A Boston doctor's ground-breaking experiments with xenotransplantation

01:19 PM EDT on Sunday, June 26, 2005

By G.WAYNE MILLER
Journal Staff Writer

I. A Golden Pig

Dr. David H. Sachs left his suburban home and headed to his Boston laboratory, a few miles distant. He was unusually excited. Overnight, experimental animal number 15502 -- a cloned, genetically engineered pig -- had arrived safely on a flight from its birthplace in Missouri.

Journal photo / Connie Grosch

Experimentally cloned pig, number 1611

A surgeon and immunologist, Sachs had distinguished himself in the field of conventional transplantation, in which human organs are used. His Transplantation Biology Research Center was a part of Massachusetts General Hospital, where he was on the staff. Sachs was a professor at Harvard Medical School and he belonged to the National Academy of Sciences's Institute of Medicine. He had written or co-written more than 700 professional articles. Science came as naturally to him as breathing.

One achievement, however, remained elusive.

For more than three decades, Sachs and other scientists had tried to find a way to get the diseased human body to accept living organs from healthy animals (pig valves used in heart surgery are not alive).

Xenotransplantation, as it is called, had the potential to save thousands of people who die every year because of a chronic shortage of human organs. Despite decades of public-education campaigns, the waiting list for all organs in the United States grew from 33,014 in 1993 to almost more than 88,000 last qweek, according to the United Network for Organ Sharing. Seventy percent sought a kidney, with almost 17,500 needing a liver, 3,628 in need of a lung, and more than 3,100 in need of a new heart. Almost 150 Rhoswe Islanders are awaiting some type of organ.

Sachs envisioned a time when patients would simply have their doctor order up an organ from the biomedical farm.

So far, the idea remained a dream.

SACHS PARKED his Saturn, then cleared the lobby guards. He needed a card key to operate the elevator and to open the outer door to his lab on a floor upstairs. Another lock secured the administrative suite, with yet one more protecting his inner office.

It was nearing 8 a.m. on Friday, Feb. 7, 2003.

Sachs checked his e-mail and put on a lab coat. He left his office and went to a separate facility, secured with another locked door. He opened it with his cardkey and stepped into a windowless domain constructed of cinderblocks painted a glossy institutional beige and lit with fluorescent bulbs.

This was the large-animal area, where experiments were conducted. Every Friday morning, Sachs gathered his scientists here for rounds. This was the weekly update on the pigs and baboons who had undergone surgery, or were about to.

STARTING IN 1973, when he was at the National Institutes of Health, Sachs had bred a line of miniature swine for use in his transplant experiments. By now, Sachs had bred more than 10,000 of the pigs, but he had never named one. It served no good purpose, he reasoned, for anyone to become emotionally attached to a creature unlikely to see ripe old age.

But number 15502 was unlike any of Sach's other pigs.

After years of trying, scientists at Immerge BioTherapeutics, a biotechnology firm with which Sachs collaborated, had succeeded in removing -- "knocking out" -- both copies of the gene that produces sugar molecules found on the surface of pig cells. These molecules are harmless to the pig -- but when a pig organ was transplanted into a baboon (or a person), the recipient's immune system recognized them as the calling cards of an invader. Within minutes, in a process called hyperacute rejection, white blood cells attacked and destroyed the organ.

Sachs also hoped that pig number 15502's unique genetic structure might provide protection against delayed rejection, which can set in weeks or months after a transplant. He hoped, too, that fewer anti-rejection drugs would be needed with this pig's organs. Eliminating the drugs -- a condition known as tolerance -- would be better still. Working with colleagues, Sachs had achieved tolerance in a small number of transplant patients who had received human organs, but the goal remained elusive in xeno research.

Sachs and Immerge collaborated with the University of Missouri, and it was scientists there who had cloned animal number 15502 from a cell lacking both copies of the sugar gene. The scientists wanted to name the pig born shortly before Thanksgiving -- the so-called double-knockout pig.

The name they chose was Goldie.

SACHS AND senior members of his staff passed through an electrically operated door into the larger of the lab's two pig chambers, home for the animals used in Sachs' conventional transplantation research. Some wore bandages from recent operations, and most had intravenous lines for administering medications and taking blood samples. The room was clean and bright with only a faint trace of odor. Sachs treated his animals well, for they were his most valuable tools. No computer could model the immune system.

Goldie stood in her cage, her ball and teddy bear at her feet.

