DOLLY'S DEMISE PUTS CLONING INTO QUESTION; FOR A FIELD MARKED MORE BY FAILURE THAN SUCCESS, DOLLY'S DEATH POINTS TO THE MIND-BOGGLING COMPLEXITY IN CLONING

THE ORLANDO SENTINEL
February 23, 2003 Sunday,
. BYLINE: Rosie Mestel, National Correspondent

For six years, she was a symbol of success -- a living, baa-ing, woolly demonstration that the cloning of mammals, long deemed impossible, was achievable after all. Now Dolly the world-famous sheep clone is dead, her life ended by a veterinarian's needle after she developed a progressive lung infection. In the weeks to come -- as her body is cut up and her organs diced, sliced and probed for answers to her relatively early death in midlife -- Dolly may take on a new, darker symbolism: that cloning may have consequences far beyond a creature's birth.

It is still unknown -- and may never be known -- if Dolly's lung infection, and her earlier problems with arthritis and obesity, have anything to do with her unnatural start in life. Middle-age sheep such as Dolly tend to develop joint problems. And Dolly's life was very unusual: The 6-year-old sheep was kept in a barn, where lung infections are more likely to take root than in the fresh outdoors. She took to standing on her hind legs and was at times overfed by journalists.

But her death is sure to strike a solemn note among cloning researchers, reminding them how complex and poorly understood the process remains. Cloning is still a field in its infancy, a craft characterized more by failure than success. In animal experiments, barely one in 100 cloned embryos survive to birth. Of those survivors, many have health problems such as heart, lung and weight abnormalities. Perhaps more seriously, a growing number of studies suggest that clones -- even the ones that make it to adulthood -- may carry within them subtle genetic abnormalities that could cause medical problems later.

Scientists say it is crucial that these subtleties be carefully explored and that many cloned animals' lives be monitored until death, so as to properly understand the scope and consequences of such problems. They say the findings underscore how irresponsible and dangerous it would be to try to clone human beings given the current state of knowledge. Beyond the ethical and moral debates about cloning, researchers say it will probably be years, if not decades, before science understands how to clone a human without introducing minuscule genetic errors that could create deformed and diseased children. "I think it's very likely that every single clone will have something wrong with it," said Davor Solter, director of the Max-Planck Institute of Immunobiology in Freiburg, Germany. "We are stuck with these problems today -- but it doesn't mean we will never know how to solve them." Cloning has long seemed to hover on the edge of possibility -- ripe for any outlandish claim -- partly because the concept is so tantalizingly simple. By replacing the genetic material of a fertilized egg with that of a body cell, you could, in theory, create an exact copy of another living organism. Achieving this goal was quite another matter.

It was only in the 1950s and 1960s that scientists successfully cloned frogs; mammals proved even tougher. In 1981, scientists reported they had cloned three mice -- but no one else could repeat the experiments, and some still suspect the claim was fraudulent. For a long while after, many believed that cloning mammals was impossible. At last, came the spectacular 1997 announcement of Dolly's birth -- and, soon after, of Cumulina the cloned mouse (named for the "cumulus cell" from which she was made), followed by cloned cows, goats, pigs, rabbits and one small kitten called Carbon Copy. Success, however, comes hard.

To get Dolly, it took 277 tries; Carbon Copy the kitten came after 86 failures. Even the most basic steps in the process remain difficult, akin to performing brain surgery with hammers, chisels and cattle prods. The process begins by drilling into the egg, removing its genetic material then replacing it with DNA from a body cell, such as a skin or mammary cell.

The next step is to trick the egg into thinking it's a bona fide, fertilized egg, so that it starts dividing -- a feat achieved with electrical or chemical triggers. Many clones fail at these early stages simply through physical damage to the nucleus or the cell's viscous innards, which are stuffed with chemicals essential for normal development. But some of the most serious problems -- the miscarriages and birth defects that make human cloning so difficult to contemplate -- run deeper than mere manhandling.

Embryo growth is a delicate chemical dance, filled with subtle cascades of gene and protein activity that are blunted, augmented or mistimed during cloning. Normally, embryo division is activated by precise changes in calcium levels, choreographed by a chemical, oscillin, which the sperm brings into the egg. In cloning, the timing of those changes is off, and the result can be errors and birth defects. Normally, eggs and early embryos are nurtured in the fallopian tubes and uterus. But in cloning, they are cultured in dishes until they're ready to be implanted. Studies show that embryo culture can cause errors even if the embryo isn't a clone. Scientists are also beginning to understand that cloned and in vitro embryos have different needs.

For example, Keith Latham, an associate professor in the Fels Institute for Cancer Research and Molecular Biology at Temple University in Philadelphia, has found that mouse clones thrive when fed glucose, while normal mouse embryos prefer other kinds of sugars. Many scientists believe that the most critical problem with cloning lies in the unnatural origin of a clone's genetic material. Normally, a fertilized egg contains genetic material from sperm and egg.

The genes in these two cells are turned on or off in a precise pattern that is in perfect readiness for the complex development of an embryo. But the genes of the body cells used in cloning are not in that state of readiness. When they are added to an egg, the egg tries its best to reprogram them.

But it wasn't built to do this job and it doesn't do it very well. "Skin cells, or whatever scientists use for cloning, are in a totally different state," said Rudolf Jaenisch, scientist at the Whitehead Institute for Biomedical Research in Cambridge, Mass. "No wonder it goes wrong. I am surprised that it works even partially." However, one cannot perform subtle behavioral tests on a cow or sheep to scan for errors of mind and mood -- the kinds of problems that might not matter as much in a cow but would be disastrous in a child. "For the life of me, I don't know if Dolly was autistic or schizophrenic -- I don't know how you assay that in a sheep," said Gerald Schatten, a University of Pittsburgh professor and director of the Pittsburgh Development Center, a biology research institute.

One also cannot know if clones have natural life spans because few have lived long enough yet. However, Jaenisch's group has examined 10,000 mouse genes in the liver and placenta of cloned mice and found that hundreds of the genes exhibited abnormal patterns of activity. "Even if clones appear normal at a younger age, they are not," Jaenisch said. "You just have to wait."