In vitro transcription of mRNA

mRNA were transcribed as previously described¹⁵ using the linearized plasmids encoding BDD-FVIII (FVIII^HSQ), the codon-optimized BDD-FVIII (CoFVIII^HSQ) and firefly luciferase (Luciferase). The Megascript T7 RNA polymerase kit (Thermo Fisher) was used for transcription, and UTP was replaced with 1-methylpseudouridine triphosphate (m1ΨTP; TriLink, San Diego, CA, USA) to generate m1Ψ-containing mRNA. All mRNA were transcribed to contain 100-nucleotide long poly(A) tails. To obtain cap1, RNA was incubated with guanylyltransferase and 2′-O-methyltransferase (Vaccinia capping system; New England Biolabs, Frankfurt, Germany). All mRNA were purified and stored at −20°C.