Biocatalytic Manufacturing of Nucleic Acid Therapeutics

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，出现了一类新的药物分子，称为核酸疗法(NAT)，它为治疗一系列遗传性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。现有的NAT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)，并以较低的产率和中等(~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。这些限制并不是目前市场上制造NAT的主要问题，因为这些NAT仅限于治疗罕见疾病，因此产量较低。然而，一种名为Inclisarin的大容量降胆固醇药物最近获得批准，目前有数百种NAT正在进行临床试验，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，因此我们现在必须为它们的生产开发新的、可持续的、可扩展的和通用的制造策略。在这一应用中，我们将开发一种绿色、低成本和真正多功能的生物催化平台，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，广泛应用于整个化学工业，利用酶(自然自身的催化剂)将原料转化为高价值的产品。与天然DNA相比，NAT包含旨在提高其有效性、选择性和代谢稳定性的化学修饰。天然酶不能很好地耐受这些化学修饰，然而，使用一种名为定向进化的技术，我们能够快速设计酶来修改它们的功能并优化它们的性质，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访问NTP构建块，这些构建块将用于后续的生物催化反应以生成NAT。然后，我们将使用最先进的分析技术与生物验证分析相结合，将使用我们的方法产生的NAT与使用标准化学方法产生的NAT进行比较。所开发的技术将使高效、可持续和具有成本效益的高纯度NAT的制造成为可能，从而使这种重要的新药物模式能够充分发挥其治疗各种疾病的潜力。

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.