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

项目摘要/摘要 自从20世纪80年代生物技术诞生以来，高效的核酸合成一直是关键 生物发现和生物制品开发的驱动力，最终导致最近出现了 合成生物学领域和核酸治疗学的快速发展。特定于序列 RNA和DNA制造在医疗保健领域产生了特别强烈的影响，FDA 第一种基于寡核苷酸的疗法在2018年获得批准，迅速导致数百人 类似的药物进入临床试验。尽管有这些积极的发展，但新的DNA和RNA 迫切需要合成技术来满足快速增长的临床级需求 寡核苷酸，因为目前的化学合成策略成本高昂，难以规模化， 低效且仅限于200个核苷酸或更少的分子。在过去的十年里，酶 为此目的，已经探索和开发了寡核苷酸合成(EOS)策略。 在第一阶段可行性项目中，Primorial Genetics Inc.演示了新型模板的使用- 独立DNA聚合酶(TIDPs)，用于控制天然、未经修饰的核苷酸与 在一个简单的过程中生长的DNA链，不需要化学解锁步骤。这 创新有可能实现强大、廉价、灵活、环境友好和 易于扩展的酶法制备用于治疗、疫苗、 诊断和研发产品。 在第二阶段小型企业创新研究(SBIR)项目中，原始遗传学 建议继续第一阶段的工作，以优化已发现的TIDP，以实现高 工业EOS所需的每个合成周期中的单核苷酸加成率(99%) 进程。该公司的遗传改良和筛查平台，具体功能 生成器™技术，将与人工智能和机器学习一起应用 我们的计算生物学合作者Koliber Biosciences改进这些酶的能力 达到理想的效率水平。将使用优化后的EOS开发原型EOS流程 作用于固定在固体载体上的寡核苷酸的酶。 原型EOS过程有可能实现更高效的DNA和RNA合成 消除目前阻碍核能发展的制造瓶颈 酸性药物。随着时间的推移，我们将调整TIDPs，以允许合成所有修饰的核苷酸 目前被纳入基于DNA和RNA的疗法、疫苗和试剂中 用于诊断、研发和基于DNA的信息存储。

Project Summary/Abstract Since 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 R&D 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, R&D and DNA-based information storage.