In just over a year, the urgent administration of over a billion doses of mRNA-based COVID-19 vaccines worldwide has placed RNA therapeutics fresh in the minds of communities around the world.
RNA therapeutics and other nucleic acid therapeutics (NATs) which operate using engineered nucleotide sequences have proven to be quick and effective alternative candidates to traditional therapies—from slowing the spread of cancers to speeding up wound healing.
Because just a few thousand of these synthetic nucleotide sequences need to be screened, as opposed to the millions of molecules in typical drug discovery processes, the speedy turnaround of RNA-based therapies narrows the gap between bench and bedside. This is particularly crucial when it comes to global emergencies like the COVID-19 pandemic.
Fresh off the crest of the world’s largest vaccine and therapeutics rollout in history, find out how A*STAR continues to ride the wave of burgeoning RNA technologies to develop novel solutions beyond battling COVID-19.
Building on the building blocks of life
Far from being a rushed development, the journey to understanding RNA and its applications began more than a century ago with the discovery and isolation of DNA in 1869 by Swiss physician Friedrich Miescher.
In 1961, Nobel laureate Sydney Brenner, who later helped place Singapore on biotech world stage, and eight other pioneering researchers detailed how cells use RNA1,2 to translate and transport information from DNA to ribosomes that synthesise proteins. It wasn’t until a decade later that scientists began to consider the possibility of harnessing one particular form of RNA—messenger RNA, or mRNA, as a tool to fight disease.
One of three types of RNA involved in protein synthesis, mRNA plays the role of messenger, as the name implies, by transcribing information from DNA and translating it into proteins by attaching to a ribosome.
The first step, transcription, is the process of creating a single-stranded copy of the target gene—smaller than a DNA strand and just the right size to slip through nuclear pores and exit the nucleus into the cytoplasm of the cell.
Like an instruction manual, the mRNA is then ‘read’ in the next major step—translation, where ribosomes in the cytoplasm use mRNA as a template to assemble specific chains of amino acids which form proteins. Here, mRNA can be synthesised to hijack the body’s natural processes and essentially ‘tell’ cells what proteins to make.
Aside from making new proteins, NATs also inhibit the creation of harmful proteins. Many currently approved NATs rely on antisense oligonucleotides (ASO), explained Prabha Sampath, a Senior Principal Investigator at the A*STAR Skin Research Labs (A*SRL). Essentially, ASOs are complementary to the relevant mRNA strand in the body and bond with it to inhibit the translation process.
“These synthetic nucleic acids modify the functions of endogenous cellular RNAs by interacting with them through Watson-Crick base pairing,” Sampath said. “All current ASO therapeutics target protein-coding RNAs and exert their effects by altering protein synthesis.”
Saying nay to cancer with NATs
One major application for NATs is using it as a tailored cancer therapy. Because cancer is a result of genetic mutation—where either tumour suppressor genes, oncogenes or DNA repair genes do not function as they should, genetic therapies like NAT have the potential to effectively target and suppress the effects of these mutations.
“Depending on how they are designed and engineered, NATs can virtually abolish or titrate the expression of a gene, edit an aberrant genetic variant or increase expression of a gene,” explained Si Hui Tan, a Senior Director at Cargene Therapeutics and a former A*STAR researcher, who received the Young Scientist Award in 2020.
“The beauty of NAT lies in its simplicity,” said Tan. “Genetic information suffices for effective drug design.”
Theoretically, once the patient’s specific mutation is discovered the relevant NAT can be deployed to combat it. Such targeted cancer therapies have been the goal of Tan from her time as an A*STAR researcher at Nick Barker’s laboratory to her current role at Cargene Therapeutics.
Tan’s previous work saw the discovery of aquaporin-5 (AQP5) as a marker for gastric cancer stem cells3. By isolating these stem cells and growing organoids to study their behaviour, researchers could potentially modify the organoids and transplant them back into the patient to repair the damaged stomach lining.
Since then, Tan has pivoted her efforts to focus on oligonucleotide therapy at Cargene Therapeutics. In particular, the biopharmaceutical company develops siRNA-based therapies—one of the frontrunners for NAT therapies next to ASOs. Small interfering RNA, or siRNA, is a double-stranded non-coding structure capable of degrading target mRNA and inducing gene silencing. Cargene’s technologies, originating from A*STAR, allow researchers to discover the relevant target mRNA, generate siRNA sequences, design stable siRNAs and effectively deliver them to target organs.
At the Genome Institute of Singapore (GIS), Group Leader Jay Shin and his team also work to uncover the therapeutic potential behind non-coding DNA and RNA through new technologies and large-scale assays. One such molecule, long non-coding RNA, or lncRNA, shows promise as a tissue-specific regulator of gene expression.
