Knocking Down Human Disease: Potential Uses of RNA Interference in Research and Gene Therapy

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A review of: Song E, Lee S-K, Wang J, Ince N, Ouyang N, min J, Chen J, Shankar P, Lieberman J 2003 RNA interference targeting Fas protects mice from fulminant hepatitis. Nat Med 9:347–351; and McCaffrey A, Nakai H, Pandey K, Huang Z, Salazar FH, Xu H, Wieland SF, Marion PL, Kay MA 2003 Nat Biotechnol 21:639–644

RNA interference (RNAi) has recently been quoted as being the “hottest topic in science.” The number of papers published on RNAi has increased over the past several years and the journal Science proclaimed it as the biggest scientific breakthrough of 2002. The importance of this discovery was based on the ability to study the function of a gene by switching it off easily in almost any organism. The clinical implications were that this tool could potentially be used to treat diseases, such as cancer or AIDS, by downregulating the genes that caused the disease (1).

RNAi is defined as sequence-specific posttranscriptional gene silencing by double-stranded RNA (dsRNA). This dsRNA is conserved throughout evolution, including plants, Neurospora, Drosophila, Caenorhabditis elegans, and mammals (2). RNAi is able to knockdown gene expression by small-interfering RNAs(siRNAs) that are produced from long dsRNAs of exogenous or endogenous origin by a ribonuclease-III type endonuclease, called Dicer. The siRNAs are about 21–23 nucleotides in length and are incorporated into a nuclease complex, the RNA-induced silencing complex (RISC), which then targets and cleaves mRNA that is complementary to the siRNA (3). RNAi is similar to anti-sense, since both act posttranscriptionally to reduce the level of the target protein. However, RNAi is more powerful than antisenseoligonucleotides and provides more effective knockdown of its targets at lower concentrations than with antisense oligonucleotides (4).

In order for RNAi to be a useful tool in research and therapy, the siRNA-mediated transcriptional silencing must be specific. Two groups have used global gene expression profiling to demonstrate that siRNA is highly specific without secondary effects (5, 6). However, Jackson et al demonstrated that there are off-target genes that were silenced and that transcript expression patterns were siRNA-specific rather than target-specific. Up to this point, studies suggest that siRNA-mediated downregulation of genes is reliable and a potentially powerful tool for understanding gene function.

The difficulty in using RNAi in mammalian cells is that siRNA-induced gene inactivation appears to be transient. In comparison to worms and plants, mammals have been thought to lack mechanisms that amplify silencing (4). To circumvent this problem, there are several plasmid vector systems that have been developed to produce short-hairpin RNAs (shRNAs) in cells that are driven by promoters dependent on RNA polymerase III (PolIII), such as U6 and H1, or on Pol II. The Pol III promoters are active in all mammalian tissues (7, 8). Kunath and colleagues demonstrated that treatment of embryonic stem cells with shRNA directed against RasGAP had the same phenotype as did the previously reported null mutation generated through homologous recombination (9). Recently, improved vectors have been designed to inducibly express shRNAs under the control of a Tet operon or hormone receptor, allowing for temporal silencing of normally lethal knockouts and analysis of gene function over a certain period of time (10).

Two landmark articles demonstrated the potential for RNAi to be used in the treatment of human disease. In one study, Song and colleagues targeted the cell surface Fas receptor using siRNA in mouse models of autoimmune hepatitis (11). Fas mediates cell death and is a key regulator of hepatocellular damage and inflammation. Duplex siRNAs directed against Fas resulted in specific downregulation of Fas mRNA in cells and in vivo. Serum alanine and aspartate aminotransferase levels were dramatically lower in the lectin-treated animals that received Fas-specific siRNA compared to control siRNAs. In addition, fibrosis was reduced in models of chronic hepatitis. Mice were protected when the Fas-specific siRNA was given after initiation of hepatic damage by lectin, suggesting that Fas siRNA may be useful in treating autoimmune hepatitis. This was the first indication that duplex siR-NAs might be effective clinically. There may be broader implications for Fas siRNA in both acute and chronic liver disease (12).

In the second study, McCaffrey and colleagues used shRNAs specific to hepatitis B virus (HBV) RNAs (13). The same group previously demonstrated that siRNA and shRNAs were powerful inhibitors of gene expression in adult mice (14). Hepatitis B infections can lead to chronic liver disease and hepatocellular carcinoma. Co-transfection experiments with HBV and shRNA plasmids in both immuno-deficient and competent mice demonstrated that shRNAs targeting viral sequences could inhibit viral replication and production of core antigen in hepatocytes. ShRNA treatment also reduced serum levels of hepatitis B surface antigen. Since this study employed an artificial method to initiate viral infections, the next question is whether RNAi can inhibit an authentic viral infection.

These proof of concept experiments support the utility of RNAi as a therapeutic modality. Recently, another group of RNA molecules known as microRNAs (miRNAs) were shown to modulate hematopoietic lineage differentiation (15). MiRNAs are an abundant class of 22 nucleotide regulatory RNAs that pair to target mRNAs and specify posttranscriptional repression of these messages. Thus, miRNAs appear to represent another layer of molecular regulation during vertebrate development.

Despite promising data, there are several challenges that need to be faced before RNAi can be used in patients. These include mode of delivery, the precise sequence of the siRNA or shRNA used, and cell type specificity. There are possible toxicities related to silencing of partially homologous genes or induction of global gene suppression by activating the interferon response. Another potential problem is the inhibition of the function of endogenous miRNAs through competition for the RNAi machinery. Despite these hurdles, RNAi provides the opportunity to pursue an exciting new therapeutic approach to treat infections, cancer, neurodegenerative diseases, and other illnesses.


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