Targeting Kupffer Cells With Short-Interfering RNA

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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.