Biocatalytic Manufacturing of Nucleic Acid Therapeutics

核酸治疗药物的生物催化制造

• 批准号: MR/W029324/1
• 负责人: Nicholas Turner
• 金额: $ 817.8万
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FW029324%2F1
• 关键词: Biocatalytic; Manufacturing; Nucleic; Acid; Therapeutics

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 nucleic acid therapeutics (NATs) 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. Existing methods of producing NATs 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. These limitations have not been a major problem for the manufacture of NATs currently on the market, as these have been limited to the treatment of rare diseases and are therefore produced in low volumes. However, a large volume cholesterol lowering drug called Inclisarin was recently approved and there are several hundred NATs under evaluation in clinical trials for the treatment of common diseases. As current chemical methods are not suitable for the large (tonne) scale synthesis of NATs, it is now essential that we develop new, sustainable, scalable and versatile manufacturing strategies for their production. In this application, we will develop a green, cost-efficient and truly versatile biocatalytic platform for manufacturing NATs and their nucleotide triphosphate (NTP) building blocks. Biocatalysis is an exciting technology which is widely used across the chemical industry, whereby enzymes (nature's own catalysts) are used to convert starting materials into high-value products. Compared to natural DNA, NATs contain chemical modifications which are designed to improve their efficacy, selectivity and metabolic stability. These chemical modifications are not well tolerated by natural enzymes, however using a technology called directed evolution we are able to quickly engineer enzymes to modify their functions and optimise their properties making them suitable for practical applications. We will use combinations of different engineered enzymes to firstly access NTP building blocks, which will be used in subsequent biocatalytic reactions to produce NATs. We will then compare NATs produced using our approaches to those produced with standard chemical approaches, using state of the art analytical techniques combined with biological validation assays. The technologies developed will allow efficient, sustainable and cost-effective manufacturing of NATs in high purity, thus allowing this important new drug modality to realise its full potential for the treatment of a wide-range of diseases.

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，出现了一类称为核酸治疗剂（NAT）的新药物分子，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。现有的生产NAT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。这些局限性对于目前市场上NAT的生产来说并不是一个主要问题，因为这些NAT仅限于治疗罕见疾病，因此产量很低。然而，一种名为Inclisarin的大容量降胆固醇药物最近获得批准，并且有数百种NAT正在临床试验中评估用于治疗常见疾病。由于目前的化学方法不适合大规模（吨）合成NAT，因此我们必须开发新的、可持续的、可扩展的和通用的生产策略。在这个应用中，我们将开发一个绿色，具有成本效益和真正通用的生物催化平台，用于制造NAT及其核苷酸三磷酸（NTP）构建块。生物催化是一项令人兴奋的技术，广泛应用于化学工业，其中酶（自然界自身的催化剂）用于将起始材料转化为高价值产品。与天然DNA相比，NAT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，以改变它们的功能并优化它们的特性，使它们适合实际应用。我们将使用不同工程酶的组合来首先获得NTP构建块，其将用于随后的生物催化反应以产生NAT。然后，我们将比较使用我们的方法产生的NAT与标准化学方法产生的NAT，使用最先进的分析技术结合生物验证测定。开发的技术将允许高效，可持续和具有成本效益的高纯度NAT制造，从而使这种重要的新药物形式能够充分发挥其治疗各种疾病的潜力。

项目成果

Engineering T7 RNA polymerases for improved manufacturing of mRNA therapeutics

工程化 T7 RNA 聚合酶以改进 mRNA 疗法的生产

• DOI: 10.1016/j.checat.2023.100559
• 发表时间: 2023
• 期刊: Chem Catalysis
• 作者: Obexer R

Biocatalytic Synthesis of Antiviral Nucleosides, Cyclic Dinucleotides, and Oligonucleotide Therapies.

• DOI: 10.1021/jacsau.2c00481
• 发表时间: 2023-01-23
• 期刊: JACS AU
• 影响因子: 8
• 作者: Van Giesen, Kyle J D;Thompson, Matthew J;Meng, Qinglong;Lovelock, Sarah L
• 通讯作者: Lovelock, Sarah L