Team:Moscow/Description

Description

Disease of the 21st century

“Disease of the 21st century” makes everyone think of the COVID-19 pandemic, which has affected 235 million people (1). Actually, another quiet disease rules the world and has no cure. This is a major depressive disorder (MDD) that affects 300 million people (2). MDD is not a low mood but a formal mental disorder, the main symptoms of which are depressed mood and diminished interest in almost all activities (3). In addition, patients have such secondary symptoms as feelings of worthlessness, excessive guilt, diminished concentration, chronic fatigue, sleep and appetite disorders, and suicidal thoughts. For MDD diagnosis the individual must be experiencing five or more symptoms during the same 2-week period and at least one of the symptoms should be the main one.

The number of people diagnosed with MDD is growing every year (Fig. 1).

Fig. 1 - Growing of the number of people diagnosed with MDD.

But the main problem remains an underdiagnosis of depressive disorder. The prevalence of undetected MDD cases varies from country to country, with about 45% in Spain (4). This is due to the lack of qualified specialists and the stigmatization of mental disorders. Moreover, even qualified doctors find it difficult to make a diagnosis, as symptoms of depression are very similar to bipolar disorder (5) and anxiety disorders (6).

Depression is connected with many areas of life.

  • Depression shortens lifespan and leads to disabilities (7)
  • The total economic burden of MDD is now estimated to be $210.5 billion per year
  • The COVID-19 pandemic has caused an increase in MDD cases (8, 9)
  • 60% of people who commit suicide have a mood disorder (10)
  • Excessive use of gadgets leads to depression as it is connected with lack of sleep and stress (11)

Our team members experienced depression. The team leader, Kirill Reshetnikov, commented on his depression “It seemed to me that my mood was not connected with the disease. Friends literally forced me to see a psychiatrist. I was diagnosed with MDD during the third psychiatrist appointment.” After analyzing the comments of specialists (link to human practice) and patients, we realized that the world needs a simple and reliable way to distinguish depression from other mental disorders.

How is depression diagnosed?

In most cases, MDD diagnosis is determined after psychiatric evaluation. Doctors base diagnosis on approved criteria for depression such as ICD-10 (12) and DSM-5 (3). Both documents contain a list of symptoms and the frequency of their occurrence. Psychiatric evaluations have significant disadvantages such as difficulty to find a mental health specialist and the reluctance of patients to set an appointment. It is because psychiatrists are wrongly considered to be biased (13).

Blood tests for depression including cortisol and BDNF level determination are less popular. An increased level of cortisol in the blood during MDD is an unreliable parameter since it depends on the type of depressive disorder and the gender of the patient (14). Blood cortisol level is also measured in the dexamethasone suppression test which was used in the last century (15). The test was helpful for diagnosing melancholic depression (16) but was hard to interpret correctly (17). Research shows that the level of BDNF (brain-derived neurotrophic factor), which is crucial for synaptic plasticity, is reduced in MDD (18). However, BDNF is inconvenient to use, since it is a biomarker of schizophrenia and bipolar disorder (19). For the diagnosis of MDD, we would need to make a panel of protein markers.

In 2018, MIT scientists developed the first context-free algorithm to detect depression by modeling audio and text sequences of an interaction between a human subject and a virtual agent (20).

More about concurrents you can read in section - Proposed Implementation

Thus, after analyzing the existing diagnostic methods, we decided to create the miPression test, which will cope with the shortcomings of current solutions. The results of our test will not be distrusted and will show higher specificity. Our test will measure the amount of depression-specific miRNAs in the blood and compare the values ​​with normal ones.

Why did we choose miRNAs?

We studied the existing theories about the correlation between depression and the concentration of various substances: BDNF, cortisol, TNF-alpha (21), IL-6 (22), C-reactive protein (23), and others. All these compounds are considered to play a role in the pathophysiology of depression.

Unfortunately, the levels of cytokines, cortisol, and BDNF can vary significantly depending on the allostatic load in both depressed and healthy people (24). Interestingly, alterations in both cytokines and cortisol levels have also been seen in non-psychiatric medical conditions, such as coronary heart disease (25, 26). Above, we have already mentioned tests for the level of cortisol and BDNF and their disadvantages.

Also, we investigated the possibility of using neurotransmitters as biomarkers of MDD, since levels of dopamine, norepinephrine, and serotonin are changed in MDD (27). It was shown that it is possible to detect depression by four markers in blood plasma (including dopamine), but it requires the use of expensive equipment for mass spectrometry (28).

