Inhibition of NF-kB with siRNA/Peptide Nanoparticles for Arthritis Treatment

 描述(申请人提供)：核因子-KB信号通路代表了一个有吸引力的治疗慢性炎症的靶点，因为它调节许多控制炎症反应的基因产物。然而，广泛抑制这一途径可能会导致严重的宿主毒性，因为核因子-KB是天然免疫和获得性免疫的主要介体。我们基于两亲性阳离子多肽蜂毒素的改良版本，设计、表征和评估了一种新型的肽-siRNA结构，它具有自我凝聚、在血清中稳定的特点，并将寡核苷酸输送到细胞质中，具有内在的内体释放机制。蜂毒素是蜂毒的一种成分，通常通过齐聚在膜上形成孔。我们发现，修饰后的蜂毒素可以与多种物质结合，并迅速将物质输送到细胞质中，以实现有效的基因沉默：多肽-siRNA。 针对核因子-kB p65规范亚单位的复合体通过泄漏的血管系统定位于炎症的脚掌，并有效地抑制类风湿关节炎小鼠模型中正在进行的炎症。大量的体内和体外研究也表明，该平台具有良好的毒性特征，在重复给药后没有诱导免疫原性。在这项提案中，我们将使用多肽-siRNA平台来探索这样的假设，即双重方法(针对NF-KB规范途径和非规范途径)将在RA的治疗中具有协同或相加的好处。为此，我们将构建和优化包含NF-KB-P65(规范)、P52/100(非规范)或两者组合的多肽-siRNA构建物，并在体外人/鼠细胞系和两种炎症性关节炎小鼠模型中测试它们对NF-KB信号通路敲除的效率。这一结果将为这一点提供活体概念验证 新的多肽-siRNA平台的成功将证明该系统将进一步开发用于其他疾病过程的适合RNA干扰治疗的系统。

 DESCRIPTION (provided by applicant): The NF-KB signaling pathway represents an attractive therapeutic target for chronic inflammation as it regulates many gene products that control the inflammatory response. However, generalized suppression of this pathway can result in serious host toxicity, as NF-KB is a major mediator of both innate and adaptive immunity. We have formulated, characterized, and evaluated a novel peptide-siRNA construct that is self-condensing, stable in serum, and delivers oligonucleotides to the cell cytoplasm with an intrinsic endosomal releasing mechanism based on a modified version of the amphipathic cationic peptide melittin, a component of bee venom that normally forms pores in membrane by oligomerization. We showed that the modified melittin could be conjugated to myriad cargos and transport materials rapidly into the cell cytoplasm for efficient gene silencing: The peptide-siRNA complexes targeting the NF-KB p65 canonical subunit homed to the inflamed paws through leaky vasculature and potently suppressed ongoing inflammation in a murine model of RA. Extensive in vivo and ex vivo studies also suggest that the platform has a favorable toxicity profile and did not elicit immunogenicity after repeated dosing. In this proposal we will use the peptide-siRNA platform to explore the hypothesis that a dual approach (targeting both NF-KB canonical and non-canonical pathways) will have synergistic or additive benefits in the treatment of RA. To that end we will formulate and optimize peptide- siRNA constructs containing NF-KB-p65 (canonical), p52/100 (non-canonical), or a combination of both and test their efficiency for NF-KB signaling pathway knockdown in vitro in human/murine cell lines and in vivo in two murine models of inflammatory arthritis. The results will provide in vivo proof-of-concept for this new peptide- siRNA platform and the success will justify further development of this system for other disease processes that are amenable to RNA interference therapy.