Targeted Delivery of TFOs for Treatment of Liver Fibrosis

描述（由申请人提供）：纤维化导致器官功能障碍，其特征在于细胞外基质（ECM）组分（即I型和III型胶原）的过量产生。虽然活化的肝星状细胞（HSC）仍然是负责肝纤维化的主要细胞类型，但来自门静脉和小叶中心静脉的成纤维细胞谱系的其他肝细胞类型也具有纤维化潜力。总的假设是，肝纤维化可以通过全身施用α 1（I）胶原基因启动子特异性三链体形成寡核苷酸（TFO）来治疗，所述寡核苷酸通过二硫键与甘露糖6-磷酸-牛血清白蛋白（M6 P-BSA）缀合。在初步研究中，对CI区特异性的反平行硫代磷酸多嘌呤TFO形成三链体，抑制α 1（I）胶原启动子的转录并改善大鼠肝纤维化。全身给药后，TFO迅速分布于全身，在肝脏中蓄积最高。当注射到肝纤维化大鼠中时，TFO在肝脏中的积累减少。枯否细胞、肝窦内皮细胞和肝星状细胞约占肝脏摄取的70%，其余30%在肝细胞中。与M6 P-BSA的生物偶联显著增强了体外HSC-T6细胞对TFO的细胞摄取，导致增强了对a1（I）型胶原转录的抑制。当将M6 P-BSA-TFO静脉内注射到纤维化大鼠中时，TFO向肝脏和HSC的递送显著增加。我们的具体目标是确定i）靶向配体与TFO的缀合是否影响三链体形成; ii）TFO通过抑制a1（I）胶原蛋白的转录和/或阻断炎症和肝纤维化细胞的活化来抑制纤维化;和iii）M6 P-BSA-TFO可以有效地递送到肝纤维化细胞并预防纤维化。这项研究的意义在于，所提出的靶向TFO递送至肝纤维化细胞将抑制a1（I）胶原蛋白的不成比例积累，这对于治疗肝纤维化至关重要。这些数据也将有利于其他器官纤维化的成功治疗。

DESCRIPTION (provided by applicant): 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.