In old-time wars which gave starring roles to cavalry, forwant of a horseshoe nail, the rider and hence the battle,was lost. In more recent, higher-tech conflicts, loss of asingle letter in deciphering a secret alphabet code mightsink a submarine.
Likewise, in the genetic code, an A (for adenosine)instead of a G (Guanine) in a strand of RNA expressing adystrophin gene spells the difference between health andmuscular dystrophy. Such point mutations abound inhereditary diseases, from cystic fibrosis to sickle-cellanemia to cancers.
Gene therapy and antisense are only two of the strategiesbeing honed to correct the dire clinical consequences ofthese inherited or acquired single-letter mutations. NowRibozyme Pharmaceuticals Inc., of Boulder, Colo., abiotech company that specializes in ribozyme therapies,has demonstrated an alternative tactic.
"Toward the therapeutic editing of mutated RNAsequences" is the title of the company's paper in thecurrent Proceedings of the National Academy ofSciences, dated Aug. 29, 1995. Its senior author, DanStinchcomb, is Ribozyme's director of biologicalresearch.
"This is a potential means for correcting defectivemessenger RNA target," Stinchcomb told BioWorldToday, "which one couldn't do by current ribozyme orantisense technologies per se."
Molecular Biologist Tod Wolf, the paper's primaryauthor, led the group at Ribozyme that mounted what hedescribed as "the first published example of this sort ofapproach."
They began, he told BioWorld Today, by synthesizing themRNA of a dystrophin gene containing a stop codon _an unwanted break in expressing the protein, created by amutant UAG base triplet where a correct sequence, suchas UGG, belonged. This point mutation afflicts a raresubset of muscular dystrophy patients.
"We chose this stop codon mutation," Wolf said,"because it provided us an easy model in which to set upa luminescent reporter gene, allowing us to detect theediting."
He and his co-authors then targeted this broken piece ofmRNA by hybridization _ base-pairing with a syntheticcomplementary oligonucleotide he calls "our fix-it pieceof RNA."
From cell nuclei they extracted an enzyme, double-stranded RNA adenosine deaminase. "This commonenzyme," Wolf explained, "recognizes the fact that theseRNAs have met, and starts changing the sequence in thatregion. By targeting this hybridization event, we targeteda cellular event to exploit it, to fix the broken gene beforeit got translated in the ribosome."
That fix worked in 35 percent of the mutated molecules,replacing the errant A in their triplet codons with themissing G. "The 35 percent," Wolf observed, "would befine for most diseases. Look at adenosine deaminase,missing in SCID [severe combined immune deficiency].Replacing a few percent of that enzyme's normal activitycorrected the disease."
He continued: "Most genes that you're missing, if youreplace 10 or 20 percent, you'll be normal. It's only whenit's totally gone that you're very sick. We were happywith 35 percent."
RNA Editing Is A Transient Correction
Wolf points out that this therapeutic editing of RNA "hasnothing to do with gene therapy. It does not involvechanging the genes, which are DNA." He added: "It's likea drug. You take it; it works while it's there. You stoptaking it; it goes away.
"RNA is transient," he said. "That's the major point ofthe thing. It's a transient way of correcting genes, asopposed to those more radical, surgical-like techniques ofgene therapy."
To which Stinchcomb added: "At the end of a very longrainbow, leading perhaps to a clinical pot of gold, onecould use either a gene therapy approach that wouldexpress the desired correcting RNA, or directly apply itand edit out the defective stop-codon message in themuscular dystrophy patient."
He made the point, however, that "For musculardystrophy, one would have to deliver the edited sequencein vivo into a sufficient number of muscle cells toeffectively correct the defect. This therapeutic editingtechnology has the same delivery hurdles that ribozymeand oligonucleotide and gene therapy face."
Looking far ahead, Stinchcomb said that "any A to Gsubstitution would appear to be theoretically correctable,from oncogene mutations to tumor suppressors."Meanwhile, he emphasized, "a lot of proof-of-principleremains to be done. One has to show it in mammaliancells, attain the best specificity and activity to correct asufficient number of the messages that would providesome wild-type [non-mutated] function."
Wolf said that this "fancy thing we found in the course ofresearch recalls a time before they had ribozymes orantisense or gene therapy, and somebody might have said,`You can throw a gene in a cell and cure diseases.' That'swhere this therapeutic editing is at." n
-- David N. Leff Science Editor
(c) 1997 American Health Consultants. All rights reserved.