Steven Webber is a pediatric heart transplant surgeon at the Monroe Carell Jr. Children’s Hospital at Vanderbilt University in Nashville, Tennessee. When he started his medical career in the mid-1980s, he says, predicting how a patient would react to drugs that prevented organ rejection was a hit-or-miss proposition: You’d try one dose and see how it worked. If it didn’t work, you’d try another dose. You’d keep doing that until the patient stabilized. Alas, that could be a dangerous and harrowing process. Sometimes, the team wouldn’t find the right dose fast enough, and the patient would die. Other times, they’d go for the Hail Mary pass and administer such a large quantity of drugs that the patient would overdose.
Soon, thanks to a genetic test being developed by Webber’s team, it may be possible to predict how a transplant patient will react to anti-rejection drugs, even before he or she goes into the operating room. In a 2015 paper published in the journal Circulation, Webber and his colleagues showed that by testing for certain gene mutations, you can predict which patients will quickly process a drug, requiring higher doses, and which will process medicines more slowly, needing much lower doses.
“The spectrum of outcome is huge,” Webber explains. “Some patients do really well, and some do miserably. The cost of getting it wrong is too high. We’re trying to understand how a patient’s genetic makeup can lead to good versus bad outcomes.”
Webber’s work is one of the many ways that the transplantation field is transforming. Some researchers are screening for particular genetic mutations that may help predict whether a patient might be prone to rejection, or whether, after transplant, the patient’s body is ramping up for rejection. Others are repurposing gene sequencers to count fragments of DNA sloughed off from transplanted organs (the more fragments, the more likely the transplanted organ is struggling). Several teams are cloning genetically modified pigs to make them more suitable as a possible source of organs for transplant in humans. Meanwhile, academic researchers and start-up companies are using 3-D printers to try synthesizing organs from scratch and are already able to manufacture simple organs like the thyroid gland.
Some researchers are screening for particular genetic mutations that may help predict whether a patient might be prone to rejection, or whether, after transplant, the patient’s body is ramping up for rejection.
“People can do better with transplanted organs long term, and do so with less medicine,” says Anthony Atala, director of the Wake Forest Institute for Regenerative Medicine in Winston-Salem, North Carolina. “And in regenerative medicine, we now have the ability to implant tissues into patients using their own cells.”
Fear of Rejection
At this writing, more than 122,000 people are awaiting transplants in the United States. Every 10 minutes, another person joins the waiting list. Despite advances in techniques and medications, barriers remain, because there are never enough organs to help all the patients who need them. While 79 patients get new organs transplanted each day, there’s a chronic shortage of organs available for transplant. About 22 patients die each day waiting for donor organs. Most have been waiting for years.
But while scarcity is a big problem, rejection can kill even those patients lucky enough to receive a transplant organ. When you take a diseased organ out of a patient and replace it with a healthy organ from another person, the patient’s body always sees the new organ as foreign. To the immune system, “foreign” means “dangerous.” And so the immune system launches a frenzied attack on the new organ by unleashing antibodies to try to attack the new organ and kill it. The response is usually intense; far more intense than one might experience with, say, a cold.
Thus, it’s not a question of if a patient’s body will reject the organ, but of when and how much, experts say. Every transplant patient is in some state of rejection, however mild (see “The DNA of Matchmaking and Rejection,” page 34). The important question is: How well can doctors control the inevitable immune response?
In the 60-plus years since the first successful transplant, a kidney donated by the identical twin of the patient, doctors have countered this immune response in a couple of ways. First, they’ve learned to screen patients for particular types of cells. For instance, before a bone marrow transplant, doctors will screen for human leukocyte antigens (HLA), markers that stud the outside of most cells. These antigens have many variations, and each person has a unique combination of them, like a molecular bar code. If the donor’s combination of antigens is close to that of the recipient, it’s more likely that the transplant will go well. But it’s difficult to find a close HLA match, let alone an exact one. A similar situation also holds for solid organ transplants.
So, there’s always some degree of rejection. Doctors have countered this with a second line of defense: drugs that suppress the immune response, or immunosuppressant drugs. The breakthrough drug was cyclosporine, approved in 1983. Since then, a related drug, tacrolimus, has been added to the transplant doctor’s tools. Both these drugs work by suppressing an enzyme that signals immune cells called T cells that something foreign is posing a threat. Another commonly used drug, CellCept (mycophenolate mofetil), works by blocking T cells from dividing and multiplying.
