Recombinant human insulin hit the drug stores 14 years ago,launched by Eli Lilly and Co. on July 5, 1983, under the trade nameHumulin. It was the first genetically engineered drug to reach themarket.

Then came Genentech Inc.'s human growth hormone (Protropin),rolled out in October 1985, followed a year later by Biogen's alphainterferon (trademarked Intron A by Schering-Plough Corp.).

Genentech unveiled its recombinant tissue plasminogen activator inOctober 1988 as Activase. Then came Amgen Inc.'s billion-dollarEpogen (human recombinant erythropoietin), approved by the FDAwith fanfare in June 1989.

This cascade of human-made human hormones put biotechnologyover the top in the 1980s _ decade of a new generation of miracledrugs. Each treats a different disease _ diabetes, dwarfism, heartattack, multiple sclerosis, anemia _ but all have one thing incommon: their injectable route of administration.

"Although largely effective," observed University of Chicagomolecular cardiologist Jeffrey Leiden, "such therapies are bothexpensive and inconvenient." He cited erythropoietin (EPO) as a casein point:

About 140,000 people in the U.S. produce too little of this blood-boosting hormone. Kidney dialysis patients and those who take AZTagainst HIV infection require two to three injections of EPO a weekfor their anemia, at a cost of about $8,000 a year. "Gene therapy,"Leiden said, "could replace those repeated treatments with a singleinjection."

All through the 1990s, gene therapists have been seeking and testingways to replace or reduce the needle as sole delivery device for theserecombinant compounds, most of which must be taken chronically orfrequently. Their viral vectors have so far proven inadequate, becausethey are a one-shot proposition that can't be re-administered.

"We had a new gene-transfer vector," Peter Hobart, director ofmolecular biology at Vical Inc. in San Diego, told BioWorld Today,"and Jeffrey Leiden sits on Vical's scientific advisory board."

Vical's plasmid, Hobart said, achieves its powerful gene delivery andexpression because "We went for simplicity; we stripped it of allunnecessary sequences, installed one of the strongest transcriptionpromoters known, that of cytomegalovirus, and did away with viralvectors to deliver this custom-built naked DNA."

Shooting Drug Genes Into Leg Muscles

Leiden and Hobart are co-senior authors of a paper in the currentProceedings of the National Academy of Sciences (PNAS), datedOct. 1, 1996. Its title: "Long-term expression of erythropoietin in thesystemic circulation of mice after intramuscular injection of aplasmid DNA vector."

Into the leg muscles of immune-deficient adult SCID mice, whichcan't reject foreign protein, they introduced their naked-DNAplasmid, loaded with human recombinant EPO genes, supplied as agift by Amgen, of Thousand Oaks, Calif. A normallyimmunocompetent strain of rodents got murine EPO DNA instead.

Both gene therapy packages stayed put in their safe-deposit muscle-cells, from which they pumped out a steady supply of EPO into theanimals' bloodstreams. The donor hormone did its job by markedlyraising the animals' hematocrit (red-blood-cell levels as a percent ofblood volume) from 48 pre-therapy to 64 at 45 days after.

A 300-microgram shot multiplied serum-EPO levels five-fold, andtemporarily boosted hematocrit levels to 79, before declining to asteady 64.

What's more, the gene therapy system kept right on going. Last May,as reported in PNAS, the plasmid was still working after 90 days.Since then, Hobart said, "in some of our recipient animals, it's outpast one year." But he added: "Some do, some don't. We're stillignorant as to why." He added, "But this is a very nice demonstrationproject."

Leiden pointed out that, "This is the first demonstration that aninjected gene, without a viral delivery system, could produce andsecrete enough protein into the blood stream to make a clinicaldifference, and keep doing it indefinitely." He added, "If thistechnique works as well in human trials _ expected to begin nextyear _ it could drastically reduce the need for erythropoietin."

Lowering The Bar To Patient Studies

Amgen's Epogen generated some $2.6 billion in sales worldwide lastyear, $885 million of it in the U.S. (See BioWorld Today, July 26,1996, p. 1.)

"Before this approach is feasible for human therapy," Leiden pointedout in his PNAS article, "several important issues remain to beaddressed." These hurdles include:

* raising the plasmid's uptake or expression efficiency, asextrapolating the DNA dose from mouse to man at present doseswould entail hiking the dollop of naked DNA 2,800 times.

* A way around this weighty obstacle would be to increase the half-life of the recombinant proteins in the patient's systemic circulation.

* Finally, a way must be found to regulate DNA expression afterinjection of the plasmid, particularly for chronic diseases such asinsulin-dependent diabetes, and hemophilia, which requires repeatedtransfusions of blood-clotting factors.

"As we reported last month to a Cold Spring Harbor symposium,"Hobart said, "we continue to dose the mice with plasmid. It showsthat re-administration brings EPO expression back up again,something the viral vectors don't do."

En route to clearing these therapeutic roadblocks, Hobart suggested,"It will be important to test our system in larger animals _ dogs, cats,rabbits, primates _ to determine the optimum relationship betweenDNA dose and recipient's body weight. We don't know yet," hecontinued; "It's still a question mark, but we'd like to think that it'snot proportional, because we're delivering a hormone, which is activeat very low doses."

Besides EPO, Hobart concluded, "our approach should work withseveral other recombinant hormones, such as leptin, insulin, growthhormone, as well as cytokines such as interferon. All of the above arecandidates." n

-- David N. Leff Science Editor

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