The magic of microRNA

The magic of microRNA

From regulating genes to aiding disease diagnosis, microRNAs’ outsized impact certainly belies their tiny proportions.

At the dawn of the 21st century, experts widely believed that humans had over 100,000 genes, which are parts of the DNA that encode proteins. It turns out that the actual number was much lower. After the completion of the Human Genome Project, scientists finally arrived at the figure of around 20,000 protein-coding genes, which comprise roughly 1 percent of the human genome.

The remaining 99 percent is composed of non-coding DNA. Once regarded as ‘junk,’ these stretches of DNA in fact have important biological functions. Some non-coding DNA, for instance, is transcribed into non-coding RNA (ncRNA) like transfer and ribosomal RNA—both of which are integral for protein translation. In this feature, we shine a spotlight on microRNA (miRNA), a small, but mighty type of ncRNA that powers MiRXES’ diagnostic products.

Small molecules, big impact

As hinted by its name, miRNAs are small RNA molecules around 22 nucleotides in length. By binding to and degrading RNA transcripts of protein-coding genes, miRNAs play a key role in gene regulation. This allows miRNA to silence gene expression where needed, making these molecules critical in maintaining regular biological processes. True enough, abnormal levels of miRNA have been associated with cancer and other diseases.

Though miRNA is typically found inside the cell, they’ve also been detected in bodily fluids like blood and saliva. Consequently, with a simple prick, clinicians can not only diagnose a patient, but also predict the disease’s progression and even the potential for recurrence. And that’s not all—miRNA profiles can also reveal an individual’s likelihood to develop a disease and predict drug response. 

Recognizing the power of miRNA as a biomarker, MiRXES has built up its pipeline of miRNA-based diagnostic tests. In 2019, we launched GASTROclear, the world’s first blood test for the early detection of gastric cancer. Blood tests for lung, breast, colorectal, ovarian and liver cancer are also in the works.

GASTROClear: the world's first miRNA blood test for gastric cancer

Unlocking the mysteries of miRNA

Through the years, miRNA has proven difficult to detect due to its small size and low abundance. There are three methods commonly used in detecting and quantifying miRNA. The first relies on next-generation sequencing (NGS), which generates sequence information for all miRNAs present in a sample. While this makes NGS useful for discovering novel miRNA variants, the technique is costly and requires large amounts of input material. Furthermore, NGS cannot accurately measure miRNA levels.

Another technique is the microarray, which compares changes in miRNA expression. Microarrays rely on the binding of miRNA to fluorescent probes, with the resulting optical signal indicating the relative quantity of DNA. The brighter the signal, the more there is of a specific miRNA. Like NGS, however, microarrays are not quantitative, making them sometimes unable to detect very low levels of miRNA. 

Unlike the two previous methods, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) has the ability to quantify the amount of miRNA in a given sample. MiRXES’ miRNA-based diagnostic products rely on RT-qPCR, albeit with a unique twist that makes our technology far more sensitive than the rest. 

As easy as one, two, three (primers)

MiRXES’ patented RT-qPCR technology relies on the three-primer approach. The first kind of primer comes into play during the initial step of RT-qPCR, called reverse transcription. In this step, the enzyme reverse transcriptase converts miRNA into complementary sequences of DNA. This is made possible by using conformation-restricted primers designed specifically for each miRNA. These primers are distinguished by their stem loop structures, which prevent the primers from detaching easily once it binds to the miRNA.

After reverse transcription, the resulting DNA is amplified with the help of forward and reverse primers that attach on either side of the sequence, further ensuring specificity. With each cycle of PCR, the amount of DNA in the sample exponentially multiplies until it reaches a detectable level.

Finally, to quantify the DNA (and by extension, the target miRNA), PCR progress is tracked by the machine in real-time using dyes that nestle in between the DNA bases. As each dye corresponds to a distinct sequence, multiple miRNA can be detected and quantified even in a single set-up. Collectively, these three primers guarantee the sensitive, specific and robust detection of miRNA in biological samples.

MiRXES' patented RT-qPCR technology allows sensitive, specific and robust detection of miRNA in biological samples.

Adopted globally for discovery and diagnostics

To facilitate the adoption of our game-changing technology by researchers in academia, the National University of Singapore (NUS) ncRNA core facility was set up in partnership with the NUS Yong Loo Lin School of Medicine. Now in its second year, the facility is an early-phase discovery center that encourages industry-academic collaboration. We have since established similar partner ncRNA core facilities at Beth Israel Deaconess Medical Center (BIDMC) and other top academic institutes worldwide.

