Contributing Writer

The children's story of "The Three Little Pigs" currently has a science fiction-like parallel in the work of Infigen Inc., which last week reported the birth of three genetically modified miniature swine.

In the updated version of this story, the wolf at the door is the lack of human organs for transplantation, a situation resulting in dozens of lives lost every day, thousands lost every year. And the little pigs in this case offer the potential for growing organs that can be transplanted into humans, with the miniature swine knockout-modified to prevent organ rejection.

The three smaller-than-normal porkers are the product of a collaboration between Infigen, of DeForest, Wis., and Immerge BioTherapeutics, a joint venture between BioTransplant Inc., of Medford, Mass., and Novartis Pharma AG, of Basel, Switzerland, formed in 2001. (See BioWorld Today, May 9, 2001.)

Immerge has provided to Infigen the vectors for specific genes and cells, with Infigen then having the responsibility to produce the pigs. The next step is to hand the pigs back to Immerge to move forward on transplantation of their cells and tissues in monkeys to determine the ability to transplant these materials into humans.

"Our whole goal has been to make these pigs," said Erik Forsberg, president of Infigen. "It's been a tough task but very rewarding."

He said that the primary hurdle was the deletion of a specific gene from "both parental copies" in a double-knockout strategy. The a-1,3-galactosyl transferase gene controls the existence of a carbohydrate on the surface of pig cells, materials not found on the cells of either humans or on those of "old world primates," Forsberg said, the type of monkeys that will be used in the preclinical trials. The existence of this carbohydrate triggers in humans the rejection of porcine cells, and the hope is that would not happen in planned animal trials or the expected work, further out, in humans. The intent, therefore, is to better control or even eliminate the rejection response.

The work to produce the pigs occurred at multiple levels, ranging from the company's work in applying nuclear transfer technology to the pigs - work published in Nature Biology in 2000 - to cloning from genetically modified cells and developing a method to remove the second copy of the specific gene of interest.

While successful production of the gene-modified pigs is the end of a long process, it is only the beginning of the next series of experiments: the implantation and development of cells and tissues in the upcoming preclinical work. "We'll see how long the cells and organs survive, with and without rejection drugs," Forsberg said

While Forsberg noted that Infigen does not control the preclinical work to be carried out by Immerge, he expects the first implantations in monkeys to be pancreatic islets that secrete insulin, and perhaps even whole kidneys. Further out are likely to be experiments with heart and lung tissues. Implantation of a liver he called "a little problematic because the liver has so many proteins."

If any of the work is successful, the miniature pigs offer the opportunity to serve as four-footed factories to produce the human organs that are so badly needed, with Infigen estimating more than 80,000 people in the U.S. alone awaiting organ transplantation of some kind.

Whether or not the transplanted cells and tissues avoid rejection and develop normally should be known in about a year, because the normal rejection - which Forsberg termed a "hyperacute rejection reaction" - occurs in a few months, if not weeks. Further out, he said, there is likely to be modifications of future pig progeny via additional genetic engineering, coupled with experiments with anti-rejection drugs.

Overall, he exercised considerable understatement in expressing the future possibilities: "We have to be cautious about the implications, long term, but they could be beneficial to humans," he said.

The work generally provides a springboard for other important company goals. One, in particular, is the production of pigs that could be engineered for a variety of disease research, much like mice and rats, with pigs frequently providing a genetic structure more like that of humans.

Meanwhile, the company is pursuing another project that is probably much closer to the hearts of medical device manufacturers - the factory-like production of human collagen and human fibrinogen, materials in short supply but much sought by the orthopedic sector.

That will be explored not with pigs but with cows.

Infigen in 1997 created Gene, the world's first cloned bull calf and now has produced a herd of more than 50 cloned cows genetically engineered to produce a variety of materials in their milk, including proteins not normally present.

Forsberg said the result is to use "the machinery of the mammary gland to produce human collagen. We introduce a transgene into the cows that has a milk promoter."

That effort was initially launched with Pharming Holding NV, of Leiden, the Netherlands, and a U.S. subsidiary, Pharming Healthcare. Pharming since then has been in and out of receivership, but Forsberg says the parties are still in discussions to move that work forward.

In general, these cows are seen as another potential set of factories to produce, on the one hand, better natural materials in the dairy category, and, on the other hand - similar to collagen and fibrinogen production - much needed, and often rare, pharmaceuticals.

These pharmaceuticals could include orphan drugs addressing a variety of complex diseases, even gene-based illnesses, the company has said.

Forsberg reports a staff of about 25 scientists in-house working on a range of projects. Infigen is self-funded, with additional support from its technology partners and The Advanced Technology Program of the National Institute of Standards and Technology in a cost-sharing structure.