By Gilang Cahya 
In everyday life, when things turn out the opposite of what you expect, it’s usually cause for frustration. In science, it’s often the starting point for discovery. This serendipitous finding, where an experiment designed to confirm an established scientific principle yielded a counterintuitive result, has opened a significant new avenue in the development of RNA-based therapeutics. Researchers at Memorial Sloan Kettering Cancer Center (MSK) and their collaborators at the Icahn School of Medicine at Mount Sinai have uncovered an unrecognized function of a protein, ALAS1, which could dramatically improve the efficacy of therapies designed to silence disease-causing genes. This breakthrough, published in the prestigious journal Science, has the potential to revolutionize treatments for a range of conditions, including certain cancers.
A Surprising Experimental Outcome
The research team, led by developmental biologist Eric Lai, PhD, and postdoctoral fellow Seungjae Lee, PhD, embarked on an investigation into the role of the enzyme ALAS1 in the production of microRNAs. MicroRNAs are short RNA molecules that play a critical role in regulating gene expression. The prevailing scientific understanding at the time was that ALAS1, a key enzyme in the heme biosynthesis pathway, was essential for the generation of these regulatory RNAs. Heme, a critical molecule involved in oxygen transport, energy production, and various cellular processes, is synthesized through a multi-step pathway, with ALAS1 initiating this process.
The researchers hypothesized that by removing ALAS1 from cells, they would observe a corresponding decrease in microRNA levels. This was a logical extension of existing knowledge, assuming a direct and positive correlation between ALAS1 activity and microRNA production. However, the experimental results defied expectations. Instead of a decline, the team observed a surprising and significant increase in microRNA levels when ALAS1 was absent from the cells.
"Sometimes you do an experiment," explained Dr. Lai, a seasoned developmental biologist, reflecting on the unexpected outcome. "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." This sentiment encapsulates the essence of scientific discovery, where the unexpected often paves the way for groundbreaking insights.
Unveiling a "Moonlighting" Enzyme
This counterintuitive finding necessitated a re-evaluation of ALAS1’s biological functions. The unexpected surge in microRNAs when ALAS1 was removed pointed to a role for the enzyme that was entirely separate from its well-established function in heme production. Further experiments conducted by Dr. Lee and his colleagues confirmed that the depletion of other enzymes in the heme biosynthesis pathway did not yield the same effect on microRNA levels. This strongly suggested that ALAS1 possessed a unique, previously unidentified "moonlighting" function – a term used in biology to describe proteins that perform multiple, distinct roles within a cell.
"This told us that ALAS1 has another job outside of helping to make heme, which no one had realized," stated Dr. Lee, emphasizing the novelty of their discovery. Dr. Lai elaborated on this concept, describing it as a "secret role regulating microRNAs that’s not connected to its normal role in heme synthesis." This revelation shifted the research focus from merely understanding heme production to exploring this newly identified regulatory mechanism.
The Power of Small RNA Snippets: A Foundation for Therapeutics
To fully appreciate the implications of this discovery, it is crucial to understand the mechanism and therapeutic potential of small RNAs. MicroRNAs, along with a related class of molecules known as small interfering RNAs (siRNAs), are short RNA strands, typically 21 to 22 nucleotides in length. Their remarkable ability lies in their capacity to bind to specific messenger RNAs (mRNAs), the molecules that carry genetic instructions from DNA to the cell’s protein-making machinery. Upon binding, microRNAs and siRNAs effectively "silence" these mRNAs, preventing them from being translated into proteins.
This gene-silencing capability has been a major focus of therapeutic development. Scientists have successfully harnessed this natural cellular process to create drugs that can specifically target and inhibit genes responsible for various diseases. The first significant milestone in this field was the U.S. Food and Drug Administration (FDA) approval of patisiran in 2018. Patisiran, an siRNA drug, treats hereditary transthyretin amyloidosis, a debilitating genetic disorder. Since then, several other siRNA drugs have received regulatory approval, with many more undergoing rigorous clinical trials. The potential applications of these RNA interference (RNAi) drugs are vast, ranging from rare genetic disorders to more common and complex diseases, including certain forms of cancer.
From Cell Culture to Animal Models: A Collaborative Endeavor
The implications of ALAS1’s "moonlighting" function were so significant that the MSK team sought to expand their research into more complex biological systems. This led to a crucial collaboration with researchers at the Icahn School of Medicine at Mount Sinai who possessed specialized expertise in heme regulation and the ALAS genes. Key figures in this collaboration included Makiko Yasuda, MD, PhD, Robert Desnick, MD, PhD, and postdoctoral fellow Sangmi Lee, PhD.
This partnership enabled the MSK researchers to transition their findings from controlled cell cultures to custom animal models developed by the Mount Sinai group. The results in these animal models mirrored those observed in cell cultures: the removal of ALAS, specifically in liver cells, led to a global increase in microRNA levels. This provided robust in vivo evidence for ALAS1’s regulatory role in microRNA production.
