In the U.S., 16 patients a day on average die while waiting in vain for a life-saving donor organ.

“They are among the 79,000 people on organ transplant waiting lists,” observed research immunologist Julia Greenstein, president and CEO of Immerge BioTherapeutics Inc. in Charlestown, Mass. “There’s probably an equal number of desperate people in end-stage organ failure who could benefit from an organ replacement. But they don’t even get on the waiting list, because they have features that exclude them. So if there were organs available in an unrestricted fashion, those people would be eligible for transplants.

“About 12,000 transplants a year take place in the U.S. alone,” Greenstein added. “The worldwide number is about twice as many. And those figures don’t even address the need for cell transplantation, for example, pancreatic islet cells for diabetic patients.”

All of the above left-out supplicants could benefit from a tribe of miniature pigs, and Greenstein’s company’s mission is to create their organs for xenotransplantation. She is lead author of a paper in this week’s online Sciencexpress dated Jan. 3, 2002. Its title: “Production of a-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning.” The article’s senior author is reproductive biologist Randall Prather, at the University of Missouri, Columbia.

“Because we have chosen to work in miniature swine,” Greenstein told BioWorld Today, “we think we’re much closer to having a viable porcine donor with size-matched organs for transplantation into human recipients. Mini-swine weigh about 2 pounds at birth, and grow to 250 or 300 pounds,” she observed. “But the domestic pig, which most other companies interested in xenotransplantation are using, can reach 1,000 pounds. That’s over five times bigger than your average human individual. So when talking about matching heart sizes, we think the miniature swine is a much better source for xenotransplantation than the domestic porker.

“Another advantage,” she added, “is that cells from this line of pigs in contrast to most others tested don’t have the capacity to spread porcine endogenous retrovirus to human cells in culture.”

But size compatibility is the least of the hurdles confronting the biotech labs and companies in the xenotransplantation race. Their No. 1 bugaboo is immune rejection of alien organ grafts, which has significantly slowed the use of animal organs.

With Organ Rejection, Nature Bats Last

“The evolutionary argument for rejection,” Greenstein explained, “is tumor biology. Your immune system is set to distinguish self from non-self, so if your body throws a cancerous mutation and creates a tumor cell, your immune system will recognize and reject it as foreign. A byproduct of that process is organ rejection from an unrelated donor.

“The perpetrator of graft rejection is a sugar 1,3-galactose,” Greenstein pointed out. “Its gene exists in every species on earth except Old World monkeys and man. It glycosylates sugar molecules a type of sugar flag on the surface of all proteins. We’re trying to take those sugars out of all the cells in the pig.

“One of the first targets for organ rejection,” she went on, “is the endothelial cell. That’s where the blood, which contains the antibody to the sugar, can meet the grafted organ. We’ve been trying to alter the sugar’s gene in the pig probably since 1992 when we started understanding how important it was in the porcine-human immune response. We tried using different methodologies; then at the publication of Dolly the sheep five years ago, switched to nuclear transfer cloning.

“Now, as we report in Science,” Greenstein continued, “we have the first nuclear-transferred litter of swine four healthy piglets which have half, so far, of the genetic modification that knocks out that sugar gene. Our four piglets, females, were born last September and October. They’re all doing wonderfully.”

Greenstein narrated her group’s approach to eliminating the graft rejection gene from their mini-pigs: “Our strategy was first of all to trace the gal transferase enzyme to its gene in the pig genome, then sequence that gene. So we constructed a recombinant targeting DNA vector homologous to the gene by almost 23 kilobases and deliberately inserted a sequence mistake, a stop codon, to knock the gene out. That way, if our mutation got inserted at an appropriate part of the genome it would be unable to make a full copy of the rejection enzyme.

“Finally, we generated a primary cell line fetal fibroblasts and exposed them to the vector. Then we selected those cell lines that have incorporated that piece of DNA in the right place in the genome. We then grew up those cells, which serve as donor of the nucleus. This was put into an enucleated oocyte, which went on to the cloning technology.”

She then transferred the genetically modified fibroblast cells to Randall Prather at the University of Missouri.

Nuclear Transfer Completes Cloning Process

“Immerge BioTherapeutics did the genetic modifications to the donor cells from the NIH miniature pigs,” Prather told BioWorld Today. “They froze the cells, then shipped them to us. We obtained mature eggs via in vitro maturation from domestic non-miniature pigs. After the eggs matured, in about 48 hours, we put them under the microscope and removed their chromosomes and mature DNA. We next transferred in the knocked-out cells that Immerge had sent us and fused the two cells together with an electric pulse that also kick-started the development program. This electrical jolt kind of tricked them into believing that they were fertilized.

“After culturing the cells overnight,” he recounted, “the next day we surgically transferred the clones to ordinary domestic sow surrogates. Then following 114 days of gestation we did a Caesarian to recover the offspring. There were seven piglets total, and three of them passed away, so we now have four healthy mini-swine. Those four knockout piglets are all heterozygotic, which means only one of their two genes carries the anti-rejection mutation. The other one is still there and functioning,” Prather observed, “so their cells still have the sugar molecule on their surface. When the piglets reach maturity,” Prather concluded, “we’ll breed them, and after some brother-sister matings, we hope to end up with animals that have both alleles that is, homozygotes making them resistant to hyper-acute graft rejection.”