575 SPECIFIC IN VIVO DELIVERY OF PROCOLLAGEN a1(I) siRNA IN LIPID-LIKE CARRIERS INHIBITS THE PROGRESSION OF BILIARY LIVER FIBROSIS

Abstract

Introduction: Hepatic fibrosis is characterized by excess production and deposition of extracellular matrix. Small interfering RNA (siRNA) is a powerful tool for post-transcriptional gene silencing in vitro. However, the systemic delivery of naked siRNAs has many obstacles, such as interaction with plasma proteins and degradation by plasma and tissue nucleases. Novel lipid-like particles (LPs) permit efficient delivery of siRNA, minimizing interference with blood cells and plasma and being devoid of side effects. Methods: Mice deficient in the phospholipid flippase (Mdr2) develop spontaneous and progressive biliary fibrosis (Popov et al, J Hepatol 2005). Optimized siRNA directed against the transcripts of the major scar tissue protein, procollagen a1(I), was encapsulated in C12–200 LPs and specifically delivered to the liver of Mdr2 knockout mice. LPs with siRNA to GFP (green fluorescent protein) served as negative controls. DiR-labeled C12–200 LPs were used for in vivo and ex vivo tracking using near infrared (NIR) and fluorescent imaging. Groups (n =7–8) of 8 week old Mdr2KO mice and their nonfibrotic wild-type controls received 4 injections of procollagen a1(I) or control (GFP) siRNA-LPs for 2 weeks (doses of 0.4 and 0.8mg siRNA per kg BW). 24h after the last injection liver collagen was quantified biochemically as hydroxyproline (Hyp) and in liver sections using Sirius red morphometry. Fibrosis related transcript levels were determined by qRT-PCR. Results: 90% of DiR-labeled C12–200 LPs were found in the liver 30 min after injection, in colocalization with hepatocytes>macrophages>myofibroblastic cells. Compared to the GFP control, procollagen a1(I) siRNA-LPs significantly suppressed the expression of its target gene in the fibrotic liver by 80%, which was accompanied by a significant reduction of collagen deposition by 25%. Parameters of hepatic inflammation (ALT and AST) and kidney function (creatinine) remained unchanged after siRNA-LPs injection, indicating the absence of proinflammatory or renal toxic side effects. Conclusion: C12–200 LPs are a specific and safe vehicle to deliver siRNAs to the liver with high efficiency. C12–200 LPs loaded with siRNA to fibrogenic transcripts are a promising approach to efficiently inhibit liver fibrosis progression in vivo.