By Gilang Cahya 
In the relentless pursuit of scientific understanding, unexpected outcomes are not failures, but often the most fertile ground for groundbreaking discoveries. This principle has once again been vividly illustrated by a collaborative effort between researchers at Memorial Sloan Kettering Cancer Center (MSK) and the Icahn School of Medicine at Mount Sinai. Their recent work, stemming from a laboratory experiment that yielded results contrary to expectations, has unveiled a novel therapeutic avenue for enhancing small RNA-based treatments, with significant implications for diseases ranging from genetic disorders to cancer.
The Unexpected Twist: A Protein’s Hidden Talent
The research team, led by developmental biologist Eric Lai, PhD, and postdoctoral fellow Seungjae Lee, PhD, from MSK’s Lai Lab, was initially focused on elucidating the precise role of the protein ALAS1 in the biogenesis of microRNAs (miRNAs). miRNAs are a class of small regulatory RNA molecules that play a critical role in controlling gene expression. The prevailing scientific hypothesis suggested that ALAS1, known for its involvement in heme synthesis—a vital component in numerous biological processes including oxygen transport and energy production—would be essential for producing these miRNAs. Consequently, the researchers anticipated that removing ALAS1 from cells would lead to a discernible decrease in miRNA levels.
However, the experimental results defied this prediction. Instead of declining, the levels of microRNAs unexpectedly surged when ALAS1 was absent. This counterintuitive finding immediately signaled a departure from their planned research trajectory and hinted at a more profound biological mechanism at play. "Sometimes you do an experiment," explained Dr. Lai, "You think you’re testing one idea, but when it doesn’t turn out the way you planned, it can lead you to find something else that’s much more interesting."
Unraveling the "Moonlighting" Function of ALAS1
This surprising observation prompted a deeper investigation into the multifaceted functions of ALAS1. Further experiments conducted by Dr. Lee and colleagues revealed that the removal of other enzymes within the heme biosynthesis pathway did not elicit the same increase in miRNA levels, strongly suggesting that ALAS1 possessed a distinct, previously unrecognized role independent of its heme-related duties. "This told us that ALAS1 has another job outside of helping to make heme, which no one had realized," stated Dr. Lee.
Dr. Lai elaborated on this discovery, characterizing it as a "moonlighting" function. This term is used in biology to describe proteins that perform more than one distinct biochemical or physiological role. "And here we discovered that ALAS1 has this secret role regulating microRNAs that’s not connected to its normal role in heme synthesis," he added. This revelation opened a new frontier in understanding gene regulation and the intricate mechanisms that govern cellular processes.
The Power of Small RNAs: A Therapeutic Revolution
To fully grasp the significance of this discovery, it is essential to understand the burgeoning field of small RNA therapeutics. Both microRNAs and their close relatives, small interfering RNAs (siRNAs), are minuscule RNA molecules, typically 21 to 22 nucleotides in length. Their primary function is to bind to specific messenger RNA (mRNA) molecules, effectively silencing the genes they carry and thereby inhibiting protein production.
This fundamental understanding of how small RNAs regulate gene expression has been ingeniously harnessed by the scientific and pharmaceutical communities to develop a novel class of drugs. These therapies leverage the ability of siRNAs to selectively target and neutralize disease-causing genes. The groundbreaking approval of the first siRNA drug, patisiran, by the U.S. Food and Drug Administration (FDA) in 2018 marked a pivotal moment, offering a treatment for the rare and debilitating genetic disorder hereditary transthyretin amyloidosis. Since then, several other siRNA drugs have received regulatory approval, with many more progressing through clinical trials. The potential applications are vast, targeting both rare genetic conditions and more prevalent diseases by interfering with the accumulation of specific messenger RNAs. These siRNA drugs are often referred to as RNAi drugs, signifying their mechanism of action—RNA interference.
Expanding the Horizon: Collaboration and In Vivo Validation
The implications of ALAS1’s dual role quickly became apparent, prompting the MSK researchers to seek expertise from specialists in heme regulation and ALAS genes at the Icahn School of Medicine at Mount Sinai. This collaboration brought together researchers Makiko Yasuda, MD, PhD, Robert Desnick, MD, PhD, and postdoctoral fellow Sangmi Lee, PhD. Their combined knowledge and resources allowed for the crucial transition from cell culture experiments to validation in custom animal models.
In these meticulously developed mouse models, the team observed a consistent phenomenon: the depletion of ALAS, particularly in liver cells, resulted in a global increase in microRNAs. This in vivo confirmation provided robust support for the existence of ALAS1’s regulatory function in microRNA production within a living organism. "The emerging picture is that ALAS acts as a brake on the production of microRNAs," Dr. Lai summarized. "So we thought, now that we know how to remove this brake, maybe we can use that to improve the efficacy of siRNA drugs and their ability to silence their target genes."
A Strategic Advantage: Enhancing siRNA Drug Efficacy
The strategic implication of this discovery lies in its potential to enhance the effectiveness of existing and future siRNA therapies. By understanding that ALAS1 acts as a natural inhibitor of miRNA production, researchers theorize that strategically removing or inhibiting ALAS1 could create a more permissive environment for siRNA drugs to exert their gene-silencing effects. This could lead to improved therapeutic outcomes for a wide range of diseases where specific genes are overexpressed or contribute to pathology.
