Antisense Making Lots Of Sense To Biotech Researchers

Research Antisense Making Lots Of Sense To Biotech Researchers Author: Joshua Fischman, p. 14, 17. In the early 1980s, at an international conference on synthetic DNA and disease, molecular biologist James Hawkins first heard what chemists proposed to do with small pieces of synthetic genetic material called antisense DNA. And he was stunned. "It was the first time," he says, "that there was a clear concept: `Let's send these molecules into a cell and regulate a gene.' This was apocalyptic--that's how profound it was for me." To some people, the concept, while profound, was also somewhat unbelievable. Indeed, Hawkins recalls, if you embraced it fully, "you were seen as a bit of a yahoo at the time, because the basic research wasn't established." It's not unbelievable anymore. What the researchers were suggesting about antisense DNA at that international conference heralded what many experts now see as the next revolution in drug design. Hawkins is now president of Synthecell Inc., a biotechnology company in Rockville, Md., whose enterprise is based on antisense technology, a field that already is large and active enough to have spawned two significant variations on its central theme--using synthetic pieces of DNA to interrupt human gene function. The classical antisense approach is carried out by researchers who create tiny bits of so-called antisense DNA to interfere with a product of a gene--called messenger RNA (mRNA)--that is implicated in a disease. In test tubes, tissue cultures, and laboratory animals, these DNA snippets have suppressed the activity of herpes and papillomaviruses, HIV, and oncogenes, genes that cause cancer. Another faction of researchers is attempting to go even further into the cell--right into the nucleus, home to the chromosomes that contain the genes that are made of DNA. If inactivating a genetic messenger is effective, these scientists reason, then inactivating the gene that sends the messenger must be better still. In order to prevent the gene from working and making mRNA, they are seeking molecules that bind to sections of the DNA double helix, forming a "triple helix." "Antisense is the first revolution in drug discovery since the discovery of the receptor," says Stanley Crooke, founder and chief executive officer of Isis Pharmaceuticals in Carlsbad, Calif., one of the new antisense ventures. "It's new chemistry. It's new biology. We've never before used these molecules as drugs. We've never used DNA hybridization as a mechanism by which drugs would bind." "The potential in therapy is virtually unlimited," says Hawkins, "because the processes of disease are all basically genetic." At least 10 biotechnology firms have sprung up in the last five years to develop these new drugs. In fact, at the end of August, one of these companies, Genta Inc. in San Diego, Calif., filed the first Investigational New Drug application based on antisense with the Food and Drug Administration (FDA), seeking to test one of these drugs--a compound to combat a form of leukemia--on humans. But while there seems to be a lot of cents and sense behind antisense, there are still a number of technical problems that need to be worked out before the revolution succeeds. Delivering the goods is perhaps the biggest obstacle for antisense researchers. Though antisense compounds find their targets with unprecedented accuracy once inside the cell, the scientists say, there's no tried-and-true method for getting them to the right cells in the body in the first place. And the compounds' vaunted specificity presents a problem for drug testing. The traditional path for drug-safety testing--from test tubes to animals to people--may not work for antisense compounds, which affect only human messenger RNA. So animals may not show any toxic effects that could appear in humans. Killing The Messenger What antisense does, essentially, is kill the genetic messenger. DNA in a cell's nucleus consists of two mirror-image strands that form the familiar double helix. One of these strands, the "sense" strand, codes for a protein. The code consists of a specific sequence of nucleic acids. Together, the two strands of DNA do nothing. But after the DNA makes a copy of its sense strand in a form known as messenger RNA--a ribonucleic acid (RNA) as opposed to a deoxyribonucleic acid, like DNA--the mRNA travels out of the nucleus. When it arrives at one of the cell's protein-making centers, which are called ribosomes, it translates its code into the protein. Antisense researchers take advantage of the fact that RNA has a complementary nature like DNA. In the world of nucleic acids, every bit of code has a complement, with code and complement fitting together like a zipper. The scientists take a small, mirror-image piece of nucleic acid, called an antisense