized equipment. It will be extremely easy to use, and all the involved steps can be carried out at room temperature in any setting.
3. Targeted Specificity and Sensitivity
Unlike existing methods for cervical cancer screening, our kit is not only highly sensitive but also extremely specific to oncogenic strains of the virus as it binds to the conserved portion of the E7 gene found in HPV16 variants only. This helps us only screen women that require immediate preventative measures and not other non-oncogenic HPV infections.
4. Rapid Diagnosis
The result can be obtained much faster as compared to standard clinical methods, which take from days to weeks. This is more suited to screening camps, and other such settings.
Potential Applications
National Screening Programs: In India, most patients with Cervical Cancer report at a late-stage of disease due to lack of awareness. A national screening program will reduce this number drastically as early detection and prevention strategies are key to tackle current problems.
Clinics: This kit can also be utilized by the doctors in hospitals and clinics for HPV detection. For people who do not wish to use the kit at home or for people who find it difficult to operate the kit can simply go to a hospital or village health nurses for assistance.
Home: We have worked on designing a suitable microfluidics chip to further simplify the usage of our kit and optimize its accessibility. The user would be enabled to test in their comfort space in order to detect HPV infection. Since it uses a vaginal swab that can be collected by the patient themselves and simply deposited into the chip along with the reagents. Any person capable of carrying out these simple steps will be able to use our kit.
References
1. Bobdey, S., Sathwara, J., Jain, A. and Balasubramaniam, G. (2016). Burden of cervical cancer and role of screening in India. Indian Journal of Medical and Paediatric Oncology : Official Journal of Indian Society of Medical & Paediatric Oncology, [online] 37(4), pp.278–285. doi:10.4103/0971-5851.195751.
2. Yim, E.-K. and Park, J.-S. (2005). The Role of HPV E6 and E7 Oncoproteins in HPV-associated Cervical Carcinogenesis. Cancer Research and Treatment, 37(6), p.319. doi:10.4143/crt.2005.37.6.319
3. Kessler, T.A. (2017). Cervical Cancer: Prevention and Early Detection. Seminars in Oncology Nursing, 33(2), pp.172–183. doi:10.1016/j.soncn.2017.02.005.
4. Chen, Z., Schiffman, M., Herrero, R., DeSalle, R., Anastos, K., Segondy, M., Sahasrabuddhe, V.V., Gravitt, P.E., Hsing, A.W., Chan, P.K.S. and Burk, R.D. (2018). Classification and Evolution of Human Papillomavirus Genome Variants: Alpha-5 (HPV26, 51, 69, 82), Alpha-6 (HPV30, 53, 56, 66), Alpha-11 (HPV34, 73), Alpha-13 (HPV54) and Alpha-3 (HPV61). Virology, [online] 516, pp.86–101. doi:10.1016/j.virol.2018.01.002
5. Burd, E.M. (2003). Human Papillomavirus and Cervical Cancer. Clinical Microbiology Reviews, 16(1), pp.1–17. doi:10.1128/cmr.16.1.1-17.2003.
6. Leung, R.K.-K., Cheng, Q.-X., Wu, Z.-L., Khan, G., Liu, Y., Xia, H.-Y. and Wang, J. (2021). CRISPR-Cas12-based nucleic acids detection systems. Methods. doi:10.1016/j.ymeth.2021.02.018.
