RESUMEN
Liver-directed adeno-associated viral (AAV) vector-mediated homology-independent targeted integration (AAV-HITI) by CRISPR-Cas9 at the highly transcribed albumin locus is under investigation to provide sustained transgene expression following neonatal treatment. We show that targeting the 3' end of the albumin locus results in productive integration in about 15% of mouse hepatocytes achieving therapeutic levels of systemic proteins in two mouse models of inherited diseases. We demonstrate that full-length HITI donor DNA is preferentially integrated upon nuclease cleavage and that, despite partial AAV genome integrations in the target locus, no gross chromosomal rearrangements or insertions/deletions at off-target sites are found. In line with this, no evidence of hepatocellular carcinoma is observed within the 1-year follow-up. Finally, AAV-HITI is effective at vector doses considered safe if directly translated to humans providing therapeutic efficacy in the adult liver in addition to newborn. Overall, our data support the development of this liver-directed AAV-based knockin strategy.
Asunto(s)
Dependovirus , Modelos Animales de Enfermedad , Vectores Genéticos , Hígado , Animales , Dependovirus/genética , Hígado/metabolismo , Hígado/patología , Ratones , Vectores Genéticos/genética , Hepatocitos/metabolismo , Humanos , Integración Viral/genética , Sistemas CRISPR-Cas/genética , Transgenes , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/terapia , Terapia Genética/métodos , Ratones Endogámicos C57BL , Albúminas/genética , Albúminas/metabolismoRESUMEN
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 clinical development. Therefore, liver gene therapy with AAV-F8-N6 intein should be considered as a potential therapeutic strategy for HemA.
