Producing a long peptide concatemer prodrug using circular mRNA

Circular mRNAs have many useful properties including greater stability in cells and the ability to have an infinite open reading frame (IORF). Using an Internal Ribosomal Entry Site (IRES) or a Shine Dalgarno sequence (in bacteria), circular mRNA can be translated in vitro or in vivo. Circular mRNAs containing no stop codon in the desired frame can have an IORF allowing the production of a long repetitive protein.
Circular mRNAs with IORFs encoding therapeutic peptides will produce long peptide concatemers containing many peptide repeats. Harbouring a protease cleavage site between each peptide unit allows for the peptide concatemer to act as a prodrug, being cleaved in the presence of its complementary protease. In many diseases, proteases are characteristically overexpressed as often they are involved in disease progression e.g. calpains and cancer. Such diseases are excellent candidates for treatment using therapeutic peptide concatemers produced from circular mRNA, as greater amounts of cleavage will occur at localised disease sites.
Using this approach, I aim to first establish the methodology of producing long peptide concatemers both in vitro and in vivo using a simple IRES/Shine Dalgarno FLAG tag sequence, then apply this method to produce a peptide concatemer prodrug functional in vivo.
IRESs recruit the ribosome independently of the 5' RNA cap of linear RNA. Circular RNAs lack this cap and therefore are useful models for IRES analysis as all ribosomal recruitment can be attributed to the IRES sequence. Using this knowledge, I aim to do a comparative study on the efficiency of ribosomal recruitment using a variety of IRESs including cellular, viral and artificial.