By David N. Leff

TV re-runs often show a 1966-vintage sci-fi film called Fantastic Voyage, starring Raquel Welch and Edmond O'Brien. It's about a team of doctors and nurses, shrunk to microscopic size, and injected into a human patient's body to treat his disease.

Of course, as is so often the case, nature got in ahead of human invention. Among the body's 60,000 to 100,000 or so genes are armies of DNA-repair enzymes, rushed to the scene of untoward slip-ups in the never-ending multiplication of cells and chromosomes. If these fixer-upper molecules can't correct errors in the errant genes, they neatly snip out the flawed sequence and replace it with a good-as-new functioning one.

All too often, of course, disease-causing mutations escape this genomic surgery, and molecular biologists strive to supplant nature's remedy with gene therapy approaches. So far, their methods employ blunt instruments - compared to DNA's own repair mechanisms.

Molecular biologist Peter Glazer, at Yale University, New Haven, Conn., has labored for a decade to create his own Fantastic Voyage-level future gene-therapy strategy. His genomic-surgery scalpel is a DNA double helix expanded to a triple helix.

"The underlying concept," Glazer told BioWorld Today, "is that a triple helix forms as mediated by an oligonucleotide [oligo] molecule that binds in the major groove of the DNA double helix. There it constitutes a lesion - a mutation - that can be recognized by the DNA repair apparatus of the cell. Our thinking," he said, "was that this might offer a tool that could be used to modify the DNA. And that modification could either trigger an error-prone pathway, including some mutations - or sensitize the target site to recombination - by recruiting repair proteins. These could stimulate gene replacement or gene-targeting events."

Precision Landing On Site-Specific Target

Glazer is senior author of a paper in today's Science, dated Oct. 20, 2000, titled: "Specific mutations induced by triplex-forming oligonucleotides in mice."

"It demonstrates," he said, "that in a living animal the genome can be targeted by small molecules such as oligos in a very site-specific way, following systemic administration. We introduced the oligos by injection to the peritoneal cavity of mice," he noted. "They were systemically absorbed and distributed to the bodily tissues, and found their way into the cells and nuclei to their target sites.

"We knew that this particular oligonucleotide binds to a target site with very high affinity," Glazer added. "It can stimulate DNA metabolism, leading to a low level of mutation at the site. That mutation is not the long-term goal of our work. We're more interested in using this approach as a tool to target genes for gene replacement.

"The mouse model we used in this in vivo experiment," he went on, "was established to contain a specialized gene construct, which allows the facile detection of mutations. It's basically a mutation detection reporter system that will report changes in at least two specific genes that are integrated within the chromosome of the mouse. We have a line of these animals, which breed true and are otherwise healthy.

"We injected the animals every day for five days," Glazer said, "with a construct consisting of an oligonucleotide 30 nucleotide bases long - a 30-mer. It had the capacity to bind one of the two reporter genes in these transgenic mice, but not the other one. The gene we targeted is called supF. It's an amber suppressor transfer RNA, and has no meaningful function in the mice, except that we could measure its activity. The other gene, cII by name, controls something in the bacteriophage lambda life cycle. Through some tricks we were able to measure its activity, too.

"About 10 days later," Glazer continued, "we looked at the various tissues, and found that in the treated animals the frequency of supF gene knockout or mutation was five-fold higher than in the untreated mice, or those which received a control oligo. In contrast, when we looked at the cII gene, the other reporter gene, we saw no effect of any oligo, because none of those - based on sequence - could have bound to this second reporter gene. There was no sequence that fit in the triple-helix binding code. We also tested some scrambled sequence of oligos, as controls, that were not targeted to either gene, and found no effect on either one."

From Dream To Therapy 'Years Away'

Although Glazer's long-term goal is genome-level gene therapy, he pointed out that, "We're years away from putting anything into a person. What we're thinking about in our dreams is that these triplex third strands - these oligos - could be part of a strategy to enhance correction of the single-base-pair or single-gene mutations that lead to certain diseases which, based on their properties, might be amenable to such correction.

"What pop into mind," he suggested, "are disorders related to hemoglobin, such as sickle cell anemia or even thalassemia. In such cases, you might be able to use this set of molecules on bone marrow cells, possibly ex vivo. That is, you might remove bone marrow from an affected patient, introduce these kinds of fancy oligos into their DNA in a certain manner, then return the bone marrow.

"Potentially," Glazer added, "this approach could also be used to treat certain diseases that might affect enzymes or proteins made in the liver, because the liver seems to take up a lot of this kind of DNA. A number of single-gene disorders, such as hemophilia, for example, lack clotting factors that are made in the liver. So that's another target.

"In those types of diseases," he pointed out, you might be able to obtain clinical amelioration by achieving only a small effect, I don't imagine in my wildest dreams that this triplex technology could achieve 100 percent gene targeting or correction in an animal model, or ex vivo in human cells. Right now we're at the level of 30 or 40 induced mutations in 100,000.

"In tissue culture cells," he said, "we can measure with these oligos about 1 percent targeting to a specific gene. So I think we're going to be in that range of a few percent." Therefore, Glazer noted, "I have to think about things like sickle cell anemia, where our study suggests that if 10 or 15 percent of the cells were synthesizing normal hemoglobin, it would make a therapeutic difference."