biostudies-literatureEsposito FEuropean Research CouncilTelethone15199https://www.ebi.ac.uk/biostudies/studies/S-EPMC9174883biostudies-literatureUnknown14(6)Liver gene therapy with adeno-associated viral (AAV) vectors is under clinical investigation for haemophilia A (HemA), the most common inherited X-linked bleeding disorder. Major limitations are the large size of the F8 transgene, which makes packaging in a single AAV vector a challenge, as well as the development of circulating anti-F8 antibodies which neutralise F8 activity. Taking advantage of split-intein-mediated protein trans-splicing, we divided the coding sequence of the large and highly secreted F8-N6 variant in two separate AAV-intein vectors whose co-administration to HemA mice results in the expression of therapeutic levels of F8 over time. This occurred without eliciting circulating anti-F8 antibodies unlike animals treated with the single oversized AAV-F8 vector under clinicaEMBO molecular medicineLiver gene therapy with intein-mediated F8 trans-splicing corrects mouse haemophilia A.PMC9174883694323TGM16MT1Tornabene PFollenzi ALyubenova HAuricchio SNusco EMerlin SEsposito FManni GOlgasi CGargaro MFallarino FAuricchio AIuliano AfalseLiver gene therapy with intein-mediated F8 trans-splicing corrects mouse haemophilia A.Liver gene therapy with adeno-associated viral (AAV) vectors is under clinical investigation for haemophilia A (HemA), the most common inherited X-linked bleeding disorder. Major limitations are the large size of the F8 transgene, which makes packaging in a single AAV vector a challenge, as well as the development of circulating anti-F8 antibodies which neutralise F8 activity. Taking advantage of split-intein-mediated protein trans-splicing, we divided the coding sequence of the large and highly secreted F8-N6 variant in two separate AAV-intein vectors whose co-administration to HemA mice results in the expression of therapeutic levels of F8 over time. This occurred without eliciting circulating anti-F8 antibodies unlike animals treated with the single oversized AAV-F8 vector under clinica2022-01-01T00:00:00Z2022 Jun2026-05-09T23:45:58.673Z2025-04-04T22:15:20.275ZS-EPMC91748833549167610.15252/emmm.202115199