In vivo expansion of gene-targeted hepatocytes enables safe and durable transgene expression

Targeted transgene insertion is a genome editing approach that could permanently correct a broad range of liver disorders. However, the utility of this strategy is currently limited by imprecise and inefficient repair mechanisms. For example, homology directed repair (HDR) requires cell division, generally limiting targeting of adult liver to 1% of hepatocytes. The goal of this work is to develop a system for selective expansion of gene-targeted hepatocytes using essential genes. Our system consists of: (1) transient conditioning of the liver by knocking down an essential gene, and (2) delivery of an untargetable version of the essential gene in cis with the therapeutic transgene. To test this approach, we used AAV vectors to insert an essential gene -fumarylacetoacetate hydrolase (FAH) - in tandem with a fluorescent marker (TdTomato) into a highly expressed locus in the liver (Apoa1). Mice were injected with AAV8 vectors encoding CRISPR/Cas9 (AAV-CRISPR) and a Donor template (AAV-Donor) with FAH and a TdTomato transgene. Monthly injections of N-acetylgalactosamine (GalNac)-modified siRNA targeting murine Fah were used as a conditioning agent to deplete untargeted hepatocytes and drive expansion of correctly repaired cells.