By David N. Leff
You can't get blood from a turnip. Everybody knows that. But how about getting hemoglobin from corn kernels?
In France, a team of government and industrial scientists are working on that. So far, they've come up with proof-of-principle quantities of the red-blood component from tobacco plants (Nicotiana tabacum var. Xanthi).
A brief item in today's Nature, dated March 6, 1997, reports this demonstrator-model cloning under the title: "Human hemoglobin from transgenic tobacco."
The paper's principal co-authors are biophysicist Michael Mardan of INSERM (the French National Institute of Health and Medical Research), in Paris, and molecular biologist Manfred Theisen, who directs the laboratory of molecular and cellular biology at Biocem Limagrain SA, one of the world's leading seed companies, in the French city of Clermont-Ferrand.
"Our main motivation in undertaking this project three years ago," Mardan told BioWorld Today, "was to avoid the problems of possible contamination in transfused blood from existing sources * human donors with AIDS or hepatitis, and bovine or eventually porcine blood products.
"The problems with AIDS and mad cow disease," he explained, "have made people think that maybe there's another unknown contaminant waiting to be discovered. People worried * and the farther away from humans, the better. So we thought there might be major advantage in switching from an animal source to a plant source."
Mardan's INSERM unit had been making recombinant hemoglobin in E. coli host cells for the past decade. So, fortified by this experience, "we prepared the initial steps two years ago, and sent the samples to Clermont-Ferrand, where Biocem introduced them into the tobacco plants, which expressed the hemoglobin in their seeds and roots."
Biocem then sent this biomass back to Mardan for extraction, purification and in vitro testing of the recombinant blood product. It checked out as functional, tetrameric human hemoglobin.
The INSERM group created their transforming vector by putting hemoglobin DNA sequences fused to those of a pea plant's chloroplastic transit peptide into a binary plasmid.
Biocem's biologists inserted this package into N. tabacum plants for co-culture with Agrobacterium tumefaciens, the classical plant transfectant of choice.
Tobacco Now, Corn Later
"This work has two aspects for us right now," Biocem's Theisen told BioWorld Today. "One, we have to make more material from the plant we're working with, which at the moment is tobacco. So we're in the process of producing more biomass for analysis in Mardan's lab.
"And the other point, for sure," he continued, "is that tobacco is not the plant of interest for large-scale output of hemoglobin. So our main effort at the moment is looking at other plants, and we have initiated work on producing it in corn seeds.
"But corn transformation is still a rather tricky business," Theisen said. "It takes nine months or so before you have your first milligrams of seeds. So it will be some time before we can have enough purified hemoglobin to go into animal experiments."
Theisen counted the ways in which corn (Zea mays) differs from N. tabacum as a host tissue for molecular pharming:
· "Agrobacterium, the vector we used to transform tobacco, which is a dicotyledon plant, won't work in corn, a monocot. So instead we must use a different approach * biolistics * to insert the DNA by gene gun.
· "Tobacco seeds are ridiculously small, so they don't express a lot of biomass, compared to much larger corn kernels.
· "In corn, we have no problems with nicotine; it's a safe, nutritionally approved plant."
Other Blood Substitutes Go Clinical
Meanwhile, Baxter Healthcare Corp., of Deerfield, Ill., has signed up the first participating U.S. hospital for a Phase III trial of its hemoglobin therapeutic, HemAssist. Late last month, the Lehigh Valley Medical Center, Allentown, Pa., enrolled in the multicenter study, the company's spokesperson, Debra Spak, told BioWorld Today. It will compare the product with current treatment in survival of patients suffering blood loss caused by severe trauma.
HemAssist consists of stabilized and purified hemoglobin derived from human donor blood, Spak said.
Another of the front-running blood-substitute developers in the U.S., Somatogen Inc., of Boulder, Colo., now has its recombinant human hemoglobin in a 400-patient Phase II trial, for intraoperative use during surgery. Launch of a second Phase II study, in coronary bypass procedures, is imminent, said the company's director of industrial relations, Nanita Waddell.
Somatogen clones its blood product, Optro, in E. coli host cells. *