mRNA Capping Technologies: Effects On Quality Attributes, Biological Effects, And Innate Immune Stimulation

By Christian Cobaugh, Kurtis Breger, Lauren Hinkel, Hana Kibe, Matt Nolan, Eli Henderson, and Kyle McKay, Vernal Biosciences

Synthetic mRNA is more potent, stable, and immuno-silent when the 5’ end contains a Cap1 structure (N7-methyl guanosine connected to the 5′ nucleotide through a 5′ to 5′ triphosphate linkage; N1 nucleotide methylated at the 2’ position). Cap1 can be added to the synthetic mRNA enzymatically using a guanylyltransferase that removes the 5’ PO4 from the 5’ of the RNA, removes two PO4 from GTP, and catalyzes the addition of the resulting GMP to the 5’ end of the mRNA. The same enzyme also methylates the newly added G at the N7 position of its base.

In the same reaction mixture, a 2’-O-methyltransferase methylates the 2’-OH of the ribose. Cap1 can also be added to the mRNA during the in vitro transcription reaction by adding a trinucleotide cap analog with a defined Cap1 structure that is transcriptionally incorporated into the 1st and 2nd positions. This study compares these two important mRNA capping technologies by evaluating the mRNA integrity, percent Cap1 content, dsRNA content, in vivo expression, and innate immune activation in mice.