Most of the viruses in the news these days seem to have "Out of Africa" on their nameplates. Field virologists tend to christen a new virus after the place where they discovered it — for example: Lassa, Ebola, Rift Valley, Semliki Forest and Sindbis.
But Africa doesn't have a viral monopoly. Take Coxsackie virus, named after a town in upstate New York, Powassan (Canada), Hantavirus (Korea), Marburg (Germany) and Sendai (Japan).
Sendai, Semliki and Sindbis viruses are not on the most-wanted list of dangerous viral pathogens. Rather, scientists use them in research. To be sure, Sindbis virus — named for a village in Egypt — can cause mild fever and musculoskeletal pain, as well as cytotoxicity in cells, but its immediate interest is as a prototype test bench for future gene therapy vectors.
A paper in the current Proceedings of the National Academy of Sciences (PNAS), dated Oct. 27, 1998, tells how its authors created harmless vectors from the cytopathic virus. Its title: "Non-cytopathic Sindbis virus RNA vectors for heterologous gene expression." Its senior author is molecular biologist Charles Rice, at Washington University, in St. Louis.
To obtain non-cytotoxic vectors, he and his co-authors first inserted an extra gene into modified fragments of Sindbis RNA. It codes for a protein that inactivates the antibiotic puromycin. They transferred this sequence into baby hamster kidney cells, in which it replicates, and exposed these to the antibiotic.
That treatment gave the cells two reasons to die: If they hadn't acquired the cytopathic viral RNA, the puromycin — which prevents mammalian cells from making protein — would kill them. If they had, the cytotoxicity would cause their death. A few of those cells exposed to the RNA managed to survive, presumably because the viral sequence had mutated and lost its toxicity.
"For us, it was a fishing expedition," Rice told BioWorld Today, "to see if we could actually select for a mutation that basically changed this RNA replication machinery from being cytopathic — cytotoxic — to something capable of surviving and replicating in the cellular environment, without apparent deleterious effects."
Rice believes gene therapy applications of a Sindbis vector are "a little farther in the future. In particular, those based on Sindbis, Semliki Forest, Venezuelan equine encephalomyelitis and other cytopathic viruses would have utility in transient therapy applications.
Sindbis Gene Delivery Prolonged, Not Permanent
Rice explained: "The issue for this cytopathic viral vector is that although its window of activity may be characterized by high levels of expression, it might be short-lived, because Sindbis kills the cell. So, these non-cytopathic derivatives may have the advantage of being able to allow longer but not permanent expression." He took vaccination as an example in which "prolonged expression would allow you to elicit a stronger immune response.
Rice pointed out that Sindbis is a non-retroviral RNA alpha virus, "so it's not going to be stably integrated into the cell. But there may be applications, for instance vaccination, where you would want to transiently deliver an antigen that corresponded to an infectious agent and elicit an immune response." He added: "If the vector delivering it then went away, so much the better."
Another area in which researchers are investigating transient gene therapy is wound healing, Rice said. He and his co-authors are now exploring "whether this strategy will work in human cells, as opposed to these hamster cells that we used in this PNAS study. We don't yet know what the adaptive mutations are, but we have some encouraging results suggesting that this same strategy will work for selecting them in human cells as well.
When HIV invades, it makes straight for the immune system's T cells and macrophages; hepatitis C virus beelines to the liver. Sindbis virus's human bull's-eye cells are still in question.
"There's not a lot of data on that," Rice said. "Probably a major target cell for replication of Sindbis would be in muscle." That supposition jibes with the aches and pains of Sindbis virus infection.
"The characteristic of any of these fever-causing alpha viruses," he pointed out, "is polyarthritis — inflammation of several joints at once — and fever. But we know very little about how Sindbis infects cells in humans, and how the viral vector might be restricted in vivo to certain cell types."
Vaccination An Apt Target
"In the case of genetic vaccination," Rice observed, "people are using either biolistics or introducing naked DNA into muscles, which take it up and transfer it to cells that can present the antigens. The same strategy can be used for delivering these replication-competent Sindbis RNAs. In that case, they're just launched in the cell when the plasma DNA gets taken up and transcribed."
Rice and his co-authors have successfully transfected several foreign genes with their disarmed viral vector, including luciferase and the hepatitis C virus gene. But they are not contemplating any in vivo tests in animals.
"We're not really in the gene therapy business," he said. "[We're] more on the basic-science side of things."
But Rice pointed out there are companies exploring the use of alpha virus-based vectors in gene delivery applications. Among these, he said, is "a company that used to be called Viagene, which was bought by Chiron [Corp., of Emeryville, Calif.] in 1995. It works on Sindbis-based vectors. Another firm just starting up, called AlphaVax, in Chapel Hill, N. C., is developing vectors based on Venezuelan equine encephalomyelitis virus. And a group in Sweden is working with Semliki Forest virus and genetic vaccination.
"I'm sure they're going to be taking a very close look at this PNAS paper," Rice concluded, "either making what we've done in their vector system, or selecting for their own adaptive mutations, using a similar strategy. Then compare those to vectors that either don't have the adaptive mutations, or other methods for gene delivery, and see how they compare."
In a commentary accompanying the PNAS paper, microbiologist Peter Palese, of Mt. Sinai School of Medicine, in New York, wrote that "the long-term expression of foreign proteins in a non-cytotoxic manner may blow new wind into the sails of our gene therapy enterprise." *