Targeted Delivery of TFOs for Treatment of Liver Fibrosis

描述(申请人提供)：纤维化导致器官功能障碍，其特征是过度产生细胞外基质(ECM)成分，即I型和III型胶原。尽管活化的肝星状细胞(HSC)仍然是导致肝纤维化的主要细胞类型，但来源于门静脉和中心小叶静脉的其他类型的肝细胞系也具有致纤维化的潜力。总的假设是，肝纤维化可以通过系统地给予A1(I)胶原基因启动子特异性的三链形成寡核苷酸(TFOS)与甘露糖6-磷酸-牛血清白蛋白(M6P-BSA)通过二硫键连接来治疗。在初步研究中，针对CI区的反平行硫代多嘌呤TFOS形成三链，抑制A1(I)胶原启动子的转录，改善大鼠肝纤维化。全身给药后，TFOS在体内迅速分布，在肝脏中蓄积最多。肝纤维化大鼠注射TFO后，肝组织中TFO蓄积减少。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%来自肝细胞。M6P-BSA生物偶联可显著增强HSC-T6细胞对TFOS的摄取，从而增强对A1(I)型胶原转录的抑制作用。肝纤维化大鼠静脉注射M6P-BSA-TFO后，TFO向肝脏和HSCs的转运明显增加。我们的具体目标是确定i)靶向配体与TFOS的结合是否影响三链的形成；ii)TFOS通过抑制A1(I)胶原的转录和/或阻断肝纤维化细胞的炎症和激活而抑制纤维化；以及iii)M6P-BSA-TFO能够有效地输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传递到肝纤维化细胞将抑制A1(I)胶原的不成比例积累，而A1(I)胶原是治疗肝纤维化所必需的。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.