Beyond The Gold Standard(s): Modernizing Oligonucleotide Synthesis

By Anna Rose Welch, Editorial & Community Director, Advancing RNA

Having covered the still nascent mRNA sector extensively for the past two years, venturing into the much more established oligo (siRNA & ASO) sector has thrown me for a bit of loop. There are 20-plus market approvals and a well-established manufacturing paradigm that meets the industry’s (current) needs. Likewise, as I wrote in my previous article summarizing several key takeaways from the OPT Conference, we’re also starting to hear the word “maturity” being used to describe the sector, especially as we continue to learn more clinically, both in trials and post-commercialization.

But to say that we are complacent in our success is far from the truth. In fact, the more I learn, the more it feels like we’re a sector on the brink of great and disruptive transformation.

A few weeks ago, I had the chance to sit down with OPT Congress speaker Phil Baran, Richard Lerner Chair Professor, Department of Chemistry, Scripps Research. Baran was slated to (and did) present a keynote on the innovations shaping the next generation of oligo synthesis. Here, I share the biggest takeaways I had from our conversation, touching on how he sees the science of oligo chemistry and manufacturing advancing in the near and far future.

The Far Future of Synthesis: Will SPOS Ever Step Aside?

In the pharma world, it can be easy to conflate the “gold standard” with “the be-all, end-all.” In the oligo world, we’ve long relied upon and espoused the merits of solid phase synthesis (SPOS) from many conference podiums. In fact, as one speaker at TIDES last year emphasized, SPOS is “unbeatable” in the discovery phase — a fact that Baran reinforced during our own conversation. “It’s definitely the fastest way to controllably make what you want,” he said.

However, as we also know, manufacturing is one area of this sector that is not standing still. Whether it be the move toward liquid-phase synthesis or hybrid enzymatic/fully enzymatic synthesis, we’re proceeding toward bigger scales and more sustainable manufacturing paradigms — a transition that raises questions about the longevity of SPOS. Though Baran believes the fully enzymatic synthesis will be the “next logical, potentially disruptive event,” he still anticipates there will always be a need for SPOS — especially because we’re still far from being able to use these next-gen manufacturing paradigms to accomplish what we’re hoping to at a molecular level.

As he went on to explain, our molecules are only getting more complicated as we strive to improve their pharmacological profiles. As such, it’s difficult to produce highly specialized/ “exotic” internucleotide bonds using an enzymatic approach. In the same vein, it can also be a challenge to incorporate “strange” monomers — namely, those that mimic natural ribose.

“If the industry can overcome these challenges, I can foresee a future in which SPOS is still used, but more sparingly,” he added, pointing specifically to the use of SPOS for R&D purposes in labs lacking enzymatic know-how.

Of course, Baran didn’t shy away from looking at the far, far future through a particularly revolutionary lens. While, in the near future, enzymatic know-how may not necessarily be accessible across different labs, in the long-term, the use of AI and robotics may help close that talent gap.

“The day that SPOS is totally unneeded will probably be the age in which we’re working in collaboration with robots,” he added.

The Complexity Paradox: Building Better Molecules by Simplifying the Cycle

As you can imagine, the title of his presentation — “Simplifying the Synthesis of Oligonucleotides” — raised several big questions in my mind about what exactly we have to simplify, and why. In fact, on the surface, it would seem we’re doing the opposite of “simplifying.” On a molecular level, the realities of biology and therapeutic effect often demand that our molecules become more complex. Baran pointed to the parallels that exist between the oligo and small molecule sectors, both sectors of which are now boasting more complicated molecules in the name of improved affinity, and PK/PD profiles.

“We see examples now in the oligo world where stereochemistry of internucleotide linkages can have dramatic effects on toxicity and efficacy,” he said. “In turn, we need to figure out how to carry out the necessary structure-activity relationship studies. How do we invent methods that address the growing need for increased diversity and complexity of internucleotide linkage? These are the kinds of synthesis conundrums that didn’t exist in the oligo world 15 years ago.”

Overall, where Baran sees the greatest need for simplification is in our own processes for building an oligo. After all, there’s a big difference between how the skeleton of an oligo (i.e., each phosphodiester/PO bond) is assembled in nature compared to our synthetic approach. While Nature herself is only concerned with making PO bonds, our own synthesis cycles comprise a variety of extra steps, including PG manipulations, redox fluctuations, and capping steps that don’t occur in biology.

“Three quarters of the steps of each synthesis cycle when you’re adding a new monomer onto a growing chain is wasteful. How do we simplify the canonical way of putting these biopolymers together?” he asked.

One of the ways he sees us better mimicking nature is by rethinking our half-a-century-long reliance on the phosphorus(III)-oxidation state P(III) in favor of revisiting P(V) (the naturally occuring oxidation state of phosphorus). While, in the past, P(V) was found to be less reactive than P(III), Baran’s recent research suggests that it’s time we reconsider our previous findings.

“We have some compelling data demonstrating that P(V) is not an end, but it can be the beginning point to access all sorts of other useful internucleotide linkages,” he offered.

In general, he sees us entering a period in which we start to more readily reimagine what is possible for internucleotide linkages. It goes without saying that the introduction of phosphorothioate into our siRNA and ASO molecules was a critical modification ensuring the “survival” of therapeutic oligos in vivo. But there’s growing interest in applying alternative compounds, such as nitrogen, to discrete spots of an oligo, especially as our goals of creating more stable, safe, targeted and/or efficacious molecules intensify.

In turn, “Simplifying access to nitrogen- or carbon-containing phosphorus-based linkages — as opposed to sulfur — that previously were viewed as too complicated to even be made is another exciting opportunity facing us moving forward,” he concluded.