It looks as if, in years to come, the pig (Sus scrofa) may displace thedog (Canis familiaris) as our best friend. Transplantationimmunologists on three continents are busy creating transgenic hogsarmed with human genes to inhibit the complement system fromrejecting subhuman donor organs.
Success in this endeavor will overcome the first hurdle that preventsxenografting of porcine organs into human recipients. (SeeBioWorld Today, Sept. 29-30, 1993, p. 1.) This hurdle, hyperacuterejection, can destroy implanted foreign tissue within minutes. Killerproteins in the body's serum complement cascade perpetrate thatxenophobic destruction by punching holes in the alien graft'sendothelial cells lining its blood vessels.
To shield the body's own cells from this tissue massacre, theimmune system provides a set of molecules that inhibit complementfrom acting. The gene that encodes one of these proteins, CD59, is afavorite of the xenografters.
In Britain, Australia and the U.S., they are constructing transgenicpigs that express human CD59 in their tissues, to forfendcomplement-triggered hyperacute rejection. The latest of these toreport progress is Stephen Squinto, vice-president of molecularresearch at Alexion Pharmaceuticals, Inc., in New Haven, Conn.
His paper in the current Proceedings of the National Academy ofSciences, (PNAS), (Nov. 8) bears the title: "Expression of afunctional human complement inhibitor in a transgenic pig as amodel for the prevention of xenogenic hyperacute organ rejection."
A Boar That Counter-Attacks Human Complement
Alexion, in collaboration with Virginia Polytechnic Institute atBlacksburg, engineered the gene for human CD59 into the embryosof mice and pigs. Of 18 porcine progeny, one boar expressed theanti-complement protein at very high levels.
"To evaluate vascularized structures in the transgenic pig withouthaving to sacrifice the founder animal," the PNAS paper reported,"tail sections were prepared and analyzed . . ." Those samplesrevealed high-level hCD59 expression on a variety of tissue types,including the vascular endothelial cells vulnerable to hyperacuterejection. Non-transgenic litter mates registered zero in this in vitrotest.
By a similar token, transgenic porcine hCD59 resisted complement-directed cell lysis by human serum complement components, as wellas the high-titer natural antibodies that unleash the complementcascade. Controls suffered lysis.
Since Squinto submitted that article to PNAS last June, Alexion'sprize transgenic boar, bred to normal sows, has sired numerousprogeny. True to genetics, "some 50 percent of these litters are inturn transgenic," as reproductive physiologist James Knight atBlacksburg told BioWorld Today.
Knight and molecular geneticist William Fodor, first author on thePNAS paper and principal scientist at Alexion, "are now sacrificingsome of the offspring of the founder animals that were the highestexpressers," Squinto told BioWorld. "If they see high levels ofexpression in some of the transgenic offsprings' hearts, livers orkidneys, then we will go ahead with a collaborator in Oklahoma, andtry some solid-organ transplantations."
One Hurdle Leads To Another
That collaborator is David Cooper, medical director of the OklahomaTransplantation Institute in Oklahoma City. He is now an externalconsultant to Alexion. Fodor plans to work with Cooper's institute,he told BioWorld, "going directly into doing heterotopic hearttransplants in baboons _ that is, not replacing the heart alreadythere."
"CD59 is going to get us across the hurdle of hyperacute rejection,"Cooper told BioWorld, "but then other hurdles will come into play."
Together with the British transplant surgeon David White, ofCambridge University and Imutran Ltd., Cooper has been doingstudies in baboons of a factor in the venom of cobras that alsoinactivates complement. "We find that we can get over thathyperacute rejection," he said, "but then we find vascular rejectionchanges beginning four or five days after the transplant."
This second hurdle, Cooper explained, is independent ofcomplement, but related to antibody complexing with antigen on theorgan-graft's blood-vessel endothelium. Once that is blocked, thethird hurdle pops up: acute, delayed cellular rejection. To counterthis third strike, the Oklahoma xenografter said, "drugs are available,such as cyclosporin."
To clear the second hurdle, Alexion is collaborating with the AustinResearch Institute of Melbourne, Australia. (See BioWorld Today,April 1, 1994, p. 5.) "We have a fairly aggressive research programgoing on there," Squinto said. "What can be disclosed," he added," isa strategy using transgenic animals to completely down-regulatewhat the Australian group believes to be the predominant xenogenicantigen."
Squinto outlined that approach last June 16 to an InternationalBusiness Communications conference in Washington on "Transgenicswine engineered to provide a universal donor."
"It aims basically," he said, "at competing out the galactosyltransferase enzyme responsible for synthesizing the predominantxenoantigen in the pig-to-human combination."
Therefore, Squinto explained, "Transgenic pig cells that we'veengineered in vitro no longer express that carbohydrate-basedepitope, and are no longer recognized by human antibodies."
Fodor and the Blacksburg group are this week "setting up tointroduce that genetic modification into pig embryos, then cross-breed the resulting animals with our CD59 complement-inhibitorswine for simultaneous anti-complement and anti-antibodyprotection."
Alexion's key competitor in the porcine-donor-organ game isPrinceton, N.J.-based DNX Corp. "As far as we know from thepublic domain, and their presentations at meetings," Squinto said,"DNX does not have an approach in place to eliminate thiscarbohydrate epitope. I think that is unique so far to Alexion."
DNX has now completed three transplants of transgenic pig organsinto primates, the company's research vice president, John Logan,told BioWorld. "That work," he added, "was submitted this week toScience." (See BioWorld Today, June 17, 1994, p.1.)
Besides CD59, the last-ditch late blocker of complement, DNXtransgenic pigs carry the gene for CD55, (a.k.a. "DAF") the earliestinhibitor of the cascade. Logan said that in the three transplants "wesee extended survival from less than 40 minutes to as much as 30hours." n
-- David N. Leff Science Editor
(c) 1997 American Health Consultants. All rights reserved.