Title:
Ten years on, has the cloning dream died? By: Aldhous, Peter, Coghlan, Andy, New Scientist, 02624079,
7/1/2006, Vol. 191, Issue 2558
This week: Cloning: International news and exclusives
The birth of Dolly the sheep heralded a biological revolution. But her legacy is not what we imagined
WERE she still with us, the most famous sheep in history would doubtless celebrate her 10th birthday on 5 July by
posing for the world's media and gobbling down a celebratory cake of barley and molasses. Dolly the cloned sheep
seemed to know she was special and, unlike her creators, loved being centre stage.
"It was fortunate for us that Dolly was such a media star," says Ian Wilmut, who headed the group at the Roslin
Institute in Midlothian, UK, that created her. "In a biological sense we realised the enormity of what we achieved.
What I didn't anticipate was the enormous interest that followed."
Dolly died in February 2003, but her iconic status as the first mammal cloned from a specialised adult cell lives on.
Yet 10 years after her euphoric birth, the hopes, and fears, that cloning would spark a revolution in biotechnology,
animal breeding and human medicine have so far proved wide of the mark.
Dolly was created by reprogramming an adult cell — in her case a sheep's udder cell. At the time, the received
biological wisdom was that a mammalian cell's developmental clock could never be turned back in this way. "There
was massive relief and surprise when it worked," says Alan Colman, former head of PPL Therapeutics, the company
collaborating with Roslin to exploit cloning technology.
From the beginning, though, politics began to cloud the issue. Headlines often concentrated not on the Roslin team's
scientific achievement, nor on the wider possible applications of cloning technology, but on concerns about cloning
humans. In response, some scientists focused on the potential benefits of "therapeutic" cloning — deriving embryonic
stem cells genetically matched to individual patients, which might be used to grow tissues to replace those lost to
disease or injury.
In fact, the Roslin team had pursued cloning primarily to improve livestock, hoping to create hordes of identical
animals from one elite individual. For PPL, cloning offered a unique means of creating genetically modified animals,
to produce drugs in their milk, or to provide organs for transplantation into people.
Fast forward, and few of these predictions have come to pass. Humans have not been cloned, despite claims by
maverick scientists. "There are no cloned Hitlers marching around," Colman observes. Therapeutic cloning has yet to
live up to its billing, and may even have muddied the debate over stem cell research , while there have been mixed
successes with applications in animal breeding . Several of the companies formed to exploit cloning technology have,
like Dolly, met their demise, and only now is cloning starting to pay its way in a few select areas.
The main problem is that creating healthy embryos by cloning has proved much harder than anticipated. To make
Dolly, Wilmut, Keith Campbell and their colleagues started by fusing nuclei from 277 cells of an adult ewe's udder
with "enucleated" sheep egg cells — those stripped of their own chromosomes. Somehow the eggs seemed to
reprogramme the genomes of the adult cells, allowing some to start developing into embryos. But only 29 grew to the
point at which they could be transferred into the uterus of a surrogate mother, and only one live lamb — Dolly — was
born.
When these results appeared in Nature in February 1997, they were seen as a proof of principle, so no one dwelled
on the numbers. Researchers assumed it would quickly be possible to boost the efficiency of cloning, by refining
techniques used to manipulate and culture the cells and embryos. Some progress has been made: by tinkering with
their methods, researchers have managed to clone about a dozen mammalian species. And those cloning livestock
now lose fewer fetuses during gestation than they used to. But there has been no leap forward to transform the
dismal numbers game.
"Essentially, the efficiencies are not much different than in the early days," says Bob Lanza of cloning company
Advanced Cell Technology (ACT) in Worcester, Massachusetts. "We really don't know what's going on."
Normal genes?
One major problem is that cloning causes abnormalities in "epigenetic" modifications to DNA, such as the addition of
methyl groups, which alter the activity of critical genes. The failure to reset the epigenetic changes present in the
adult cell used for cloning may undermine the survival of the resulting cloned embryo.
So researchers are still looking for the magic recipe to turn cloning into a routine procedure. Last December, for
instance, a team led by Teruhiko Wakayama of the RIKEN Center for Developmental Biology in Kobe, Japan,
announced that it could boost the number of implantable mouse embryos up to fivefold by treating them with a drug
called trichostatin A, which wipes clean many epigenetic changes to DNA.
