We developed the test to address the needs of patients. From talking to medical professionals and MS patients, we discovered that there is a significant group of patients (10-30%) for whom diagnosing MS is challenging. For these patients, the burden of the diagnostic process is very high, as it can feel like a never-ending road of tests. This is especially exhausting when the needed tests are expensive, invasive and painful. We aim to support patients through this process by offering a minimally invasive test.

Final product design

Our finalised test detects a specific combination of miRNAs in a cell-free, paper-based test platform and comes with the following features:

Long-term storage
Easy distribution
Blood-based test
High accuracy (95%)

To ensure that the test is simple to use, all test components should be supplied in a ready-to-use kit. Therefore, in our test, the necessary materials and reagents for the isothermal amplification of miRNA will be provided, along with the correct positive controls. The logic circuit and threshold module are already embedded in the paper during assembly. The following items are included in the kit:

Reagents for one-pot NASBA
Amplification mastermix
RNase-free tubes
RNase decontamination solution
Reference miRNA (positive and negative control)
Paper test system
User guide

Practical use

Preparing the test

The development of the test starts with the identification of the miRNAs (Figure 1). Our miRNA search model finds specific miRNAs that distinguish MS from diseases with similar symptoms. This information is fed into miRADAR’s logic circuit algorithm, which designs an optimal circuit based on the number and importance of the selected miRNAs. The miRNA sequences are used to design primers for the isothermal amplification step. The amplified miRNAs serve as the input for our threshold system, which relies on Toehold-Mediated Strand Displacement (TMSD). The output of the threshold module acts as the trigger for the toehold circuit. Our toehold design software generates the best toehold switch structure to detect the trigger. Following computational optimisation, the DNA template containing the threshold system and the logic circuit can be synthesised, cloned into an expression plasmid and isolated. The template is then used to assemble the cell-free reactions on paper and freeze-dried. The tests can be constructed in a 384-well plate, facilitating easy handling and distribution.

Flow of assembly of miRADAR’s diagnostic test kit. After optimisation using the miRADAR models, the test can be experimentally validated and prepared for distribution.

Performing the test

Within the diagnostic procedure, this test is implemented when a patient has already been referred to a neurologist by their general practitioner. Since it is a specialised test, it will be ordered by the neurologist when the patient’s symptoms could be indicative of MS or when previous test results are inconclusive and the McDonald criteria are not met.¹

The McDonald criteria are a set of diagnostic criteria for MS.^1,2 Clinical, laboratory and imaging data is combined to accurately diagnose patients with MS when other possibilities have been ruled out. The most important criteria that should be fulfilled are dissemination in space (DIS), referring to the development in lesions (areas of inflammation) in the central nervous system, and dissemination in time (DIT), referring to the appearance of new lesions in the central nervous system over time. The most common methods used to investigate these criteria are magnetic resonance imaging (MRI) to visualise lesions and lumbar punctures to analyse the cerebrospinal fluid (CSF).

The test itself is carried out by a lab specialist, who only requires a blood sample from the patient. This blood sample first needs to be processed to isolate the miRNA. The first step in this procedure is to separate the serum from the plasma through centrifugation, which is standard procedure in most medical facilities. The miRNA extraction from the plasma fraction is possible with commercially available kits, which commonly require a volume of 200 \muL plasma.³ Isolated miRNA is amplified with the miRADAR test kit, allowing detection by the threshold module. Our test platform itself is pre-prepared and only the amplification product is required to reactivate the cell-free expression system. To ensure accurate testing, a set of controls is included in the test. In total, each test contains three discs (Figure 2). The two control discs contain a threshold module coupled to a toehold switch for a supplied reference miRNA with known concentrations. The first disc is rehydrated with the reference miRNA concentration above the threshold. This should activate the threshold system and is used as positive control, generating a purple colour when activated. The second disc is rehydrated with the reference miRNA concentration below the threshold. This should not activate the threshold system and is used as the negative control, remaining yellow. The third disc contains the threshold module and the MS-specific logic circuit and will be activated by the miRNA amplified from a blood sample. When the paper is rehydrated, the threshold system and the logic circuit are transcribed. Only the miRNAs that are present above the threshold will be able to activate the logic circuit, leading to the translation of lacZ. The reaction proceeds within two to three hours at room temperature.

Overview of the controls included in the miRADAR test kit. Disc 1: threshold module + toehold switch for reference miRNA, the disc should turn purple upon rehydration with reference miRNA above threshold; Disc 2: threshold module + toehold switch for reference miRNA, the disc should remain yellow upon rehydration with reference miRNA below threshold; Disc 3: threshold module + MS-specific logic circuit, the disc is rehydrated with miRNA amplified from a blood sample.

Test results and next steps

Our test was designed to generate an easily detectable qualitative output. When the test is negative, the levels of specific miRNAs in the patient sample are below a certain threshold associated with non-MS patients. A positive result shows that the miRNA levels of the patient are above this threshold and are considered suspicious. Based on the result, the neurologist will decide to continue with additional tests to confirm the diagnosis or to move on to different possibilities. Unlike current methods, our test is minimally invasive and easy to perform, lowering the burden on the patient regardless of the outcome.

