A versatile biocatalytic platform for scalable therapeutic oligonucleotide manufacturing

蛋白质控制着人体内几乎所有的生化过程。这些生物大分子编码在我们的DNA中，首先转录为mRNA，随后翻译为蛋白质。传统的小分子药物被设计成选择性地结合靶蛋白以调节其功能。虽然这种方法已被证明是非常有效的，但有许多疾病很难或不可能以这种方式治疗。近年来，出现了一类称为治疗性寡核苷酸的新型药物分子，其为治疗广泛的遗传病症和疾病提供了潜在的通用方法。这些分子是短的修饰的DNA序列，其被设计为结合mRNA并直接调节疾病相关蛋白的产生。尽管目前有超过160种不同的寡核苷酸疗法正在进行临床试验，但迄今为止，美国食品和药物管理局（FDA）批准的唯一治疗方法都是针对罕见疾病的，而且非常昂贵。为了将治疗性寡核苷酸作为更常见疾病的可行且具有成本效益的治疗方法，我们现在必须为其生产开发可持续，可扩展和通用的制造策略。现有的生产寡核苷酸的方法依赖于化学合成，其需要大量过量的昂贵试剂、大量的有机溶剂（每千克产物1吨乙腈）并且以低收率和适度（~90%）纯度提供最终产物。反应在固体支持物或柱上进行，这限制了工艺的可扩展性，意味着这些方法仅适用于生产<10 Kg批次的寡核苷酸。简而言之，目前的化学方法不适合合成治疗常见疾病所需的高容量寡核苷酸产物。 在本申请中，我将开发一种绿色、具有成本效益和真正通用的生物催化方法来大规模生产治疗性寡核苷酸。生物催化是一项令人兴奋的技术，广泛应用于化学工业，酶用于将起始材料转化为高价值产品。在自然界中，DNA被称为聚合酶的酶复制或“扩增”。我将利用这些聚合酶作为生物催化剂来扩增可重复使用的催化DNA模板，以在环境友好的条件下以高产率和纯度生产大量的治疗性寡核苷酸。与天然DNA相比，治疗性寡核苷酸含有化学修饰，旨在提高其功效，选择性和代谢稳定性。这些化学修饰不能被天然聚合酶很好地耐受，但是使用一种称为定向进化的技术，我们能够快速地改造酶以修改它们的功能并优化它们的特性，使它们适合实际应用。我将使用定向进化来设计聚合酶，使其在工艺条件下有效地产生含有治疗应用所需的化学修饰的寡核苷酸。这种方法将使用价值数十亿美元的治疗性寡核苷酸Spinraza作为初始合成靶标进行开发和优化，该靶标将以多克规模制备，以展示这种生物催化平台的力量，并吸引更多的投资用于多公斤规模的工艺。

Proteins control almost all biochemical processes in the human body. These biological macromolecules are encoded in our DNA, which is first transcribed to mRNA and subsequently translated to proteins. Traditional, small molecule pharmaceuticals are designed to selectively bind to a target protein in order to modulate its function. While this approach has proven very powerful, there are numerous diseases which are difficult or not possible to treat in this manner. In recent years, a new class of drug molecules called therapeutic oligonucleotides have emerged, which offer a potentially versatile approach for the treatment of a wide range of genetic disorders and diseases. These molecules are short modified DNA sequences, which are designed to bind to mRNA and directly modulate the production of disease related proteins. Despite the fact that there are currently more than 160 different oligonucleotide therapies in clinical trials, to date the only treatments approved by the Food and Drug Administration (FDA) have been for rare diseases and are extremely expensive. To establish therapeutic oligonucleotides as viable and cost-effective treatments for more common diseases, it is now essential that we develop sustainable, scalable and versatile manufacturing strategies for their production. Existing methods of producing oligonucleotides rely on chemical synthesis, which requires large excesses of expensive reagents, huge volumes of organic solvent (1 ton of acetonitrile per Kg of product) and deliver the final products with low yield and modest (~90%) purity. Reactions are performed on solid supports or columns, which limits the process scalability meaning that these methods are only suitable for producing oligonucleotides in <10 Kg batches. In short, current chemical methods are not suitable for the synthesis of high volume oligonucleotide products required for the treatment of common diseases. In this application, I will develop a green, cost-efficient and truly versatile biocatalytic approach to manufacture therapeutic oligonucleotides on a large scale. Biocatalysis is an exciting technology which is widely used across the chemical industry, whereby enzymes are used to convert starting materials into high-value products. In nature, DNA is copied or 'amplified' by enzymes called polymerases. I will exploit these polymerases as biocatalysts to amplify a reusable catalytic DNA template to produce large quantities of therapeutic oligonucleotides in high yield and purity under environmentally friendly conditions. Compared to natural DNA, therapeutic oligonucleotides contain chemical modifications which are designed to improve their efficacy, selectivity and metabolic stability. These chemical modifications are not well tolerated by natural polymerases, however using a technology called directed evolution we are able to quickly engineer enzymes to modify their functions and optimize their properties to make them suitable for practical applications. I will use directed evolution to engineer polymerases to operate under process conditions to efficiently produce oligonucleotides containing the chemical modifications needed for therapeutic applications. This approach will be developed and optimized using the billion dollar therapeutic oligonucleotide Spinraza as an initial synthetic target, which will be prepared on a multi gram scale to showcase the power of this biocatalytic platform and to attract further investment for multi-kilo scale processes.