In a classic atomic bomb, the uranium-isotope payload exists in twoseparate unequal chunks. Upon firing, a detonator mechanism ramsthe smaller charge into the larger one. This critical mass then sets offthe explosive chain reaction.

A somewhat similar set-up animates a system that proposes toresuscitate the flagging field of gene therapy by controlling DNAexpression in vivo. This concept, just validated in mice, relies on asmall, orally bioavailable molecule to achieve critical mass bybridging the two halves of the transcription-factor protein complexthat sets off DNA expression.

Ariad Gene Therapeutics Inc. (AGTI), of Cambridge, Mass., hasnearly completed synthesis of that small molecule in the form of a pillsuitable for human gene therapy protocols. That drug, rapamycin,already is in clinical studies elsewhere as an immunosuppressant, toprevent graft rejection of organ transplants.

Just as that atomic bomb detonator pushed together the two separatealiquots of uranium, so rapamycin connects up two molecularcomponents in the immune system that destroyed its ability toactivate T lymphocytes in its own defense.

Rapamycin actually got its start in life as an antifungal. A soilbacterium, Streptomycetes hygroscopicus, secretes the molecule todisarm its natural enemies, such as fungi.

In short, rapamycin is capable of dimerization _ doubling up twoprotein molecules to act as one.

AGTI is converting this secret weapon deployed by rapamycin into apill that gene therapy patients may some day swallow to regulateexpression of their previously inserted therapeutic transgenes.

An article in the September 1996 Nature Medicine reports the first invivo trial of this concept under the title: "A humanized system forpharmacologic control of gene expression." Its senior author ismolecular biologist Michael Gilman, scientific director of AGTI, asubsidiary of Ariad Pharmaceuticals Inc.

Moving Gene Therapy From Experimental To Routine

"Ariad started this project," Gilman told BioWorld Today, "becausewe believed that one of the key missing pieces of technology holdingback the gene therapy field was the ability to regulate the dosing ofthe transgenes. Obviously," he continued, "all the medicines that youand I take are subject to dosage control, and genes are going to be nodifferent. There has to be a way to terminate and restart therapy,which is difficult to do in current gene therapies, given existingtechnologies."

Ariad's technology, Gilman explained, "takes a transcription factor _the proteins inside cells that normally are in the business of turninggenes on and off _ and simply breaks it conceptually into two pieces,so it doesn't work any more. And then uses rapamycin to glue thosepieces back together again."

In actual practice, the Ariad team synthesized two individualfragments. "Your basic transcription factor is composed of twodomains, Gilman went on. "One is the so-called DNA-bindingdomain; its job is to bring the protein to the target gene in thenucleus. The second, the activation domain, is then responsible forkick-starting the transcription apparatus."

With these two synthetic recombinant genes in hand, the team thenmade a third construct, which contained their target gene therapysequence, recombinant human growth hormone, and packaged allthree constructs into a human fibrosarcoma cell line.

These two engineered transcription-factor fragments, Gilman said,"float about in the nucleus of the cell, and in the absence of drug arenever really aware of one another. But once those cells seerapamycin, the drug can bind simultaneously to both, with its DNAbinding proteins on the one hand; its activation-domain proteins onthe other. Thereby, rapamycin can assemble an active transcriptionfactor, which now will switch on the target gene."

That's what it did in the mouse demonstrator experiment described inNature Medicine.

System Works In Rodents

Nude mice, which can't reject foreign cells, received injections of thetriple-gene engineered cells in their leg muscles. After a single shotof synthetic rapamycin to a tail vein, they promptly began expressinghuman growth hormone in their bloodstream. This secretioncontinued for 48 hours, whereas conventionally delivered humangrowth hormone has a half-life in mice of only three minutes.

When drug dosage stopped, so did production of the transgenehormone.

"There is a very nice dose relationship," Gilman observed, "betweenthe amount of drug given the animals and the amount of growthhormone that appears in their blood." He added, "if you take therapamycin away, it decays from the bloodstream with a half-life ofabout 11 hours in animals, considerably longer in humans."

Trials in humans await two challenges, Gilman said, one technical,the other, business-related. He explained:

"The business challenge for us is to select a delivery technology,either developed in-house or through a partner, and then actuallymove toward clinical trials.

"It's hard to say with certainty," he added, "what kind of study wouldcome first, because it will depend a lot on the partner we end upworking with. I would say that growth hormone is very high on thelist, because it has applications outside the dwarfism area, such as forcachexia in AIDS and cancer patients."

Two technical challenges, he said, are near solution: creating arapamycin analogue stripped of its immunosuppressive properties,and realizing its inherent potential for oral administration. The latteralready has checked out in mice.

Ariad, Gilman revealed, "now is speaking to many of the majorbiotech and pharmaceutical companies that have an active interest ingene therapy. Also we spend a lot of time talking to other smallbiotech companies who specialize in gene therapy, and who haveessentially complementary technology to ours." n

-- David N. Leff Science Editor

(c) 1997 American Health Consultants. All rights reserved.