"Should be a different color or something, don't you think?" said a scientist. But she was a pretty shade of pink like the other pigs, and she was cute. She bore a resemblance to Babe, the movie pig.

Sachs put on rubber gloves and reached into her cage. Goldie came to him without hesitation.

Sachs patted the animal, felt her ears, and tickled her snout. The double-knockout snorted agreeably. Sachs said nothing. He was listening to her breathing to confirm that her lungs were clear -- that she was healthy.

"A lot of hopes are riding on this pig," Sachs said.

They were -- not only for him and his field but for a $25-billion company based in Switzerland.

Journal photo / Connie Grosch

Dr. David H. Sachs

Novartis, seventh-largest drug firm in the world, was the principal investor in Immerge, a major source of funding for Sachs' lab. How long Novartis would continue without a breakthrough was often on Sachs' mind. Encouraged by a Salomon Brothers study that foresaw a $6-billion xeno market by 2010, the company (then known as Sandoz) had spent untold millions of dollars in the 1990s on Imutran, an English xeno firm. But Novartis closed Imutran in 2000 when its scientists failed to get pig organs to survive long enough in baboons to justify human trials.

Novartis' investment in Immerge was $10 million a year for three years, with an option to renew.

This was February of year three.

II. Animal O.R.

Goldie ate her last supper, a treat of dog food and raisins, early the evening of Tuesday, Feb. 18.

When attendant Shannon Moran arrived at 7 o'clock the next morning, the double-knockout was rooting around for breakfast, but Moran, following the standard pre-operative orders, could not oblige. She administered a sedative and soon Goldie settled down onto the softness of her lambswool. Moran swaddled the half-groggy pig in a sheet and hefted her up.

"All right, let's go, baby," she said.

Moran carried the pig to a small washroom between the two operating rooms and lowered her onto a stainless-steel table, where technician Meaghan Sheils shaved Goldie's abdomen with a well-worn set of barber clippers.

At one point, Goldie stirred.

"Almost done! Go back to sleep!" Sheils said.

Goldie's hair was vacuumed away and the pig was transferred to the operating room -- to a veterinarian's operating table. After tying the pig's limbs to the corners of the table, Shiels attached pulse and blood-pressure monitors, started an intravenous line, and connected Goldie to the anesthesia machine. The bellows began their mechanized breathing.

It was 7:40 a.m.

Dr. Kazuhiko Yamada, 43, one of Sachs' chief scientists, walked into the operating room. He would transplant Goldie's kidneys. Another of Sachs' chief researchers, Dr. David K.C. Cooper, 62, a British surgeon who had transplanted hundreds of human hearts, would transplant Goldie's heart. A third doctor would transplant Goldie's bone marrow.

"Big day!" said Kaz, as the Japanese surgeon was often called.

Kaz scrubbed Goldie's hairless belly with alcohol and two types of sterilizing surgical soap, then draped the pig; except for a glimpse of tail and two hind hooves, it could have been a small child beneath the sheet. Kaz photographed his assistant, Yosuke Hisashi, standing by Goldie, and then Hisashi photographed Kaz.

Kaz put his camera away, and he and Hisashi put on fresh pairs of sterile gloves.

ACROSS THE central corridor -- behind yet another locked, windowless door -- things were busy in the baboon room.

Like the pigs, the baboons lived alone in their cages -- but these cages were padlocked, and people approached cautiously. Baboons were strong and fast and sometimes threatening: make eye contact and they might snarl and try to grab you through the bars of their cages. And they were unpleasant-looking, with angry faces and brown or gray fur. No moviemaker had ever told the story of a cuddly baboon. No pig room featured a Pig Bite Kit.

Most of the baboons were not headed for surgery today, and an attendant was feeding them a breakfast of oranges, apples, bananas, and sweet potatoes. A videotape played on a TV monitor. The baboon library included Babe the Pig, ironically, but this morning's presentation was People of the Forest: the Chimps of Gombe. It was difficult to determine if the animals were watching. Too much else was going on that caught their attention.

Like all but one of the pigs now, the baboons had no names, only numbers. B214 watched quietly from a perch on the side of its cage as research fellow Dr. Kenji Kawaki, who would assist Cooper today, drew a blood sample from the intravenous line that ran inside a tether that was secured to the animal with a nylon jacket. At thirty pounds, B214 was an average-size baboon, a male born at a Florida breeding center on June 5, 1998.

