Artificial microRNA mediated Tgfbr2 and Pdgfrb co-silencing ameliorates CCl4-induced hepatic fibrosis in mice.

Hepatic stellate cell (HSC) is the primary cell type responsible for liver fibrogenesis. TGF-β1 and PDGF are key profibrotic cytokines that regulate HSCs activation and proliferation with functional convergence. Dual RNA interference against their receptors may achieve therapeutic effects. We have devised a novel RNAi strategy based on HSCs specific GFAP promoter driven and lentivirally expressed artificial microRNAs (amiRNAs) that consist of microRNA-30a backbone and effective shRNAs against mouse Pdgfrβ and Tgfbr2, and tested their antifibrotic efficacy in primary and cultured HSCs and in mice affected with CCl4-induced hepatic fibrosis. Here we show that amiRNA-mediated Pdgfrβ and Tgfbr2 co-silencing inhibits HSCs activation and proliferation. After the recombinant lentivirus particles were delivered into liver through tail vein injection, the therapeutic amiRNAs are preferentially expressed in HSCs, efficiently co-knockdown the expressions in situ of Tgfbr2 and Pdgfrβ, which correlates with down-regulated expression of target or effector genes of their signaling such as Pai-1, P70S6K and D-cyclins. The amiRNA-based HSCs-specific co-silencing of Tgfbr2 and Pdgfrβ significantly suppresses hepatic expressions of fibrotic markers α-Sma and Col1a1, extracellular matrix regulators Mmps and Timp1, and phenotypically ameliorates liver fibrosis as evidenced by remarkable reductions in serum alanine aminotransferase activity, collagen deposition, and strength of α-Sma-positive staining. Our findings provide a proof-of-concept for the use of amiRNA-mediated co-silencing of two profibrogenic pathways in liver fibrosis treatment, and highlight therapeutic potential of concatenated amiRNAs in gene therapy.