ASGCT slides MDG May4.pptx
Targeted integration of therapeutic transgenes into a common genomic site could be used to treat a broad range of liver diseases. Recently, we showed that the Apolipoprotein A1 (Apoa1) locus could be targeted with adeno-associated viral (AAV) delivery of CRISPR-Cas9 for transgene expression in the liver. We achieved a rate of ~6% of targeted hepatocytes through homology-directed repair. However, a significant number of liver diseases will require higher targeting efficiencies for therapeutic benefit. The goal of this work is to develop a robust and modular system for selective expansion of gene-targeted hepatocytes using essential genes. We developed an expansion strategy consisting of: (1) transient conditioning of the liver by knocking down an essential gene and (2) delivery of an untargetable version of the essential gene (selectable marker) in cis with the therapeutic gene of interest. To test this approach, we generated AAV-based targeting vectors to insert an essential gene - fumarylacetoacetate hydrolase (FAH) - in tandem with a fluorescent marker (TdTomato) into the Apoa1 locus. We screened siRNAs to identify sequences capable of mediating highly efficient knockdown of endogenous murine Fah in vivo using N-acetylgalactosamine (GalNac)-modification. Mice were injected with AAV vectors encoding CRISPR/Cas9 (AAV-CRISPR) and a Donor template (AAV-Donor) to insert the TdTomato reporter cassette at the Apoa1 locus. Following genome editing, mice were injected with siRNA targeting Fah on a monthly basis for four months. Mice receiving only AAV-CRISPR and AAV-Donor showed ~3% of targeted hepatocytes at the endpoint. This was dramatically increased 5-fold in animals subjected to selective expansion with the conditioning siRNA, reaching ~16% of all hepatocytes. Expansion of correctly targeted Apoa1 alleles was confirmed by sequencing, droplet digital PCR, and western blotting for secreted apoA1 with a 2A epitope tag. We also show that selective pressure can be further increased using a high protein diet, where up to 25% of hepatocytes express TdTomato after three months. This system achieves efficient in vivo expansion of gene-targeted hepatocytes, through transient conditioning of the liver with siRNA, without the use of exogenous drug resistance genes. Importantly, correctly targeted cells do not contain permanent disruption of other genes. This approach should broaden the range of liver diseases correctable with gene therapy using targeted transgene insertion.