LIKE AN operating room for people, this one, like its companion next door, was bright and clean -- except for the trash can, which was filling with bloodied sponges. The only sounds were the suctioning, the anesthesia machine, and the occasional soft exchange in Japanese between Kaz and his assistant. Ordinarily, the technicians would have the radio on or would be playing a CD -- '70s pop and U2 were favorites -- but this was no ordinary occasion.

No one had attempted anything like this before: five animals, six doctors, six technicians and assistants, one veterinarian and one operating room supervisor, all on one day.

Kaz's hands were deep inside Goldie when, shortly before 11 a.m., Sachs materialized at the O.R. door. Sachs no longer operated, and he had vowed not to come today; he feared his presence would be disruptive.

But in the end, he could not resist being an eyewitness to history.

"I couldn't not come!" he said, peeking in.

No one acknowledged him, if anyone noticed that he was there. Sachs fell silent. He was still watching when the door opened and Dr. Frank J.M.F. Dor, a Dutch research fellow, entered with baboon 214 in his arms. The animal was sedated, but not completely unconscious.

Dor lay the baboon on the operating room's second table, and he and the technicians tied its arms and legs and hooked up the monitors. B214's blood pressure cuff was green and labeled "Child Cuff." It fit well on a skinny primate's arm.

A technician placed a mask over B214's face and the animal fell asleep. B214 was washed and draped, and Dor began an incision into the baboon's belly.

Kaz had spent much of the morning freeing up the lobes of Goldie's thymus, buried in fat and tissue in the animal's neck. As it came into view, the thymus revealed itself to be small and squishy, like the inside of an oyster. Unappealing though it looked, the thymus was one of the most vital parts of the immune system. In other experiments, the Sachs group had demonstrated its potential for helping to achieve tolerance -- the elimination of immunosupressive drugs.

"Bowl," Kaz said.

He dropped a thymus lobe into a bowl of sterile saline water and brought it to a table where he and his assistant began to trim. B212, the baboon who would receive it, was already anesthetized and opened in the adjacent operating room. Kaz finished and went next door, bowl in hand.

It was 11:15. They were right on schedule.

With Kaz out of the way, Cooper and Kawaki stepped up to Goldie.

"Good luck," Sachs said.

"Thank you," said Cooper.

Sachs left, and Cooper and Kawaki prepared to remove Goldie's heart.

BY NOON, they were ready.

Cooper instructed technician Crystal Dugan to infuse Goldie with heparin, which would prevent coagulated blood from gumming up the heart's four housechambers once they had been stilled. Ten minutes later, Cooper finished sewing a tube into an artery running directly into Goldie's heart.

He asked Dugan to administer a dose of cardioplegia, a mix of chemicals that temporarily stops beating.

"It's running," Dugan said.

"Cold saline," Cooper said. He poured two bowlfuls directly onto the heart, and it gave up its beat in seconds. Dugan turned the ventilator off. An alarm beeped. "Equipment Alert. No Pulses Detected," the monitor read.

Kawaki cut the major vessels and lifted the organ out.

Goldie was gone.

KAZ, MEANTIME, had completed transplanting the thymus lobe into B212 and he was back with Goldie, set to harvest the kidney and other thymus lobe that he would transplant together into another baboon, B113, which lay anesthetized on the second table in the second operating room.

Others were taking more samples from the still-warm pig: slivers of pancreas, ear, liver, lymph nodes, tonsils, skin, stomach, large and small intestine, and salivary glands. "This is not the fun part," said Dor, assigned to take bone marrow, which necessitated sawing and cracking open bones. He said it reminded him of the butcher.

It was a few minutes past 1 o'clock. They were running a little behind schedule.

COOPER REMOVED the bandage that had been protecting B214's insides and felt around inside the abdominal cavity. Goldie's heart would not replace the baboon's: the purpose of this experiment was to study rejection, not determine if a pig heart could sustain a baboon's life. And so it would be a redundant heart -- a secondary organ of no value to B214, only to medical science, nestled inside the baboon in an area near the intestines where two major vessels would supply it blood. Even if Goldie's heart was rejected, B214 would still have its own and it might live to see another day.

Cooper and Kawaki needed less than an hour to connect the heart and unclamp the vessels, allowing the baboon's blood into it. So far, there was no sign of hyperacute rejection. But it was too soon to rule out.