Recently, Shin’s lab mapped out lineage-specific lncRNAs in human dermal lymphatic and blood vascular endothelial cells, known as LECs and BECs, to discover their potential role in cancer progression, chronic inflammatory diseases and diseases that lead to blindness.
Interestingly, the team was able to identify LETR1 as a modulator of essential genes and gatekeeper of the LEC transcriptome—indicating that every cell type could express precise lncRNA signatures to control lineage-specific regulatory programmes4.
However, even with an in-depth understanding of various non-coding RNAs, targeted cancer therapies are easier said than done. According to Tan, NAT-based cancer therapies face three major challenges—recurrence, delivery and cancer with epigenetic or post-translational causes. The first, recurrence, happens when the bulk of a tumour is eliminated but a minor population harbouring its own set of mutations remains resistant. This minor population eventually grows to form a new tumour.
“This is where tumour sequencing and NAT can synergise to deliver precision medicine,” explained Tan. “Based on the patient’s tumour genetic profile, effective NATs could be prescribed or designed when needed.”
Similarly, precise NATs tailored to individuals can also tackle cancers with epigenetic or post-translational causes. Despite having a less direct effect, as researchers gain a deeper understanding of the pathways in which affected genes act, the relevant molecular interactors
can be identified and targeted with NAT.
Finally, to send such therapies to tumour cells, specific and efficient delivery agents must be developed—a field that A*STAR’s bioengineers are extensively looking into.
“NAT is a very promising class of drugs with a great deal of global momentum across academia, start-ups and pharmaceutical companies,” said Tan. “I foresee NAT benefitting cancer patients on a large scale in the next decade.”
A micro molecule with massive impact
Another class of non-coding RNA, microRNA (miRNA), has a major role to play in the NAT landscape. These short, single-stranded molecules can be harnessed to bind to the relevant mRNA sections of harmful genes and inhibit protein synthesis.
At A*SRL, Sampath and her team investigate miRNA’s role in skin cancer, inflammation and wound healing. In particular, the researchers analysed the difference in miRNA and mRNA expression between individuals with and without atopic dermatitis, commonly known as eczema. Their study not only reaffirmed the role of miRNA in eczema but also identified several specific miRNA involved5.
Building on their discovery, Sampath’s team sought to identify the underlying mechanisms behind skin barrier defects that contribute to the disease’s development. They found miR-335 to be the most consistently downregulated miRNA in patients with eczema. Responsible for keratinocyte differentiation and cornification, miR-335 represses SOX6 and is essential for proper skin barrier maturation6.
“We are looking for small molecules that restore miRNA expression in the epidermis, the outermost layer of skin on the body,” explained Sampath. “Compound effects are assayed by imaging skin cells expressing a fluorescence-based biosensor we developed in-house. Candidate substances identified through screening will be subjected to in vitro, ex vivo and in vivo testing to validate their effects on skin maturation.”
One such candidate drug Sampath’s team has identified is belinostat, which was found to consistently induce miR-335 expression to repair barrier defects in the epidermis7. With further investigation into its applications and delivery, belinostat has the potential to be a frontrunner in alleviating the uncomfortable effects of eczema, according to Sampath.
When it comes to wound healing, miRNA research holds similar promise but runs into unique challenges. “Resolving wound healing defects often involves a fine balancing act,” explained Sampath. “Many of the cellular and molecular pathways that encourage wound healing can potentially trigger malignant pathways if inappropriately activated.”
Sampath’s team is particularly familiar with the intricate relationship between wound healing and epithelial cancers that attack the exterior surfaces of skin and internal organs8. Beneficial processes like keratinocyte migration, upregulation of cell proliferation and neo-angiogenesis can encourage wound healing, but with the risk of being hijacked by tumour cells for cancer progression.
Similar to their work with eczema, Sampath’s team has identified a potentially relevant miRNA, miR-198, for its role in encouraging safe wound healing9.
miRNA also has applications in potentially treating neurodegeneration and movement disorders—a field Sherry Aw, a Principal Investigator at the Institute of Molecular and Cell Biology (IMCB), has been involved in extensively.
Along with her team, Aw works closely with computer scientists, chemists and engineers within the A*STAR ecosystem to study how defects in miRNA biology can lead to neurodegeneration, tremor and movement disorders.
One significant achievement from the lab was the development of the RNA biosensor Pandan10. It builds on a fluorescent RNA known as Spinach by altering the ends of the structure and including an additional stem loop. The new structure allows for a switch-on in fluorescent signal intensity when the biosensor binds to target miRNA, allowing direct miRNA detection within complex RNA mixtures. Currently, Aw’s lab is working to develop additional RNA sensors that can function as new diagnostic and therapeutic modalities.