Accessibility

A study by the MS International Foundation on the clinical management of MS described the most important factors that are hindering the early diagnosis of MS.⁴ For many countries, especially those that are on the lower-income scale, access to specialised equipment and tests is limited. This means that getting an accurate MS diagnosis is not only more complicated, but can also be more expensive when travel is required. To tackle these problems, we designed our test to be suitable for standard laboratory equipment. By generating a visible output, no imagers are necessary. The freeze-dried tests are stable at room temperature, so they are readily stored and distributed.^5,6 Furthermore, the activation of the test can be carried out at room temperature. We use filter paper as the test matrix, so miRADAR is also cost-effective.⁷ Moreover, we investigated an alternative signal output, DNAzymes, that could contribute the the accessibility of the test in the future. In comparison to the conventional protein enzymes that we now use in the miRADAR diagnostic test, DNAzymes offer several advantages. Most importantly, using DNAzymes in cell-free systems completely omits the need for transcription and translation machinery. Simple DNAzyme-based systems require nucleic acids, common and cheap cofactors and reagents, and simple salt buffers, resulting in significantly lower costs due to the lack of purified proteins and required components (NTPs, amino acids, etc.).^8–13

DNAzymes are very small in size; consisting of short DNA oligos (minimally 15 nt) meaning they are easy and inexpensive to synthesise. They are adaptable for rational design in sensing platforms by triggering the formation of the G4/hemin structure in a reaction. One major example is by linking them to probes in order to directly target nucleic acid sequences. Other options include blocking and releasing (part of) the sequence to inhibit/promote secondary structure folding (similar to RNA aptamers), or synthesis by amplification techniques. They are also remarkably physiologically tolerant; the ability to self-assemble in suitable conditions shows they can be denatured and renatured without losing catalytic activity. This also means they are compatible with freeze-drying and are functional in a range of different conditions. All of this makes them easy to handle and suitable for storage and shipping, thus fitting our accessibility criteria. Currently, miRADAR relies on using PURExpress, a commercial high-performing transcription/translation system, which increases the cost per test. Cheaper alternatives such as E. coli TXTL or ’home-made’ OnePot PURE are laborious to make, have batch-to-batch variation and still require addition of molecules such as tRNA and cofactors.^14,15

So far, our DNAzymes showed a level of background signal, which should be avoided for a diagnostic platform. Additionally, we managed to generate a coloured output for detection of DNA, but not yet for RNA. For highly sensitive detections the signal to noise (S/N) ratio can be improved by implementing (signal) amplification strategies such as a hybridisation chain reaction (HCR) in which probes, when activated, hybridise with each other to form chains containing many catalytic regions (Figure 3) Buffer compositions may also be further optimised, such as the addition of NH4Cl as recently discovered by Chen et al. (2020) improves the colour formation and retention, which we also observed in the lab, as well as optimising the hemin and antioxidant concentrations and ratios. ⁹

Example for sensor design with the hybridisation chain reaction (HCR) amplification strategy.

Furthermore, the G4/hemin complex can convert alternative substrates, such as the colourimetric TMB (3,3',5,5'-Tetramethylbenzidine) or the chemiluminescent luminol, and more.¹⁶ Besides the type described above, there are other well investigated DNAzymes to be applied, for example those with cleavage activity, which may allow for dual output signals when using fluorophore quenchers in addition to the colourimetric systems. Using different colours and outputs could expand the testing platform beyond one binary standard and account for different patient or disease (sub)types, or allow to system to be broken down into composite components to lessen the burden and improve robustness.

With the use of DNAzymes perhaps other modes of action should be considered as well. A plethora of G4/hemin biosensors have been developed over the last decade, with many focussing on miRNA detection.^10,¹⁶ Many of these are fully based on synthetic DNA oligos designed to function according to strand displacement principles (Figure 4).^10,^17,¹² Such systems are fully enzyme-free, making them even more durable for storage, shipping and handling by hospital workers, as it avoids handling precarious RNA in an RNAse free setup, prevents possible denaturing of proteins and in many cases is more cost effective as very few ingredients are required. With the increasing efforts made by many in the field we may in the future also design complex logic circuits.¹⁸

A G4-hemin miRNA sensor based on strand displacement by miRNA and a hemin-aptamer. Adapted from X. Li et al (2017).¹⁷

Outside of these properties, the main feature of our test is that it is minimally invasive. This could make patients more willing to take the test in the early stages of their diagnostic procedure. We aim to contribute to a quicker diagnosis of MS, by providing a test that does not rely on specialised personnel or equipment. Therefore, patients can be prescribed medication or other therapeutic intervention sooner, to maintain or improve their quality of life.

Lastly, the lack of awareness of MS and its symptoms among healthcare professionals and the general public can complicate the diagnostic process.⁴ With this project, we strive to spread more awareness about the various symptoms of MS, and the difficulty associated with diagnosing complex diseases.

