13th BIC International Conference

Background: First generation AAV-based gene therapies are approved for the treatment of congenital hemophilia A (HA), however in most instances FVIII plasma levels have been observed to decline over time.Endonucleasemediated genomic insertion of an AAV-delivered FVIII transgene into a safe-harbor locus in the patient's genome may protect the transgene from loss and/or silencing thereby improve durability of FVIII expression.Aim: The aim was to develop a durable, in vivo genomic transgene insertion therapy for patients with HA. Materials and Methods: A site-specific megaTAL endonuclease was engineered to induce double stranded DNA breaks in the albumin gene, and delivered to hepatocytes as lipid nanoparticle-encapsulated mRNA.AAV vector donors encoding promoter-less, B-domain truncated FVIII transgene variants were developed to enable FVIII expression following targeted genomic insertion and optimized for levels of circulating, functional FVIII.In vivo efficacy and tolerability were evaluated in F8 -/-/ Rag2 -/-mice and cynomolgus monkeys following simultaneous treatment with both components.Results: Administration of AAV donor vector encoding human FVIII and LNP-encapsulated mRNA encoding the megaTAL nuclease enabled targeted insertion of the FVIII transgene into the albumin genomic locus in mice and cynomolgus monkeys.In F8 -/-/Rag2 -/-HA mice, hepatic expression of human FVIII rescued the bleeding phenotype.Assays specific for human FVIII, the truncated B-domain and human FVIII heavy/ light chain, respectively, allowed for quantification of human FVIII in cynomolgus plasma containing endogenous cynomolgus FVIII.Genomic insertion of human FVIII transgene in cynomolgus monkeys resulted in human FVIII plasma levels within the normal range, however anti-drug antibody responses against the human protein complicated long-term studies.Design of surrogate AAV vectors, encoding cynomolgus FVIII, allowed for durable FVIII transgene expression in a 12 week study (Fig. 1).Conclusions: Targeted genomic insertion of FVIII transgene has shown promise in pre-clinical models with the potential to become a treatment option for people with haemophilia A with a life-long effect.