Biocatalytic Manufacturing of 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.

Nucleic acid therapeutics (NAT) are a new drug modality with the potential to treat a wide range of disease areas. While NATs currently on the market are for the treatment of rare diseases, there are many more potential therapeutics for common diseases currently under evaluation in clinical trials. In this regard Inclisirin has recently been approved as a cholesterol lowering treatment. Many of these drugs are projected to require metric ton scale synthesis, which creates a significant manufacturing challenge as existing methods of chemical synthesis are restricted to 10Kg batches and are not suitable for large scale applications (>100Kg). This proposal will develop scalable biocatalytic approaches to the manufacture of nucleotide triphosphate (NTP) building blocks and nucleic acid therapeutics. Our processes operate under aqueous conditions, do not require prohibitively large volumes of acetonitrile and are more atom efficient than standard phosphoramidite chemistry as minimal protecting groups are required. Biocatalytic reactions are typically characterized by high efficiencies, selectivities and minimal by-product formation, so our approaches are expected to deliver products with higher purity and yields than current methods. Our strategies also have the benefit of providing access to single stereoisomers of phosphorothioate modified NATs. We will compare NATs produced using our approaches to those produced with standard chemical methods, using newly developed analytical methods and established biological validation assays.