AAV-mediated silencing of Sox4 leads to long-term amelioration of liver phenotypes in mouse models of Alagille syndrome (scRNA-Seq)

BACKGROUND & AIMS: In patients with Alagille syndrome (ALGS), bile duct paucity often leads to severe cholestatic phenotypes that can only be cured with liver transplantation. However, no mechanism-based strategies exist to enhance biliary development in ALGS or other diseases associated with bile duct paucity. We aimed to identify a therapeutic target to address this unmet clinical need. METHODS: Preclinical mouse models of ALGS lacking one copy of Jag1 in germline with or without conditional deletion of one or both copies of Sox9 with Albumin-Cre were used. Sox4 levels were reduced in these models genetically or by using adeno-associated virus serotype 8 (AAV8) vectors driving a Sox4-silencing sequence. Liver histology, biliary tree ink injection, serum chemistry, RNAscope, and single-cell RNA-sequencing (scRNA-Seq) were performed for phenotypic characterization and gene expression analysis. RESULTS: Conditional removal of one copy of Sox4 in the liver significantly improved the liver phenotypes in ALGS mouse models in a Sox9-dependent manner. RNAscope experiments showed an increase in Sox4 expression level and relative abundance of Sox4-Sox9 double-positive cells in early postnatal livers of Jag1-heterozygous mice. scRNA-Seq revealed the appearance of an intermediate hepatobiliary cluster co-expressing Sox4 and Sox9 in Jag1-heterozygous livers. AAV8-mediated Sox4 knockdown, ubiquitously or driven by the hepatocyte-specific TBG promoter, led to long-term improvement of ALGS liver phenotypes upon one injection at postnatal day 1 (P1). An AAV8 injection at P15, when the liver damage response is activated, also led to phenotypic improvement. CONCLUSIONS: Our preclinical studies provide proof of principle for AAV-mediated Sox4 knockdown in TBG+ cells as a therapeutic approach for ALGS liver disease.