While these drugs have greatly increased the success of transplants, prescribing them is a delicate balancing act. We all need our immune system. If we didn’t have a functional immune response, we’d be in danger of dying from every little virus or bacteria that happened by. In transplant cases, doctors use these drugs to suppress the immune system just enough to allow the transplanted organ to function, but not so much that the patient is too susceptible to infection or that the side effects from the drugs become intolerable.
Searching for Clues
One of the problems with rejection is that sometimes the reaction builds silently and slowly, only to explode suddenly in a barrage of terrible, life-threatening side effects. If you could detect signs of rejection early, doctors say, you could greatly improve the long-term health and survival of transplant patients.
One early detection approach involves repurposing gene sequencers to be fancy, ultrafast counting machines. Stephen Quake, a professor of bioengineering at Stanford University, has developed a way to use sequencers to measure the amount of foreign DNA in a blood sample, rather than conducting a painful biopsy, the current gold standard used to detect transplant rejection.
Imagine a process a bit like ripping up an old-fashioned phone book, throwing it into an aqueduct, then using the sequencer to reconstruct the directory. All blood contains what’s called cell-free DNA, bits of genetic code that have been sloughed off from whole cells. If you have sequenced the patient’s full genetic record, or genome, then you can match the patient’s DNA to that genomic record. Whatever doesn’t match is the foreign DNA, and the more of it there is, the more likely that the transplanted organ is struggling and may be on the cusp of being rejected.
Recently, a noninvasive blood test was shown to accurately predict lung transplant rejection as well as screen for potential infection. This process builds upon another 2014 study of 65 adult and pediatric heart transplant patients conducted by Quake’s team. They found that elevated levels of a certain kind of donor DNA could signal the beginning of a serious rejection episode.
“We see it as a way to prevent rejection. Doctors can respond by increasing immunosuppressant drugs,” Quake explains. “Substantial clinical tests still need to be done, but everyone gets that it’s better to have a simple blood test than a painful biopsy.”
In a different approach, Megan Sykes, the director of the Columbia Center for Translational Immunology, has developed a way to analyze the genetics of the immune system T cells. Patients’ T cells recognizing the donor are identified by specific genetic rearrangements; those patients showing a deletion of this specific subset of T cells seem to tolerate transplanted organs better than those patients whose T cells are not deleted.
“We think this method has potential to have a more specific biomarker than any identified for predicting outcomes, distinguishing patients who may be tolerant from those who might be about to reject,” Sykes says. “It may even have the potential to diagnose rejection. It’s the only assay [test] that looks specifically at the cells that are responding to the donor.”
In recent years, some scientists have gone off on a completely different tack: What if you could find a source of transplant organs that doesn’t cause all these problems? What if you could somehow modify, or create, a transplant organ that doesn’t send the patient’s body into a state of immune hysteria?
The first efforts along these lines were made by experimenting with pigs. Worries about transmission of viruses from the donor pigs to humans made this research controversial at first, but these concerns have been laid to rest.
Pigs and humans are actually fairly similar genetically. Some of our organs are about the same size, and conveniently, pigs grow fast. Using mechanical pig parts, such as heart valves, is already standard practice. Since their function is mostly mechanical, these smaller bits can be stripped of living cells, leaving a structure that works but doesn’t trigger the recipient’s immune system. Insulin-producing cells from a piglet have been transplanted into diabetic patients.
Doing this with larger organs is more complicated. Pig cells have several antigens that make primate immune systems go bananas. Often, a severe reaction may be triggered within seconds.
So, at the National Institutes of Health, and elsewhere, scientists have developed pig strains whose genes for certain antigens have been “knocked out.” For instance, some teams have replaced the pig genes for certain factors involved in coagulating blood with human genes, in an attempt to reduce rejection.
“We are using the cloning technique, just like Dolly the sheep,” explains Muhammad Mohiuddin, chief of the transplant section at the Cardiothoracic Surgery Research National Heart, Lung and Blood Institute in Washington, D.C. “We, along with our collaborator, Revivicor, take human genes and insert them into the pig genomes. Then we clone those pigs. We have transplanted hearts from these pigs into the abdomens of primates. The longest survival we have is almost three years. We are very close.”
Just Press “Print”
For the last 10 or 20 years, doctors have also dreamed of an even more pie-in-the-sky goal: manufacturing functioning organs from a patient’s own cells. Since the cells are the patient’s own, the immune system doesn’t go straight to red alert, and you don’t create the problems attendant with immune response and the side effects of immunosuppressant drugs, such as diarrhea, nausea, rash, depression, and even diabetes and cancer.
