siRNAs Knock Down Hepatitis







Phillip D. Zamore, Neil Aronin







The power of small RNAs to shut down specific gene activities has now been brought to bear on an animal model of hepatitis. Mice infused with an siRNA against a cell death receptor recover liver function after experimentally induced injury (pages 347-351).



Successful drugs have high specificity for their molecular targets. In theory, nucleic acids, whose base-pairing interactions are highly specific, could be effective drugs. The challenge has been to create artificial nucleic acids, such as antisense oligonucleotides and ribozymes, that reach cells in adequate amounts to regulate specific gene function, yet are nontoxic. Efforts to date have focused on altering the phosphodiester linkages or modifying the ribose sugar to improve the stability, efficacy or cellular uptake of nucleic acids.



The work of Song et al. in this issue suggests that one type of entirely natural nucleic acid, small interfering RNAs (siRNAs), may hold promise as a therapeutic agent even without further engineering. These investigators provide the first in vivo evidence that infusion of siRNAs into an animal can alleviate disease, in this case hepatitis.



siRNAs are double-stranded RNA (dsRNA) molecules of 21-23 nucleotides with characteristic 2-nucleotide over hanging 3' ends . They act as intermediates in the RNA interference (RNAi) pathway, which is thought to protect cells from harmful transposons and highly repetitive sequences by targeting their RNA transcripts for endonucleolytic cleavage and subsequent exonucleolytic degradation (Figure 1). In culture, exogenously added siRNAs can protect mammalian cells from infection by a variety of viruses , but it is not yet known whether RNAi is a component of the mammalian antiviral response. In contrast, siRNA-directed RNA degradation is central to the antiviral response in plants, where it represents a potent form of sequence-based immunity.



The ribonuclease Dicer generates siRNAs by cleaving long dsRNAs. In siRNA-directed RNA interference, the two strands of an siRNA separate in a process probably mediated by specific protein 'helicases,' after which the individual siRNA strands associate with another set of proteins to form a protein-enzyme complex -- the RNA-induced silencing complex (RISC). The RISC has the remarkable property of using the sequence of its tightly bound siRNA strand to direct the complex to mRNAs with perfect or nearly perfect complementarity. Tethered to the mRNA by its siRNA guide, the RISC destroys the mRNA by cutting it once, across from the centerof the siRNA. Cleavage completed, the RISC departs, with the unperturbed siRNA ready to begin anew the cycle of target recognition and cleavage.



The power of siRNAs springs from the cellular biochemistry of the RNAi pathway. Like antisense oligonucleotides, siRNAs use sequence complementarity to target an mRNA for destruction. Unlike the antisense pathway, the RNAi pathway couples the specificity of an RNA guide to the stability and efficiency of a multiple-turnover protein enzyme.



Assembly of an siRNA strand into a RISC seems to protect it from rapid degradation, the normal fate of small single-stranded RNA in cells. With this durability in mind, Song et al. set out to test whether direct infusion of siRNAs into mice might protect them from fulminant hepatitis. Both mice and humans with this disease suffer severe hepatic failure, with consequent encephalopathy, cerebral edema, metabolic imbalance and organ collapse. Fulminant hepatitis leads to death in over two-thirds of patients who do not receive liver transplants.



Song et al. chose as their target a cell surface receptor, Fas, required for cell death in the liver. Fas mediates cell death by both apoptotic and necrotic pathways . A variety of experimental insults can trigger Fas-dependent hepatic cell death in mice. For example, exposure to concanavalin A triggers cell death and fulminant hepatitis.



It is not surprising, then, that blocking Fas expression or function can prevent death of liver cells, a finding borne out by several studies. An antisense oligonucleotide directed against Fas mRNA can abrogate fulminant hepatitis caused by agonistic Fas-specific monoclonal antibodies and mitigate acetaminophen-induced hepatitis . Antibody neutralization of Fas ligand alleviates hepatitis B-induced chronic hepatitis in mice . In addition, patients with fulminant hepatitis have higher levels of hepatic Fas expression and circulating soluble Fas ligand . Together, these data suggest that reducing Fas will provide therapeutic benefit in humans.



Song et al. pretreated mice with Fas siRNA and then induced fulminant hepatitis by exposing the mice to concanavalin A or agonistic Fas-specific antibodies. They found that siRNA treatment blocked the development of fulminant hepatitis and improved survival -- even if siRNA was delivered after initiating hepatic insult. Thus, reduction of Fas expression early in the course of hepatitis could prevent its progression to more virulent disease.



In these studies, Fas expression returned to normal levels 20 days after siRNA treatment. This finding offers encouragement that siRNA directed to the Fas gene might be used safely, as Fas-mediated apoptosis regulates the production of lymphocytes. Mice lacking either Fas or Fas ligand develop a lymphoproliferative syndrome, and patients with dominant-negative Fas mutations have autoimmune lymphoproliferative disease . Long-term silencing of Fas could therefore have deleterious consequences, and the risks of even a temporary reduction in Fas will need to be monitored.



As therapeutic agents, siRNAs have enticing properties. Their actions appear to be short-lived in mammals; they are sequence specific; and they are natural, cellular products and may therefore not produce toxic metabolites. Nonetheless, caveats for clinical use remain. Delivering siRNAs to the appropriate cells is a major challenge. siRNAs have thus far only been administered intravenously to mice by 'hydrodynamic transfection,' the rapid infusion of siRNA in a volume one-tenth the mass of the animal. Furthermore, the liver seems to be particularly receptive to exogenous RNA. Better delivery methods -- such as formulation of siRNAs with compounds that promote transit across cell membranes -- are clearly required before siRNAs can be used in therapy, especially to suppress gene expression in tissues other than the liver. Nonetheless, the results of Song et al. begin to reveal the power of siRNAs in a disease model.