"OK, let's have a look at this, Kenji," Cooper said. "Perfect. OK. Good. No bleeding from the suture line. Kenji will get paid this week!"

THE PHONE rang. O.R. supervisor Jim Winter answered. It was Sachs.

"No problems whatsoever," Winter said. "It's run quite smoothly actually."

Winter hung up the phone.

"Let's have the paddles," Cooper said.

Winter switched on the defibrillator and handed Cooper the electrodes. Cooper held them on opposite sides of Goldie's heart.

"OK," he said.

Winter hit the button but the heart did not budge.

"Try again, Jim."

"Twenty?"

"Twenty."

Winter turned the power up.

The second shock prompted the atria to quiver, but the heart's other two chambers did not go along.

"OK, we'll try again, Jim," Cooper said.

"Whenever you're ready."

The third zap failed, too.

"Try one more."

The fourth shock lifted the still-unconscious B214 off the table.

Goldie's heart began beating properly.

Blood coursed through it, and Cooper watched with satisfaction, remembering what hyperacute rejection looked like: within a few minutes, as you watched in growing disappointment, the transplanted organ would darken and die. The immune system was mighty indeed.

"I remember thinking: are we ever going to get over this? This is such a violent thing," Cooper said.

But this double-knockout heart was a marvelous pink.

AT 2:20 P.M., as Cooper adjusted B214's medications, Goldie was wheeled into the other operating room, where Dor would continue to harvest bone marrow -- this batch to be transplanted, by catheter, into another baboon later in the day. Soon, there would be precious little of the golden pig left.

Cooper was satisfied with the baboon's condition.

"You can wake him up," he said.

The technician shut off the anesthesia. B214 quivered momentarily, but did not come to. The beat of its native heart fluctuated wildly: a pulse of 100, then 88 a few seconds later, then 110. Cooper wiggled the animal's left arm while Winter stroked its face and ear, to no response.

"He's got rather large, dilated, fixed pupils, which worries me," Cooper said.

The pulse was dropping: from 77 to 51 in less than a minute.

But suddenly, B214's right leg began to move. Its pulse and blood pressure began climbing, and it was at this point that Meaghan Sheils arrived.

Sheils held the baboon's hand and stroked its face.

"Wanna wake up?" she said.

The animal remained still. Sheils scratched his ear and head, and tickled his nose and tongue.

"Put your silly tongue in!"

The animal stirred.

It was 5:34 p.m.

"OK, you can disconnect him," Cooper said.

They sat the animal up on the table. It looked like a child emerging bewildered from a dream.

"Hi, handsome!" Sheils said. "Say, 'That's what I always look like.' You're cute though, huh? Very cute!"

"You can take him back now," Cooper said.

Kawaki carried the animal out of the operating room and across the hall to the baboon room, where a fresh lambswool had been placed in its cage. B214 went slowly to its perch and sat quietly. Pain medication was administered.

"I think he's OK," Cooper said.

A short while later, Winter telephoned Sachs.

"Everything went very well," he said. "All of the baboons are back. All are sitting up. All are perched. So they look quite well."

"I'm elated!" Sachs told Winter. "That's the call I've been waiting for!"

The technicians cleaned the operating rooms and what remained of Goldie was placed into a plastic bag that would be stored in a freezer until a company that incinerated medical waste took it away.

"There's really not much left," a German research fellow commented. "Did you take the brain?"

"No," said veterinarian Mike Duggan. "Nobody wanted the brain."

III. Unprecedented Results

On Wednesday, March 19, 2003, Sachs's surgeons transplanted the organs from a second double-knockout pig into a new round of baboons.

The pig was numbered but not named; Goldie would be the first and only one to have that honor. The second pig had been cloned at Infigen, a Wisconsin biotechnology firm that also collaborated with Immerge and Sachs.

At large animal rounds on Friday, March 21, Sachs's scientists reported on the Wednesday xenotransplants. The operations had gone smoothly, hyperacute rejection had not occurred in any animal, and all of the baboons were recuperating nicely, the researchers said.

Thirty days after the first double-knockout transplants, the news regarding Goldie's baboons was equally encouraging.

Just the day before, they had brought B113 to the operating room to biopsy the kidney transplanted from Goldie. Unlike in the heart experiment, this organ was providing function: Kaz had removed one of the baboon's native kidneys, replacing it with Goldie's, and then he had tied off the baboon's other native kidney. He had also removed the baboon's thymus, replacing it with a lobe of Goldie's thymus. The aim was to induce tolerance.

