In vivo expansion of gene-targeted hepatocytes through transient inhibition of an essential gene

Homology directed repair (HDR)-based genome editing is an approach that could permanently correct a broad range of genetic diseases. However, its utility is limited by inefficient and imprecise DNA repair mechanisms in terminally differentiated tissues. Here, we tested “Repair Drive”, a platform technology for selectively expanding HDR-corrected hepatocytes in vivo. Repair Drive involves transient conditioning of the liver by knocking down an essential gene – Fumarylacetoacetate hydrolase (Fah), and delivery of an untargetable version of the essential gene in cis with a therapeutic transgene. We show that Repair Drive dramatically increases the percentage of correctly targeted hepatocytes, up to 25%. This resulted in a five-fold increased expression of a therapeutic transgene – human Factor IX (FIX). Repair Drive was well-tolerated and did not induce toxicity or tumorigenesis in long-term follow up. This approach will broaden the range of liver diseases that can be treated with somatic genome editing. Overall design: To investigate the hepatocyte population expanded through Repair Drive we isolated nuclei from ~100 mg of frozen liver samples. The liver samples were homogenized using Dounce homogenizers. Nuclei were isolated by using strainers and sucrose gradient centrifugation. Nuclei samples were diluted to obtain 15000 - 18000 nuclei for single cell library construction according to the 10X single cell gene expression workflow. Libraries were sequenced on an Illumina NovaSeq 6000 instrument.