The potential applications are particularly exciting in the context of cancer, where oncogenes—genes that promote cell growth and division—can become abnormally activated. By enhancing the ability of siRNA drugs to silence these oncogenes, this newfound understanding could pave the way for novel cancer therapies. "Potentially this could include oncogenes known to drive cancer," Dr. Lai noted.
However, he tempered this enthusiasm with a pragmatic assessment of the current limitations. "But we’re not quite there yet," he cautioned. "Therapeutic siRNA drugs don’t work well enough against all targets and are currently limited in where they can be used in the body." A significant hurdle has been the challenge of effectively delivering siRNA drugs to specific tissues beyond the liver. Currently, all six FDA-approved siRNA drugs target hepatocytes, the primary cells of the liver, due to the organ’s role as the body’s natural filter, making drug delivery relatively straightforward.
Proof of Concept: Boosting siRNA Activity in the Liver
To demonstrate the practical applicability of their findings, the research team conducted a proof-of-concept experiment. They successfully depleted ALAS in mouse liver cells, observing the predicted increase in microRNAs. Crucially, this intervention also enhanced the silencing efficacy of a model siRNA compound administered to the same mice. This experiment provided tangible evidence that modulating ALAS1 activity can indeed amplify the effectiveness of siRNA therapeutics.
Adding a layer of serendipity to their work, the researchers noted that one of the existing FDA-approved siRNA drugs, givosiran, functions by inhibiting ALAS1 to treat acute hepatic porphyrias, a group of rare genetic disorders. Drs. Yasuda and Desnick were instrumental in the preclinical and clinical development of givosiran. The fact that an siRNA targeting ALAS1 is already proven to be effective and safe in humans significantly bolsters the prospect of combining such an agent with other siRNA drugs to achieve synergistic therapeutic effects. Dr. Lai expressed optimism, stating that this strategy "could be generally applicable to any siRNA."
The broader implications of making siRNA drugs more potent are substantial. Enhanced efficacy could translate to lower required dosages, potentially reducing the incidence of side effects and improving the overall safety profile of these therapies. Furthermore, it could expand the therapeutic window, making siRNA drugs viable for a wider array of diseases and potentially enabling their delivery to cell types beyond the liver, thereby broadening their reach and impact.
The Enduring Value of Discovery Science
This remarkable scientific journey, sparked by an unexpected laboratory result, underscores the profound importance of curiosity-driven research, often referred to as discovery science. The recent Nobel Prize in Physiology or Medicine awarded to Harvard geneticist Gary Ruvkun, PhD, and Victor Ambros, PhD, for their pioneering discovery of microRNAs in the early 1990s, serves as a powerful testament to this principle. Dr. Lai, who conducted his undergraduate thesis research in Dr. Ruvkun’s lab, credits his mentor for igniting his passion for scientific inquiry.
"Dr. Ruvkun didn’t start out looking for microRNAs," Dr. Lai reflected. "Like Dr. Ambros, he was investigating the development of nematodes, these tiny worms that live in the soil. And not only did this unveil an entirely new paradigm for how genes are controlled, the field they started eventually resulted in a novel class of human therapies." This historical perspective highlights how fundamental research into seemingly esoteric biological systems can ultimately lead to transformative medical breakthroughs.
In an era where research funding often faces scrutiny and debate, Dr. Lai emphasized the critical need to sustain support for foundational scientific exploration. "When people ask why we’re not spending all of our research dollars directly studying diseases like cancer, why we’re funding research into cells and processes in model organisms like fruit flies, yeast, and bacteria — this is a great example of how discovery science fuels the biggest breakthroughs," he argued. He further stressed the importance of fostering this dialogue, especially given societal and governmental uncertainties surrounding the allocation of public funds for scientific research. "And I think it is especially critical to keep this conversation active, given how much uncertainty and disagreement there is in society and government about how much to publicly fund scientific research and in what areas. Hopefully, there will be continued support to keep the engine of foundational research strong."
Funding and Future Prospects
The research that led to these pivotal findings was supported by grants from the National Institutes of Health (R01DK134783, R01-GM083300, P30-CA008748), a Cooperative Centers of Excellence in Hematology pilot grant (10040500-05S1), and a NYSTEM training award (C32559GG).
In recognition of the potential impact of their discovery, the researchers have filed a patent application for their methods of enhancing RNAi therapy by targeting ALAS1/ALAS2 (WO2024148236A1). Furthermore, Drs. Yasuda and Desnick are co-inventors on a patent related to RNAi therapy for acute hepatic porphyrias and have reported pharmaceutical consulting work. These steps indicate a clear trajectory toward translating these scientific findings into tangible therapeutic advancements. The ongoing exploration of ALAS1’s "moonlighting" function represents a significant leap forward in the field of RNA therapeutics, promising a future where gene silencing drugs are more potent, more accessible, and more effective in combating a wider spectrum of human diseases.