Other researchers doubt whether such simple treatments will solve the problems with efficiency. "They can never
ensure that a nucleus is completely reprogrammed into a normal embryonic state," says Konrad Hochedlinger, a
cloning specialist at the Massachusetts General Hospital in Boston.
Then there is the related question of whether clones can ever be "normal". The large numbers of cloned animals that
die during gestation or around birth bear testament to serious problems, and whether those that survive into
adulthood are truly healthy is still a matter of debate.
In the early days, cloning specialists worried that cloned animals would age prematurely due to shortened telomeres
— caps on the ends of mammalian chromosomes that protect genes but erode as cells divide. Today, this is not
thought to be a major problem, and ACT and other firms have found no obvious clinical abnormalities in their cloned
animals. But it may be a different story at the cellular level, because of the epigenetic abnormalities.
At the Massachusetts Institute of Technology, researchers led by Rudolf Jaenisch have compared the epigenetic
profiles of cloned and normal mice, and found massive differences that affect the activity of a wide variety of genes. "I
don't think that cloned animals will ever be completely normal," says Hochedlinger, who worked on the experiments
with Jaenisch.
Pharming dreams
Dolly's legacy is not completely dominated by disappointment, however. PPL hoped cloning would usher in the era of
"pharming" — genetically modifying animals to produce useful proteins in their milk. The company also hoped to help
many people waiting for organ transplants by engineering pigs whose organs would not immediately be rejected by
the human immune system.
PPL went bust in 2004. Its plans to market alpha-1-antitrypsin, an enzyme to treat the lung disease emphysema,
sank due to inconclusive results and a lack of money. Rather than embracing the idea of making proteins in animals,
the biotech industry has concentrated on other methods, such as cultures of genetically modified mammalian cells,
which pose fewer regulatory hurdles.
But cloning is finally starting to be accepted as a way to deliver some pharmed products, because it offers a much
more precise way of making genetically engineered animals. Traditionally, transgenic animals were made by injecting
gene copies into a new embryo — an inefficient and chaotic process. Genes can land anywhere in the genome,
disrupting other genes. Now researchers can insert DNA at a precise position into a single adult cell, and then clone it
to create the desired animals.
GTC Biotherapeutics in Framingham, Massachusetts, is now using cloning to make animals that produce novel
proteins, and cloning is starting to make its mark for applications such as gene "knockouts", where it is the only
option.
For example, Hematech, a company in Sioux Falls, South Dakota, has created cows that can make human
antibodies, by knocking out the animals' own antibody genes and adding the human equivalents. Company president
Jim Robl says that cloning was necessary both for the knockouts and also to "rejuvenate" the cells between rounds of
genetic engineering.
And the dream of creating pigs that can serve as organ donors is still alive, with a descendant of PPL called
Revivicor in Blacksburg, Virginia. It has made "double knockout" pigs in which both copies of a gene for the enzyme
alpha 1,3 galactosyl transferase are disabled. This enzyme adorns pig cells with a sugar against which the human
immune system mounts a massive attack, causing rapid "hyperacute" rejection. Kidneys and hearts from the
knockout pigs transplanted into baboons lasted up to four months. The organs were still rejected, so Revivicor has
added two other genes to its knockout pigs: one to prevent blood clots in the transplanted organs; the other to mop
up antibodies against the organs. The next step is to transplant organs from these animals into primates.
Finally, cloning can be used to create knockout animals in which to study human disease. For instance, Randall
Prather of the University of Missouri-Columbia is trying to clone pigs missing the CFTR gene, which causes cystic
fibrosis when faulty.
Some of these applications would not immediately have sprung to mind with the birth of Dolly exactly 10 years ago.
Dolly remains an icon, and one whose legacy remains to be fully written. She may yet turn out to be the trigger for
many ground-breaking innovations — which isn't at all bad for a rather ordinary-looking sheep that just happened to
love the limelight.
