BioWorld International Correspondent

LONDON - The task of understanding the roles of all the hitherto unknown genes in the human genome appears to have just become a whole lot easier. A team of researchers working in Germany reports in Nature how it is now possible to turn off single genes in mammalian cells at will.

Thomas Tuschl, a researcher at the Department of Cellular Biochemistry at the Max Planck Institute for Biophysical Chemistry in Gottingen, told BioWorld International: "Any cell biologist who wants to study gene function in tissue culture will use this method in the future."

The technique involves adding to the cell short pieces of double-stranded ribonucleic acid (dsRNA) that are homologous in sequence to the messenger RNA of the gene of interest. The result, which is known as RNA interference, is similar to that of a conventional gene knockout experiment involving homologous recombination, in mice or somatic cells, but is much simpler and easier to bring about.

Researchers are likely to apply the method to help them answer a huge range of questions. As well as finding out what happens to cells in which a particular gene is silenced, they will be able to target mutated tumor suppressor genes, or genes that are overexpressed in certain diseases, and thereby speed up assessment of whether these are valid targets for candidate drugs.

Applying the technique to silence one or more genes of various viruses, for example HIV, could give rise to new sequence-specific drugs for treating infectious diseases. Any disease caused by the inheritance of one dominant copy of a mutated gene also could be amenable to treatment by RNA interference, although, as always, researchers will need to find efficient ways of delivering the therapeutic RNA.

Tuschl predicted a boom in the demand for reagents containing short sequences of RNA from researchers wanting to take advantage of the new method. Patent specialists at the Massachusetts Institute of Technology in Boston, where Tuschl initiated the developmental work for the discovery while he was a postdoctoral fellow, have discussed granting a license to a company specializing in RNA synthesis for the distribution of RNA sequences of the correct size. The patents also cover the therapeutic applications of the technique, and its use as a tool for the analysis of gene function in an industrial setting.

A paper describing the experiments carried out by Tuschl and his colleagues published in Nature is titled "Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells."

Two years ago, Tuschl, working with Phillip Zamore, then of the University of Massachusetts Medical School in Worcester, developed a biochemical system using Drosophila embryos to analyze the mechanism of RNA interference, a phenomenon that had already been reported by other researchers. They knew from the earlier studies that adding dsRNA that was several hundred base pairs in length to cells of the worm Caenorhabditis elegans resulted in silencing of genes homologous in sequence to the dsRNA. When similar lengths of dsRNA were added to mammalian cells in culture, however, the cells responded as if they had been infected with a virus, by making huge amounts of the cytokine interferon and turning off all gene expression.

Tuschl told BioWorld International: "We found that when you add long dsRNA to Drosophila lysate, it is chopped up within the cells into short dsRNA segments, which we have called short interfering RNAs, or siRNAs. When we added these siRNAs back to lysate, we found that we could get specific mRNA degradation."

Further work showed that the siRNAs had to be of a particular length and format in order to be effective. Each strand had to be 21 nucleotides in length, annealed to its partner for a length of 19 base pairs, with an overhang of two nucleotides at each 3' end.

Tuschl said his team's "real breakthrough" came when it realized that adding the equivalent mammalian siRNAs to mammalian cells in culture also gave rise to "very efficient and sequence-specific gene silencing" and bypassed the so-called "interferon reaction."

The paper in Nature describes how they silenced several genes in mammalian cell culture, which they had targeted with the appropriate siRNAs. The authors wrote that their experiments indicate "that siRNAs are extraordinarily powerful reagents for mediating gene silencing, and that siRNAs are effective at concentrations that are several orders of magnitude below the concentrations applied in conventional antisense or ribozyme gene-targeting experiments."

Tuschl expects that a genome consortium or company will now embark on knocking out each of the genes in the human genome in turn with siRNAs. "Our own work now will concentrate on identifying the protein components involved in the process of generating siRNAs in the cell under normal circumstances, and characterizing that machinery," he said. "We also want to see how far we can go in trying to get this to work therapeutically, perhaps by introducing an siRNA into a mouse model of a disease that would theoretically be amenable to treatment by this approach."

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