Once considered as genetic junk, miRNA has since transformed into a diagnostic mainstay thanks to MiRXES. Now, that is the magic of miRNA. 

MiRXES ID3EAL suite of discovery tools has been adopted by researchers in academia and industry for discovery and diagnostic applications.

Data from pulmonary hypertension collaboration with Janssen-J&J presented at ERS Congress 2020

SINGAPORE – September 8, 2020 – MiRXES Pte Ltd, a leading microRNA (miRNA)a diagnostic company headquartered in Singapore, in collaboration with Actelion Pharmaceuticals Ltd, one of the Janssen Pharmaceutical Companies of Johnson & Johnson, has presented the preliminary results of a study revealing that miRNA biomarker signatures have the potential to support early identification and diagnosis of pulmonary hypertension (PH). The data were presented today at the annual European Respiratory Society (ERS) International Congress.1

There is currently no simple, non-invasive test to identify and diagnose PH, a serious condition which results in high blood pressure in the blood vessels that supply the lungs.2,3 Pulmonary arterial hypertension (PAH) is a progressive form of PH that takes, on average, two years to diagnose from the onset of symptoms. Many patients are diagnosed at an advanced stage of the disease while many others remain unidentified.4,5 As PAH is progressive, delays in diagnosis can prevent early treatment and impact on patients’ prognoses, worsening clinical outcomes and survival.6

The positive results of this study show the potential of miRNA-based diagnostic signatures as a tool to help identify those in the early stages of PH,” said Aaron Waxman, M.D., Ph.D., Director of the Pulmonary Vascular Disease Program at Brigham and Women’s Hospital, Boston, USA, and Associate Professor of Medicine at Harvard Medical School.

Assessment of the biomarker N-terminal pro-brain natriuretic peptide (NTproBNP) is routinely used in PH centers today, but this measure is not specific for PH and can be elevated in patients with almost any type of heart disease, making its utility in detecting PH very limited, especially in the early stages.2,7

Increasing evidence suggests that patients with borderline PH (bPH)b and exercise PH (ePH)c may represent those in the early stages of PH.8 This study aimed to assess miRNA-based biomarker signatures in patients in the early stage of the disease, as well as those with established PH using MiRXES’ proprietary assay technology and biomarker discovery platform. In total, 245 plasma samples were collected from bPHb, (ePH)c, established PH, and non-PH symptomatic patients. All samples were assessed for NTproBNP and 600 miRNAs. The performance of NT-proBNP and miRNAs, alone or in combination, for distinguishing PH, bPH and ePH were analyzed.1

The analysis showed that miRNAs outperformed NT-proBNP for distinguishing non-PH symptomatic patients from bPH+ePH, bPH, and PH+bPH+ePH, with an area under the curved (AUC) of 0.71 vs 0.59; 0.60 vs 0.56; and 0.75 vs 0.70 respectively, suggesting that miRNAs may uniquely identify early stages of PH, prior to disease progression.1 The results also showed that combining miRNA with NTproBNP allows distinguishing of non-PH symptomatic patients from established PH patients.1

These results build on promising data from an earlier phase of the global research collaboration conducted in the UK and presented last month at the American Thoracic Society International Conference.9 The UK study demonstrated the feasibility of developing miRNA-based diagnostics for early detection of PH using 1,521 samples from PH patients, symptomatic non-PH patients, and healthy people collected at 3 UK National PH centres. AUC of miRNA signatures in identifying PH patients from healthy people and symptomatic non-PH patients were 0.94 and 0.75, respectively.

Building on these data, Janssen has initiated the CIPHER trial11, the design of which is also being presented at ERS. The CIPHER trial is an ongoing, prospective, multi-centre study that aims to identify miRNA biomarker signatures for early detection of pulmonary hypertension.

We are very excited with the promising early PH miRNA biomarker data generated from the current studies. Having launched our first regulatory-approved blood-based miRNA oncology test in 2019, MiRXES is committed to leveraging our end-to-end In Vitro Diagnostic (IVD) test development and manufacturing capabilities to support the translation of the PH miRNA biomarker signatures from bench to bedside,” said Lihan Zhou, Ph.D., CEO of MiRXES Pte Ltd.

Additional clinical studies are being planned in Asia, starting with Singapore and Japan, to provide further evidence to support the development and validation of the miRNA-based diagnostic test for early identification and diagnosis of PH.

