Promoterless gene targeting without nucleases ameliorates haemophilia B in mice

Site-specific gene addition can allow stable transgene expression for gene therapy. When possible, this is preferred over the use of promiscuously integrating vectors, which are sometimes associated with clonal expansion(1) and oncogenesis(2). Site-specific endonucleases that can induce high rates of targeted genome editing are finding increasing applications in biological discovery and gene therapy(3). However, two safety concerns persist: (1) endonuclease-associated adverse effects, both on(4) and off-target(5,6); and (2) oncogene activation caused by promoter integration, even without nucleases(7). Here, we perform recombinant adeno-associated virus (rAAV) mediated promoterless gene targeting without nucleases and demonstrate amelioration of the bleeding diathesis in haemophilia B mice. In particular, we target a promoterless human coagulation factor IX (hF9) gene to the liver-expressed albumin (Alb) locus. hF9 is targeted, along with a preceding 2A-peptide coding sequence, to be integrated just upstream to the Alb stop codon. While hF9 is fused to Alb at the DNA and RNA levels, two separate proteins are synthesized by way of ribosomal skipping. Thus, hF9 expression is linked to robust hepatic albumin expression without disrupting it. We injected an AAV8-hF9 vector into neonatal and adult mice and achieved on-target integration into ~0.5% of the albumin alleles in hepatocytes. We established that hF9 was produced only from on-target integration, and ribosomal skipping was highly efficient. Stable hF9 plasma levels at 7–20% of normal were obtained, and treated factor IX deficient mice had normal coagulation times. In conclusion, transgene integration as a 2A-fusion to a highly expressed endogenous gene may obviate the requirement for nucleases and/or vector-borne promoters. This method may allow for safe and efficacious gene targeting in both infants and adults by greatly diminishing off-target effects while still providing therapeutic levels of expression from integration.