Porcine Possibilities

Courtesy of PPL TherapeuticsA new generation of pigs. Headlines in late summer 2000 introduced long-awaited reports on pig cloning and retroviral transmission to mice, pig cells healing rat spinal cords, and a gaff by Dolly dad Ian Wilmut erroneously heralding halt of xenograft work at the Roslin Institute near Edinburgh, Scotland. So it seemed that the question of whether pigs can pass their retroviruses to humans might finally be on the road to resolution. Not quite. Pigs, as the purveyors of new influenza strains, do not have a stellar reputation in epidemiological circles. Experiments reveal that porcine endogenous retroviruses (PERVs) nestled in the pig genome can indeed find their way into cells of other species, although they do not seem to do much once there. "Swine fever, hepatitis E, and the Nipah virus have been linked to pigs, however all are exogenous viruses and can be excluded from a herd which is bred in a high biosecurity unit. There are no known viruses in the pig genome that have caused disease in man," says Khazal Paradis, the director of clinical research at Imutran Ltd. in Cambridge, U.K. Pigs are prime candidates for organ donors due to their size and anatomical similarities to humans. One difference, however, sets the stage for potential disaster. The endothelial cells that line the insides of porcine blood vessels sport galactose chains. Humans and Old World monkeys, unlike other mammals, have mutations in the gene that encodes the enzyme that places galactose on cells (1,3-galactosyltransferase), rendering human cells free of the sugar. As a result, human immune systems regard galactose-tagged cells as foreign, and launch a fast and furious hyperacute rejection. But transgenic technology can perhaps prevent hyperacute rejection in two ways: stripping galactose from pig cells by knocking out the gene for 1,3-galactosyltransferase, or by endowing pig cells with human complement inhibitory protein, which suppresses our hyperacute response.1 Jeffery Kocsis and his colleagues at the Yale University Neuroscience Research Center and Alexion Pharmaceuticals Inc. in New Haven, Conn., took the complement blocking approach in rats. The researchers implanted Schwann cells and olfactory ensheathing cells (OECs) from pigs' snouts into severed spinal cords in rats. Seven of 10 rats regenerated neurons sporting normal fatty sheaths, as well as restored neurotransmission that was even faster than normal, thanks to the natural capacity of the OECs. Photo: Ricki LewisPig cloning could be a boon to the pork industry by reducing the time it takes to get food products to market. The Cloning Connection Cloning can have an impact on the feasibility of xenotransplantation by decreasing the number of steps to make a transgenic animal. Pigs have been slow to join the list of cloned mammals, mainly because a sow must maintain four healthy embryos to stay pregnant. But once this early hurdle is overcome, pig cloning may go smoothly. "Large birthweights will not be as problematic in pigs as it is in cattle, and a shorter gestation in pigs allows for quicker answers to research questions," says Steve Stice, Chief Scientific Officer of ProLinia Inc. in Athens, Ga., and a professor of animal science at the University of Georgia. The first cloned piglets, from PPL Therapeutics Inc. of Blacksburg, Va., and Midlothian, Scotland, greeted the world on the Today show in March 2000. The top-secret details of their creation were published five months later in electronic form in Nature in mid-August2 to coincide with another pig cloning claim in Science.3 PPL Therapeutics used a new approach: two nuclear transfers. The researchers first injected the donor nucleus, from an adult cell, into an unfertilized egg. Then, once the nucleus had expanded to form a structure called a pronucleus, they transferred it to a fertilized egg. The investigators hypothesize that factors present in the pronucleus, plus not needing to artificially activate an unfertilized egg, contributed to the success. But still, cloning remains less-than-efficient. The five little piggies of Today show fame resulted from 401 implanted fertilized ova. The odds encountered in producing the lone porcine reported in Science, named Xena, were similar. Xena resulted from microinjection of a fetal fibroblast nucleus using the "Honolulu" technique pioneered on cloned mice. Her team, led by Akira Onishi of the National Institute of Animal Industry in Tsukuba, Japan, attributes the success to transferring only DNA, rather than fusing cells as had been done to