By David N. Leff

Porcine donor organs - for xenotransplantation into humans - came six cloned piglets closer to reality this week.

A litter of five females emerged into the world on March 5, 2000, at PPL Therapeutics Inc. in Blacksburg, Va. However, the eagerly awaited novel, beyond-Dolly procedure of their cloning was made known only Wednesday. It emerged in a special, electronically released Nature article, titled: "Cloned pigs produced by nuclear transfer from adult somatic cells." (It originally was slotted for publication in Nature's Aug. 24, 2000 issue.)

The sixth cloned piglet, also female, was born July 2 in Japan's Tskuba "science city," at the National Institute of Animal Industry. Its different cloning technology (see related story, "Japanese Use Skin Cells To Clone Their Porcine Progeny") will be reported in Friday's issue of Science, dated Aug. 18, 2000, under the title: "Pig cloning by microinjection of fetal fibroblast nuclei." (Science advanced its media embargo on this report by a day, upon learning of Nature's electronic one-upmanship.)

Molecular embryologist Alan Colman, at PPL in Roslin, Scotland, a principal co-author of the Nature paper, is senior vice president of research at the multinational company's facilities in Virginia and New Zealand. PPL put livestock cloning on the map three years ago with Dolly, the cloned ewe, born on July 5, 1997. Since then, other centers have followed suit with cloned cows and goats - but until now, no pigs.

Colman described to BioWorld Today the technological advance his collaborators have made between cloning Dolly the sheep and producing this year's porcine quintuplets:

"For Dolly," he began, "we took a somatic cell and transferred its nucleus to an unfertilized egg, or oocyte, from which the genetic material had been removed. That's called a reconstructed embryo. Next we used electrical impulses to simulate the fertilization stimulus that normally comes in with the sperm. Then you have a developing embryo - if you're lucky.

"We found that our Dolly approach did not work for pigs by the conventional cloning technology. We tried quite hard. What we did in the pigs," Colman continued, "and what is different from Dolly, is we took that first step, where we put a somatic cell nucleus into an unfertilized egg, and simulate activating it. What happens then," he went on, "is that the nucleus swells up a bit - this always happens - because it thinks it's an embryonic germ line nucleus, not a somatic one any more."

'Double Reconstructed Embryo' Clones Piglets

"Then we actually took it out of that unfertilized egg, also a reconstructed embryo, and put it into a normally fertilized embryo, from which we'd removed all genetic information. So this is our novel technology, called 'double nuclear transfer.'

"And the reason we did that," Colman went on, "is we argued that the normal fertilized embryo receives its activation stimulus from the sperm, and what we were doing was really just taking advantage of that, by getting rid of the zygote nucleus - the result of the egg nucleus and the sperm nucleus combining. We substituted for it the somatic cell nucleus, which we'd pretreated in another oocyte.

"And this worked pretty well," Colman noted. "That's why we got five animals, which are still very healthy at 4 or 5 months old."

The porcine quints answer to five carefully chosen monikers, fraught with meaning:

Millie stands for the new millennium; Christa honors heart surgeon Christian Barnard, who launched human heart transplantation in 1967; Alexis and Carrel are for Franco-U.S. surgeon Alexis Carrel (1873-1944), a Nobel prize-winner in 1912 for his contribution to transplantation. "Dotcom is the unexpected fifth piglet"- so-called, deadpans PPL Managing Director Ron James, "Because any association with dot-coms right now seems to have a very positive influence on a company's valuation."

The solo Japanese piglet is named Xena - for "stranger," in Greek, and "xenotransplantation" in English - transferring donor organs from animals into human recipients. Cloned pigs are regarded as a limitless mine of donor organs because of their size and similarity to human physiology. But at least two hurdles bar the path to that Golconda - one immunological, the other retroviral.

Even transplanted human donor organs are quickly destroyed by the immune system's hyperacute rejection. That's why recipients must take immunosuppressant medication all their lives. The target of this rejection is an enzyme, a-1,3-galactosyl transferase, which parks a specific molecule on the surface of porcine cells. When the human immune system sees this antigen, it goes ballistic, and dispatches antibodies to wipe out the intruding antigens - ergo, hyperacute rejection.

PPL has a $2 million grant from the U.S. National Institute of Standards and Technology, expressly to produce a "knockout" pig that inactivates the gene for a-1,3-galactosyl transferase. "The ability to clone pigs," Colman observed, is the first essential step in achieving this objective. Now," he added, "we're about to revisit that problem."

Go Slow: Infectious Virus Ahead!

A longer-range hurdle is defined in a companion paper released Aug. 16, 2000, by Nature. Its title signals that caveat: "Infection by porcine endogenous retrovirus after islet cell xenotransplantation in SCID mice." Its co-authors, at the Scripps Research Institute in La Jolla, Calif., warn, "All pigs contain several copies of porcine endogenous retroviruses (PERV)."

They set up an in vitro experiment simulating porcine insulin-secreting islet cells xenotransplanted into diabetic mice, and reported detecting "ongoing viral expression and infection of several tissue compartments." They concluded: "These results show that a concern for PERV infection risk associated with pig islet xenotransplantation in immunosuppressed human patients may be justified."

Colman concurs - strongly:

"In a general way," he said. "I believe there is legitimate public concern. It's an issue that can't be ignored. The public has a right to feel that clinical trials of porcine donor organs do not endanger the public at large. So we take it very seriously.

"The fact is, however," Colman pointed out, "that pig organs won't be even technically suitable for going near a human recipient for four to five years. If all our work went as well as anyone could have expected, I do not believe that we could be in a position to move into humans any sooner than that."

He explained why: "Because I think the knockout of the a-1,3-galactosyl transferase gene is not the only thing you have to do in terms of preparing an organ so it's not rejected. Therefore I think that gives us ample time - us and others - to see how serious this retrovirus problem really is going to be - if it is going to be a problem - and develop strategies to avoid it."

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