Targeting Kupffer Cells With Short-Interfering RNA

描述(申请人提供)：酒精性肝病(ALD)影响美国约20%的酗酒者。据报道，在美国循环中，酒精性肝硬变患者的内毒素水平升高。在动物模型系统中，已证明内毒素(脂多糖)极大地加强了酒精介导的肝损伤，内毒素是炎性细胞因子如肿瘤坏死因子α(TNF-α)、白介素IL-1、IL-6、IL-8和前列腺素的有效诱导者。在这项应用中，有人提议测试最近发现的新一代高度序列特异性的基因沉默分子siRNA(短干扰RNA)对抗肿瘤坏死因子-α产生的有效性。在我们的第一个目标中，将在大鼠Kupffer细胞的原代培养中测试针对肿瘤坏死因子-αmRNA特定区域的21碱基对双链siRNAs的有效性。将siRNAs导入Kupffer细胞后，通过测量脂多糖诱导的肿瘤坏死因子-α产生的抑制程度来评估siRNAs的效果。在鉴定出高效的siRNA结构后，具有1-4个碱基对错配的siRNA的效率将为 进行类似的测试以确定序列特异性。随后，在我们的第二个目标中，将确定最有效的siRNAs的体内有效性。 对于体内研究，高效的siRNA构建体将被包裹在对pH敏感的脂质体中，并通过静脉注射进行隔离，主要由Kupffer细胞和其他巨噬细胞进行。静脉注射后，在预定的间隔(1、2、3和5天)，将通过测量构建体阻断内毒素诱导的细胞因子(TNF-α)产生的抑制能力来确定siRNA构建体的体内有效性。通过测量肿瘤坏死因子-α的信使核糖核酸水平将进一步评估siRNAs的体内有效性。 由于siRNA构建物将使用设计用于破坏内体膜稳定性的pH敏感脂质体递送，因此预计相对较低剂量的siRNA将足以在Kupffer细胞/巨噬细胞隔离后将药理上显著数量的siRNA递送到细胞质。预计脂质体系统向Kupffer细胞运送siRNA的效率至少是在没有载体的情况下的5-10倍。 这项研究的成功结果将极大地增强我们使用极低浓度的基因特异性分子治疗酒精性肝病等疾病的能力。

DESCRIPTION (provided by applicant): Alcoholic liver disease (ALD) affects about 20% of the alcoholics the States. Circulating in United Increased levels of endotoxin have been reported in patients with alcoholic cirrhosis. In animal model systems, it has been demonstrated that alcohol-mediated liver injury is greatly potentiated by endotoxins (lipopolysaccharides), that are potent inducers of inflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha), interleukins (IL-1, IL-6, IL-8) and prostanoids. In this application, it is proposed to test the efficacy of a recently identified new generation of a highly sequence-specific gene-silencing molecule called siRNA (short interfering RNA) against TNF-alpha production. In our first objective, the efficacy of 21-base-pair double-stranded siRNAs targeted to defined regions of TNF-alpha mRNA will be tested in primary cultures of rat Kupffer cells. Efficacies of siRNAs will be evaluated by measuring the extent of inhibition of lipopolysaccharide-induced production of TNF-alpha following transfection of Kupffer cells with siRNAs. After identifying siRNA constructs with high efficacy, the efficacies of siRNAs with 1-4 base-pair mismatches will be tested similarly to ascertain sequence-specificity. Subsequently, in our second objective, the in vivo efficacy of the most effective siRNAs will be determined. For in vivo studies, highly efficacious siRNA constructs will be encapsulated in pH-sensitive liposomes and injected intravenously for sequestration, primarily by Kupffer cells and other macrophages. After the intravenous injection, at predetermined intervals (1, 2, 3 and 5 days) the in vivo efficacies of the siRNA constructs will be determined by measuring the inhibitory ability of the constructs to block lipopolysaccharide-induced production of the cytokine (TNF-alpha). The in vivo efficacies of siRNAs will be further assessed by measuring the mRNA levels of TNF-alpha. Since the siRNA constructs will be delivered using pH-sensitive liposomes, which are designed to destabilize the endosomal membrane, it is expected that relatively low doses of siRNAs will be sufficient to deliver pharmacologically significant amounts of the siRNAs to the cytosol following sequestration by Kupffer cells/macrophages. It is expected that the liposomal system will deliver siRNAs at least 5-10 times more efficiently to Kupffer cells than in the absence of a delivery vehicle. A successful outcome of this study will greatly enhance our ability to use very low concentrations of gene-specific molecules for the treatment of disease such as alcoholic liver disease.