Project supervisor: Dr John Le Quesne, Department of Cancer Studies and Professor Anne Willis
Industrial collaborator: Catherine Betts and Stewart Jones, AstraZeneca
University of registration: University of Leicester
Project title: To explore mechanisms to enhance protein production from novel modified therapeutic in vitro transcribed RNAs (IVTmRNAs)
The use of in vitro transcribed modified mRNAs (mIVTmRNAs) holds promise for the safe, transient delivery of transgenes. However, there are limitations in the efficacy of IVTmRNAs due to inflammatory reactions and relatively low levels of transgene expression. This is due to antiviral mechanisms that are mediated via the delivery of naked mRNAs. To devise new strategies to overcome these issues and examine the complex dose:response relationship between mRNA burden and protein expression in single cells, we will develop i) multiplex immunohistochemical assays to detect mIVTmRNAs by in situ hybridisation (ISH), quantification of the transgene product by immunohistochemistry (IHC), and further IHC to simultaneously quantify stress response proteins. ii) Use ISH and IHC to examine the induction of inflammatory signalling pathways. iii) Examine the tissue cellular immune response by assaying for markers for transfected mRNA/protein with immune cell types. These assays will be applied to experimental tissues supplied by AstraZeneca. Based on these data we will iv) optimise mIVTmRNAs including by modifying nucleotide content, altering codon optimisation, and modification of mRNA stability elements/initiation efficiency modifiers. Treated model tissues will be supplied and re-examined and information gained will enhance the transfer of IVTmRNA technologies into a clinical setting.
Contact: Dr John Le Quesne, University of Leicester