Synthetic Biology of mRNA

Given the diverse role of mRNA in major life science applications from vaccination via gene therapy, chemistry, and diagnostics to pest control in agriculture there is a huge opportunity to improve the outcome of medical therapies, broaden chemical catalysis, advance medical diagnostics, and reduce the human footprint in agriculture. With a focus on medical applications, NEWmRNA proposes to follow a synthetic biology approach to redraw and transform biological mass production of mRNAs.
In the course of the project, we will implement a novel cellular assay to rapidly assess the impact of non-canonical forms of mRNA on translation and cellular signaling, re-engineer enzymatic mRNA synthesis via T7 RNA polymerase, introduce the concept of cloaked bioproduction in bacteria – the production of protected forms of instable molecules, coupled to enzymatic recovery post production – and introduce crucial steps for industrial-scale production of mRNA.

Why RNA?

RNA is no longer viewed as having a passive role of communicating information from DNA to protein synthesis. Instead it is now thought to dynamically coordinate and instruct cellular function. Furthermore, advantages of RNA for therapeutic purposes have become clear: When transferring an mRNA, the molecule only needs to reach the cytoplasm to exert its function (not, as DNA, the nucleus), which makes the response rapid and delivery of the molecule inherently simpler.

The expression of information is only transient, which could be either an advantage or disadvantage. The information-carrying molecule is not DNA nor is it integrating or replicating, so there is no potential for neoplastic behaviour. Any encoded protein will be endowed with the proper posttranslational modifications. Finally, as natural molecules, mRNAs possess, in general, low toxicity and immunogenicity (in particular if they are properly modified before application).

Therapeutic RNA

These properties have laid the foundation for a broad variety of applications in the life sciences. Delivering mRNAs can treat monogenic diseases (in particular metabolic diseases), or function as replacement therapy, in particular if the target tissue is the liver or the lungs. Interventions on genomes can be done using mRNA-encoded CAS9 co-delivered with the guide a. Delivery of antibody-coding mRNA can be used as therapy against toxins or infectious disease by way of passive immunization. Synthetic mRNAs provide an unequivocally “footprint-free” method to reprogram differentiated cells into induced pluripotent stem cells.

A current focus is on vaccines, as mRNA molecules are easy-to-program, rapid, and create robustly accessible vaccines, as evidenced by RNA vaccine companies such as Moderna (Boston, USA), BioNTech (Mainz, DE) and CureVac (Tübingen, DE) who have developed anti-COVID19 mRNA-based vaccines.

Limitations of Synthetic RNA and mRNA

These compelling properties of synthetic mRNA are countered by a set of disadvantages:

1. RNA has typically a short half-life in cells due to a variety of RNase enzymes that patrol through all major delivery channels and in all cells.
2. RNAs as polyanionic molecules cannot reach the cytoplasm without help, but RNA delivery has over the last decades benefitted from tremendous progress in formulation, in particular the development of dedicated lipid nanoparticles (LNPs).
3. Synthetic mRNA production is largely limited to canonical nucleotides and enzymes. As opposed to chemically synthesised RNA oligonucleotides that can be endowed with many modifications which tune central properties, such as resistance to RNases, synthetic mRNAs remain largely unmodified. They are produced enzymatically, in reactions in which the canonical ribonucleotides (rNTPs) are polymerized by a suitable RNA polymerase using a DNA-template. Consequently, the fundamental degree of chemical freedom in the production of mRNAs is determined by the flexibility with which for the RNAPol can polymerize non-canonical monomers into an mRNA polymer.

Towards therapeutic mRNA production

Making vaccine-grade mRNA is possible, but vaccine campaigns should be accessibly to as many people as possible, as quickly as possible. As a result, manufacturing costs are to be kept small. Furthermore, the manufacturing of long RNAs is difficult. Current mRNA manufacturing is a cell-free enzymatic process based on the substrate scope of the RNAPol and the availability of mostly still expensive triphosphates.

When looking at life-long treatments outside the domain of vaccines (and, in general, applications outside pharma), the current economics and scope of the production process seem unsustainable. In contrast, proteins are produced in purified form at the costs of a few USD per kg, and even in the pharmaceutical domain, with insulin being produced in and purified from Escherichia coli by Sanofi on the ton/year-level. As such, bacterial production has become a crucial part of the success of the product.

However, any cell-based strategy needs to deal with the fact that the problem of degradation is much more severe for RNA than for proteins, half-life times for both bioproducts – minutes in the case of mRNA versus hours to days and longer for proteins. Furthermore, overproduction of proteins is a process in which a cell is already prepared for (50% of its dry biomass is protein) – but overproduction of mRNAs would occur against a background of only 4 % of mRNAs in an average bacterial cell (20% of total RNA, including rRNA). Consequently, the microbial cell might be less adapted to mass production of mRNA than for mass production of proteins.

NEWmRNA

We propose our science-to-technology breakthrough to master RNA synthesis, which rests on 4 pillars:

1. A fundamental reengineering of a T7 RNAPol, the engine room of mRNA synthesis, to efficiently produce mRNAs with partially or entirely non-canonical nucleotides.
2. A redrawing of the map of chemical flexibility of eukaryotic translation.
3. A novel design of the manufacturing of non-canonical mRNA in bacteria.
4. Designing the tools for the upscaling of mRNA production.

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