St. Louis, Oct. 21, 1998 -- Scientists often use the genetic material of viruses to smuggle foreign genes into cells. But such vectors frequently kill the cells they enter, limiting their long-term utility. For the first time, researchers at Washington University School of Medicine in St. Louis have devised a way to create harmless vectors from a harmful virus. In a paper in the Oct. 27 issue of Proceedings of the National Academy of Sciences, they show that the vectors are efficient couriers of genes.
"A similar strategy might work for modifying other cytotoxic viruses that are being used as gene vectors," says Charles M. Rice, Ph.D., professor of molecular microbiology and head of the research team. Postdoctoral fellow Eugene V. Agapov, M.D., Ph.D., and Ilya Frolov, Ph.D., research assistant professor of molecular microbiology, were the paper's lead authors.
By transferring foreign genes into cultured cells, scientists can study the regulation and functions of those genes under controlled conditions. They also can alter the activities of cells, to compensate for a defective gene, for example. Commenting on Rice's paper in an accompanying article, Peter Palese, Ph.D., of Mount Sinai School of Medicine, says that the "long-term expression of foreign proteins in a noncytotoxic manner may blow new wind into the sails of our gene therapy enterprise." This new type of vector might eventually be used to deliver DNA vaccines, he also suggests.
Rice and colleagues worked with Sindbis virus, which produces fever, headache and musculoskeletal symptoms. The virus's genetic material is a single strand of RNA, which Washington University research groups engineered into a gene vector in 1989.
To obtain vectors that will not kill cells, the researchers first inserted an extra gene into modified pieces of Sindbis RNA. The gene coded for a protein that inactivates puromycin, an antibiotic. They transferred this altered RNA into hamster kidney cells, which then were exposed to the antibiotic. The cells now had two reasons to die. If they hadn't acquired the viral RNA, they would be killed by puromycin, which prevents mammalian cells from making proteins. If they had, they would die from the toxic effects of the viral RNA, though not from the antibiotic.
A small proportion of the cells survived, however. These cells seemed to have acquired the viral RNA because they survived their contact with puromycin. But that RNA must have mutated so that it no longer was toxic to cells.
The researchers cultured the survivors and showed that they did indeed contain pieces of Sindbis RNA. They also found that this RNA was very efficient at making other hamster cells resistant to puromycin.
The researchers mapped the adaptive mutations and cloned the mutant RNAs for use as noncytotoxic gene expression vectors. They also made vectors that could be launched from DNA copies of the RNA. "The basic idea is that if you transfect cells with Sindbis vectors that have these adaptive mutations, Sindbis will establish replication, confer puromycin resistance, and the cells will be perfectly happy and capable of normal growth," Rice says.
The Sindbis vectors contain two regulatory regions. One regulates the gene for puromycin resistance; the other controls a foreign gene of choice. To test the effectiveness of the vectors, the researchers tried out several foreign genes, including luciferase, the enzyme that makes fireflies glow. Some of the genes were expressed at high levels, even after the host cells had divided 30 to 40 times.
In unpublished work, the group also has used these vectors to express proteins of hepatitis C virus, an important human pathogen for which no vaccine exists.
"These vectors should be very useful scientific tools because, in a matter of days, people will be able to make cell populations that express a gene of interest," Rice says. "The transfected cells are normal and capable of division. This is a tremendous advantage in studies where you don't want the vector to perturb the biology of the cell."
Agapov EV, Frolov I, Lindenbach B, Pragai BM, Schlesinger S, Rice CM (1998). Noncytopathic Sindbis virus RNA vectors for heterologous gene expression. Proceedings of the National Academy of Sciences, 95.
Grants from the Public Health Service supported this research.