Targeting Kupffer Cells With Short-Interfering RNA

描述（由申请人提供）：酒精性肝病（ALD）影响着美国约20%的酗酒者。据报道，酒精性肝硬化患者内毒素水平升高。在动物模型系统中，已经证明酒精介导的肝损伤被内毒素（脂多糖）大大增强，内毒素是炎症细胞因子如肿瘤坏死因子α (tnf - α)、白细胞介素（IL-1、IL-6、IL-8）和前列腺素的有效诱导性因子。在这个应用中，我们建议测试最近发现的新一代高度序列特异性基因沉默分子siRNA（短干扰RNA）对tnf - α产生的功效。在我们的第一个目标中，针对tnf - α mRNA特定区域的21碱基对双链sirna的功效将在大鼠Kupffer细胞的原代培养中进行测试。sirna的有效性将通过测量转染Kupffer细胞后脂多糖诱导的tnf - α产生的抑制程度来评估。在鉴定出高效siRNA构建体后，1-4个碱基对错配的siRNA的效率将为

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.