siRNA-mediated Timp1 silencing induces fibrosis regression in a murine model of Primary Biliary Cholangitis

Autoimmune liver diseases are chronic inflammatory disorders that lead to severe impairment of liver function. Among them, Primary Biliary Cholangitis (PBC) is characterized by the progressive destruction of small intrahepatic bile ducts, causing liver damage through bile acid retention. This damage results in sustained inflammation, cholestasis, and fibrosis, eventually leading to cirrhosis. Current first-line treatment (UDCA) is ineffective in 40% of patients, resulting in increased risk of death or need for transplantation. To investigate therapeutic intervention in PBC, we utilized a murine model of the disease, the ARE Del-/- mouse. This model is characterized by a deletion in the 3’ untranslated region (3’ UTR) adenylate uridylate-rich element (ARE) of the IFN- γ gene. This deletion causes chronic and prolonged expression of IFN-γ, leading to the development of liver lesions, up-regulation of bile acids, as well as female predominance, mimicking human PBC. In this pathological context, MMP (matrix metalloproteinase) activity, which degrades ECM, is impaired by an overproduction of its specific inhibitors, Timp (tissue inhibitors of metalloproteinases), causing the replacement of the liver parenchyma by fibrotic scar tissue. This study aimed to develop an innovative gene-silencing strategy targeting Timp1, exploiting siRNA delivery via lipid nanoparticles (LNPs) in the ARE Del mouse model. Given the critical role of Timp1 in the ECM balance, the targeted suppression of this gene in the liver could facilitate proteolytic ECM remodelling and alleviate cholestasis injury, improving the clinical picture of PBC. Four LNP formulations with varying lipid compositions were screened for hepatic tropism. The lead LNP candidate, characterized by a high cholesterol concentration, was loaded with a siRNA targeting Timp1. The impact of chronic siTIMP1-LNP administration on the clinical and pathological progression of PBC was assessed in the ARE-Del-/- model. Therapeutic efficacy was evaluated through serum biochemical analysis, histological staining, and gene expression to monitor the hepatocellular function, lipid metabolism, and cholestasis. Treatment with siTIMP1-LNP successfully reduced the expression of TIMP1 while upregulating MMP9 levels. This shift in the proteolytic balance resulted in a significant reduction of the histological fibrotic area and the degradation of basement membrane components. Functionally, serum total bile acids (TBA), a dynamic biomarker of disease severity, were significantly reduced, indicating restoration of bile metabolism from the cholestatic state. At the immunological level, Timp1 silencing also induced a shift in the hepatic microenvironment. Chronic treatment induced a reduction of macrophage infiltration and downregulation of pro-inflammatory chemokines, paired with an increase in the resolving cytokine IL-10. Collectively, these results demonstrate that LNP-mediated Timp1 silencing in the context of chronic liver diseases exerts a dual therapeutic effect by reactivating ECM degradation and by reprogramming the immune environment from a state of chronic inflammation to one of active resolution. Overall, these effects converge to mitigate fibrogenic progression within a cholestatic setting, validating the therapeutic efficacy for targeting TIMP-1 as a viable strategy to treat chronic hepatic pathologies.