"Kidney is pink. Looks good," said one of Kaz's assistants; measurements of kidney function showed that Goldie's organ was performing as well as a native one. There was no sign of rejection.

"This is the first time we've seen a pig kidney looking beautiful in a baboon this far out!" said Sachs.

One of Cooper's assistants gave the report on B214, the animal with Goldie's heart. "Beating well," said the researcher. "Shows no rejection."

Sachs was thrilled. With more cloned double-knockout pigs on the way, he wanted to ramp up the experiments. Having room for only 16 baboons at his facility, he was hunting around the Boston area for another place to house baboons.

THE SPRING of 2003 came, and kidney-baboon B113 flourished.

Some of the medications the animal had been receiving in the immediate post-operative period were being tapered off, with the hope that the pig's thymus would help the baboon's immune system to accept Goldie's kidney and the immunosupressives could be discontinued altogether.

At rounds on Friday, April 18, researchers noted that it had been 58 days since the baboon had received Goldie's kidney. "This is the longest we've ever had a functioning xeno kidney!" Sachs said.

But the update on heart-baboon B214 was less optimistic.

Cooper reported that Goldie's heart was still beating well -- if you looked closely, you could see it -- but the baboon didn't seem itself. Five days before, Cooper had noticed that the animal was walking unsteadily, wasn't eating well, and didn't respond to eye contact. B214 was receiving a multitude of medications, and Cooper theorized that the animal might be experiencing a drug reaction. So he adjusted the meds, to no significant effect.

"He certainly doesn't look quite right," Cooper said. He wondered aloud if they should give steroids.

"Do what you think is right," Sachs said. His only other advice was to get a biopsy of Goldie's heart -- soon. He did not want B214 to die -- especially not before a biopsy, which would provide valuable clues into what had been going on inside B214 for two months.

Later that Friday, one of Cooper's assistants could find no pulse on B214's abdomen. First thing the next morning, Cooper opened the animal and confirmed that Goldie's heart had, indeed, stopped beating. It had become enlarged and unnaturally firm, and it was mottled, a sign of rejection; the hyperacute variety hadn't claimed it, but a more drawn-out type apparently had. Cooper cut the lifeless organ out. The baboon survived and recovered.

"Very disappointed," Sachs said. After two months, he'd started to believe that Goldie's heart might beat for many more.

April neared its end, bringing another disappointment, with B113, the xeno kidney record-holder: the animal contracted an infection, apparently through a contaminated intravenous line. Antibiotics did not clear the infection and on Monday, April 28, Kaz took the animal into the operating room to replace the line.

An anesthesia mistake killed the animal.

But the kidney was fine -- no signs of rejection or compromised function. It had lasted twice as long as the very best of the pre-double knockout transplants, and twice as long as control experiments transplanting double-knockout kidneys without thymus.

Xeno tolerance seemed within reach.

"I am excited and delighted with the results so far," Sachs said. "I'm not yet convinced -- but very encouraged -- that this will make the kind of difference that I had hoped."

THE MONTH of June approached and new double-knockout experiments began with more cloned pigs and new baboons.

Sachs's scientists were fine-tuning their protocols: varying the dosages and duration of familiar drugs, substituting new drugs for old, using or not using pre-transplant radiation, trying a different procedure with the thymus. Weeks before a transplant, Kaz would open a double-knockout and graft a piece of thymus onto the kidney. The resulting organ, a so-called thymo-kidney, would then be transplanted into a baboon.

Having lost B113 when its kidney was still so good, Sachs had taken a special interest in B117 and B118, baboons that had received thymo-kidneys on the last day of April.

Both did well immediately after their transplants -- but then, on May 16, B117 died. The animal had been observed coughing before being found dead, and researchers hypothesized that it had aspirated a peanut. But no peanut was found on necropsy, and the cause would remain a mystery.

"It's another tragic loss -- the kidney was looking good," Sachs said. "But this doesn't discourage me or set me back. It makes me sad."

With B113 and B117 gone now, Sachs had taken an almost obsessive interest in thymo-kidney baboon B118. Every morning, he asked Koji Yazawa, one of Kaz's assistants, for the animal's condition. He had never monitored an animal so closely before.

At rounds on June 6, Koji Yazawa presented B118. Yazawa said that 37 days after the transplant, the baboon's creatinine level -- a measure of kidney function obtained by analzying a serum sample -- remained stable at about .85, a healthy reading.

