By David N. Leff

During this 20th century, now ending, we humans have shot ourselves in both feet.

In one case, by all-out burning of fossil fuel, mankind is demolishing the ozone layer that protects us from cancer. In the other, by excessive abuse of antibiotics, we are unilaterally spiking our guns against bacterial infection.

Since the early 1980s, physicians have observed that more and more of the 160 or so antibiotics they prescribe are falling before increasingly drug-resistant bacteria. This leads them to fall back on more expensive antibacterials, with more severe side effects.

One example is Haemophilus influenzae (no relation to the flu), which used to be treated routinely with ampicillin. Today, about 20 percent of these infections are resistant to the drug.

Just as Willy Sutton robbed banks because that's where the money is, infectious microorganisms congregate in hospitals, because that's where the patients are. Two other pistons that drive the spread of drug-resistant germs are the over-prescribing of antibiotics for viral infections (especially the common cold), for which such drugs are useless, and their availability in many countries over the counter.

Besides antibiotic-blunting bacteria and fungi, human cells are developing multidrug resistance genes, especially against cancer chemotherapeutics.

These twin challenges have led Yale University Nobelist Sidney Altman to develop a nucleic-acid weapon for disabling the drug-resistance genes in infective pathogens.

Building on the work that won him a 1989 Nobel Prize in chemistry, Altman has devised strings of synthetic genes that encode catalytic oligonucleotides, which target messenger RNA and prevent it from expressing resistance factors. His latest progress report in this quest appears in today's Proceedings of the National Academy of Sciences (PNAS), dated Aug. 5, 1997. Its title: "Phenotypic conversion of drug-resistant bacteria to drug sensitivity."

Designated Hitter Disables Drug-Disabling Genes

Altman calls his search-and-destroy gene fragments "External Guide Sequences," or EGS. Once these molecules bind to their target in a designated bacterium, they cause an RNA enzyme called ribonuclease P to destroy the messenger RNA to which they are bound. This process turns the EGS loose to repeat the action.

The PNAS paper reports experiments in which Altman and his co-authors aimed their EGS at genes in Escherichia coli that encode two drug-resistance enzymes, chloramphenicol acetyl transferase and beta lactamase. These are resistant, respectively, to two major antibiotics, chloramphenicol and ampicillin.

Expression of one EGS-containing plasmid inhibited the bacterium's anti-antibiotic resistance very well. Two provided even more efficient cutting of the RNA, hence even better functioning of the antibiotics.

Yale has licensed Altman's EGS technology in exclusivity to Innovir Laboratories Inc., of New York. That company's chairman and CEO, Alan Goldberg, told BioWorld Today: "We have been working to make a chemical analogue of what Altman — who is on our scientific advisory board — reports in this PNAS paper. We have actually been able to truncate the first oligonucleotides he described from about 65 nucleotides down to 13.

"The EGS," Goldberg explained, "looks like a piece of a particular type of normal RNA for ribonuclease P, an enzyme found in every cell. We were able to engineer that piece of RNA to hybridize to any target RNA that we wish. When that EGS does so," he continued, "it looks to the enzyme like its own natural substrate. So then the ribonuclease P cuts the target RNA, thereby inactivating it and preventing expression of a particular protein.

"It's very generalizable," Goldberg went on, "from bacteria all through to every human cell. You can target EGSs to literally any RNA, and our business here at Innovir has been to find which sites on any RNA are most susceptible to cutting. Then we make these molecules, put them into cells and target particular EGSs, using the enzyme in every living cell."

Goldberg likes to compare this intracellular effect to disabling an automobile: "Say you have a piece of the car that's absolutely essential, whether it's the distributor, an axle or the fuel pump — whatever. If you hit the fuel pump, clearly the car won't go any more. On the other hand, if you stop a window from going up and down, it won't affect the car's ability to go from point A to point B."

Driving home his point, he added, "There are critically essential and non-essential genes, as there are parts in cars. If you can determine which are the essential genes, you might be able to make a general drug, which would be different from an antibiotic."

EGS Hits Malignancies As Well As Infections

Here his example is "human cancer cells becoming resistant to chemotherapeutic drugs, frequently through the expression of multidrug resistance genes (MDRG). The drug gets into the tumor cells, and MDRGs immediately pump them back out. So that would be a great target for EGS technology."

Not so, another form of antibiotic resistance.

"In some cases," Goldberg observed, "EGS may not be appropriate. For example, a bacterium can become resistant because a receptor to the antibiotic on its cell surface has mutated, and there's no way for it to bring the antibiotic into the cell. Our EGS technology can't target that. It can inhibit only a gene that has evolved to break down the antibiotic."

The Innovir executive envisages a number of clinical scenarios, once EGS therapy has been found safe and effective in humans.

"Bacterial infections come in many different flavors," he pointed out. "It could be enteric — in the gut — or a skin infection. For instance, Pseudomonas is a bacterial infection that frequently accompanies burn injuries. And there are a lot of drug-resistant Pseudomonas strains now, especially since burn victims spent a lot of time in hospitals, where there's a lot of antibiotics around."

For some gut infections, "the EGSs would probably be swallowed as a plasmid in the context of a pill, or as a synthetic drug."

Innovir, he added, "has been able to get EGSs into bacteria using a drug-delivery compound or facilitator we have developed. We don't know yet if it will be appropriate for treatment of antibiotic-resistant patients. That's something we would want to explore, preferably with a pharmaceutical partner, and/or a genomics company, to find the genes most important for a particular syndrome." *