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。这些生物大分子在我们的DNA中编码，首先被转录成信使核糖核酸，然后被翻译成蛋白质。传统的小分子药物被设计成选择性地与目标蛋白结合，以调节其功能。虽然这种方法被证明非常有效，但有许多疾病很难或不可能用这种方式治疗。近年来，出现了一种名为治疗性寡核苷酸的新型药物分子，它为治疗一系列遗传疾病和疾病提供了一种潜在的通用方法。这些分子是经过修饰的短DNA序列，旨在与信使核糖核酸结合，直接调节疾病相关蛋白的产生。尽管目前有160多种不同的寡核苷酸疗法正在进行临床试验，但到目前为止，美国食品和药物管理局(FDA)批准的唯一疗法是针对罕见疾病的，而且极其昂贵。为了建立治疗性寡核苷酸作为治疗更常见疾病的可行和经济有效的方法，现在至关重要的是，我们必须为它们的生产开发可持续、可扩展和通用的制造策略。现有的寡核苷酸生产方法依赖于化学合成，这需要大量昂贵的试剂和大量的有机溶剂(每公斤产品需要1吨乙腈)，并以低产率和中等(~90%)的纯度提供最终产品。反应是在固体载体或柱上进行的，这限制了过程的可扩展性，这意味着这些方法只适用于生产10公斤批次的寡核苷酸。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大容量寡核苷酸产物。在这项应用中，我将开发一种绿色、低成本和真正多功能的生物催化方法来大规模制造治疗性寡核苷酸。生物催化是一项令人兴奋的技术，广泛应用于整个化学工业，利用酶将原料转化为高价值的产品。在自然界中，DNA是被称为聚合酶的酶复制或‘放大’的。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，在环境友好的条件下高产率和高纯度地生产大量治疗性寡核苷酸。与天然DNA相比，治疗性寡核苷酸包含旨在提高其有效性、选择性和代谢稳定性的化学修饰。天然聚合酶不能很好地耐受这些化学修饰，然而，使用一种称为定向进化的技术，我们能够快速设计酶来修饰它们的功能并优化它们的性质，使它们适合实际应用。我将使用定向进化来设计聚合酶，使其在工艺条件下运行，从而有效地生产包含治疗应用所需的化学修饰的寡核苷酸。这一方法将使用价值数十亿美元的治疗性寡核苷酸Spinraza作为初始合成目标进行开发和优化，该目标将在多克规模上制备，以展示这种生物催化平台的力量，并为数千克规模的过程吸引更多投资。

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.