produce Dolly. Xena was one of 110 attempts. Photo: Ricki Lewis Tracking PERVs Although pigs seem to provide perfect stand-ins for human organs, there is concern that their viruses could enter human cells and either cause disease directly, or recombine with other viral sequences to generate a new killer. "We know that some viral diseases, such as AIDS, can jump species. So people are getting nervous and are raising a red flag," says Max Rothschild, a distinguished professor of animal science at Iowa State University who is the facilitator for the U.S. Pig Genome Project. Such concern has fueled experiments to see just what PERVs do beyond pigs. Veterinarians who care for pigs have known about PERVs since the 1970s, says Daniel R. Salomon, an associate professor in the department of molecular and experimental medicine at the Scripps Research Institute in La Jolla, Calif. "But the vets quickly lost interest because PERVs didn't cause disease in pigs. The field remained dormant," he says. Enter biotechnology. "Five or six years ago, the first transgenic pigs from biotech companies came on the scene, and there was big money from big pharma. Many people became excited about going forward with clinical trials" of xenotransplants, he adds. With the very real prospect of pig-to-human transplants on the horizon, tracking PERVs became paramount. In 1997, Robin A. Weiss, at the Windeyer Institute of Medical Sciences at University College, London, exposed human cells in culture to two types of PERVs and detected viral envelope genes in the cells.4 "Robin Weiss, in a beautiful study, showed the theoretical risk. That study, and more general concerns about transplants from pigs to humans, set the stage for the public debate and discussion in the various health agencies," explains Salomon. "Should we go forward with clinical trials or should there be a moratorium?" It wasn't so much the idea of a virus flitting from one species to another, which happens naturally, but the unusual route that has alerted researchers and public health officials. "There had been transmission routes from domesticated animals to humans for centuries, and in the wild for thousands of years. But a transplant is different," Salomon says, adding that, despite the possibilities, nary a pig nor human has yet to become ill from a PERV. This Little (Cloned) Piggie Went to Market While epidemiologists and ethicists debate the risks and benefits of pig parts as transplants, cloning of the animals is proceeding in another, more traditional area: producing pork, the "other white meat." And unlike xenotransplantation fears, pig tissue as a ham sandwich doesn't introduce the possibility of retroviral stowaways. Americans consume about 14 million pigs a year. Cloning the animals would accelerate the timetable of developing and commercializing new breeds from the present six to nine years, down to two to three years. And that is the business plan of ProLinia Inc., a new agricultural biotech genetics supplier spun from expertise in the animal science department of the University of Georgia. Smithfield Foods Inc., which hogs the market, invested $1 million in ProLinia in June 2000, to support application of proprietary cloning methods to jumpstart mass production of certain elite breeds. ProLinia has been working independently of the two groups that recently announced successful pig cloning. "We are developing our own technology to clone adult animals. We aren't interested in fetal or embryo cell cloning," says Steve Stice, Chief Scientific Officer of ProLinia. And in contrast to research groups concentrating on xenotransplantation, ProLinia sees a bigger genetic picture--honing cloning in the near future is just the start. "It might take three to eight years to get cloning integrated into commercial hog producer systems. We see ourselves as providing better genetics to the producer through cloning technology," Stice says. The company will eventually supply Smithfield Foods with cloned embryos bearing certain combinations of genes. Rather than creating the pigs-in-human-clothing that transgenesis offers, agricultural efforts focus on such traits as meat quality and fat distribution. "The economic value of pig cloning in the pork industry could be considerable," says Max Rothschild, a distinguished professor of animal science at Iowa State University. Cloning could greatly speed the process of transforming pig to pork. "It starts with a 'genetic nucleus farm,' which is a farm that uses quantitative genetics and molecular genetics to get a particular combination of traits