A hand-held remote controller can turn a TV set on and off fromacross the room. Gene therapists dream of a comparable controller toturn tissues or drugs on or off inside a patient.
Their dream is a bit closer to coming true some day, thanks to a"surprising" experiment, which plugged and unplugged ribozymes atwill, as reported in the current Proceedings of the National Academyof Sciences (PNAS), dated Oct. 15, 1996.
Ribozymes are short RNA nucleotides that act like conventionalprotein enzymes _ catalyzing and accelerating chemical reactions _but with certain potential advantages.
The PNAS paper's title suggests how the molecular on/off switchmight work: "Rescue of abasic hammerhead ribozymes by exogenousaddition of specific bases." Its senior author, biochemist DanielHerschlag, at Stanford University, sits on the scientific advisoryboard of Ribozyme Pharmaceuticals Inc. (RPI), in Boulder, Colo.
That company's vice president of research, nucleic acid chemistNassim Usman, is a co-author of the PNAS article. He told BioWorldToday: "We did the chemistry and Herschlag did the enzymology."
What they were able to do in vitro, Usman explained, "was to removeone entire base component from a certain class of ribozymes, calledhammerhead ribozymes, and show that you can add thosecomponents back in as a separate piece, and restore the ribozymes'function."
First they stripped out one or another of RNA's four nucleotide bases_ adenine, cytosine, guanine and uracil _ from 13 synthesizedvariant nucleotides that make up the catalytic core of hammerheadribozymes, (which are the smallest known RNA catalysts). Thosefour bases give RNA molecules their shape and specificity.
Thus spayed, the now-abasic ribozyme molecules lost all activity. AsHerschlag put it, "If you remove one piece of the puzzle, the wholething collapses."
But when he reinserted the subtracted bases, he told BioWorldToday, "the result was a great surprise to us. We didn't expect theexperiment to work, but when we added back the bases that had beenremoved, we were able to see rescue activity at four of the 13positions."
One "quite speculative but interesting" implication of this unexpectedoutcome, Herschlag continued, "is in the therapeutic realm,especially for people who are thinking about gene therapyapproaches. One of their biggest problems is how to keep somethingin the patient `off.' What we have been able to do now is makeribozymes, in which the same strategy works. You don't need to havean abasic, just natural RNA will do, to introduce a structural defect.For example, introducing a mismatch of a base pair, that can then berescued by adding back the missing base."
Extending this scenario, Herschlag then added: "That introduces thepossibility of having a ribozyme in vivo that's `off', and that you turnon either by adding a co-drug, or _ in another imaginative rendition_ it can even be designed to be turned on by a metabolite that'sprevalent in a disease tissue, such as a tumor or a viral infection. Youmight be able to build in some specificity for the target tissue."
On this score, Usman noted that "all standard anti-cancer drugs arepoisons; they all do the same thing. They shut down DNA replicationone way or another, and then the cells die. And hopefully, yourcancer cells are dividing much more quickly than your normal cells.
"What's unstandard about our ribozyme contribution is that you get asort of dual effect. Those normal cytotoxic agents could do their job,or they could help out the ribozymes to go and shut down harmfulmessenger RNAs that are causing the tumor."
Beyond such a cancer-specific performance, Usman continued,"there's no reason to believe you couldn't use it in another setting,where you want to have a drug that you turn on and off in general."To achieve this selective control, he suggested, "You give them aribozyme with one of the bases missing. Then you give thatseparately to the patient when you want to turn it on, and take it awayto turn it off. It's a neat trick."
Herschlag emphasized: "This is not something that's in the pipelinefor going into production," and observed, "I'm gratified when thingshave potential therapeutic spin-offs into the therapeutic side, but whatUsman and I focus on are the structure/function aspects of theribozyme molecule."
He pointed out that in their novel "subtractive mutagenesis" approachto functional analysis, "Instead of having to make 20 differentribozymes to test 20 different types of groups at a position _ a taskthat might take months _ basically, that experiment can be done nowin a day."
The Stanford biochemist noted that "three or four companies, and anumber of academic labs as well, are devoted to trying to useribozymes as therapeutics. I hope others will take our findings and tryto apply them in this field."
At RPI, Usman agreed. "I think the next step would be to repeat ourexperiment in cells. We've done it so far only with naked nucleotidesin vitro." His company, however, he pointed out, "has more urgentpriorities in its research clinical programs in place that we may haveto drive forward. We just can't do everything at once, and I've got abusiness to run here too." (See BioWorld Today, May 31, 1996, p.3.) n
-- David N. Leff Science Editor
(c) 1997 American Health Consultants. All rights reserved.