Challenges and opportunities

The miRNA expression data availability for different diseases varies widely. For MS specifically, there is a lack of data on the miRNA levels of patients and healthy individuals. Before this technology can fully be established, we need more data to find specific miRNA combinations with diagnostic value. This also goes for many other diseases that could benefit from (an additional) diagnostic tool. The field of miRNA diagnostics has been steadily growing, and their suitability as biomarkers is frequently reported.^16–18 miRADAR contributes to this field, showing a way to do multiplex miRNA diagnostics through model-guided customisation.

Regulatory approval is still required before the test could be implemented in the diagnostic procedure. We acknowledge that this is a crucial step moving forward and should be investigated further. miRADAR is based in Europe, so the test should be compliant with the In Vitro Diagnostic Medical Devices Regulation (IVDR).¹⁹ This legislation describes the safety and performance requirements for an in vitro diagnostic test, as well as the considerations for introducing such a test to the market. The evaluation of miRADAR in this context would therefore be the priority. It would also be valuable to reach out to insurance companies to understand what properties are required for the test to be covered by insurance. However, this varies widely per country and thus requires research on a larger scale.

Another crucial aspect is that the test should have an accuracy above 95%. False positives will needlessly worry patients, whereas false negatives prevent them from getting the care they need. We have not yet shown this in the lab for our final test design. This therefore requires additional optimisation in the future.

The accuracy of a diagnostic test is dependent on two values: the sensitivity and specificity.²³ The sensitivity is the probability that the test will be positive for people with disease, whereas the specificity is the probability that the test will be negative for those without disease. Therefore, by investigating the number of false positives and false negatives, the sensitivity and specificity can be determined, as well as the positive and negative predictive values (Figure 5). These values indicate how good a diagnostic test can predict a true positive or true negative, respectively.

Assessment of accuracy for diagnostic tests. Respective parameters are shown for the sensitivity, specificity, positive predictive value and negative predictive value. Based on Mediratta et al. (2023).²³

In our project, we designed an experiment to determine these values for an MS-specific miRNA toehold switch. We wanted to perform this experiment by assembling the cell-free paper-based tests in a 384-well plate, as a sample size of 300 is in most cases enough to evaluate the sensitivity and specificity of a diagnostic test.²⁴ In the experimental setup, we decided on several test conditions related to the presence/absence and concentration of trigger miRNA. Additionally, we wanted to test the toehold switch in the presence of a different miRNA, to make sure our toehold switch was specific only to hsa-miR-484, a miRNA that was found to be upregulated in Relapse-Remitting MS.²⁵ A very low concentration of miRNA, similar to the levels in blood, could be used to show that amplification is necessary before a toehold switch can be activated. Unfortunately, we were not able to fully realise this experiment, but this setup could be used in the future to determine the accuracy of our diagnostic test.

Despite these challenges (Figure 6), miRADAR’s modular design makes it possible to expand this technology towards the detection of other diseases, requiring only the identification of disease-specific miRNAs. With more information, miRNA diagnostics could cement itself as a minimally invasive test-platform for the accurate diagnosis of (complex) diseases.

The potential of the technology at the core of miRADAR also raises some questions about the field of diagnostics as a whole. With technology constantly improving and evolving, there are more and more possibilities to diagnose diseases. However, MS spokesperson Hanneke Kool taught us that getting an early diagnosis is not always best for patients. When there are no treatment options, the weight of a diagnosis can be heavy. It can also make mundane tasks difficult, like buying a house, getting a mortgage or finding suitable insurance. On the contrary, as we heard from students during our lecture about the ethical considerations of our project, having a diagnosis early might help patients prepare for what’s to come, or in best-case scenarios give them more treatment options. During the development of novel diagnostic tests, it is important to consider the practicalities associated with having a diagnosis or not.

Overview of the SWOT (Strengths, Weaknesses, Opportunities, Threats) analysis of miRADAR, showcasing the key aspects visually in the four quadrants.

Future perspectives

Recent studies have also shown that a pattern of dysregulation can be detected even before traditional symptoms begin.²¹ This initial phase, known as the prodrome, is much more subtle, with ambiguous symptoms such as fatigue, sleep problems and headaches. The cerebrospinal fluid (CSF) of individuals who went on to receive an MS diagnosis showed elevated demyelination roughly six years before diagnosis.²² This implies that MS could be diagnosed in the prodrome stage. miRADAR could prove to be a critical tool for early detection of MS, especially for minimally invasive screening on a larger scale.

Ultimately, the use of miRNA in diagnostics is extremely promising. As biomarkers, miRNAs are highly specific, allowing for accurate testing, and stable in blood, allowing for minimally invasive access.²³ miRADAR was designed to contribute to the diagnostic procedure for MS, and to prevent unnecessary stress on patients. By using synthetic biology, we have provided a framework for the identification of disease-specific miRNAs and the custom design of genetic circuits using toehold switches. In this iGEM project, miRADAR has taken an important step forward in miRNA diagnostics and simplifying the diagnosis of complex MS cases.

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