Whenever an 18-wheeler rolls over across a through-way at rushhour, traffic backs up for miles. By a similar but smaller token, if atooth breaks loose halfway down a zipper, that garment is stuck.
And on a submicroscopic scale, certain gene mutations create a false"stop" signal along a stretch of RNA that's busy translating anencoded protein.
When this premature termination happens on the cystic fibrosistransmembrane conductance regulator gene, CFTR, it stopsexpression of that protein dead in its ribosomal tracks.
Among the 400-odd gene mutations discovered so far that cause CF,the widest spread is one designated DF508; it knocks out the aminoacid phenylalanine from the healthy CFTR protein. That mutant aloneis responsible for 70 percent of all CF cases.
Three other mutations perpetrate unwanted stop codons, whichexpress those functionless, amputated proteins. Two such mutant stopcodons account for five percent of all CF cases. The third afflicts 60percent of a particular population group _ Jewish people of EasternEuropean Ashkenazi genetic origin.
Whatever the mutational etiology, CF patients all suffer from thesame suffocating symptomology _ lungs clogged with thick mucus.Besides its strangulating effect, this mucus build-up renders the lungshelpless against life-threatening infection by a particularly predatorystrain of the Pseudomonas aeruginosa bacterium. (See BioWorldToday, Jan. 5, 1996, p. 1.)
Which is why patients inhale an aerosolized antibiotic, gentamycin,to fight off P. aeruginosa.
Gentamycin belongs to a class of antibiotics called aminoglycosides.Other members include streptomycin, neomycin and tobramycin.
From Fighting Infection To Frustrating Mutation
At the University of Alabama in Birmingham, molecular geneticistDavid Bedwell mused over the known fact that aminoglycosides alsohave a specific genomic property: they knock out stop codons. "Thatraises the intriguing possibility," he told BioWorld Today, "thatbesides killing pseudomonas, which it normally does in CF patients,gentamycin could be inducing some low-level expression of therestored full-length functioning protein.
"The normal stop-codon mechanism," Bedwell explained,"terminates translation in the ribosome, where the process is carriedout. A premature stop mutation blocks that read-through process partway. What the aminoglycoside antibiotics appear to be doing," headded, "is allowing synthesis of the protein to go all the way to theend, by misincorporating an amino acid at the stop codon's position."
Accordingly, he and Alabama physiologist Raymond Frizzell set up aseries of in vitro experiments to test the ability of these antibiotics tosquelch the free-loading stop codons on CFTR genes, andpresumably correct the symptoms of the disease.
Their report of this work appears today in the April issue of NatureMedicine. Its title: "Aminoglycoside antibiotics restore CFTRfunction by overcoming premature stop mutations."
Bedwell summed up: "We tested two antibiotics that appeared toshow some activity, one weak, the other strong. The strong one,unfortunately," he went on, "is an experimental aminoglycoside, G-418. It's used for cell culture in research labs, but is not approved forhuman use.
"The other antibiotic," he continued, "is gentamycin. It was veryweak, but at least we could see the full-length protein it corrected."
Bedwell has received approval for a clinical trial of gentamycin, totest its ability to abolish the mid-stream mutant stop codons in thecells of CF patients. "G-418," he observed, "would be more ideal butwe have to go through a rigorous approval process before we can dohuman studies on that drug."
Human Trials This Year
The regional CF centers around Birmingham have a number ofpatients with premature-stop mutations. In subjects drawn from thiscohort, Bedwell said, "What we're going to do initially is putgentamycin in their nose, and look for a change in the nasal electricpotential difference measurements." This determines the expressionof functioning protein.
He explained: "It turns out that nasal epithelium is very similar tolung epithelia. So it's easier to show correction in the nose than in thelung _ and the nose is less invasive too."
Testing his human protocol will begin "within a few months."
The antibiotic will be sprayed into one nostril, with the other as acontrol. The trial, Bedwell said, "will also compare patients with astop codon against people carrying other CF mutations.
But Bedwell doesn't stop there. "Any loss-of-function disease," hepointed out, "could essentially be caused by premature stops. Takecancer: "Many genes can get mutations that cause cancer, p53 forexample. One could essentially use this aminoglycoside approach tosuppress the five percent of premature stop mutations."
Frizzell, and the Nature Medicine paper's first author, molecularbiologist Marybeth Howard, moved six months ago fromBirmingham to the University of Pittsburg. There, Frizell chairs thedepartment of cell biology and physiology. "I'm really a channelperson," he told BioWorld Today, "who became interested in CFbecause of its ion-channel-defect implication."
Still pursuing the antibiotic strategy for stopping stop codons, hesaid, "We have been constructing an assay system that will allow usto screen different aminoglycosides for inducing read-through. Weare forming connections with several pharmaceutical companiesinvolved in developing these antibiotics," Frizzell continued, "to tryto obtain compounds that would promote read-through, yet berelatively safe."
As for human trials, "one simply needs to obtain the appropriateapprovals, and we have a clinical protocol under consideration byPittsburgh's internal review board."
He and Bedwell both view their antibiotic approach as a validalternative to gene therapy for CF, which has yet to prove itself. "Idon't think our aminoglycoside strategy interfaces very much withthe gene-delivery aspects of treating the disease," Frizzell observed."I think this work would be more of interest to the pharmaceuticalindustry, which has thousands of aminoglycoside antibiotics sittingon its shelves, than to the biotechnology community."
Bedwell made the point that "Pharmacology as an alternative to genetherapy might work faster, at least in the short term. I think that's asmart way to think about it." n
-- David N. Leff Science Editor
(c) 1997 American Health Consultants. All rights reserved.