Artificial Microrna-Mediated Tgfbr2 And Pdgfrb Co-Silencing Ameliorates Carbon Tetrachloride-Induced Hepatic Fibrosis In Mice

Hepatic stellate cells (HSCs) are the primary cell type responsible for liver fibrogenesis. Transforming growth factor beta 1 (TGF-1) and platelet-derived growth factor (PDGF) are key profibrotic cytokines that regulate HSC activation and proliferation with functional convergence. Dual RNA interference against their receptors may achieve therapeutic effects. A novel RNAi strategy based on HSC-specific GFAP promoter-driven and lentiviral-expressed artificial microRNAs (amiRNAs) was devised that consists of an microRNA-30a backbone and effective shRNAs against mouse Pdgfr and Tgfbr2. Then, its antifibrotic efficacy was tested in primary and cultured HSCs and in mice affected with carbon tetrachloride-induced hepatic fibrosis. The study shows that amiRNA-mediated Pdgfr and Tgfbr2 co-silencing inhibits HSC activation and proliferation. After recombinant lentiviral particles were delivered into the liver via tail-vein injection, therapeutic amiRNAs were preferentially expressed in HSCs and efficiently co-knocked down in situTgfbr2 and Pdgfr expression, which correlates with downregulated expression of target or effector genes of their signaling, which include Pai-1, P70S6K, and D-cyclins. amiRNA-based HSC-specific co-silencing of Tgfbr2 and Pdgfr significantly suppressed hepatic expression of fibrotic markers -Sma and Col1a1, extracellular matrix regulators Mmps and Timp1, and phenotypically ameliorated liver fibrosis, as indicated by reductions in serum alanine aminotransferase activity, collagen deposition, and -Sma-positive staining. The findings provide proof of concept for the use of amiRNA-mediated co-silencing of two profibrogenic pathways in liver fibrosis treatment and highlight the therapeutic potential of concatenated amiRNAs for gene therapy.