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Introducing Proteins Into the Body's Cells

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Science  03 Sep 1999:
Vol. 285, Issue 5433, pp. 1466b-1467b
DOI: 10.1126/science.285.5433.1466b

To pharmaceutical chemists and basic researchers, proteins are a bit like protégés who never quite fulfill their potential. Despite their wealth of biochemical talents, they generally lack the one skill scientists need to put those talents to work: the ability to make their way through the fatty membrane that surrounds cells. One answer is to coax the target cells to make the protein themselves, by inserting the corresponding gene, but so far no one's figured out how to deliver nucleic acids efficiently to cells in animals or humans. Now researchers may have hit on a powerful strategy: fusing foreign proteins to a segment of another protein, derived from the AIDS virus, that has an unusual ability to cross cell membranes.

On page 1569, molecular biologist Steven Dowdy of Washington University School of Medicine in St. Louis and his colleagues report that such tagged molecules can infiltrate all the tissues of living mice. “This is an entirely novel and apparently powerful approach for introducing proteins into the brain and throughout the body,” says Raymond Bartus, a neuroscientist at Alkermes Inc. in Cambridge, Massachusetts.

If the method works with other proteins, it might be used to combat inherited diseases and other conditions caused by a malfunctioning or absent intracellular protein. Researchers might, for example, introduce a tumor suppressor gene into cancer cells to help stop their abnormal growth or to add back the enzyme that's defective in the hereditary neurodegenerative disease, Tay-Sachs disease. “It really is intriguing and unexpected … that you can get proteins so pervasively into cells,” says Bert Vogelstein, a cancer geneticist at Johns Hopkins University School of Medicine in Baltimore. Still, Bartus cautions, “a lot of the details have to be worked out, and it will take some time before [the method] is harnessed for therapy in humans.”

To devise the method, Dowdy and his colleagues exploited the 10-year-old discovery that an AIDS virus protein known as TAT (for trans-activating protein) enters cells without aids such as cell surface receptors. Researchers don't know how TAT does that, but in 1994, investigators at Biogen in Cambridge, Massachusetts, showed that it could ferry other proteins into cells. They chemically attached a bacterial enzyme called β-galactosidase to a large piece of TAT that included its “protein transduction domain” (PTD), a stretch of 11 amino acids that helps TAT traverse the cell membrane. When they injected the cross-linked protein into mice, they detected hints of its presence in several tissues. “[The method] was inefficient, but it did work,” Dowdy recalls. “We thought to ourselves, ‘This has tremendous merit’ and picked up the literature trail.”

To try to improve the efficiency, the group took what Dowdy calls a “biochemically blasphemous” approach. Unfolded, “denatured” proteins lose their activity. But reasoning that a partially unfolded protein would have more of its oily interior amino acids exposed and might therefore slide more easily through the lipid-rich cell membrane, the researchers denatured test proteins that carried the TAT PTD before incubating them with cultured cells. As the group reported last December in Nature Medicine, denatured PTD-containing proteins enter cells more efficiently than do the native versions. “Other molecules in the neighborhood don't go in, and nothing appears to leak out,” says Dowdy. But with the denatured protein and its attached PTD, “it's like the parting of the Red Sea. No one knows how it happens.”

The group has used this strategy to transport over 50 proteins ranging widely in size into a variety of human and mouse cell types in culture. Once inside, they regain their activity, presumably because they can access the cell's normal protein-folding machinery, says Dowdy. Now the team has extended the method to live animals.

Steven Schwarze, a postdoc in Dowdy's lab, engineered a protein that contains the PTD from the TAT protein attached to β-galactosidase. After partially unfolding the protein, the team injected it into the abdominal cavities of mice, while control animals got a version without the TAT sequence. Four hours later, the researchers found little or no detectable β-galactosidase in the tissues of the controls. But the protein joined to the TAT PTD showed up throughout every tissue they looked at—blood, spleen, liver, kidney, heart, lung, and even brain—and it had regained its enzymatic activity. “Not only do you know that the whole protein got in, but you know it refolded properly,” says Joan Brugge, a cell biologist at Harvard Medical School in Boston.

The technique should give basic researchers an extremely efficient way of introducing proteins into cultured cells to see how they affect cell function. And ultimately it might be used in treating human diseases as well. But as Bartus and others point out, there are potential pitfalls. The PTD could elicit an immune response or the method could produce other toxic effects, although no signs of problems have appeared yet. And the very efficiency of the method could cause trouble. “One important issue is that if there's a spill, the aerosol could be taken up by the lungs and then spread quickly in the body,” Brugge says. “So for experimental use, investigators have to be really careful.”

Dowdy says that it probably won't be possible to target proteins carrying the TAT PTD to particular cells, but the group has already begun to cope with the delivery system's promiscuity by designing proteins to act only in certain cellular environments. As scientists tune the basic scheme, they'll no doubt find many ways to help proteins reach their full potential.

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