A Systemically Deliverable RNAi-based Approach for Alcoholic Liver Disease

描述（由申请人提供）：长期饮酒可导致进行性肝损伤，导致脂肪性肝炎和肝硬化，这可能导致其他更致命的并发症，如肝细胞癌（HCC）。酒精性肝病（ALD）的进展，像许多慢性形式的肝脏疾病，是由肝细胞损伤，促进炎症反应和肝星状细胞（HSC）的活化成基质生产肌成纤维细胞（MF）驱动的。我们的合作者已经表明，Hedgehog（Hh）信号通路的激活在受损肝脏如何应对急性和慢性损伤（包括ALD）中起着关键作用。在受损肝脏中产生基质的MF的活化被认为是慢性肝病进展为肝硬化的关键步骤，并且是一个在肝硬化中调节的过程。 部分通过Hh信令。现在有令人信服的证据表明，胚胎发育Hh通路在成人肝损伤期间重新激活，并在伤口愈合反应中起关键作用。Hh通路的失调发生在肝硬化和许多原发性肝癌中。研究表明，Smoothened基因（SMO）（Hh通路的一个组成部分）的拮抗剂具有治疗脂肪肝和酒精诱导的肝硬化的潜力。在该提案中，将探索SMO的RNA沉默作为治疗酒精性肝脏病的高度特异性疗法。虽然有几家公司正试图利用RNA干扰（RNAi）过程来创造有效和特异性的治疗方法，但该领域进展的关键障碍是系统性地提供持久的基于RNAi的治疗方法的能力。目前，siRNA通过静脉内（IV）注射，如果不与潜在毒性和免疫原性脂质包封载体一起配制，则它们从体内快速清除。在这项提议中，我们将我们的新型载体技术应用于一类称为sdRNA的新型siRNA，这种siRNA可以有效地沉默基因，并且能够在没有制剂的情况下穿透细胞。我们的载体分子，当与肽和蛋白质结合时，通过防止肾脏清除和增加皮下（SC）生物利用度，显著改善了药代动力学（PK）特性。使用这种载体与sdRNA可以允许它们是长效的，并且具有SC而不是IV递送的潜力。如果成功的话，这将是真正使这类药物。在本文提出的研究中，将鉴定对SMO特异性的修饰的sdRNA，将其与载体缀合，然后在体外进行检查，以确认它们的细胞穿透和沉默特性在缀合后没有改变。然后将在大鼠的PK研究中测试sdRNA缀合物的半衰期和生物利用度。我们预计这些sdRNA缀合物现在将是长效的，并且能够全身递送，可能是SC。最后，将在ALD的动物模型中测试sdRNA缀合物的功效。该I期提案旨在证明SMO特异性sdRNA缀合物作为治疗酒精性肝病的方法的可行性。然后，基于载体的技术可以应用于其他类别的寡核苷酸疗法，并且可以是用于体内功能基因组和靶点验证研究的一般方法。

DESCRIPTION (provided by applicant): Chronic alcohol consumption can cause progressive liver damage, resulting in steatohepatitis and cirrhosis, which can lead to other more fatal complications such as hepatocellular carcinoma (HCC). Progression of alcoholic liver disease (ALD), like many chronic forms of liver disease, is driven by hepatocyte damage that promotes inflammatory responses and the activation of hepatic stellate cells (HSC) into matrix-producing myofibroblasts (MF). Our collaborator has shown that activation of the Hedgehog (Hh) signaling pathway plays a critical role in how damaged livers respond to acute and chronic injuries, including ALD. Activation of matrix- producing MF in damaged livers is considered the critical step in the progression of chronic liver disease to cirrhosis and is a process that is regulated in part by Hh signaling. There is now compelling evidence that the embryonic developmental Hh pathway becomes reactivated during adult liver injury and plays a key role in the wound healing response. Deregulation of the Hh pathway occurs during cirrhosis and in many primary liver cancers. Studies have shown that antagonists of the Smoothened gene (SMO), a component of the Hh pathway, have the potential to treat fatty liver disease and alcohol-induced cirrhosis. In this proposal, RNA silencing of SMO will be explored as a highly specific therapy for ALD. While several companies are attempting to harness the RNA interference (RNAi) process to create potent and specific therapeutics, the critical barrier to progress in the field is the ability to systemically deliver long-lasting RNAi-based therapeutics. Currently, siRNAs are injected intravenously (IV) where they undergo rapid clearance from the body if not formulated with potentially toxic and immunogenic lipid-encapsulating vehicles. In this proposal, we apply our novel carrier technology to a new class of siRNA called sdRNAs, which can efficiently silence genes and are capable of penetrating cells without formulation. Our carrier molecule, when conjugated to peptides and proteins, significantly improved pharmacokinetic (PK) properties by preventing kidney clearance and increasing subcutaneous (SC) bioavailability. Use of this carrier with sdRNAs may allow them to be long-acting and have the potential to be delivered SC rather than IV. If successful, this would be truly enabling for this drug class. In the studies proposed here, modified sdRNAs specific for SMO will be identified, conjugated to the carrier, then examined in vitro to confirm that their cell-penetrating and silencing properties have not been altered following conjugation. The half-life and bioavailability of the sdRNA conjugates will then be tested in a PK study in rats. We expect that these sdRNA conjugates will now be long-acting and able to be systemically delivered, potentially SC. Finally, the sdRNA conjugates will be tested for efficacy in an animal model of ALD. This Phase I proposal aims to demonstrate the feasibility of SMO-specific sdRNA conjugates as an approach to treat alcoholic liver disease. The carrier-based technology can then be applied to other classes of oligonucleotide therapeutics and could be a general approach for in vivo functional genomic and target validation studies.