Frontiers Fall 1998

After 15 million years, Sleeping Beauty awakes

U researchers have reconstructed a once-defunct fish gene that opens doors to better gene therapy and new methods of genetic research

by Deane Morrison

Most cells are like children: They won't swallow anything they don't like. Sugarcoating usually does the trick with children, but scientists trying to get genes into cells – let alone into chromosomes – must resort to subterfuges like hiding the gene in a virus that infects the target cells. Viral infections are risky, but without a transport vehicle for genetic material, scientists have little hope of replacing defective genes with good ones or modifying chromosomes in other ways that will reveal how the genetic machinery works.

Now, a new vehicle is coming off the assembly line, thanks to Perry Hackett, a professor in the Department of Genetics and Cell Biology, and his colleagues. It's called Sleeping Beauty because it represents a "reawakening" of a once – defunct fish gene that had lain dormant for about 15 million years.

"We have to have a system to deliver genes to cells," says Hackett. "We believe we've developed a nonviral system to enhance integration of foreign genes into human chromosomes. It may be an alternative to using viruses." Hackett developed Sleeping Beauty with collaborators Zsuzsanna Izsvák and Zoltán Ivics.

In beginning the work, Hackett asked whether the chromosomes of fish possessed anything like certain DNA sequences found in simple animals like worms and flies. Known as transposable elements, or transposons, those elements can hop from one chromosome to another. They do it by directing cells to make a special enzyme that cuts the transposon free and moves it to a new chromosomal address. But studies of vertebrates had never yielded such functional DNA sequences – only remnants of elements that were active many millions of years ago.

Examining the genetic makeup of many species of fish, Hackett's group found recurring patterns of DNA that looked enough like known transposons – albeit heavily mutated and nonfunctional transposons – that they felt confident they could make what they were looking for. The researchers used statistical methods to figure out which of those patterns were probably part of the ancient, functioning fish transposon and, through a process that essentially turned back the evolutionary clock, used them to construct a new one, which Izsvák named Sleeping Beauty.

To test the system, they stitched genes for antibiotic resistance into the transposon and treated cells of zebrafish (a common experimental animal for geneticists) and humans. Sure enough, the genes were transferred and cells acquired antibiotic resistance, a sign that Sleeping Beauty had delivered its cargo. The first known vertebrate transposon was born.

Sleeping Beauty has garnered Hackett a lot of attention in the year since he published an account of it. Yet he came to the study of fish genetics reluctantly about 13 years ago, when he was persuaded by Tony Faras, then head of the Medical School's Institute of Human Genetics, and Kevin Guise of the animal science department. Guise and Faras announced they were going to genetically engineer fish to grow bigger for sport fishing and aquaculture purposes and asked for Hackett's help.

"Kevin and Tony told me this would be my chance to explain to my parents just exactly what I was doing," Hackett says. He joined on, with Anne Kapuscinski of the fisheries and wildlife department as the project head. Working with walleye, northern pike, salmon, and trout, the team managed to produce some bigger fish. But the Minnesota Environmental Quality Board issued rules that effectively prevented the fish from being kept in outdoor facilities, and Hackett decided it was time to get out of the aquaculture business. By 1996 the last of the "superfish" were gone.

One spinoff from the project remained: molecular genetics in zebrafish.

Although genetic engineering of commercial fish seemed out of the question, genetically engineered zebrafish weren't seen as an ecological threat, says Hackett. Therefore, zebrafish made an appealing organism for work aimed at improving the study of fish genetics. Zebrafish were brought into the aquaculture project because they spawn much more frequently than local sport fish and so more quickly display the results of genetic engineering. They proved valuable for all sorts of genetic research and so were chosen for the Sleeping Beauty work.

Sleeping Beauty may someday see a variety of uses. For example, it could help in determining what genes do. That would work as follows: If the transposon inserts itself into the DNA of an animal, it might land in the middle of a gene and inactivate the gene, causing a defect. By locating Sleeping Beauty in DNA from the animal, researchers could remove the inactivated gene for further study. Meanwhile, the nature of the defect would give a clue as to what function the disrupted gene normally performed. Sleeping Beauty may also one day be used for gene therapy, in which healthy genes are delivered to cells with genetic defects as a means to cure disease or birth defects.

Several other University researchers are collaborating with Hackett to further develop the Sleeping Beauty system. Steve Ekker, a developmental biologist in the biochemistry department, is working with Hackett to improve Sleeping Beauty and use it to discover the function of genes involved in embryonic development. David Largaespada, a 1986 CBS graduate and assistant professor of laboratory medicine and pathology, wants to use it to identify genes that suppress the development of cancerous tumors.

Scott McIvor, director of the University's Gene Therapy Program, is testing the system's ability to transfer genes to cells of bone marrow, lung, and liver. "It could be a way of efficiently introducing new therapeutic genes without viruses," he says. Clifford Steer and Mendel Tuchman in the Medical School are also collaborating in determining the efficacy of the transposon system for human gene therapy.

As the transposon undergoes more study and refinement, its potential uses are bound to multiply. Certainly Hackett has his work cut out for him. And there'll be no rest for Sleeping Beauty.

Ekker, Hackett, Largaespada, and McIvor will all be faculty of the new Department of Genetics, Cell Biology, and Development effective July 1, 1999.

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