Research on anti-sense agents is at an early stage but there is already agreement on some key technical problems that must be dealt with. Targeting the various anti-sense agents into cells is a top concern.
“Many are vulnerable to attack by enzymes in the cell,” says Dr. Melton. “Their stability has to be improved. With conventional drug therapy, delivery is easy and specificity is a problem. Here [with anti-sense agents], specificity is exquisite and delivery is a problem.”
Another hurdle is the need to develop reasonably priced scale-up processes. “Machines presently in use do not make sufficient quantity for large-scale therapy,” says Dr. Melton.
A first practical triumph for anti-sense technology came late last summer when Dr. Donald Grierson, School of Agriculture, University of Nottingham, U.K., published in Nature, a system for using an anti-sense agent to turn down the production of polygalacturonase (PG). This enzyme is responsible for breaking down cell walls and softening in tomatoes. Better control over this mushiness stimulant should allow better marketing of the more flavorful vine-ripened tomatoes, as opposed to the bland variety, which are picked green to avoid the destructive effects of galacturonase. This advance should also reduce losses to tomato processors, who discard bruised fruit.
“We [used anti-sense techniques to] reduce gene expression by 90 percent,” says Dr. Grierson, “and we hope to refine the system to reduce it by 99 percent. The PG gene is one of 80,000 in the tomato. We are delighted to have selectively tampered with one gene with no nasty side effects.” His group’s work is sponsored by the U.K’s Science and Engineering Research Council (SERC) and ICI Seeds, Berkshire, U.K.
In the U.S., Davis, CA-based Calgene, Inc. is also using anti-sense techniques to tinker with the production of PG. Their research, in press at the Proceedings of the National Academy of Sciences, has been aided by the Campbell Soup Company.
In fact, since Drs. Melton and Harold Weintraub, of Seattle’s Fred Hutchinson Cancer Research Center, published their landmark papers on anti-sense agents in 1984-85, a number of other academic research labs, and a few industrial groups, including, for example, Glaxo, DuPont, and Monsanto, have invested in anti-sense studies. Two privately-held companies dedicated to the field are the previously mentioned Gilead and Antivirals of Corvallis, Oregon. Key goals of these various groups are to expand the repertoire of anti-sense agents available and to define how they work.
Three anti-sense techniques are now actively discussed: anti-sense RNA; anti-sense DNA; and, the use of synthetic oligonucleotides to bind RNA or DNA.
The tomato research used anti-sense DNA. The Nottingham team identified and sequenced the 8,500 bases of the complementary DNA for the PG gene, then inserted it, using Ti technique, back into the plant chromosome. This new bit of DNA, the anti-sense gene, started making anti-sense RNA, that is, backwards RNA. It has been hypothesized that the RNA produced by the anti-sense gene binds, and inactivates the RNA produced by the normal DNA. Without functioning cytoplasmic RNA for polygalacturonase, this enzyme can’t be synthesized.
“But locking up the RNA is only one element of the process,” says Dr. Grierson. “Something more complicated is going on,” he adds, alluding to his recent findings that the quantity of mRNA is reduced by tinkering with anti-sense agents. Some researchers believe that anti-sense agents may also inhibit enzymes associated with replicating and translating the genetic code.
“No one knows in great detail how anti-sense genes work,” stresses Dr. Grierson.
Another stratagem is to use RNA as an anti-sense agent. If machine-made RNA could be made easily and cheaply, RNA fragments might be supplied externally to block gene action. Because RNA is normally found in the cytoplasm, it might be less vulnerable to enzymatic destruction than DNA, which is routinely chewed up when it leaves the nucleus. Cells might also be manipulated so that they supply their own, endogenous supply of anti-sense RNA. Infecting cells with virus that produces RNA may yield the desired results.
Ideas on the use of synthetic oligonucleotides to disrupt single-stranded RNA function were published 10 years ago. “But the field slumbered, until recently,” says Dr. P.C. Zamecnik, of the Worcester Foundation for Experimental Biology in Shrewsbury, MA, one of the early workers in the field. Now, he says, there are about a dozen labs working in the area.