Cloning Trevor

Granted rare access to the labs of Advanced Cell Technology, the only U.S. group openly pursuing human cloning research for medical purposes, our correspondent spent six months tracking highly experimental work on the cells of a young boy with a life-threatening genetic disorder
A Shot in the Dark

In the cloning lab Kate had added several of Trevor Ross's skin cells to the droplet of medium that contained the egg she had just worked on. The cells floated in the solution, a hundredth the size of the egg itself. She drew a promising-looking cell up into the needle, slipped it through the tiny window already drilled in the egg's zona, and deposited it between the egg and the zona. She nudged the zona with the needle, trying to push the egg and the skin cell into contact, but apparently without luck. Kate frowned. "Let me get a better angle," she said, rotating the egg a little, after which she could see that the cells were touching ever so slightly.

Contact was essential, because the plan was to fuse the two cells with a pulse of electricity. Electricity also helps to "activate" the egg—a process, normally performed by sperm, that kick-starts embryonic development. ACT had tried fusing human cells only once before this—and all the eggs had died. Cibelli was apprehensive. "Fusion is kind of a shot in the dark," he told me. "There's nothing you can do with your hands, like going in mechanically and taking the chromosomes out. Here you have to rely on physics."

Before the fusion attempt, Kate repeated the entire extraction procedure with the other egg. Everything went smoothly, and this time she was able to tuck a skin cell securely between the egg and its zona, with lots of contact. "Yeah! Nice!" she exclaimed, giving a thumbs-up and looking at Cibelli. "See? See?" she said.

She turned the operation over to Cibelli. "Okay," she said, beginning an incantation: "Please fuse. Please fuse. Please fuse." She ran both hands over her face. "Who'd think two eggs could be so stressful?"

Cibelli set up at a second microscope—a much simpler one, designed to provide basic illumination from below. On the microscope platform he placed a fusion chamber—a round, shallow plastic dish with two wires lined up along the bottom, separated by a gap of only half a millimeter. He would soon place the eggs, one at a time, between these two wires. He rolled up his sleeves and attached a black lead to one electrode and a red lead to the other, completing the circuit. Then he did a test on the electrical-pulse generator that had been wheeled up beside him on a stainless-steel cart. "Let's play it safe," he said and set the generator for a pulse of ninety volts. Kate asked, "What do you think about being a little bit on the high side for this one, and the second one we drop it down to eighty?" Cibelli agreed and then placed the first of the two eggs in the fusion chamber. Under the microscope the egg looked good—round, with uniform cytoplasm and a healthy space between it and its zona.

If for some reason the egg and the skin cell didn't fuse, there was always plan B: isolating the skin cell's nucleus—the crucial packet containing its DNA—and injecting it directly into the egg. Another member of the cloning team, an expert in injection, was standing by in case this became necessary. But Cibelli was hoping not to have to resort to injection. Isolating a nucleus requires strong handling. "I'm always concerned that will be detrimental," Cibelli said. "We're not sure which method is going to be the one that is going to pay off. Too many things to try, and too few eggs!"

Using a tiny glass rod shaped like a miniature fencing foil, with a bulbous tip that can prod without piercing, Cibelli positioned the egg between the two electrodes at the bottom of the dish. His goal was to line it up so that the wires would send maximum current directly through the two cells, pushing them toward each other and confusing their membranes enough to make them fuse. Too little current and the cells wouldn't fuse; too much and the egg would be "fried," so to speak. With cow eggs the parameters had been well worked out, but with human cells it wasn't clear how much current was best.

If all went well with the cloning procedure, the nucleus of the reconstructed egg would have enlarged dramatically by the next morning. The genes that made the skin cell a skin cell would be silenced; others that had been laid away unneeded would be unpacked; and the genes that initiate early embryonic development would be activated. The process is not well understood, however, and frequently goes awry—which is one reason for the oft-cited fact that it took 277 cloning attempts to produce just one Dolly.

Cibelli flipped the switch, and a mechanical beep sounded as the machine sent a pulse of electricity through the cells. Everybody waited as Cibelli looked through his microscope. There was no camera mounted on this microscope, so only he could see what was going on. "How'd it do?" Kate asked.

"The cell is running away from the egg," he said.

"What?!" she said. "It's not supposed to do that."

Quickly they gave the second egg its pulse of electricity, so that they could then examine both closely.

"I don't see the zona," Cibelli said, peering at one egg. Kate took a look through the microscope. Each egg's zona had disappeared. This had never happened before. "They had a zona before," she said. "Where'd it go?"

"I don't think we got fusion," Cibelli said to Kate. They took a few minutes to load both eggs up again with DNA-binding dye, and then checked them with UV light under the other microscope. "There's no DNA, for sure," Cibelli said. "There's nothing there. There's no DNA."

