Scientists devise new 'gene silencing' strategy: Genome, biomedical research expected to flourish

April 14, 2002

Treatments for cancer, AIDS and a host of other diseases may lie in scraps of hairpin-shaped RNA.

Researchers at Cold Spring Harbor Laboratory have successfully co-opted a natural cellular mechanism to shut down the activity of specific genes in mammalian cells cheaply and efficiently. The new technique should speed the exploration of gene function for many research and clinical applications, and may eventually be used in gene therapy to selectively kill cancer cells, to block HIV infection, and for several other purposes (for figure, see accompanying PDF).

"There hasn't been a tool this sharp in a long time," says Doug Conklin who, along with Greg Hannon, led the Cold Spring Harbor research team.

The new technique is published in the April 15 issue of Genes and Development. It stems from work in several laboratories, including a discovery made four years ago by Andy Fire of the Carnegie Institute of Washington and Craig Mello of the University of Massachusetts. These scientists found that they could block the expression of a gene in nematode worms by injecting the animals with double stranded RNA corresponding to the gene, or even by simply feeding the animals the double stranded RNA. (Genes are made of DNA. RNA is a chemical cousin of DNA, but has different structure and properties).

Such blockage of gene expression by double stranded RNA was termed "RNA interference"--RNAi, for short. RNAi is now known to operate in humans, mice and other mammals, as well as in fungi, flies, and plants. An early incarnation of the phenomenon was uncovered in 1990 by a horticultural researcher who, when trying to create more purple petunias, achieved an unexpected opposite result (i.e. more white petunias!).

"RNAi took the worm world by storm," says Hannon. Moreover, scientists quickly realized that such a deceptively simple and effective way to block individual genes might be an invaluable tool both for basic research and for treating disease.

Whereas RNAi proved mighty handy to worm geneticists, applying it in human cells has been fraught with challenges. For example, researchers soon found that long stretches of double stranded RNA cause a global, non-specific shutdown of gene activity in many mammalian cell types, culminating in cell suicide. It wasn't until Hannon and others began to explore the precise mechanism of RNAi that they gleaned clues for circumventing the global response and efficiently silencing select genes.

RNAi begins when an enzyme--which Hannon and colleague Emily Bernstein discovered and aptly named Dicer--encounters double stranded RNA and chops it into pieces called small interfering or siRNAs. A complex of proteins gathers up these RNA scraps and uses their code as a guide to search out and destroy any RNAs in the cell with a matching sequence, such as messenger RNA.

Because messenger RNA serves as a template for the production of the proteins that carry out gene function, genes ultimately no longer function if the messenger RNA they code for is targeted for destruction by RNAi.

After scientists learned that long stretches of double stranded RNA frequently trigger cell suicide instead of RNAi, Tom Tushl's group of the Max Planck Institute reasoned that using smaller double stranded RNAs--akin to those produced naturally by Dicer--should do the trick. Indeed, such small RNAs have since become the molecules of choice for RNAi in many laboratories.

Driven in part by their desire for a longer-lasting alternative to small RNAs, Hannon and his Cold Spring Harbor Laboratory colleagues tried using RNAs folded over like hairpins to quash the function of specific genes. They were inspired to try these short hairpin RNAs (shRNAs) by their finding, in collaboration with Dutch worm researcher Ronald Plasterk, that some genes naturally regulate other genes--through RNAi--by coding for just such hairpin-shaped pieces of RNA.

In the new study, Hannon and lead author Patrick Paddison showed that in human cells, shRNAs silenced genes as efficiently as siRNAs. The study also established that such short hairpin activated gene silencing (which the researchers playfully termed "SHAGging") operates in a variety of normal and cancer cell lines, and in mouse cells as well as in human cells.

Significantly, the researchers also generated transgenic cell lines bearing chromosomal genes that code for engineered shRNAs. In doing so, they successfully programmed the cells to synthesize the engineered shRNAs and thereby created long-lasting gene silencing that was stably passed on to progeny cells as the cultured cells multiplied.

Hannon has begun to harness the power of RNAi as a tool for gene discovery in cancer research. By using shRNAs to systematically block the expression of thousands of different human genes individually, he aims to identify genes that are essential for the survival of cancer cells, but that are not required for the survival of normal cells. Such genes represent an "Achilles' heel" of cancer cells against which highly selective cancer therapies can be targeted--therapies that would destroy cancer cells while leaving normal cells unscathed (see figure, below).

Hannon is also devising ways to apply RNAi directly as a therapeutic tool. In one approach, he is using short hairpin RNAs to silence known cancer-causing genes ("oncogenes") and thereby control the growth of cancer cells. Other researchers are testing whether shRNAs can silence expression of the cell surface receptor that enables HIV to slip inside cells.

"In many cases, this strategy could translate from benchtop, to animal model, to clinic much more quickly than traditional medicinal chemistry," says Hannon. He predicts that short hairpin RNAs could be incorporated into novel therapeutic approaches within a few years, although additional time would be required for such treatments make their way to the clinic.

Cold Spring Harbor Laboratory

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