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Feature Articles : Mar 15, 2011 ( )
GEN's 30th Anniversary: Antisense
Recent news stories have noted that RNAi technology, once thought so promising among many in the life science community and especially big pharma, is now falling out of favor. The biggest hit took place last November when Roche dropped out of RNAi R&D after plunking down a half a billion bucks to explore the field over a four-year period. Delivery issues and the lack of a sooner-than-later expected payback helped lead to the Roche decision.
In the meantime, industry reports point out that large pharma firms have been in-licensing antisense technology, which was developed over 20 years ago. What’s ironic is that antisense, just like RNAi, is intended to shut down the activity of specific genes. And similar to RNAi, antisense has had its own share of delivery problems.
But unlike RNAi, antisense has a long history of trial-and-error and experimentation with lessons learned along the way. That’s probably why some biotech and pharma firms are giving it another closer look.
This issue’s article commemorating GEN’s 30th anniversary is reprinted from November/December 1988. It is very timely in that it provides one of the first in-depth profiles of antisense technology and its range of potential applications.
This story, like all the old stories that we are reprinting, demonstrates a particular significance and relevance for the life science research community.
—John Sterling, Editor in Chief
"As Seen in GEN—Flashback" Volume 8, Number 10 November/December 1988
Researchers Pursue “Anti-Sense” Technology In Quest for Novel Drugs and Agriproducts
By Anne Simon Moffat
More flavorful fruits and vegetables, new treatments for a range of genetic diseases and a cure for AIDS are possible prizes for researchers pursuing a new technology, known as “anti-sense agents.”
“If anti-sense approaches work, they will usher in a new era of drug therapy, and a new wave of [pharmaceutical] companies,” says Dr. Douglas Melton of Harvard University and a pioneer in the field. He believes that anti-sense approaches may offer our best hope for curing AIDS.
“During the past two years, progress in anti-sense research has been asymptotically vertical,” says Michael Riordan, president of Gilead Sciences, Inc. (Foster City, CA), one of two U.S. companies dedicated to the development of anti-sense agents. The worldwide market for new products that stem from anti-sense research has been estimated to be in the range of $2 to $20 billion annually.
Anti-sense agents block gene function. It has been known for about 10 years that anti-sense mechanisms can control gene expression in bacteria. But within the past few years, it has been shown that such agents may also be manipulated in eukaryotes to inhibit the action of “bad genes”, such as those that code for an enzyme that accelerates food spoilage or those needed for virus replication.
However, no one has proven that anti-sense agents can perform in man. Still, because many human diseases can be attributed to a single deleterious gene, researchers are hopeful that anti-sense agents can be used therapeutically, to clamp down on problem genes.
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.
However, Dr. Peter Dervan, of the California Institute of Technology (Pasadena, CA), has taken the field in a new direction by developing oligonucleotides that bind double-stranded DNA. The technique is known as the triple helix, and it is based on sophisticated organic chemistry. Dr. Dervan and his colleagues have found that a short, synthetic oligonucleotide, 15 to 25 base pairs in length, will bind specifically to a segment of double helix, forming a short triple helix. The oligonucleotide both binds and cleaves DNA, which could prevent normal gene expression.
“As molecular biology defines specific disease states at the DNA level, a chemotherapeutic strategy of ‘artificial repressors’ based on triple helix forming DNA become a possibility,” says Dr. Dervan.
The triple helix stratagem is still far from being fully developed, he admits. First, synthetic oligonucleotides are now only available for recognizing guanine and adenine.
“Assuming we will find oligonucleotides that recognize all four base pairs, we still have to construct them to be nuclease resistant and to be permeable to cell membranes,” says Dr. Dervan. “But I am optimistic that [such DNA analogs] will be created. The anti-sense area is taking off.”
Indeed, some of the most exciting results have come from research teams at the Worcester Foundation for Experimental Biology, the National Cancer Institute at the National Institutes of Health, and Northwestern University, which have helped to show that anti-sense agents can block the damage of the human immunodeficiency virus.
Gilead president Michael Riordan says his company is working on the three previously mentioned anti-sense stratagems and told GEN that, “There are others under wraps. We are doing broad-based genetic targeting.”
The company, he says, “of about 16 staff,” was organized “about a year ago” with $2 million in funding from Menlo Ventures in Menlo Park, CA, a company for which Riordan worked earlier. Equipped with a Hopkins M.D., and a Harvard MBA, Riordan, 31, has spent most of his professional life not in the clinics or at the lab bench, but at business meetings. But, by the accounts of both colleagues and competitors, he has succeeded in recruiting top chemists to his company. Their efforts are key to determining the commercial future of anti-sense agents.
An early goal of the company is to develop nucleic acid analogs that bind mRNA, and block gene translation. At the same time, work must be done to advance automated synthesis to reduce the costs of making anti-sense compounds.
A subsequent goal is to develop agents that act directly on the target gene, such as an oncogene, protooncogene, or a gene that expresses proteins that trigger autoimmune disease.
Dr. James Summerton, president of Antivirals, the other privately held company dedicated to anti-sense agents, claims to have been working on the anti-sense concept since 1969, and to have started bench work in his home basement, while working with Oregon State University scientists. “A paper I submitted in 1973 to the Journal of Experimental Biology on anti-sense technique was rejected,” says Dr. Summerton, adding that a rewritten version was published in 1979.
His company is developing two techniques, neu-genes (for neutral genes), which bind to single-stranded genetic sequences. Antivirals was founded in 1980 and, for the last four years, has been financed at an average rate of $200,000 annually through federal grants, equity investments and support from Du Pont (which dropped out in May 1987). Antivirals is in advanced negotiations with Glaxo and Mitsui for further funding. The State of Oregon is also assisting.
Dr. Summerton says that his anti-sense agents are superior to those of others because, “We have a more radical design of oligonucleotides that allows for the use of inexpensive technology. We can use cheaper reagents and we can build (the oligonucleotides) more easily.”
In particular, they have built anti-sense agents whose component subunits are derived from the ribonucleosides, rather than the more expensive deoxyribonucleosides. “The major starting materials for our agents would run about three percent of the cost of starting materials for competing agents,” says Dr. Summerton.
Moreover, he adds that anti-sense agents built from these starting materials have the virtues of easily penetrating tissues and of being insensitive to destructive nucleases; that is, they are tough and can be targeted.
Dr. Summerton admits that, “for the desired commercial success we will require a prompt financial investment in our program, estimated on the order of $14 million over four years, plus a close collaboration with a major partner…”
Clearly, both Gilead and Antivirals are trying to accelerate their development program in the face of newly gained “bandwagon status” for anti-sense research.
What Dr. Summerton does not want is, “a commercial situation, much like that today in the field of recombinant DNA, where multiple companies are all trying to commercialize the same products…”
Dr. Riordan is not sure that dilemma can be avoided. “All indications are that the field is too elegant and direct to be ignored,” he says.
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