molecule, in the hope that by binding it to mRNA, they prevent the mRNA from participating in making the disease-causing protein in a cell. The binding of the two molecules alters the mRNA structure enough to prevent it from doing its normal job at the ribosomes. The ability to home in on and neutralize a particular bit of genetic code is what gives antisense its power. Most drugs in use today start working later in the disease process; they usually target proteins, and their targeting ability is fairly crude. They often miss their mark entirely, and even if they hit it, they are liable to hit other proteins as well, causing unwanted and often dangerous side effects. "If I didn't know drugs worked, I wouldn't believe it," says Crooke, who was vice president at the Philadelphia-based pharmaceutical company SmithKline Beecham Inc. before starting Isis. "It's remarkable that a drug that couldn't discriminate between a mouse, a dog, and a human could--we hope--discriminate between a healthy and a diseased human." The specificity of antisense offers a solution to these problems. "This is all great news for the patient. For the first time, we have the ability to design drugs that are major therapeutic advances," says Crooke. Thomas Adams, CEO of Genta, the first company to apply to test an antisense compound on humans, says: "I think that in 15 years, 20 percent of the world pharmaceutical market might be antisense-based. That's tens of billions of dollars." Genesis Of Antisense Biochemists began building antisense strands of DNA, called oligonucleotides ("oligos," for short) in the 1960s and 1970s. Oligos are synthetic pieces of single-stranded DNA around 15 bases long. (Bases are the building blocks of DNA responsible for its coding or information content.) In test tubes filled with DNA, the researchers found, the oligos were very good at finding and binding specific genetic sequences. But living cells, they learned to their chagrin, were a different matter. "Nobody could get these things to work inside the cells," says Hawkins. Cells have enzymes, called nucleases, that break down nucleic acids. "If a cell sees a piece of nucleic acid that it thinks has gone a little astray, it takes care of it very quickly," Hawkins says. Chemists also had trouble getting the oligos into the cell through the cell membrane. All that began to change when the researchers began to restructure the oligo design. In the early 1980s, biochemists Paul Miller and Paul Ts'o of Johns Hopkins University in Baltimore substituted a methyl group for one of the phosphate groups that constitute the backbone of the oligo. This made the oligo electrically neutral, which enabled it to slip through the cell membrane. It also prevented it from being attacked and broken down by nucleases. Other scientists tried other substitutions, such as sulfur, and found similar improvements. "Essentially, you could characterize these as backbone modifications," Hawkins says. "There are about a dozen different variations on the molecule, most on the backbone. And whereas regular DNA would be degraded within minutes, these would survive for days. What the backbone compounds did was legitimize antisense research at the level of tissue culture." In 1987, Hawkins put out the first antisense bibliography, and shortly thereafter started a journal, Antisense Research and Development, published by Mary Ann Liebert Inc. in New York. New Companies Form That's when the antisense companies started springing up, most based on a particular type of modification. Genta, for example, was founded in 1989 around the work of Miller and Ts'o, who serve as the company's scientific advisers. "We made a bet early on that the Miller-Ts'o group was the leading academic group, and we decided to form a company around the Hopkins technology," Adams says. "And we immediately started looking for applications." Like most companies, Genta wanted to focus on a well-defined target disease, one with a well-understood genetic sequence. They decided to go after chronic myologenous leukemia (CML) in human bone marrow cells, which is caused by a translocation of part of chromosome 9 to chromosone 22. "It's a very unique sequence," says Adams. In test-tube experiments with bone marrow taken from CML patients, Genta researchers have added an antisense oligo that binds to this sequence and ties up the cancerous gene. Within three days, Adams says, normal bone marrow cells in the culture outgrow the CML cells, which disappear. Now Genta is ready to try this treatment on humans, given FDA approval. The company plans to have doctors at M.D. Anderson Cancer Center in Houston remove bone marrow from 10 CML patients and treat it with the Genta oligo. The patients would get massive radiation and chemotherapy doses to kill their remaining bone marrow, and then the treated, cancer-free marrow