"This animal, knock on wood," Sachs said, "looks very healthy, has no signs of infection. That's just great, Koji. I'm very happy."

He was also very worried.

SACHS HAD just learned that after investing more than $100 million in years of xeno research, executives in the Swiss headquarters of Novartis had decided to stop all spending. The loss to Sachs would be over $1 million dollars a year -- about 10 percent of his annual budget.

"The results, while extremely interesting from a scientific point of view, were still not close enough to a practical application," said Paul Herrling, head of corporate research for Novartis. "We are not a university who is doing science for science's sake -- at the end of the day when we invest, we need to have a product coming out of it."

After more than a decade of xeno research, Novartis questioned whether the xeno puzzle would ever be solved.

"Who knows?" Herrling said. "It's very, very difficult to predict."

Sachs could make no guarantees, either, but he believed he was closer than anyone ever had been: by October 2003, he had a large body of data demonstrating the advantages of his double-knockout pigs.

Hyperacute rejection had been decisively solved.

B118, the baboon with the thymo-kidney that Sachs had watched so carefully, had lived 81 days, a world record.

The other kidney recipients had not lived that long -- but most had died of complications, not rejection. Most of the heart transplants had survived more than two months. The double-knockout hearts seemed to require less immunosupression, which in itself was significant.

Sachs wanted to expand his experiments, not be forced to curtail them.

"My major concern right now is not whether the biology and the immunology are capable of overcoming the rest of the problems," he said. "It's whether or not we're going to be able to continue to get sufficient funding for these studies."

Sachs was working tirelessly on federal grants and he had assigned a member of his staff to search for philanthropists. He continued to work with Immerge as the company sought new investors. He went to the Mass. General Hospital administration. He wrote to the president of Novartis.

But the money wasn't there, and so the last double-knockout transplants for 2003 took place on Monday, Nov. 17. At large-animal rounds the following Friday, the scientists reported that all of the the baboons were doing well. So were the baboons from the previous round, which had taken place on Nov. 6.

One of those animals was B228, recipient of a double-knockout heart.

IV. A World Record

On the afternoon of Feb. 9, 2004, Sachs gathered his scientists for an update on the few remaining double-knockout experiments.

No new experiments were scheduled for at least two months: with Novartis' money gone, Sachs's collaborators had stopped producing double-knockout pigs. Only four of the animals remained, and Sachs wanted to use them for breeding, in the hope that nature would pick up where the cloners had left off. Sachs had no expertise in cloning, or money now to hire someone who did; he needed to establish a line of double-knockouts through normal reproduction.

The Feb. 9 meeting brought mixed news.

Encouragement came from the report on the last remaining heart baboon, B228, which had received the pig organ in a transplant on Nov. 6. Ninety-five days after the operation, a recent biopsy had disclosed, the heart remained healthy; of the eight transplants Cooper had performed using double-knockout hearts, B228's was now the second-longest survivor. Only B223, whose pig heart had lasted 110 days, had gone longer.

The discouraging news concerned B134, the last baboon to have received one of Kaz's thymo-kidneys. What happened was an unpleasant surprise, given the beginning history.

The Nov. 17 transplant operation had proceeded smoothly, not a hint of hyperacute rejection, and as the weeks had passed, B134 had become something of a celebrity. Kaz had been using a minimum of drugs as he progressed toward tolerance, and tests of the kidney's creatinine levels had consistently revealed normal function, with no evidence of longer-term rejection. This was the sort of outcome that prompted visions of clinical trials: tests on people.

The scientists were so pleased with B134 that just two days before, they had shot a video of the animal. They showed it at the meeting. The animal sat contentedly in its cage, eating a banana and grinning. This was about as close to cute as a baboon could get.

But just yesterday, B134 had been found dead in its cage.

"It's a great disappointment," Sachs said.

The animal had quickly been necropsied, and slides of various organs were projected on the conference room screen. The animal's lungs had swelled, one slide showed, but another slide revealed that the thymo-kidney was a pure and healthy pink. B134's other, native, kidney had been tied off, and Kaz's composite organ had been supporting the baboon's life since the transplant. It had done so for 83 days, a world record.

"Kidney looks perfect," said Meagan Sykes, another of Sachs' principal scientists. "What a tragedy."

One of Kaz's assistants projected a slide of the baboon's heart. It showed a scar, suggesting that acute myocardial infarction had killed B134.

