New and versatile chemical approaches for the synthesis of mRNA and tRNA

The central dogma of molecular biology states that "DNA makes RNA makes proteins." We are interested in the possibility of intercepting the second step "RNA makes proteins." Two different RNA molecules, messenger RNA (mRNA) and transfer RNA (tRNA) are required in this step; they combine to convert the four letter code of DNA into the sequence of a protein. In principle this process could be manipulated by chemists who can assemble RNA by automated solid-phase methods. This would make it possible to produce chemically modified RNA and in turn modified proteins with new and improved properties by including unnatural building blocks (amino-acids). However, making the required long modified RNA strands to achieve this is beyond the scope of current chemical methods which are based on solid-phase synthesis. Therefore we will develop a new methodology, solid-phase synthesis of RNA combined with chemical ligation, to join together several chemically synthesised RNA strands in a chain to make mRNA molecules that are large enough to be used as therapeutic molecules and which can also be used to make useful proteins. Our synthetic mRNA (chemRNA) will have many advantages over biologically produced RNA. In particular it can be made on a very large scale, and it is possible to introduce many exotic chemical modifications that are not available to biological systems to enhance its stability and improve its ability to target particular organs in the body. This is an important objective because biochemically-derived, mRNA is starting to show promise in immunotherapy, infectious disease control and cancer therapy. Indeed, some mRNAs have even entered clinical trials. However, their development is limited by the difficulty in modifying them to make them stable in the human body. ChemRNA could also be used in combination with chem-tRNA to make proteins that cannot be made by current biochemical or biological methods. Chemical evolution in a test tube could then be used to produce new proteins with enhanced properties for use in many fields including biotechnology and synthetic biology.

Current gene synthesis methods rely on PCR amplification which is highly developed, but do not allow for the site-specific inclusion of modified nucleotides e.g. for epigenetic studies. Unfortunately, ligation-based nucleic acid assembly does not provide a solution due to the limitations of ligase enzymes. To address this problem we have recently combined automated solid-phase synthesis with chemical ligation to synthesize an epigenetically modified gene. Taking this as proof of principle we now propose to pursue a more ambitious target, the chemical synthesis of large RNA constructs including mRNA (chemRNA) and tRNA (chem-tRNA). This is an important objective because biochemically-derived, in vitro transcribed mRNA (IVT-mRNA) is showing promise in immunotherapy, infectious disease control and cancer therapy. Indeed, some mRNAs have entered clinical trials. However, production of IVT-mRNA suffers from several problems; it relies on labour intensive production of plasmid templates, scale-up is challenging, and the production methods are incompatible with many RNA modifications that would confer therapeutic benefits. This is because RNA polymerases incorporate modified nucleotides with low efficiency, and even the ones they can utilize cannot be introduced site-specifically into RNA. An efficient scalable non-enzymatic (chemical) method for mRNA production could solve these problems. In this project, automated solid-phase RNA synthesis chemistry will be used to assemble RNA strands (~70mers) which will then be ligated together to produce entirely synthetic chemRNA. This approach should enhance the therapeutic benefits of mRNA by making it possible to introduce multiple modified bases, sugars, and backbone linkages in defined positions. Therapeutic applications of chemRNA will be explored, and chemRNA will be used in conjunction with chem-tRNA for the synthesis of natural and unnatural peptides and proteins for applications in biotechnology and synthetic biology.

Academic
The results of this project will be of particular interest to the international scientific community working in chemical biology, nanotechnology, protein chemistry, synthetic biology and cancer research. Establishing chemRNA and chem-tRNA as tools in these disciplines will be a major academic breakthrough. We will publish our results in high impact journals and present them at international conferences. We will also promote our research on social media to increase awareness and to encourage discussion. We will make our novel RNA constructs available to collaborators. The training that the postdoctoral researchers will receive in this interdisciplinary field will provide them with a broad set of skills, making them highly sought after. This research falls within three of the BBSRC strategic priority areas; New strategic approaches to industrial biotechnology, synthetic biology and technical development for the biosciences.

Economic
The development of new scalable chemical approaches to modified mRNA, tRNA and to the synthesis of proteins containing non-canonical amino acids will overcome some of the key constraints of current methodologies. This will have an impact in the fields of biotechnology and therapeutics. It will provide new IP and licencing opportunities for Oxford University and the SME (ATDBio) which will be pursued through interactions with Oxford Innovation and via presentations at conferences with high level industrial participation, as well as by direct approaches to Pharma and Biotech companies with interests in the diagnostic and therapeutic fields. The commercial potential is huge; practical applications are not restricted to humans; synthetic RNA and modified proteins also have applications in animal health and in plant sciences.

Societal
This is generic tehcnology that has potential applications in many areas, one of which is the cancer field. Cancer is one of the leading causes of deaths worldwide with over 14 million new cases annually. There are 160,000 deaths from cancer in the UK annually (CRUK) and the annual NHS budget for cancer care is £6 billion and rising. New and improved therapeutic RNA-based approaches in the Oncology field could eventually come from the new methodology and this would have an impact on society in terms of cost, quality of life and survival rates. Macromolecular therapies based on RNA and proteins are also relevant to many other diseases, so the overall societal impact of advances coming from this project are potentially major.