 

Definitions

aMicroRNAs (miRNAs) are a class of small non-coding RNAs that play an important role in regulating gene expression. Deregulation of miRNA expression can lead to pathological processes such as PAH.12

bBorderline pulmonary hypertension (bPH) in this study was defined as having a mean pulmonary artery pressure (mPAP) between 21 and 24mmHg.1,13

cExercise pulmonary hypertension (ePH) in this study was defined as having a normal mPAP at rest and a mPAP of >30mmHg during exercise.1,14

dArea Under the ROC Curve (AUC) is a parameter that compares the usefulness of tests. The receiver operating characteristic (ROC) curve allows researchers to graphically compare the connection between clinical sensitivity and specificity. The AUC of the perfect test is 1, a value of 0.5 indicates there is no difference.15

 

About Pulmonary Hypertension (PH) and Pulmonary Arterial Hypertension (PAH)

PH is elevated pressure in the blood vessels of the lungs, which causes the heart to work harder to pump blood through the lungs.2 It is a serious, progressive disease and can lead to heart failure and early death. There are five groups of PH: group 1 includes PAH which could be inherited, caused by drugs or toxins or related to other conditions; group 2 refers to PH caused by a disease of the left side of the heart; group 3 includes PH resulting from lung disease and reduced oxygen in the body; group 4 refers to chronic thromboembolic pulmonary hypertension (CTEPH) and group 5 includes PH of unclear origin and mechanisms.16,17

PAH (group 1) causes the walls of the pulmonary arteries (blood vessels leading from the right side of the heart to the lungs) to become thick and stiff, narrowing the space for blood to flow, and causing an increased blood pressure to develop within the lungs. PAH is a serious, progressive disease with a variety of aetiologies, and has a major impact on patients’ functioning, as well as their physical, psychological and social well-being. There is currently no cure for PH and it is often fatal.2,3,17 However, the last decade has seen significant advances in the understanding of the pathophysiology of PAH, transforming the prognosis for PAH patients from symptomatic improvements in exercise tolerance 10 years ago, to delayed disease progression today.

 

References

  1. Waxman A, et al. Presented at European Respiratory Society Conference 2020 (Abstract 22823).
  2. Galiè N, et al. 2015 ESC/ERS Guidelines for the diagnosis and treatment of pulmonary hypertension. Eur Heart J 2016;37:67–119.
  3. Vachiéry JL and Gaine S. Challenges in the diagnosis and treatment of pulmonary arterial hypertension. Eur Respir Rev 2012;21:313–20.
  4. Prins KW, et al. WHO Group I Pulmonary Hypertension: Epidemiology and Pathophysiology. Cardiol Clin 2016;34:363–74.
  5. Humbert M, et al. Arterial Hypertension in France. Results from a National Registry. Am J Respir Crit Care Med 2006;173:1023–30.
  6. Brown LM, et al. Delay in Recognition of Pulmonary Arterial Hypertension. Factors Identified From the REVEAL Registry. CHEST 2011;140:19–26.
  7. Cao Z, et al. BNP and NT-proBNP as Diagnostic Biomarkers for Cardiac Dysfunction in Both Clinical and Forensic Medicine. Int J Mol Sci. 2019;20:1820.
  8. Simonneau G, et al. Haemodynamic definitions and updated clinical classification of pulmonary hypertension. Eur Respir J. 2019;53:1801913.
  9. Lawrie A, et al. Feasibility of microRNA-based signatures for early detection of pulmonary hypertension using machine learning methods. Am J Respir Crit Care Med 2020;201:A6352.
  10. Howard L, et al. Presented at European Respiratory Society Conference 2020 (Abstract 24264).
  11. gov. A Study for the Identification of Biomarker Signatures for Early Detection of Pulmonary Hypertension (PH) (CIPHER). Identifier NCT04193046. Available at https://www.clinicaltrials.gov/ct2/show/NCT04193046?term=NCT04193046&draw=2&rank=1. (Last accessed July 2020).
  12. Lee A, et al. Therapeutic implications of microRNAs in pulmonary arterial hypertension. BMB Rep. 2014;47:311–
  13. Nemoto K, et al. Borderline pulmonary hypertension is associated with exercise intolerance and increased risk for acute exacerbation in patients with interstitial lung disease. BMC Pulm Med. 2019;19:167.
  14. Wallace, WD et al. Treatment of exercise pulmonary hypertension improves pulmonary vascular distensibility. Pulm Circ. 2018;8:2045894018787381.
  15. Acute Care Testing. ROC curves – what are they and how are they used? Available at https://acutecaretesting.org/en/articles/roc-curves-what-are-they-and-how-are-they-used. (Last accessed July 2020).
  16. Rose-Jones LJ, Mclaughlin VV. Pulmonary hypertension: types and treatments. Curr Cardiol Rev. 2015;11:73–
  17. About Pulmonary Hypertension. Types of PH. Available at https://phassociation.org/types-pulmonary-hypertension-groups/. (Last accessed July 2020).
  18. Hoeper MG and Gibbs SR. The changing landscape of pulmonary arterial hypertension and implications for patient care. Eur Respir Rev 2014;23:450–7.