"We have to figure out why this heart attack occurred and what we're going to do about it," Sachs said.

He sounded and looked tired. A drug reaction could have caused the heart attack, he theorized, although he later speculated that a small clot that had formed inside a catheter might have broken loose.

Almost a year had passed since the excitement of Goldie.

After some two dozen experiments, only one baboon, the heart-bearing B228, survived with a double-knockout organ.

THE REPORT on B228 at an April 2 conference was bittersweet: the animal's pig heart continued to beat, but it seemed to be hardening. It had been transplanted into the baboon 148 days before.

"This is now the world's record, isn't it?" Sachs said.

Kaz confirmed that it was, by a single day: the previous record, he said, was 147 days, held by the Mayo Clinic's Dr. Christopher G. A. McGregor.

"It is starting to fail but it still is very nice," Sachs said. "We'll see what happens now."

Sachs scheduled a biopsy of the organ for May 4 to get a definitive analysis, but by the end of April, the baboon's health was declining. Rather than lose the host animal, Sachs decided to remove the pig heart. Surgeons took it out on May 3, 179 days after it was implanted.

It had lasted half a year, longer than any xeno heart ever.

Since returning to Harvard in 1991, Sachs had tried many approaches in his pig-to-primate experiments. He had perfused monkey blood through pig livers, flowed baboon blood through a silicon matrix, used pigs similar to those developed at Imutran before it went under. "All those things helped but nothing has worked as well as these knockouts," Sachs said. They probably needed some further genetic engineering and the protocols might need to be further refined, but these pigs were the answer, Sachs was convinced.

"This is the time really to be doing it all," he said. "We should be expanding with the liver, the heart, lung, kidney. At this point, we should be raising large numbers of these pigs. We should be getting various groups working on it together and going in all these directions. This is the time for this thing to really be moving forward.

"Instead, we're cutting back -- I'm narrowing down to the minimum I can do to prove that this will work. It's so frustrating -- so exciting but frustrating. If it hadn't worked, I could very easily have walked away from it. There's lots of other things I have here that are working that I'm excited about. I could spend (more of) my time on that instead of beating my head against the wall to get funding."

But the dream still cast its spell.

V. Conviction

Starting with Goldie, pig-to-baboon operations were virtually a monthly exercise during most of 2003. But nearly half a year had passed since the last such operation when, on the morning of May 12, 2004, technician Sheils went to fetch a pig from one of the pig rooms. It was a double-knockout -- among the first that Sachs had bred from a herd of single-knockouts, which had been created before the Goldie line.

The animal had no name, just the number 16013, etched on a tag clipped to its ear -- but pig number 16013 had elicited an unusual degree of affection from the animal-care and operating room staffs. Perhaps it was because the animal had a crossed eye.

"Hey buddy," said Sheils, as she stroked the animal's head.

Another technician gave 16013 a sedative, and it drifted off. Soon, it was on an operating room table, awaiting Kaz's scalpel.

A few minutes later, a shaved and groggy baboon was brought into the room and placed on the other table.

"Hey, big guy!" Sheils said.

The baboon was settled onto the table, and a technician started the anesthesia.

"Ready, buddy?" Sheils said as she held the animal's hand.

The baboon went under quickly.

At 9 a.m. exactly, Kaz arrived. He shot photographs of the pig and the baboon, then cleaned and sterilized it and began to open it up. This was Kaz's second operation on the animal: six weeks before, he had grafted a piece of its own thymus onto one of its kidneys, creating the composite thymo-kidney he would put into B138 today.

Removing the thymo-kidney took little more than an hour. Kaz placed it in a bowl of saline water, trimmed it, and announced: "Ready for transplant."

He brought the organ to B138. Other surgeons had already removed one of the baboon's kidneys, and Kaz set about sewing his creation into place. Then he tied off the animal's native kidney. He worked without hesitation or break.

"He's fast," said a technician.

"Very fast!" Kaz agreed.

By noon, he was done. Pink Floyd's "Brain Damage" played on the radio. Kaz surveyed his transplant and said: "Nice." Then he asked a technician to take a photograph of him and his assistant by the baboon.

"Smile!" the technician said

"One more, please," Kaz said. "Transplant kidney is producing urine."

