Coupling AAV-mediated promoterless gene targeting to SaCas9 nuclease to efficiently correct liver metabolic diseases

Nonintegrative AAV-mediated gene therapy in the liver is effective in adult patients but faces limitations in pediatric settings because of episomal DNA loss during hepatocyte proliferation. Gene targeting is a promising approach as it results in the permanent modification of the genome. We previously rescued neonatal lethality in Crigler-Najjar mice by inserting a promoterless human uridine glucuronosyl transferase A1 (UGT1A1) cDNA in exon 14 of the albumin gene, without the use of nucleases. To increase the recombination rate and therapeutic efficacy, we used CRISPR/SaCas9. Neonatal mice were transduced with 2 AAVs: one expressing the SaCas9 and sgRNA and one containing a promoterless cDNA flanked by albumin homology regions. Targeting efficiency increased approximately 26-fold with an EGFP reporter cDNA, reaching up to 24% of EGFP-positive hepatocytes. Next, we fully corrected the diseased phenotype of Crigler-Najjar mice by targeting the hUGT1A1 cDNA. Treated mice had normal plasma bilirubin up to 10 months after administration, hUGT1A1 protein levels were approximately 6-fold higher than in WT liver, with a 90-fold increase in recombination rate. Liver histology, inflammatory markers, and plasma albumin were normal in treated mice, with no off-targets in predicted sites. Thus, the improved efficacy and reassuring safety profile support the potential application of the proposed approach to other liver diseases.