Rational design of sequence determinants of therapeutic mRNA stability

Therapeutic mRNAs are an emerging class of biological drugs and vaccines currently in clinical trials. They may be more versatile and easier to produce than therapeutic proteins and do not alter the genetic content of the recipient cell in contrast to DNA-based therapeutics. One of the challenges to overcome, is to predict and modify the intracellular stability of mRNA therapeutics and design molecules with desirable stability characteristics.
A general model has emerged in which the rate-limiting step of mRNA degradation is the removal of the polyA tail by the Ccr4-Not deadenylase. In this model, mRNA degradation is initiated by binding of RNA binding proteins to specific sequence elements in the 3' end, which in turn recruit the Ccr4-Not nuclease leading to removal of the polyA tail and degradation of the mRNA. We hypothesise that the intracellular stability of mRNAs can be determined by the quality of designed sequence elements that direct the recruitment of specific RNA-binding proteins to the mRNA 3' end.
To test this model, a reconstituted system will be used to determine the effects of sequence elements on the removal of the polyA tail by Ccr4-Not. In parallel, cells will be transfected using in vitro transcribed mRNA to correlate mRNA stability in defined biochemical and cellular systems.
The project will provide new information about the regulation of mRNA stability that is relevant for the design of mRNA therapeutics. Training includes protein expression and purification, in vitro transcription, mammalian cell culture, RNA transfection and reporter assays.