A Novel Single Subunit RNA Polymerases for Commercial RNA Manufacturing
Lead Research Organisation:
University of Edinburgh
Department Name: Sch of Biological Sciences
Abstract
The use of enzymes called RNA polymerases for the efficient production of mRNA is enormously important for the burgeoning field of cell-free protein production and use in an expanding range of biosensor applications, however, the largest market opportunity is in the RNA therapeutics and vaccines arena. mRNA holds the potential to revolutionise vaccination, protein replacement therapies, and the treatment of genetic diseases. The use of mRNA for the expression of therapeutic proteins holds the potential to treat or prevent a wide range of diseases including (1) restoration of the function of a single protein for rare monogenic diseases; (2) cell reprogramming and (3) immunotherapies where mRNA encoded transcripts provoke immune responses against targets such as tumour cells and (4) RNA vaccines, currently the largest market.
Most traditional vaccines are made from proteins produced by infectious microbes, or from weakened forms of the microbes themselves. RNA vaccines however work by introducing an mRNA sequence encoding a disease specific antigen that gets translated into protein as soon as it gets into the cell cytoplasm. Once produced within the body, the antigen is recognised by the immune system, triggering recognition of the disease. RNA vaccines offer many advantages, including the ability to design a rapid response manufacturing platform. Their ease of production would allow distributed, localised manufacturing systems to meet the challenges of any emerging disease epidemic within a relatively short time and in the geography where it is needed. RNA based vaccines are also safer for the patient, as they are not produced using infectious elements.
The global RNA drugs market is forecast to exceed $10 billion by 2024 (based on an analysis carried out using the GlobalData Plc database), highlighting the significant commercial potential of this emerging class of therapeutics.
Currently T7 RNA polymerase is the gold standard for industrial mRNA production but there is great interest in improved alternative RNA polymerases. In our preliminary work we have identified a novel single subunit RNA polymerase and cognate synthetic promoters. In this project we aims to further characterise and further develop our novel RNA polymerase (and its mutant derivatives) in order to establish a strong patent position for licencing to industry. A new efficient RNAP could potentially be highly disruptive and would reduce the costs of RNA manufacturing and use in R and D bringing more affordable products to the mRNA vaccine market for the benefit of patients.
Most traditional vaccines are made from proteins produced by infectious microbes, or from weakened forms of the microbes themselves. RNA vaccines however work by introducing an mRNA sequence encoding a disease specific antigen that gets translated into protein as soon as it gets into the cell cytoplasm. Once produced within the body, the antigen is recognised by the immune system, triggering recognition of the disease. RNA vaccines offer many advantages, including the ability to design a rapid response manufacturing platform. Their ease of production would allow distributed, localised manufacturing systems to meet the challenges of any emerging disease epidemic within a relatively short time and in the geography where it is needed. RNA based vaccines are also safer for the patient, as they are not produced using infectious elements.
The global RNA drugs market is forecast to exceed $10 billion by 2024 (based on an analysis carried out using the GlobalData Plc database), highlighting the significant commercial potential of this emerging class of therapeutics.
Currently T7 RNA polymerase is the gold standard for industrial mRNA production but there is great interest in improved alternative RNA polymerases. In our preliminary work we have identified a novel single subunit RNA polymerase and cognate synthetic promoters. In this project we aims to further characterise and further develop our novel RNA polymerase (and its mutant derivatives) in order to establish a strong patent position for licencing to industry. A new efficient RNAP could potentially be highly disruptive and would reduce the costs of RNA manufacturing and use in R and D bringing more affordable products to the mRNA vaccine market for the benefit of patients.
Organisations
People |
ORCID iD |
| Susan Rosser (Principal Investigator) |
| Description | We characterised the RNA polymerase (RNAP) from Klebsiella phage 32, exhibiting different characteristics to T7 RNAP which is currently the gold standard. The utilisation of both KP32 WT RNAP and T7 RNAP in tandem could enable new genetic circuits to be investigated. We have found that both the KP32 RNAP optimally transcribes RNA at 30 °C, while reducing the temperature to 21 °C maintains 71.4% of production. This high activity rate over a wider temperature range than T7 RNAP (temperature optima of 37 °C) opens the possibility of utilising KP32 RNAP within cell free reactions, or transcription-based biosensors in the field, without the need for temperature-controlled incubation of the transcription reaction to yield an output in a timely manner. A broader pH tolerance range in comparison to T7 RNAP further enables this real-world use potential. Lastly, KP32 RNAP has been found to be more tolerant to NaCl than T7 RNAP. This characteristic again lends KP32 WT RNAP to the use within real world samples where monovalent salt ion concentrations cannot be easily controlled. Lastly, the use of 2'F modified nucleotides has increased the potential for small therapeutic RNAs to have improved stability in vitro or in vivo. Here we have shown KP32 RNAP to have an increased capacity for 2'F modified nucleotide incorporation, relative to that observed with T7 RNAP. A significant use case for RNAPs following the success of the COVID-19 vaccines is mRNA vaccine production. Our results indicate KP32 RNAP could be a useful tool for the efficient large-scale production of therapeutic mRNA. We showed that the KP32 RNAP has an increased tolerance to iPP which would mean less iPPase is required within the reaction, thus reducing production costs. These production cost savings could be further increased through the need for less RNAP to obtain the same amount of final mRNA. Of critical importance to mRNA therapies is the use of me1?-UTP as this lowers the immune response to the mRNA, thereby increasing mRNA stability and increasing translation of the mRNAClick or tap here to enter text.. We have found that KP32 RNAP incorporates me1?-UTP into mRNA products at an equal efficiency when compared to reactions using standard UTP, at optimum reaction temperatures. KP32 WT RNAP therefore has significant potential for real-world applications within biotechnology. |
| Exploitation Route | The novel RNA Polymerases are being assessed for use by industrial partners |
| Sectors | Manufacturing including Industrial Biotechology |
| Description | An invention disclosure form has been submitted to our tech transfer office. We are in early stage discussions with Moderna about the potential of the technology. |
| First Year Of Impact | 2024 |
| Sector | Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
| Impact Types | Economic |