There’s a sort of boom-and-bust hype cycle in this “bioprinting” field, but solid results are finally starting to appear. Several companies have already succeeded in growing thin layers of living cells that can be used clinically. It’s now possible to grow thin sheets of functional muscle or skin cells. While not as thick as normal skin, with its deep-seated hair follicles, the lab-grown skin can be used on burn victims and greatly reduces the risk of deadly infection.
The breakthrough technology in the “grow-your-own organ” field has been 3-D printing. Ever more exact scanning technologies, such as MRI, computer-aided design, and 3-D printers, have already made it possible to create body parts out of nonliving tissue: exact replicas of a hip joint, fashioned in titanium, or exact models of external ear tissue. In 2012, an American baby, who had a birth defect that caused a weak windpipe, received the first 3-D–printed synthetic trachea, developed by researchers at the University of Michigan. And in China in 2015, a 3-year-old who had a skull three times the normal size as a result of hydrocephalus, or too much water on the brain, received a normal-size skull 3-D–printed in titanium. Researchers have also bioprinted external ears, spinal inserts, breast tissue, nipples for women who’ve had lumpectomies or mastectomies, and even lab-grown vaginas for women who have lost tissue due to cancer, trauma, or congenital abnormalities.
Again, as with pig heart valves, these are essentially mechanical parts or prosthetics, not organs with complex biochemical functions and structure. Early efforts to build replacement organs with a patient’s own cells usually involve harvesting stem cells and then growing them on a “scaffold” of a material that’s neutral or that will dissolve once the patient’s cells have grown numerous enough to take over. Simple organs such as a bladder — essentially a pocket for urine — have already been constructed this way.
Earlier this year, a Russian company announced that it had 3-D–printed a “construct” of a thyroid gland, a small and relatively simple organ. The researchers said they were now testing transplants of the gland in mice.
A biotech start-up, Organovo in San Diego, this year announced that it had 3-D–printed a thin layer of complex liver cells. This product, called exVive3D, has the same depth as five sheets of printer paper and contains several types of liver cells, not just one. A sheet so thin couldn’t be transplanted as a functional liver, but it can be used to test drug reactions, and backers say it’s the first step toward creating a functional liver from living cells.
Just the Beginning
There are, of course, many challenges in 3-D printing an organ as complex as a kidney or a liver. How do you replicate the complex structure of an organ and get the cells to talk to each other in the right way? Some groups have managed to print functioning but super-miniaturized hearts (less than a millimeter in diameter) and livers (just over 2.5 centimeters in diameter). How do you go from that microstunt to a human-size organ?
There’s also the challenge of making sure that the organ can interact with the rest of the body. How do you fashion blood vessels to get to all the cells that need blood? Jennifer Lewis, a professor of biologically inspired engineering at Harvard University, has developed a “bio-ink” process that makes it possible to print rudimentary blood vessels.
Much work remains to be done, of course, but many experts say the revolution in transplant medicine has only just begun.
“The main thing we’ve seen is a major increase in the number of tissues that we can put into patients,” says Atala, of Wake Forest. “In the next 10 years, we will see more and more things that can be treated by these techniques, and more patients will benefit from them. The main thing is that we’re already seeing cell therapies — that’s already here.”
The DNA of Matchmaking and Rejection
For now, the most common methods for matching donors and patients is through human leukocyte antigen (HLA) testing. HLAs are proteins that stud the outside of most cells. Each person has a unique combination of antigens that identifies a person’s cells as “self.” More than 100 different antigens have been identified, but six antigens seem to be crucial in transplantation.
Of these six, we inherit three from each parent. Except in cases of identical twins and some siblings, it’s rare (about 1 in 100,000) to find two unrelated people with the same six antigens.
The importance of the antigens varies. In bone marrow transplants, doctors try to find as close a match as possible. But kidneys can be transplanted even if no antigens match. However, the more antigens that match, the less likely a rejection becomes. There are three types of rejection:
Hyper-acute rejection happens when there’s a large mismatch between the antigens of the donor and the recipient. This kind of rejection can happen within seconds. It often occurs if a patient is given the wrong type of blood or in experimental transplants from one species to another. In organ transplants, the rejection may interfere with the blood supply to the donor organ and may cause it to die from lack of oxygen.
Acute rejection may occur at any time from the first week until three months after transplant. It often affects endothelial cells, which line blood vessels and lymph vessels. All patients exhibit some amount of acute rejection, which is treated with immunosuppressant drugs.
Chronic rejection occurs months to years later as the immune system slowly damages the donor organ, usually by a gradual buildup of fibrous tissue in blood vessels that eventually blocks the blood supply to the transplanted organ.