By David N. Leff

Cloning for human-body spare parts has been both discussed and decried as a spinoff of Dolly-triggered debate on cloning whole humans. Laws forbidding such genetic duplication of entire persons are pending at the state and federal levels. Some of this drafted legislation is feared to be so broad that it will put a damper on cloning animals for medical research as well.

Meanwhile, since 1988, an estimated 200 American patients with Parkinson's disease (PD) have received transplanted human fetal brain cells, to replace the dopamine-producing neurons that have died off. Loss of those cells inflicts disabling tremors, rigidity, unsteady gait and other hallmarks of PD.

The National Institute of Neurological Diseases and Stroke (NINDS) funded the first federal grant for human fetal tissue transplantation research in November 1994. It was awarded to neurosurgeon Curt Freed, at the University of Colorado, in Denver, who pioneered such transplants in the U.S. a decade ago. (See BioWorld Today, Feb. 7, 1994, p. 1.)

Cow Tissue Potential Transplantable Cell Source

But, in addition pursuing the still-experimental human fetal tissue therapy, Freed has dropped another shoe: cloning such dopamine-rich tissue in cows, as a potential future source of transplantable cells.

Freed is senior author of a report in this month's Nature Medicine, published May 1, 1998, and titled: "Somatic cell cloned transgenic bovine neurons for transplantation in parkinsonian rats."

The experiment it describes began with inputs from the University of Massachusetts (U. Mass), at Amherst, and Advanced Cell Technology Inc. (ACT), of Worcester, Mass. Colorado neuroscientist Michael Zawada is lead co-author of Freed's article.

"First," Freed told BioWorld Today, "scientists at U. Mass transfected fetal bovine somatic fibroblasts with a marker gene. Then, at ACT, cell nuclei from these fibroblasts were cloned into enucleated bovine oocytes, cultured for a week, and inserted in the uteri of recipient cows."

After six to seven weeks of gestation, the calf embryos — by then about four inches long — were collected and shipped to Colorado for implantation in the brains of rats that had been rendered unilaterally parkinsonian. (See BioWorld Today, Jan. 21, 1998, p. 1.)

Rats don't contract PD the way people do, but a chemical dripped onto their mid-brains, which poisons dopamine neurons, can create a reasonable, and quantifiable, facsimile of parkinsonianism. Rodents thus brain-damaged rotate in aimless circles. (See BioWorld Today, March 10, 1997, p. 1.)

Through millimeter-wide holes drilled in their skulls while the rats were under anesthesia, groups of the PD-mimicking rats in Denver received infusions of the cloned bovine dopaminergic neurons, aimed precisely at their brains' striatal region. Only one side of their mid-brains had had the striatal cells poisoned, so the rats spun in one direction only.

One month later, these animals showed significant remissions in their rotational motor behavior. Specifically, their unilateral circling was down to 58 percent of the pre-transplant rate. But animals given sham infusions stayed at 97 percent or more of their rotations. These contrasting performances persisted; after another month, rats treated with cloned bovine tissue were down to 52 percent of pre-transplant numbers; the control animals were up to 107 percent.

At that time, postmortem exams of clone-treated rats revealed large dopamine cell grafts in their striata, which paralleled their performance improvement. Controls had no such neurons.

"The use of cloned tissue," Freed commented, "is an improvement over the use of existing animal transplantation technologies, because it provides us with large quantities of identical cells. While this early-stage research shows promising results, it will be some time before we begin human studies."

Zawada said that, during this time, the combined team must overcome a "major hurdle" — namely, immune rejection of the bovine cells by the rats, and eventually by human recipients.

Rats in the experiment just reported got daily doses of cyclosporin A, the immunosuppressant that human graft recipients routinely receive, to guard against adverse immune attacks on donor organs.

"Dr. Freed predominantly transplants his human PD patients without cyclosporin," Zawada said. "If there is a rejection of their fetal human tissue, it would be very minor." But the bovine and porcine xenotransplantation grafts now on various drawing boards would pose a problem.

Double-Barreled Bead On Rejection

"When we transplant a bovine tissue into a rat or a different species," Zawada pointed out, "the problem is that the antigens on the surface of the transplants themselves will be recognized by the antibodies of the host." For future cloned tissues, he sees two possible paradigms:

One would be knocking out the surface antigens on bovine cells. Another would be introducing human markers — or rat markers when testing in rats. Or doing both, perhaps."

He added: "Whenever we talk about multiple genetic manipulations, then it becomes compoundedly difficult. Trying to lessen the immune response from the host, and so reduce the risk of rejection, is going to be our next step.

"I can't tell you the specific antigenic surface molecules that we will go after," Zawada said, "because we haven't yet decided."

Among Freed's 40 or more PD patients transplanted with human fetal tissues, Zawada recalled, the therapeutic effect has been "very variable. And this is why we hope that cloning tissue and having identical cells would help, perhaps." *