The double-knockout had not been left to die: Kaz had taken only the one, composite, organ, and the pig could live with its remaining kidney, which the surgeon might use in another experiment. The animal was brought back to its cage. Assistant Shannon Moran, who had shepherded Goldie off to surgery more than a year before, climbed in with it.

Feeling the aftermath of anesthesia, the pig tried unsuccessfully to stand. Moran cradled it on her lap.

"I know -- it's been a tough day," Moran said. "It's all right. I'm right here."

AS SUMMER passed and no investors signed on, Sachs grew increasingly disappointed with Novartis. He was dismayed that the firm had spent so much on another company whose pigs had not proved to be the answer -- and now they had given up on him, the scientist whose pigs had proved superior.

But Sachs' efforts in getting money were paying off: he was winning new grants and renewing existing ones. It was enough to keep his breeding program going and to conduct a few more xeno experiments, but not enough to bring him to the next milestone any time soon.

The next milestone would be FDA approval for clinical trials. The FDA had already drawn up guidelines for human tests. With restoration of funding to the Immerge-era level, Sachs envisioned a scenario in which trials could begin in as soon as two years.

"We would have to get pigs that the FDA would say were adequate for us to put into patients. And that would mean doing some Caesarian sections and having clean animals -- verified that they don't have any bacteria, etc. We'd have to set up our screening program for (the pig virus) PERV, which Novartis had set up but now is canceled. And then we'd have to convince the FDA we were ready to do it. You're talking about several years -- at least two or three years. And that's if everything started working today.

"But that's a lot less than I used to say. I used to say 10 years, so it's getting closer."

ON JUNE 27, 2004, B138 died of gastrointestinal bleeding, a complication unrelated to its thymo-kidney.

The summer passed, without the arrival of a white knight or the performance of another knockout experiment. Sachs kept his fellows busy on non-xeno research, especially tolerance.

On a Monday in late August, Sachs met with Kaz, veterinarian Duggan, and Scott Arn, who tracked the breeding programs. The topic was the small number of double-knockout pigs that had been produced from the breeding program Sachs had established. Sachs looked weary. Kaz was unusually subdued.

Arn handed out a sheet of paper that displayed the status of 40 pigs. Starting in the late summer of 2002, Sachs had begun mating single-knockouts, in the hope of producing double-knockouts. This was a hedge, a wise one, as it now happened: if cloning double-knockouts had not succeeded, inbreeding single-knockouts eventually would deliver double-knockouts that he could use in experiments -- and also to establish a natural line. This was basic Mendellian genetics.

Arn's sheet showed only 10 living double-knockouts from the breeding program. None had yet proved fertile. A double-knockout line had yet to be established.

"My overall, number-one priority has to be not to lose the knockouts," Sachs said. "At this point, we have to take the route of postponing experiments and save the line."

That pleased Duggan and Arn, who at this point did not want to use a double-knockout for anything other than breeding. Kaz, however, had conducted only one thymo-kidney experiment all year. He was anxious for more.

But Kaz did not put up a fuss: he simply asked when he might expect to have a pig.

Sachs looked at the sheet and his eye settled on animal number 16188, a female double-knockout born on July 18. He gave Kaz permission to use it. Kaz would create two thymo-kidneys inside the animal on Sept. 17, and transplant them into baboons in the fall.

"It's a risk, but we're going to do it," Sachs said.

THE MEETING ended, and Sachs returned to his corner office, which had such a magnificent view of Boston Harbor. The sun sparkled off the water.

"That was depressing," he said. "But the last thing I want to do is lose the animals."

Sachs was not beaten -- discouraged, but not beaten.

The xeno puzzle, he insisted, would be solved -- if not by him, then by someone else. "I can't believe we won't get there," he said. "I just hope it doesn't take longer than I've got to put into it."

EPILOGUE

Since August, Sachs has concentrated on establishing his line of double-knockouts, but it has been slow going. "It will be several more months before we have enough pigs to start the experiments again in earnest," he said recently.

Sachs continues to seek a new commercial partner that would allow him to expand his xeno program. Meanwhile, he likely will win renewal of a grant that will give him $13 million over the next five years. "This will enable us to continue the most exciting current studies," Sachs said, "but is not enough to expand the work as I would like to do at this phase."

G. Wayne Miller's latest book is THE XENO CHRONICLES, an expanded version of this story. Contact Miller at gwmiller [at] projo.com

Digital Extra: Browse previous reports by Journal staff writer G. Wayne Miller on pioneering medical technologies, at:

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