cells would be returned to the patients. The trial would take about a year. Other genetically characterized maladies, like viral diseases, are popular targets for antisense since their molecular biology is understood and their genetic sequences are known. Isis expects to ask for FDA approval within the next few months for human trials of antisense compounds for human papillomavirus and herpes simplex virus. Genta is also working on a herpes compound. A number of companies are developing compounds that are active against HIV. In addition, Isis is working on compounds against a variety of inflammatory diseases and the ras oncogene, which is implicated in some cancers. The Triple-Helix Method But for some scientists, antisense does not go far enough, and they want compounds that interfere with diseases at an even more fundamental level: the gene itself. Two companies, Triplex Pharmaceutical Corp. in Houston and Gilead Sciences Inc. in Foster City, Calif., are developing oligos that enter the nucleus and bind to the DNA double helix at a particular sequence. According to Joseph Zendegui, assistant director of molecular biology at Triplex, the oligo, or the third DNA strand, nestles right into a groove in the double helix. It occupies sites that normally attract proteins that promote the activity of a gene. With the third strand masking those sites, which are called promoters, genes can't be switched on to make mRNA. Zendegui says the triple-helix approach has the simple logic of numbers behind it: "In antisense you're targeting messenger RNA, and there may be hundreds or even thousands of copies of target mRNA in a cell. And the source of these mRNAs hasn't been affected by therapy. [With a] triple helix, you're going to the source and turning it off. In general, there are only going to be a few copies of a gene in a cell." Therefore, he notes, one needs much less of a triple helix compound in a cell for the treatment to be effective. Antisense Vs. Triplex Antisense researchers disagree with this analysis. "It's not the numbers of targets; it's the kinetics of targets that are important," says Crooke. The gene will be there for a long time. If the third strand comes loose and the gene becomes active again, he asks, what good have scientists done? By contrast, mRNA turns over very rapidly in a cell, so the antisense oligo has to bind to it for only a short time to be effective. Another problem with the triple helix, he says, is that genes are covered with proteins that restrict oligos from binding to the genes. Zendegui acknowledges that the stability of the triple helix might be a problem, but he denies that access to a gene presents any difficulty. "Let's look at it this way: If a gene is being transcribed, it's not covered up," he says. "It has to be available for proteins to interact with if it's a promoter site. It's been demonstrated in cell cultures that triple helix works. It's not just a theory." Triplex is currently refining compounds for use against HIV and herpes simplex viruses. `Black Holes' Remain A central issue that confronts both triple-helix and antisense companies is the aforementioned question of how to test their drugs for safety before beginning human trials. The traditional method has been to test drugs on animals for toxic effects. But since antisense oligos are designed to interact with unique genetic sequences, such as the human papillomavirus--which doesn't infect animals--what they do in animals may have no relation to what they do in people. An animal test would have to involve a different viral sequence. Genta's CML compound sidesteps this testing issue by treating cells outside the body. And Crooke believes that for antisense compounds that will be applied topically, such as a papillomavirus drug, test tube trials provide a margin of safety. "Since we're going to be treating locally, in many ways a tissue culture model is as relevant as an animal model using a different virus," he says. It remains to be seen whether or not regulatory agencies will agree with him. Drug delivery presents another problem for the antisense researchers. Topical treatments limit the choice of disease; extracorporeal treatments, such as Genta's CML compound, are very costly. "Oral delivery is the next problem that has to be solved in order to translate this technology into one that would be broadly applicable," says Adams. Researchers need to keep enough of the compound from being destroyed by the digestive system. "There are still a lot of black holes in `antiscience,' " Hawkins says. Nonetheless, Zendegui adds, "the whole idea of targeting genes and gene products is obviously an idea whose time has come." Joshua Fischman is senior editor of Discover magazine in New York.

Antisense Making Lots Of Sense To Biotech Researchers

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