A Biocatalytic Platform for Therapeutic Oligonucleotide Synthesis

Therapeutic oligonucleotides are a powerful drug modality with the potential to treat many diseases. The rapidly growing number of therapies approved and in advanced clinical trials will place unprecedented demands on our capacity to manufacture oligonucleotides at scale. Solid phase phosphoramidite chemistry has underpinned small scale DNA synthesis for decades, however this approach was not developed with large scale applications in mind and the method suffers from inherent limitations that restrict its scalability and sustainability. During Phase I of my Future Leaders Fellowship, my team developed a transformative biocatalytic approach to efficiently produce oligonucleotides in a single operation, which contrasts with the iterative rounds of chain extension, capping, oxidation and deprotection associated with established methods [Science 2023, 381, 754; Science 2024, 384, eadl4015]. Using engineered enzymes, we were also able to produce diverse oligonucleotide sequences containing a range of pharmaceutically relevant modifications [manuscript in preparation].
In Phase II of the research program, we will extend our approach to the production of real-world therapeutics with increased structural complexity. To this end, our cascade process will be adapted to produce oligonucleotides equipped with bioorthogonal reactive handles (e.g. amines, alkynes) to allow facile downstream conjugations to a variety of drug delivery vehicles, including glycans, antibodies and peptides. Such conjugations are commonly needed to ensure effective delivery of the oligonucleotide therapy to the target organ or cell. Finally, we will develop convergent oligonucleotide assembly strategies where enzymatically synthesized fragments produced in parallel are assembled using DNA ligases to produce sequences containing a greater variety of chemical modifications.