DNA 3.0: Development of a novel, efficient and cost-effective enzymatic process for synthesis of DNA oligonucleotides

Project Summary/AbstractSince the birth of biotechnology in the 1980s, efficient nucleic acid synthesis has been a key driver of biological discovery and bio-product development, culminating in the recent emergence of the ‘synthetic biology’ field and rapid development of nucleic acid therapeutics. Sequence-specific RNA and DNA manufacturing is having an especially strong impact in healthcare, where FDA approval of the first oligonucleotide-based therapeutics in 2018 quickly led to many hundreds of similar drugs entering clinical trials. Despite these positive developments, new DNA and RNA synthesis technologies are urgently needed to meet the rapidly rising demand for clinical-grade oligonucleotides, because the current chemical synthesis strategies are costly, difficult to scale, inefficient and limited to molecules of 200 nucleotides or less in length. In the past decade, enzymatic oligonucleotide synthesis (EOS) strategies have been explored and developed for this purpose.In a Phase I feasibility project, Primordial Genetics Inc. demonstrated the use of novel template- independent DNA polymerases (TIDPs) for controlled addition of natural, unmodified nucleotides to a growing DNA strand in a simple process that does not require a chemical deblocking step. This innovation has the potential to enable a robust, inexpensive, flexible, environmentally friendly and easily scalable enzymatic route to manufacturing DNA and RNA used in therapeutics, vaccines, diagnostics and RandD products.In this Phase II Small Business Innovation Research (SBIR) project, Primordial Genetics proposes to continue the Phase I work to optimize the already discovered TIDPs to enable a high rate (99%) of single nucleotide addition in each synthesis cycle that is needed for an industrial EOS process. The company’s genetic improvement and screening platform, specifically the Function Generator™ technology, will be applied together with the artificial intelligence and machine learning capabilities of our computational biology collaborator Koliber Biosciences to improve these enzymes to the desired level of efficiency. A prototype EOS process will be developed using the optimized enzymes acting on oligonucleotides immobilized on a solid support.The prototype EOS process has the potential to enable more efficient DNA and RNA synthesis and remove the manufacturing bottlenecks that are currently holding back the development of nucleic acid medicines. Over time, we will adapt the TIDPs to allow synthesis of all modified nucleotides currently being incorporated into DNA- and RNA-based therapeutics, vaccines, as well as reagents for diagnostics, RandD and DNA-based information storage.