By David N. Leff

Like environmentally conscious families and forward-looking communities, nature recycles its genomic junk.

When a gene gets set to pull itself together from segments of its sequence - called exons - strewn along the genome, these leave behind the intervening segments, called introns. Presumably, they're junk to be discarded.

But that presumption - increasingly old hat of late - leaves evolution out of the equation. Today's issue of Science, dated July 21, 2000, reports a discovery tacitly implying that if nature had a bumper sticker, it would read "Waste not, want not." The paper is titled: "Group II introns designed to insert into therapeutically relevant DNA target sites in human cells."

Its senior author, molecular biologist Alan Lambowitz at the University of Texas, Austin, told BioWorld Today: "We discovered that intron RNAs move and duplicate themselves, and go from one place to another by inserting themselves directly into double-stranded DNA. It was not a mechanism that anyone had ever seen before.

"This new method for doing gene targeting," he added, "is based on the ability of group II introns to insert directly into double-stranded DNA by a mechanism in which it's possible to control the target site. Group II introns are found in lower organisms - in bacteria, fungi and also plants. And they turn up sporadically in a number of bacteria.

"These are catalytic introns," Lambowitz pointed out. "They can self-splice in vitro and act as ribozymes. Group IIs are believed to be the evolutionary ancestors of the introns found today in the nuclear genes of higher organisms.

"They are often considered 'junk DNA' - a phrase used frequently," he went on. "But it's a type of junk, at least for group II introns, that can reproduce itself and move itself from one gene into another. Introns can spread and multiply in genomes - but respect the policy that it's desirable not to kill the host you're residing in.

"In fact," Lambowitz observed, "introns are very polite. They're found in the middle of gene-coding sequences, then just remove themselves and ligate the exons together."

He explained this insertion trick: "The intron encodes a protein, which turns out to be a reverse transcriptase. So the first step results in an RNA integrated between two DNA exons. Then that protein just copies the intron RNA back into double-stranded DNA."

Turning Introns Loose Against AIDS Virus

To test the intron's precise gene-targeting ability, Lambowitz and his co-authors carried out experiments on the HIV provirus, and its co-conspirator in penetration of the T-cell - the human CCR5 receptor. "CCR5," he pointed out, "is an important target site in HIV therapy. Individuals who are defective in this gene are naturally resistant to the virus, so if you can knock it out, then you can confer immunity to AIDS.

"Our ultimate plan for doing that," Lambowitz said, "is to use a particular intron to try to knock out the CCR5 gene in T cells isolated from AIDS-infected individuals. Then we would reintroduce them into the patients, thus providing them with a population of T cells resistant to HIV. We're doing this in collaboration with other people at the university, here in Austin.

"What we have yet to show in the next phase of our work," he added, "is that we can actually target the introns to chromosomal genes in humans." (Lambowitz is a professor, and director of the university's Institute of Cellular and Molecular Biology.)

"The procedure works very well for chromosomal gene targeting in bacteria," he recounted, "and there are a number of bacterial applications that are possible now, especially in functional genomics and genetic engineering. These introns have a very wide host range; the same one is functional in both Gram-negative and Gram-positive bacteria.

"A lot of bacterial genomes have been sequenced recently," Lambowitz pointed out, "so it's desirable to have an efficient way of knocking out genes to assess their functions. And introns offer a very efficient way of doing that in a wide variety of microorganisms, including pathogenic bacteria, where there are no good genetic systems.

"In addition," he continued, "we could also use them to introduce genes into bacteria for production purposes, or to knock out specific genes that may interfere with certain commercial processes. For example, one thing that we're doing, in collaboration with people at the University of California-Davis, "is to try to engineer wine-producing strains of bacteria, so that introns delete genes that are deleterious for wine production."

Reverting to human therapy, Lambowitz observed, "We also hope to make attenuated bacterial strains for vaccines by doing gene knockouts. We have some experiments planned in collaboration with people at the Wadsworth Center of the New York State Health Department in Albany to target specific genes for Mycobacterium tuberculosis, which causes TB pathogenicity.

"If this intron approach works as planned," he said, "then it is potentially applicable to gene therapy for a wide variety of diseases - knocking out deleterious genes, like oncogenes, and incorporating functional copies of required genes.

"Unlike random incorporation, this affords the opportunity to target a specific location, so there's no danger that the insertion will do something harmful to the cell - for example by inadvertently inserting the intron into a tumor suppressor gene, or activating an oncogene. In addition the approach could be used for antiviral therapies, for DNA viruses, such as herpes, hepatitis B, human papilloma virus associated with cervical cancer. These viruses could all be potentially inactivated by directing introns to insert into them."

Back To The Future - Bound For Phase I

"Getting efficient chromosomal insertion of the introns requires developing methods for efficient introduction into animal cells or human cells," he said. "We're really gearing up now to try microinjection, and actively pursuing in vitro expression of the introns. Once that's accomplished, then we would really be in a position to proceed to Phase I clinical trials."

The university holds five U.S. patents on the intron technology, issued between December 1998 and February 2000, with additional filings pending. "The claims are very broad," Lambowitz observed. "Within the last two months," he added," the patents have been licensed to a small start-up company called InGex LLC in St. Louis. One option is to sell products based on the technology," he concluded, "and I think that is part of their business plan."

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