We all stared at the eggs, which now glowed implacably on the video monitor. Without a zona, the eggs were completely unusable. Twenty-two thousand dollars and three months of waiting had apparently come to naught. The team couldn't try fusion again, because without a zona there was nothing to hold a skin cell in contact with an egg. They couldn't try injection either, because the technique requires anchoring an egg in one place with the holding pipette while a skin-cell nucleus is inserted. The zona's resilience makes it easy to grab with the pipette, but the egg inside is much more delicate. A holding pipette might well simply suck up the egg, destroying it in the process.

The team conferred briefly and came up with a last-ditch plan to save the eggs: using zonas from the cow eggs left over in the cow-cloning lab next door. Within half an hour Kate had vacuumed the eggs out of four or five cow zonas using a much bigger needle. The plan at this point was to suck the human eggs up into this needle and transfer them into empty cow zonas. "Well, if it works, it works," she said, "and if it doesn't, we're going to kill them."

She tried pulling one of the eggs up into the needle, with no luck—its outer membrane ruptured, and cytoplasm rushed through the tear. Kate groaned. "It lysed. Shoot." Looking at Cibelli apprehensively, she asked, "What do you think? Should I try the other one?" He nodded.

With the second egg she proceeded more slowly. As suction drew it into the needle, the egg elongated and distorted beyond all recognition. "It isn't going to like this at all," Kate said. When the egg had inched all the way into the tube, she moved it over to the empty cow zona and began extruding it. As the egg exited the tube, it began to resume its normal round shape.

"Oh, I don't know, Jose, I think this doesn't look good," Kate said.

"Keep going, you can do it," Cibelli replied.

The egg was almost all the way out of the tube, almost safe in the zona, when it suddenly burst. Cytoplasm flooded the microscope field. Everybody in the room let out an anguished yell.

"You tried," Cibelli said to Kate. "Okay, party's over."

It was midnight, and the dispirited team began breaking down the equipment. "We'll have more eggs," Cibelli said, to nobody in particular. "Hopefully, anyway."

This past January, as ACT's scientists were preparing to work with Trevor Ross's skin cells, a newly created presidential advisory group known as the Council on Bioethics met for the first time. President Bush had created the panel to advise him on controversial matters such as stem-cell research and cloning. "You can help be the conscience of the country," the President told his new advisers, adding that they would "help people ... come to grips with how medicine and science interface [with] the dignity of life, and the notion that life is—you know, that there is a Creator."

Bush's remarks set off alarm bells in the scientific community. So did the composition of the council. Expressing a widely held view, a prominent bioethicist, Arthur Caplan, wrote in an editorial that the new bioethics council was "stacked" with members likely to support the President's opposition to cloning research of any kind. More generally, he argued that the council "will rely on religious rather than secular principles to navigate its course."

At this writing the fate of therapeutic-cloning research remains uncertain. A complete ban in the United States is a distinct possibility. At the end of March the President signaled his ongoing opposition to human embryo research of almost any kind by describing Elias Zerhouni, his highly respected new nominee to head the National Institutes of Health, as a man who "shares my view that human life is precious and should not be exploited or destroyed for the benefits of others." And the Bush Administration even appeared to have international aspirations for its anti-cloning agenda: in February a U.S. delegate to the United Nations proposed a "global and comprehensive" ban on all human cloning research.

That proposal met with firm opposition, however, from other countries. In large part this was because therapeutic-cloning research like that being done at ACT is already under way elsewhere around the world. Britain's House of Lords, for instance, has given the go-ahead for therapeutic-cloning research to proceed, with government oversight and funding. And researchers in China claim that they have used therapeutic-cloning techniques to create human blastocysts, and that they have isolated stem cells from embryos created using human cells and rabbit eggs.

In the United States the broad consensus in the scientific community is that therapeutic-cloning research merits significant exploration, and that real progress is likely only with government funding and support. "Such research," Harold Varmus, the former NIH director, wrote last year in The New York Times, "is vital not just to biotechnology companies and their investors, but to the nation as a whole. By structuring our system so that only those with private funds or a commercial motive do this pioneering work, we curb our full capacity to expand our scientific understanding." To put it another way: as long as a federal-funding ban remains in place, the organizations most likely to move forward with therapeutic-cloning research will be companies like ACT—which, despite generally noble intentions, are bedeviled by the need to raise money, generate buzz, and please investors.

Last summer, in recognition of the importance of government support (but with considerable reluctance), Bush announced a small exception to the federal-funding ban: he authorized federal funds for work with a limited number of human embryonic-stem-cell lines already in existence. But he quickly went on to point out that the government would not fund experiments involving the further destruction of embryos. Cells later obtained from embryos—which would include, obviously, cloned embryos—would remain off limits to federally funded scientists. And Congress may, of course, end up banning cloning research altogether.

What is clear is that the potential fruits of therapeutic-cloning research will not come soon enough for Trevor Ross. In February, doctors detected the first signs of childhood cerebral onset of ALD. All hopes of developing an experimental cure for Trevor were dashed; time had run out. The Rosses immediately scheduled a more traditional cord-blood transplant, fully aware of the risks and of the odds of failure.

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