In vivo delivery of siRNA to key transcipts of fibrogenesis in lipid-like carriers inhibits development liver fibrosis

Introduction: Antifibrotic therapies have to be specific (on target), efficient and free of major side effects. C12–200 lipid-like particles (LPs) consisting of epoxide terminated alkyl chains and secondary amine monomers permit efficient delivery of siRNA, minimizing interference with blood cells and plasma and being devoid of side effects. Intravenous C12–200 LPs allow efficient and safe delivery of small interfering RNAs (siRNAs) to the liver (Akinc et al, Nat Biotechnol 2008).

Methods: Optimized siRNAs targeting the fibrogenic genes procollagen α1(I), TGFß1 and Smad2/3 were linked to C12–200 LPs. siGFP (green fluorescent protein)-LPs served as negative controls. Groups of 8 mice were treated with CCl4 by oral gavage two times at days 1 and 4, and LPs were injected i.v. at 0.1mg/kg BW at day 8. After 24h liver collagen content was quantified biochemically (hydroxyproline, Hyp), collagen on liver sections was assessed using Sirius red morphometry, and fibrosis related transcript levels were determined by RT-PCR.

Results: Compared to the GFP controls, procollagen α1(I)-, TGFß1- and Smad2/3 siRNA-LPs significantly suppressed the expression of their respective target genes by 62%, 34% and 25%, respectively. C12–200-siTGFß1 also significantly downregulated the expression of procollagen α1(I) mRNA by 56%. The 2-fold increased deposition of total collagen was significantly reduced by 58%, 75% and 73% after treatment with procollagen α1(I)-, TGFß1- and Smad2/3 siRNA-LPs, respectively. Parameters of hepatic inflammation (ALT and AST) remained unchanged in all treatment groups compared to siGFP-LPs or buffer treated CCl4-fibrotic controls, indicating the absence of proinflammatory side effects.

Conclusions: These are the first results demonstrating efficient in vivo knockdown of profibrogenic genes using optimized siRNA-LPs with liver specific delivery, resulting in a significant reduction of liver fibrosis. These Lipid-like particles may be lead to a promising, liver specific and on-target antifibrotic treatment in which different targets can be combined without increased side effects.