Nucleoside Modifications and Poly(A) Tail Length Greatly Influence Protein Expression from In Vitro-Transcribed mRNA in a Salmonid Cell Line

Background/Objectives: It is challenging to develop efficient vaccines against intracellular pathogens such as viruses, and since viral infections are one of the main challenges for farmed salmon, a novel vaccine strategy is needed. mRNA vaccines are optimized and approved for humans, but for fish, the mRNA technology is new, and optimization is required to ensure efficient protein expression. We made an mRNA tailored to salmon and studied the effect of modified nucleosides and the length of the poly(A) tail on protein expression from in vitro-transcribed mRNA in CHSE-214 cells, using enhanced green fluorescent protein (EGFP) as a reporter. Methods: Different lengths of the poly(A) tail were tested, and various modified nucleotides were incorporated in the mRNA during in vitro transcription, including pseudouridine (Ψ), N1-methylpseudouridine (m1Ψ), N6-methyladenosine (m6A), 5-methyluridine (m5U), and 5-methylcytidine (m5C). Protein expression was observed in fluorescence microscopy and quantified using flow cytometry. Results: mRNA containing Ψ resulted in the strongest EGFP expression 1–3 days post-transfection (dpt), while EGFP expression from m5C mRNA was high throughout the experiment (<10 dpt). m5U-containing mRNA had low EGFP expression until 6 dpt, but reached the level of m5C mRNA at 10 dpt. The m5U mRNA, however, expressed EGFP at much higher intensity than all the other mRNAs at all time points. Poly(A) tails with lengths of 40, 100, and >100 were tested, and the one with >100 adenines showed the highest expression. The effects of phosphatase treatment and purification of the mRNA were also investigated. Furthermore, EGFP expression was observed in yolk-sac salmon larvae following micro-injection. Conclusions: Our study provides an important basis for the development of efficient mRNA-based vaccines in the future.