Full size image available through contact |
St. Louis, Sept. 3, 1999--For decades, pharmaceutical companies have struggled to overcome the molecular equivalent of the Great Wall of China: the outer membrane of cells, which prevents all but the tiniest of proteins from entering. Now researchers have slipped a protein that's more than 200 times larger than the average drug into the cells of living mice and shown that the protein functions.
"For the very first time, we've introduced a large, biologically active protein into every cell of the body-- including cells in the brain that are normally protected by the blood-brain barrier," says Steven F. Dowdy, Ph.D., who led the research team at Washington University School of Medicine in St. Louis. The group published its results in today's issue of Science.
Dowdy is an assistant investigator of the Howard Hughes Medical Institute and an assistant professor of pathology and medicine. Steven R. Schwarze, Ph.D., a postdoctoral fellow in his laboratory, was lead author of the paper.
Getting full-sized, therapeutic proteins into cells would be advantageous because smaller drugs tend to interact with unintended targets. Larger proteins fit only onto the molecules for which they were designed, so they could be given in substantially lower doses, resulting in fewer side effects.
Dowdy led a previous research team that used test-tube experiments to smuggle an enzyme into HIV-infected cells. The results, reported in Nature Medicine last December and January focused on a human enzyme that makes cells self-destruct. The enzyme was modified to include a string of 11 amino acids that served as a passport for crossing a cell's outer membrane. But the researchers needed to prove that large proteins could slip into cells in model animals before considering human applications.
In the Science study, Dowdy and fellow investigators first attached a molecular passport known as a protein transduction domain (PTD) to a compound whose uptake by cells could be monitored. The compound was a dye called fluorescein, which turns green when exposed to fluorescent lighting. It normally doesn't enter cells because of its size-- 2,000 daltons compared with the 500 dalton limit placed on most drugs.
The researchers injected mice with this combined PTD-fluorescein protein and isolated cells from the animals' blood and spleen. All the cells fluoresced green when checked 20 minutes after the injection. Cells in muscle and brain tissue also had soaked up the combined protein. "It was very encouraging to discover that we could get a mouse with an entirely green brain," Dowdy says, noting that the blood-brain barrier, a layer of cells lining the brain's blood vessels, normally prevents most proteins from entering.
Dowdy and colleagues then linked a bacterial enzyme to the PTD and fluorescein. Their fluorescent analysis revealed that the 120,000 dalton enzyme, beta-galactosidase, entered all the cell types tested.
Beta-galactosidase was chosen because its activity could reveal whether an enzyme could continue to function after it had been transported into cells by the PTD. Cells take up proteins better if they are at least partially unfoled, as was true for the enzyme.
Dowdy's team tested whether beta-galactosidase trapped inside cells of injected mice converted the enzyme's clear chemical target into a blue dye. A vibrant blue image of the kidney of the first mouse tissue they evaluated is pinned on a bulletin board above Dowdy's desk. "Once we got this first result, we realized that the protein would be biologically active in the rest of the body," Dowdy says.
The liver, lung, and other tissues of the injected mice also turned blue when exposed to the enzyme's target. The animals' entire brains also stained blue within four hours of injection, indicating that a lot of the bacterial enzyme had refolded there by then. Importantly, the PTD didn't work its magic in the brain by destroying the blood-brain barrier. And the animals had no visible behavioral changes or other differences compared with untreated mice.
Dowdy has since tested modified versions of the PTD that should allow proteins to enter brain cells and other cells even more rapidly. And he is using his new technology to determine whether a malfunctioning protein helps jump-start cancer.
He notes that the laboratory's protein-targeting technology also may enable companies to create drugs that act only in disease-related cells, opening up a completely novel avenue of therapeutic approaches. "We can now do things in normal cells of mice that you could never even dream of doing with any reliability a year ago," Dowdy says.
GRAPHIC: Image of mouse brain tissue showing active beta-galactosidase available upon request.
Schwarze SR, Ho A, Vocero-Akbani A, Dowdy SF. In Vivo Protein Transduction: Delivery of a Biologically Active Protein into the Mouse. Science, 285, 1569-1572. Sept. 3, 1999.
This research was supported by the Howard Hughes Medical Institute and the National Institutes of Health.
The technology from Dowdy's laboratory has been licensed to IDUN Pharmaceuticals of La Jolla, Calif., and Life Technologies of Rockville, Md.
Journal
Science