"At the time Dolly was created the received wisdom was that an adult cell's developmental clock could never be
turned back"
"Cloning is starting to be accepted as a way to deliver some products, as it is a more precise way to engineer
animals"
TEN YEARS OF CLONING 1996
 July 1996 Dolly born, the first mammal cloned from an adult cell. Named after singer Dolly Parton
1997
 February 1997 Dolly announced to the world in Nature (vol 380, p 64)
 December 1997 World Health Organization demands ban on cloning humans
1998
 December 1998 Dolly procedure repeated in mice Clones of clones made
1999
 April 1999 First cloned goat born
2000
 March 2000 First cloned pig born
2001
 January 2001 Fertility specialists Panos Zavos and Severino Antinori announce plans to clone a human
January 2001 Dolly revealed to have arthritis
 September 2001 France and Germany propose UN treaty to ban reproductive cloning
November 2001 Advanced Cell Technology claims to have cloned human embryo that grows to six cells
December 2001 first cloned cat born
2002
 December 2002 Raelians claim on Christmas day to have cloned humans. No evidence ever produced to support
claim
2003
 February 2003 Dolly dies from pulmonary adenomatosis, a common viral disease in sheep kept indoors
May 2003 First cloned horse, deer and mule born
November 2003 US Food and Drug Administration says studies indicate meat and milk from clones is safe, but no
policy yet introduced that allows cloned livestock to be eaten. Effective moratorium remains in place today
2004
 February 2004 Woo Suk Hwang's team at Seoul University in South Korea claims to have made first human
embryonic stem cells from cloned human embryos
November 2004 Plans of UN treaty banning cloning abandoned
December 2004 First cloned pet cat sold
2005
 November 2005 Korean cloning scandal begins as Hwang's team accused of procuring eggs unethically
April 2005 First cloned dog, Snuppy, born to Hwang's team
May 2005 Hwang claims he has produced 11 customised cultures of human embryonic stem cells from cloned
human embryos
2006
 January 2006 Science withdraws two key cloning papers authored by Hwang
June 2006 Hwang's trial begins. If found guilty he faces 5 years in jail
May 2006 Hwang and some colleagues indicted on fraud and embezzlement charges, and charged with breaching
bioethics laws
PHOTO (COLOR): A star is born
~~~~~~~~
By Peter Aldhous and Andy Coghlan
THE PATTER OF CLONED HOOVES
According to some predictions, farmyards should already be rattling with the sound of cloned hooves. But agricultural
applications have so far been limited by the low efficiency of animal cloning — and by the fact that regulatory
agencies have yet to approve the meat and milk of clones and their offspring for human consumption.
Even the most optimistic cloners did not expect the technique to be routinely used to create animals destined for our
dinner plates. Rather, they wanted a way to copy genetically valuable stud animals, which would then be used for
breeding in the normal way. At the moment this is not a viable prospect. "The efficiency is still too low for commercial
agricultural applications," says Xiangzhong Yang of the University of Connecticut in Storrs.
ViaGen of Austin, Texas, can clone a stud bull for about $14,000. Company president Mark Walton hopes that this
can be halved, but says a bigger obstacle is the fact that the US Food and Drug Administration (FDA) has not yet
decided whether cloning poses a risk to the food supply. Studies have not revealed any problems, and Walton is
hoping for an FDA ruling by the end of the year. ViaGen then aims to start cloning around 500 stud bulls a year — a
tiny fraction of the total US market.
For now, ViaGen is concentrating on niche applications, such as bucking bulls used in rodeo contests. Its main
money spinner, though, is cloning horses. ViaGen charges about $150,000 to clone a horse, but for breeders in
lucrative events such as cutting, in which a rider splits cattle off from a herd, that may be seen as worth their while —
especially if champion cutters have been gelded and so cannot be put out to stud.
In Sausalito, California, Genetic Savings and Clone is exploiting another small niche: cloning much-loved cats for
customers for whom a price tag of $30,000 is no obstacle. The company is also trying to clone dogs; a feat so far
achieved only by the South Korean team led by Woo Suk Hwang.
Cloning has also been touted as a last chance for rescuing endangered species. The main difficulty is finding a
supply of suitable eggs. "For livestock, we go to the slaughterhouse and collect ovaries," says Randall Prather of the
University of Missouri-Columbia. "You can't go to the endangered species slaughterhouse."
The company Advanced Cell Technology is trying to get round this problem by inserting the genetic material of
endangered species into the eggs of related non-endangered animals. It cloned two endangered Asian bovines: a
gaur, which later diedof dysentery, and a banteng, which is still alive. But attempts to clone an Asian buffalo — the
anoa — failed, presumably because this species is too distantly related to domestic cattle.