Promoterless gene targeting without nucleases ameliorates haemophilia B in mice

Abstract

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 expansion1 and oncogenesis2. Site-specific endonucleases that can induce high rates of targeted genome editing are finding increasing applications in biological discovery and gene therapy3. However, two safety concerns persist: (1) endonuclease-associated adverse effects, both on4 and off-target5,6; and (2) oncogene activation caused by promoter integration, even without nucleases7. 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.

Therapeutic editing of hepatocyte genome in vivo

“…One alternative strategy to increase the expression of restored genes is to integrate them in the albumin locus downstream of the endogenous albumin promoter. 82,83 Albumin is highly expressed in hepatocytes and the integration of the wild-type gene into this locus would ensure high expression.…”

Regenerative medicine: targeted genome editing in vivo

“…A recent study raised an intriguing possibility of in vivo promoterless gene targeting without the use of nucleases, thus greatly diminishing the risk of adverse off-target effects [5]. Comparably, however, the promoterless nuclease-independent site-specific gene targeting strategy illustrated by Barzel et al [5] is effective in only certain therapeutic effects conferred by gene targeting.…”

To cleave or not to cleave: therapeutic gene editing with and without programmable nucleases

“…Although such approaches were pioneered in the 1990s 3 , before the advent of the nuclease-based gene editing platforms, their use and progression to clinical trials since has been held back by issues including frequencies of gene editing that are typically well below those needed for potential therapeutic applications. However, in the past 2 years, results with a diverse group of gene editing platforms -ranging from recombinogenic adeno-associated viral (AAV) vectors, to various oligonucleotide-only approaches, to nucleobase modification (TABLE 1) -have demonstrated substantially improved gene editing efficiencies [4][5][6][7][8][9] , spurring renewed interest in such approaches. Moreover, these platforms do not require cleavage of the targeted DNA to achieve gene editing -a characteristic of nuclease-based mechanisms that has raised concerns about random insertion and deletions (indels) that occur in some proportion of the targeted cleavage sites and their potential for off-target genotoxicity.…”

“…Although the published efficiency of this technology is only in the 1% range, the long repair template allows for site-directed gene replacement. By directing integration of a replacement factor IX 'gene' (cDNA) to the highly expressed albumin locus with a variant of recombinant AAV in a mouse haemophilia model, 7-20% of normal factor IX was expressed in the serum, resulting in correction of the mutant phenotype without exogenous programmable nucleases 4 .…”

“…The authors obtained up to 14% edited cells in culture per treatment of haematopoietic stem cells and ~4% editing in a mouse model of beta-thalassaemia after four intravenous administrations of oligonucleotides, which resulted in long-term phenotypic correction 6 . Recombinogenic AAV It has recently been demonstrated that much longer single-stranded donor DNA delivered by a variant of recombinant AAV vectors can also lead to editing in cell culture and in animal models without the need for exogenous nucleases 4 . Although the published efficiency of this technology is only in the 1% range, the long repair template allows for site-directed gene replacement.…”