As a result, we chose microRNAs as biomarkers for MDD. These short non-coding RNAs with a length of 18-25 nucleotides are involved in the regulation of gene expression, including neurogenesis (29) and synaptic plasticity (30). Interest in these molecules as a diagnostic marker has grown significantly over the past 10 years: from 891 articles in 2010 to 5716 articles in 2020 (PubMed, search request “miRNA in diagnostics).

Fig. 2 - A number of articles, which are devoted to the diagnostic role of miRNA.

A study in 2016 found 5 miRNAs that were associated with depression but did not correlate with bipolar disorder (31). It led us to work with a similar system. We decided to extract miRNAs from blood plasma since a similar study has already been successfully carried out. It diagnosed depression in blood plasma by a depressive disorder-specific miR-134 with 79% sensitivity and 84% specificity compared to controls (32).

miRNAs are already used to diagnose thyroid cancer (33). In the near future, these biomarkers will be used for many types of cancer such as colorectal, lung, prostate, and breast (34). Panels for the diagnosis of Alzheimer's disease, heart failure, and hepatotoxicity are developing (35).

Selecting a detection system

One of the main methods for detecting miRNAs is qPCR (quantitative polymerase chain reaction), which is widely used due to its sensitivity. However, qPCR can lead to false-positive results (36) associated with the small size of miRNAs, which reduces the chance of correct annealing of primers. In addition, the high homology of miRNAs within the family reduces the specificity of the method (37).

As a part of the competition, we need to use the methods of synthetic biology, so we considered the use of Toehold Switches (38). Such a system has high sensitivity and the possibility of multiplex detection (39). However, the analysis of Toehold Switchers showed that laboratory work with this method will cost a lot due to the cell-free synthesis system (40). Moreover, in Russia, there are no laboratories and experts in the system, so this task would be too risky.

As a result, we settled on the CRISPR/Cas system, since we have enough experience, materials, and specialists in the technology. More importantly, the sensitivity of nucleic acid detection with CRISPR/Cas is much higher compared to Toehold Switchers (10 ^(-18) M vs 10^(-9) M, respectively) (41). We also compared different variants of Cas proteins and chose dCas9 (42) as it suits all our requirements.

Thus, Cas12 and Cas14 have non-specific collateral activity (43, 44), which means that it is impossible to make a multiplex detector system with it. Cas13 suits for multiplex detection only with the use of proteins from different organisms and reporter nucleotides, but there are only 3 such variants (and we are targeting a panel of at least 10 miRNAs) (45). Moreover, the isolation of proteins from different organisms can be difficult and very noisy.

Our system

The inspiration for miPression is the iGEM NUDT CHINA project, 2016 (46). Our main goal was to improve the system and make it multiplex while maintaining its sensitivity. The main difficulties in the development of the system were associated with our biomarkers since miRNAs are present in samples in small quantities and their length is only 18-25 nucleotides.

Our system combines rolling ring amplification (RCA) and dCas proteins coupled with split fluorescent proteins. We decided to prove the concept by developing a system with a single fluorescent protein. We use GFP (green fluorescent protein) to validate our system. The system is based on bimolecular fluorescence complementation, which means that GFP is split into two halves and dCas9 carries one of the halves.

Experiment design:

  • All miRNAs are isolated from the patient's blood plasma with the use of a special extraction kit. The resulting miRNAs are added to a test tube containing a pre-ligated dumbbell probe and phi29 polymerase.
  • Target microRNA and the dumbbell probe hybridization triggers Rolling circle amplification. Non-compliment miRNA cannot bind the probe and thus produce no amplification products.
  • After amplification, a long DNA molecule is formed. It consists of alternating linear structures and hairpin loops.
  • dCas9 conjugated with the N-terminus of GFP, interacts with the linear region, while dCas9 with the C-terminus of GFP binds the hairpin.
  • If the amplification is successful and the proteins bind to the product correctly, then we will be able to register the glow of GFP. Fluorescence can be quantified on a fluorometer and based on the data the concentration of the target microRNA can be quantified.
    • Fig. 3 - Our system.

However, there is a chance that the bimolecular system will not perform well in vitro and the rate of maturation will not be quite high. Therefore, we consider upgrading the miPression to a tripartite Split-GFP System (47).

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