Adeno-associated Virus-mediated Silencing of Sox4 Leads to Long-Term Amelioration of Liver Phenotypes in Mouse Models of Alagille Syndrome.

Abstract

In patients with Alagille syndrome (ALGS), bile duct paucity often leads to severe cholestatic phenotypes for which liver transplantation remains the only definitive treatment. No Food and Drug Administration-approved mechanism-based strategies exist to enhance biliary development in ALGS or other diseases with bile duct paucity. We aimed to identify a therapeutic target to address this unmet need. Preclinical ALGS mouse models lacking 1 copy of Jag1 with or without conditional deletion of 1 or both copies of Sox9 were used. Sox4 levels were reduced genetically or with adeno-associated virus 8 (AAV8) vectors driving a Sox4-silencing sequence. Liver histology, biliary tree ink injection, serum chemistry, RNAscope (on mouse and human livers), mouse liver single-cell RNA-sequencing, and reanalysis of published human liver bulk RNA-sequencing were performed. Conditional removal of 1 copy of Sox4 in mouse liver significantly improved the ALGS liver phenotypes in a Sox9-dependent manner. An increase in Sox4/SOX4 expression in early postnatal Jag1-heterozygous mouse livers and ALGS patient livers was observed. single-cell RNA-sequencing 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 thyroxine-binding globulin promoter, led to long-term improvement of ALGS liver phenotypes upon injection at postnatal day 1. AAV8 injection at postnatal day 15-after the appearance of liver necrosis-led to the incorporation of some transduced cells into bile ducts and phenotypic improvement. Preclinical studies provide proof of principle for postnatal AAV-mediated Sox4 knockdown in thyroxine-binding globulin⁺ cells as a therapeutic approach for ALGS liver disease.