Fibrosis leads to organ dysfunction and is characterized by an excessive production of extracellular matrix (ECM) components, namely type I and III collagens. Although activated hepatic stellate cell (HSC) remains the principal cell type responsible for liver fibrosis, other liver cell type of fibroblast lineage derived from portal and centrolobular veins also have fibrogenic potential. The overall hypothesis is that liver fibrosis can be treated by systemic administration of a1(I) collagen gene promoter specific triplex forming oligonucleotides (TFOs) conjugated with mannose 6-phosphate-bovine serum albumin (M6P-BSA) via a disulfide bond. In preliminary studies, antiparallel phosphorothioate polypurine TFOs specific for CI region formed triplexes, inhibited transcription of a1(I) collagen promoter and improved rat liver fibrosis. TFOs rapidly distributed throughout the body after systemic administration, with the highest accumulation in the liver. TFO accumulation in the liver was decreased when injected into liver fibrotic rats. Kupffer, sinusoidal endothelial and hepatic stellate cells accounted for approximately 70% of the liver uptake, and the remaining 30% in the hepatocytes. Bioconjugation with M6P-BSA significantly enhanced the cellular uptake of the TFOs by HSC-T6 cells in vitro, leading to enhanced inhibition of type a1(I) collagen transcription. TFO delivery to the liver and to the HSCs was significantly increased when M6P-BSA-TFO was injected intravenously into fibrotic rats.
Our specific aims are to determine whether i) conjugation of targeting ligands to TFOs affect triplex formation;ii) TFOs inhibit fibrosis by inhibiting transcription of a1(I) collagen and/or blocking inflammation and activation of liver fibrogenic cells;and iii) M6P-BSA-TFO can be delivered efficiently to liver fibrogenic cells and prevent fibrosis. The significance of this research is that the proposed targeted TFO delivery to liver fibrogenic cells will inhibit disproportionate accumulation of a1(I) collagen, which is essential for the treatment of liver fibrosis. The data will also be beneficial to successful treatment of other organ fibrosis.
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