Team:Moscow/Poster

Poster: Moscow



HaploSense
Presented by Team Moscow 2020

Daria Smolyarova, Marina Marchenko, Vera Sysoeva, Anastasia Kislova, Anna Lazareva, Nadezhda Korostyleva, Roman Novikov, Nikolay Kristovsky, Maria Medvedeva, Daria Koshkina, Ekaterina Kravchuk, Alexandra Greshnova, Anastasia Ivanova, Arina Kholkina, Viktoria Ovchinnikova.

Abstract

As viruses mutate quickly, different genotypes among one species can develop. For some viral diseases, genotype detection is crucial for successful treatment, and hepatitis C is one of them. Nobel Prize 2020 was awarded for the fight against hepatitis С, a major global health problem that causes cirrhosis and liver cancer.

Hepatitis C is caused by hepatitis C virus (HCV) that has at least six different genotypes. Recombinant variant RF2k/1b is common in Russia, but it is often mistaken for genotype 1 that leads to incorrect treatment. However, a lot of infected people in Russia do not know their HCV genotype because of expensive testing or the impossibility to perform tests in peripheral regions.

So, we are developing fast, specific and easy-to-use portable Cas-based device “HaploSense” to solve this problem. HaploSense can be optimized for genotype detection of any RNA viruses, including HIV or Sars-CoV-2, and may become a universal detection platform.

Project Goals
• Design a device for RNA-viruses’ genotypes detection

We designed both Hardware and Molecular system for RNA-viruses’ genotypes detection that are visualized in Poster.

• Approve and improve concept through Human Practices and Modeling

We asked researchers, health workers, officials and BIOCAD company about the detection object choice, our molecular system improvements and hardware mechanism.
Also we selected SNPs and made a kinetic model to prove that HaploSence will work as expected.

• Share knowledge about synthetic biology

We put a lot of time into teaching, public lectures and BioLego Go bioengineering game.

Human Practices: From SARS-CoV-2 to HCV


From SARS-CoV-2 to HCV

Human Practices cycles changed the entire concept of the project: the detection system initially focused only on genetic variants of SARS-CoV-2, turned into a detector of hepatitis C virus (HCV) genotypes. Because there is a specific genetic variant of HCV in Russia that is hardly determined by existing test systems.

Interaction with BIOCAD

The BIOCAD experts gave us feedback on almost every aspect of our work: molecular system, hardware, product introduction in the healthcare system, and patent law. As we describe on the Engineering Success page, we considered every answer and changed the design of the system and our workflow, respectively. The patent law experts gave us an extremely valuable webinar about the Russian & world patent law basics and product patenting workflow.

Collaboration with the nonprofit organization “Together Against Hepatitis”

When we switched to HCV we found a nonprofit organization - “Together against hepatitis.” This organization unites physicians, patients, and volunteers to help patients in Russia get full information about the disease, testing, and treatment possibilities. We met the NPO president to ask him about patients’ view on hepatitis C and its treatment. The meetings showed us that we could help not only by creating an easy-to-use and relatively inexpensive virus genotype detection platform but also by organizing an HCV awareness campaign. In collaboration with the nonprofit organization, our team has started a series of informational posts on social media, and we are looking forward to the future possibilities in this field.

Modeling

During modeling we solved two main problems:

1. For detection CasX needs a guide RNA with complementary sequence to target DNA. So we made a list of SNPs of SARS-CoV-2 and hepatitis C virus to select the region for detection based on CasX properties.

2. Then we created a detailed kinetic model to ensure system performance, optimal conditions, reaction time, potential sensitivity and to prove efficiency of our system. As a result, we determined that additional HUDSON step is an essential stage because it kills viruses, reduces RNA degradation and, thus, increases sensitivity and safety (see graph).

Test System

1. Blood collection by medical staff.

2. HUDSON: virus inactivation at 95 degrees Celsius.

3. RT-LAMP: RNA is converted into DNA and amplified to increase sensitivity.



4. Detection of target sequence: CasX cuts the reporter molecule labeled with biotin and the FAM fluorophore from opposite sides.

5. Visualization: resulting solution is placed on a test strip with two binding zones and visualized with the help of a fluorimeter.

6. Result interpretation:

2 bands → presence of a specific viral genotype;
1 band → no target sequence.



Hardware
Device is aimed to detect fluorescence on the test-strip. To do so we designed a fluorimeter with a replaceable and washable unit. Test strip after addition of the resulting solution is placed in a removable unit and fixed in a fluorimeter that emits blue light to induce green fluorescence of FAM and, finally, measurement results will be displayed on the screen.
Implementation
Proposed end users

Developing our project, we primarily aimed to create a test system that could be used in Russia and CIS countries. By developing an easy-to-use and relatively cheap test system, we wanted to make HCV genotyping more affordable for infected people in Russia to make their treatment more effective.
As blood samples are required for HCV testing, we assume that our test system will not require a well-equipped laboratory and trained staff, other than nursing personnel. So, our system can be implemented by junior medical staff in any medical facility (hospitals, polyclinics, etc.)

Why is HaploSense potentially better than existing methods?
  • PCR is quite sensitive but requires several hours to extract the RNA and expensive equipment to perform the testing.
  • ELISA is simple and inexpensive but sacrifices sensitivity. False readings occur with high frequency.
  • HaploSense has potentially high sensitivity and speed, allowing accurate detection of small amounts of a pathogen potentially in 1 hour. The method requires simple and cheap equipment: a small portable fluorometer that is part of the hardware and an additional simple thermostat that can hold temperatures up to 95 degrees.
Education

Education

In order to spread principles of synthetic biology in Russia and worldwide we gave lectures, seminars, workshops, and even went on the radio! Experts in biology, students, schoolchildren, and people, who heard about synbio for the first time, interacted with us. Due to all this we found the best format for learning about synthetic biology - a game.

We created an online game - BioLego Go. It teaches the basics of genetic circuits design, uses the terms of synbio logic, has a nice and simple design, and imitates the familiar lego bricks game.

Want to perform your own experiments in synbio? Just try BioLego Go!

Future Direction

During this iGEM cycle, we performed modeling and theoretical research for our system. In future, we will have to deal with the next crucial step, experimental validation of our system, and to compare its parameters with the calculated ones.

As we never had to deal with the implementation of such a big project before, we asked BIOCAD experts about patenting and marketing. So, after iGEM we plan to:

  1. Make a prototype of our molecular detection system and hardware;
  2. (If prototype works as expected) to take care about patenting in advance;
  3. Proof system’s efficiency on test synthesized sequences through comparison with real-time PCR ;
  4. Validate and verificate the test system;
  5. Register our test-system and implement it in medical facilities.

Our product might become the first CRISPR/Cas based testing method in Russia when it is introduced into the market.

Acknowledgements and Sponsors






















References
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  3. Max J. Kellner et al., “SHERLOCK: Nucleic Acid Detection with CRISPR Nucleases,” Nature Protocols 14, no. 10 (October 2019): 2986–3012, https://doi.org/10.1038/s41596-019-0210-2.

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