"The emerging picture is that ALAS acts as a brake on the production of microRNAs," Dr. Lai articulated, drawing an analogy to a braking system. "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."
Enhancing siRNA Drug Efficacy: A Path to Better Treatments
The scientific insight that ALAS1 acts as a brake on microRNA production opened up a compelling therapeutic strategy. If ALAS1 inhibits microRNA synthesis, then its removal could potentially enhance the activity of gene-silencing therapies that rely on microRNAs or related molecules like siRNAs. This could be particularly impactful for diseases where target genes are overexpressed, such as certain oncogenes driving cancer progression.
"In theory, this knowledge might help boost the activity of siRNA drugs against any problematic gene that is overactive in disease," Dr. Lai explained. The potential applications are broad, and the researchers believe this strategy could be particularly beneficial in overcoming limitations currently faced by siRNA therapeutics.
Despite the remarkable progress in RNAi technology, challenges remain. "We’re not quite there yet," Dr. Lai 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 is the delivery of these drugs to specific cells and tissues. Currently, all six FDA-approved siRNA drugs target hepatocytes, the primary cells of the liver, due to the liver’s role as a natural filter for the body, making it relatively straightforward to deliver drugs to this organ.
To demonstrate the practical utility of their discovery, the team conducted a proof-of-concept experiment. They successfully depleted ALAS in mouse liver cells, confirming the anticipated increase in microRNAs. Crucially, they also showed that this depletion enhanced the gene-silencing activity of a model siRNA compound administered to the mice. This experiment provided tangible evidence that manipulating ALAS1 levels could indeed improve the effectiveness of siRNA-based therapies.
A Fortuitous Connection: The Givosiran Case Study
Adding another layer of significance to their findings, the researchers noted a remarkable coincidence: one of the existing FDA-approved siRNA drugs, givosiran, works by turning off ALAS1 to treat acute hepatic porphyrias. Drs. Yasuda and Desnick, key collaborators on the current study, were instrumental in the preclinical and clinical trials for givosiran. This existing drug serves as a powerful validation that targeting ALAS1 with an siRNA is not only feasible but also safe and effective in humans.
The existence of givosiran suggests a potential pathway for combining therapies. The strategy of using an siRNA agent to enhance other siRNA drugs by targeting ALAS1 could be broadly applicable to a wide range of siRNA therapeutics. "Since an siRNA against ALAS1 works effectively and safely in humans, this raises the possibility of combining such an agent to enhance other siRNA drugs," Dr. Lai observed.
The implications of this combined therapeutic approach are substantial. If siRNA drugs can be made more effective, it could lead to several positive outcomes:
- Improved Cost-Effectiveness: Higher efficacy might allow for lower drug dosages, potentially reducing manufacturing costs and making treatments more accessible.
- Reduced Side Effects: Using lower doses of siRNA drugs could minimize off-target effects and improve patient tolerance.
- Expanded Therapeutic Reach: Enhanced efficacy might enable siRNA drugs to target cell types beyond the liver, opening up new treatment possibilities for a wider array of diseases.
The Enduring Importance of Discovery Science
This research journey, born from an unexpected experimental result, underscores the profound importance of curiosity-driven, foundational science. The recent awarding of the Nobel Prize in Physiology or Medicine to Harvard geneticist Gary Ruvkun, PhD, and Victor Ambros, PhD, for their discovery of microRNA in the early 1990s, serves as a powerful testament to this principle. Dr. Lai himself noted that his undergraduate thesis research was conducted in Dr. Ruvkun’s lab, crediting his mentor for igniting his passion for developmental biology and small RNAs.
"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."
The implications of this discovery extend beyond the immediate therapeutic potential. It highlights the critical role of "discovery science" – research pursued for its own sake, driven by fundamental questions about the natural world. Such research, often conducted in model organisms like fruit flies, yeast, and bacteria, may not have immediate applications, but it lays the groundwork for revolutionary breakthroughs.
"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… this is a great example of how discovery science fuels the biggest breakthroughs," Dr. Lai emphasized. He further stressed the importance of continued public and governmental support for foundational research, especially in a climate of societal and political uncertainty regarding scientific funding. "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 Directions
The groundbreaking research 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).
The researchers have filed a patent application on their methods for enhancing the efficacy of RNAi therapy by targeting ALAS1/ALAS2 (WO2024148236A1), signaling their intent to translate these findings into practical applications. Drs. Yasuda and Desnick also have co-inventor status on a patent related to RNAi therapy for acute hepatic porphyrias and report pharmaceutical consulting work.
The discovery that ALAS1 plays a critical, previously unrecognized role in regulating microRNA production represents a significant leap forward in our understanding of gene regulation. By uncovering this "moonlighting" function, scientists have not only gained deeper insights into fundamental biological processes but have also identified a promising new strategy to enhance the effectiveness of life-saving siRNA therapies, potentially bringing us closer to more potent and accessible treatments for a wide spectrum of diseases.