Now try this multiple-choice quiz:
Q: How many life-threatened Americans are on waiting lists to receive an organ transplant?
(a) 19,000; (b) 79,000; (c) 7,900; (d) 790.
A: The envelope, doctor? The answer is "b," 79,000. That averages 16 patients a day who die of their disease, while waiting in vain for a life-saving donor organ.
One of their last best hopes is the domesticated hog, Sus scrofa. This familiar farm animal has a lot going for it as provider of replacement body parts, but also some hang-ups working against porcine donor organs.
The name of that Russian roulette gamble is xenotransplantation - putting nonhuman mammalian parts into recipient patients. One live bullet is that pigs harbor hidden pathogenic viruses. Another is graft rejection, when the recipient's immune defenses lash out against the well-meaning but antigenic organ. Immunologists and gene therapists the world over have been fighting these hazards with the whole dicey arsenal of transgenic animals and cloning - la Dolly the sheep. (See BioWorld Today, Jan. 7, 2002, p. 1.)
A paper in the Proceedings of the National Academy of Sciences (PNAS) released online Oct. 22, 2002, carries the tell-all title: "Efficient production by sperm-mediated gene transfer of hDAF transgenic pigs for xenotransplantation." Its lead author is immunologist/pathologist Marialuisa Lavitrano at Bicocca University in Milan, Italy.
"In this PNAS paper we described the results we obtained using a new kind of approach to transgenesis," she told BioWorld Today, "which is the sperm-gene transfer. We produced transgenic animals in a different way," Lavitrano continued. "We did not use the microinjection technique, or the cloning technique or other better-known methodologies. We developed the approach of sperm-mediated gene transfer over the past 15 years. Our first paper appeared in Cell in 1989. It reported our discovery of the natural ability of stem cells and exogenous DNA for fertilizing eggs in vitro or artificially. We used the sperm for transferring the new genes, to yield transgenic animals."
Sperm + DNA Inhibit Immune Rejection
"What we did in these past 15 years," Lavitrano went on, "was to understand by which mechanism the sperm can bind, internalize and integrate the DNA into the genome. Since we know the mechanism, we now can use in a reproducible and very efficient way the sperms of farm animals, and possibly other animals, for transferring the DNA into the egg at the moment of fertilization. The beauty of this technique," she added, "is that it's kind of natural. In the sense that we do not microinject, we have no risk of disrupting or aborting the embryos. We do not need to reprogram the genome of somatic cells as is currently done during cloning.
"Given our interest in xenotransplantation," Lavitrano observed, "and the possibility that a donor animal will need to express several transgenes, we have used sperm-mediated gene transfer to produce pigs transgenic for the human decay accelerating factor (hDAF). This transgene," she added, "has been shown to help overcome hyperacute rejection of a pig organ by nonhuman primates.
"One might say," she suggested, "that every fertilization could be considered as a risky event for the genetic identity of the species. But that's not true in nature, and in fact when the sperms can come out they are together with the seminal fluid. We found that there is an inhibitory factor present in seminal fluid, which totally prevents binding of exogenous DNA to the sperm. So by nature the sperms are fully protected. We optimize the conditions of the sperm-DNA interaction for the pigs, with the final goal of obtaining the highest sperm percentage carrying the exogenous DNA, which of course needs to be realized for fertilizing eggs."
Lavitrano recounted her in vitro lab experiments: "We selected two sperm-donor boars, collected their semen, and washed it thoroughly to totally flush away this inhibitory factor present in the seminal fluid. Then we incubated the purified semen in the fertilization medium together with the DNA - this mini-gene coding for the DAF protein. Then we used these sperms for artificial inseminations, as is done in every farm.
"We inseminated 15 sows," Lavitrano said, "and from them got 93 piglets. Of these, 57 were genetically modified with a percentage of success ranging from 46 [percent] to 88 percent. Then we selected 34 founder mini-pigs expressing the transgene for a next breeding and got a transgenic swine second generation. The transgene transmitted to the progeny was also expressed to the progeny of the progeny.
"When we then tested the function of the transgene, we found that the protein it encodes is functional. We challenged the monocytes and endothelial cells with human serum containing complement. The cells from the transgenic animals were not killed by the human serum, while cells from the conventional wild-type animals were killed 100 percent in a few minutes."
Complement Rejection Cascade Blocked
"This demonstrated that the hDAF-encoding protein is functional. It modulates complement activation and blocks the complement cascade. It was needed to add the human protein on the surface of the cells of pig organs, in case of xenotransplantation." She explained: "In xenotransplantation, when an animal organ is transplanted into a primate, immediately there is a rejection reaction which is very quick and very important. And this reaction is called hyperacute rejection.
"Our goal now," Lavitrano observed, "is to produce multi-transgenic animals, using xenotransplantation research. Blocking the hyperacute rejection is only the first step. What we need in order to use animal organs for transplantation in humans is to have more transgenes expressed in the pigs. We think we need now to add at least 10 different genes to our human DAF pigs.
"The second phase of rejection," she noted, "called acute vascular rejection, cannot be blocked by immunosuppressants. There are two ways to approach xenotransplantation at this moment. One is to develop immune tolerance. The second is to add more human genes to the donor pig, in order to have an organ which can be totally compatible with its human recipient.
"At this point, I think our technique - our approach to transgenesis - can be very helpful, first of all because it's efficient, second because it is not so expensive. So," Lavitrano concluded, "we can afford this effort."