selected for in the breeding population. The nucleus farm is the site of genetic improvement. Then, multiplication farms increase the number of pigs, which then go to commercial farms. If you can clone an animal from a nucleus farm, you can go straight to the commercial level, and skip the multiplication phase. This does away with what is called "genetic lag"--the time it takes to get improvements into a population." --Ricki Lewis Another telling report was published in 1998,5 from Paradis' group at Imutran and a team from the Centers for Disease Control and Prevention in Atlanta and the University of Glasgow. The researchers used PCR with PERV primers to screen for pig retroviral sequences in the blood of 160 patients who had received pig tissue implants over the past 12 years. None showed evidence of lingering PERVs, with an interesting exception: 23 patients whose blood had been circulated through pig spleens for an hour, 81/2 years ago in the Soviet Union. These patients had microchimerism, the persistence of pig cells in their bodies. The PERV RNA detected in the study was in these cells, as might be expected. The Imutran/CDC study had the limitations of being retrospective, and tracking PERV in blood, a tissue it does not infect in human cells in culture. An animal model of PERV transmission across species could help to clarify the virus' ability to jump species. Salomon's group turned to SCID (immunodeficient) mice, with a neat genetic twist developed by colleagues Edward Otto and Christopher Lockey.6 The researchers transplanted pig pancreatic islet cells into the mice, then looked for viral expression and infection. But following a clue from the microchimerism noted in the Imutran/CDC study, they calculated ratios of PERV DNA to DNA from pig chromosome centromeres. The idea was that ratios would remain constant only if the PERV DNA in the mice came only from remaining pig cells; the ratio would spike if PERV DNA had escaped the porcine cells and was now being cranked out by mouse cells. Of 17 chimeric mice, eight showed the telltale increased ratio indicating that PERV had ventured beyond the pig cells. Plus, the viral replication came from mouse epithelial cells near the implants, indicating that the virus had indeed reached nearby mouse tissue. On the surface, PERV jumping species may seem alarming, but Salomon puts it into perspective. "None of the animals was sick. When the virus gets into cells, it replicates once or twice and then becomes latent, a pattern seen for a lot of viruses. So we can say, even in a SCID mouse, there is an unimpressive host response to the presence of the virus. As an M.D., I'd say that the risk is real, but at this point, there is no evidence that it is great." Meanwhile, the possibility of creating PERV-free pigs looms, if only in researchers' imaginations for now. Weiss and Rothschild see breeding virus-free pigs as an enormous task, if it's possible at all. "Groups are trying to map all the retroviral elements. Why not create a pig without these elements? That's not a trivial question, because there are 20 to 30 retroviral sites. It would be hard to breed them all out. Another way would be to knock them out, but so far there are no knockouts in pigs. I don't see an easy way out," says Rothschild. But Paradis thinks it could happen. "There is a lot of polymorphism for PERVs--that is, the site and number of insertions of PERV DNA varies from pig to pig. If only a few sites of the PERV DNA encode infectious virus, it may be possible to breed PERV negative pigs, or to knock the genes out." Ricki Lewis (rickilewis@nasw.org) is a contributing editor for The Scientist. References 1. T. Imaizumi et al., "Xenotransplantation of transgenic pig olfactory ensheathing cells promotes axonal regeneration in rat spinal cord," Nature Biotechnology, 18:949-63, September 2000. 2. I. Polejaeva et al., "Cloned pigs produced by nuclear transfer from adult somatic cells," Nature, 407:86-90, Sept. 7, 2000. 3. M. Iwamoto et al. "Pig cloning by microinjection of fetal fibroblast nuclei," Science, 289:1188-90, Aug. 18, 2000. 4. R. A. Weiss. "Two sets of human-tropic pig retrovirus," Nature, 389:681-2, Oct. 16, 1997. 5. K. Paradis et al. "Search for cross-species transmission of porcine endogenous retrovirus in patients treated with living pig tissue," Science, 285:1236-41, 1999. 6. L. J. W. Van der Laan et al. "Infection by porcine endogenous retrovirus after islet xenotransplantation in SCID mice," Nature, 407:90-4, Sept. 7, 2000.

Porcine Possibilities

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