Determining the right delivery
After understanding mRNA’s role and developing the right RNA solution, the NATs need to be sent to where they can make an impact—in the bodies of patients. Yi Yan Yang, covering Executive Director of the Institute of Bioengineering and Bioimaging (IBB), explains that in addition to using viral vectors to deliver genetic material to target cells, there are two main types of nanoparticles used to transport NATs—biopolymer-based nanoparticles and lipid nanoparticles.
Biopolymers include synthetic biodegradable polymers, which have been used to formulate nanoparticles for the delivery of various NATs11, 12, 13. Meanwhile, lipid nanoparticles, perhaps most notably utilised in COVID-19 vaccines, can be used to encapsulate mRNA to protect it against degradation and to deliver it to a desired cell type14.
In their study, Yang and her team condense NATs into various nanoparticle types to be taken up by cells. They also develop nanocarriers to deliver NATs to the right tissues or cells. To date, in this field, Yang’s lab holds the highest number of polymer-based NAT delivery patents in Singapore.
One particularly interesting project Yang’s team is currently working on is a patch that functions as a potential replacement for injections. “NATs-loaded nanoparticles are contained within microneedles in the patch. Once it is applied to the skin, the nanoparticles will be released under the skin and travel to a target site like the lymph nodes—it’s a painless application,” Yang shared.
Yang was recently awarded a contract as a Co-principal Investigator from the prestigious Wellcome Leap R3 funding programme with GIS Principal Investigator, Yue Wan, to further accelerate the development and accessibility of mRNA technology.
Wan’s lab primarily explores the complex secondary and tertiary structures of RNA after it folds to further understand its function in the human body.
Notably, RNA structures are notoriously difficult to crystallise and visualise. To that end, Wan’s team developed a strategy called parallel analysis of RNA structures (PARS)15 to examine thousands of RNA structures. PARS has been applied to study the transcriptomes of yeast16 and humans17 as well as the impact of temperature and mutations on RNA structure.
As part of the Wellcome Leap R3 programme, the team intends to develop circular RNA designs for mRNA vaccines which would increase and stabilise the amount of protein created—resulting in smaller doses and lower vaccine costs.
“It is a huge privilege to be able to be part of the programme,” said Wan. “As different teams are working on a range of problems such as RNA manufacturing, design and downstream applications, we get to interact and plug into many of the newer technologies.”
Entering a sea of opportunity
In the wake of the rapid progress being made, A*STAR has shown a concerted effort to advance the implementation of RNA-based medicine in Singapore with the launch of an official NAT Programme, led by Senior Group Leader Kevin White, who is also a professor of precision medicine and biochemistry at the Yong Loo Lin School of Medicine, National University of Singapore.
Arriving with an extensive background in precision medicine, White was formerly the President and Chief Scientific Officer of Tempus, a prominent precision medicine company based in the United States. Before launching and helping to build Tempus into a multibillion-dollar company, he was a professor of human genetics at the University of Chicago and the Founding Director of the Institute for Genomics and Systems Biology, which he led for a decade.
White will lead the NAT Programme alongside Boon Tong Koh, Executive Director of the Bioprocessing Technology Institute (BTI) at A*STAR. “BTI’s programme is focused on the biomanufacturing aspects of mRNA,” said Koh. “Our contributions will be towards improving manufacturing processes, removing existing operations bottlenecks, improving quality and reducing the cost of drugs.”
While White believes that implementing NAT research in clinics and hospitals will generally face the same obstacles as most other drugs and vaccines—namely delivery, toxicity and efficacy—as an emerging field, NAT developers must also address regulatory uncertainty.
“As a relatively new set of modalities, there is a steep learning curve for most regulatory agencies,” explained White. “It is especially crucial for NAT developers to have clear and frequent lines of communication with regulators.”
Nonetheless, as the field continues to grow, A*STAR is in a strong position to ride the wave of mRNA therapeutics. According to Andre Choo, Executive Director of the Biomedical Research Council at A*STAR, this potential can be attributed to the diverse expertise of A*STAR’s scientists that spans RNA design, protein engineering, process manufacturing and more. “This collaborative effort across the institutes gives us both breadth and depth to make a lasting impact in this new space,” said Choo. “At the same time, the entire ecosystem is ripe for progress with the inclusion of A*STAR start-ups, industry partnerships and academic collaborations.”
“I hope to see a strong integration between local SMEs, investors and the academic sector in Singapore in the area of NATs,” White added. “I believe it could be a model to demonstrate how innovations funded by the public sector can be rapidly developed into useful products for humanity.”