Onwards and upwards with MiRXES

Onwards and upwards with MiRXES

Now a trusted name in biotechnology manufacturing, MiRXES is hoping to boost its capabilities by tapping upon local talent and the defining technologies of Industry 4.0.

It’s been over two centuries since the first Industrial Revolution proved that technology can be harnessed to increase economic output. Now, we are in the midst of another great transformation in the way we live, work and manufacture products.

As the fourth Industrial Revolution (Industry 4.0) unfolds, we are seeing how productivity can be further enhanced by going beyond simple automation. Through emerging technologies like machine learning and the Internet of Things (IoT), manufacturers are now able to set up smart factories that get smarter over time. The end result? Interconnected systems that can communicate with each other to independently make decisions that increase efficiency and productivity.

While all this may sound straight out of science fiction, homegrown company MiRXES is hoping to make its own smart factories a reality. It’s the natural progression for a company that started with just a three-man team, but has since gone on to create the world’s first RNA blood test for early cancer detection and manufacture millions of COVID-19 diagnostic test kits in record time. In the second article of this two-part series, we focus on MiRXES’ ongoing efforts to surpass the status quo as a leading, Singapore-proud company in the era of Industry 4.0.

 

Creating more with less

As the threat of COVID-19 grew apparent earlier this year, MiRXES was granted the license to manufacture and commercialize the Fortitude diagnostic kit developed by the Agency for Science, Technology and Research (A*STAR) and Tan Tock Seng Hospital. Without a doubt, being called upon to support the mass production of Fortitude Kit was perhaps the company’s greatest chance so far to showcase its capabilities and prove itself on the world stage.

But there was a catch: at the time, the MiRXES production team was small, running a lean operation supported by the quality assurance, regulatory affairs and logistics teams. Almost overnight, the company’s compact team had to supply the whole of Singapore and countries abroad with the Fortitude kit. To cope with the sudden demand for test kits, the team had to practically overhaul their entire operational set-up.

Far from being intimidated, however, it was the opportune time for the MiRXES team to apply the capabilities they had worked to build over the past six years. After all, the team had invested much time and energy into understanding crucial matters like quality management as well as manufacturing processes and the science behind them. This lean and agile mindset allowed the company to adapt and deliver COVID-19 test kits just when Singapore needed them the most.

Part of the MiRXES manufacturing team

Taking MiRXES to the next level

Having gained confidence in the ability of its team to rise to the challenge, MiRXES is now aiming even higher. Today, the company’s goal is to use its combined R&D, manufacturing, and commercialization expertise to become a one-stop shop for Singapore’s vibrant biotechnology ecosystem. Moving forward, MiRXES is seeking to diversify its operations into areas like manufacturing raw materials for diagnostics, contract manufacturing and even product development for external companies.

Accordingly, MiRXES is now hiring for a variety of roles including process and systems engineers as well as facility executives. Amidst the retrenchments caused by COVID-19’s economic fallout, the company is hoping to create new opportunities for local talent. Moreover, MiRXES is also building a clinical laboratory and expanding their manufacturing facility by five times.

To scale its production output, MiRXES is turning to the emerging technologies that define Industry 4.0. The aim of implementing Industry 4.0 is to create a more versatile manufacturing line across multiple products, with a scalable platform capable of handling “any mix, any volume” manufacturing. This versatility will support different manufacturing demands, from prototyping to large-scale production.

After having invested close to S$2 million in building up its infrastructure, the company is now introducing automation modules into its existing manufacturing facilities. This involves automating processes for better efficiency and round the clock verification of product, machine and process quality. These modules are set to drive productivity and efficiency to the next level, enabling the company to face even bigger challenges and opportunities in the future.

Digitalisation is another key factor in Industry 4.0 where MiRXES will be incorporating processes from Demand planning all the way to delivery of products. This would include the harmonisation of CRM, ERP, MES and QMS modules electronically, allowing ease of use and “on the spot” trouble shooting, and performance monitoring over long and short periods of time.

Taken altogether, all these efforts are shaping MiRXES into a stronger and more resilient solution-oriented manufacturing company for Singapore and the world.

Watch this space: a brand new Industry 4.0 manufacturing facility coming soon.