(7) Many thanks to co-first authors @tanjarothgangl and Eleonora Ioannidi! We are also thankful for our @schwanklab members @YanikWeber, @BoeckDesiree, Elina Villiger, @marquark, @lukas_villiger, @p_kulcsar and collaborators from the #NorbertPardi and #JohannesHäberle lab.

(6) Encouraged by these results, we returned to the full RNA-LNPs delivery using PE7 mRNA and synthetic La-accessible pegRNAs. We achieved 8% correction, lowering Phe below the therapeutic level of 360 µM. Finally, we edited 8/11 common human PKU mutations with ≥Pah-enu efficiency in cells.

(5) We next exchanged the PEmax mRNA with the PE7 mRNA developed at @bsadamson lab. This increased editing efficiencies to 20%, bringing Phe levels below 360 µmol/L—the therapeutic threshold for children.

(4) Hypothesizing that the pegRNA is the limiting factor, we turned to a hybrid approach: we delivered the pegRNA via scAAV and only PEmax via mRNA-LNPs. This resulted in 4% editing and Phe reduction below 600 µmol/L—the therapeutic threshold for adults.

(3) Applying the same strategy to the pathogenic Pah-enu mutation in PKU mice resulted in editing efficiencies below 1% in isolated hepatocytes, insufficient to reduce blood phenylalanine to therapeutic levels.

(2) We set out to develop a fully RNA-based prime editing strategy for treating PKU in a mouse model. First, we optimized the dosing at the Dnmt1 locus. LNP-mediated co-delivery of PEmax mRNA with pegRNA - rather than subsequent delivery (first mRNA then pegRNA) - resulted in highest editing.