Targeting Kupffer Cells With Short-Interfering RNA

描述（由申请人提供）：酒精性肝病（ALD）影响了美国约20%的酗酒者.酒精性肝硬化患者体内内毒素水平升高已被报道。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，内毒素是炎性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）和前列腺素类的有效诱导剂。在本申请中，提出测试最近鉴定的新一代高度序列特异性基因沉默分子（称为siRNA（短干扰RNA））针对TNF-α产生的功效。在我们的第一个目标中，将在大鼠枯否细胞的原代培养物中测试靶向TNF-α mRNA的限定区域的21个碱基对双链siRNA的功效。通过测量用siRNA转染枯否细胞后脂多糖诱导的TNF-α产生的抑制程度来评价siRNA的效力。在鉴定出具有高功效的siRNA构建体之后，将评估具有1-4个碱基对错配的siRNA的功效。 类似地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siRNA的体内功效。 对于体内研究，将高效siRNA构建体包封在pH敏感性脂质体中，并静脉内注射用于隔离，主要通过枯否细胞和其他巨噬细胞。静脉内注射后，以预定的间隔（1、2、3和5天），通过测量构建体阻断脂多糖诱导的细胞因子（TNF-α）产生的抑制能力来确定siRNA构建体的体内效力。siRNA的体内功效将通过测量TNF-α的mRNA水平来进一步评估。 由于siRNA构建体将使用pH敏感性脂质体递送，所述脂质体被设计成使内体膜不稳定，因此预期相对低剂量的siRNA将足以在库普弗细胞/巨噬细胞螯合后将显著量的siRNA递送至胞质溶胶。预期脂质体系统将比在不存在递送载体的情况下更有效地将siRNA递送至库普弗细胞至少5-10倍。 这项研究的成功结果将大大提高我们使用非常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.