While COVID-19 vaccines introduced many people to RNA-based medicines, RNA oligonucleotides have already been on the market for years to treat diseases such as Duchenne muscular dystrophy and amyloidosis. RNA therapies offer many advantages over traditional small-molecule drugs, including their ability to target nearly any genetic component within cells and guide gene-editing tools like CRISPR to their targets.
However, the promise of RNA is currently limited by the fact that growing global demand is outpacing the industry’s ability to manufacture it. The standard method of chemically synthesizing RNA was invented in the 1980s and requires specialized equipment and labor-intensive processes. Chemical synthesis methods are also limited in the variety of nucleotide building blocks they can incorporate into RNA molecules, and they produce metric tons of toxic chemical byproducts that create environmental hazards and limit factory production capacity. These problems will only increase as RNA production scales up in response to demand.
A team of scientists at Harvard University’s Wyss Institute and Harvard Medical School (HMS) has created a solution to this problem: a new RNA synthesis process that expands the therapeutic design space for RNA and unlocks the potential for rapid scale-up that chemical synthesis cannot achieve. Their novel method can produce RNA with efficiencies and purities comparable to current industry standards using water and enzymes instead of the toxic solvents and explosive catalysts that plague current manufacturing. It can also incorporate all of the common molecular modifications found in RNA drugs today, and has the potential to incorporate new RNA chemistries for new types of therapies. The achievement is described in a paper published today in Biotechnology of nature.
“As demand for RNA drugs continues to grow and more products come to market, we will outgrow the current global supply of acetonitrile, the organic solvent used in chemical RNA synthesis methods,” said co-lead author Jonathan Rittichier, Ph.D., a former postdoctoral fellow at Wyss and HMS. He and his colleague, first author and former Wyss research scientist Daniel Wiegand, M.S.Ch.E., Wyss faculty member George Church, Ph.D., and others co-founded EnPlusOne Biosciences to commercialize their technology. “Delivering RNA drugs to the world at these scales requires a paradigm shift to renewable aqueous synthesis, and we believe our proprietary enzyme technology will enable that shift.”
A better and greener way
In the Church lab, Rittichier, Wiegand and co-author Dr. Erkin Kuru recognized that the pharmaceutical industry was in the midst of an RNA revolution. The lab had previously devised a way to synthesize DNA using enzymes and hypothesized that they could do the same with RNA.
The scientists started with an enzyme from a strain of yeast, Schizosaccharomyces pombewhich is known to link nucleotide molecules together to form RNA chains. They designed the enzyme to be more efficient and able to incorporate non-standard nucleotides into RNA. This was especially important for building a useful drug development platform, since every FDA-approved RNA drug contains nucleotides that have been modified from their original form to increase their stability in the body or endow them with new functions.
They then focused on the nucleotides themselves. In standard chemical synthesis of RNA,
Nucleotides are then given “protecting groups” – a sort of chemical bubble wrap that prevents the molecule from being damaged by the harsh reaction conditions. These protecting groups must be removed after synthesis for the RNA to work, and this process requires an additional round of chemical reactions that can damage the RNA while it is being built. The gentler conditions of EnPlusOne synthesis eliminate the need for any bulky bubble wrap, ultimately leading to better manufacturing.
But while it solved one problem, the team’s enzyme introduced another: its natural activity linked nucleotides uncontrollably, resulting in inaccurate RNA sequences. To solve this problem, they modified their nucleotides with a “blocker”—a chemical group that blocks the enzyme, allowing only one nucleotide to be added at a time. Once the desired nucleotide has been added, the blocker is removed to allow the next nucleotide in the sequence to bind, resulting in a two-step process that is simpler and requires fewer reagents than the typical four-step chemical synthesis method.
The researchers showed that their new process incorporated nucleotides with 95% efficiency, comparable to chemical synthesis. The team then iteratively repeated cycles of enzymatic RNA synthesis to build molecules 10 nucleotides long. They can now routinely build molecules 23 nucleotides long, which is the size of many of the most successful RNA therapeutics.
From molecules to medicines
The key to turning RNA into useful drugs is modifying naturally occurring nucleotides. The team also showed that their enzymatic synthesis method could successfully produce RNA chains with multiple types of modified nucleotides with the same ability as naturally occurring nucleotides. “Natural RNA is made up of four letters: A, U, C, and G, but we can expand this simple alphabet with synthetic biology,” said Kuru, who is a postdoctoral fellow at HMS. “Our process essentially increases the number of keys we have on our ‘RNA typewriter’ to a much richer alphabet that we can use to write RNA with new functions and properties.”
This work formed the basis of a validation project at the Wyss Institute in 2019 and 2020, when it was de-risked and ready for commercialization. Also in 2020, the project became the first to receive support through the Wyss Institute’s partnership with Northpond Labs. through The Bioengineering Research and Innovation Lab, based on its potential for significant real-world impact, EnPlusOne Biosciences was launched in 2022 to commercialize the novel approach and bring enzymatic RNA synthesis to the world. Funding was led by Northpond Ventures with participation from Breakout Ventures and Coatue.
“Enzymatic nucleotide synthesis technologies offer many advantages as an alternative to chemical methods. This platform can help unlock the immense potential of RNA therapeutics in a sustainable manner, especially the manufacturing of high-quality guide RNA molecules for CRISPR/Cas gene editing,” said co-corresponding author Church, who is also the Robert Winthrop Professor of Genetics at HMS.
EnPlusOne is also using its platform to manufacture small interfering RNAs (siRNAs) at laboratory scale that could be used to treat a wide variety of diseases.
“RNA drugs offer a powerful new treatment approach for a wide range of diseases. However, current manufacturing methods for these drugs are limited in terms of the chemical diversity they can produce, the amount of material that can be produced at a reasonable cost, and their negative impact on the environment due to the harsh chemicals they require. EnPlusOne’s elegant bioinspired enzymatic synthesis alternative offers a way to overcome all of these limitations and could help the RNA therapeutics industry soar,” said Wyss Founding Principal Don Ingber, M.D., Ph.D. Ingber is also the Professor of Vascular Biology Judah Folkman at HMS and Boston Children’s Hospital, and the Hansjörg Wyss Professor of Bioinspired Engineering at the Harvard John A. Paulson School of Engineering and Applied Sciences (SEAS).
Additional authors of the Biotechnology of nature The paper’s authors include Ella Meyer, Howon Lee, Nicholas J. Conway, Daniel Ahlstedt, Zeynep Yurtsever, and Dominic Rainone. Lee and Ahlstedt are also co-founders of EnPlusOne. This work was supported by the U.S. Department of Energy under grant DE-FG02-02ER63445 and the Wyss Institute at Harvard University.