By simultaneously attacking two genes that are found in all strains of the influenza virus, Yale researchers have succeeded in curbing reproduction of the virus in mouse cells in tissue cultures. If successful in animal and human studies, Yale's general method for thwarting the virus could bring relief to millions suffering from flu and other viral diseases, who now have few options other than letting the diseases run their course.
"Because flu viruses spontaneously mutate at a high rate, we targeted two essential genes -- the polymerase and nucleocapsid genes -- which contain DNA sequences that are highly conserved in different flu strains," explains Sidney Altman, Sterling Professor of Biology, who announced the finding in the June 23 issue of the Proceedings of the National Academy of Sciences.
Altman, along with former graduate student Debora Plehn-Dujowich, used a method based on research that earned him the 1989 Nobel Prize in chemistry. His discovery that RNA is not just a passive carrier of genetic code, but also can be an enzyme that actively engages in chemical reactions, triggered a new branch of genetic engineering aimed at treating lethal viruses and drug-resistant bacteria, as well as repairing genetic defects.
"Our success in inhibiting gene action in flu viruses with an enzyme called RNase P and small strings of RNA nucleotides called external guide sequences (EGSs) is further proof that this general method can be used against human viruses," says Altman. His laboratory also has used this technology to prevent the expression of genes that make bacteria resistant to two widely used antibiotics, chloramphenicol and ampicillin. Research is now underway in animals to see whether the method can restore the full usefulness of these frontline antibiotics.
Tailoring genetic codes to virus. To attack the flu virus in cell cultures, the Yale biologists crafted synthetic genes coding for specific strings of RNA and introduced them into the cells via small circular pieces of DNA called plasmids. Once inside the cells, the synthetic genes produced the EGSs.
To fight both viruses and bacteria, EGSs are engineered to bind to targeted "messenger RNA" (mRNA), a family of compounds that play a key role in controlling body chemistry. Once the EGS molecules attach to their target, they cause an RNA enzyme called RNase P to destroy the mRNA to which they are bound. The EGS molecules are then freed to repeat the process. The EGS technology's therapeutic value lies in the fact that it can be used to seek out and destroy the mRNAs associated with particular diseases -- or the mRNAs associated with resistance to specific drugs.
This research, which was funded by the National Institutes of Health, confirms the effectiveness of simultaneously targeting two or more genes when preventing virus replication or bacterial drug resistance with Altman's method. In the realm of basic research, scientists also can use EGSs to inactivate mRNA molecules in a highly selective way to gain a better understanding of how cellular chemistry functions.
Determining best delivery method. The next step in boosting the disease-fighting capability of EGSs is to find the best method of getting EGSs inside bacteria or infected human cells, Altman says. Instead of using plasmids as he did, which would require exposing patients to yet another bacterium, researchers most likely will find a chemical package that can readily enter the target bacteria or human cells. Then the method must be tested in animals and humans.
It is a relatively simple matter to design the EGS sequence itself, Altman adds, because the methods are "all pretty well worked out." In fact, a specific EGS can be designed in a matter of hours or days, and then produced with a machine called an RNA synthesizer. The entire process of finding and testing the effectiveness of a specific EGS takes only a few weeks or months.
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