By David N. Leff
A drunken driver, whose out-of-control pickup truck killed 27 people on a church bus, was released Thursday from a Kentucky prison after serving more than nine years of a 16-year sentence for manslaughter. He got that time off for good behavior behind the bars - signs that the convict had become a reformed character.
Prisons have long called themselves reformatories, houses of correction, penitentiaries. Nowadays, people convicted of crimes are often condemned to community service instead of incarceration.
It's high time that one of the world's most egregious serial killers - the AIDS virus - got a chance to do some good works to atone for its pandemic mayhem. Today's issue of Science, dated Sept. 3, 1999, reports research that gives HIV a role in saving, rather than taking, human lives. The paper's title - "In vivo protein transduction: Delivery of a biologically active protein into the mouse" - scarcely hints at the viral benefactor that made this potential therapy possible.
The name of that experimental molecular do-gooder is an HIV protein named TAT - standing for transactivating protein. Molecular oncologist Steven Dowdy, the Science paper's senior author, abducted that TAT sequence from HIV's genome in order to smuggle outsize proteins into target cells for therapeutic purposes. Dowdy is a Howard Hughes Medical Institute investigator at Washington University in St. Louis. (See BioWorld Today, Jan. 8, 1999, p. 1.)
"What that HIV TAT protein is supposed to do biologically in terms of virus production," he told BioWorld Today, "is, in some unknown way, to stabilize the viral message. But TAT was never evolutionarily selected for its ability to transduce and enter cells."
However, when an HIV virion breaks and enters its target T cell, TAT goes in along with the other elements of the viral genome. Some scientists had surmised that it serves to activate the host's immune system cells, thus making it possible for the virus to replicate.
Dowdy and his co-authors saw the TAT protein as a molecular locomotive for hauling large proteins of interest into a cell, across its fatty outer membrane, rather than as a battering ram for forcing their freight's entry. They were looking for a way to break the logjam that frustrates drug designers seeking to squeeze sizeable candidate compounds through cell walls.
Resurrecting Discarded Oversized Drugs
"This paper," Dowdy pointed out, "demonstrates the potential of being able to introduce not only protein transduction therapy - in terms of cancers, infectious, neurodegenerative and hereditary diseases - but the ability to deliver prospective molecules that pharmaceutical companies have on their shelves. In fact," he added, "when they do a drug screening, they throw away 70 percent of the compounds because they're too big. Most drug-discovery firms don't look at anything higher than 500 Daltons [units of atomic mass] because it just couldn't enter the cells with any efficiency to treat the disease."
Like a prestidigitator transducing a rabbit from a hat, Dowdy assembled a molecular freight train that transported an unprecedentedly gigantic protein - bacterial beta-galactosidase - into virtually every cell of a mouse's body. "We used the mouse for obvious reasons," he explained, "as the preferred genetic model for human disease.
"We chose beta-gal," he went on, "because it's an easy enzyme to assay for. At 120,000 Daltons, it contains over 1,000 amino acids, whereas the current bioavailability inside of a human body is five or six amino acids - that's 500 or 600 Daltons at most. So beta-gal is 200 times larger than the current pharmaceutical bioavailability wall. Beta-galactoside's enzymic activity consists in cleaving complex carbohydrates. When the protein's X-galactosidase substrate is cleaved to its colorimetric form, its surroundings turn blue."
The team coupled its immense string of beta-gal container cars to a stripped down, synthetic 11-amino-acid segment of HIV's TAT protein, and injected this protein package into the abdominal cavities of mice.
In every mouse cell type they tested, they found, within minutes of injection, blue evidence of the exploratory enzyme's penetration.
"In the brain," Dowdy pointed out, "it takes longer to detect enzymatic activity. At two hour, we could see it principally around the cerebral blood vessels. At four hours - with maximum enzymatic activity, not just transduction - the entire brains were completely blue. So you're not blue unless you have beta-gal activity there. Then at eight hours, the enzyme localized to the cerebral cell bodies, apparently because of nuclear localization on the protein.
"So there are two rate-limiting steps here," Dowdy noted. "First, transduction; then refolding the protein to its active form." He made the point that the cerebral dissemination of the enzyme did not break through the blood-brain barrier. "It behaved like a self-sealing tire," he recalled. "We call it 'the gamma ray,' because it just goes through.
"So if you're interested in doing experiments in animals," the Washington University scientist observed, "especially for eventual protein transduction therapy in humans, you want to leave that barrier intact. And one critical aspect of this paper," he said, "is that the protein was refolded and enzymatically active in every tissue of the body we assayed, including across the blood-brain barrier."
Poised To Treat Genome Project's Disease Genes
"The beauty of transduction," Dowdy pointed out, "is that it can deliver extremely large proteins. Transduction does not require energy, doesn't require ATP, or a transporter or a reporter. It appears to interact directly with the lipid bilayer of the cell membrane, and goes right through it - literally like parting of the Red Sea waters.
"The human genome," he reflected, "will be sequenced in a year, and give us four or five thousand disease genes that are important for therapy in humans. How are we going to deliver them? By gene therapy? It's got a big efficiency wall. It can't get even close to reaching 100 percent of target cells. And in two to four months after treatment, most of those cells will have dropped their expression to nothing, or very low levels.
"So gene therapy," Dowdy continued, "has two big problems, which have existed for 10 years. I think they're solvable, and once those problems are solved, you can cure disease in one shot. On the other hand, we know with this Science paper that protein transduction can express very big molecules in 100 percent of target cells. And," he concluded, "I don't think 120,000 Daltons is the upper limit."