Infectious Diseases

The first approach aiming at the use of RNAi in therapeutic applications was the silencing of viral genes. With regard to the number of outbreaks and people infected worldwide, several groups of viruses are currently recognized as the most important human pathogens. Among those, aquired immunodeficiency syndrome (AIDS), hepatitis, and influenza are viral diseases of global dimension, bringing along high morbidity and mortality. Influenza even occurs in annual epidemics and in pandemics of infrequent occurrence but very high attack rates (151). Although RNAi is an ancient antiviral defense in plants and invertebrates, it does not play an important role in mammals. Nevertheless, the RNAi machinery is present in mammals and might be exploited to inhibit viral infections upon triggering by siRNAs.

So far, many groups have been successful in activating the pathway against viral targets with diverse replication strategies, including HIV (136,152-162), hepatitis B and C (HBV/ HCV) (163-170), human papilloma virus (HPV) (171-173), polio virus (174,175), herpes virus (176), human T-cell leukemia virus-1 (177), respiratory syncytial virus (178), influenza (179), Epstein-Barr virus (180), and, finally, corona viruses such as SARS (181-185).

At the beginning, mostly genes of the respective virus were targeted. Bitko and Barik were the first to observe specific antiviral effects of siRNA against the respiratory syncytial virus. Shortly after that, the same effects were reported for poliovirus in infected human cells (174) and HIV-1 using siRNAs directed against different regions ofthe HIV-1 genome, including its long terminal repeat and the genes encoding for the highly conserved genes vifand nef(152). siRNA-treated cells were particularly immune to subsequent HIV-1 infection in short-term experiments. Novina and co-workers used a contrary approach that notably reduced the ability of the HIV-1 to enter cells by targeting the CD-4 receptor, which is responsible for HIV-1 entry into the host cells. This indicates that the viral infectivity can also be stopped if host genes involved in the viral life cycle are targeted (162).

These studies clearly show that RNAi will hold its promise to become an efficient tool in the treatment of viral and other infectious diseases in human. However, before RNAi can make its way to therapeutic applications, an appropriate siRNA delivery system is required to guide the active species to the target organs. Interestingly, siRNA delivery to mice by hydrodynamic injection into the tail vein has led to a gene downregulation of 90% in the majority of liver cells. However, this technique requires high pressure and large volumes of siRNA solutions, making it inappropriate for application in humans (142,143). On the other hand, after the coinjection of luciferase-targeting siRNA and plasmid-luciferase into the tail vein ofmice, gene silencing was observed in several organs, including the liver, spleen, kidney and lung. Mice have been also healed for 10 d from Fas-induced fulminant hepatitis by the injection of the appropriate siRNAs (168).

More striking results were obtained on HBV. RNAi could inhibit the production ofHBV replicative intermediates in cell culture and in immunocompetent and immunodeficient mice transfected with an HBV plasmid (186). Upon siRNA treatment, substantially reduced levels ofHBV RNAs and replicated HBV genomes were found in mouse liver. In these studies, most ofthe targets are genes that are relatively conserved between different viral strains and exhibit a low mutation activity, as found for the genes essential for replication. Likewise, host genes required for viral entry or playing an essential role in the viral life cycle are also potential targets (187).

In spite of the great excitement created by those studies, it has to be kept in mind that most ofthe experiments were performed in cell culture or model animals using laboratory strains of viruses that are well characterized.

Some recent studies indicated that a therapeutic treatment of viral infection does not stand up to its expectations (188,189). In the long term, siRNA directed against the highly conserved Nef gene (siRNA-Nef) confers resistance to HIV-1 replication. However, the inhibition of replication is not complete. After several weeks of culture, HIV-1 escape variants appeared that were no longer inhibited by siRNA-Nefbecause ofnucleotide substitutions or deletions in the Nefgene that altered the homology to the siRNA-Nef sequence (188,189). To minimize the risk of viral escape, several viral and host genes must be targeted simultaneously.

Other infectious diseases such as those spread by parasites are also the subject to therapeutical RNAi approaches (190-195). Protozoan parasites and pathogenic fungi often resist manipulation by standard molecular genetic approaches. The discovery of RNAi in Trypanosoma brucei provides a convenient method for generating knockout pheno-types of the parasite (196). Further, erythrocyte-infecting stages of the malaria parasite Plasmodium falciparum were successfully treated with dsRNA encoding the dihydro-orotate dehydrogenase (DHODH) (192).

The application of RNAi to fight diseases reaches its limit when it comes to bacterial infections, because prokaryotes are not amenable to silencing by siRNA. Yet, the manipulation of the host genes involved in bacterial invasion or in immune response might help to immunize cells and tissues against bacterial infections (197). The recent discovery of the DNA or RNA editing roles of prokaryotic PIN domains, which are strikingly numerous in thermophiles and in organisms such as Mycobacterium tuberculosis now supports the idea that, similar to their eukaryotic counterparts, bacterial PIN domains participate in a related RNA silencing mechanism and nonsense-mediated RNA degradation (198). After an initial flood of mainly descriptive reports about the effects of bacterial infection, transcriptome and proteome studies are now becoming more refined in their approach and are shedding light on the role of pathogen-host interactions, which eventually can be targeted by RNAi (199). First, RNAi experiments targeting pro-inflammatory cytokines (interleukin-1 [IL-1], or tumor necrosis factor a [TNF-a]) successfully reduced the sepsis triggered by lipopolysaccharide (LPS) in mice (200). The intervention with the peroxisome proliferation-activated receptor (PPAR)-y-dependent anti-inflammatory mechanism for the treatment of chronic inflammation is only one of many further possibilities (201).

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